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  1. Inhibition of Geranylgeranyl Transferase-I Decreases Cell Viability of HTLV-1-Transformed Cells

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    Cynthia A. Pise-Masison

    2011-10-01

    Full Text Available Human T-cell leukemia virus type-1 (HTLV-1 is the etiological agent of adult T-cell leukemia (ATL, an aggressive and highly chemoresistant malignancy. Rho family GTPases regulate multiple signaling pathways in tumorigenesis: cytoskeletal organization, transcription, cell cycle progression, and cell proliferation. Geranylgeranylation of Rho family GTPases is essential for cell membrane localization and activation of these proteins. It is currently unknown whether HTLV-1-transformed cells are preferentially sensitive to geranylgeranylation inhibitors, such as GGTI-298. In this report, we demonstrate that GGTI-298 decreased cell viability and induced G2/M phase accumulation of HTLV-1-transformed cells, independent of p53 reactivation. HTLV-1-LTR transcriptional activity was inhibited and Tax protein levels decreased following treatment with GGTI-298. Furthermore, GGTI-298 decreased activation of NF-κB, a downstream target of Rho family GTPases. These studies suggest that protein geranylgeranylation contributes to dysregulation of cell survival pathways in HTLV-1-transformed cells.

  2. Arecoline decreases interleukin-6 production and induces apoptosis and cell cycle arrest in human basal cell carcinoma cells

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    Huang, Li-Wen [Department of Medical Laboratory Science and Biotechnology, Kaohsiung Medical University, Kaohsiung 80708, Taiwan (China); Hsieh, Bau-Shan; Cheng, Hsiao-Ling [Department of Biochemistry, Faculty of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung 80708, Taiwan (China); Hu, Yu-Chen [Graduate Institute of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung 80708, Taiwan (China); Chang, Wen-Tsan [Graduate Institute of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung 80708, Taiwan (China); Division of Hepatobiliarypancreatic Surgery, Department of Surgery, Kaohsiung Medical University Hospital, Kaohsiung 80708, Taiwan (China); Chang, Kee-Lung, E-mail: Chang.KeeLung@msa.hinet.net [Department of Biochemistry, Faculty of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung 80708, Taiwan (China); Graduate Institute of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung 80708, Taiwan (China)

    2012-01-15

    Arecoline, the most abundant areca alkaloid, has been reported to decrease interleukin-6 (IL-6) levels in epithelial cancer cells. Since IL-6 overexpression contributes to the tumorigenic potency of basal cell carcinoma (BCC), this study was designed to investigate whether arecoline altered IL-6 expression and its downstream regulation of apoptosis and the cell cycle in cultured BCC-1/KMC cells. BCC-1/KMC cells and a human keratinocyte cell line, HaCaT, were treated with arecoline at concentrations ranging from 10 to 100 μg/ml, then IL-6 production and expression of apoptosis- and cell cycle progress-related factors were examined. After 24 h exposure, arecoline inhibited BCC-1/KMC cell growth and decreased IL-6 production in terms of mRNA expression and protein secretion, but had no effect on HaCaT cells. Analysis of DNA fragmentation and chromatin condensation showed that arecoline induced apoptosis of BCC-1/KMC cells in a dose-dependent manner, activated caspase-3, and decreased expression of the anti-apoptotic protein Bcl-2. In addition, arecoline induced progressive and sustained accumulation of BCC-1/KMC cells in G2/M phase as a result of reducing checkpoint Cdc2 activity by decreasing Cdc25C phosphatase levels and increasing p53 levels. Furthermore, subcutaneous injection of arecoline led to decreased BCC-1/KMC tumor growth in BALB/c mice by inducing apoptosis. This study demonstrates that arecoline has potential for preventing BCC tumorigenesis by reducing levels of the tumor cell survival factor IL-6, increasing levels of the tumor suppressor factor p53, and eliciting cell cycle arrest, followed by apoptosis. Highlights: ► Arecoline has potential to prevent against basal cell carcinoma tumorigenesis. ► It has more effectiveness on BCC as compared with a human keratinocyte cell line. ► Mechanisms involved including reducing tumor cells’ survival factor IL-6, ► Decreasing Cdc25C phosphatase, enhancing tumor suppressor factor p53, ► Eliciting G2/M

  3. Tumor necrosis factor (cachetin) decreases adipose cell differentiation in primary cell culture

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    Martin, R.J.; Jones, D.D.; Jewell, D.E.; Hausman, G.J.

    1986-01-01

    Cachetin has been shown to effect gene product expression in the established adipose cell line 3T3-L1. Expression of messenger RNA for lipoprotein lipase is suppressed in cultured adipocytes. The purpose of this study was to determine the effect of Cachetin on adipose cell differentiation in primary cell culture. Stromalvascular cells obtained from the inguinal fat pad of 4-5 week old Sprague-Dawley rats were grown in culture for two weeks. During the proliferative growth phase all cells were grown on the same medium and labelled with 3 H-thymidine. Cachetin treatment (10 -6 to 10 -10 M) was initiated on day 5, the initial phase of preadipocyte differentiation. Adipocytes and stromal cells were separated using density gradient, and 3 H-thymidine was determined for both cell types. Thymidine incorporation into adipose cells was decreased maximally (∼ 50%) at 10 -10 M. Stromalvascular cells were not influenced at any of the doses tested. Adipose cell lipid content as indicated by oil red-O staining was decreased by Cachetin. Esterase staining by adipose cells treated with Cachetin was increased indicating an increase in intracellular lipase. These studies show that Cachetin has specific effects on primary adipose cell differentiation

  4. Adipose Tissue Inflammation Induces B Cell Inflammation and Decreases B Cell Function in Aging

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    Daniela Frasca

    2017-08-01

    Full Text Available Aging is the greatest risk factor for developing chronic diseases. Inflamm-aging, the age-related increase in low-grade chronic inflammation, may be a common link in age-related diseases. This review summarizes recent published data on potential cellular and molecular mechanisms of the age-related increase in inflammation, and how these contribute to decreased humoral immune responses in aged mice and humans. Briefly, we cover how aging and related inflammation decrease antibody responses in mice and humans, and how obesity contributes to the mechanisms for aging through increased inflammation. We also report data in the literature showing adipose tissue infiltration with immune cells and how these cells are recruited and contribute to local and systemic inflammation. We show that several types of immune cells infiltrate the adipose tissue and these include macrophages, neutrophils, NK cells, innate lymphoid cells, eosinophils, T cells, B1, and B2 cells. Our main focus is how the adipose tissue affects immune responses, in particular B cell responses and antibody production. The role of leptin in generating inflammation and decreased B cell responses is also discussed. We report data published by us and by other groups showing that the adipose tissue generates pro-inflammatory B cell subsets which induce pro-inflammatory T cells, promote insulin resistance, and secrete pathogenic autoimmune antibodies.

  5. Piperlongumine decreases cell proliferation and the expression of cell cycle-associated proteins by inhibiting Akt pathway in human lung cancer cells.

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    Seok, Jin Sil; Jeong, Chang Hee; Petriello, Michael C; Seo, Han Geuk; Yoo, Hyunjin; Hong, Kwonho; Han, Sung Gu

    2018-01-01

    Piperlongumine (PL) is an alkaloid of a pepper plant found in Southeast Asia. PL is known to induce selective toxicity towards a variety of cancer cell types. To explore the possible anti-lung cancer effects of PL, A549 cells were treated with PL (0-40 μM) for 24 h. Alterations in the expression of cell cycle-associated proteins (cyclin D1, cyclin-dependent kinase 4 (CDK4), CDK6 and retinoblastoma (Rb)) and intracellular signaling molecules (extracellular signal receptor-activated kinase 1/2 (ERK1/2), Akt, p38 and nuclear factor-κB (NF-κB)) were examined in cells following treatment of PL using Western blot analysis. Results showed that proliferation of cells were significantly decreased by PL in a dose-dependent manner. Flow cytometry results demonstrated increased number of cells in G1 phase in PL (40 μM)-treated group. Reactive oxygen species was significantly increased in cells treated with PL at 20-40 μM. The expression of cyclin D1, CDK4, CDK6 and p-Rb were markedly decreased in cells treated with PL at 40 μM. Treatment of cells with PL suppressed phosphorylation of Akt but increased ERK1/2 phosphorylation. Treatment of PL significantly decreased nuclear translocation of NF-κB p65 in cells. These results suggest that PL possesses antiproliferative properties in A549 cells. Copyright © 2017 Elsevier Ltd. All rights reserved.

  6. Bone stroma-derived cells change coregulators recruitment to androgen receptor and decrease cell proliferation in androgen-sensitive and castration-resistant prostate cancer cells

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    Villagran, Marcelo A.; Gutierrez-Castro, Francisco A.; Pantoja, Diego F.; Alarcon, Jose C.; Fariña, Macarena A.; Amigo, Romina F.; Muñoz-Godoy, Natalia A.; Pinilla, Mabel G.; Peña, Eduardo A.; Gonzalez-Chavarria, Ivan; Toledo, Jorge R.; Rivas, Coralia I.; Vera, Juan C.; McNerney, Eileen M.; Onate, Sergio A.

    2015-01-01

    Prostate cancer (CaP) bone metastasis is an early event that remains inactive until later-stage progression. Reduced levels of circulating androgens, due to andropause or androgen deprivation therapies, alter androgen receptor (AR) coactivator expression. Coactivators shift the balance towards enhanced AR-mediated gene transcription that promotes progression to androgen-resistance. Disruptions in coregulators may represent a molecular switch that reactivates latent bone metastasis. Changes in AR-mediated transcription in androgen-sensitive LNCaP and androgen-resistant C4-2 cells were analyzed for AR coregulator recruitment in co-culture with Saos-2 and THP-1. The Saos-2 cell line derived from human osteosarcoma and THP-1 cell line representing human monocytes were used to display osteoblast and osteoclast activity. Increased AR activity in androgen-resistant C4-2 was due to increased AR expression and SRC1/TIF2 recruitment and decreased SMRT/NCoR expression. AR activity in both cell types was decreased over 90% when co-cultured with Saos-2 or THP-1 due to dissociation of AR from the SRC1/TIF2 and SMRT/NCoR coregulators complex, in a ligand-dependent and cell-type specific manner. In the absence of androgens, Saos-2 decreased while THP-1 increased proliferation of LNCaP cells. In contrast, both Saos-2 and THP-1 decreased proliferation of C4-2 in absence and presence of androgens. Global changes in gene expression from both CaP cell lines identified potential cell cycle and androgen regulated genes as mechanisms for changes in cell proliferation and AR-mediated transactivation in the context of bone marrow stroma cells. - Highlights: • Decreased corepressor expression change AR in androgen-resistance prostate cancer. • Bone stroma-derived cells change AR coregulator recruitment in prostate cancer. • Bone stroma cells change cell proliferation in androgen-resistant cancer cells. • Global gene expression in CaP cells is modified by bone stroma cells in co

  7. Bone stroma-derived cells change coregulators recruitment to androgen receptor and decrease cell proliferation in androgen-sensitive and castration-resistant prostate cancer cells

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    Villagran, Marcelo A.; Gutierrez-Castro, Francisco A.; Pantoja, Diego F.; Alarcon, Jose C.; Fariña, Macarena A.; Amigo, Romina F.; Muñoz-Godoy, Natalia A. [Molecular Endocrinology and Oncology Laboratory, University of Concepcion, Concepcion (Chile); Pinilla, Mabel G. [Department of Medical Specialties, School of Medicine, University of Concepcion, Concepcion (Chile); Peña, Eduardo A.; Gonzalez-Chavarria, Ivan; Toledo, Jorge R.; Rivas, Coralia I.; Vera, Juan C. [Department of Physiopathology, School of Biological Sciences, University of Concepcion, Concepcion (Chile); McNerney, Eileen M. [Molecular Endocrinology and Oncology Laboratory, University of Concepcion, Concepcion (Chile); Onate, Sergio A., E-mail: sergio.onate@udec.cl [Molecular Endocrinology and Oncology Laboratory, University of Concepcion, Concepcion (Chile); Department of Medical Specialties, School of Medicine, University of Concepcion, Concepcion (Chile); Department of Urology, State University of New York at Buffalo, NY (United States)

    2015-11-27

    Prostate cancer (CaP) bone metastasis is an early event that remains inactive until later-stage progression. Reduced levels of circulating androgens, due to andropause or androgen deprivation therapies, alter androgen receptor (AR) coactivator expression. Coactivators shift the balance towards enhanced AR-mediated gene transcription that promotes progression to androgen-resistance. Disruptions in coregulators may represent a molecular switch that reactivates latent bone metastasis. Changes in AR-mediated transcription in androgen-sensitive LNCaP and androgen-resistant C4-2 cells were analyzed for AR coregulator recruitment in co-culture with Saos-2 and THP-1. The Saos-2 cell line derived from human osteosarcoma and THP-1 cell line representing human monocytes were used to display osteoblast and osteoclast activity. Increased AR activity in androgen-resistant C4-2 was due to increased AR expression and SRC1/TIF2 recruitment and decreased SMRT/NCoR expression. AR activity in both cell types was decreased over 90% when co-cultured with Saos-2 or THP-1 due to dissociation of AR from the SRC1/TIF2 and SMRT/NCoR coregulators complex, in a ligand-dependent and cell-type specific manner. In the absence of androgens, Saos-2 decreased while THP-1 increased proliferation of LNCaP cells. In contrast, both Saos-2 and THP-1 decreased proliferation of C4-2 in absence and presence of androgens. Global changes in gene expression from both CaP cell lines identified potential cell cycle and androgen regulated genes as mechanisms for changes in cell proliferation and AR-mediated transactivation in the context of bone marrow stroma cells. - Highlights: • Decreased corepressor expression change AR in androgen-resistance prostate cancer. • Bone stroma-derived cells change AR coregulator recruitment in prostate cancer. • Bone stroma cells change cell proliferation in androgen-resistant cancer cells. • Global gene expression in CaP cells is modified by bone stroma cells in co

  8. 4-Aminopyridine Decreases Progesterone Production by Porcine Granulosa Cells

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    Mitchell Brianna M

    2003-04-01

    Full Text Available Abstract Background Ion channels occur as large families of related genes with cell-specific expression patterns. Granulosa cells have been shown to express voltage-gated potassium channels from more than one family. The purpose of this study was to determine the effects of 4-aminopyridine (4-AP, an antagonist of KCNA but not KCNQ channels. Methods Granulosa cells were isolated from pig follicles and cultured with 4-AP, alone or in combination with FSH, 8-CPT-cAMP, estradiol 17β, and DIDS. Complimentary experiments determined the effects of 4-AP on the spontaneously established pig granulosa cell line PGC-2. Granulosa cell or PGC-2 function was assessed by radio-immunoassay of media progesterone accumulation. Cell viability was assessed by trypan blue exclusion. Drug-induced changes in cell membrane potential and intracellular potassium concentration were documented by spectrophotometric determination of DiBAC4(3 and PBFI fluorescence, respectively. Expression of proliferating cell nuclear antigen (PCNA and steroidogenic acute regulatory protein (StAR was assessed by immunoblotting. Flow cytometry was also used to examine granulosa cell viability and size. Results 4-AP (2 mM decreased progesterone accumulation in the media of serum-supplemented and serum-free granulosa cultures, but inhibited cell proliferation only under serum-free conditions. 4-AP decreased the expression of StAR, the production of cAMP and the synthesis of estradiol by PGC-2. Addition of either 8-CPT-cAMP or estradiol 17β to serum-supplemented primary cultures reduced the inhibitory effects of 4-AP. 4-AP treatment was also associated with increased cell size, increased intracellular potassium concentration, and hyperpolarization of resting membrane potential. The drug-induced hyperpolarization of resting membrane potential was prevented either by decreasing extracellular chloride or by adding DIDS to the media. DIDS also prevented 4-AP inhibition of progesterone production

  9. Hypoxia Decreases Invasin-Mediated Yersinia enterocolitica Internalization into Caco-2 Cells.

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    Zeitouni, Nathalie E; Dersch, Petra; Naim, Hassan Y; von Köckritz-Blickwede, Maren

    2016-01-01

    Yersinia enterocolitica is a major cause of human yersiniosis, with enterocolitis being a typical manifestation. These bacteria can cross the intestinal mucosa, and invade eukaryotic cells by binding to host β1 integrins, a process mediated by the bacterial effector protein invasin. This study examines the role of hypoxia on the internalization of Y. enterocolitica into intestinal epithelial cells, since the gastrointestinal tract has been shown to be physiologically deficient in oxygen levels (hypoxic), especially in cases of infection and inflammation. We show that hypoxic pre-incubation of Caco-2 cells resulted in significantly decreased bacterial internalization compared to cells grown under normoxia. This phenotype was absent after functionally blocking host β1 integrins as well as upon infection with an invasin-deficient Y. enterocolitica strain. Furthermore, downstream phosphorylation of the focal adhesion kinase was also reduced under hypoxia after infection. In good correlation to these data, cells grown under hypoxia showed decreased protein levels of β1 integrins at the apical cell surface whereas the total protein level of the hypoxia inducible factor (HIF-1) alpha was elevated. Furthermore, treatment of cells with the HIF-1 α stabilizer dimethyloxalylglycine (DMOG) also reduced invasion and decreased β1 integrin protein levels compared to control cells, indicating a potential role for HIF-1α in this process. These results suggest that hypoxia decreases invasin-integrin-mediated internalization of Y. enterocolitica into intestinal epithelial cells by reducing cell surface localization of host β1 integrins.

  10. Down-regulation of Rab5 decreases characteristics associated with maintenance of cell transformation

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    Silva, Patricio; Soto, Nicolás; Díaz, Jorge; Mendoza, Pablo; Díaz, Natalia; Quest, Andrew F.G.; Torres, Vicente A.

    2015-01-01

    The early endosomal protein Rab5 is highly expressed in tumor samples, although a causal relationship between Rab5 expression and cell transformation has not been established. Here, we report the functional effects of targeting endogenous Rab5 with specific shRNA sequences in different tumor cell lines. Rab5 down-regulation in B16-F10 cells decreased tumor formation by subcutaneous injection into C57/BL6 mice. Accordingly, Rab5 targeting in B16-F10 and A549, but not MDA-MB-231 cells was followed by decreased cell proliferation, increased apoptosis and decreased anchorage-independent growth. These findings suggest that Rab5 expression is required to maintain characteristics associated with cell transformation. - Highlights: • Rab5 is important to the maintenance of cell transformation characteristics. • Down-regulation of Rab5 decreases cell proliferation and increases apoptosis in different cancer cells. • Rab5 is required for anchorage-independent growth and tumorigenicity in-vivo

  11. Transgenic overexpression of active calcineurin in beta-cells results in decreased beta-cell mass and hyperglycemia.

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    Ernesto Bernal-Mizrachi

    2010-08-01

    Full Text Available Glucose modulates beta-cell mass and function through an initial depolarization and Ca(2+ influx, which then triggers a number of growth regulating signaling pathways. One of the most important downstream effectors in Ca(2+ signaling is the calcium/Calmodulin activated serine threonine phosphatase, calcineurin. Recent evidence suggests that calcineurin/NFAT is essential for beta-cell proliferation, and that in its absence loss of beta-cells results in diabetes. We hypothesized that in contrast, activation of calcineurin might result in expansion of beta-cell mass and resistance to diabetes.To determine the role of activation of calcineurin signaling in the regulation of pancreatic beta-cell mass and proliferation, we created mice that expressed a constitutively active form of calcineurin under the insulin gene promoter (caCn(RIP. To our surprise, these mice exhibited glucose intolerance. In vitro studies demonstrated that while the second phase of Insulin secretion is enhanced, the overall insulin secretory response was conserved. Islet morphometric studies demonstrated decreased beta-cell mass suggesting that this was a major component responsible for altered Insulin secretion and glucose intolerance in caCn(RIP mice. The reduced beta-cell mass was accompanied by decreased proliferation and enhanced apoptosis.Our studies identify calcineurin as an important factor in controlling glucose homeostasis and indicate that chronic depolarization leading to increased calcineurin activity may contribute, along with other genetic and environmental factors, to beta-cell dysfunction and diabetes.

  12. Estradiol decreases iodide uptake by rat thyroid follicular FRTL-5 cells

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    Furlanetto T.W.

    2001-01-01

    Full Text Available Estradiol has well-known indirect effects on the thyroid. A direct effect of estradiol on thyroid follicular cells, increasing cell growth and reducing the expression of the sodium-iodide symporter gene, has been recently reported. The aim of the present investigation was to study the effect of estradiol on iodide uptake by thyroid follicular cells, using FRTL-5 cells as a model. Estradiol decreased basal iodide uptake by FRTL-5 cells from control levels of 2.490 ± 0.370 to 2.085 ± 0.364 pmol I-/µg DNA at 1 ng/ml (P<0.02, to 1.970 ± 0.302 pmol I-/µg DNA at 10 ng/ml (P<0.003, and to 2.038 ± 0.389 pmol I-/µg DNA at 100 ng/ml (P<0.02. In addition, 4 ng/ml estradiol decreased iodide uptake induced by 0.02 mIU/ml thyrotropin from 8.678 ± 0.408 to 7.312 ± 0.506 pmol I-/µg DNA (P<0.02. A decrease in iodide uptake by thyroid cells caused by estradiol has not been described previously and may have a role in goiter pathogenesis.

  13. Decreased expression of cell adhesion genes in cancer stem-like cells isolated from primary oral squamous cell carcinomas.

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    Mishra, Amrendra; Sriram, Harshini; Chandarana, Pinal; Tanavde, Vivek; Kumar, Rekha V; Gopinath, Ashok; Govindarajan, Raman; Ramaswamy, S; Sadasivam, Subhashini

    2018-05-01

    The goal of this study was to isolate cancer stem-like cells marked by high expression of CD44, a putative cancer stem cell marker, from primary oral squamous cell carcinomas and identify distinctive gene expression patterns in these cells. From 1 October 2013 to 4 September 2015, 76 stage III-IV primary oral squamous cell carcinoma of the gingivobuccal sulcus were resected. In all, 13 tumours were analysed by immunohistochemistry to visualise CD44-expressing cells. Expression of CD44 within The Cancer Genome Atlas-Head and Neck Squamous Cell Carcinoma RNA-sequencing data was also assessed. Seventy resected tumours were dissociated into single cells and stained with antibodies to CD44 as well as CD45 and CD31 (together referred as Lineage/Lin). From 45 of these, CD44 + Lin - and CD44 - Lin - subpopulations were successfully isolated using fluorescence-activated cell sorting, and good-quality RNA was obtained from 14 such sorted pairs. Libraries from five pairs were sequenced and the results analysed using bioinformatics tools. Reverse transcription quantitative polymerase chain reaction was performed to experimentally validate the differential expression of selected candidate genes identified from the transcriptome sequencing in the same 5 and an additional 9 tumours. CD44 was expressed on the surface of poorly differentiated tumour cells, and within the The Cancer Genome Atlas-Head and Neck Squamous Cell Carcinoma samples, its messenger RNA levels were higher in tumours compared to normal. Transcriptomics revealed that 102 genes were upregulated and 85 genes were downregulated in CD44 + Lin - compared to CD44 - Lin - cells in at least 3 of the 5 tumours sequenced. The upregulated genes included those involved in immune regulation, while the downregulated genes were enriched for genes involved in cell adhesion. Decreased expression of PCDH18, MGP, SPARCL1 and KRTDAP was confirmed by reverse transcription quantitative polymerase chain reaction. Lower expression of

  14. Mitofusin2 decreases intracellular cholesterol of oxidized LDL-induced foam cells from rat vascular smooth muscle cells.

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    He, Chao; Chen, Ying; Liu, Chun; Cao, Ming; Fan, Yu-jin; Guo, Xiao-mei

    2013-04-01

    Mitofusin2 (Mfn2) plays a pivotal role in the proliferation and apoptosis of vascular smooth muscle cells (VSMCs). The purpose of this study was to investigate the effects of Mfn2 on the trafficking of intracellular cholesterol in the foam cells derived from rat VSMCs (rVSMCs) and also to investigate the effects of Mfn2 on the expression of adenosine triphosphate-binding cassette subfamily A member 1 (ABCA1), adenosine triphosphate-binding cassette subfamily G member 1 (ABCG1) and peroxisome proliferator-activated receptor gamma (PPARγ). The rVSMCs were co-cultured with oxidized low density lipoprotein (LDL, 80 μg/mL) to produce foam cells and cholesterol accumulation in cells. Before oxidized LDL treatment, different titers (20, 40 and 60 pfu/cell) of recombinant adenovirus containing Mfn2 gene (Adv-Mfn2) were added into the culture medium for 24 h to transfect the Mfn2 gene into the rVSMCs. Then the cells were harvested for analyses. The protein expression of Mfn2 was significantly higher in Adv-Mfn2-transfected group than in untransfected group (PLDL treatment, rVSMCs became irregular and their nuclei became larger, and their plasma abounded with red lipid droplets. However, the number of red lipid droplets was significantly decreased in Adv-Mfn2-transfected group as compared with untransfected group. At 48 h after oxidized LDL treatment, the intracellular cholesterol in rVSMCs was significantly increased (P0.05), the phosporylation levels of PPARγ were significantly decreased in Adv-Mfn2-transfected group as compared with untransfected group (Pcholesterol in oxidized LDL-induced rVSMCs possibly by decreasing PPARγ phosporylation and then increasing protein expression levels of ABCA1 and ABCG1, which may be helpful to suppress the formation of foam cells.

  15. Decreased cisplatin uptake by resistant L1210 leukemia cells

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    Hromas, R.A.; North, J.A.; Burns, C.P.

    1987-01-01

    Cisplatin resistance remains poorly understood compared to other forms of anti-neoplastic drug resistance. In this report radiolabelled cisplatin and rapid separation techniques were used to compare drug uptake by L1210 leukemia cells that are sensitive (K25) or resistant (SCR9) to cisplatin. Uptake of cisplatin by both cell lines was linear without saturation kinetics up to 100 μM. The resistant ZCR9 cells had 36-60% reduced drug uptake as compared to its sensitive parent line, K25. In contrast, there was no difference in the rate of efflux. We conclude that a decreased rate of uptake is one possible mechanism of cellular cisplatin resistance. (Author)

  16. Differentiation-associated decrease in muscarinic receptor sensitivity in human neuroblastoma cells

    International Nuclear Information System (INIS)

    Heikkilae, J.E.; Scott, J.G.; Suominen, L.A.; Akerman, K.E.O.

    1987-01-01

    Muscarinic receptor-linked increases in intracellular free Ca 2+ as measured with quin-2 and Ca 2+ release from monolayers of cells have been measured in the human neuroblastoma cell line SH-SY5Y. Induction of differentiation with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) leads to a decrease in the sensitivity of the cells to low concentrations of agonists with respect to the induced increase in cytosolic free Ca 2+ and stimulation of Ca 2+ efflux. No decrease in agonist binding affinity was observed when the displacement of a labelled antagonist, 3 H-NMS, by a non-labelled agonist was studied

  17. Decreased immunoglobulin production by a human lymphoid cell line following melphalan treatment

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    Griffin, G.D. (Oak Ridge National Lab., TN); Owen, B.A.; Atchley, C.E.; Novelli, G.D.; Solomon, A.

    1982-11-01

    The effect of melphalan on immunoglobulin G (IgG) production by a human lymphoblastoid cell line (BF) was studied. The amount of secreted IgG and the percentage of cells containing cytoplasmic IgG were measured by immunoassay and cytofluorometry, respectively. Dose-response studies indicated that melphalan concentrations of 2 x 10/sup -8/ M had no effect, while concentrations of 8 x 10/sup -7/ M were totally toxic, after 72-h exposures to the drug. Statistically significant, persistent, alterations in both synthesis and secretion of IgG by BF cells were observed following treatment for 72 h with 4 x 10/sup -7/ M melphalan, and there was an increase in population-doubling time from 24 to 72 h in these drug-treated cells. The percentage of IgG-containing cells in melphalan-treated cultures was significantly decreased as compared to control cultures. IgG secretion was also decreased in these cultures, and the variation in IgG secretion as a function of cellular growth was significantly altered following melphalan treatment. Decreased IgG production following melphalan treatment may be related to altered cell cycle kinetics. Based on immunological analysis, there was no evident alteration in the IgG secreted by melphalan-treated cells, nor did melphalan treatment produce a cellular population lacking IgG entirely.

  18. Total glucosides of paeony inhibits Th1/Th17 cells via decreasing dendritic cells activation in rheumatoid arthritis.

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    Lin, Jinpiao; Xiao, Lianbo; Ouyang, Guilin; Shen, Yu; Huo, Rongfen; Zhou, Zhou; Sun, Yue; Zhu, Xianjin; Zhang, Jie; Shen, Baihua; Li, Ningli

    2012-12-01

    Total glucoside of paeony (TGP), an active compound extracted from paeony root, has been used in therapy for rheumatoid arthritis (RA). Th1 and Th17 cells are now believed to play crucial roles in the lesions of RA. However, the molecular mechanism of TGP in inhibition of Th1 and Th17 cells remains unclear. In this study, we found that TGP treatment significantly decreased percentage and number of Th1 and Th17 cells in collagen induced arthritis (CIA) mice. Consistently, treatment with TGP decreased expression of T-bet and RORγt as well as phosphorylation of STAT1 and STAT3. In particular, TGP treatment inhibited dendritic cells (DCs) maturation and reduced production of IL-12 and IL-6. Moreover, TGP-treatment RA patients showed shank population of matured DCs and IFN-γ-, IL-17-producing cells. Taken together, our results demonstrated that TGP inhibited maturation and activation of DCs, which led to impaired Th1 and Th17 differentiation in vivo. Copyright © 2013 Elsevier Inc. All rights reserved.

  19. Density increment and decreased survival of rat red blood cells induced by cadmium

    International Nuclear Information System (INIS)

    Kunimoto, M.; Miura, T.

    1986-01-01

    Male Wistar rats were injected with CdCl 2 subcutaneously to examine in vivo effects of Cd on density and survival of red blood cells. During the 7 days after administration of 1.0 mg Cd/kg, the following sequence of events occurred: (1) a progressive increase in the amount of more dense red blood cells concomitant with a decrease in that of light red blood cells from the first to the third day; (2) an increase in the spleen weight at the third day; (3) a decrease in the hematocrit value and an increase in the amount of light red blood cells at the fifth day; and (4) a recovery of the hematocrit value at the seventh day. Five days after administration, the hematocrit value decreased in a dose-dependent mode and the decrease was significant at the 1% level at 1.0 and 1.5 mg Cd/kg. A highly significant splenomegaly was also observed at 0.5 to 1.5 mg Cd/kg. In order to label red blood cells in vivo, [ 3 H] diisopropylfluorophosphate ([ 3 H]DFP) was injected into rats. At Day 11, Cd at either 0.5 or 1.0 mg/kg was administered to [ 3 H]DFP-prelabeled animals. Cd administration accelerated 3 H-labeled red cell clearance from the blood. Six days after Cd administration, the radioactivity of red blood cells was 76 and 68% of the control at 0.5 and 1.0 mg Cd/kg, respectively. In vitro treatment of rat red density and accelerated in vivo clearance of red blood cells from the recipient circulation. These results show that Cd at low dose can cause anemia by increasing red cell density and by accelerating red cell sequestration, presumably in the spleen

  20. Localized decrease of β-catenin contributes to the differentiation of human embryonic stem cells

    International Nuclear Information System (INIS)

    Lam, Hayley; Patel, Shyam; Wong, Janelle; Chu, Julia; Li, Adrian; Li, Song

    2008-01-01

    Human embryonic stem cells (hESC) are pluripotent, and can be directed to differentiate into different cell types for therapeutic applications. To expand hESCs, it is desirable to maintain hESC growth without differentiation. As hESC colonies grow, differentiated cells are often found at the periphery of the colonies, but the underlying mechanism is not well understood. Here, we utilized micropatterning techniques to pattern circular islands or strips of matrix proteins, and examined the spatial pattern of hESC renewal and differentiation. We found that micropatterned matrix restricted hESC differentiation at colony periphery but allowed hESC growth into multiple layers in the central region, which decreased hESC proliferation and induced hESC differentiation. In undifferentiated hESCs, β-catenin primarily localized at cell-cell junctions but not in the nucleus. The amount of β-catenin in differentiating hESCs at the periphery of colonies or in multiple layers decreased significantly at cell-cell junctions. Consistently, knocking down β-catenin decreased Oct-4 expression in hESCs. These results indicate that localized decrease of β-catenin contributes to the spatial pattern of differentiation in hESC colonies

  1. Vascular endothelial growth factor modified macrophages transdifferentiate into endothelial-like cells and decrease foam cell formation.

    Science.gov (United States)

    Yan, Dan; He, Yujuan; Dai, Jun; Yang, Lili; Wang, Xiaoyan; Ruan, Qiurong

    2017-06-30

    Macrophages are largely involved in the whole process of atherosclerosis from an initiation lesion to an advanced lesion. Endothelial disruption is the initial step and macrophage-derived foam cells are the hallmark of atherosclerosis. Promotion of vascular integrity and inhibition of foam cell formation are two important strategies for preventing atherosclerosis. How can we inhibit even the reverse negative role of macrophages in atherosclerosis? The present study was performed to investigate if overexpressing endogenous human vascular endothelial growth factor (VEGF) could facilitate transdifferentiation of macrophages into endothelial-like cells (ELCs) and inhibit foam cell formation. We demonstrated that VEGF-modified macrophages which stably overexpressed human VEGF (hVEGF 165 ) displayed a high capability to alter their phenotype and function into ELCs in vitro Exogenous VEGF could not replace endogenous VEGF to induce the transdifferentiation of macrophages into ELCs in vitro We further showed that VEGF-modified macrophages significantly decreased cytoplasmic lipid accumulation after treatment with oxidized LDL (ox-LDL). Moreover, down-regulation of CD36 expression in these cells was probably one of the mechanisms of reduction in foam cell formation. Our results provided the in vitro proof of VEGF-modified macrophages as atheroprotective therapeutic cells by both promotion of vascular repair and inhibition of foam cell formation. © 2017 The Author(s).

  2. Live Cells Decreased Methane Production in Intestinal Content of Pigs

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    Y. L. Gong

    2013-06-01

    Full Text Available An in vitro gas production technique was used in this study to elucidate the effect of two strains of active live yeast on methane (CH4 production in the large intestinal content of pigs to provide an insight to whether active live yeast could suppress CH4 production in the hindgut of pigs. Treatments used in this study include blank (no substrate and no live yeast cells, control (no live yeast cells and yeast (YST supplementation groups (supplemented with live yeast cells, YST1 or YST2. The yeast cultures contained 1.8×1010 cells per g, which were added at the rates of 0.2 mg and 0.4 mg per ml of the fermented inoculum. Large intestinal contents were collected from 2 Duroc×Landrace×Yorkshire pigs, mixed with a phosphate buffer (1:2, and incubated anaerobically at 39°C for 24 h using 500 mg substrate (dry matter (DM basis. Total gas and CH4 production decreased (p<0.05 with supplementation of yeast. The methane production reduction potential (MRP was calculated by assuming net methane concentration for the control as 100%. The MRP of yeast 2 was more than 25%. Compared with the control group, in vitro DM digestibility (IVDMD and total volatile fatty acids (VFA concentration increased (p<0.05 in 0.4 mg/ml YST1 and 0.2 mg/ml YST2 supplementation groups. Proportion of propionate, butyrate and valerate increased (p<0.05, but that of acetate decreased (p<0.05, which led to a decreased (p<0.05 acetate: propionate (A: P ratio in the both YST2 treatments and the 0.4 mg/ml YST 1 supplementation groups. Hydrogen recovery decreased (p<0.05 with yeast supplementation. Quantity of methanogenic archaea per milliliter of inoculum decreased (p<0.05 with yeast supplementation after 24 h of incubation. Our results suggest that live yeast cells suppressed in vitro CH4 production when inoculated into the large intestinal contents of pigs and shifted the fermentation pattern to favor propionate production together with an increased population of acetogenic

  3. Methamphetamine decreases dentate gyrus stem cell self-renewal and shifts the differentiation towards neuronal fate

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    Sofia Baptista

    2014-09-01

    Full Text Available Methamphetamine (METH is a highly addictive psychostimulant drug of abuse that negatively interferes with neurogenesis. In fact, we have previously shown that METH triggers stem/progenitor cell death and decreases neuronal differentiation in the dentate gyrus (DG. Still, little is known regarding its effect on DG stem cell properties. Herein, we investigate the impact of METH on mice DG stem/progenitor cell self-renewal functions. METH (10 nM decreased DG stem cell self-renewal, while 1 nM delayed cell cycle in the G0/G1-to-S phase transition and increased the number of quiescent cells (G0 phase, which correlated with a decrease in cyclin E, pEGFR and pERK1/2 protein levels. Importantly, both drug concentrations (1 or 10 nM did not induce cell death. In accordance with the impairment of self-renewal capacity, METH (10 nM decreased Sox2+/Sox2+ while increased Sox2−/Sox2− pairs of daughter cells. This effect relied on N-methyl-d-aspartate (NMDA signaling, which was prevented by the NMDA receptor antagonist, MK-801 (10 μM. Moreover, METH (10 nM increased doublecortin (DCX protein levels consistent with neuronal differentiation. In conclusion, METH alters DG stem cell properties by delaying cell cycle and decreasing self-renewal capacities, mechanisms that may contribute to DG neurogenesis impairment followed by cognitive deficits verified in METH consumers.

  4. Using CRISPR/Cas9 to Knock out Amylase in Acinar Cells Decreases Pancreatitis-Induced Autophagy

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    Kohei Yasunaga

    2018-01-01

    Full Text Available Pancreatic cancer is a malignant neoplasm that originates from acinar cells. Acinar cells get reprogrammed to become duct cells, resulting in pancreatic cancer. Pancreatitis is an acinar cell inflammation, leading to “impaired autophagy flux”. Pancreatitis promotes acinar-to-ductal transdifferentiation. Expression of amylase gets eliminated during the progression of pancreatic cancer. Amylase is considered as an acinar cell marker; however, its function in cells is not known. Thus, we investigated whether amylase affects the acinar cell autophagy and whether it plays any role in development of pancreatitis. Here, we knocked out ATG12 in a pancreatic cancer cells and acinar cells using CRISPR/Cas9. Autophagy inhibition led to an increase in the expression of duct cell markers and a simultaneous decrease in that of acinar cell markers. It also caused an increase in cell viability and changes in mitochondrial morphology. Next, we knocked out amylase in acinar cells. Amylase deficiency decreased autophagy induced by pancreatitis. Our results suggest that amylase controls pancreatitis-induced autophagy. We found that eliminating amylase expression contributes to pancreatic cancer etiology by decreasing autophagy. Furthermore, our results indicate that amylase plays a role in selective pancreatitis-induced autophagy of pancreatic enzyme vesicles.

  5. Curcumin and Viscum album Extract Decrease Proliferation and Cell Viability of Soft-Tissue Sarcoma Cells: An In Vitro Analysis of Eight Cell Lines Using Real-Time Monitoring and Colorimetric Assays.

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    Harati, K; Behr, B; Daigeler, A; Hirsch, T; Jacobsen, F; Renner, M; Harati, A; Wallner, C; Lehnhardt, M; Becerikli, M

    2017-01-01

    The cytostatic effects of the polyphenol curcumin and Viscum album extract (VAE) were assessed in soft-tissue sarcoma (STS) cells. Eight human STS cell lines were used: fibrosarcoma (HT1080), liposarcoma (SW872, T778, MLS-402), synovial sarcoma (SW982, SYO1, 1273), and malignant fibrous histiocytoma (U2197). Primary human fibroblasts served as control cells. Cell proliferation, viability, and cell index (CI) were analyzed by BrdU assay, MTT assay, and real-time cell analysis (RTCA). As indicated by BrdU and MTT, curcumin significantly decreased the cell proliferation of five cell lines (HT1080, SW872, SYO1, 1273, and U2197) and the viability of two cell lines (SW872 and SW982). VAE led to significant decreases of proliferation in eight cell lines (HT1080, SW872, T778, MLS-402, SW982, SYO1, 1293, and U2197) and reduced viability in seven STS lines (HT1080, SW872, T778, MLS-402, SW982, SYO1, and 1273). As indicated by RTCA for 160 h, curcumin decreased the CI of all synovial sarcoma cell lines as well as T778 and HT1080. VAE diminished the CI in most of the synovial sarcoma (SW982, SYO1) and liposarcoma (SW872, T778) cell lines as well as HT1080. Primary fibroblasts were not affected adversely by the two compounds in RTCA. Curcumin and VAE can inhibit the proliferation and viability of STS cells.

  6. T315 Decreases Acute Myeloid Leukemia Cell Viability through a Combination of Apoptosis Induction and Autophagic Cell Death

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    Chang-Fang Chiu

    2016-08-01

    Full Text Available T315, an integrin-linked kinase (ILK inhibitor, has been shown to suppress the proliferation of breast cancer, stomach cancer and chronic lymphocytic leukemia cells. Here we demonstrate that T315 decreases cell viability of acute myeloid leukemia (AML cell lines (HL-60 and THP-1 and primary leukemia cells from AML patients in a dose-responsive manner. Normal human bone marrow cells are less sensitive than leukemia cells to T315. T315 down regulates protein kinase B (Akt and p-Akt and induces caspase activation, poly-ADP-ribose polymerase (PARP cleavage, apoptosis and autophagy through an ILK-independent manner. Interestingly, pretreatment with autophagy inhibitors rescues cells from apoptosis and concomitant PARP cleavage, which implicates a key role of autophagic cell death in T315-mediated cytotoxicity. T315 also demonstrates efficacy in vivo, suppressing the growth of THP-1 xenograft tumors in athymic nude mice when administered intraperitoneally. This study shows that autophagic cell death and apoptosis cooperatively contribute to the anticancer activity of T315 in AML cells. In conclusion, the complementary roles of apoptotic and autophagic cell death should be considered in the future assessment of the translational value of T315 in AML therapy.

  7. Decreased stability of DNA in cells treated with alkylating agents

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    Frankfurt, O.S. (Cedars Medical Center, Miami, FL (United States))

    1990-12-01

    A modified highly sensitive procedure for the evaluation of DNA damage in individual cells treated with alkylating agents is reported. The new methodology is based on the amplification of single-strandedness in alkylated DNA by heating in the presence of Mg{sup 2+}. Human ovarian carcinoma cells A2780 were treated with nitrogen mustard (HN2), fixed in methanol, and stained with monoclonal antibody (MOAB) F7-26 generated against HN2-treated DNA. Binding of MOAB was measured by flow cytometry with indirect immunofluorescence. Intensive binding of MOAB to control and drug-treated cells was observed after heating in Tris buffer supplemented with MgCl{sub 2}. Thus, the presence of phosphates and MgCl{sub 2} during heating was necessary for the detection of HN2-induced changes in DNA stability. Fluorescence of HN2-treated cells decreased to background levels after treatment with single-strand-specific S{sub 1} nuclease. MOAB F7-26 interacted with single-stranded regions in DNA and did not bind to dsDNA or other cellular antigens. It is suggested that alkylation of guanines decreased the stability of the DNA molecule and increased the access of MOAB F7-26 to deoxycytidines on the opposite DNA strand.

  8. ACh-induced hyperpolarization and decreased resistance in mammalian type II vestibular hair cells.

    Science.gov (United States)

    Poppi, Lauren A; Tabatabaee, Hessam; Drury, Hannah R; Jobling, Phillip; Callister, Robert J; Migliaccio, Americo A; Jordan, Paivi M; Holt, Joseph C; Rabbitt, Richard D; Lim, Rebecca; Brichta, Alan M

    2018-01-01

    In the mammalian vestibular periphery, electrical activation of the efferent vestibular system (EVS) has two effects on afferent activity: 1) it increases background afferent discharge and 2) decreases afferent sensitivity to rotational stimuli. Although the cellular mechanisms underlying these two contrasting afferent responses remain obscure, we postulated that the reduction in afferent sensitivity was attributed, in part, to the activation of α9- containing nicotinic acetylcholine (ACh) receptors (α9*nAChRs) and small-conductance potassium channels (SK) in vestibular type II hair cells, as demonstrated in the peripheral vestibular system of other vertebrates. To test this hypothesis, we examined the effects of the predominant EVS neurotransmitter ACh on vestibular type II hair cells from wild-type (wt) and α9-subunit nAChR knockout (α9 -/- ) mice. Immunostaining for choline acetyltransferase revealed there were no obvious gross morphological differences in the peripheral EVS innervation among any of these strains. ACh application onto wt type II hair cells, at resting potentials, produced a fast inward current followed by a slower outward current, resulting in membrane hyperpolarization and decreased membrane resistance. Hyperpolarization and decreased resistance were due to gating of SK channels. Consistent with activation of α9*nAChRs and SK channels, these ACh-sensitive currents were antagonized by the α9*nAChR blocker strychnine and SK blockers apamin and tamapin. Type II hair cells from α9 -/- mice, however, failed to respond to ACh at all. These results confirm the critical importance of α9nAChRs in efferent modulation of mammalian type II vestibular hair cells. Application of exogenous ACh reduces electrical impedance, thereby decreasing type II hair cell sensitivity. NEW & NOTEWORTHY Expression of α9 nicotinic subunit was crucial for fast cholinergic modulation of mammalian vestibular type II hair cells. These findings show a multifaceted

  9. Decreased memory B cells and increased CD8 memory T cells in blood of breastfed children: the generation R study.

    Science.gov (United States)

    Jansen, Michelle A E; van den Heuvel, Diana; van Zelm, Menno C; Jaddoe, Vincent W V; Hofman, Albert; de Jongste, Johan C; Hooijkaas, Herbert; Moll, Henriette A

    2015-01-01

    Breastfeeding provides a protective effect against infectious diseases in infancy. Still, immunological evidence for enhanced adaptive immunity in breastfed children remains inconclusive. To determine whether breastfeeding affects B- and T-cell memory in the first years of life. We performed immunophenotypic analysis on blood samples within a population-based prospective cohort study. Participants included children at 6 months (n=258), 14 months (n=166), 25 months (n=112) and 6 years of age (n=332) with both data on breastfeeding and blood lymphocytes. Total B- and T-cell numbers and their memory subsets were determined with 6-color flow cytometry. Mothers completed questionnaires on breastfeeding when their children were aged 2, 6, and 12 months. Multiple linear regression models with adjustments for potential confounders were performed. Per month continuation of breastfeeding, a 3% (95% CI -6, -1) decrease in CD27+IgM+, a 2% (95 CI % -5, -1) decrease in CD27+IgA+ and a 2% (95% CI -4, -1) decrease in CD27-IgG+ memory B cell numbers were observed at 6 months of age. CD8 T-cell numbers at 6 months of age were 20% (95% CI 3, 37) higher in breastfed than in non-breastfed infants. This was mainly found for central memory CD8 T cells and associated with exposure to breast milk, rather than duration. The same trend was observed at 14 months, but associations disappeared at older ages. Longer breastfeeding is associated with increased CD8 T-cell memory, but not B-cell memory numbers in the first 6 months of life. This transient skewing towards T cell memory might contribute to the protective effect against infectious diseases in infancy.

  10. Decreased memory B cells and increased CD8 memory T cells in blood of breastfed children: the generation R study.

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    Michelle A E Jansen

    Full Text Available Breastfeeding provides a protective effect against infectious diseases in infancy. Still, immunological evidence for enhanced adaptive immunity in breastfed children remains inconclusive.To determine whether breastfeeding affects B- and T-cell memory in the first years of life.We performed immunophenotypic analysis on blood samples within a population-based prospective cohort study. Participants included children at 6 months (n=258, 14 months (n=166, 25 months (n=112 and 6 years of age (n=332 with both data on breastfeeding and blood lymphocytes. Total B- and T-cell numbers and their memory subsets were determined with 6-color flow cytometry. Mothers completed questionnaires on breastfeeding when their children were aged 2, 6, and 12 months. Multiple linear regression models with adjustments for potential confounders were performed.Per month continuation of breastfeeding, a 3% (95% CI -6, -1 decrease in CD27+IgM+, a 2% (95 CI % -5, -1 decrease in CD27+IgA+ and a 2% (95% CI -4, -1 decrease in CD27-IgG+ memory B cell numbers were observed at 6 months of age. CD8 T-cell numbers at 6 months of age were 20% (95% CI 3, 37 higher in breastfed than in non-breastfed infants. This was mainly found for central memory CD8 T cells and associated with exposure to breast milk, rather than duration. The same trend was observed at 14 months, but associations disappeared at older ages.Longer breastfeeding is associated with increased CD8 T-cell memory, but not B-cell memory numbers in the first 6 months of life. This transient skewing towards T cell memory might contribute to the protective effect against infectious diseases in infancy.

  11. Dendritic cells decreased the concomitant expanded Tregs and Tregs related IL-35 in cytokine-induced killer cells and increased their cytotoxicity against leukemia cells.

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    Ying Pan

    Full Text Available Regulatory T cells (Tregs are potent immunosuppressive cells and essential for inducing immune tolerance. Recent studies have reported that Tregs and Tregs related cytokines can inhibit the antitumor activity of cytokine-induced killer (CIK cells, but dendritic cells co-cultured CIK (DC-CIK cells can be used for induction of a specific immune response by blocking of Tregs and TGF-β, IL-10. As a novel identified cytokine, IL-35 is specially produced by Tregs and plays an essential role in immune regulation. However, it remains unknown whether IL-35 roles in tumor immunotherapy mediated by CIK and DC-CIK cells. In this study, we cultured CIK and DC-CIK cells from the same healthy adult samples, and investigated their phenotype, proliferation, cytotoxic activity against leukemia cell lines K562 and NB4 by FCM and CCK-8, measured IL-35, TGF-β and IL-10 protein by ELISA, detected Foxp3, IL-35 and IL-35 receptor mRNA by Real-time PCR, respectively. We found Tregs and IL-35 concomitantly expanded by a time-dependent way during the generation of CIK cells, but DC significantly down-regulated the expression of them and simultaneously up-regulated the proliferation ability as well as cytotoxic activity of CIK cells against leukemia cell lines. Therefore, our data suggested that DC decreased concomitant expanded Tregs and Tregs related IL-35 in CIK cells and might contribute to improve their cytotoxicity against leukemia cells in vitro.

  12. Activation of p44/42 in Human Natural Killer Cells Decreases Cell-surface Protein Expression: Relationship to Tributyltin-induced alterations of protein expression

    Science.gov (United States)

    Dudimah, Fred D.; Abraha, Abraham; Wang, Xiaofei; Whalen, Margaret M.

    2010-01-01

    Tributyltin (TBT) activates the mitogen activated protein kinase (MAPK), p44/42 in human natural killer (NK) cells. TBT also reduces NK cytotoxic function and decreases the expression of several NK-cell proteins. To understand the role that p44/42 activation plays in TBT-induced loss of NK cell function, we have investigated how selective activation of p44/42 by phorbol 12-myristate 13-acetate (PMA) affects NK cells. Previously we showed that PMA caused losses of lytic function similar to those seen with TBT exposures. Here we examined activation of p44/42 in the regulation of NK-cell protein expression and how this regulation may explain the protein expression changes seen with TBT exposures. NK cells exposed to PMA were examined for levels of cell-surface proteins, granzyme mRNA, and perforin mRNA expression. The expression of CD11a, CD16, CD18, and CD56 were reduced, perforin mRNA levels were unchanged and granzyme mRNA levels were increased. To verify that activation of p44/42 was responsible for the alterations seen in CD11a, CD16, CD18, and CD56 with PMA, NK cells were treated with the p44/42 pathway inhibitor (PD98059) prior to PMA exposures. In the presence of PD98059, PMA caused no decreases in the expression of the cell-surface proteins. Results of these studies indicate that the activation of p44/42 may lead to the loss of NK cell cytotoxic function by decreasing the expression of CD11a, CD16, CD18, and CD56. Further, activation of p44/42 appears to be at least in part responsible for the TBT-induced decreases in expression of CD16, CD18, and CD56. PMID:20883105

  13. Pentoxifylline sensitizes human cervical tumor cells to cisplatin-induced apoptosis by suppressing NF-kappa B and decreased cell senescence

    International Nuclear Information System (INIS)

    Hernandez-Flores, Georgina; Bravo-Cuellar, Alejandro; Ortiz-Lazareno, Pablo C; Lerma-Diaz, Jose Manuel; Dominguez-Rodriguez, Jorge R; Jave-Suarez, Luis F; Aguilar-Lemarroy, Adriana del C; Celis-Carrillo, Ruth de; Toro-Arreola, Susana del; Castellanos-Esparza, Yessica C

    2011-01-01

    Worldwide, cervical cancer is the second most common causes of cancer in women and represents an important mortality rate. Cisplatin (CIS) is a very important antitumoral agent and can lead tumor cells toward two important cellular states: apoptosis and senescence. In some types of cancers pentoxifylline (PTX) sensitizes these cells to the toxic action of chemotherapeutics drugs such as adriamycin, inducing apoptosis. In the present work, we studied in vitro whether PTX alone or in combination with CIS induces apoptosis and/or senescence in cervix cancer HeLa and SiHa cell lines infected with HPV types 16 and 18, respectively, as well as in immortalized keratinocytyes HaCaT cells. HeLa (HPV 18+), SiHa (HPV 16+) cervix cancer cells and non-tumorigenic immortalized HaCaT cells (control) were treated with PTX, CIS or both. The cellular toxicity and survival fraction of PTX and CIS were determinate by WST-1 and clonogenic assays respectively. Apoptosis, caspase activation and phosphorylation of ERK1/2, p38, p65 (NF-κB), Bcl-2 and Bcl-XL anti-apoptotic proteins were determinated by flow cytometry. Senescence by microscopy. Phosphorylation of IκBα and IκB total were measured by ELISA. Pro-apoptotic, anti-apoptotic and senescence genes, as well as HPV-E6/7 mRNA expression, were detected by RT-PCR. Our results show that after 24 hours of incubation PTX per se is toxic for cancer cells affecting cell viability and inducing apoptosis. The toxicity in HaCaT cells was minimal. CIS induces apoptosis in HeLa and SiHa cells and its effect was significantly increases when the cells were treated with PTX + CIS. In all studies there was a direct correlation with levels of caspases (-3, -6, -7, -9 and -8) activity and apoptosis. CIS induces important levels of senescence and phosphorylation of ERK1/2, p38, p65/RELA, and IκBα, and decreased the expression of anti-apoptotic protein Bcl-XL. Surprisingly these levels were significantly reduced by PTX in tumor cells, and at the same

  14. Professional killer cell deficiencies and decreased survival in pulmonary arterial hypertension.

    Science.gov (United States)

    Edwards, Adrienne L; Gunningham, Sarah P; Clare, Geoffrey C; Hayman, Matthew W; Smith, Mark; Frampton, Christopher M A; Robinson, Bridget A; Troughton, Richard W; Beckert, Lutz E L

    2013-11-01

    Increasing evidence implicates lymphocytes in pulmonary arterial hypertension (PAH) pathogenesis. Rats deficient in T-lymphocytes show increased propensity to develop PAH but when injected with endothelial progenitor cells are protected from PAH (a mechanism dependent on natural killer (NK) cells). A decreased quantity of circulating cytotoxic CD8+ T-lymphocytes and NK cells are now reported in PAH patients; however, the effect of lymphocyte depletion on disease outcome is unknown. This prospective study analysed the lymphocyte profile and plasma brain natriuretic peptide (BNP) levels of patients with idiopathic PAH (IPAH), connective tissue disease-associated PAH (CTD-APAH) and matched healthy controls. Lymphocyte surface markers studied include: CD4+ (helper T-cell marker), CD8+ (cytotoxic T-cell marker), CD56/CD16 (NK cell marker) and CD19+ (mature B-cell marker). Lymphocyte deficiencies and plasma BNP levels were then correlated with clinical outcome. Fourteen patients with PAH (9 IPAH, 5CTD) were recruited. Three patients were deceased at 1-year follow-up; all had elevated CD4 : CD8 ratios and deficiencies of NK cells and cytotoxic CD8+ T-lymphocytes at recruitment. Patients with normal lymphocyte profiles at recruitment were all alive a year later, and none were on the active transplant list. As univariate markers, cytotoxic CD8+ T-cell and NK cell counts were linked to short-term survival. Deficiencies in NK cells and cytotoxic CD8+ T-cells may be associated with an increased risk of death in PAH patients. Further research is required in larger numbers of patients and to elucidate the mechanism of these findings. © 2013 The Authors. Respirology © 2013 Asian Pacific Society of Respirology.

  15. Nonspecific suppressor T cells cause decreased mixed lymphocyte culture reactivity in bone marrow transplant patients

    International Nuclear Information System (INIS)

    Harada, M.; Ueda, M.; Nakao, S.; Kondo, K.; Odaka, K.; Shiobara, S.; Matsue, K.; Mori, T.; Matsuda, T.

    1986-01-01

    Decreased reactivity in mixed lymphocyte culture (MLC) was observed in patients within 1 yr after allogeneic and autologous bone marrow transplantation. Suppressor activity of peripheral blood mononuclear cells (PBMC) from transplant patients was studied by adding these cells as modulator cells to a bidirectional MLC with cells from normal individuals. PBMC from transplant patients markedly suppressed MLC reactivity in a dose-dependent manner. Suppressor activity was present in cells forming rosettes with sheep erythrocytes. Treatment of modulator cells with monoclonal antibodies against T cell differentiation antigens (OKT8, OKIa1) and complement completely abolished suppression of MLC. Suppressor activity was unaffected by 30 Gy irradiation. Suppressor activity declined gradually after transplantation and was inversely correlated with MLC reactivity of each patient at a significant level (p less than 0.01). These observations suggest that OKT8+ Ia+ radioresistant suppressor T cells play a role in the development of decreased MLC reactivity observed during the early post-transplant period

  16. Childhood tuberculosis is associated with decreased abundance of T cell gene transcripts and impaired T cell function.

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    Cheryl Hemingway

    Full Text Available The WHO estimates around a million children contract tuberculosis (TB annually with over 80 000 deaths from dissemination of infection outside of the lungs. The insidious onset and association with skin test anergy suggests failure of the immune system to both recognise and respond to infection. To understand the immune mechanisms, we studied genome-wide whole blood RNA expression in children with TB meningitis (TBM. Findings were validated in a second cohort of children with TBM and pulmonary TB (PTB, and functional T-cell responses studied in a third cohort of children with TBM, other extrapulmonary TB (EPTB and PTB. The predominant RNA transcriptional response in children with TBM was decreased abundance of multiple genes, with 140/204 (68% of all differentially regulated genes showing reduced abundance compared to healthy controls. Findings were validated in a second cohort with concordance of the direction of differential expression in both TBM (r2 = 0.78 p = 2x10-16 and PTB patients (r2 = 0.71 p = 2x10-16 when compared to a second group of healthy controls. Although the direction of expression of these significant genes was similar in the PTB patients, the magnitude of differential transcript abundance was less in PTB than in TBM. The majority of genes were involved in activation of leucocytes (p = 2.67E-11 and T-cell receptor signalling (p = 6.56E-07. Less abundant gene expression in immune cells was associated with a functional defect in T-cell proliferation that recovered after full TB treatment (p<0.0003. Multiple genes involved in T-cell activation show decreased abundance in children with acute TB, who also have impaired functional T-cell responses. Our data suggest that childhood TB is associated with an acquired immune defect, potentially resulting in failure to contain the pathogen. Elucidation of the mechanism causing the immune paresis may identify new treatment and prevention strategies.

  17. Decreased glucose uptake by hyperglycemia is regulated by different mechanisms in human cancer cells and monocytes

    International Nuclear Information System (INIS)

    Kim, Chae Kyun; Chung, June Key; Lee, Yong Jin; Hong, Mee Kyoung; Jeong, Jae Min; Lee, Dong Soo; Lee, Myung Chul

    2002-01-01

    To clarify the difference in glucose uptake between human cancer cells and monocytes, we studied ( 18 F) fluorodeoxyglucose (FDG) uptake in three human colon cancer cell lines (SNU-C2A, SNU-C4, SNU-C5), one human lung cancer cell line (NCI-H522), and human peripheral blood monocytes. The FDG uptake of both cancer cells and monocytes was increased in glucose-free medium, but decreased in the medium containing 16.7 mM glucose (hyperglycemic). The level of Glut1 mRNA decreased in human colon cancer cells and NCI-H522 under hyperglycemic condition. Glut1 protein expression was also decreased in the four human cancer cell lines under hyperglycemic condition, whereas it was consistently undetectable in monocytes. SNU-C2A, SNU-C4 and NCI-H522 showed a similar level of hexokinase activity (7.5-10.8 mU/mg), while SNU-C5 and moncytes showed lower range of hexokinase activity (4.3-6.5 mU/mg). These data suggest that glucose uptake is regulated by different mechanisms in human cancer cells and monocytes

  18. Knockdown of XBP1 by RNAi in Mouse Granulosa Cells Promotes Apoptosis, Inhibits Cell Cycle, and Decreases Estradiol Synthesis

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    Nan Wang

    2017-05-01

    Full Text Available Granulosa cells are crucial for follicular growth, development, and follicular atresia. X-box binding protein 1 (XBP1, a basic region-leucine zipper protein, is widely involved in cell differentiation, proliferation, apoptosis, cellular stress response, and other signaling pathways. In this study, RNA interference, flow cytometry, western blot, real-time PCR, Cell Counting Kit (CCK8, and ELISA were used to investigate the effect of XBP1 on steroidogenesis, apoptosis, cell cycle, and proliferation of mouse granulosa cells. ELISA analysis showed that XBP1 depletion significantly decreased the concentrations of estradiol (E2. Additionally, the expression of estrogen synthesis enzyme Cyp19a1 was sharply downregulated. Moreover, flow cytometry showed that knockdown of XBP1 increased the apoptosis rate and arrests the cell cycle in S-phase in granulosa cells (GCs. Further study confirmed these results. The expression of CCAAT-enhancer-binding protein homologous protein (CHOP, cysteinyl aspartate specific proteases-3 (caspase-3, cleaved caspase-3, and Cyclin E was upregulated, while that of Bcl-2, Cyclin A1, and Cyclin B1 was downregulated. Simultaneously, CCK8 analysis indicated that XBP1 disruption inhibited cell proliferation. In addition, XBP1 knockdown also alters the expression of Has2 and Ptgs2, two essential genes for folliculogenesis. Collectively, these data reveal a novel critical role of XBP1 in folliculogenesis by regulating the cell cycle, apoptosis, and steroid synthesis of mouse granulosa cells.

  19. L-carnitine significantly decreased aging of rat adipose tissue-derived mesenchymal stem cells.

    Science.gov (United States)

    Mobarak, Halimeh; Fathi, Ezzatollah; Farahzadi, Raheleh; Zarghami, Nosratollah; Javanmardi, Sara

    2017-03-01

    Mesenchymal stem cells are undifferentiated cells that have the ability to divide continuously and tissue regeneration potential during the transplantation. Aging and loss of cell survival, is one of the main problems in cell therapy. Since the production of free radicals in the aging process is effective, the use of antioxidant compounds can help in scavenging free radicals and prevent the aging of cells. The aim of this study is evaluate the effects of L-carnitine (LC) on proliferation and aging of rat adipose tissue-derived mesenchymal stem cells (rADSC). rADSCs were isolated from inguinal region of 5 male Rattus rats. Oil red-O, alizarin red-S and toluidine blue staining were performed to evaluate the adipogenic, osteogenic and chondrogenic differentiation of rADSCs, respectively. Flow cytometric analysis was done for investigating the cell surface markers. The methyl thiazol tetrazolium (MTT) method was used to determine the cell proliferation of rADSCs following exposure to different concentrations of LC. rADSCs aging was evaluated by beta-galactosidase staining. The results showed significant proliferation of rADSCs 48 h after treatment with concentrations of 0.2 mM LC. In addition, in the presence of 0.2 mM LC, rADSCs appeared to be growing faster than control group and 0.2 mM LC supplementation could significantly decrease the population doubling time and aging of rADSCs. It seems that LC would be a good antioxidant to improve lifespan of rADSCs due to the decrease in aging.

  20. Non-steroidal anti-inflammatory drugs decrease E2F1 expression and inhibit cell growth in ovarian cancer cells.

    Directory of Open Access Journals (Sweden)

    Blanca L Valle

    Full Text Available Epidemiological studies have shown that the regular use of non-steroidal anti-inflammatory (NSAIDs drugs is associated with a reduced risk of various cancers. In addition, in vitro and experiments in mouse models have demonstrated that NSAIDs decrease tumor initiation and/or progression of several cancers. However, there are limited preclinical studies investigating the effects of NSAIDs in ovarian cancer. Here, we have studied the effects of two NSAIDs, diclofenac and indomethacin, in ovarian cancer cell lines and in a xenograft mouse model. Diclofenac and indomethacin treatment decreased cell growth by inducing cell cycle arrest and apoptosis. In addition, diclofenac and indomethacin reduced tumor volume in a xenograft model of ovarian cancer. To identify possible molecular pathways mediating the effects of NSAID treatment in ovarian cancer, we performed microarray analysis of ovarian cancer cells treated with indomethacin or diclofenac. Interestingly, several of the genes found downregulated following diclofenac or indomethacin treatment are transcriptional target genes of E2F1. E2F1 was downregulated at the mRNA and protein level upon treatment with diclofenac and indomethacin, and overexpression of E2F1 rescued cells from the growth inhibitory effects of diclofenac and indomethacin. In conclusion, NSAIDs diclofenac and indomethacin exert an anti-proliferative effect in ovarian cancer in vitro and in vivo and the effects of NSAIDs may be mediated, in part, by downregulation of E2F1.

  1. Selective decreases of nicotinic acetylcholine receptors in PC12 cells exposed to fluoride

    International Nuclear Information System (INIS)

    Chen Jia; Shan, K.-R.; Long, Y.-G.; Wang, Y.-N.; Nordberg, Agneta; Guan, Z.-Z.

    2003-01-01

    In an attempt to elucidate the mechanism by which excessive fluoride damages the central nervous system, the effects of exposure of PC12 cells to different concentrations of fluoride for 48 h on nicotinic acetylcholine receptors (nAChRs) were characterized here. Significant reductions in the number of binding sites for both [ 3 H]epibatidine and [ 125 I]α-bungarotoxin, as well as a significant decrease in the B max value for the high-affinity of epibatidine binding site were observed in PC12 cells subjected to high levels of fluoride. On the protein level, the α3 and α7 subunits of nAChRs were also significantly decreased in the cells exposed to high concentrations of fluoride. In contrast, such exposure had no significant effect on the level of the β2 subunit. These findings suggest that selective decreases in the number of nAChRs may play an important role in the mechanism(s) by which fluoride causes dysfunction of the central nervous system

  2. RNA interference-mediated c-MYC inhibition prevents cell growth and decreases sensitivity to radio- and chemotherapy in childhood medulloblastoma cells

    International Nuclear Information System (INIS)

    Bueren, André O von; Shalaby, Tarek; Oehler-Jänne, Christoph; Arnold, Lucia; Stearns, Duncan; Eberhart, Charles G; Arcaro, Alexandre; Pruschy, Martin; Grotzer, Michael A

    2009-01-01

    With current treatment strategies, nearly half of all medulloblastoma (MB) patients die from progressive tumors. Accordingly, the identification of novel therapeutic strategies remains a major goal. Deregulation of c-MYC is evident in numerous human cancers. In MB, over-expression of c-MYC has been shown to cause anaplasia and correlate with unfavorable prognosis. To study the role of c-MYC in MB biology, we down-regulated c-MYC expression by using small interfering RNA (siRNA) and investigated changes in cellular proliferation, cell cycle analysis, apoptosis, telomere maintenance, and response to ionizing radiation (IR) and chemotherapeutics in a representative panel of human MB cell lines expressing different levels of c-MYC (DAOY wild-type, DAOY transfected with the empty vector, DAOY transfected with c-MYC, D341, and D425). siRNA-mediated c-MYC down-regulation resulted in an inhibition of cellular proliferation and clonogenic growth, inhibition of G1-S phase cell cycle progression, and a decrease in human telomerase reverse transcriptase (hTERT) expression and telomerase activity. On the other hand, down-regulation of c-MYC reduced apoptosis and decreased the sensitivity of human MB cells to IR, cisplatin, and etoposide. This effect was more pronounced in DAOY cells expressing high levels of c-MYC when compared with DAOY wild-type or DAOY cells transfected with the empty vector. In human MB cells, in addition to its roles in growth and proliferation, c-MYC is also a potent inducer of apoptosis. Therefore, targeting c-MYC might be of therapeutic benefit when used sequentially with chemo- and radiotherapy rather than concomitantly

  3. RNA interference-mediated c-MYC inhibition prevents cell growth and decreases sensitivity to radio- and chemotherapy in childhood medulloblastoma cells

    Directory of Open Access Journals (Sweden)

    Arcaro Alexandre

    2009-01-01

    Full Text Available Abstract Background With current treatment strategies, nearly half of all medulloblastoma (MB patients die from progressive tumors. Accordingly, the identification of novel therapeutic strategies remains a major goal. Deregulation of c-MYC is evident in numerous human cancers. In MB, over-expression of c-MYC has been shown to cause anaplasia and correlate with unfavorable prognosis. Methods To study the role of c-MYC in MB biology, we down-regulated c-MYC expression by using small interfering RNA (siRNA and investigated changes in cellular proliferation, cell cycle analysis, apoptosis, telomere maintenance, and response to ionizing radiation (IR and chemotherapeutics in a representative panel of human MB cell lines expressing different levels of c-MYC (DAOY wild-type, DAOY transfected with the empty vector, DAOY transfected with c-MYC, D341, and D425. Results siRNA-mediated c-MYC down-regulation resulted in an inhibition of cellular proliferation and clonogenic growth, inhibition of G1-S phase cell cycle progression, and a decrease in human telomerase reverse transcriptase (hTERT expression and telomerase activity. On the other hand, down-regulation of c-MYC reduced apoptosis and decreased the sensitivity of human MB cells to IR, cisplatin, and etoposide. This effect was more pronounced in DAOY cells expressing high levels of c-MYC when compared with DAOY wild-type or DAOY cells transfected with the empty vector. Conclusion In human MB cells, in addition to its roles in growth and proliferation, c-MYC is also a potent inducer of apoptosis. Therefore, targeting c-MYC might be of therapeutic benefit when used sequentially with chemo- and radiotherapy rather than concomitantly.

  4. Stable SREBP-1a knockdown decreases the cell proliferation rate in human preadipocyte cells without inducing senescence

    International Nuclear Information System (INIS)

    Alvarez, María Soledad; Fernandez-Alvarez, Ana; Cucarella, Carme; Casado, Marta

    2014-01-01

    Highlights: • SGBS cells mostly expressed SREBP-1a variant. • SREBP-1a knockdown decreased the proliferation of SGBS cells without inducing senescence. • We have identified RBBP8 and CDKN3 genes as potential SREBP-1a targets. - Abstract: Sterol regulatory element binding proteins (SREBP), encoded by the Srebf1 and Srebf2 genes, are important regulators of genes involved in cholesterol and fatty acid metabolism. Whereas SREBP-2 controls the cholesterol synthesis, SREBP-1 proteins (-1a and -1c) function as the central hubs in lipid metabolism. Despite the key function of these transcription factors to promote adipocyte differentiation, the roles of SREBP-1 proteins during the preadipocyte state remain unknown. Here, we evaluate the role of SREBP-1 in preadipocyte proliferation using RNA interference technology. Knockdown of the SREBP-1a gene decreased the proliferation rate in human SGBS preadipocyte cell strain without inducing senescence. Furthermore, our data identified retinoblastoma binding protein 8 and cyclin-dependent kinase inhibitor 3 genes as new potential SREBP-1 targets, in addition to cyclin-dependent kinase inhibitor 1A which had already been described as a gene regulated by SREBP-1a. These data suggested a new role of SREBP-1 in adipogenesis via regulation of preadipocyte proliferation

  5. Stable SREBP-1a knockdown decreases the cell proliferation rate in human preadipocyte cells without inducing senescence

    Energy Technology Data Exchange (ETDEWEB)

    Alvarez, María Soledad [Instituto de Biomedicina de Valencia (IBV-CSIC), Jaime Roig 11, E-46010 Valencia (Spain); Fernandez-Alvarez, Ana [Fundación Instituto Leloir, IIBBA-CONICET, Av. Patricias Argentinas 435, Ciudad Autónoma de Buenos Aires C1405BWE (Argentina); Cucarella, Carme [Instituto de Biomedicina de Valencia (IBV-CSIC), Jaime Roig 11, E-46010 Valencia (Spain); Casado, Marta, E-mail: mcasado@ibv.csic.es [Instituto de Biomedicina de Valencia (IBV-CSIC), Jaime Roig 11, E-46010 Valencia (Spain)

    2014-04-25

    Highlights: • SGBS cells mostly expressed SREBP-1a variant. • SREBP-1a knockdown decreased the proliferation of SGBS cells without inducing senescence. • We have identified RBBP8 and CDKN3 genes as potential SREBP-1a targets. - Abstract: Sterol regulatory element binding proteins (SREBP), encoded by the Srebf1 and Srebf2 genes, are important regulators of genes involved in cholesterol and fatty acid metabolism. Whereas SREBP-2 controls the cholesterol synthesis, SREBP-1 proteins (-1a and -1c) function as the central hubs in lipid metabolism. Despite the key function of these transcription factors to promote adipocyte differentiation, the roles of SREBP-1 proteins during the preadipocyte state remain unknown. Here, we evaluate the role of SREBP-1 in preadipocyte proliferation using RNA interference technology. Knockdown of the SREBP-1a gene decreased the proliferation rate in human SGBS preadipocyte cell strain without inducing senescence. Furthermore, our data identified retinoblastoma binding protein 8 and cyclin-dependent kinase inhibitor 3 genes as new potential SREBP-1 targets, in addition to cyclin-dependent kinase inhibitor 1A which had already been described as a gene regulated by SREBP-1a. These data suggested a new role of SREBP-1 in adipogenesis via regulation of preadipocyte proliferation.

  6. Inhibition of EGFR nuclear shuttling decreases irradiation resistance in HeLa cells.

    Science.gov (United States)

    Wei, Hong; Zhu, Zijie; Lu, Longtao

    2017-01-01

    Cervical cancer is a leading cause of mortality in women worldwide. The resistance to irradiation at the advanced stage is the main reason for the poor prognosis and high mortality. This work aims to elucidate the molecular mechanism underlying the radio-resistance. In this study, we determined the pEGFR-T654 and pDNA-PK-T2609 expression level changes in irradiated HeLa cells treated with T654 peptide, a nuclear localization signal (NLS) inhibitor, to inhibit EGFR nuclear transport. Cell viability, cell cycle and migratory capacity were analyzed. Xenograft animal model was used to evaluate the effect of EGFR nuclear transport inhibition on the tumor growth in vivo. The enhanced translocation of nuclear EGFR in the irradiated HeLa cells correlated with the increasing level of pEGFR-T654 and pDNA-PK-T2609. Inhibition of EGFR nuclear translocation by NLS peptide inhibitor attenuated DNA damage repair in the irradiated HeLa cells, decreased cell viability and promoted cell death through arrest at G0 phase. NLS peptide inhibitor impaired the migratory capacity of irradiated HeLa cells, and negatively affected tumorigenesis in xenograft mice. This work puts forward a potential molecular mechanism of the irradiation resistance in cervical cancer cells, providing a promising direction towards an efficient therapy of cervical cancer.

  7. Force via integrins but not E-cadherin decreases Oct3/4 expression in embryonic stem cells

    Energy Technology Data Exchange (ETDEWEB)

    Uda, Yuhei [Department of Mechanical Science and Engineering, University of Illinois at Urbana-Champaign, Urbana, IL 61801 (United States); Department of Biomedical Engineering, Graduate School of Biomedical Engineering, Tohoku University, 6-6-01, Aramaki-aoba, Aoba-ward, Sendai City (Japan); Poh, Yeh-Chuin; Chowdhury, Farhan; Wu, Douglas C. [Department of Mechanical Science and Engineering, University of Illinois at Urbana-Champaign, Urbana, IL 61801 (United States); Tanaka, Tetsuya S. [Department of Animal Sciences, University of Illinois at Urbana-Champaign, Urbana, IL 61801 (United States); Sato, Masaaki [Department of Biomedical Engineering, Graduate School of Biomedical Engineering, Tohoku University, 6-6-01, Aramaki-aoba, Aoba-ward, Sendai City (Japan); Wang, Ning, E-mail: nwangrw@illinois.edu [Department of Mechanical Science and Engineering, University of Illinois at Urbana-Champaign, Urbana, IL 61801 (United States)

    2011-11-18

    Highlights: Black-Right-Pointing-Pointer Force via integrins or cadherins induces similar cell stiffening responses. Black-Right-Pointing-Pointer Force via integrins but not cadherins induces cell spreading. Black-Right-Pointing-Pointer Force via integrins but not cadherins induces differentiation of embryonic stem cells. -- Abstract: Increasing evidence suggests that mechanical factors play a critical role in fate decisions of stem cells. Recently we have demonstrated that a local force applied via Arg-Gly-Asp (RGD) peptides coated magnetic beads to mouse embryonic stem (ES) cells increases cell spreading and cell stiffness and decreases Oct3/4 (Pou5f1) gene expression. However, it is not clear whether the effects of the applied stress on these functions of ES cells can be extended to natural extracellular matrix proteins or cell-cell adhesion molecules. Here we show that a local cyclic shear force applied via fibronectin or laminin to integrin receptors increased cell spreading and stiffness, downregulated Oct3/4 gene expression, and decreased cell proliferation rate. In contrast, the same cyclic force applied via cell-cell adhesion molecule E-cadherin (Cdh1) had no effects on cell spreading, Oct3/4 gene expression, and the self-renewal of mouse ES cells, but induced significant cell stiffening. Our findings demonstrate that biological responses of ES cells to force applied via integrins are different from those to force via E-cadherin, suggesting that mechanical forces might play different roles in different force transduction pathways to shape early embryogenesis.

  8. Decreased glucose uptake by hyperglycemia is regulated by different mechanisms in human cancer cells and monocytes

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Chae Kyun; Chung, June Key; Lee, Yong Jin; Hong, Mee Kyoung; Jeong, Jae Min; Lee, Dong Soo; Lee, Myung Chul [College of Medicine, Seoul National Univ., Seoul (Korea, Republic of)

    2002-04-01

    To clarify the difference in glucose uptake between human cancer cells and monocytes, we studied ({sup 18}F) fluorodeoxyglucose (FDG) uptake in three human colon cancer cell lines (SNU-C2A, SNU-C4, SNU-C5), one human lung cancer cell line (NCI-H522), and human peripheral blood monocytes. The FDG uptake of both cancer cells and monocytes was increased in glucose-free medium, but decreased in the medium containing 16.7 mM glucose (hyperglycemic). The level of Glut1 mRNA decreased in human colon cancer cells and NCI-H522 under hyperglycemic condition. Glut1 protein expression was also decreased in the four human cancer cell lines under hyperglycemic condition, whereas it was consistently undetectable in monocytes. SNU-C2A, SNU-C4 and NCI-H522 showed a similar level of hexokinase activity (7.5-10.8 mU/mg), while SNU-C5 and moncytes showed lower range of hexokinase activity (4.3-6.5 mU/mg). These data suggest that glucose uptake is regulated by different mechanisms in human cancer cells and monocytes.

  9. Bee Venom Decreases LPS-Induced Inflammatory Responses in Bovine Mammary Epithelial Cells.

    Science.gov (United States)

    Jeong, Chang Hee; Cheng, Wei Nee; Bae, Hyojin; Lee, Kyung Woo; Han, Sang Mi; Petriello, Michael C; Lee, Hong Gu; Seo, Han Geuk; Han, Sung Gu

    2017-10-28

    The world dairy industry has long been challenged by bovine mastitis, an inflammatory disease, which causes economic loss due to decreased milk production and quality. Attempts have been made to prevent or treat this disease with multiple approaches, primarily through increased abuse of antibiotics, but effective natural solutions remain elusive. Bee venom (BV) contains a variety of peptides ( e.g. , melittin) and shows multiple bioactivities, including prevention of inflammation. Thus, in the current study, it was hypothesized that BV can reduce inflammation in bovine mammary epithelial cells (MAC-T). To examine the hypothesis, cells were treated with LPS (1 μg/ml) to induce an inflammatory response and the anti-inflammatory effects of BV (2.5 and 5 μg/ml) were investigated. The cellular mechanisms of BV against LPS-induced inflammation were also investigated. Results showed that BV can attenuate expression of an inflammatory protein, COX2, and pro-inflammatory cytokines such as IL-6 and TNF-α. Activation of NF-κB, an inflammatory transcription factor, was significantly downregulated by BV in cells treated with LPS, through dephosphorylation of ERK1/2. Moreover, pretreatment of cells with BV attenuated LPS-induced production of intracellular reactive oxygen species ( e.g. , superoxide anion). These results support our hypothesis that BV can decrease LPS-induced inflammatory responses in bovine mammary epithelial cells through inhibition of oxidative stress, NF-κB, ERK1/2, and COX-2 signaling.

  10. Analysis of Hereditary Elliptocytosis with Decreased Binding of Eosin-5-maleimide to Red Blood Cells

    Directory of Open Access Journals (Sweden)

    Shin-ichiro Suemori

    2015-01-01

    Full Text Available Flow cytometric test for analyzing the eosin-5-maleimide (EMA binding to red blood cells has been believed to be a specific method for diagnosing hereditary spherocytosis (HS. However, it has been reported that diseases other than HS, such as hereditary pyropoikilocytosis (HPP and Southeast Asian ovalocytosis (SAO, which are forms in the category of hereditary elliptocytosis (HE, show decreased EMA binding to red blood cells. We analyzed EMA binding to red blood cells in 101 healthy control subjects and 42 HS patients and obtained a mean channel fluorescence (MCF cut-off value of 36.4 (sensitivity 0.97, specificity 0.95. Using this method, we also analyzed 12 HE patients. Among them, four HE patients showed the MCF at or below the cut-off value. It indicates that some HE patients have decreased EMA binding to red blood cells. Two of these four HE patients were classified as common HE, and two were spherocytic HE with reduced spectrin. This study demonstrates that, in addition to patients with HPP or SAO, some HE patients have decreased EMA binding to red blood cells.

  11. Decreased IL-33 Production Contributes to Trophoblast Cell Dysfunction in Pregnancies with Preeclampsia

    Directory of Open Access Journals (Sweden)

    Hong Chen

    2018-01-01

    Full Text Available Preeclampsia (PE is a life-threatening pregnancy complication which is related to aggradation of risk regarding fetal and maternal morbidity and mortality. Dysregulation of systemic inflammatory response and dysfunction of trophoblast cells have been proposed to be involved in the development and progression of PE. Some studies have demonstrated that interleukin-33 (IL-33 is an immunomodulatory cytokine that is associated with the immune regulation of tumor cells. However, little is known whether IL-33 and its receptor ST2/IL-1 R4 could regulate trophoblast cells, which are associated with the pathogenesis of PE. In this study, our target is to explore the impact of IL-33 on trophoblast cells and elucidate its underlying pathophysiological mechanisms. Placental tissues from the severe PE group (n=11 and the normotensive pregnant women’s group (n=11 were collected for the protein expression and distribution of IL-33 along with its receptor ST2/IL-1 R4 via Western blot analysis and immunohistochemistry, respectively. We discovered that the level of IL-33 was decreased in placental tissues of pregnant women with PE, while no distinction was observed in the expression of ST2/IL-1 R4. These results were further verified in villous explants which were treated with sodium nitroprusside with different concentrations, to simulate the pathological environment of PE. To investigate IL-33 effects on trophoblast cells separately, IL-33 shRNA was introduced into HTR8/SVneo cells and villi. IL-33 shRNA weakened the proliferation, migration, and invasion capacity of HTR8/SVneo cells. The migration distance of villous explants was also markedly decreased. The reduced invasion of trophoblast cells is a result of IL-33 knockdown which could be related to the decline of MMP2/9 activity and the increased utterance of TIMP1/2. Overall, our findings demonstrated that the reduction of IL-33 production was connected with the reduced functional capability of

  12. Decreased numbers of CD4+ naive and effector memory T cells, and CD8+ naïve T cells, are associated with trichloroethylene exposure

    Directory of Open Access Journals (Sweden)

    H Dean eHosgood

    2012-01-01

    Full Text Available Trichloroethylene (TCE is a volatile chlorinated organic compound that is commonly used as a solvent for lipophilic compounds. Although recognized as an animal carcinogen, TCE’s carcinogenic potential in humans is still uncertain. We have carried out a cross-sectional study of 80 workers exposed to TCE and 96 unexposed controls matched on age and sex in Guangdong, China to study TCE’s early biologic effects. We previously reported that the total lymphocyte count and each of the major lymphocyte subsets (i.e., CD4+ T cells, CD8+ T cells, natural killer (NK cells, and B cells were decreased in TCE-exposed workers compared to controls, suggesting a selective effect on lymphoid progenitors and/or lymphocyte survival. To explore which T lymphocyte subsets are affected, we investigated the effect of TCE exposure on the numbers of CD4+ naïve and memory T cells, CD8+ naïve and memory T cells, and regulatory T cells by FACS analysis. Linear regression of each subset was used to test for differences between exposed workers and controls adjusting for potential confounders. We observed that CD4+ and CD8+ naïve T cell counts were about 8% (p = 0.056 and 17% (p = 0.0002 lower, respectively, among exposed workers. CD4+ effector memory T cell counts were decreased by about 20% among TCE exposed workers compared to controls (p = 0.001. The selective targeting of TCE on CD8+ naïve and possibly CD4+ naive T cells, and CD4+ effector memory T cells, provide further insights into the immunosuppression-related response of human immune cells upon TCE exposure.

  13. MDMA (Ecstasy) Decreases the Number of Neurons and Stem Cells in Embryonic Cortical Cultures

    DEFF Research Database (Denmark)

    Kindlundh-Högberg, Anna M S; Pickering, Chris; Wicher, Grzegorz

    2010-01-01

    Ecstasy, 3,4-methylenedioxymetamphetamine (MDMA), is a recreational drug used among adolescents, including young pregnant women. MDMA passes the placental barrier and may therefore influence fetal development. The aim was to investigate the direct effect of MDMA on cortical cells using dissociated...... CNS cortex of rat embryos, E17. The primary culture was exposed to a single dose of MDMA and collected 5 days later. MDMA caused a dramatic, dose-dependent (100 and 400 muM) decrease in nestin-positive stem cell density, as well as a significant reduction (400 muM) in NeuN-positive cells. By q......PCR, MDMA (200 muM) caused a significant decrease in mRNA expression of the 5HT3 receptor, dopamine D(1) receptor, and glutamate transporter EAAT2-1, as well as an increase in mRNA levels of the NMDA NR1 receptor subunit and the 5HT(1A) receptor. In conclusion, MDMA caused a marked reduction in stem cells...

  14. Prion protein-deficient mice exhibit decreased CD4 T and LTi cell numbers and impaired spleen structure.

    Science.gov (United States)

    Kim, Soochan; Han, Sinsuk; Lee, Ye Eun; Jung, Woong-Jae; Lee, Hyung Soo; Kim, Yong-Sun; Choi, Eun-Kyoung; Kim, Mi-Yeon

    2016-01-01

    The cellular prion protein is expressed in almost all tissues, including the central nervous system and lymphoid tissues. To investigate the effects of the prion protein in lymphoid cells and spleen structure formation, we used prion protein-deficient (Prnp(0/0)) Zürich I mice generated by inactivation of the Prnp gene. Prnp(0/0) mice had decreased lymphocytes, in particular, CD4 T cells and lymphoid tissue inducer (LTi) cells. Decreased CD4 T cells resulted from impaired expression of CCL19 and CCL21 in the spleen rather than altered chemokine receptor CCR7 expression. Importantly, some of the white pulp regions in spleens from Prnp(0/0) mice displayed impaired T zone structure as a result of decreased LTi cell numbers and altered expression of the lymphoid tissue-organizing genes lymphotoxin-α and CXCR5, although expression of the lymphatic marker podoplanin and CXCL13 by stromal cells was not affected. In addition, CD3(-)CD4(+)IL-7Rα(+) LTi cells were rarely detected in impaired white pulp in spleens of these mice. These data suggest that the prion protein is required to form the splenic white pulp structure and for development of normal levels of CD4 T and LTi cells. Copyright © 2015. Published by Elsevier GmbH.

  15. Progesterone receptor blockade in human breast cancer cells decreases cell cycle progression through G2/M by repressing G2/M genes.

    Science.gov (United States)

    Clare, Susan E; Gupta, Akash; Choi, MiRan; Ranjan, Manish; Lee, Oukseub; Wang, Jun; Ivancic, David Z; Kim, J Julie; Khan, Seema A

    2016-05-23

    The synthesis of specific, potent progesterone antagonists adds potential agents to the breast cancer prevention and treatment armamentarium. The identification of individuals who will benefit from these agents will be a critical factor for their clinical success. We utilized telapristone acetate (TPA; CDB-4124) to understand the effects of progesterone receptor (PR) blockade on proliferation, apoptosis, promoter binding, cell cycle progression, and gene expression. We then identified a set of genes that overlap with human breast luteal-phase expressed genes and signify progesterone activity in both normal breast cells and breast cancer cell lines. TPA administration to T47D cells results in a 30 % decrease in cell number at 24 h, which is maintained over 72 h only in the presence of estradiol. Blockade of progesterone signaling by TPA for 24 h results in fewer cells in G2/M, attributable to decreased expression of genes that facilitate the G2/M transition. Gene expression data suggest that TPA affects several mechanisms that progesterone utilizes to control gene expression, including specific post-translational modifications, and nucleosomal organization and higher order chromatin structure, which regulate access of PR to its DNA binding sites. By comparing genes induced by the progestin R5020 in T47D cells with those increased in the luteal-phase normal breast, we have identified a set of genes that predict functional progesterone signaling in tissue. These data will facilitate an understanding of the ways in which drugs such as TPA may be utilized for the prevention, and possibly the therapy, of human breast cancer.

  16. Low Doses of Curcuma longa Modulates Cell Migration and Cell-Cell Adhesion.

    Science.gov (United States)

    de Campos, Paloma Santos; Matte, Bibiana Franzen; Diel, Leonardo Francisco; Jesus, Luciano Henrique; Bernardi, Lisiane; Alves, Alessandro Menna; Rados, Pantelis Varvaki; Lamers, Marcelo Lazzaron

    2017-09-01

    Cell invasion and metastasis are involved in clinical failures in cancer treatment, and both events require the acquisition of a migratory behavior by tumor cells. Curcumin is a promising natural product with anti-proliferative activity, but its effects on cell migration are still unclear. We evaluated the effects of curcumin on the proliferation, apoptosis, migration, and cell-cell adhesion of keratinocyte, oral squamous cell carcinoma (OSCC), and fibroblast cell lines, as well as in a xenograft model of OSCC. Curcumin (2 μM) decreased cell proliferation in cell lines with mesenchymal characteristics, while cell death was detected only at 50 μM. We observed that highly migratory cells showed a decrease on migration speed and directionality when treated with 2 or 5 μM of curcumin (50% and 40%, respectively, p curcumin dose dependently decreased cell-cell adhesion, especially on tumor-derived spheroids. Also, in a xenograft model with patient-derived OSCC cells, the administration of curcumin decreased tumor growth and aggressiveness when compared with untreated tumors, indicating the potential antitumor effect in oral cancer. These results suggest that lower doses of curcumin can influence several steps involved in tumorigenesis, including migration properties, suggesting a possible use in cancer therapy. Copyright © 2017 John Wiley & Sons, Ltd. Copyright © 2017 John Wiley & Sons, Ltd.

  17. Butyrate decreases its own oxidation in colorectal cancer cells through inhibition of histone deacetylases.

    Science.gov (United States)

    Han, Anna; Bennett, Natalie; Ahmed, Bettaieb; Whelan, Jay; Donohoe, Dallas R

    2018-06-05

    Colorectal cancer is characterized by an increase in the utilization of glucose and a diminishment in the oxidation of butyrate, which is a short chain fatty acid. In colorectal cancer cells, butyrate inhibits histone deacetylases to increase the expression of genes that slow the cell cycle and induce apoptosis. Understanding the mechanisms that contribute to the metabolic shift away from butyrate oxidation in cancer cells is important in in understanding the beneficial effects of the molecule toward colorectal cancer. Here, we demonstrate that butyrate decreased its own oxidation in cancerous colonocytes. Butyrate lowered the expression of short chain acyl-CoA dehydrogenase, an enzyme that mediates the oxidation of short-chain fatty acids. Butyrate does not alter short chain acyl-CoA dehydrogenase levels in non-cancerous colonocytes. Trichostatin A, a structurally unrelated inhibitor of histone deacetylases, and propionate also decreased the level of short chain acyl-CoA dehydrogenase, which alluded to inhibition of histone deacetylases as a part of the mechanism. Knockdown of histone deacetylase isoform 1, but not isoform 2 or 3, inhibited the ability of butyrate to decrease short chain acyl-CoA dehydrogenase expression. This work identifies a mechanism by which butyrate selective targets colorectal cancer cells to reduce its own metabolism.

  18. Macrocyclic peptides decrease c-Myc protein levels and reduce prostate cancer cell growth.

    Science.gov (United States)

    Mukhopadhyay, Archana; Hanold, Laura E; Thayele Purayil, Hamsa; Gisemba, Solomon A; Senadheera, Sanjeewa N; Aldrich, Jane V

    2017-08-03

    The oncoprotein c-Myc is often overexpressed in cancer cells, and the stability of this protein has major significance in deciding the fate of a cell. Thus, targeting c-Myc levels is an attractive approach for developing therapeutic agents for cancer treatment. In this study, we report the anti-cancer activity of the macrocyclic peptides [D-Trp]CJ-15,208 (cyclo[Phe-D-Pro-Phe-D-Trp]) and the natural product CJ-15,208 (cyclo[Phe-D-Pro-Phe-Trp]). [D-Trp]CJ-15,208 reduced c-Myc protein levels in prostate cancer cells and decreased cell proliferation with IC 50 values ranging from 2.0 to 16 µM in multiple PC cell lines. [D-Trp]CJ-15,208 induced early and late apoptosis in PC-3 cells following 48 hours treatment, and growth arrest in the G2 cell cycle phase following both 24 and 48 hours treatment. Down regulation of c-Myc in PC-3 cells resulted in loss of sensitivity to [D-Trp]CJ-15,208 treatment, while overexpression of c-Myc in HEK-293 cells imparted sensitivity of these cells to [D-Trp]CJ-15,208 treatment. This macrocyclic tetrapeptide also regulated PP2A by reducing the levels of its phosphorylated form which regulates the stability of cellular c-Myc protein. Thus [D-Trp]CJ-15,208 represents a new lead compound for the potential development of an effective treatment of prostate cancer.

  19. DeltaFosB induces osteosclerosis and decreases adipogenesis by two independent cell-autonomous mechanisms

    DEFF Research Database (Denmark)

    Kveiborg, Marie; Sabatakos, George; Chiusaroli, Riccardo

    2004-01-01

    establishes that the skeletal phenotype is cell autonomous to the osteoblast lineage and independent of adipocyte formation. It also strongly suggests that the decreased fat phenotype of NSE-DeltaFosB mice is independent of the changes in the osteoblast lineage. In vitro, overexpression of Delta......Osteoblasts and adipocytes may develop from common bone marrow mesenchymal precursors. Transgenic mice overexpressing DeltaFosB, an AP-1 transcription factor, under the control of the neuron-specific enolase (NSE) promoter show both markedly increased bone formation and decreased adipogenesis...... of DeltaFosB on adipocyte differentiation appears to occur at early stages of stem cell commitment, affecting C/EBPbeta functions. It is concluded that the changes in osteoblast and adipocyte differentiation in DeltaFosB transgenic mice result from independent cell-autonomous mechanisms....

  20. Nickel decreases cellular iron level and converts cytosolic aconitase to iron-regulatory protein 1 in A549 cells

    International Nuclear Information System (INIS)

    Chen Haobin; Davidson, Todd; Singleton, Steven; Garrick, Michael D.; Costa, Max

    2005-01-01

    Nickel (Ni) compounds are well-established carcinogens and are known to initiate a hypoxic response in cells via the stabilization and transactivation of hypoxia-inducible factor-1 alpha (HIF-1α). This change may be the consequence of nickel's interference with the function of several Fe(II)-dependent enzymes. In this study, the effects of soluble nickel exposure on cellular iron homeostasis were investigated. Nickel treatment decreased both mitochondrial and cytosolic aconitase (c-aconitase) activity in A549 cells. Cytosolic aconitase was converted to iron-regulatory protein 1, a form critical for the regulation of cellular iron homeostasis. The increased activity of iron-regulatory protein 1 after nickel exposure stabilized and increased transferrin receptor (Tfr) mRNA and antagonized the iron-induced ferritin light chain protein synthesis. The decrease of aconitase activity after nickel treatment reflected neither direct interference with aconitase function nor obstruction of [4Fe-4S] cluster reconstitution by nickel. Exposure of A549 cells to soluble nickel decreased total cellular iron by about 40%, a decrease that likely caused the observed decrease in aconitase activity and the increase of iron-regulatory protein 1 activity. Iron treatment reversed the effect of nickel on cytosolic aconitase and iron-regulatory protein 1. To assess the mechanism for the observed effects, human embryonic kidney (HEK) cells over expressing divalent metal transporter-1 (DMT1) were compared to A549 cells expressing only endogenous transporters for inhibition of iron uptake by nickel. The inhibition data suggest that nickel can enter via DMT1 and compete with iron for entry into the cell. This disturbance of cellular iron homeostasis by nickel may have a great impact on the ability of the cell to regulate a variety of cell functions, as well as create a state of hypoxia in cells under normal oxygen tension. These effects may be very important in how nickel exerts phenotypic

  1. Daphnetin inhibits invasion and migration of LM8 murine osteosarcoma cells by decreasing RhoA and Cdc42 expression

    Energy Technology Data Exchange (ETDEWEB)

    Fukuda, Hiroki [Department of Clinical and Translational Physiology, Kyoto Pharmaceutical University, Kyoto (Japan); Nakamura, Seikou [Department of Pharmacognosy, Kyoto Pharmaceutical University, Kyoto (Japan); Chisaki, Yugo [Education and Research Center for Clinical Pharmacy, Kyoto Pharmaceutical University, Kyoto (Japan); Takada, Tetsuya [Department of Clinical and Translational Physiology, Kyoto Pharmaceutical University, Kyoto (Japan); Toda, Yuki [Department of Medicinal Chemistry, Kyoto Pharmaceutical University, Kyoto (Japan); Murata, Hiroaki [Department of Orthopedics, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Kyoto (Japan); Department of Orthopedic Surgery, Matsushita Memorial Hospital, Osaka (Japan); Itoh, Kazuyuki [Department of Biology, Osaka Medical Center of Cancer and Cardiovascular Diseases, Osaka (Japan); Yano, Yoshitaka [Education and Research Center for Clinical Pharmacy, Kyoto Pharmaceutical University, Kyoto (Japan); Takata, Kazuyuki [Department of Clinical and Translational Physiology, Kyoto Pharmaceutical University, Kyoto (Japan); Ashihara, Eishi, E-mail: ash@mb.kyoto-phu.ac.jp [Department of Clinical and Translational Physiology, Kyoto Pharmaceutical University, Kyoto (Japan)

    2016-02-26

    Daphnetin, 7,8-dihydroxycoumarin, present in main constituents of Daphne odora var. marginatai, has multiple pharmacological activities including anti-proliferative effects in cancer cells. In this study, using a Transwell system, we showed that daphnetin inhibited invasion and migration of highly metastatic murine osteosarcoma LM8 cells in a dose-dependent manner. Following treatment by daphnetin, cells that penetrated the Transwell membrane were rounder than non-treated cells. Immunofluorescence analysis revealed that daphnetin decreased the numbers of intracellular stress fibers and filopodia. Moreover, daphnetin treatment dramatically decreased the expression levels of RhoA and Cdc42. In summary, the dihydroxycoumarin derivative daphnetin inhibits the invasion and migration of LM8 cells, and therefore represents a promising agent for use against metastatic cancer. - Highlights: • Daphnetin, a coumarin-derivative, inhibited invasion and migration of LM8 cells. • Stress fibers and filopodia were decreased by daphnetin treatment. • Daphnetin decreased RhoA and Cdc42 protein expression.

  2. Daphnetin inhibits invasion and migration of LM8 murine osteosarcoma cells by decreasing RhoA and Cdc42 expression

    International Nuclear Information System (INIS)

    Fukuda, Hiroki; Nakamura, Seikou; Chisaki, Yugo; Takada, Tetsuya; Toda, Yuki; Murata, Hiroaki; Itoh, Kazuyuki; Yano, Yoshitaka; Takata, Kazuyuki; Ashihara, Eishi

    2016-01-01

    Daphnetin, 7,8-dihydroxycoumarin, present in main constituents of Daphne odora var. marginatai, has multiple pharmacological activities including anti-proliferative effects in cancer cells. In this study, using a Transwell system, we showed that daphnetin inhibited invasion and migration of highly metastatic murine osteosarcoma LM8 cells in a dose-dependent manner. Following treatment by daphnetin, cells that penetrated the Transwell membrane were rounder than non-treated cells. Immunofluorescence analysis revealed that daphnetin decreased the numbers of intracellular stress fibers and filopodia. Moreover, daphnetin treatment dramatically decreased the expression levels of RhoA and Cdc42. In summary, the dihydroxycoumarin derivative daphnetin inhibits the invasion and migration of LM8 cells, and therefore represents a promising agent for use against metastatic cancer. - Highlights: • Daphnetin, a coumarin-derivative, inhibited invasion and migration of LM8 cells. • Stress fibers and filopodia were decreased by daphnetin treatment. • Daphnetin decreased RhoA and Cdc42 protein expression.

  3. Curcumin-mediated decrease in the expression of nucleolar organizer regions in cervical cancer (HeLa) cells.

    Science.gov (United States)

    Lewinska, Anna; Adamczyk, Jagoda; Pajak, Justyna; Stoklosa, Sylwia; Kubis, Barbara; Pastuszek, Paulina; Slota, Ewa; Wnuk, Maciej

    2014-09-01

    Curcumin, the major yellow-orange pigment of turmeric derived from the rhizome of Curcuma longa, is a highly pleiotropic molecule with the potential to modulate inflammation, oxidative stress, cell survival, cell secretion, homeostasis and proliferation. Curcumin, at relatively high concentrations, was repeatedly reported to be a potent inducer of apoptosis in cancer cells and thus considered a promising anticancer agent. In the present paper, the effects of low concentrations of curcumin on human cervical cancer (HeLa) cells were studied. We found curcumin-mediated decrease in the cell number and viability, and increase in apoptotic events and superoxide level. In contrast to previously shown curcumin cytotoxicity toward different cervical cancer lines, we observed toxic effects when even as low as 1 μM concentration of curcumin was used. Curcumin was not genotoxic to HeLa cells. Because argyrophilic nucleolar protein (AgNOR protein) expression is elevated in malignant cells compared to normal cells reflecting the rapidity of cancer cell proliferation, we evaluated curcumin-associated changes in size (area) and number of silver deposits. We showed curcumin-induced decrease in AgNOR protein pools, which may be mediated by global DNA hypermethylation observed after low concentration curcumin treatment. In summary, we have shown for the first time that curcumin at low micromolar range may be effective against HeLa cells, which may have implications for curcumin-based treatment of cervical cancer in humans. Copyright © 2014 Elsevier B.V. All rights reserved.

  4. Upregulating Nonneuronal Cholinergic Activity Decreases TNF Release from Lipopolysaccharide-Stimulated RAW264.7 Cells

    Directory of Open Access Journals (Sweden)

    Yi Lv

    2014-01-01

    Full Text Available Nonneuronal cholinergic system plays a primary role in maintaining homeostasis. It has been proved that endogenous neuronal acetylcholine (ACh could play an anti-inflammatory role, and exogenous cholinergic agonists could weaken macrophages inflammatory response to lipopolysaccharide (LPS stimulation through activation of α7 subunit-containing nicotinic acetylcholine receptor (α7nAChR. We assumed that nonneuronal cholinergic system existing in macrophages could modulate inflammation through autocrine ACh and expressed α7nAChR on the cells. Therefore, we explored whether LPS continuous stimulation could upregulate the nonneuronal cholinergic activity in macrophages and whether increasing autocrine ACh could decrease TNF release from the macrophages. The results showed that, in RAW264.7 cells incubated with LPS for 20 hours, the secretion of ACh was significantly decreased at 4 h and then gradually increased, accompanied with the enhancement of α7nAChR expression level. The release of TNF was greatly increased from RAW264.7 cells at 4 h and 8 h exposure to LPS; however, it was suppressed at 20 h. Upregulating choline acetyltransferase (ChAT expression through ChAT gene transfection could enhance ACh secretion and reduce TNF release from the infected RAW264. 7cells. The results indicated that LPS stimulation could modulate the activity of nonneuronal cholinergic system of RAW264.7 cells. Enhancing autocrine ACh production could attenuate TNF release from RAW264.7 cells.

  5. Potassium-chloride cotransporter 3 interacts with Vav2 to synchronize the cell volume decrease response with cell protrusion dynamics.

    Directory of Open Access Journals (Sweden)

    Adèle Salin-Cantegrel

    Full Text Available Loss-of-function of the potassium-chloride cotransporter 3 (KCC3 causes hereditary motor and sensory neuropathy with agenesis of the corpus callosum (HMSN/ACC, a severe neurodegenerative disease associated with defective midline crossing of commissural axons in the brain. Conversely, KCC3 over-expression in breast, ovarian and cervical cancer is associated with enhanced tumor cell malignancy and invasiveness. We identified a highly conserved proline-rich sequence within the C-terminus of the cotransporter which when mutated leads to loss of the KCC3-dependent regulatory volume decrease (RVD response in Xenopus Laevis oocytes. Using SH3 domain arrays, we found that this poly-proline motif is a binding site for SH3-domain containing proteins in vitro. This approach identified the guanine nucleotide exchange factor (GEF Vav2 as a candidate partner for KCC3. KCC3/Vav2 physical interaction was confirmed using GST-pull down assays and immuno-based experiments. In cultured cervical cancer cells, KCC3 co-localized with the active form of Vav2 in swelling-induced actin-rich protruding sites and within lamellipodia of spreading and migrating cells. These data provide evidence of a molecular and functional link between the potassium-chloride co-transporters and the Rho GTPase-dependent actin remodeling machinery in RVD, cell spreading and cell protrusion dynamics, thus providing new insights into KCC3's involvement in cancer cell malignancy and in corpus callosum agenesis in HMSN/ACC.

  6. An anthocyanin rich strawberry extract induces apoptosis and ROS while decreases glycolysis and fibrosis in human uterine leiomyoma cells.

    Science.gov (United States)

    Islam, Md Soriful; Giampieri, Francesca; Janjusevic, Milijana; Gasparrini, Massimiliano; Forbes-Hernandez, Tamara Y; Mazzoni, Luca; Greco, Stefania; Giannubilo, Stefano Raffaele; Ciavattini, Andrea; Mezzetti, Bruno; Capocasa, Franco; Castellucci, Mario; Battino, Maurizio; Ciarmela, Pasquapina

    2017-04-04

    Uterine leiomyomas are highly prevalent benign tumors in reproductive aged women. Unfortunately, medical treatments are still limited and no preventive therapies have been developed. In the present study, we investigated the therapeutic effects of strawberry extract on uterine leiomyoma cells. Leiomyoma and myometrial cells were treated with strawberry (cultivar Alba) extract (250 μg/ml) for 48 h to measure apoptosis, reactive oxygen species (ROS), oxidative phosphorylation (OCR, oxygen consumption rate) and glycolysis (ECAR, extracellular acidification rate) as well as fibrosis associated gene and/or protein expression. In leiomyoma cells, strawberry increased the percentage of apoptotic and dead cells. Strawberry significantly increased ROS concentration in leiomyoma cells, while decreased it in myometrial cells. After strawberry treatment, leiomyoma cells showed a significant decreased rate of ECAR, while OCR was unchanged in both myometrial and leiomyoma cells. Strawberry significantly decreased collagen1A1, fibronectin and versican mRNA expression in leiomyoma cells. The reduced protein expression of fibronectin was observed by strawberry extract in leiomyoma cells as well. Furthermore, strawberry was able to reduce activin A induced fibronectin, collagen1A1, and versican as well as activin A and PAI-1 mRNA expression in leiomyoma cells. This study suggests that strawberry can be developed as therapeutic and/or preventive agent for uterine leiomyomas.

  7. Active Prompting to Decrease Cell Phone Use and Increase Seat Belt Use While Driving

    OpenAIRE

    Clayton, Michael; Helms, Bridgett; Simpson, Cathy

    2006-01-01

    Automobile crashes are the leading cause of death for those aged 3 to 33, with 43,005 (118 per day) Americans killed in 2002 alone. Seat belt use reduces the risk of serious injury in an accident, and refraining from using a cell phone while driving reduces the risk of an accident. Cell phone use while driving increases accident rates, and leads to 2,600 U.S. fatalities each year. An active prompting procedure was employed to increase seat belt use and decrease cell phone use among drivers ex...

  8. HIV-1 transgenic rat CD4+ T cells develop decreased CD28 responsiveness and suboptimal Lck tyrosine dephosphorylation following activation

    International Nuclear Information System (INIS)

    Yadav, Anjana; Pati, Shibani; Nyugen, Anhthu; Barabitskaja, Oxana; Mondal, Prosanta; Anderson, Michael; Gallo, Robert C.; Huso, David L.; Reid, William

    2006-01-01

    Impaired CD4+ T cell responses, resulting in dysregulated T-helper 1 (Th1) effector and memory responses, are a common result of HIV-1 infection. These defects are often preceded by decreased expression and function of the α/β T cell receptor (TCR)-CD3 complex and of co-stimulatory molecules including CD28, resulting in altered T cell proliferation, cytokine secretion and cell survival. We have previously shown that HIV Tg rats have defective development of T cell effector function and generation of specific effector/memory T cell subsets. Here we identify abnormalities in activated HIV-1 Tg rat CD4+ T cells that include decreased pY505 dephosphorylation of Lck (required for Lck activation), decreased CD28 function, reduced expression of the anti-apoptotic molecule Bcl-xL, decreased secretion of the mitogenic lympokine interleukin-2 (IL-2) and increased activation induced apoptosis. These events likely lead to defects in antigen-specific signaling and may help explain the disruption of Th1 responses and the generation of specific effector/memory subsets in transgenic CD4+ T cells

  9. Progesterone receptor blockade in human breast cancer cells decreases cell cycle progression through G2/M by repressing G2/M genes

    International Nuclear Information System (INIS)

    Clare, Susan E.; Gupta, Akash; Choi, MiRan; Ranjan, Manish; Lee, Oukseub; Wang, Jun; Ivancic, David Z.; Kim, J. Julie; Khan, Seema A.

    2016-01-01

    The synthesis of specific, potent progesterone antagonists adds potential agents to the breast cancer prevention and treatment armamentarium. The identification of individuals who will benefit from these agents will be a critical factor for their clinical success. We utilized telapristone acetate (TPA; CDB-4124) to understand the effects of progesterone receptor (PR) blockade on proliferation, apoptosis, promoter binding, cell cycle progression, and gene expression. We then identified a set of genes that overlap with human breast luteal-phase expressed genes and signify progesterone activity in both normal breast cells and breast cancer cell lines. TPA administration to T47D cells results in a 30 % decrease in cell number at 24 h, which is maintained over 72 h only in the presence of estradiol. Blockade of progesterone signaling by TPA for 24 h results in fewer cells in G2/M, attributable to decreased expression of genes that facilitate the G2/M transition. Gene expression data suggest that TPA affects several mechanisms that progesterone utilizes to control gene expression, including specific post-translational modifications, and nucleosomal organization and higher order chromatin structure, which regulate access of PR to its DNA binding sites. By comparing genes induced by the progestin R5020 in T47D cells with those increased in the luteal-phase normal breast, we have identified a set of genes that predict functional progesterone signaling in tissue. These data will facilitate an understanding of the ways in which drugs such as TPA may be utilized for the prevention, and possibly the therapy, of human breast cancer. The online version of this article (doi:10.1186/s12885-016-2355-5) contains supplementary material, which is available to authorized users

  10. Decreased soluble cell adhesion molecules after tirofiban infusion in patients with unstable angina pectoris

    Directory of Open Access Journals (Sweden)

    Aliyev Emil

    2004-04-01

    Full Text Available Abstract Aim The inflammatory response, initiated by neutrophil and monocyte adhesion to endothelial cells, is important in the pathogenesis of acute coronary syndromes. Platelets play an important role in inflammatory process by interacting with monocytes and neutrophils. In this study, we investigated the effect of tirofiban on the levels of cell adhesion molecules (soluble intercellular adhesion molecule-1, sICAM-1, and vascular cell adhesion molecule-1, sVCAM-1 in patients with unstable angina pectoris (AP. Methods Thirty-five patients with unstable AP (Group I, ten patients with stable AP (Group II and ten subjects who had angiographycally normal coronary arteries (Group III were included the study. Group I was divided into two subgroups for the specific treatment regimens: Group IA (n = 15 received tirofiban and Group IB (n = 20 did not. Blood samples for investigating the cell adhesion molecules were drawn at zero time (baseline; 0 h in all patients and at 72 h in Group I. Results The baseline levels of sICAM-1 and sVCAM-1 were higher in Group I than in Groups II and III. They were higher in Group IA than in Group IB. However, the sICAM-1 and sVCAM-1 levels decreased significantly in Group IA after tirofiban infusion. In contrast, these levels remained unchanged or were increased above the baseline value in Group IB at 72 h. Conclusion The levels of cell adhesion molecules in patients with unstable AP decreased significantly after tirofiban infusion. Inhibition of platelet function by specific glycoprotein IIb/IIIa antagonists may decrease platelet-mediated inflammation and the ischemic end-point.

  11. Cytochalasin E alters the cytoskeleton and decreases ENaC activity in Xenopus 2F3 cells.

    Science.gov (United States)

    Reifenberger, Matthew S; Yu, Ling; Bao, Hui-Fang; Duke, Billie Jeanne; Liu, Bing-Chen; Ma, He-Ping; Alli, Ahmed A; Eaton, Douglas C; Alli, Abdel A

    2014-07-01

    Numerous reports have linked cytoskeleton-associated proteins with the regulation of epithelial Na(+) channel (ENaC) activity. The purpose of the present study was to determine the effect of actin cytoskeleton disruption by cytochalasin E on ENaC activity in Xenopus 2F3 cells. Here, we show that cytochalasin E treatment for 60 min can disrupt the integrity of the actin cytoskeleton in cultured Xenopus 2F3 cells. We show using single channel patch-clamp experiments and measurements of short-circuit current that ENaC activity, but not its density, is altered by cytochalasin E-induced disruption of the cytoskeleton. In nontreated cells, 8 of 33 patches (24%) had no measurable ENaC activity, whereas in cytochalasin E-treated cells, 17 of 32 patches (53%) had no activity. Analysis of those patches that did contain ENaC activity showed channel open probability significantly decreased from 0.081 ± 0.01 in nontreated cells to 0.043 ± 0.01 in cells treated with cytochalasin E. Transepithelial current from mpkCCD cells treated with cytochalasin E, cytochalasin D, or latrunculin B for 60 min was decreased compared with vehicle-treated cells. The subcellular expression of fodrin changed significantly, and several protein elements of the cytoskeleton decreased at least twofold after 60 min of cytochalasin E treatment. Cytochalasin E treatment disrupted the association between ENaC and myristoylated alanine-rich C-kinase substrate. The results presented here suggest disruption of the actin cytoskeleton by different compounds can attenuate ENaC activity through a mechanism involving changes in the subcellular expression of fodrin, several elements of the cytoskeleton, and destabilization of the ENaC-myristoylated alanine-rich C-kinase substrate complex. Copyright © 2014 the American Physiological Society.

  12. Keratin23 (KRT23 knockdown decreases proliferation and affects the DNA damage response of colon cancer cells.

    Directory of Open Access Journals (Sweden)

    Karin Birkenkamp-Demtröder

    Full Text Available Keratin 23 (KRT23 is strongly expressed in colon adenocarcinomas but absent in normal colon mucosa. Array based methylation profiling of 40 colon samples showed that the promoter of KRT23 was methylated in normal colon mucosa, while hypomethylated in most adenocarcinomas. Promoter methylation correlated with absent expression, while increased KRT23 expression in tumor samples correlated with promoter hypomethylation, as confirmed by bisulfite sequencing. Demethylation induced KRT23 expression in vitro. Expression profiling of shRNA mediated stable KRT23 knockdown in colon cancer cell lines showed that KRT23 depletion affected molecules of the cell cycle and DNA replication, recombination and repair. In vitro analyses confirmed that KRT23 depletion significantly decreased the cellular proliferation of SW948 and LS1034 cells and markedly decreased the expression of genes involved in DNA damage response, mainly molecules of the double strand break repair homologous recombination pathway. KRT23 knockdown decreased the transcript and protein expression of key molecules as e.g. MRE11A, E2F1, RAD51 and BRCA1. Knockdown of KRT23 rendered colon cancer cells more sensitive to irradiation and reduced proliferation of the KRT23 depleted cells compared to irradiated control cells.

  13. Reduced expression levels of PTEN are associated with decreased sensitivity of HCC827 cells to icotinib.

    Science.gov (United States)

    Zhai, Yang; Zhang, Yanjun; Nan, Kejun; Liang, Xuan

    2017-05-01

    The clinical resistance of non-small cell lung cancer (NSCLC) to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) has been linked to EGFR T790M resistance mutations or MET amplifications. Additional mechanisms underlying EGFR-TKI drug resistance remain unclear. The present study demonstrated that icotinib significantly inhibited the proliferation and increased the apoptosis rate of HCC827 cells; the cellular mRNA and protein expression levels of phosphatase and tensin homolog (PTEN) were also significantly downregulated. To investigate the effect of PTEN expression levels on the sensitivity of HCC827 cells to icotinib, PTEN expression was silenced using a PTEN-specific small interfering RNA. The current study identified that the downregulation of PTEN expression levels may promote cellular proliferation in addition to decreasing the apoptosis of HCC827 cells, and may reduce the sensitivity of HCC827 cells to icotinib. These results suggested that reduced PTEN expression levels were associated with the decreased sensitivity of HCC827 cells to icotinib. Furthermore, PTEN expression levels may be a useful marker for predicting icotinib resistance and elucidating the resistance mechanisms underlying EGFR-mutated NSCLC.

  14. Mast cell stabilization decreases cardiomyocyte and LV function in dogs with isolated mitral regurgitation.

    Science.gov (United States)

    Pat, Betty; Killingsworth, Cheryl; Chen, Yuanwen; Gladden, James D; Walcott, Greg; Powell, Pamela C; Denney, Thomas; Gupta, Himanshu; Desai, Ravi; Tillson, Michael; Dillon, A Ray; Dell'italia, Louis J

    2010-09-01

    Mast cells are increased in isolated mitral regurgitation (MR) in the dog and may mediate extracellular matrix loss and left ventricular (LV) dilatation. We tested the hypothesis that mast cell stabilization would attenuate LV remodeling and improve function in the MR dog. MR was induced in adult dogs randomized to no treatment (MR, n = 5) or to the mast cell stabilizer, ketotifen (MR + MCS, n = 4) for 4 months. LV hemodynamics were obtained at baseline and after 4 months of MR and magnetic resonance imaging (MRI) was performed at sacrifice. MRI-derived, serial, short-axis LV end-diastolic (ED) and end-systolic (ES) volumes, LVED volume/mass ratio, and LV 3-dimensional radius/wall thickness were increased in MR and MR + MCS dogs compared with normal dogs (n = 6) (P < .05). Interstitial collagen was decreased by 30% in both MR and MR + MCS versus normal dogs (P < .05). LV contractility by LV maximum time-varying elastance was significantly depressed in MR and MR + MCS dogs. Furthermore, cardiomyocyte fractional shortening was decreased in MR versus normal dogs and further depressed in MR + MCS dogs (P < .05). In vitro administration of ketotifen to normal cardiomyocytes also significantly decreased fractional shortening and calcium transients. Chronic mast cell stabilization did not attenuate eccentric LV remodeling or collagen loss in MR. However, MCS therapy had a detrimental effect on LV function because of a direct negative inotropic effect on cardiomyocyte function. Published by Elsevier Inc.

  15. Hypoxia-activated prodrug TH-302 decreased survival rate of canine lymphoma cells under hypoxic condition.

    Science.gov (United States)

    Yamazaki, Hiroki; Lai, Yu-Chang; Tateno, Morihiro; Setoguchi, Asuka; Goto-Koshino, Yuko; Endo, Yasuyuki; Nakaichi, Munekazu; Tsujimoto, Hajime; Miura, Naoki

    2017-01-01

    We tested the hypotheses that hypoxic stimulation enhances growth potentials of canine lymphoma cells by activating hypoxia-inducible factor 1α (HIF-1α), and that the hypoxia-activated prodrug (TH-302) inhibits growth potentials in the cells. We investigated how hypoxic culture affects the growth rate, chemoresistance, and invasiveness of canine lymphoma cells and doxorubicin (DOX)-resistant lymphoma cells, and influences of TH-302 on survival rate of the cells under hypoxic conditions. Our results demonstrated that hypoxic culture upregulated the expression of HIF-1α and its target genes, including ATP-binding cassette transporter B1 (ABCB1), ATP-binding cassette transporter G2 (ABCG2), platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF), and survivin, and enhanced the growth rate, DOX resistance, and invasiveness of the cells. Additionally, TH-302 decreased the survival rate of the cells under hypoxic condition. Our studies suggest that hypoxic stimulation may advance the tumorigenicity of canine lymphoma cells, favoring malignant transformation. Therefore, the data presented may contribute to the development of TH-302-based hypoxia-targeting therapies for canine lymphoma.

  16. Casticin impairs cell growth and induces cell apoptosis via cell cycle arrest in human oral cancer SCC-4 cells.

    Science.gov (United States)

    Chou, Guan-Ling; Peng, Shu-Fen; Liao, Ching-Lung; Ho, Heng-Chien; Lu, Kung-Wen; Lien, Jin-Cherng; Fan, Ming-Jen; La, Kuang-Chi; Chung, Jing-Gung

    2018-02-01

    Casticin, a polymethoxyflavone, present in natural plants, has been shown to have biological activities including anti-cancer activities. Herein, we investigated the anti-oral cancer activity of casticin on SCC-4 cells in vitro. Viable cells, cell cycle distribution, apoptotic cell death, reactive oxygen species (ROS) production, and Ca 2+ production, levels of ΔΨ m and caspase activity were measured by flow cytometric assay. Cell apoptosis associated protein expressions were examined by Western blotting and confocal laser microscopy. Results indicated that casticin induced cell morphological changes, DNA condensation and damage, decreased the total viable cells, induced G 2 /M phase arrest in SCC-4 cells. Casticin promoted ROS and Ca 2+ productions, decreases the levels of ΔΨ m , promoted caspase-3, -8, and -9 activities in SCC-4 cells. Western blotting assay demonstrated that casticin affect protein level associated with G2/M phase arrest and apoptosis. Confocal laser microscopy also confirmed that casticin increased the translocation of AIF and cytochrome c in SCC-4 cells. In conclusion, casticin decreased cell number through G 2 /M phase arrest and the induction of cell apoptosis through caspase- and mitochondria-dependent pathways in SCC-4 cells. © 2017 Wiley Periodicals, Inc.

  17. Decreased cell survival and DNA repair capacity after UVC irradiation in association with down-regulation of GRP78/BiP in human RSa cells

    International Nuclear Information System (INIS)

    Zhai Ling; Kita, Kazuko; Wano, Chieko; Wu Yuping; Sugaya, Shigeru; Suzuki, Nobuo

    2005-01-01

    In contrast to extensive studies on the roles of molecular chaperones, such as heat shock proteins, there are only a few reports about the roles of GRP78/BiP, an endoplasmic reticulum (ER) stress-induced molecular chaperone, in mammalian cell responses to DNA-damaging stresses. To investigate whether GRP78/BiP is involved in resistance to a DNA-damaging agent, UVC (principally 254 nm in wavelength), we established human cells with down-regulation of GRP78/BiP by transfection of human RSa cells with antisense cDNA for GRP78/BiP. We found that the transfected cells showed higher sensitivity to UVC-induced cell death than control cells transfected with the vector alone. In the antisense-cDNA transfected cells, the removal capacities of the two major types of UVC-damaged DNA (thymine dimers and (6-4) photoproducts) in vivo and DNA synthesis activity of whole cell extracts to repair UVC-irradiated plasmids in vitro were remarkably decreased compared with those in the control cells. Furthermore, the antisense-cDNA transfected cells also showed slightly higher sensitivity to cisplatin-induced cell death than the control cells. Cisplatin-induced DNA damage is primarily repaired by nucleotide excision repair, like UVC-induced DNA damage. The present results suggest that GRP78/BiP plays a protective role against UVC-induced cell death possibly via nucleotide excision repair, at least in the human RSa cells tested

  18. Secreted Human Adipose Leptin Decreases Mitochondrial Respiration in HCT116 Colon Cancer Cells

    Science.gov (United States)

    Yehuda-Shnaidman, Einav; Nimri, Lili; Tarnovscki, Tanya; Kirshtein, Boris; Rudich, Assaf; Schwartz, Betty

    2013-01-01

    Obesity is a key risk factor for the development of colon cancer; however, the endocrine/paracrine/metabolic networks mediating this connection are poorly understood. Here we hypothesize that obesity results in secreted products from adipose tissue that induce malignancy-related metabolic alterations in colon cancer cells. Human HCT116 colon cancer cells, were exposed to conditioned media from cultured human adipose tissue fragments of obese vs. non-obese subjects. Oxygen consumption rate (OCR, mostly mitochondrial respiration) and extracellular acidification rate (ECAR, mostly lactate production via glycolysis) were examined vis-à-vis cell viability and expression of related genes and proteins. Our results show that conditioned media from obese (vs. non-obese) subjects decreased basal (40%, prespiration and function in HCT116 colon cancer cells, an effect that is at least partly mediated by leptin. These results highlight a putative novel mechanism for obesity-associated risk of gastrointestinal malignancies, and suggest potential new therapeutic avenues. PMID:24073224

  19. Chrysophanol-induced cell death (necrosis) in human lung cancer A549 cells is mediated through increasing reactive oxygen species and decreasing the level of mitochondrial membrane potential.

    Science.gov (United States)

    Ni, Chien-Hang; Yu, Chun-Shu; Lu, Hsu-Feng; Yang, Jai-Sing; Huang, Hui-Ying; Chen, Po-Yuan; Wu, Shin-Hwar; Ip, Siu-Wan; Chiang, Su-Yin; Lin, Jaung-Geng; Chung, Jing-Gung

    2014-05-01

    Chrysophanol (1,8-dihydroxy-3-methylanthraquinone) is one of the anthraquinone compounds, and it has been shown to induce cell death in different types of cancer cells. The effects of chrysophanol on human lung cancer cell death have not been well studied. The purpose of this study is to examine chrysophanol-induced cytotoxic effects and also to investigate such influences that involved apoptosis or necrosis in A549 human lung cancer cells in vitro. Our results indicated that chrysophanol decreased the viable A549 cells in a dose- and time-dependent manner. Chrysophanol also promoted the release of reactive oxygen species (ROS) and Ca(2+) and decreased the levels of mitochondria membrane potential (ΔΨm ) and adenosine triphosphate in A549 cells. Furthermore, chrysophanol triggered DNA damage by using Comet assay and DAPI staining. Importantly, chrysophanol only stimulated the cytocheome c release, but it did not activate other apoptosis-associated protein levels including caspase-3, caspase-8, Apaf-1, and AIF. In conclusion, human lung cancer A549 cells treated with chrysophanol exhibited a cellular pattern associated with necrotic cell death and not apoptosis in vitro. © 2012 Wiley Periodicals, Inc. Environ Toxicol 29: 740-749, 2014. Copyright © 2012 Wiley Periodicals, Inc., a Wiley company.

  20. Low Oxygen Modulates Multiple Signaling Pathways, Increasing Self-Renewal, While Decreasing Differentiation, Senescence, and Apoptosis in Stromal MIAMI Cells

    Science.gov (United States)

    Rios, Carmen; D'Ippolito, Gianluca; Curtis, Kevin M.; Delcroix, Gaëtan J.-R.; Gomez, Lourdes A.; El Hokayem, Jimmy; Rieger, Megan; Parrondo, Ricardo; de las Pozas, Alicia; Perez-Stable, Carlos; Howard, Guy A.

    2016-01-01

    Human bone marrow multipotent mesenchymal stromal cell (hMSC) number decreases with aging. Subpopulations of hMSCs can differentiate into cells found in bone, vasculature, cartilage, gut, and other tissues and participate in their repair. Maintaining throughout adult life such cell subpopulations should help prevent or delay the onset of age-related degenerative conditions. Low oxygen tension, the physiological environment in progenitor cell-rich regions of the bone marrow microarchitecture, stimulates the self-renewal of marrow-isolated adult multilineage inducible (MIAMI) cells and expression of Sox2, Nanog, Oct4a nuclear accumulation, Notch intracellular domain, notch target genes, neuronal transcriptional repressor element 1 (RE1)-silencing transcription factor (REST), and hypoxia-inducible factor-1 alpha (HIF-1α), and additionally, by decreasing the expression of (i) the proapoptotic proteins, apoptosis-inducing factor (AIF) and Bak, and (ii) senescence-associated p53 expression and β-galactosidase activity. Furthermore, low oxygen increases canonical Wnt pathway signaling coreceptor Lrp5 expression, and PI3K/Akt pathway activation. Lrp5 inhibition decreases self-renewal marker Sox2 mRNA, Oct4a nuclear accumulation, and cell numbers. Wortmannin-mediated PI3K/Akt pathway inhibition leads to increased osteoblastic differentiation at both low and high oxygen tension. We demonstrate that low oxygen stimulates a complex signaling network involving PI3K/Akt, Notch, and canonical Wnt pathways, which mediate the observed increase in nuclear Oct4a and REST, with simultaneous decrease in p53, AIF, and Bak. Collectively, these pathway activations contribute to increased self-renewal with concomitant decreased differentiation, cell cycle arrest, apoptosis, and/or senescence in MIAMI cells. Importantly, the PI3K/Akt pathway plays a central mechanistic role in the oxygen tension-regulated self-renewal versus osteoblastic differentiation of progenitor cells. PMID:27059084

  1. Smoking decreases the level of circulating CD34+ progenitor cells in young healthy women - a pilot study

    Directory of Open Access Journals (Sweden)

    Baumann Gert

    2010-05-01

    Full Text Available Abstract Background Decreased levels of circulating bone marrow-derived progenitor cells have been associated with risk factors and cardiovascular diseases. Smoking is the most important modifiable risk factor for atherosclerosis in young women. The aim of this pilot study was to assess in healthy premenopausal women without other risk factors for cardiovascular disease the influence of nicotine abuse on the number of circulating progenitor cells in relation to endothelial function. Methods The number of endothelial progenitor cells, measured as colony-forming units in a cell-culture assay (EPC-CFU and the number of circulating CD34 + and CD34 + /CD133 + cells, measured by flow cytometry, was estimated in 32 women at the menstrual phase of the menstrual cycle. In addition, flow-mediated dilation (FMD was assessed as a marker for vascular function. In a subgroup of these women (n = 20, progenitor cells were also investigated at the mid-follicular and luteal phases of the menstrual cycle. Results Compared to non-smokers, the abundance of circulating CD34 + cells was significantly lower in smoking women in the menstrual, mid-luteal, and mid-follicular phases of the menstrual cycle. The number of CD34 + progenitor cells was revealed to have significant positive correlation with FMD in young healthy women, whereas CD34 + /CD133 + progenitor cells and EPC-CFU showed no significant correlation. Conclusion The number of CD34 + progenitor cells positively correlates with FMD in young healthy women and is decreased by smoking.

  2. The suppression of manganese superoxide dismutase decreased the survival of human glioblastoma multiforme T98G cells

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    Novi S. Hardiany

    2017-05-01

    Full Text Available Background: Glioblastoma multiforme (GBM is a primary malignant brain tumor which has poor prognosis. High incidence of oxidative stress-based therapy resistance could be related to the high antioxidant status of GBM cells. Our previous study has reported that manganese superoxide dismutase (MnSOD antioxidant expression was significantly higher in high grade glioma than in low grade. The aim of this study was to analyze the impact of MnSOD suppression toward GBM cell survival.Methods: This study is an experimental study using human glioblastoma multiforme T98G cell line. Suppression of MnSOD expression was performed using in vitro transfection MnSOD-siRNA. The MnSOD expression was analyzed by measuring the mRNA using real time RT-PCR, protein using ELISA technique, and specific activity of enzyme using inhibition of xantine oxidase. Concentration of reactive oxygen species (ROS intracellular was determined by measuring superoxide radical and hydrogen peroxide. Cell survival was analyzed by measuring viability, proliferation, and cell apoptosis.Results: In vitro transfection of MnSOD-siRNA suppressed the mRNA, protein, and specific activity of MnSOD. This treatment significantly increased the concentration of superoxide radical; however, it did not influence the concentration of hydrogen peroxide. Moreover, viability MnSOD-suppressing cell significantly decreased, accompanied by increase of cell apoptosis without affecting cell proliferation.Conclusion: The suppression of MnSOD expression leads to decrease glioblastoma multiforme cell survival, which was associated to the increase of cell apoptotic.

  3. Paradoxical sleep deprivation decreases serum testosterone and Leydig cells in male rats

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    Fitranto Arjadi

    2014-04-01

    Full Text Available Background Chronic stress increases glucocorticoid levels and accelerates reduction in Leydig cells functions and numbers. Chronic stress models in the working place comprise sleep deprivation, sedentary stress, and physical stress. The aim of this study was to evaluate the effect of various work stress models, such as stress from paradoxical sleep deprivation (PSD, immobilization, and footshock, on serum testosterone levels and number of Leydig cells in male albino rats. Methods This study was of experimental randomized post-test only with control group design using 24 male Wistar albino rats (Rattus norvegicus. The sample was divided into 4 groups: K1 (control, K2 (PSD, K3 (immobilization and K4 (footshock, receiving treatment for 25 days. Measured parameters were serum testosterone level and Leydig cell number. Analysis of variance (ANOVA was used for statistical analysis, followed by post hoc LSD. Results Mean serum testosterone levels (0.07 ± 0.08 ng/mL and Leydig cell numbers (4.22 ± l0.96 were lowest in the PSD stress model. Serum testosterone levels differed significantly between controls and PSD group (p=0.014, while there was a significant difference in numbers of Leydig cells between footshock stress and PSD (p=0.011 and between the three stress groups and controls (p=0.006. Conclusion This study demonstrated that PSD, immobilization and footshock stress significantly decreased serum testosterone levels and number of Leydig cells in male albino rats (Rattus norvegicus. The mechanism by which PSD affects serum testosterone is still unclear.

  4. GPR30 decreases cardiac chymase/angiotensin II by inhibiting local mast cell number

    International Nuclear Information System (INIS)

    Zhao, Zhuo; Wang, Hao; Lin, Marina; Groban, Leanne

    2015-01-01

    Chronic activation of the novel estrogen receptor GPR30 by its agonist G1 mitigates the adverse effects of estrogen (E2) loss on cardiac structure and function. Using the ovariectomized (OVX) mRen2.Lewis rat, an E2-sensitive model of diastolic dysfunction, we found that E2 status is inversely correlated with local cardiac angiotensin II (Ang II) levels, likely via Ang I/chymase-mediated production. Since chymase is released from cardiac mast cells during stress (e.g., volume/pressure overload, inflammation), we hypothesized that GPR30-related cardioprotection after E2 loss might occur through its opposing actions on cardiac mast cell proliferation and chymase production. Using real-time quantitative PCR, immunohistochemistry, and immunoblot analysis, we found mast cell number, chymase expression, and cardiac Ang II levels were significantly increased in the hearts of OVX-compared to ovary-intact mRen2.Lewis rats and the GPR30 agonist G1 (50 mg/kg/day, s.c.) administered for 2 weeks limited the adverse effects of estrogen loss. In vitro studies revealed that GPR30 receptors are expressed in the RBL-2H3 mast cell line and G1 inhibits serum-induced cell proliferation in a dose-dependent manner, as determined by cell counting, BrdU incorporation assay, and Ki-67 staining. Using specific antagonists to estrogen receptors, blockage of GPR30, but not ERα or ERβ, attenuated the inhibitory effects of estrogen on BrdU incorporation in RBL-2H3 cells. Further study of the mechanism underlying the effect on cell proliferation showed that G1 inhibits cyclin-dependent kinase 1 (CDK1) mRNA and protein expression in RBL-2H3 cells in a dose-dependent manner. - Highlights: • GPR30 activation limits mast cell number in hearts from OVX mRen2.Lewis rats. • GPR30 activation decreases cardiac chymase/angiotensin II after estrogen loss. • GPR30 activation inhibits RBL-2H3 mast cell proliferation and CDK1 expression

  5. GPR30 decreases cardiac chymase/angiotensin II by inhibiting local mast cell number

    Energy Technology Data Exchange (ETDEWEB)

    Zhao, Zhuo [Department of Anesthesiology, Wake Forest School of Medicine, Medical Center Boulevard, Winston-Salem, NC 27159-1009 (United States); Department of Cardiology, Jinan Central Hospital, Affiliated with Shandong University, 105 Jiefang Road, Jinan, 250013 (China); Wang, Hao; Lin, Marina [Department of Anesthesiology, Wake Forest School of Medicine, Medical Center Boulevard, Winston-Salem, NC 27159-1009 (United States); Groban, Leanne, E-mail: lgroban@wakehealth.edu [Department of Anesthesiology, Wake Forest School of Medicine, Medical Center Boulevard, Winston-Salem, NC 27159-1009 (United States); Hypertension and Vascular Disease Center, Wake Forest School of Medicine, Medical Center Boulevard, Winston-Salem, NC 27157 (United States); Office of Women in Medicine and Science, Wake Forest School of Medicine, Medical Center Boulevard, Winston-Salem, NC 27157 (United States)

    2015-03-27

    Chronic activation of the novel estrogen receptor GPR30 by its agonist G1 mitigates the adverse effects of estrogen (E2) loss on cardiac structure and function. Using the ovariectomized (OVX) mRen2.Lewis rat, an E2-sensitive model of diastolic dysfunction, we found that E2 status is inversely correlated with local cardiac angiotensin II (Ang II) levels, likely via Ang I/chymase-mediated production. Since chymase is released from cardiac mast cells during stress (e.g., volume/pressure overload, inflammation), we hypothesized that GPR30-related cardioprotection after E2 loss might occur through its opposing actions on cardiac mast cell proliferation and chymase production. Using real-time quantitative PCR, immunohistochemistry, and immunoblot analysis, we found mast cell number, chymase expression, and cardiac Ang II levels were significantly increased in the hearts of OVX-compared to ovary-intact mRen2.Lewis rats and the GPR30 agonist G1 (50 mg/kg/day, s.c.) administered for 2 weeks limited the adverse effects of estrogen loss. In vitro studies revealed that GPR30 receptors are expressed in the RBL-2H3 mast cell line and G1 inhibits serum-induced cell proliferation in a dose-dependent manner, as determined by cell counting, BrdU incorporation assay, and Ki-67 staining. Using specific antagonists to estrogen receptors, blockage of GPR30, but not ERα or ERβ, attenuated the inhibitory effects of estrogen on BrdU incorporation in RBL-2H3 cells. Further study of the mechanism underlying the effect on cell proliferation showed that G1 inhibits cyclin-dependent kinase 1 (CDK1) mRNA and protein expression in RBL-2H3 cells in a dose-dependent manner. - Highlights: • GPR30 activation limits mast cell number in hearts from OVX mRen2.Lewis rats. • GPR30 activation decreases cardiac chymase/angiotensin II after estrogen loss. • GPR30 activation inhibits RBL-2H3 mast cell proliferation and CDK1 expression.

  6. Preferential antitumor effect of the Src inhibitor dasatinib associated with a decreased proportion of aldehyde dehydrogenase 1-positive cells in breast cancer cells of the basal B subtype

    Directory of Open Access Journals (Sweden)

    Watanabe Mika

    2010-10-01

    Full Text Available Abstract Background Recent studies have suggested that the Src inhibitor dasatinib preferentially inhibits the growth of breast cancer cells of the basal-like subtype. To clarify this finding and further investigate combined antitumor effects of dasatinib with cytotoxic agents, a panel of breast cancer cell lines of various subtypes was treated with dasatinib and/or chemotherapeutic agents. Methods Seven human breast cancer cell lines were treated with dasatinib and/or seven chemotherapeutic agents. Effects of the treatments on c-Src activation, cell growth, cell cycle, apoptosis and the proportion of aldehyde dehydrogenase (ALDH 1-positive cells were examined. Results The 50%-growth inhibitory concentrations (IC50s of dasatinib were much lower in two basal B cell lines than those in the other cell lines. The IC50s of chemotherapeutic agents were not substantially different among the cell lines. Dasatinib enhanced antitumor activity of etoposide in the basal B cell lines. Dasatinib induced a G1-S blockade with a slight apoptosis, and a combined treatment of dasatinib with etoposide also induced a G1-S blockade in the basal B cell lines. Dasatinib decreased the expression levels of phosphorylated Src in all cell lines. Interestingly, dasatinib significantly decreased the proportion of ALDH1-positive cells in the basal B cell lines but not in the other cell lines. Conclusions The present study indicates that dasatinib preferentially inhibits the growth of breast cancer cells of the basal B subtype associated with a significant loss of putative cancer stem cell population. A combined use of dasatinib with etoposide additively inhibits their growth. Further studies targeting breast cancers of the basal B subtype using dasatinib with cytotoxic agents are warranted.

  7. TNF-α decreases VEGF secretion in highly polarized RPE cells but increases it in non-polarized RPE cells related to crosstalk between JNK and NF-κB pathways.

    Directory of Open Access Journals (Sweden)

    Hiroto Terasaki

    Full Text Available Asymmetrical secretion of vascular endothelial growth factor (VEGF by retinal pigment epithelial (RPE cells in situ is critical for maintaining the homeostasis of the retina and choroid. VEGF is also involved in the development and progression of age-related macular degeneration (AMD. We studied the effect of tumor necrosis factor-α (TNF-α on the secretion of VEGF in polarized and non-polarized RPE cells (P-RPE cells and N-RPE cells, respectively in culture and in situ in rats. A subretinal injection of TNF-α caused a decrease in VEGF expression and choroidal atrophy. Porcine RPE cells were seeded on Transwell™ filters, and their maturation and polarization were confirmed by the asymmetrical VEGF secretion and trans electrical resistance. Exposure to TNF-α decreased the VEGF secretion in P-RPE cells but increased it in N-RPE cells in culture. TNF-α inactivated JNK in P-RPE cells but activated it in N-RPE cells, and TNF-α activated NF-κB in P-RPE cells but not in N-RPE cells. Inhibition of NF-κB activated JNK in both types of RPE cells indicating crosstalk between JNK and NF-κB. TNF-α induced the inhibitory effects of NF-κB on JNK in P-RPE cells because NF-κB is continuously inactivated. In N-RPE cells, however, it was not evident because NF-κB was already activated. The basic activation pattern of JNK and NF-κB and their crosstalk led to opposing responses of RPE cells to TNF-α. These results suggest that VEGF secretion under inflammatory conditions depends on cellular polarization, and the TNF-α-induced VEGF down-regulation may result in choroidal atrophy in polarized physiological RPE cells. TNF-α-induced VEGF up-regulation may cause neovascularization by non-polarized or non-physiological RPE cells.

  8. Triclosan treatment decreased the antitumor effect of sorafenib on hepatocellular carcinoma cells

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    Wu M

    2018-05-01

    Full Text Available Man Wu,1,2 Guanren Zhao,2 Xiaomei Zhuang,1 Tianhong Zhang,1 Ce Zhang,2 Wenpeng Zhang,1 Zhenqing Zhang1 1State Key Laboratory of Toxicology and Medical Countermeasures, Beijing Institute of Pharmacology and Toxicology, Beijing, China; 2Department of Pharmacy, The 309th Hospital of PLA, Beijing, China Background: Triclosan is a widely applied antimicrobial agent which affects the endocrine system and homeostasis; it may also promote the cirrhosis and hepatocellular carcinoma (HCC growth in a mice model. The exact roles of triclosan in regulating human hepatocellular carcinoma development and treatment remain unknown. Methods: MHCC97-H, a highly aggressive HCC cell line, was treated with indicated concentration of triclosan or sorafenib. The expression of drug-resistance genes was examined by qPCR. The clearance or metabolism of sorafenib was determined by liquid chromatograph-mass spectrometer/mass spectrometer (LC-MS/MS. MTT assay was used to examine the MHCC97-H cell proliferation. Nude mice were used to exam the anti-tumor effect of sorafenib on subcutaneous and intrahepatic growth of MHCC97-H cells. Results: In the present study, triclosan could induce the expression of drug-resistance genes in MHCC97-H cells (a highly aggressive HCC cell line, accelerate the clearance of sorafenib, and attenuate the anti-proliferation effect of this molecular targeted agent in MHCC97-H cells. Triclosan decreased the antitumor effect of sorafenib on subcutaneous and intrahepatic growth of MHCC97-H in nude mice. Conclusion: By discovering the fact that triclosan treatment enhances sorafenib resistance in HCC cells, this work suggests exposure of triclosan is detrimental to HCC patients during chemotherapy. Keywords: HCC, triclosan, sorafenib resistance, drug clearance 

  9. CURCUMIN DECREASES SPECIFICITY PROTEIN (Sp) EXPRESSION IN BLADDER CANCER CELLS

    OpenAIRE

    Chadalapaka, Gayathri; Jutooru, Indira; Chintharlapalli, Sudhakar; Papineni, Sabitha; Smith, Roger; Li, Xiangrong; Safe, Stephen

    2008-01-01

    Curcumin is the active component of tumeric, and this polyphenolic compound has been extensively investigated as an anticancer drug that modulates multiple pathways and genes. In this study, 10 – 25 µM curcumin inhibited 253JB-V and KU7 bladder cancer cell growth, and this was accompanied by induction of apoptosis and decreased expression of the proapoptotic protein survivin and the angiogenic proteins vascular endothelial growth factor (VEGF) and VEGF receptor 1 (VEGFR1). Since expression of...

  10. Decreased Siglec-9 Expression on Natural Killer Cell Subset Associated With Persistent HBV Replication

    Directory of Open Access Journals (Sweden)

    Di Zhao

    2018-05-01

    Full Text Available Siglec-9 is an MHC-independent inhibitory receptor selectively expressed on CD56dim NK cells. Its role in infection diseases has not been investigated yet. Here, we studied the potential regulatory roles of NK Siglec-9 in the pathogenesis of chronic hepatitis B (CHB infection. Flow cytometry evaluated the expression of Siglec-9 and other receptors on peripheral NK cells. Immunofluorescence staining was used to detect Siglec-9 ligands on liver biopsy tissues and cultured hepatocyte cell lines. Siglec-9 blocking assay was carried out and cytokine synthesis and CD107a degranulation was detected by flow cytometry. Compared to healthy donors, CHB patients had decreased Siglec-9+ NK cells, which reversely correlated with serum hepatitis B e antigen and HBV DNA titer. Siglec-9 expression on NK cells from patients achieving sustained virological response recovered to the level of normal donors. Neutralization of Siglec-9 restored cytokine synthesis and degranulation of NK cells from CHB patients. Immunofluorescence staining showed increased expression of Siglec-9 ligands in liver biopsy tissues from CHB patients and in hepatocyte cell lines infected with HBV or stimulated with inflammatory cytokines (IL-6 or TGF-β. These findings identify Siglec-9 as a negative regulator for NK cells contributing to HBV persistence and the intervention of Siglec-9 signaling might be of potentially translational significance.

  11. Cisplatin-induced mesenchymal stromal cells-mediated mechanism contributing to decreased antitumor effect in breast cancer cells.

    Science.gov (United States)

    Skolekova, Svetlana; Matuskova, Miroslava; Bohac, Martin; Toro, Lenka; Durinikova, Erika; Tyciakova, Silvia; Demkova, Lucia; Gursky, Jan; Kucerova, Lucia

    2016-01-12

    Cells of the tumor microenvironment are recognized as important determinants of the tumor biology. The adjacent non-malignant cells can regulate drug responses of the cancer cells by secreted paracrine factors and direct interactions with tumor cells. Human mesenchymal stromal cells (MSC) actively contribute to tumor microenvironment. Here we focused on their response to chemotherapy as during the treatment these cells become affected. We have shown that the secretory phenotype and behavior of mesenchymal stromal cells influenced by cisplatin differs from the naïve MSC. MSC were more resistant to the concentrations of cisplatin, which was cytotoxic for tumor cells. They did not undergo apoptosis, but a part of MSC population underwent senescence. However, MSC pretreatment with cisplatin led to changes in phosphorylation profiles of many kinases and also increased secretion of IL-6 and IL-8 cytokines. These changes in cytokine and phosphorylation profile of MSC led to increased chemoresistance and stemness of breast cancer cells. Taken together here we suggest that the exposure of the chemoresistant cells in the tumor microenvironment leads to substantial alterations and might lead to promotion of acquired microenvironment-mediated chemoresistance and stemness.

  12. Aging increases microglial proliferation, delays cell migration, and decreases cortical neurogenesis after focal cerebral ischemia.

    Science.gov (United States)

    Moraga, Ana; Pradillo, Jesús M; García-Culebras, Alicia; Palma-Tortosa, Sara; Ballesteros, Ivan; Hernández-Jiménez, Macarena; Moro, María A; Lizasoain, Ignacio

    2015-05-10

    Aging is not just a risk factor of stroke, but it has also been associated with poor recovery. It is known that stroke-induced neurogenesis is reduced but maintained in the aged brain. However, there is no consensus on how neurogenesis is affected after stroke in aged animals. Our objective is to determine the role of aging on the process of neurogenesis after stroke. We have studied neurogenesis by analyzing proliferation, migration, and formation of new neurons, as well as inflammatory parameters, in a model of cerebral ischemia induced by permanent occlusion of the middle cerebral artery in young- (2 to 3 months) and middle-aged mice (13 to 14 months). Aging increased both microglial proliferation, as shown by a higher number of BrdU(+) cells and BrdU/Iba1(+) cells in the ischemic boundary and neutrophil infiltration. Interestingly, aging increased the number of M1 monocytes and N1 neutrophils, consistent with pro-inflammatory phenotypes when compared with the alternative M2 and N2 phenotypes. Aging also inhibited (subventricular zone) SVZ cell proliferation by decreasing both the number of astrocyte-like type-B (prominin-1(+)/epidermal growth factor receptor (EGFR)(+)/nestin(+)/glial fibrillary acidic protein (GFAP)(+) cells) and type-C cells (prominin-1(+)/EGFR(+)/nestin(-)/Mash1(+) cells), and not affecting apoptosis, 1 day after stroke. Aging also inhibited migration of neuroblasts (DCX(+) cells), as indicated by an accumulation of neuroblasts at migratory zones 14 days after injury; consistently, aged mice presented a smaller number of differentiated interneurons (NeuN(+)/BrdU(+) and GAD67(+) cells) in the peri-infarct cortical area 14 days after stroke. Our data confirm that stroke-induced neurogenesis is maintained but reduced in aged animals. Importantly, we now demonstrate that aging not only inhibits proliferation of specific SVZ cell subtypes but also blocks migration of neuroblasts to the damaged area and decreases the number of new interneurons in

  13. Gestational lead exposure selectively decreases retinal dopamine amacrine cells and dopamine content in adult mice

    Energy Technology Data Exchange (ETDEWEB)

    Fox, Donald A., E-mail: dafox@uh.edu [College of Optometry, University of Houston, Houston, TX (United States); Department of Biology and Biochemistry, University of Houston, Houston, TX (United States); Department of Pharmacology and Pharmaceutical Sciences, University of Houston, Houston, TX (United States); Hamilton, W. Ryan [Department of Biology and Biochemistry, University of Houston, Houston, TX (United States); Johnson, Jerry E. [Department of Natural Sciences, University of Houston-Downtown, Houston, TX (United States); Xiao, Weimin [College of Optometry, University of Houston, Houston, TX (United States); Chaney, Shawntay; Mukherjee, Shradha [Department of Biology and Biochemistry, University of Houston, Houston, TX (United States); Miller, Diane B.; O' Callaghan, James P. [Toxicology and Molecular Biology Branch, Health Effects Research Laboratory, Centers for Disease Control and Prevention-NIOSH, Morgantown, WV USA (United States)

    2011-11-15

    Gestational lead exposure (GLE) produces supernormal scotopic electroretinograms (ERG) in children, monkeys and rats, and a novel retinal phenotype characterized by an increased number of rod photoreceptors and bipolar cells in adult mice and rats. Since the loss of dopaminergic amacrine cells (DA ACs) in GLE monkeys and rats contributes to supernormal ERGs, the retinal DA system was analyzed in mice following GLE. C57BL/6 female mice were exposed to low (27 ppm), moderate (55 ppm) or high (109 ppm) lead throughout gestation and until postnatal day 10 (PN10). Blood [Pb] in control, low-, moderate- and high-dose GLE was {<=} 1, {<=} 10, {approx} 25 and {approx} 40 {mu}g/dL, respectively, on PN10 and by PN30 all were {<=} 1 {mu}g/dL. At PN60, confocal-stereology studies used vertical sections and wholemounts to characterize tyrosine hydroxylase (TH) expression and the number of DA and other ACs. GLE dose-dependently and selectively decreased the number of TH-immunoreactive (IR) DA ACs and their synaptic plexus without affecting GABAergic, glycinergic or cholinergic ACs. Immunoblots and confocal revealed dose-dependent decreases in retinal TH protein expression and content, although monoamine oxidase-A protein and gene expression were unchanged. High-pressure liquid chromatography showed that GLE dose-dependently decreased retinal DA content, its metabolites and DA utilization/release. The mechanism of DA selective vulnerability is unknown. However, a GLE-induced loss/dysfunction of DA ACs during development could increase the number of rods and bipolar cells since DA helps regulate neuronal proliferation, whereas during adulthood it could produce ERG supernormality as well as altered circadian rhythms, dark/light adaptation and spatial contrast sensitivity. -- Highlights: Black-Right-Pointing-Pointer Peak [BPb] in control, low-, moderate- and high-dose newborn mice with gestational lead exposure: {<=} 1, {<=} 10, 25 and 40 {mu}g/dL Black

  14. Gestational lead exposure selectively decreases retinal dopamine amacrine cells and dopamine content in adult mice

    International Nuclear Information System (INIS)

    Fox, Donald A.; Hamilton, W. Ryan; Johnson, Jerry E.; Xiao, Weimin; Chaney, Shawntay; Mukherjee, Shradha; Miller, Diane B.; O'Callaghan, James P.

    2011-01-01

    Gestational lead exposure (GLE) produces supernormal scotopic electroretinograms (ERG) in children, monkeys and rats, and a novel retinal phenotype characterized by an increased number of rod photoreceptors and bipolar cells in adult mice and rats. Since the loss of dopaminergic amacrine cells (DA ACs) in GLE monkeys and rats contributes to supernormal ERGs, the retinal DA system was analyzed in mice following GLE. C57BL/6 female mice were exposed to low (27 ppm), moderate (55 ppm) or high (109 ppm) lead throughout gestation and until postnatal day 10 (PN10). Blood [Pb] in control, low-, moderate- and high-dose GLE was ≤ 1, ≤ 10, ∼ 25 and ∼ 40 μg/dL, respectively, on PN10 and by PN30 all were ≤ 1 μg/dL. At PN60, confocal-stereology studies used vertical sections and wholemounts to characterize tyrosine hydroxylase (TH) expression and the number of DA and other ACs. GLE dose-dependently and selectively decreased the number of TH-immunoreactive (IR) DA ACs and their synaptic plexus without affecting GABAergic, glycinergic or cholinergic ACs. Immunoblots and confocal revealed dose-dependent decreases in retinal TH protein expression and content, although monoamine oxidase-A protein and gene expression were unchanged. High-pressure liquid chromatography showed that GLE dose-dependently decreased retinal DA content, its metabolites and DA utilization/release. The mechanism of DA selective vulnerability is unknown. However, a GLE-induced loss/dysfunction of DA ACs during development could increase the number of rods and bipolar cells since DA helps regulate neuronal proliferation, whereas during adulthood it could produce ERG supernormality as well as altered circadian rhythms, dark/light adaptation and spatial contrast sensitivity. -- Highlights: ► Peak [BPb] in control, low-, moderate- and high-dose newborn mice with gestational lead exposure: ≤ 1, ≤ 10, 25 and 40 μg/dL ► Gestational lead exposure dose-dependently decreased the number of TH

  15. Effect of tributyltin (TBT) on ATP levels in human natural killer (NK) cells: relationship to TBT-induced decreases in NK function.

    Science.gov (United States)

    Dudimah, Fred D; Odman-Ghazi, Sabah O; Hatcher, Frank; Whalen, Margaret M

    2007-01-01

    The purpose of this study was to investigate the role that tributyltin (TBT)-induced decreases in ATP levels may play in TBT-induced decreases in the tumor lysing (lytic) function of natural killer (NK) cells. NK cells are a subset of lymphocytes that act as an initial immune defense against tumor cells and virally infected cells. TBT is an environmental contaminant that has been detected in human blood, which has been shown to interfere with ATP synthesis. Previous studies have shown that TBT is able to decrease very significantly the lytic function of NK cells. In this study NK cells were exposed to various concentrations of TBT and to two other compounds that interfere with ATP synthesis (rotenone a complex I inhibitor and oligomycin an ATP synthase inhibitor) for various lengths of time before determining the levels of ATP and lytic function. Exposures of NK cells to 10, 25, 50 and 100 nm TBT did not significantly reduce ATP levels after 24 h. However, these same exposures caused significant decreases in cytotoxic function. Studies of brief 1 h exposures to a range of TBT, rotenone and oligomycin concentrations followed by 24 h, 48 h and 6 day periods in compound-free media prior to assaying for ATP levels or cytotoxic function showed that each of the compounds caused persistent decreases in ATP levels and lytic function of NK cells. Exposures to 0.05-5 microm rotenone or oligomycin for 1 h reduced ATP levels by 20-25% but did not have any measurable effect on the ability of NK cells to lyse tumor cells. ATP levels were also decreased by about 20-25% after 24 h or 48 h exposures to rotenone or oligomycin (0.5 microm ), and the lytic function was decreased by about 50%. The results suggest that TBT-induced decreases in ATP levels were not responsible for the loss of cytotoxic function seen at 1 h and 24 h. However, TBT-induced decreases of NK-ATP levels may be at least in part responsible for losses of NK-cytotoxic function seen after 48 h and 6 day exposures

  16. Nocodazole treatment decreases expression of pluripotency markers Nanog and Oct4 in human embryonic stem cells

    DEFF Research Database (Denmark)

    Kallas, Ade; Pook, Martin; Maimets, Martti

    2011-01-01

    in the expression of transcription markers Nanog and Oct4 as well as SSEA-3 and SSEA-4 in human embryonic cells after their treatment with nocodazole. Multivariate permeabilised-cell flow cytometry was applied for characterising the expression of Nanog and Oct4 during different cell cycle phases. Among untreated h......ESC we detected Nanog-expressing cells, which also expressed Oct4, SSEA-3 and SSEA-4. We also found another population expressing SSEA-4, but without Nanog, Oct4 and SSEA-3 expression. Nocodazole treatment resulted in a decrease of cell population positive for all four markers Nanog, Oct4, SSEA-3, SSEA-4....... Nocodazole-mediated cell-cycle arrest was accompanied by higher rate of apoptosis and upregulation of p53. Twenty-four hours after the release from nocodazole block, the cell cycle of hESC normalised, but no increase in the expression of transcription markers Nanog and Oct4 was detected. In addition...

  17. CD56+ immune cell infiltration and MICA are decreased in breast lobules with fibrocystic changes.

    Science.gov (United States)

    Kerekes, Daniel; Visscher, Daniel W; Hoskin, Tanya L; Radisky, Derek C; Brahmbhatt, Rushin D; Pena, Alvaro; Frost, Marlene H; Arshad, Muhammad; Stallings-Mann, Melody; Winham, Stacey J; Murphy, Linda; Denison, Lori; Carter, Jodi M; Knutson, Keith L; Degnim, Amy C

    2018-02-01

    While the role of natural killer (NK) cells in breast cancer therapy has been investigated, little information is known about NK cell function and presence in nonmalignant and premalignant breast tissue. Here, we investigate and quantify NK cell marker CD56 and activating ligand MICA in breast tissue with benign breast disease. Serial tissue sections from 88 subjects, 44 with benign breast disease (BBD) who remained cancer-free, and 44 with BBD who later developed cancer, were stained with H&E, anti-MICA, and anti-CD56. Up to ten representative lobules were identified on each section. Using digital image analysis, MICA and CD56 densities were determined for each lobule, reported as percent of pixels in the lobule that registered as stained by each antibody. Analyses were performed on a per-subject and per-lobule basis. Per-subject multivariate analyses showed associations of CD56 and MICA with age: CD56 was increased in older subjects (p = 0.03), while MICA was increased in younger subjects (p = 0.005). Per-lobule analyses showed that CD56 and MICA levels were both decreased in lobules with fibrocystic change, with median levels of CD56 and MICA staining, respectively, at 0.31 and 7.0% in fibrocystic lobules compared to 0.76 and 12.2% in lobules without fibrocystic change (p fibrocystic lobules, proliferative/atypical lobules showed significantly lower expression compared to nonproliferative lobules for MICA (p = 0.02) but not for CD56 (p = 0.80). Levels of CD56+ NK cells and activating ligand MICA were decreased in breast lobules with fibrocystic change, and MICA levels showed a significant stepwise decrease with increasing histopathologic abnormality. MICA levels were also significantly decreased in older subjects, who generally have higher risk of developing cancer. These findings advance a model in which MICA promotes cytotoxic activity in CD56+ NK cells to protect against tumorigenesis in breast lobules, and suggest further research is warranted.

  18. Valproic acid decreases urothelial cancer cell proliferation and induces thrombospondin-1 expression

    Directory of Open Access Journals (Sweden)

    Byler Timothy K

    2012-08-01

    Full Text Available Abstract Background Prevention of bladder cancer recurrence is a central challenge in the management of this highly prevalent disease. The histone deacetylase inhibitor valproic acid (sodium valproate has anti-angiogenic properties and has been shown to decrease bladder cancer growth in model systems. We have previously shown reduced expression of thrombospondin-1 in a mouse model and in human bladder cancer relative to normal urothelium. We speculated that inhibition of angiogenesis by valproate might be mediated by this anti-angiogenic protein. Methods Bladder cancer cell lines UMUC3 and T24 were treated with valproate or another histone deacetylase inhibitor, vorinostat, in culture for a period of three days. Proliferation was assessed by alamar blue reduction. Gene expression was evaluated by reverse transcription of RNA and quantitative PCR. Results Proliferation assays showed treatment with valproate or vorinostat decreased proliferation in both cell lines. Histone deacetylase inhibition also increased relative expression of thrombospondin-1 up to 8 fold at 5 mM valproate. Conclusions Histone deacetylase inhibitors warrant further study for the prevention or treatment of bladder cancer.

  19. Decreased Intracellular pH Induced by Cariporide Differentially Contributes to Human Umbilical Cord-Derived Mesenchymal Stem Cells Differentiation

    Directory of Open Access Journals (Sweden)

    Wei Gao

    2014-01-01

    Full Text Available Background/Aims: Na+/H+ exchanger 1 (NHE1 is an important regulator of intracellular pH (pHi. High pHi is required for cell proliferation and differentiation. Our previous study has proven that the pHi of mesenchymal stem cells is higher than that of normal differentiated cells and similar to tumor cells. NHE1 is highly expressed in both mesenchymal stem cells and tumor cells. Targeted inhibition of NHE1 could induce differentiation of K562 leukemia cells. In the present paper we explored whether inhibition of NHE1 could induce differentiation of mesenchymal stem cells. Methods: MSCs were obtained from human umbilical cord and both the surface phenotype and functional characteristics were analyzed. Selective NHE1 inhibitor cariporide was used to treat human umbilical cord-derived mesenchymal stem cells (hUC-MSCs. The pHi and the differentiation of hUC-MSCs were compared upon cariporide treatment. The putative signaling pathway involved was also explored. Results: The pHi of hUC-MSCs was decreased upon cariporide treatment. Cariporide up-regulated the osteogenic differentiation of hUC-MSCs while the adipogenic differentiation was not affected. For osteogenic differentiation, β-catenin expression was up-regulated upon cariporide treatment. Conclusion: Decreased pHi induced by cariporide differentially contributes to hUC-MSCs differentiation.

  20. Single cell time-lapse analysis reveals that podoplanin enhances cell survival and colony formation capacity of squamous cell carcinoma cells.

    Science.gov (United States)

    Miyashita, Tomoyuki; Higuchi, Youichi; Kojima, Motohiro; Ochiai, Atsushi; Ishii, Genichiro

    2017-01-06

    Tumor initiating cells (TICs) are characterized by high clonal expansion capacity. We previously reported that podoplanin is a TIC-specific marker for the human squamous cell carcinoma cell line A431. The aim of this study is to explore the molecular mechanism underlying the high clonal expansion potential of podoplanin-positive A431cells using Fucci imaging. Single podoplanin-positive cells created large colonies at a significantly higher frequency than single podoplanin-negative cells, whereas no difference was observed between the two types of cells with respect to cell cycle status. Conversely, the cell death ratio of progenies derived from podoplanin-positive single cell was significantly lower than that of cells derived from podoplanin-negative cells. Single A431 cells, whose podoplanin expression was suppressed by RNA interference, exhibited increased cell death ratios and decreased frequency of large colony forming. Moreover, the frequency of large colony forming decreased significantly when podoplanin-positive single cells was treated with a ROCK (Rho-associated coiled-coil kinase) inhibitor, whereas no difference was observed in single podoplanin-negative cells. Our current study cleared that high clonal expansion capacity of podoplanin-positive TICs populations was the result of reduced cell death by podoplanin-mediated signaling. Therefore, podoplanin activity may be a therapeutic target in the treatment of squamous cell carcinomas.

  1. Decreased astroglial cell adhesion and proliferation on zinc oxide nanoparticle polyurethane composites

    Science.gov (United States)

    Seil, Justin T; Webster, Thomas J

    2008-01-01

    Nanomaterials offer a number of properties that are of interest to the field of neural tissue engineering. Specifically, materials that exhibit nanoscale surface dimensions have been shown to promote neuron function while simultaneously minimizing the activity of cells such as astrocytes that inhibit central nervous system regeneration. Studies demonstrating enhanced neural tissue regeneration in electrical fields through the use of conductive materials have led to interest in piezoelectric materials (or those materials which generate a transient electrical potential when mechanically deformed) such as zinc oxide (ZnO). It has been speculated that ZnO nanoparticles possess increased piezoelectric properties over ZnO micron particles. Due to this promise in neural applications, the objective of the present in vitro study was, for the first time, to assess the activity of astroglial cells on ZnO nanoparticle polymer composites. ZnO nanoparticles embedded in polyurethane were analyzed via scanning electron microscopy to evaluate nanoscale surface features of the composites. The surface chemistry was characterized via X-ray photoelectron spectroscopy. Astroglial cell response was evaluated based on cell adhesion and proliferation. Astrocyte adhesion was significantly reduced on ZnO nanoparticle/polyurethane (PU) composites with a weight ratio of 50:50 (PU:ZnO) wt.%, 75:25 (PU:ZnO) wt.%, and 90:10 (PU:ZnO) wt.% in comparison to pure PU. The successful production of ZnO nanoparticle composite scaffolds suitable for decreasing astroglial cell density demonstrates their potential as a nerve guidance channel material with greater efficiency than what may be available today. PMID:19337420

  2. Nitrogen deficiency inhibits leaf blade growth in Lolium perenne by increasing cell cycle duration and decreasing mitotic and post-mitotic growth rates.

    Science.gov (United States)

    Kavanová, Monika; Lattanzi, Fernando Alfredo; Schnyder, Hans

    2008-06-01

    Nitrogen deficiency severely inhibits leaf growth. This response was analysed at the cellular level by growing Lolium perenne L. under 7.5 mM (high) or 1 mM (low) nitrate supply, and performing a kinematic analysis to assess the effect of nitrogen status on cell proliferation and cell growth in the leaf blade epidermis. Low nitrogen supply reduced leaf elongation rate (LER) by 43% through a similar decrease in the cell production rate and final cell length. The former was entirely because of a decreased average cell division rate (0.023 versus 0.032 h(-1)) and thus longer cell cycle duration (30 versus 22 h). Nitrogen status did not affect the number of division cycles of the initial cell's progeny (5.7), and accordingly the meristematic cell number (53). Meristematic cell length was unaffected by nitrogen deficiency, implying that the division and mitotic growth rates were equally impaired. The shorter mature cell length arose from a considerably reduced post-mitotic growth rate (0.033 versus 0.049 h(-1)). But, nitrogen stress did not affect the position where elongation stopped, and increased cell elongation duration. In conclusion, nitrogen deficiency limited leaf growth by increasing the cell cycle duration and decreasing mitotic and post-mitotic elongation rates, delaying cell maturation.

  3. Increase in Dye:Dendrimer Ratio Decreases Cellular Uptake of Neutral Dendrimers in RAW Cells.

    Science.gov (United States)

    Vaidyanathan, Sriram; Kaushik, Milan; Dougherty, Casey; Rattan, Rahul; Goonewardena, Sascha N; Banaszak Holl, Mark M; Monano, Janet; DiMaggio, Stassi

    2016-09-12

    Neutral generation 3 poly(amidoamine) dendrimers were labeled with Oregon Green 488 (G3-OG n ) to obtain materials with controlled fluorophore:dendrimer ratios (n = 1-2), a mixture containing mostly 3 dyes per dendrimer, a mixture containing primarily 4 or more dyes per dendrimer ( n = 4+), and a stochastic mixture ( n = 4 avg ). The UV absorbance of the dye conjugates increased linearly as n increased and the fluorescence emission decreased linearly as n increased. Cellular uptake was studied in RAW cells and HEK 293A cells as a function of the fluorophore:dendrimer ratio (n). The cellular uptake of G3-OG n ( n = 3, 4+, 4 avg ) into RAW cells was significantly lower than G3-OG n ( n = 1, 2). The uptake of G3-OG n ( n = 3, 4+, 4 avg ) into HEK 293A cells was not significantly different from G3-OG 1 . Thus, the fluorophore:dendrimer ratio was observed to change the extent of uptake in the macrophage uptake mechanism but not in the HEK 293A cell. This difference in endocytosis indicates the presence of a pathway in the macrophage that is sensitive to hydrophobicity of the particle.

  4. Lactoferricin treatment decreases the rate of cell proliferation of a human colon cancer cell line.

    Science.gov (United States)

    Freiburghaus, C; Janicke, B; Lindmark-Månsson, H; Oredsson, S M; Paulsson, M A

    2009-06-01

    Food components modify the risk of cancer at a large number of sites but the mechanism of action is unknown. In the present investigation, we studied the effect of the peptide lactoferricin derived from bovine milk lactoferrin on human colon cancer CaCo-2 cells. The cells were either untreated or treated with 2.0, 0.2, or 0.02 microM lactoferricin. Cell cycle kinetics were investigated with a bromodeoxyuridine DNA flow cytometric method. The results show that lactoferricin treatment slightly but significantly prolonged the S phase of the cell cycle. Lactoferricin treatment lowered the level of cyclin E1, a protein involved in the regulation of genes required for G(1)/S transition and consequently for efficient S phase progression. The slight prolongation of the S phase resulted in a reduction of cell proliferation, which became more apparent after a long treatment time.

  5. Glucose-6-phosphate dehydrogenase activity decreases during storage of leukoreduced red blood cells

    NARCIS (Netherlands)

    Peters, Anna L.; van Bruggen, Robin; de Korte, Dirk; van Noorden, Cornelis J. F.; Vlaar, Alexander P. J.

    2016-01-01

    During storage, the activity of the red blood cell (RBC) antioxidant system decreases. Glucose-6-phosphate dehydrogenase (G6PD) is essential for protection against oxidative stress by producing NADPH. G6PD function of RBC transfusion products is reported to remain stable during storage, but activity

  6. CHEMERIN (RARRES2) decreases in vitro granulosa cell steroidogenesis and blocks oocyte meiotic progression in bovine species.

    Science.gov (United States)

    Reverchon, Maxime; Bertoldo, Michael J; Ramé, Christelle; Froment, Pascal; Dupont, Joëlle

    2014-05-01

    CHEMERIN, or RARRES2, is a new adipokine that is involved in the regulation of adipogenesis, energy metabolism, and inflammation. Recent data suggest that it also plays a role in reproductive function in rats and humans. Here we studied the expression of CHEMERIN and its three receptors (CMKLR1, GPR1, and CCRL2) in the bovine ovary and investigated the in vitro effects of this hormone on granulosa cell steroidogenesis and oocyte maturation. By RT-PCR, immunoblotting, and immunohistochemistry, we found CHEMERIN, CMKLR1, GPR1, and CCRL2 in various ovarian cells, including granulosa and theca cells, corpus luteum, and oocytes. In cultured bovine granulosa cells, INSULIN, IGF1, and two insulin sensitizers-metformin and rosiglitazone-increased rarres2 mRNA expression whereas they decreased cmklr1, gpr1, and cclr2 mRNA expression. Furthermore, TNF alpha and ADIPONECTIN significantly increased rarres2 and cmklr1 expression, respectively. In cultured bovine granulosa cells, human recombinant CHEMERIN (hRec, 200 ng/ml) reduced production of both progesterone and estradiol, cholesterol content, STAR abundance, CYP19A1 and HMGCR proteins, and the phosphorylation levels of MAPK3/MAPK1 in the presence or absence of FSH (10(-8) M) and IGF1 (10(-8) M). All of these effects were abolished by using an anti-CMKLR1 antibody. In bovine cumulus-oocyte complexes, the addition of hRec (200 ng/ml) in the maturation medium arrested most oocytes at the germinal vesicle stage, and this was associated with a decrease in MAPK3/1 phosphorylation in both oocytes and cumulus cells. Thus, in cultured bovine granulosa cells, hRec decreases steroidogenesis, cholesterol synthesis, and MAPK3/1 phosphorylation, probably through CMKLR1. Moreover, in cumulus-oocyte complexes, it blocked meiotic progression at the germinal vesicle stage and inhibited MAPK3/1 phosphorylation in both the oocytes and cumulus cells during in vitro maturation. © 2014 by the Society for the Study of Reproduction, Inc.

  7. Snail modulates cell metabolism in MDCK cells

    Energy Technology Data Exchange (ETDEWEB)

    Haraguchi, Misako, E-mail: haraguci@m3.kufm.kagoshima-u.ac.jp [Department of Biochemistry and Molecular Biology, Graduate School of Medical and Dental Sciences, Kagoshima University, Kagoshima 890-8544 (Japan); Indo, Hiroko P. [Department of Maxillofacial Radiology, Graduate School of Medical and Dental Sciences, Kagoshima University, Kagoshima 890-8544 (Japan); Iwasaki, Yasumasa [Health Care Center, Kochi University, Kochi 780-8520 (Japan); Iwashita, Yoichiro [Department of Maxillofacial Radiology, Graduate School of Medical and Dental Sciences, Kagoshima University, Kagoshima 890-8544 (Japan); Fukushige, Tomoko [Department of Dermatology, Graduate School of Medical and Dental Sciences, Kagoshima University, Kagoshima 890-8544 (Japan); Majima, Hideyuki J. [Department of Maxillofacial Radiology, Graduate School of Medical and Dental Sciences, Kagoshima University, Kagoshima 890-8544 (Japan); Izumo, Kimiko; Horiuchi, Masahisa [Department of Environmental Medicine, Graduate School of Medical and Dental Sciences, Kagoshima University, Kagoshima 890-8544 (Japan); Kanekura, Takuro [Department of Dermatology, Graduate School of Medical and Dental Sciences, Kagoshima University, Kagoshima 890-8544 (Japan); Furukawa, Tatsuhiko [Department of Molecular Oncology, Graduate School of Medical and Dental Sciences, Kagoshima University, Kagoshima 890-8544 (Japan); Ozawa, Masayuki [Department of Biochemistry and Molecular Biology, Graduate School of Medical and Dental Sciences, Kagoshima University, Kagoshima 890-8544 (Japan)

    2013-03-22

    Highlights: ► MDCK/snail cells were more sensitive to glucose deprivation than MDCK/neo cells. ► MDCK/snail cells had decreased oxidative phosphorylation, O{sub 2} consumption and ATP content. ► TCA cycle enzyme activity, but not expression, was lower in MDCK/snail cells. ► MDCK/snail cells showed reduced PDH activity and increased PDK1 expression. ► MDCK/snail cells showed reduced expression of GLS2 and ACLY. -- Abstract: Snail, a repressor of E-cadherin gene transcription, induces epithelial-to-mesenchymal transition and is involved in tumor progression. Snail also mediates resistance to cell death induced by serum depletion. By contrast, we observed that snail-expressing MDCK (MDCK/snail) cells undergo cell death at a higher rate than control (MDCK/neo) cells in low-glucose medium. Therefore, we investigated whether snail expression influences cell metabolism in MDCK cells. Although gylcolysis was not affected in MDCK/snail cells, they did exhibit reduced pyruvate dehydrogenase (PDH) activity, which controls pyruvate entry into the tricarboxylic acid (TCA) cycle. Indeed, the activity of multiple enzymes involved in the TCA cycle was decreased in MDCK/snail cells, including that of mitochondrial NADP{sup +}-dependent isocitrate dehydrogenase (IDH2), succinate dehydrogenase (SDH), and electron transport Complex II and Complex IV. Consequently, lower ATP content, lower oxygen consumption and increased survival under hypoxic conditions was also observed in MDCK/snail cells compared to MDCK/neo cells. In addition, the expression and promoter activity of pyruvate dehydrogenase kinase 1 (PDK1), which phosphorylates and inhibits the activity of PDH, was increased in MDCK/snail cells, while expression levels of glutaminase 2 (GLS2) and ATP-citrate lyase (ACLY), which are involved in glutaminolysis and fatty acid synthesis, were decreased in MDCK/snail cells. These results suggest that snail modulates cell metabolism by altering the expression and activity of

  8. Decreased Regulatory T Cells in Vulnerable Atherosclerotic Lesions: Imbalance between Pro- and Anti-Inflammatory Cells in Atherosclerosis

    Directory of Open Access Journals (Sweden)

    Ilonka Rohm

    2015-01-01

    Full Text Available Atherosclerosis is a chronic inflammatory disease of the arterial wall in which presentation of autoantigens by dendritic cells (DCs leads to the activation of T cells. Anti-inflammatory cells like Tregs counterbalance inflammation in atherogenesis. In our study, human carotid plaque specimens were classified as stable (14 and unstable (15 according to established morphological criteria. Vessel specimens (n=12 without any signs of atherosclerosis were used as controls. Immunohistochemical staining was performed to detect different types of DCs (S100, fascin, CD83, CD209, CD304, and CD123, proinflammatory T cells (CD3, CD4, CD8, and CD161, and anti-inflammatory Tregs (FoxP3. The following results were observed: in unstable lesions, significantly higher numbers of proinflammatory cells like DCs, T helper cells, cytotoxic T cells, and natural killer cells were detected compared to stable plaques. Additionally, there was a significantly higher expression of HLA-DR and more T cell activation (CD25, CD69 in unstable lesions. On the contrary, unstable lesions contained significantly lower numbers of Tregs. Furthermore, a significant inverse correlation between myeloid DCs and Tregs was shown. These data suggest an increased inflammatory state in vulnerable plaques resulting from an imbalance of the frequency of local pro- and anti-inflammatory immune cells.

  9. Effects of DDT and Triclosan on Tumor-cell Binding Capacity and Cell-Surface Protein Expression of Human Natural Killer Cells

    Science.gov (United States)

    Hurd-Brown, Tasia; Udoji, Felicia; Martin, Tamara; Whalen, Margaret M.

    2012-01-01

    1,1,1-trichloro-2,2-bis(4-chlorophenyl)ethane (DDT) and triclosan (TCS) are organochlorine (OC) compounds that contaminate the environment, are found in human blood, and have been shown to decrease the tumor-cell killing (lytic) function of human natural killer (NK) cells. NK cells defend against tumor cells and virally infected cells. They bind to these targets, utilizing a variety of cell surface proteins. This study examined concentrations of DDT and TCS that decrease lytic function for alteration of NK binding to tumor targets. Levels of either compound that caused loss of binding function were then examined for effects on expression of cell-surface proteins needed for binding. NK cells exposed to 2.5 μM DDT for 24 h (which caused a greater than 55% loss of lytic function) showed a decrease in NK binding function of about 22%, and a decrease in CD16 cell-surface protein of 20%. NK cells exposed to 5 μM TCS for 24 h showed a decrease in ability to bind tumor cells of 37% and a decrease in expression of CD56 of about 34%. This same treatment caused a decrease in lytic function of greater than 87%. These results indicated that only a portion of the loss of NK lytic function seen with exposures to these compounds could be accounted for by loss of binding function. They also showed that loss of binding function is accompanied by a loss cell-surface proteins important in binding function. PMID:22729613

  10. Shading Contributes to the Reduction of Stem Mechanical Strength by Decreasing Cell Wall Synthesis in Japonica Rice (Oryza sativa L.

    Directory of Open Access Journals (Sweden)

    Longmei Wu

    2017-05-01

    Full Text Available Low solar radiation caused by industrial development and solar dimming has become a limitation in crop production in China. It is widely accepted that low solar radiation influences many aspects of plant development, including slender, weak stems and susceptibility to lodging. However, the underlying mechanisms are not well understood. To clarify how low solar radiation affects stem mechanical strength formation and lodging resistance, the japonica rice cultivars Wuyunjing23 (lodging-resistant and W3668 (lodging-susceptible were grown under field conditions with normal light (Control and shading (the incident light was reduced by 60% with a black nylon net. The yield and yield components, plant morphological characteristics, the stem mechanical strength, cell wall components, culm microstructure, gene expression correlated with cellulose and lignin biosynthesis were measured. The results showed that shading significantly reduced grain yield attributed to reduction of spikelets per panicles and grain weight. The stem-breaking strength decreased significantly under shading treatment; consequently, resulting in higher lodging index in rice plant in both varieties, as revealed by decreased by culm diameter, culm wall thickness and increased plant height, gravity center height. Compared with control, cell wall components including non-structural carbohydrate, sucrose, cellulose, and lignin reduced quite higher. With histochemical straining, shading largely reduced lignin deposition in the sclerenchyma cells and vascular bundle cells compared with control, and decreased cellulose deposition in the parenchyma cells of culm tissue in both Wuyunjing23 and W3668. And under shading condition, gene expression involved in secondary cell wall synthesis, OsPAL, OsCOMT, OsCCoAOMT, OsCCR, and OsCAD2, and primary cell wall synthesis, OsCesA1, OsCesA3, and OsCesA8 were decreased significantly. These results suggest that gene expression involved in the reduction of

  11. Shading Contributes to the Reduction of Stem Mechanical Strength by Decreasing Cell Wall Synthesis in Japonica Rice (Oryza sativa L.).

    Science.gov (United States)

    Wu, Longmei; Zhang, Wujun; Ding, Yanfeng; Zhang, Jianwei; Cambula, Elidio D; Weng, Fei; Liu, Zhenghui; Ding, Chengqiang; Tang, She; Chen, Lin; Wang, Shaohua; Li, Ganghua

    2017-01-01

    Low solar radiation caused by industrial development and solar dimming has become a limitation in crop production in China. It is widely accepted that low solar radiation influences many aspects of plant development, including slender, weak stems and susceptibility to lodging. However, the underlying mechanisms are not well understood. To clarify how low solar radiation affects stem mechanical strength formation and lodging resistance, the japonica rice cultivars Wuyunjing23 (lodging-resistant) and W3668 (lodging-susceptible) were grown under field conditions with normal light (Control) and shading (the incident light was reduced by 60%) with a black nylon net. The yield and yield components, plant morphological characteristics, the stem mechanical strength, cell wall components, culm microstructure, gene expression correlated with cellulose and lignin biosynthesis were measured. The results showed that shading significantly reduced grain yield attributed to reduction of spikelets per panicles and grain weight. The stem-breaking strength decreased significantly under shading treatment; consequently, resulting in higher lodging index in rice plant in both varieties, as revealed by decreased by culm diameter, culm wall thickness and increased plant height, gravity center height. Compared with control, cell wall components including non-structural carbohydrate, sucrose, cellulose, and lignin reduced quite higher. With histochemical straining, shading largely reduced lignin deposition in the sclerenchyma cells and vascular bundle cells compared with control, and decreased cellulose deposition in the parenchyma cells of culm tissue in both Wuyunjing23 and W3668. And under shading condition, gene expression involved in secondary cell wall synthesis, OsPAL, OsCOMT, OsCCoAOMT, OsCCR , and OsCAD2 , and primary cell wall synthesis, OsCesA1, OsCesA3 , and OsCesA8 were decreased significantly. These results suggest that gene expression involved in the reduction of lignin and

  12. Suppressed histamine release from rat peritoneal mast cells by ultraviolet B irradiation: decreased diacylglycerol formation as a possible mechanism

    International Nuclear Information System (INIS)

    Danno, K.; Fujii, K.; Tachibana, T.; Toda, K.; Horio, T.

    1988-01-01

    This study was designed to investigate the effect of ultraviolet B (UVB) irradiation on mast cell functions. Purified mast cells obtained from rat peritoneal cavity were irradiated with UVB and subsequently exposed to a degranulator, compound 48/80, or the calcium ionophore A-23187. The amount of histamine released from mast cells measured by the enzyme isotopic assay was significantly decreased by UVB irradiation (100-400 mJ/cm2). Within this dose range, UVB alone was not cytotoxic to the cells because it did not induce histamine release. The suppression was observed when mast cells were subjected to degranulation without intervals after UVB irradiation, and even after 5 h postirradiation. The wavelength of 300 nm from a monochromatic light source showed the maximum effect. When mast cells prelabeled with [ 3 H]arachidonate were irradiated and challenged by compound 48/80, label accumulation in diacylglycerol produced by the phosphatidylinositol cycle was considerably decreased by UVB irradiation. From these results, we hypothesize that, within an adequate irradiation dose, UVB irradiation suppresses histamine release from mast cells, probably by causing noncytotoxic damage to the membrane phospholipid metabolism, which is tied to the degranulation mechanisms

  13. Increased circulating follicular helper T cells with decreased programmed death-1 in chronic renal allograft rejection.

    Science.gov (United States)

    Shi, Jian; Luo, Fengbao; Shi, Qianqian; Xu, Xianlin; He, Xiaozhou; Xia, Ying

    2015-11-03

    Chronic antibody-mediated rejection is a major issue that affects long-term renal allograft survival. Since follicular helper T (Tfh) cells promote the development of antigen-specific B cells in alloimmune responses, we investigated the potential roles of Tfh cells, B cells and their alloimmune-regulating molecules in the pathogenesis of chronic renal allograft rejection in this study. The frequency of Tfh, B cells and the levels of their alloimmune-regulating molecules including chemokine receptor type 5 (CXCR5), inducible T cell co-stimulator (ICOS), programmed death-1 (PD-1), ICOSL, PDL-1 and interleukin-21 (IL-21), of peripheral blood were comparatively measured in 42 primary renal allograft recipients within 1-3 years after transplantation. Among them, 24 patients had definite chronic rejection, while other 18 patients had normal renal function. Tfh-cell ratio was significantly increased with PD-1 down-regulation in the patients with chronic renal allograft rejection, while B cells and the alloimmune-regulating molecules studied did not show any appreciable change in parallel. The patients with chronic renal allograft rejection have a characteristic increase in circulating Tfh cells with a decrease in PD-1 expression. These pathological changes may be a therapeutic target for the treatment of chronic renal allograft rejection and can be useful as a clinical index for monitoring conditions of renal transplant.

  14. Lycopene inhibits IGF-I signal transduction and growth in normal prostate epithelial cells by decreasing DHT-modulated IGF-I production in co-cultured reactive stromal cells.

    Science.gov (United States)

    Liu, Xunxian; Allen, Jeffrey D; Arnold, Julia T; Blackman, Marc R

    2008-04-01

    Prostate stromal and epithelial cell communication is important in prostate functioning and cancer development. Primary human stromal cells from normal prostate stromal cells (PRSC) maintain a smooth muscle phenotype, whereas those from prostate cancer (6S) display reactive and fibroblastic characteristics. Dihydrotestosterone (DHT) stimulates insulin-like growth factor-I (IGF-I) production by 6S but not PSRC cells. Effects of reactive versus normal stroma on normal human prostate epithelial (NPE or PREC) cells are poorly understood. We co-cultured NPE plus 6S or PRSC cells to compare influences of different stromal cells on normal epithelium. Because NPE and PREC cells lose androgen receptor (AR) expression in culture, DHT effects must be modulated by associated stromal cells. When treated with camptothecin (CM), NPE cells, alone and in stromal co-cultures, displayed a dose-dependent increase in DNA fragmentation. NPE/6S co-cultures exhibited reduced CM-induced cell death with exposure to DHT, whereas NPE/PRSC co-cultures exhibited CM-induced cell death regardless of DHT treatment. DHT blocked CM-induced, IGF-I-mediated, NPE death in co-cultured NPE/6S cells without, but not with, added anti-IGF-I and anti-IGF-R antibodies. Lycopene consumption is inversely related to human prostate cancer risk and inhibits IGF-I and androgen signaling in rat prostate cancer. In this study, lycopene, in dietary concentrations, reversed DHT effects of 6S cells on NPE cell death, decreased 6S cell IGF-I production by reducing AR and beta-catenin nuclear localization and inhibited IGF-I-stimulated NPE and PREC growth, perhaps by attenuating IGF-I's effects on serine phosphorylation of Akt and GSK3beta and tyrosine phosphorylation of GSK3. This study expands the understanding of the preventive mechanisms of lycopene in prostate cancer.

  15. Lipophilic fractions from the marine sponge Halichondria sitiens decrease secretion of pro-inflammatory cytokines by dendritic cells and decrease their ability to induce a Th1 type response by allogeneic CD4+ T cells.

    Science.gov (United States)

    Di, Xiaxia; Oskarsson, Jon T; Omarsdottir, Sesselja; Freysdottir, Jona; Hardardottir, Ingibjorg

    2017-12-01

    Halichondria (Halichondriidae) marine sponges contain components possessing various biological activities, but immunomodulation is not among the ones reported. This study evaluated the immunomodulatory effects of fractions/compounds from Halichondria sitiens Schmidt. Crude dichloromethane/methanol extracts of H. sitiens were subjected to various chromatographic techniques to obtain fractions/compounds with immunomodulatory activity, using bioassay-guided isolation. The effects of the fractions/compounds were determined by measuring secretion of cytokines and expression of surface molecules by dendritic cells (DCs) and their ability to stimulate and modify cytokine secretion by allogeneic CD4 + T cells. The bioactive fractions were chemically analyzed to identify the immunomodulatory constituents by 1D, 2D NMR, and HRMS data. Several lipophilic fractions from H. sitiens at 10 μg/mL decreased secretion of the pro-inflammatory cytokines IL-12p40 and IL-6 by the DCs, with maximum inhibition being 64% and 25%, respectively. In addition, fractions B3b3F and B3b3J decreased the ability of DCs to induce T cell secretion of IFN-γ. Fraction B3b3 induced morphological changes in DCs, characterized by extreme elongation of dendrites and cell clustering. Chemical screening revealed the presence of glycerides and some minor unknown constituents in the biologically active fractions. One new glyceride, 2,3-dihydroxypropyl 2-methylhexadecanoate (1), was isolated from one fraction and two known compounds, 3-[(1-methoxyhexadecyl)oxy]propane-1,2-diol (2) and monoheptadecanoin (3), were identified in another, but none of them had immunomodulatory activity. These results demonstrate that several lipophilic fractions from H. sitiens have anti-inflammatory effects on DCs and decrease their ability to induce a Th1 type immune response.

  16. Decreased astroglial cell adhesion and proliferation on zinc oxide nanoparticle polyurethane composites

    Directory of Open Access Journals (Sweden)

    Justin T Seil

    2008-11-01

    Full Text Available Justin T Seil, Thomas J WebsterLaboratory for Nanomedicine Research, Division of Engineering, Brown University, Providence, RI, USAAbstract: Nanomaterials offer a number of properties that are of interest to the field of neural tissue engineering. Specifically, materials that exhibit nanoscale surface dimensions have been shown to promote neuron function while simultaneously minimizing the activity of cells such as astrocytes that inhibit central nervous system regeneration. Studies demonstrating enhanced neural tissue regeneration in electrical fields through the use of conductive materials have led to interest in piezoelectric materials (or those materials which generate a transient electrical potential when mechanically deformed such as zinc oxide (ZnO. It has been speculated that ZnO nanoparticles possess increased piezoelectric properties over ZnO micron particles. Due to this promise in neural applications, the objective of the present in vitro study was, for the first time, to assess the activity of astroglial cells on ZnO nanoparticle polymer composites. ZnO nanoparticles embedded in polyurethane were analyzed via scanning electron microscopy to evaluate nanoscale surface features of the composites. The surface chemistry was characterized via X-ray photoelectron spectroscopy. Astroglial cell response was evaluated based on cell adhesion and proliferation. Astrocyte adhesion was significantly reduced on ZnO nanoparticle/polyurethane (PU composites with a weight ratio of 50:50 (PU:ZnO wt.%, 75:25 (PU:ZnO wt.%, and 90:10 (PU:ZnO wt.% in comparison to pure PU. The successful production of ZnO nanoparticle composite scaffolds suitable for decreasing astroglial cell density demonstrates their potential as a nerve guidance channel material with greater efficiency than what may be available today.Keywords: zinc oxide, nanoparticles, astrocytes, neural tissue, nervous system, biomaterials

  17. Cholesterol pathways affected by small molecules that decrease sterol levels in Niemann-Pick type C mutant cells.

    Directory of Open Access Journals (Sweden)

    Madalina Rujoi

    2010-09-01

    Full Text Available Niemann-Pick type C (NPC disease is a genetically inherited multi-lipid storage disorder with impaired efflux of cholesterol from lysosomal storage organelles.The effect of screen-selected cholesterol lowering compounds on the major sterol pathways was studied in CT60 mutant CHO cells lacking NPC1 protein. Each of the selected chemicals decreases cholesterol in the lysosomal storage organelles of NPC1 mutant cells through one or more of the following mechanisms: increased cholesterol efflux from the cell, decreased uptake of low-density lipoproteins, and/or increased levels of cholesteryl esters. Several chemicals promote efflux of cholesterol to extracellular acceptors in both non-NPC and NPC1 mutant cells. The uptake of low-density lipoprotein-derived cholesterol is inhibited by some of the studied compounds.Results herein provide the information for prioritized further studies in identifying molecular targets of the chemicals. This approach proved successful in the identification of seven chemicals as novel inhibitors of lysosomal acid lipase (Rosenbaum et al, Biochim. Biophys. Acta. 2009, 1791:1155-1165.

  18. When larger brains do not have more neurons: Increased numbers of cells are compensated by decreased average cell size across mouse individuals

    Directory of Open Access Journals (Sweden)

    Suzana eHerculano-Houzel

    2015-06-01

    Full Text Available There is a strong trend toward increased brain size in mammalian evolution, with larger brains composed of more and larger neurons than smaller brains across species within each mammalian order. Does the evolution of increased numbers of brain neurons, and thus larger brain size, occur simply through the selection of individuals with more and larger neurons, and thus larger brains, within a population? That is, do individuals with larger brains also have more, and larger, neurons than individuals with smaller brains, such that allometric relationships across species are simply an extension of intraspecific scaling? Here we show that this is not the case across adult male mice of a similar age. Rather, increased numbers of neurons across individuals are accompanied by increased numbers of other cells and smaller average cell size of both types, in a trade-off that explains how increased brain mass does not necessarily ensue. Fundamental regulatory mechanisms thus must exist that tie numbers of neurons to numbers of other cells and to average cell size within individual brains. Finally, our results indicate that changes in brain size in evolution are not an extension of individual variation in numbers of neurons, but rather occur through step changes that must simultaneously increase numbers of neurons and cause cell size to increase, rather than decrease.

  19. Epidermal growth factor decreases PEPT2 transport capacity and expression in the rat kidney proximal tubule cell line SKPT0193 cl.2

    DEFF Research Database (Denmark)

    Bravo, Silvina A; Nielsen, Carsten Uhd; Amstrup, Jan

    2004-01-01

    by studies of apical uptake of [14C]glycylsarcosine, rPepT2 mRNA levels, and immunostaining of SKPT cells with a rPEPT2-specific antibody. On the contrary, apical uptake of glucose and lysine was increased in EGF-treated cells, indicating that EGF was not acting generally to decrease apical nutrient uptake...... mechanisms in the proximal tubule cells. Our findings indicate that EGF decreases rPEPT2 expression by lowering transcription of the rat PepT2 gene or by decreasing rat PepT2 mRNA stability. Previous investigators routinely used SKPT cell culture media with a high (10 ng/ml) EGF concentration. Our study...

  20. Carbon nanotubes enhance the internalization of drugs by cancer cells and decrease their chemoresistance to cytostatics

    Science.gov (United States)

    Mahmood, M.; Xu, Y.; Dantuluri, V.; Mustafa, T.; Zhang, Y.; Karmakar, A.; Casciano, D.; Ali, S.; Biris, A.

    2013-02-01

    Etoposide is a semisynthetic, chemotherapeutic drug widely recommended to treat an extensive range of human cancers. Our studies indicate that, while etoposide is capable of killing human cancer cells, exposure to single-walled carbon nanotubes (SWCNTs) and etoposide results in enhanced cell death that appears to be synergistic and not merely additive. In this study, we used high pressure liquid chromatography and mass spectrometry to quantify the internal effective dose of etoposide when the human pancreatic cancer cell (PANC-1) was exposed to the combination of these agents. Our results unequivocally indicate that SWCNTs improve etoposide uptake and increase its capacity to kill cancer cells. We suggest that a combination of SWCNTs and etoposide may prove to be a more efficient chemotherapeutic protocol, especially because of the potential to lower toxic drug doses to levels that may be useful in decreasing adverse side effects, as well as in lowering the probability of inducing chemoresistance in exposed cancer cells.

  1. Carbon nanotubes enhance the internalization of drugs by cancer cells and decrease their chemoresistance to cytostatics

    International Nuclear Information System (INIS)

    Mahmood, M; Xu, Y; Dantuluri, V; Mustafa, T; Karmakar, A; Casciano, D; Biris, A; Zhang, Y; Ali, S

    2013-01-01

    Etoposide is a semisynthetic, chemotherapeutic drug widely recommended to treat an extensive range of human cancers. Our studies indicate that, while etoposide is capable of killing human cancer cells, exposure to single-walled carbon nanotubes (SWCNTs) and etoposide results in enhanced cell death that appears to be synergistic and not merely additive. In this study, we used high pressure liquid chromatography and mass spectrometry to quantify the internal effective dose of etoposide when the human pancreatic cancer cell (PANC-1) was exposed to the combination of these agents. Our results unequivocally indicate that SWCNTs improve etoposide uptake and increase its capacity to kill cancer cells. We suggest that a combination of SWCNTs and etoposide may prove to be a more efficient chemotherapeutic protocol, especially because of the potential to lower toxic drug doses to levels that may be useful in decreasing adverse side effects, as well as in lowering the probability of inducing chemoresistance in exposed cancer cells. (paper)

  2. A decrease in ubiquitination and resulting prolonged life-span of KIT underlies the KIT overexpression-mediated imatinib resistance of KIT mutation-driven canine mast cell tumor cells.

    Science.gov (United States)

    Kobayashi, Masato; Kuroki, Shiori; Kurita, Sena; Miyamoto, Ryo; Tani, Hiroyuki; Tamura, Kyoichi; Bonkobara, Makoto

    2017-10-01

    Overexpression of KIT is one of the mechanisms that contributes to imatinib resistance in KIT mutation-driven tumors. Here, the mechanism underlying this overexpression of KIT was investigated using an imatinib-sensitive canine mast cell tumor (MCT) line CoMS, which has an activating mutation in KIT exon 11. A KIT-overexpressing imatinib-resistant subline, rCoMS1, was generated from CoMS cells by their continuous exposure to increasing concentrations of imatinib. Neither a secondary mutation nor upregulated transcription of KIT was detected in rCoMS1 cells. A decrease in KIT ubiquitination, a prolonged KIT life-span, and KIT overexpression were found in rCoMS1 cells. These events were suppressed by withdrawal of imatinib and were re-induced by re‑treatment with imatinib. These findings suggest that imatinib elicited overexpression of KIT via suppression of its ubiquitination. These results also indicated that imatinib-induced overexpression of KIT in rCoMS1 cells was not a permanently acquired feature but was a reversible response of the cells. Moreover, the pan deubiquitinating enzyme inhibitor PR619 prevented imatinib induction of KIT overexpression, suggesting that the imatinib-induced decrease in KIT ubiquitination could be mediated by upregulation and/or activation of deubiquitinating enzyme(s). It may be possible that a similar mechanism of KIT overexpression underlies the acquisition of imatinib resistance in some human tumors that are driven by KIT mutation.

  3. Curcumin targets fibroblast–tumor cell interactions in oral squamous cell carcinoma

    International Nuclear Information System (INIS)

    Dudás, József; Fullár, Alexandra; Romani, Angela; Pritz, Christian; Kovalszky, Ilona; Hans Schartinger, Volker; Mathias Sprinzl, Georg; Riechelmann, Herbert

    2013-01-01

    Co-culture of periodontal ligament fibroblasts (PDLs) and SCC-25 oral squamous carcinoma cells (OSCC) results in conversion of PDLs into carcinoma-associated fibroblasts (CAFs) and induces epithelial-to mesenchymal transition (EMT) of OSCC tumor cells. We hypothesized that Curcumin targets this dynamic mutual interaction between CAFs and tumor cells. Normal and 2 μM Curcumin-treated co-culture were performed for 4 days, followed by analysis of tumor cell invasivity, mRNA/protein expression of EMT-markers and mediators, activity measure of matrix metalloproteinase 9 (MMP-9), and western blot analysis of signal transduction in tumor cells and fibroblasts. In Curcumin-treated co-culture, in tumor cells, the levels of nuclear factor κB (NFκBα) and early response kinase (ERK)—decreased, in fibroblasts, integrin αv protein synthesis decreased compared to corresponding cells in normal co-culture. The signal modulatory changes induced by Curcumin caused decreased release of EMT-mediators in CAFs and reversal of EMT in tumor cells, which was associated with decreased invasion. These data confirm the palliative potential of Curcumin in clinical application. - Graphical abstract: Co-culture of periodontal ligament fibroblasts (PDLs) and SCC-25 oral squamous carcinoma cells (OSCC) results in conversion of PDLs into carcinoma-associated fibroblasts (CAFs) and induces epithelial-to mesenchymal transition (EMT) of tumor cells. Curcumin targets this dynamic mutual interaction between CAFs and tumor cells by inhibiting the production of EMT mediators in CAFs and by modification of intracellular signaling in tumor cells. This causes less invasivity and reversal of EMT in tumor cells. Highlights: ► Curcumin targets tumor–fibroblast interaction in head and neck cancer. ► Curcumin suppresses mediators of epithelial–mesenchymal transition. ► Curcumin decreases the invasivity of tumor cells

  4. Curcumin targets fibroblast–tumor cell interactions in oral squamous cell carcinoma

    Energy Technology Data Exchange (ETDEWEB)

    Dudás, József, E-mail: jozsef.dudas@i-med.ac.at [Department of Otorhinolaryngology and Head and Neck Surgery, Medical University Innsbruck, Anichstrasse 35, A-6020 Innsbruck (Austria); Fullár, Alexandra, E-mail: fullarsz@gmail.com [Department of Otorhinolaryngology and Head and Neck Surgery, Medical University Innsbruck, Anichstrasse 35, A-6020 Innsbruck (Austria); 1st Department of Pathology and Experimental Cancer Research, Semmelweis University, Üllői út 26, 1085 Budapest (Hungary); Romani, Angela, E-mail: angela.romani@i-med.ac.at [Department of Otorhinolaryngology and Head and Neck Surgery, Medical University Innsbruck, Anichstrasse 35, A-6020 Innsbruck (Austria); Pritz, Christian, E-mail: christian.pritz@i-med.ac.at [Department of Otorhinolaryngology and Head and Neck Surgery, Medical University Innsbruck, Anichstrasse 35, A-6020 Innsbruck (Austria); Kovalszky, Ilona, E-mail: koval@korb1.sote.hu [1st Department of Pathology and Experimental Cancer Research, Semmelweis University, Üllői út 26, 1085 Budapest (Hungary); Hans Schartinger, Volker, E-mail: volker.schartinger@i-med.ac.at [Department of Otorhinolaryngology and Head and Neck Surgery, Medical University Innsbruck, Anichstrasse 35, A-6020 Innsbruck (Austria); Mathias Sprinzl, Georg, E-mail: georg.sprinzl@i-med.ac.at [Department of Otorhinolaryngology and Head and Neck Surgery, Medical University Innsbruck, Anichstrasse 35, A-6020 Innsbruck (Austria); Riechelmann, Herbert, E-mail: herbert.riechelmann@i-med.ac.at [Department of Otorhinolaryngology and Head and Neck Surgery, Medical University Innsbruck, Anichstrasse 35, A-6020 Innsbruck (Austria)

    2013-04-01

    Co-culture of periodontal ligament fibroblasts (PDLs) and SCC-25 oral squamous carcinoma cells (OSCC) results in conversion of PDLs into carcinoma-associated fibroblasts (CAFs) and induces epithelial-to mesenchymal transition (EMT) of OSCC tumor cells. We hypothesized that Curcumin targets this dynamic mutual interaction between CAFs and tumor cells. Normal and 2 μM Curcumin-treated co-culture were performed for 4 days, followed by analysis of tumor cell invasivity, mRNA/protein expression of EMT-markers and mediators, activity measure of matrix metalloproteinase 9 (MMP-9), and western blot analysis of signal transduction in tumor cells and fibroblasts. In Curcumin-treated co-culture, in tumor cells, the levels of nuclear factor κB (NFκBα) and early response kinase (ERK)—decreased, in fibroblasts, integrin αv protein synthesis decreased compared to corresponding cells in normal co-culture. The signal modulatory changes induced by Curcumin caused decreased release of EMT-mediators in CAFs and reversal of EMT in tumor cells, which was associated with decreased invasion. These data confirm the palliative potential of Curcumin in clinical application. - Graphical abstract: Co-culture of periodontal ligament fibroblasts (PDLs) and SCC-25 oral squamous carcinoma cells (OSCC) results in conversion of PDLs into carcinoma-associated fibroblasts (CAFs) and induces epithelial-to mesenchymal transition (EMT) of tumor cells. Curcumin targets this dynamic mutual interaction between CAFs and tumor cells by inhibiting the production of EMT mediators in CAFs and by modification of intracellular signaling in tumor cells. This causes less invasivity and reversal of EMT in tumor cells. Highlights: ► Curcumin targets tumor–fibroblast interaction in head and neck cancer. ► Curcumin suppresses mediators of epithelial–mesenchymal transition. ► Curcumin decreases the invasivity of tumor cells.

  5. Deferoxamine compensates for decreases in B cell counts and reduces mortality in enterovirus 71-infected mice.

    Science.gov (United States)

    Yang, Yajun; Ma, Jing; Xiu, Jinghui; Bai, Lin; Guan, Feifei; Zhang, Li; Liu, Jiangning; Zhang, Lianfeng

    2014-07-07

    Enterovirus 71 is one of the major causative agents of hand, foot and mouth disease in children under six years of age. No vaccine or antiviral therapy is currently available. In this work, we found that the number of B cells was reduced in enterovirus 71-infected mice. Deferoxamine, a marine microbial natural product, compensated for the decreased levels of B cells caused by enterovirus 71 infection. The neutralizing antibody titer was also improved after deferoxamine treatment. Furthermore, deferoxamine relieved symptoms and reduced mortality and muscle damage caused by enterovirus 71 infection. This work suggested that deferoxamine has the potential for further development as a B cell-immunomodulator against enterovirus 71.

  6. Collective cell streams in epithelial monolayers depend on cell adhesion

    International Nuclear Information System (INIS)

    Czirók, András; Varga, Katalin; Méhes, Előd; Szabó, András

    2013-01-01

    We report spontaneously emerging, randomly oriented, collective streaming behavior within a monolayer culture of a human keratinocyte cell line, and explore the effect of modulating cell adhesions by perturbing the function of calcium-dependent cell adhesion molecules. We demonstrate that decreasing cell adhesion induces narrower and more anisotropic cell streams, reminiscent of decreasing the Taylor scale of turbulent liquids. To explain our empirical findings, we propose a cell-based model that represents the dual nature of cell–cell adhesions. Spring-like connections provide mechanical stability, while a cellular Potts model formalism represents surface-tension driven attachment. By changing the relevance and persistence of mechanical links between cells, we are able to explain the experimentally observed changes in emergent flow patterns. (paper)

  7. SIRT1 overexpression decreases cisplatin-induced acetylation of NF-κB p65 subunit and cytotoxicity in renal proximal tubule cells

    International Nuclear Information System (INIS)

    Jung, Yu Jin; Lee, Jung Eun; Lee, Ae Sin; Kang, Kyung Pyo; Lee, Sik; Park, Sung Kwang; Lee, Sang Yong; Han, Myung Kwan; Kim, Duk Hoon; Kim, Won

    2012-01-01

    Highlights: ► Cisplatin increases acetylation of NF-κB p65 subunit in HK2 cells. ► SIRT1 overexpression decreases cisplatin-induced p65 acetylation and -cytotoxicity. ► Resveratrol decreased cisplatin-induced cell viability through deacetylation of p65. -- Abstract: As the increased acetylation of p65 is linked to nuclear factor-κB (NF-κB) activation, the regulation of p65 acetylation can be a potential target for the treatment of inflammatory injury. Cisplatin-induced nephrotoxicity is an important issue in chemotherapy of cancer patients. SIRT1, nicotinamide adenine dinucleotide (NAD + )-dependent protein deacetylase, has been implicated in a variety of cellular processes such as inflammatory injury and the control of multidrug resistance in cancer. However, there is no report on the effect of SIRT1 overexpression on cisplatin-induced acetylation of p65 subunit of NF-κB and cell injury. To investigate the effect of SIRT1 in on cisplatin-induced acetylation of p65 subunit of NF-κB and cell injury, HK2 cells were exposed with SIRT1 overexpression, LacZ adenovirus or dominant negative adenovirus after treatment with cisplatin. While protein expression of SIRT1 was decreased by cisplatin treatment compared with control buffer treatment, acetylation of NF-κB p65 subunit was significantly increased after treatment with cisplatin. Overexpression of SIRT1 ameliorated the increased acetylation of p65 of NF-κB during cisplatin treatment and cisplatin-induced cytotoxicity. Further, treatment of cisplatin-treated HK2 cells with resveratrol, a SIRT1 activator, also decreased acetylation of NF-κB p65 subunit and cisplatin-induced increase of the cell viability in HK2 cells. Our findings suggests that the regulation of acetylation of p65 of NF-κB through SIRT1 can be a possible target to attenuate cisplatin-induced renal cell damage.

  8. Chemotherapy-induced cognitive impairment is associated with decreases in cell proliferation and histone modifications

    Directory of Open Access Journals (Sweden)

    Briones Teresita L

    2011-12-01

    Full Text Available Abstract Background In this study, we examined the effects of cyclophosphamide, methothrexate, and 5-Fluorouracil (CMF drug combination on various aspects of learning and memory. We also examined the effects of CMF on cell proliferation and chromatin remodeling as possible underlying mechanisms to explain chemotherapy-associated cognitive dysfunction. Twenty-four adult female Wistar rats were included in the study and had minimitter implantation for continuous activity monitoring two weeks before the chemotherapy regimen was started. Once baseline activity data were collected, rats were randomly assigned to receive either CMF or saline injections given intraperitoneally. Treatments were given once a week for a total of 4 weeks. Two weeks after the last injection, rats were tested in the water maze for spatial learning and memory ability as well as discrimination learning. Bromodeoxyuridine (BrdU injection was given at 100 mg/Kg intraperitoneally 4 hours prior to euthanasia to determine hippocampal cell proliferation while histone acetylation and histone deacetylase activity was measured to determine CMF effects on chromatin remodeling. Results Our data showed learning and memory impairment following CMF administration independent of the drug effects on physical activity. In addition, CMF-treated rats showed decreased hippocampal cell proliferation, associated with increased histone acetylation and decreased histone deacetylase activity. Conclusions These results suggest the negative consequences of chemotherapy on brain function and that anti-cancer drugs can adversely affect the self-renewal potential of neural progenitor cells and also chromatin remodeling in the hippocampus. The significance of our findings lie on the possible usefulness of animal models in addressing the clinical phenomenon of 'chemobrain.'

  9. Decreased HIV diversity after allogeneic stem cell transplantation of an HIV-1 infected patient: a case report

    Directory of Open Access Journals (Sweden)

    Thielen Alexander

    2010-03-01

    Full Text Available Abstract The human immunodeficiency virus type 1 (HIV-1 coreceptor use and viral evolution were analyzed in blood samples from an HIV-1 infected patient undergoing allogeneic stem cell transplantation (SCT. Coreceptor use was predicted in silico from sequence data obtained from the third variable loop region of the viral envelope gene with two software tools. Viral diversity and evolution was evaluated on the same samples by Bayesian inference and maximum likelihood methods. In addition, phenotypic analysis was done by comparison of viral growth in peripheral blood mononuclear cells and in a CCR5 (R5-deficient T-cell line which was controlled by a reporter assay confirming viral tropism. In silico coreceptor predictions did not match experimental determinations that showed a consistent R5 tropism. Anti-HIV directed antibodies could be detected before and after the SCT. These preexisting antibodies did not prevent viral rebound after the interruption of antiretroviral therapy during the SCT. Eventually, transplantation and readministration of anti-retroviral drugs lead to sustained increase in CD4 counts and decreased viral load to undetectable levels. Unexpectedly, viral diversity decreased after successful SCT. Our data evidence that only R5-tropic virus was found in the patient before and after transplantation. Therefore, blocking CCR5 receptor during stem cell transplantation might have had beneficial effects and this might apply to more patients undergoing allogeneic stem cell transplantation. Furthermore, we revealed a scenario of HIV-1 dynamic different from the commonly described ones. Analysis of viral evolution shows the decrease of viral diversity even during episodes with bursts in viral load.

  10. Benzyl isothiocyanate alters the gene expression with cell cycle regulation and cell death in human brain glioblastoma GBM 8401 cells.

    Science.gov (United States)

    Tang, Nou-Ying; Chueh, Fu-Shin; Yu, Chien-Chih; Liao, Ching-Lung; Lin, Jen-Jyh; Hsia, Te-Chun; Wu, King-Chuen; Liu, Hsin-Chung; Lu, Kung-Wen; Chung, Jing-Gung

    2016-04-01

    Glioblastoma multiforme (GBM) is a highly malignant devastating brain tumor in adults. Benzyl isothiocyanate (BITC) is one of the isothiocyanates that have been shown to induce human cancer cell apoptosis and cell cycle arrest. Herein, the effect of BITC on cell viability and apoptotic cell death and the genetic levels of human brain glioblastoma GBM 8401 cells in vitro were investigated. We found that BITC induced cell morphological changes, decreased cell viability and the induction of cell apoptosis in GBM 8401 cells was time-dependent. cDNA microarray was used to examine the effects of BITC on GBM 8401 cells and we found that numerous genes associated with cell death and cell cycle regulation in GBM 8401 cells were altered after BITC treatment. The results show that expression of 317 genes was upregulated, and two genes were associated with DNA damage, the DNA-damage-inducible transcript 3 (DDIT3) was increased 3.66-fold and the growth arrest and DNA-damage-inducible α (GADD45A) was increased 2.34-fold. We also found that expression of 182 genes was downregulated and two genes were associated with receptor for cell responses to stimuli, the EGF containing fibulin-like extracellular matrix protein 1 (EFEMP1) was inhibited 2.01-fold and the TNF receptor-associated protein 1 (TRAP1) was inhibited 2.08-fold. BITC inhibited seven mitochondria ribosomal genes, the mitochondrial ribosomal protein; tumor protein D52 (MRPS28) was inhibited 2.06-fold, the mitochondria ribosomal protein S2 (MRPS2) decreased 2.07-fold, the mitochondria ribosomal protein L23 (MRPL23) decreased 2.08-fold, the mitochondria ribosomal protein S2 (MRPS2) decreased 2.07-fold, the mitochondria ribosomal protein S12 (MRPS12) decreased 2.08-fold, the mitochondria ribosomal protein L12 (MRPL12) decreased 2.25-fold and the mitochondria ribosomal protein S34 (MRPS34) was decreased 2.30-fold in GBM 8401 cells. These changes of gene expression can provide the effects of BITC on the genetic level and are

  11. The volume of Purkinje cells decreases in the cerebellum of acrylamide-intoxicated rats, but no cells are lost

    DEFF Research Database (Denmark)

    Larsen, Jytte Overgaard; Tandrup, T; Braendgaard, H

    1994-01-01

    The effects of acrylamide intoxication on the numbers of granule and Purkinje cells and the volume of Purkinje cell perikarya have been evaluated with stereological methods. The analysis was carried out in the cerebella of rats that had received a dose of 33.3 mg/kg acrylamide, twice a week, for 7.......5 weeks. The total numbers of cerebellar granule and Purkinje cells were estimated using the optical fractionator and the mean volume of the Purkinje cell perikarya was estimated with the vertical rotator technique. The volumes of the molecular layer, the granular cell layer and the white matter were...... estimated using the Cavalieri principle. The mean weight of the cerebellum of the intoxicated rats was 7% lower than that of the control rats (2P = 0.001). The numbers of the Purkinje cells and granule cells were the same in both groups, but the mean volume of the perikarya of the Purkinje cells...

  12. Decreased N-TAF1 expression in X-linked dystonia-parkinsonism patient-specific neural stem cells

    Directory of Open Access Journals (Sweden)

    Naoto Ito

    2016-04-01

    Full Text Available X-linked dystonia-parkinsonism (XDP is a hereditary neurodegenerative disorder involving a progressive loss of striatal medium spiny neurons. The mechanisms underlying neurodegeneration are not known, in part because there have been few cellular models available for studying the disease. The XDP haplotype consists of multiple sequence variations in a region of the X chromosome containing TAF1, a large gene with at least 38 exons, and a multiple transcript system (MTS composed of five unconventional exons. A previous study identified an XDP-specific insertion of a SINE-VNTR-Alu (SVA-type retrotransposon in intron 32 of TAF1, as well as a neural-specific TAF1 isoform, N-TAF1, which showed decreased expression in post-mortem XDP brain compared with control tissue. Here, we generated XDP patient and control fibroblasts and induced pluripotent stem cells (iPSCs in order to further probe cellular defects associated with this disease. As initial validation of the model, we compared expression of TAF1 and MTS transcripts in XDP versus control fibroblasts and iPSC-derived neural stem cells (NSCs. Compared with control cells, XDP fibroblasts exhibited decreased expression of TAF1 transcript fragments derived from exons 32-36, a region spanning the SVA insertion site. N-TAF1, which incorporates an alternative exon (exon 34′, was not expressed in fibroblasts, but was detectable in iPSC-differentiated NSCs at levels that were ∼threefold lower in XDP cells than in controls. These results support the previous findings that N-TAF1 expression is impaired in XDP, but additionally indicate that this aberrant transcription might occur in neural cells at relatively early stages of development that precede neurodegeneration.

  13. Cerebral ischemic injury decreases α-synuclein expression in brain tissue and glutamate-exposed HT22 cells.

    Science.gov (United States)

    Koh, Phil-Ok

    2017-09-01

    α-Synuclein is abundantly expressed in neuronal tissue, plays an essential role in the pathogenesis of neurodegenerative disorders, and exerts a neuroprotective effect against oxidative stress. Cerebral ischemia causes severe neurological disorders and neuronal dysfunction. In this study, we examined α-synuclein expression in middle cerebral artery occlusion (MCAO)-induced cerebral ischemic injury and neuronal cells damaged by glutamate treatment. MCAO surgical operation was performed on male Sprague-Dawley rats, and brain samples were isolated 24 hours after MCAO. We confirmed neurological behavior deficit, infarction area, and histopathological changes following MCAO injury. A proteomic approach and Western blot analysis demonstrated a decrease in α-synuclein in the cerebral cortices after MCAO injury. Moreover, glutamate treatment induced neuronal cell death and decreased α-synuclein expression in a hippocampal-derived cell line in a dose-dependent manner. It is known that α-synuclein regulates neuronal survival, and low levels of α-synuclein expression result in cytotoxicity. Thus, these results suggest that cerebral ischemic injury leads to a reduction in α-synuclein and consequently causes serious brain damage.

  14. Decreased proliferative, migrative and neuro-differentiative potential of postnatal rat enteric neural crest-derived cells during culture in vitro

    International Nuclear Information System (INIS)

    Yu, Hui; Pan, Wei-Kang; Zheng, Bai-Jun; Wang, Huai-Jie; Chen, Xin-Lin; Liu, Yong; Gao, Ya

    2016-01-01

    A growing body of evidence supports the potential use of enteric neural crest-derived cells (ENCCs) as a cell replacement therapy for Hirschsprung's disease. Based on previous observations of robust propagation of primary ENCCs, as opposed to their progeny, it is suggested that their therapeutic potential after in vitro expansion may be restricted. We therefore examined the growth and differentiation activities and phenotypic characteristics of continuous ENCC cultures. ENCCs were isolated from the intestines of postnatal rats and were identified using an immunocytochemical approach. During continuous ENCC culture expansion, proliferation, migration, apoptosis, and differentiation potentials were monitored. The Cell Counting Kit-8 was used for assessment of ENCC vitality, Transwell inserts for cell migration, immunocytochemistry for cell counts and identification, and flow cytometry for apoptosis. Over six continuous generations, ENCC proliferation potency was reduced and with prolonged culture, the ratio of migratory ENCCs was decreased. The percentage of apoptosis showed an upward trend with prolonged intragenerational culture, but showed a downward trend with prolonged culture of combined generations. Furthermore, the percentage of peripherin"+ cells decreased whilst the percentage of GFAP"+ cells increased with age. The results demonstrated that alterations in ENCC growth characteristics occur with increased culture time, which may partially account for the poor results of proposed cell therapies. - Highlights: • Differences were identified between primary and daughter ENCCs. • Daughter ENCCs had reduced proliferation, migration and differentiation. • Daughter ENCCs also had increased apoptosis. • These altered characteristics warrant further investigation.

  15. Decreased proliferative, migrative and neuro-differentiative potential of postnatal rat enteric neural crest-derived cells during culture in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Yu, Hui [Department of Pediatric Surgery, the Second Affiliated Hospital, Xi’an Jiaotong University, No 157, Xi Wu Road, Xi’an 710004, Shaanxi (China); Institute of Neurobiology, Environment and Genes Related to Diseases Key Laboratory of Chinese Ministry of Education, Xi’an Jiaotong University, No 96, Yan Ta Xi Road, Xi’an 710061, Shaanxi (China); Pan, Wei-Kang; Zheng, Bai-Jun; Wang, Huai-Jie [Department of Pediatric Surgery, the Second Affiliated Hospital, Xi’an Jiaotong University, No 157, Xi Wu Road, Xi’an 710004, Shaanxi (China); Chen, Xin-Lin; Liu, Yong [Institute of Neurobiology, Environment and Genes Related to Diseases Key Laboratory of Chinese Ministry of Education, Xi’an Jiaotong University, No 96, Yan Ta Xi Road, Xi’an 710061, Shaanxi (China); Gao, Ya, E-mail: ygao@mail.xjtu.edu.cn [Department of Pediatric Surgery, the Second Affiliated Hospital, Xi’an Jiaotong University, No 157, Xi Wu Road, Xi’an 710004, Shaanxi (China)

    2016-05-01

    A growing body of evidence supports the potential use of enteric neural crest-derived cells (ENCCs) as a cell replacement therapy for Hirschsprung's disease. Based on previous observations of robust propagation of primary ENCCs, as opposed to their progeny, it is suggested that their therapeutic potential after in vitro expansion may be restricted. We therefore examined the growth and differentiation activities and phenotypic characteristics of continuous ENCC cultures. ENCCs were isolated from the intestines of postnatal rats and were identified using an immunocytochemical approach. During continuous ENCC culture expansion, proliferation, migration, apoptosis, and differentiation potentials were monitored. The Cell Counting Kit-8 was used for assessment of ENCC vitality, Transwell inserts for cell migration, immunocytochemistry for cell counts and identification, and flow cytometry for apoptosis. Over six continuous generations, ENCC proliferation potency was reduced and with prolonged culture, the ratio of migratory ENCCs was decreased. The percentage of apoptosis showed an upward trend with prolonged intragenerational culture, but showed a downward trend with prolonged culture of combined generations. Furthermore, the percentage of peripherin{sup +} cells decreased whilst the percentage of GFAP{sup +} cells increased with age. The results demonstrated that alterations in ENCC growth characteristics occur with increased culture time, which may partially account for the poor results of proposed cell therapies. - Highlights: • Differences were identified between primary and daughter ENCCs. • Daughter ENCCs had reduced proliferation, migration and differentiation. • Daughter ENCCs also had increased apoptosis. • These altered characteristics warrant further investigation.

  16. Deferoxamine Compensates for Decreases in B Cell Counts and Reduces Mortality in Enterovirus 71-Infected Mice

    OpenAIRE

    Yang, Yajun; Ma, Jing; Xiu, Jinghui; Bai, Lin; Guan, Feifei; Zhang, Li; Liu, Jiangning; Zhang, Lianfeng

    2014-01-01

    Enterovirus 71 is one of the major causative agents of hand, foot and mouth disease in children under six years of age. No vaccine or antiviral therapy is currently available. In this work, we found that the number of B cells was reduced in enterovirus 71-infected mice. Deferoxamine, a marine microbial natural product, compensated for the decreased levels of B cells caused by enterovirus 71 infection. The neutralizing antibody titer was also improved after deferoxamine treatment. Furthermore...

  17. Deferoxamine Compensates for Decreases in B Cell Counts and Reduces Mortality in Enterovirus 71-Infected Mice

    Directory of Open Access Journals (Sweden)

    Yajun Yang

    2014-07-01

    Full Text Available Enterovirus 71 is one of the major causative agents of hand, foot and mouth disease in children under six years of age. No vaccine or antiviral therapy is currently available. In this work, we found that the number of B cells was reduced in enterovirus 71-infected mice. Deferoxamine, a marine microbial natural product, compensated for the decreased levels of B cells caused by enterovirus 71 infection. The neutralizing antibody titer was also improved after deferoxamine treatment. Furthermore, deferoxamine relieved symptoms and reduced mortality and muscle damage caused by enterovirus 71 infection. This work suggested that deferoxamine has the potential for further development as a B cell-immunomodulator against enterovirus 71.

  18. Metformin inhibits cell cycle progression of B-cell chronic lymphocytic leukemia cells.

    Science.gov (United States)

    Bruno, Silvia; Ledda, Bernardetta; Tenca, Claudya; Ravera, Silvia; Orengo, Anna Maria; Mazzarello, Andrea Nicola; Pesenti, Elisa; Casciaro, Salvatore; Racchi, Omar; Ghiotto, Fabio; Marini, Cecilia; Sambuceti, Gianmario; DeCensi, Andrea; Fais, Franco

    2015-09-08

    B-cell chronic lymphocytic leukemia (CLL) was believed to result from clonal accumulation of resting apoptosis-resistant malignant B lymphocytes. However, it became increasingly clear that CLL cells undergo, during their life, iterative cycles of re-activation and subsequent clonal expansion. Drugs interfering with CLL cell cycle entry would be greatly beneficial in the treatment of this disease. 1, 1-Dimethylbiguanide hydrochloride (metformin), the most widely prescribed oral hypoglycemic agent, inexpensive and well tolerated, has recently received increased attention for its potential antitumor activity. We wondered whether metformin has apoptotic and anti-proliferative activity on leukemic cells derived from CLL patients. Metformin was administered in vitro either to quiescent cells or during CLL cell activation stimuli, provided by classical co-culturing with CD40L-expressing fibroblasts. At doses that were totally ineffective on normal lymphocytes, metformin induced apoptosis of quiescent CLL cells and inhibition of cell cycle entry when CLL were stimulated by CD40-CD40L ligation. This cytostatic effect was accompanied by decreased expression of survival- and proliferation-associated proteins, inhibition of signaling pathways involved in CLL disease progression and decreased intracellular glucose available for glycolysis. In drug combination experiments, metformin lowered the apoptotic threshold and potentiated the cytotoxic effects of classical and novel antitumor molecules. Our results indicate that, while CLL cells after stimulation are in the process of building their full survival and cycling armamentarium, the presence of metformin affects this process.

  19. IL-6 Inhibition With MEDI5117 Decreases The Fraction of Head and Neck Cancer Stem Cells and Prevents Tumor Recurrence

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    Kelsey A. Finkel

    2016-05-01

    Full Text Available Head and neck squamous cell carcinomas (HNSCC exhibit a small population of uniquely tumorigenic cancer stem cells (CSC endowed with self-renewal and multipotency. We have recently shown that IL-6 enhances the survival and tumorigenic potential of head and neck cancer stem cells (i.e. ALDHhighCD44high cells. Here, we characterized the effect of therapeutic inhibition of IL-6 with a novel humanized anti-IL-6 antibody (MEDI5117 using three low-passage patient-derived xenograft (PDX models of HNSCC. We observed that single agent MEDI5117 inhibited the growth of PDX-SCC-M1 tumors (P < .05. This PDX model was generated from a previously untreated HNSCC. In contrast, MEDI5117 was not effective at reducing overall tumor volume for PDX models representing resistant disease (PDX-SCC-M0, PDX-SCC-M11. Low dose MEDI5117 (3 mg/kg consistently decreased the fraction of cancer stem cells in PDX models of HNSCC when compared to IgG-treated controls, as follows: PDX-SCC-M0 (P < .001, PDX-SCC-M1 (P < .001, PDX-SCC-M11 (P = .04. Interestingly, high dose MEDI5117 (30 mg/kg decreased the CSC fraction in the PDX-SCC-M11 model (P = .002, but not in PDX-SCC-M0 and PDX-SCC-M1. MEDI5117 mediated a dose-dependent decrease in the number of orospheres generated by ALDHhighCD44high cells cultured in ultra-low attachment plates (P < .05, supporting an inhibitory effect on head and neck cancer stem cells. Notably, single agent MEDI5117 reduced the overall recurrence rate of PDX-SCC-M0, a PDX generated from the local recurrence of human HNSCC. Collectively, these data demonstrate that therapeutic inhibition of IL-6 with low-dose MEDI5117 decreases the fraction of cancer stem cells, and that adjuvant MEDI5117 inhibits recurrence in preclinical models of HNSCC.

  20. Differentiation of Human Induced Pluripotent or Embryonic Stem Cells Decreases the DNA Damage Repair by Homologous Recombination

    Directory of Open Access Journals (Sweden)

    Kalpana Mujoo

    2017-11-01

    Full Text Available The nitric oxide (NO-cyclic GMP pathway contributes to human stem cell differentiation, but NO free radical production can also damage DNA, necessitating a robust DNA damage response (DDR to ensure cell survival. How the DDR is affected by differentiation is unclear. Differentiation of stem cells, either inducible pluripotent or embryonic derived, increased residual DNA damage as determined by γ-H2AX and 53BP1 foci, with increased S-phase-specific chromosomal aberration after exposure to DNA-damaging agents, suggesting reduced homologous recombination (HR repair as supported by the observation of decreased HR-related repair factor foci formation (RAD51 and BRCA1. Differentiated cells also had relatively increased fork stalling and R-loop formation after DNA replication stress. Treatment with NO donor (NOC-18, which causes stem cell differentiation has no effect on double-strand break (DSB repair by non-homologous end-joining but reduced DSB repair by HR. Present studies suggest that DNA repair by HR is impaired in differentiated cells.

  1. Down-regulation of human endogenous retrovirus type K (HERV-K) viral env RNA in pancreatic cancer cells decreases cell proliferation and tumor growth

    Science.gov (United States)

    Li, Ming; Radvanyi, Laszlo; Yin, Bingnan; Li, Jia; Chivukula, Raghavender; Lin, Kevin; Lu, Yue; Shen, JianJun; Chang, David Z.; Li, Donghui; Johanning, Gary L.; Wang-Johanning, Feng

    2017-01-01

    Purpose We investigated the role of the human endogenous retrovirus type K (HERV-K) envelope (env) gene in pancreatic cancer (PC). Experimental Design shRNA was employed to knockdown (KD) the expression of HERV-K in PC cells. Results HERV-K env expression was detected in seven PC cell lines and in 80% of PC patient biopsies, but not in two normal pancreatic cell lines or uninvolved normal tissues. A new HERV-K splice variant was discovered in several PC cell lines. RT activity and virus-like particles were observed in culture media supernatant obtained from Panc-1 and Panc-2 cells. HERV-K viral RNA levels and anti-HERV-K antibody titers were significantly higher in PC patient sera (N=106) than in normal donor sera (N=40). Importantly, the in vitro and in vivo growth rates of three PC cell lines were significantly reduced after HERV-K KD by shRNA targeting HERV-K env, and there was reduced metastasis to lung after treatment. RNA-seq results revealed changes in gene expression after HERV-K env KD, including RAS and TP53. Furthermore, downregulation of HERV-K Env protein expression by shRNA also resulted in decreased expression of RAS, p-ERK, p-RSK, and p-AKT in several PC cells or tumors. Conclusion These results demonstrate that HERV-K influences signal transduction via the RAS-ERK-RSK pathway in PC. Our data highlight the potentially important role of HERV-K in tumorigenesis and progression of PC, and indicate that HERV-K viral proteins may be attractive biomarkers and/or tumor-associated antigens, as well as potentially useful targets for detection, diagnosis and immunotherapy of PC. PMID:28679769

  2. Cell-cell interactions mediate cytoskeleton organization and collective endothelial cell chemotaxis.

    Science.gov (United States)

    Shamloo, Amir

    2014-09-01

    This study investigates the role of cell-cell and cell-ligand interactions in cytoskeleton organization of endothelial cells (ECs) and their directional migration within a microfluidic device. The migration of ECs in response to a biochemical factor was studied. Mathematical analysis of the cell migration pathways and cellular cytoskeleton revealed that directional migration, migration persistence length, migration speed, and cytoskeletal stress fiber alignment can be mediated by the level of cell contacts as well as the presence or absence of a biochemical polarizing factor. It was shown that in the presence of a biochemical polarizing factor, higher cell density and more frequent cell contacts has a reinforcing effect on collective cell chemotaxis. In contrast, in the absence of a polarizing factor, high cell density can decrease or suppress the ability of the cells to migrate. Also, the correlation of actin stress fiber organization and alignment with directional migration of ECs was investigated. It was shown that in the presence of a biochemical polarizing factor, stress fibers within the cytoskeleton of ECs can be significantly aligned parallel to the gradient direction when the cells have higher level of contacts. The results also show that the organization and alignment of actin stress fibers is mediated by cell adhesion junctions during collective cell migration and introduce cell-cell interactions as a key factor during collective cell chemotaxis. © 2014 Wiley Periodicals, Inc.

  3. Colon Cancer Chemoprevention by Sage Tea Drinking: Decreased DNA Damage and Cell Proliferation.

    Science.gov (United States)

    Pedro, Dalila F N; Ramos, Alice A; Lima, Cristovao F; Baltazar, Fatima; Pereira-Wilson, Cristina

    2016-02-01

    Salvia officinalis and some of its isolated compounds have been found to be preventive of DNA damage and increased proliferation in vitro in colon cells. In the present study, we used the azoxymethane model to test effects of S. officinalis on colon cancer prevention in vivo. The results showed that sage treatment reduced the number of ACF formed only if administered before azoxymethane injection, demonstrating that sage tea drinking has a chemopreventive effect on colorectal cancer. A decrease in the proliferation marker Ki67 and in H2 O2 -induced and azoxymethane-induced DNA damage to colonocytes and lymphocytes were found with sage treatment. This confirms in vivo the chemopreventive effects of S. officinalis. Taken together, our results show that sage treatment prevented initiation phases of colon carcinogenesis, an effect due, at least in part, to DNA protection, and reduced proliferation rates of colon epithelial cell that prevent mutations and their fixation through cell replication. These chemopreventive effects of S. officinalis on colon cancer add to the many health benefits attributed to sage and encourage its consumption. Copyright © 2015 John Wiley & Sons, Ltd.

  4. Reduction in cardiolipin decreases mitochondrial spare respiratory capacity and increases glucose transport into and across human brain cerebral microvascular endothelial cells.

    Science.gov (United States)

    Nguyen, Hieu M; Mejia, Edgard M; Chang, Wenguang; Wang, Ying; Watson, Emily; On, Ngoc; Miller, Donald W; Hatch, Grant M

    2016-10-01

    Microvessel endothelial cells form part of the blood-brain barrier, a restrictively permeable interface that allows transport of only specific compounds into the brain. Cardiolipin is a mitochondrial phospholipid required for function of the electron transport chain and ATP generation. We examined the role of cardiolipin in maintaining mitochondrial function necessary to support barrier properties of brain microvessel endothelial cells. Knockdown of the terminal enzyme of cardiolipin synthesis, cardiolipin synthase, in hCMEC/D3 cells resulted in decreased cellular cardiolipin levels compared to controls. The reduction in cardiolipin resulted in decreased mitochondrial spare respiratory capacity, increased pyruvate kinase activity, and increased 2-deoxy-[(3) H]glucose uptake and glucose transporter-1 expression and localization to membranes in hCMEC/D3 cells compared to controls. The mechanism for the increase in glucose uptake was an increase in adenosine-5'-monophosphate kinase and protein kinase B activity and decreased glycogen synthase kinase 3 beta activity. Knockdown of cardiolipin synthase did not affect permeability of fluorescent dextran across confluent hCMEC/D3 monolayers grown on Transwell(®) inserts. In contrast, knockdown of cardiolipin synthase resulted in an increase in 2-deoxy-[(3) H]glucose transport across these monolayers compared to controls. The data indicate that in hCMEC/D3 cells, spare respiratory capacity is dependent on cardiolipin. In addition, reduction in cardiolipin in these cells alters their cellular energy status and this results in increased glucose transport into and across hCMEC/D3 monolayers. Microvessel endothelial cells form part of the blood-brain barrier, a restrictively permeable interface that allows transport of only specific compounds into the brain. In human adult brain endothelial cell hCMEC/D3 monolayers cultured on Transwell(®) plates, knockdown of cardiolipin synthase results in decrease in mitochondrial

  5. Decreased Cytotoxicity of Peripheral and Peritoneal Natural Killer Cell in Endometriosis.

    Science.gov (United States)

    Jeung, InCheul; Cheon, Keunyoung; Kim, Mee-Ran

    2016-01-01

    Endometriosis causes significant chronic pelvic pain, dysmenorrhea, and infertility and affects 10% of all women. In endometriosis, ectopic endometrium surviving after retrograde menstruation exhibits an abnormal immune response characterized by increased levels of activated macrophages and inflammatory cytokines. Particularly, dysfunctional natural killer (NK) cells play an important role in the pathogenesis of the disease by either facilitating or inhibiting the survival, implantation, and proliferation of endometrial cells. NK cells in the peritoneum and peritoneal fluid exhibit reduced levels of cytotoxicity in women with endometriosis. Several cytokines and inhibitory factors in the serum and peritoneal fluid also dysregulate NK cell cytotoxicity. Additionally, increased numbers of immature peripheral NK cells and induction of NK cell apoptosis are evident in the peritoneal fluid of women with endometriosis. The high rate of endometriosis recurrence after pharmaceutical or surgical treatment, which is associated with dysfunctional NK cells, indicates that new immunomodulatory management strategies are required. A good understanding of immune dysfunction would enable improvement of current treatments for endometriosis.

  6. Decreased Cytotoxicity of Peripheral and Peritoneal Natural Killer Cell in Endometriosis

    Directory of Open Access Journals (Sweden)

    InCheul Jeung

    2016-01-01

    Full Text Available Endometriosis causes significant chronic pelvic pain, dysmenorrhea, and infertility and affects 10% of all women. In endometriosis, ectopic endometrium surviving after retrograde menstruation exhibits an abnormal immune response characterized by increased levels of activated macrophages and inflammatory cytokines. Particularly, dysfunctional natural killer (NK cells play an important role in the pathogenesis of the disease by either facilitating or inhibiting the survival, implantation, and proliferation of endometrial cells. NK cells in the peritoneum and peritoneal fluid exhibit reduced levels of cytotoxicity in women with endometriosis. Several cytokines and inhibitory factors in the serum and peritoneal fluid also dysregulate NK cell cytotoxicity. Additionally, increased numbers of immature peripheral NK cells and induction of NK cell apoptosis are evident in the peritoneal fluid of women with endometriosis. The high rate of endometriosis recurrence after pharmaceutical or surgical treatment, which is associated with dysfunctional NK cells, indicates that new immunomodulatory management strategies are required. A good understanding of immune dysfunction would enable improvement of current treatments for endometriosis.

  7. [Thyroid C cells are decreased in experimental CDH].

    Science.gov (United States)

    Martínez, L; De Ceano-Vivas, M; González-Reyes, S; Fernández-Dumont, V; Calonge, W M; Ruiz, E; Rodríguez, J I; Tovar, J A

    2006-04-01

    Experimental CDH is often associated with malformations of neural crest origin. Several of these features are present in human CDH and therefore likely similar pathogenic mechanisms should be explored. The aim of the present study is to examine whether thyroid C-cells, another neural crest derivative, are abnormal in this rat model. Pregnant rats were exposed either to 100 mg of 2-4-dichlorophenyl-p-nitrophenyl ether (nitrofén) or vehicle (controls) on 9.5 day of gestation. Fetuses were recovered on day 21st and the thyroids of those with CDH (68%) were immuno-histochemically stained with anti-calcitonin antibody. The number of positively stained cells per high power field were counted using a computer-assisted image analysis method in at least 5 sections per thyroid. The distribution of the cells within the gland was assessed as well. Comparisons between CDH and control rats were made by non-parametric tests with a significance threshold of pcraneo-facial malformations in some babies with CDH strongly support further research in this field.

  8. SIRT1 overexpression decreases cisplatin-induced acetylation of NF-{kappa}B p65 subunit and cytotoxicity in renal proximal tubule cells

    Energy Technology Data Exchange (ETDEWEB)

    Jung, Yu Jin; Lee, Jung Eun; Lee, Ae Sin [Department of Internal Medicine, Chonbuk National University Medical School, Jeonju (Korea, Republic of); Kang, Kyung Pyo; Lee, Sik; Park, Sung Kwang [Department of Internal Medicine, Chonbuk National University Medical School, Jeonju (Korea, Republic of); Institute for Medical Sciences, Chonbuk National University Medical School, Jeonju (Korea, Republic of); Lee, Sang Yong [Department of Diagnostic Radiology, Chonbuk National University Medical School, Jeonju (Korea, Republic of); Institute for Medical Sciences, Chonbuk National University Medical School, Jeonju (Korea, Republic of); Han, Myung Kwan [Department of Microbiology, Institute for Medical Sciences, Chonbuk National University Medical School, Jeonju (Korea, Republic of); Kim, Duk Hoon [Division of Forensic Medicine, National Forensic Service, Seoul (Korea, Republic of); Kim, Won, E-mail: kwon@jbnu.ac.kr [Department of Internal Medicine, Chonbuk National University Medical School, Jeonju (Korea, Republic of); Institute for Medical Sciences, Chonbuk National University Medical School, Jeonju (Korea, Republic of)

    2012-03-09

    Highlights: Black-Right-Pointing-Pointer Cisplatin increases acetylation of NF-{kappa}B p65 subunit in HK2 cells. Black-Right-Pointing-Pointer SIRT1 overexpression decreases cisplatin-induced p65 acetylation and -cytotoxicity. Black-Right-Pointing-Pointer Resveratrol decreased cisplatin-induced cell viability through deacetylation of p65. -- Abstract: As the increased acetylation of p65 is linked to nuclear factor-{kappa}B (NF-{kappa}B) activation, the regulation of p65 acetylation can be a potential target for the treatment of inflammatory injury. Cisplatin-induced nephrotoxicity is an important issue in chemotherapy of cancer patients. SIRT1, nicotinamide adenine dinucleotide (NAD{sup +})-dependent protein deacetylase, has been implicated in a variety of cellular processes such as inflammatory injury and the control of multidrug resistance in cancer. However, there is no report on the effect of SIRT1 overexpression on cisplatin-induced acetylation of p65 subunit of NF-{kappa}B and cell injury. To investigate the effect of SIRT1 in on cisplatin-induced acetylation of p65 subunit of NF-{kappa}B and cell injury, HK2 cells were exposed with SIRT1 overexpression, LacZ adenovirus or dominant negative adenovirus after treatment with cisplatin. While protein expression of SIRT1 was decreased by cisplatin treatment compared with control buffer treatment, acetylation of NF-{kappa}B p65 subunit was significantly increased after treatment with cisplatin. Overexpression of SIRT1 ameliorated the increased acetylation of p65 of NF-{kappa}B during cisplatin treatment and cisplatin-induced cytotoxicity. Further, treatment of cisplatin-treated HK2 cells with resveratrol, a SIRT1 activator, also decreased acetylation of NF-{kappa}B p65 subunit and cisplatin-induced increase of the cell viability in HK2 cells. Our findings suggests that the regulation of acetylation of p65 of NF-{kappa}B through SIRT1 can be a possible target to attenuate cisplatin-induced renal cell damage.

  9. Number of Langerhans cells is decreased in premalignant keratosis and skin cancers.

    Science.gov (United States)

    Shevchuk, Z; Filip, A; Shevchuk, V; Kashuba, E

    2014-03-01

    It was shown earlier that a number of CD207 positive Langerhans cells was lower in basal cell carcinomas than in the normal epidermis. Moreover, benign skin lesions presented a higher number of Langerhans cells when they were compared to malignant tumors. To count Langerhans cells, assessing expression levels of CD1A and CD207 markers in actinic keratosis, basal and squamous cell carcinomas, compared with the normal skin. Comparison of Langerhans cells might give a valuable prognostic marker for skin cancer. Immunohistochemistry and methods of statistics were used. Expression of CD1A and CD207 markers was assessed in tumor samples of actinic keratosis, cutaneous basal and squamous cell carcinomas, in comparison with the normal skin. In each cohort there were 40 patients (and 11 healthy individuals). We have shown that the number of Langerhans cells is considerably lower in cutaneous basal and squamous cell carcinomas, compared with their number in the normal skin (p keratosis, basal and squamous cell carcinoma. This may suggest an alteration of Langerhans cells phenotype in skin neoplastic diseases, making the number of Langerhans cells a valuable prognostic factor for skin tumors.

  10. Cystatin C deficiency suppresses tumor growth in a breast cancer model through decreased proliferation of tumor cells.

    Science.gov (United States)

    Završnik, Janja; Butinar, Miha; Prebanda, Mojca Trstenjak; Krajnc, Aleksander; Vidmar, Robert; Fonović, Marko; Grubb, Anders; Turk, Vito; Turk, Boris; Vasiljeva, Olga

    2017-09-26

    Cysteine cathepsins are proteases that, in addition to their important physiological functions, have been associated with multiple pathologies, including cancer. Cystatin C (CstC) is a major endogenous inhibitor that regulates the extracellular activity of cysteine cathepsins. We investigated the role of cystatin C in mammary cancer using CstC knockout mice and a mouse model of breast cancer induced by expression of the polyoma middle T oncoprotein (PyMT) in the mammary epithelium. We showed that the ablation of CstC reduced the rate of mammary tumor growth. Notably, a decrease in the proliferation of CstC knockout PyMT tumor cells was demonstrated ex vivo and in vitro , indicating a role for this protease inhibitor in signaling pathways that control cell proliferation. An increase in phosphorylated p-38 was observed in CstC knockout tumors, suggesting a novel function for cystatin C in cancer development, independent of the TGF-β pathway. Moreover, proteomic analysis of the CstC wild-type and knockout PyMT primary cell secretomes revealed a decrease in the levels of 14-3-3 proteins in the secretome of knock-out cells, suggesting a novel link between cysteine cathepsins, cystatin C and 14-3-3 proteins in tumorigenesis, calling for further investigations.

  11. Ion channels involved in cell volume regulation: effects on migration, proliferation, and programmed cell death in non adherent EAT cells and adherent ELA cells.

    Science.gov (United States)

    Hoffmann, Else Kay

    2011-01-01

    This mini review outlines studies of cell volume regulation in two closely related mammalian cell lines: nonadherent Ehrlich ascites tumour cells (EATC) and adherent Ehrlich Lettre ascites (ELA) cells. Focus is on the regulatory volume decrease (RVD) that occurs after cell swelling, the volume regulatory ion channels involved, and the mechanisms (cellular signalling pathways) that regulate these channels. Finally, I shall also briefly review current investigations in these two cell lines that focuses on how changes in cell volume can regulate cell functions such as cell migration, proliferation, and programmed cell death. Copyright © 2011 S. Karger AG, Basel.

  12. Inhibition of myeloperoxidase decreases vascular oxidative stress and increases vasodilatation in sickle cell disease mice.

    Science.gov (United States)

    Zhang, Hao; Xu, Hao; Weihrauch, Dorothee; Jones, Deron W; Jing, Xigang; Shi, Yang; Gourlay, David; Oldham, Keith T; Hillery, Cheryl A; Pritchard, Kirkwood A

    2013-11-01

    Activated leukocytes and polymorphonuclear neutrophils (PMN) release myeloperoxidase (MPO), which binds to endothelial cells (EC), is translocated, and generates oxidants that scavenge nitric oxide (NO) and impair EC function. To determine whether MPO impairs EC function in sickle cell disease (SCD), control (AA) and SCD mice were treated with N-acetyl-lysyltyrosylcysteine-amide (KYC). SCD humans and mice have high plasma MPO and soluble L-selectin (sL-selectin). KYC had no effect on MPO but decreased plasma sL-selectin and malondialdehyde in SCD mice. MPO and 3-chlorotyrosine (3-ClTyr) were increased in SCD aortas. KYC decreased MPO and 3-ClTyr in SCD aortas to the levels in AA aortas. Vasodilatation in SCD mice was impaired. KYC increased vasodilatation in SCD mice more than 2-fold, to ∼60% of levels in AA mice. KYC inhibited MPO-dependent 3-ClTyr formation in EC proteins. SCD mice had high plasma alanine transaminase (ALT), which tended to decrease in KYC-treated SCD mice (P = 0.07). KYC increased MPO and XO/XDH and decreased 3-ClTyr and 3-nitrotyrosine (3-NO₂Tyr) in SCD livers. These data support the hypothesis that SCD increases release of MPO, which generates oxidants that impair EC function and injure livers. Inhibiting MPO is an effective strategy for decreasing oxidative stress and liver injury and restoring EC function in SCD.

  13. Boron neutron capture therapy induces cell cycle arrest and cell apoptosis of glioma stem/progenitor cells in vitro

    International Nuclear Information System (INIS)

    Sun, Ting; Zhang, Zizhu; Li, Bin; Chen, Guilin; Xie, Xueshun; Wei, Yongxin; Wu, Jie; Zhou, Youxin; Du, Ziwei

    2013-01-01

    Glioma stem cells in the quiescent state are resistant to clinical radiation therapy. An almost inevitable glioma recurrence is due to the persistence of these cells. The high linear energy transfer associated with boron neutron capture therapy (BNCT) could kill quiescent and proliferative cells. The present study aimed to evaluate the effects of BNCT on glioma stem/progenitor cells in vitro. The damage induced by BNCT was assessed using cell cycle progression, apoptotic cell ratio and apoptosis-associated proteins expression. The surviving fraction and cell viability of glioma stem/progenitor cells were decreased compared with differentiated glioma cells using the same boronophenylalanine pretreatment and the same dose of neutron flux. BNCT induced cell cycle arrest in the G2/M phase and cell apoptosis via the mitochondrial pathway, with changes in the expression of associated proteins. Glioma stem/progenitor cells, which are resistant to current clinical radiotherapy, could be effectively killed by BNCT in vitro via cell cycle arrest and apoptosis using a prolonged neutron irradiation, although radiosensitivity of glioma stem/progenitor cells was decreased compared with differentiated glioma cells when using the same dose of thermal neutron exposure and boronophenylalanine pretreatment. Thus, BNCT could offer an appreciable therapeutic advantage to prevent tumor recurrence, and may become a promising treatment in recurrent glioma

  14. Knockdown of αII spectrin in normal human cells by siRNA leads to chromosomal instability and decreased DNA interstrand cross-link repair

    International Nuclear Information System (INIS)

    McMahon, Laura W.; Zhang Pan; Sridharan, Deepa M.; Lefferts, Joel A.; Lambert, Muriel W.

    2009-01-01

    Nonerythroid α-spectrin (αIISp) is a structural protein involved in repair of DNA interstrand cross-links and is deficient in cells from patients with Fanconi anemia (FA), which are defective in ability to repair cross-links. In order to further demonstrate the importance of the role that αIISp plays in normal human cells and in the repair defect in FA, αIISp was knocked down in normal cells using siRNA. Depletion of αIISp in normal cells by siRNA resulted in chromosomal instability and cellular hypersensitivity to DNA interstrand cross-linking agents. An increased number of chromosomal aberrations were observed and, following treatment with a DNA interstrand cross-linking agent, mitomycin C, cells showed decreased cell growth and survival and decreased formation of damage-induced αIISp and XPF nuclear foci. Thus depletion of αIISp in normal cells leads to a number of defects observed in FA cells, such as chromosome instability and a deficiency in cross-link repair.

  15. The epigenetic agents suberoylanilide hydroxamic acid and 5‑AZA‑2' deoxycytidine decrease cell proliferation, induce cell death and delay the growth of MiaPaCa2 pancreatic cancer cells in vivo.

    Science.gov (United States)

    Susanto, Johana M; Colvin, Emily K; Pinese, Mark; Chang, David K; Pajic, Marina; Mawson, Amanda; Caldon, C Elizabeth; Musgrove, Elizabeth A; Henshall, Susan M; Sutherland, Robert L; Biankin, Andrew V; Scarlett, Christopher J

    2015-05-01

    Despite incremental advances in the diagnosis and treatment for pancreatic cancer (PC), the 5‑year survival rate remains <5%. Novel therapies to increase survival and quality of life for PC patients are desperately needed. Epigenetic thera-peutic agents such as histone deacetylase inhibitors (HDACi) and DNA methyltransferase inhibitors (DNMTi) have demonstrated therapeutic benefits in human cancer. We assessed the efficacy of these epigenetic therapeutic agents as potential therapies for PC using in vitro and in vivo models. Treatment with HDACi [suberoylanilide hydroxamic acid (SAHA)] and DNMTi [5‑AZA‑2' deoxycytidine (5‑AZA‑dc)] decreased cell proliferation in MiaPaCa2 cells, and SAHA treatment, with or without 5‑AZA‑dc, resulted in higher cell death and lower DNA synthesis compared to 5‑AZA‑dc alone and controls (DMSO). Further, combination treatment with SAHA and 5‑AZA‑dc significantly increased expression of p21WAF1, leading to G1 arrest. Treatment with epigenetic agents delayed tumour growth in vivo, but did not decrease growth of established pancreatic tumours. In conclusion, these data demonstrate a potential role for epigenetic modifier drugs for the management of PC, specifically in the chemoprevention of PC, in combination with other chemotherapeutic agents.

  16. Decreased survival of prostate cancer cells in vitro by combined treatment of heat and an antioxidant inhibitor diethyldithiocarbamate (DDC).

    Science.gov (United States)

    Moriyama-Gonda, Nobuko; Igawa, Mikio; Shiina, Hiroaki; Urakami, Shinji; Terashima, Masaharu

    2003-11-01

    The aim of this study was to examine a modulation of thermotolerance by treatment with combination of heat and the antioxidant inhibitor diethyldithiocarbamate (DDC) of the PC-3 prostate cancer cells. To determine thermotolerance, cells were heated once or twice. Two 1 h exposures at 43 degrees C, with a recovery period in between, revealed better survival/recovery of cells after the second exposure than after the first (fig. 1A + 1B). Additional experiments were performed, heating cells twice (fig. 1B + 1C). First, cells were heated at 43 degrees C for 1 h and, after various recovery times (intervals) at 37 degree C, subsequently reheated at 44 degrees C for 1 h. To ensure effective cell killing, efficiency of the combined treatments of 1 mM DDC and heating at 43 or 44 degrees C for 1 h was estimated by measuring cell survival, reactive oxygen species (ROS) generation, superoxide dismutase (SOD) activity and heat shock protein 70 (hsp 70) expression. To obtain a more effective method for subsequent heat exposure, cells were heated twice after a 24 h interval in the presence or absence of 1 mM DDC. ROS generation and SOD activity immediately increased correlating with duration of heating, but their levels gently decreased with time after discontinuation of heating. On the other hand, hsp 70 levels slowly increased, also correlating with duration of heating but continued to increase with time after discontinuation of heating for a certain period. DDC administration coupled with heating at 43 or 44 degrees C significantly decreased cell survival compared to heating alone (p DDC as compared to heat alone at 43 and 44 degrees C (p DDC could have potential benefits in the treatment of prostate cancer.

  17. Methyl gallate from Acer barbinerve decreases melanin synthesis in Mel-Ab cells.

    Science.gov (United States)

    Kim, In Wook; jeong, Hyo-Soon; Kim, Jin Kyu; Lee, Jin-Koo; Kim, Hak Rim; Yun, Hye-Young; Baek, Kwang Jin; Kwon, Nyoun Soo; Park, Kyoung-Chan; Kim, Dong-Seok

    2015-01-01

    Methyl gallate (MG) was isolated from the bark of Acer barbinerve, which has traditionally been used in Oriental medicine. In the present study, we examined the effects of MG on melanin synthesis in Mel-Ab melanocyte cells. MG decreased melanin pigmentation in a concentration-dependent manner, but did not directly inhibit tyrosinase activity. Further analysis showed that MG had no effect on extracellular signal-regulated kinase (ERK) activation, but induced phosphorylation of glycogen synthase kinase (GSK)3β, which is known to increase β-catenin accumulation. Accordingly, the β-catenin level was increased by MG. However, a specific GSK3β inhibitor did not rescue the MG-induced inhibition of melanogenesis. Additionally, MG decreased the protein expression of microphthalmia-associated transcription factor (MITF) and tyrosinase, which regulate melanin synthesis. Based on these results, we conclude that MG inhibits melanogenesis by decreasing the expression of MITF and tyrosinase.

  18. IGF-1 decreases collagen degradation in diabetic NOD mesangial cells: implications for diabetic nephropathy.

    Science.gov (United States)

    Lupia, E; Elliot, S J; Lenz, O; Zheng, F; Hattori, M; Striker, G E; Striker, L J

    1999-08-01

    Nonobese diabetic (NOD) mice develop glomerulosclerosis shortly after the onset of diabetes. We showed that mesangial cells (MCs) from diabetic mice exhibited a stable phenotypic switch, consisting of both increased IGF-1 synthesis and proliferation (Elliot SJ, Striker LJ, Hattori M, Yang CW, He CJ, Peten EP, Striker GE: Mesangial cells from diabetic NOD mice constitutively secrete increased amounts of insulin-like growth factor-I. Endocrinology 133:1783-1788, 1993). Because the extracellular matrix (ECM) accumulation in diabetic glomerulosclerosis may be partly due to decreased degradation, we examined the effect of excess IGF-1 on collagen turnover and the activity of metalloproteinases (MMPs) and tissue inhibitors of metalloproteinase (TIMPs) in diabetic and nondiabetic NOD-MC. Total collagen degradation was reduced by 58 +/- 18% in diabetic NOD-MCs, which correlated with a constitutive decrease in MMP-2 activity and mRNA levels, and nearly undetectable MMP-9 activity and mRNA. TIMP levels were slightly decreased in diabetic NOD-MC. The addition of recombinant IGF-1 to nondiabetic NOD-MC resulted in a decrease in MMP-2 and TIMP activity. Furthermore, treatment of diabetic NOD-MC with a neutralizing antibody against IGF-1 increased the latent form, and restored the active form, of MMP-2. In conclusion, the excessive production of IGF-1 contributes to the altered ECM turnover in diabetic NOD-MC, largely through a reduction of MMP-2 activity. These data suggest that IGF-1 could be a major contributor to the development of diabetic glomerulosclerosis.

  19. Mechanism for the decrease in the FIP1L1-PDGFRalpha protein level in EoL-1 cells by histone deacetylase inhibitors.

    Science.gov (United States)

    Ishihara, Kenji; Kaneko, Motoko; Kitamura, Hajime; Takahashi, Aki; Hong, Jang Ja; Seyama, Toshio; Iida, Koji; Wada, Hiroshi; Hirasawa, Noriyasu; Ohuchi, Kazuo

    2008-01-01

    Acetylation and deacetylation of proteins occur in cells in response to various stimuli, and are reversibly catalyzed by histone acetyltransferase and histone deacetylase (HDAC), respectively. EoL-1 cells have an FIP1L1-PDGFRA fusion gene that causes transformation of eosinophilic precursor cells into leukemia cells. The HDAC inhibitors apicidin and n-butyrate suppress the proliferation of EoL-1 cells and induce differentiation into eosinophils by a decrease in the protein level of FIP1L1-PDGFRalpha without affecting the mRNA level for FIP1L1-PDGFRA. In this study, we analyzed the mechanism by which the protein level of FIP1L1-PDGFRalpha is decreased by apicidin and n-butyrate. EoL-1 cells were incubated in the presence of the HDAC inhibitors apicidin, trichostatin A or n-butyrate. The protein levels of FIP1L1-PDGFRalpha and phosphorylated eIF-2alpha were determined by Western blotting. Actinomycin D and cycloheximide were used to block RNA synthesis and protein synthesis, respectively, in the chasing experiment of the amount of FIP1L1-PDGFRalpha protein. When apicidin- and n-butyrate-treated EoL-1 cells were incubated in the presence of actinomycin D, the decrease in the protein level of FIP1L1-PDGFRalpha was significantly enhanced when compared with controls. In contrast, the protein levels were not changed by cycloheximide among these groups. Apicidin and n-butyrate induced the continuous phosphorylation of eIF-2alpha for up to 8 days. The decrease in the level of FIP1L1-PDGFRalpha protein by continuous inhibition of HDAC may be due to the decrease in the translation rate of FIP1L1-PDGFRA. Copyright 2008 S. Karger AG, Basel.

  20. Docosahexaenoic acid and other fatty acids induce a decrease in pHi in Jurkat T-cells

    OpenAIRE

    Aires, Virginie; Hichami, Aziz; Moutairou, Kabirou; Khan, Naim Akhtar

    2003-01-01

    Docosahexaenoic acid (DHA) induced rapid (t1/2=33 s) and dose-dependent decreases in pHi in BCECF-loaded human (Jurkat) T-cells. Addition of 5-(N,N-dimethyl)-amiloride, an inhibitor of Na+/H+ exchanger, prolonged DHA-induced acidification as a function of time, indicating that the exchanger is implicated in pHi recovery.Other fatty acids like oleic acid, arachidonic acid, eicosapentaenoic acid, but not palmitic acid, also induced a fall in pHi in these cells.To assess the role of calcium in t...

  1. High Glucose-Induced PC12 Cell Death by Increasing Glutamate Production and Decreasing Methyl Group Metabolism

    Directory of Open Access Journals (Sweden)

    Minjiang Chen

    2016-01-01

    Full Text Available Objective. High glucose- (HG- induced neuronal cell death is responsible for the development of diabetic neuropathy. However, the effect of HG on metabolism in neuronal cells is still unclear. Materials and Methods. The neural-crest derived PC12 cells were cultured for 72 h in the HG (75 mM or control (25 mM groups. We used NMR-based metabolomics to examine both intracellular and extracellular metabolic changes in HG-treated PC12 cells. Results. We found that the reduction in intracellular lactate may be due to excreting more lactate into the extracellular medium under HG condition. HG also induced the changes of other energy-related metabolites, such as an increased succinate and creatine phosphate. Our results also reveal that the synthesis of glutamate from the branched-chain amino acids (isoleucine and valine may be enhanced under HG. Increased levels of intracellular alanine, phenylalanine, myoinositol, and choline were observed in HG-treated PC12 cells. In addition, HG-induced decreases in intracellular dimethylamine, dimethylglycine, and 3-methylhistidine may indicate a downregulation of methyl group metabolism. Conclusions. Our metabolomic results suggest that HG-induced neuronal cell death may be attributed to a series of metabolic changes, involving energy metabolism, amino acids metabolism, osmoregulation and membrane metabolism, and methyl group metabolism.

  2. B cells and B cell products-helping to restore cellular immunity?

    OpenAIRE

    Cascalho, Marilia; Platt, Jeffrey L.

    2006-01-01

    T cells that provide vital protection against tumors, viruses and intracellular bacteria are thought to develop independently of B cells. However, recent discoveries suggest that development of T cells depends on B cells. One way B cells promote T cell development is by providing diverse peptides that may promote positive selection of thymocytes. Diverse peptides and B cells help in diversification of the T cell receptor repertoire and may decrease cross-reactivity in the mature T cell compar...

  3. Xanthophylls and alpha-tocopherol decrease UVB-induced lipid peroxidation and stress signaling in human lens epithelial cells.

    Science.gov (United States)

    Chitchumroonchokchai, Chureeporn; Bomser, Joshua A; Glamm, Jayme E; Failla, Mark L

    2004-12-01

    Epidemiological studies suggest that consumption of vegetables rich in the xanthophylls lutein (LUT) and zeaxanthin (ZEA) reduces the risk for developing age-related cataract, a leading cause of vision loss. Although LUT and ZEA are the only dietary carotenoids present in the lens, direct evidence for their photoprotective effect in this organ is not available. The present study examined the effects of xanthophylls and alpha-tocopherol (alpha-TC) on lipid peroxidation and the mitogen-activated stress signaling pathways in human lens epithelial (HLE) cells following ultraviolet B light (UVB) irradiation. When presented with LUT, ZEA, astaxanthin (AST), and alpha-TC as methyl-beta-cyclodextrin complexes, HLE cells accumulated the lipophiles in a concentration- and time-dependent manner with uptake of LUT exceeding that of ZEA and AST. Pretreatment of cultures with either 2 micromol/L xanthophyll or 10 micromol/L alpha-TC for 4 h before exposure to 300 J/m(2) UVB radiation decreased lipid peroxidation by 47-57% compared with UVB-treated control HLE cells. Pretreatment with the xanthophylls and alpha-TC also inhibited UVB-induced activation of c-JUN NH(2)-terminal kinase (JNK) and p38 by 50-60 and 25-32%, respectively. There was substantial inhibition of UVB-induced JNK and p38 activation for cells containing xanthophylls/mg, respectively, whereas >2.3 nmol alpha-TC/mg protein was required to significantly decrease UVB-induced stress signaling. These data suggest that xanthophylls are more potent than alpha-TC for protecting human lens epithelial cells against UVB insult.

  4. Radiation Exposure Decreases the Quantity and Quality of Cardiac Stem Cells in Mice

    Science.gov (United States)

    Luo, Lan; Urata, Yoshishige; Yan, Chen; Hasan, Al Shaimaa; Goto, Shinji; Guo, Chang-Ying; Tou, Fang-Fang; Xie, Yucai; Li, Tao-Sheng

    2016-01-01

    Radiation exposure may increase cardiovascular disease risks; however, the precise molecular/cellular mechanisms remain unclear. In the present study, we examined the hypothesis that radiation impairs cardiac stem cells (CSCs), thereby contributing to future cardiovascular disease risks. Adult C57BL/6 mice were exposed to 3 Gy γ-rays, and heart tissues were collected 24 hours later for further experiments. Although c-kit-positive cells were rarely found, radiation exposure significantly induced apoptosis and DNA damage in the cells of the heart. The ex vivo expansion of CSCs from freshly harvested atrial tissues showed a significantly lower production of CSCs in irradiated mice compared with healthy mice. The proliferative activity of CSCs evaluated by Ki-67 expression was not significantly different between the groups. However, compared to the healthy control, CSCs expanded from irradiated mice showed significantly lower telomerase activity, more 53BP1 foci in the nuclei, lower expression of c-kit and higher expression of CD90. Furthermore, CSCs expanded from irradiated mice had significantly poorer potency in the production of insulin-like growth factor-1. Our data suggest that radiation exposure significantly decreases the quantity and quality of CSCs, which may serve as sensitive bio-parameters for predicting future cardiovascular disease risks. PMID:27195709

  5. Radiation Exposure Decreases the Quantity and Quality of Cardiac Stem Cells in Mice.

    Directory of Open Access Journals (Sweden)

    Lan Luo

    Full Text Available Radiation exposure may increase cardiovascular disease risks; however, the precise molecular/cellular mechanisms remain unclear. In the present study, we examined the hypothesis that radiation impairs cardiac stem cells (CSCs, thereby contributing to future cardiovascular disease risks. Adult C57BL/6 mice were exposed to 3 Gy γ-rays, and heart tissues were collected 24 hours later for further experiments. Although c-kit-positive cells were rarely found, radiation exposure significantly induced apoptosis and DNA damage in the cells of the heart. The ex vivo expansion of CSCs from freshly harvested atrial tissues showed a significantly lower production of CSCs in irradiated mice compared with healthy mice. The proliferative activity of CSCs evaluated by Ki-67 expression was not significantly different between the groups. However, compared to the healthy control, CSCs expanded from irradiated mice showed significantly lower telomerase activity, more 53BP1 foci in the nuclei, lower expression of c-kit and higher expression of CD90. Furthermore, CSCs expanded from irradiated mice had significantly poorer potency in the production of insulin-like growth factor-1. Our data suggest that radiation exposure significantly decreases the quantity and quality of CSCs, which may serve as sensitive bio-parameters for predicting future cardiovascular disease risks.

  6. Cell surface alteration in Epstein-Barr virus-transformed cells from patients with extreme insulin resistance

    International Nuclear Information System (INIS)

    Gorden, D.L.; Robert, A.; Moncada, V.Y.; Taylor, S.I.; Muehlhauser, J.C.; Carpentier, J.L.

    1990-01-01

    An abnormality was detected in the morphology of the cell surface of Epstein-Barr virus-transformed lymphocytes of patients with genetic forms of insulin resistance. In cells from two patients with leprechaunism and two patients with type A extreme insulin resistance, scanning electron microscopy demonstrated a decrease in the percentage of the cell surface occupied by microvilli in cells from the patients with leprechaunism and type A insulin resistance compared with control cells. When cells from a healthy control subject and one of the patients with leprechaunism (Lep/Ark-1) were incubated with 125 I-labeled insulin, there was a decrease in the percentage of 125 I-insulin associated with microvilli on the cell surface. Thus, the decreased localization of insulin receptors with the microvillous region of the cell surface was in proportion to the decrease in microvilli

  7. Expression of FK506 binding protein 65 (FKBP65) is decreased in epithelial ovarian cancer cells compared to benign tumor cells and to ovarian epithelium

    DEFF Research Database (Denmark)

    Henriksen, Rudi; Sørensen, Flemming Brandt; Orntoft, Torben Falck

    2011-01-01

    to be followed by a strongly increased risk of ovarian cysts. We performed the present study to reveal how FKBP65 is expressed in the ovary and in ovarian tumors and to see if this expression might be related to ovarian tumor development, a relationship we have found in colorectal cancer. Biopsies from...... prospectively collected samples from ovaries and benign, borderline, and invasive ovarian tumors were analyzed for expression of FKBP65 by immunohistochemistry. The expression was compared to survival and several clinicopathological parameters. FKBP65 is strongly expressed in ovarian epithelium and in benign...... ovarian tumor cells. In the ovary, a positive staining was also found in endothelial cells of blood vessels. In non-invasive and in invasive malignant tumor cells, a decreased staining was observed, which was not correlated to stage, histology, or survival. A significant inversed correlation to expression...

  8. Lack of CD4+ T cell percent decrease in alemtuzumab-treated multiple sclerosis patients with persistent relapses.

    Science.gov (United States)

    Rolla, Simona; De Mercanti, Stefania Federica; Bardina, Valentina; Horakova, Dana; Habek, Mario; Adamec, Ivan; Cocco, Eleonora; Annovazzi, Pietro; Vladic, Anton; Novelli, Francesco; Durelli, Luca; Clerico, Marinella

    2017-12-15

    Alemtuzumab, a highly effective treatment for relapsing remitting multiple sclerosis (RRMS), induces lymphopenia especially of CD4+ T cells. Here, we report the atypical CD4+ T population behaviour of two patients with persistent disease activity despite repeated alemtuzumab treatments. Whereas lymphocytes count decreased and fluctuated accordingly to alemtuzumab administration, their CD4+ cell percentage was not or just mildly affected and was slightly below the lowest normal limit already before alemtuzumab. These cases anticipate further studies aimed to investigate whether the evaluation of the CD4+ cell percentage could represent a helpful tool to address the individual clinical response to alemtuzumab. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

  9. Arsenite reduces insulin secretion in rat pancreatic β-cells by decreasing the calcium-dependent calpain-10 proteolysis of SNAP-25

    International Nuclear Information System (INIS)

    Diaz-Villasenor, Andrea; Burns, Anna L.; Salazar, Ana Maria; Sordo, Monserrat; Hiriart, Marcia; Cebrian, Mariano E.; Ostrosky-Wegman, Patricia

    2008-01-01

    An increase in the prevalence of type 2 diabetes has been consistently observed among residents of high arsenic exposure areas. We have previously shown that in rat pancreatic β-cells, low arsenite doses impair the secretion of insulin without altering its synthesis. To further study the mechanism by which arsenite reduces insulin secretion, we evaluated the effects of arsenite on the calcium-calpain pathway that triggers insulin exocytosis in RINm5F cells. Cell cycle and proliferation analysis were also performed to complement the characterization. Free [Ca 2+ ]i oscillations needed for glucose-stimulated insulin secretion were abated in the presence of subchronic low arsenite doses (0.5-2 μM). The global activity of calpains increased with 2 μM arsenite. However, during the secretion of insulin stimulated with glucose (15.6 mM), 1 μM arsenite decreased the activity of calpain-10, measured as SNAP-25 proteolysis. Both proteins are needed to fuse insulin granules with the membrane to produce insulin exocytosis. Arsenite also induced a slowdown in the β cell line proliferation in a dose-dependent manner, reflected by a reduction of dividing cells and in their arrest in G2/M. Data obtained showed that one of the mechanisms by which arsenite impairs insulin secretion is by decreasing the oscillations of free [Ca 2+ ]i, thus reducing calcium-dependent calpain-10 partial proteolysis of SNAP-25. The effects in cell division and proliferation observed with arsenite exposure can be an indirect consequence of the decrease in insulin secretion

  10. Reduced LAK cytotoxicity of peripheral blood mononuclear cells in patients with bladder cancer: Decreased LAK cytotoxicity caused by a low incidence of CD56+ and CD57+ mononuclear blood cells

    International Nuclear Information System (INIS)

    Hermann, G.G.; Petersen, K.R.; Steven, K.; Zeuthen, J.

    1990-01-01

    The cytotoxicity of unstimulated peripheral blood mononuclear cells (US-PBMC), phytohemagglutinin (PHA)-stimulated PBMC (PS-PBMC) and interleukin-2 (IL-2)-activated PBMC (LAK cells) was assessed in patients with noninvasive and invasive transitional-cell bladder cancer and compared with those determined in healthy controls. The differences in the cytotoxicities were correlated with specific changes in the subsets of peripheral blood mononuclear cells (PBMC). PBMC from 37 patients and 13 healthy controls were tested against the bladder cancer cell line T24 in 51 Cr-release assays. The PBMC subsets were analyzed using monoclonal antibodies against T cells, natural killer (NK) -cells, monocytes, and activation markers. The cytotoxicities of US-PBMC, PS-PBMC, and LAK cells were all significantly lower in the cancer patients than in the controls (P less than 0.05). The percentages of PBMC positive for the NK-cell markers CD56 and CD57 were lowest in the patients and were correlated to the decrease in cytotoxicity. Depletion of CD56+ or CD57+ cells from PBMC prior to or after 2 days stimulation with IL-2 demonstrated that these cells are the major source of LAK-cell cytotoxicity and showed that the reduced ability of bladder cancer patient PBMC to develop LAK-cell cytotoxicity is a result of a low incidence of CD56+ and CD57+ cells in the blood. These findings indicate that IL-2 therapy alone might not be a sufficient therapy of bladder cancer patients

  11. Multiplexed quantitative high content screening reveals that cigarette smoke condensate induces changes in cell structure and function through alterations in cell signaling pathways in human bronchial cells

    International Nuclear Information System (INIS)

    Carter, Charleata A.; Hamm, Jonathan T.

    2009-01-01

    Human bronchial cells are one of the first cell types exposed to environmental toxins. Toxins often activate nuclear factor-κB (NF-κB) and protein kinase C (PKC). We evaluated the hypothesis that cigarette smoke condensate (CSC), the particulate fraction of cigarette smoke, activates PKC-α and NF-κB, and concomitantly disrupts the F-actin cytoskeleton, induces apoptosis and alters cell function in BEAS-2B human bronchial epithelial cells. Compared to controls, exposure of BEAS-2B cells to doses of 30 μg/ml CSC significantly activated PKC-α, while CSC doses above 20 μg/ml CSC significantly activated NF-κB. As NF-κB was activated, cell number decreased. CSC treatment of BEAS-2B cells induced a decrease in cell size and an increase in cell surface extensions including filopodia and lamellipodia. CSC treatment of BEAS-2B cells induced F-actin rearrangement such that stress fibers were no longer prominent at the cell periphery and throughout the cells, but relocalized to perinuclear regions. Concurrently, CSC induced an increase in the focal adhesion protein vinculin at the cell periphery. CSC doses above 30 μg/ml induced a significant increase in apoptosis in BEAS-2B cells evidenced by an increase in activated caspase 3, an increase in mitochondrial mass and a decrease in mitochondrial membrane potential. As caspase 3 increased, cell number decreased. CSC doses above 30 μg/ml also induced significant concurrent changes in cell function including decreased cell spreading and motility. CSC initiates a signaling cascade in human bronchial epithelial cells involving PKC-α, NF-κB and caspase 3, and consequently decreases cell spreading and motility. These CSC-induced alterations in cell structure likely prevent cells from performing their normal function thereby contributing to smoke-induced diseases.

  12. Decreased CXCL12 is associated with impaired alveolar epithelial cell migration and poor lung healing after lung resection.

    Science.gov (United States)

    Kanter, Jacob A; Sun, Haiying; Chiu, Stephen; DeCamp, Malcolm M; Sporn, Peter H S; Sznajder, Jacob I; Bharat, Ankit

    2015-10-01

    Prolonged air leak (PAL) is an important cause of morbidity and mortality after lung resection, but its pathogenesis has not been elucidated. Migration of alveolar type II epithelial cells is essential for lung wound repair. Here we determined the role of C-X-C motif chemokine 12 (CXCL12) on alveolar epithelial cell migration and lung wound healing. CXCL12 in the pleural fluid of patients was analyzed using enzyme-linked immunosorbent assay. Human A549 and murine MLE12 alveolar epithelial cell lines were used for wound closure, cell migration, and proliferation assays. Western blot was used to analyze Rac1 and cofilin. Pleural CXCL12 was decreased in patients with PAL (1,389 ± 192 vs 3,270 ± 247 pg/mL; P alveolar epithelial cell migration by binding to its receptor CXCR4 and may have a role in lung healing. CXCL12-mediated alveolar epithelial cell migration is associated with Rac1 and cofilin activation. Copyright © 2015 Elsevier Inc. All rights reserved.

  13. Heat Shock Protein 90 Inhibitor (17-AAG) Induces Apoptosis and Decreases Cell Migration/Motility of Keloid Fibroblasts.

    Science.gov (United States)

    Yun, In Sik; Lee, Mi Hee; Rah, Dong Kyun; Lew, Dae Hyun; Park, Jong-Chul; Lee, Won Jai

    2015-07-01

    The regulation of apoptosis, proliferation, and migration of fibroblasts is altered in keloids. The 90-kDa heat shock protein (heat shock protein 90) is known to play a key role in such regulation. Therefore, the authors investigated whether the inhibition of heat shock protein 90 in keloid fibroblasts could induce apoptosis and attenuate keloid fibroblast proliferation and migration. The authors evaluated heat shock protein 90 expression in keloid tissues with immunohistochemistry. The authors used cell viability [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assays and annexin V/propidium iodide staining for apoptosis, a wound healing model and cell tracking system to assess cell migration, and Akt Western blotting analysis in keloid fibroblasts after inhibition of heat shock protein 90 with 17-allylaminodemethoxygeldanamycin (17-AAG). The expression of heat shock protein 90 in keloid tissues was significantly increased compared with normal tissues. The 17-AAG-treated keloid fibroblasts showed significantly decreased proliferation, promotion of apoptosis, and decreased expression of Akt. Furthermore, a dose-dependent decrease in cell migration was noted after 17-AAG treatment of keloid fibroblasts. The 17-AAG-treated keloid fibroblasts had less directionality to the wound center and migrated a shorter distance. The authors confirmed that the inhibition of heat shock protein 90 in keloid fibroblasts could promote apoptosis and attenuate proliferation and migration of keloid fibroblasts. Therefore, the authors think that the inhibition of heat shock protein 90 is a key factor in the regulation of biological processes in keloids. With further preclinical study, the authors will be able to apply these results clinically for keloid treatment.

  14. NKT Cell Responses to B Cell Lymphoma.

    Science.gov (United States)

    Li, Junxin; Sun, Wenji; Subrahmanyam, Priyanka B; Page, Carly; Younger, Kenisha M; Tiper, Irina V; Frieman, Matthew; Kimball, Amy S; Webb, Tonya J

    2014-06-01

    Natural killer T (NKT) cells are a unique subset of CD1d-restricted T lymphocytes that express characteristics of both T cells and natural killer cells. NKT cells mediate tumor immune-surveillance; however, NKT cells are numerically reduced and functionally impaired in lymphoma patients. Many hematologic malignancies express CD1d molecules and co-stimulatory proteins needed to induce anti-tumor immunity by NKT cells, yet most tumors are poorly immunogenic. In this study, we sought to investigate NKT cell responses to B cell lymphoma. In the presence of exogenous antigen, both mouse and human NKT cell lines produce cytokines following stimulation by B cell lymphoma lines. NKT cell populations were examined ex vivo in mouse models of spontaneous B cell lymphoma, and it was found that during early stages, NKT cell responses were enhanced in lymphoma-bearing animals compared to disease-free animals. In contrast, in lymphoma-bearing animals with splenomegaly and lymphadenopathy, NKT cells were functionally impaired. In a mouse model of blastoid variant mantle cell lymphoma, treatment of tumor-bearing mice with a potent NKT cell agonist, α-galactosylceramide (α-GalCer), resulted in a significant decrease in disease pathology. Ex vivo studies demonstrated that NKT cells from α-GalCer treated mice produced IFN-γ following α-GalCer restimulation, unlike NKT cells from vehicle-control treated mice. These data demonstrate an important role for NKT cells in the immune response to an aggressive hematologic malignancy like mantle cell lymphoma.

  15. Dihydropyridines decrease X-ray-induced DNA base damage in mammalian cells

    Energy Technology Data Exchange (ETDEWEB)

    Wojewodzka, M., E-mail: marylaw@ichtj.waw.pl [Center of Radiobiology and Biological Dosimetry, Institute of Nuclear Chemistry and Technology, Warszawa (Poland); Gradzka, I.; Buraczewska, I.; Brzoska, K.; Sochanowicz, B. [Center of Radiobiology and Biological Dosimetry, Institute of Nuclear Chemistry and Technology, Warszawa (Poland); Goncharova, R.; Kuzhir, T. [Institute of Genetics and Cytology, Belarussian National Academy of Sciences, Minsk (Belarus); Szumiel, I. [Center of Radiobiology and Biological Dosimetry, Institute of Nuclear Chemistry and Technology, Warszawa (Poland)

    2009-12-01

    Compounds with the structural motif of 1,4-dihydropyridine display a broad spectrum of biological activities, often defined as bioprotective. Among them are L-type calcium channel blockers, however, also derivatives which do not block calcium channels exert various effects at the cellular and organismal levels. We examined the effect of sodium 3,5-bis-ethoxycarbonyl-2,6-dimethyl-1,4-dihydropyridine-4-carboxylate (denoted here as DHP and previously also as AV-153) on X-ray-induced DNA damage and mutation frequency at the HGPRT (hypoxanthine-guanine phosphoribosyl transferase) locus in Chinese hamster ovary CHO-K1 cells. Using formamido-pyrimidine glycosylase (FPG) comet assay, we found that 1-h DHP (10 nM) treatment before X-irradiation considerably reduced the initial level of FPG-recognized DNA base damage, which was consistent with decreased 8-oxo-7,8-dihydro-2'-deoxyguanosine content and mutation frequency lowered by about 40%. No effect on single strand break rejoining or on cell survival was observed. Similar base damage-protective effect was observed for two calcium channel blockers: nifedipine (structurally similar to DHP) or verapamil (structurally unrelated). So far, the specificity of the DHP-caused reduction in DNA damage - practically limited to base damage - has no satisfactory explanation.

  16. Pancreatic stellate cells promote epithelial-mesenchymal transition in pancreatic cancer cells

    International Nuclear Information System (INIS)

    Kikuta, Kazuhiro; Masamune, Atsushi; Watanabe, Takashi; Ariga, Hiroyuki; Itoh, Hiromichi; Hamada, Shin; Satoh, Kennichi; Egawa, Shinichi; Unno, Michiaki; Shimosegawa, Tooru

    2010-01-01

    Research highlights: → Recent studies have shown that pancreatic stellate cells (PSCs) promote the progression of pancreatic cancer. → Pancreatic cancer cells co-cultured with PSCs showed loose cell contacts and scattered, fibroblast-like appearance. → PSCs decreased the expression of epithelial markers but increased that of mesenchymal markers, along with increased migration. → This study suggests epithelial-mesenchymal transition as a novel mechanism by which PSCs contribute to the aggressive behavior of pancreatic cancer cells. -- Abstract: The interaction between pancreatic cancer cells and pancreatic stellate cells (PSCs), a major profibrogenic cell type in the pancreas, is receiving increasing attention. There is accumulating evidence that PSCs promote the progression of pancreatic cancer by increasing cancer cell proliferation and invasion as well as by protecting them from radiation- and gemcitabine-induced apoptosis. Because epithelial-mesenchymal transition (EMT) plays a critical role in the progression of pancreatic cancer, we hypothesized that PSCs promote EMT in pancreatic cancer cells. Panc-1 and SUIT-2 pancreatic cancer cells were indirectly co-cultured with human PSCs isolated from patients undergoing operation for pancreatic cancer. The expression of epithelial and mesenchymal markers was examined by real-time PCR and immunofluorescent staining. The migration of pancreatic cancer cells was examined by scratch and two-chamber assays. Pancreatic cancer cells co-cultured with PSCs showed loose cell contacts and a scattered, fibroblast-like appearance. The expression of E-cadherin, cytokeratin 19, and membrane-associated β-catenin was decreased, whereas vimentin and Snail (Snai-1) expression was increased more in cancer cells co-cultured with PSCs than in mono-cultured cells. The migration of pancreatic cancer cells was increased by co-culture with PSCs. The PSC-induced decrease of E-cadherin expression was not altered by treatment with anti

  17. Dichloroacetate Decreases Cell Health and Activates Oxidative Stress Defense Pathways in Rat Alveolar Type II Pneumocytes

    Directory of Open Access Journals (Sweden)

    Alexis Valauri-Orton

    2015-01-01

    Full Text Available Dichloroacetate (DCA is a water purification byproduct that is known to be hepatotoxic and hepatocarcinogenic and to induce peripheral neuropathy and damage macrophages. This study characterizes the effects of the haloacetate on lung cells by exposing rat alveolar type II (L2 cells to 0–24 mM DCA for 6–24 hours. Increasing DCA concentration and the combination of increasing DCA concentration plus longer exposures decrease measures of cellular health. Length of exposure has no effect on oxidative stress biomarkers, glutathione, SOD, or CAT. Increasing DCA concentration alone does not affect total glutathione or its redox ratio but does increase activity in the SOD/CAT oxidative stress defense pathway. These data suggest that alveolar type II cells rely on SOD and CAT more than glutathione to combat DCA-induced stress.

  18. Current Smoking Dose-Dependently Associated with Decreased β-Cell Function in Chinese Men without Diabetes

    Directory of Open Access Journals (Sweden)

    Chun Wang

    2015-01-01

    Full Text Available The aim of this study was to evaluate the associations between chronic smoking and insulin resistance and β-cell function in Chinese men without diabetes. A total of 1,568 participants were recruited by multistage sampling. Using homeostatic model assessment (HOMA, geometric means of insulin resistance (HOMA-IR and β-cell function (HOMA-β with 95% confidence interval (CI were calculated by general linear model. Odds ratios (ORs with 95% CI were estimated to evaluate the associations between smoking status and insulin resistance and β-cell deficiency under a logistic regression model. Current smokers had higher levels of 2 h glucose (6.66 versus 6.48 mmol/L for oral glucose tolerance test and lower levels of fasting insulin (5.68 versus 6.03 mU/L than never smokers. The adjusted means for HOMA-β (% were 54.86 in current smokers and 58.81 in never smokers (P=0.0257. Current smoking was associated with β-cell deficiency (OR 1.29, 95% CI 1.01–1.64 compared to never smoking. The β-cell function gradually decreased with increasing smoking intensity (Ptrend=0.0026, and the differences were statistically significant when the pack-year of smoking was 20 or above. No association was observed between smoking status and HOMA-IR. Our study suggested that chronic smoking may dose-dependently suppress insulin secretion in Chinese men.

  19. Three-dimensional culture conditions lead to decreased radiation induced cytotoxicity in human mammary epithelial cells

    International Nuclear Information System (INIS)

    Sowa, Marianne B.; Chrisler, William B.; Zens, Kyra D.; Ashjian, Emily J.; Opresko, Lee K.

    2010-01-01

    For both targeted and non-targeted exposures, the cellular responses to ionizing radiation have predominantly been measured in two-dimensional monolayer cultures. Although convenient for biochemical analysis, the true interactions in vivo depend upon complex interactions between cells themselves and the surrounding extracellular matrix. This study directly compares the influence of culture conditions on radiation induced cytotoxicity following exposure to low-LET ionizing radiation. Using a three-dimensional (3D) human mammary epithelial tissue model, we have found a protective effect of 3D cell culture on cell survival after irradiation. The initial state of the cells (i.e., 2D versus 3D culture) at the time of irradiation does not alter survival, nor does the presence of extracellular matrix during and after exposure to dose, but long term culture in 3D which offers significant reduction in cytotoxicity at a given dose (e.g. ∼4-fold increased survival at 5 Gy). The cell cycle delay induced following exposure to 2 and 5 Gy was almost identical between 2D and 3D culture conditions and cannot account for the observed differences in radiation responses. However the amount of apoptosis following radiation exposure is significantly decreased in 3D culture relative to the 2D monolayer after the same dose. A likely mechanism of the cytoprotective effect afforded by 3D culture conditions is the down regulation of radiation induced apoptosis in 3D structures.

  20. Fragrance chemicals lyral and lilial decrease viability of HaCat cells' by increasing free radical production and lowering intracellular ATP level: protection by antioxidants.

    Science.gov (United States)

    Usta, Julnar; Hachem, Yassmine; El-Rifai, Omar; Bou-Moughlabey, Yolla; Echtay, Karim; Griffiths, David; Nakkash-Chmaisse, Hania; Makki, Rajaa Fakhoury

    2013-02-01

    We investigate in this study the biochemical effects on cells in culture of two commonly used fragrance chemicals: lyral and lilial. Whereas both chemicals exerted a significant effect on primary keratinocyte(s), HaCat cells, no effect was obtained with any of HepG2, Hek293, Caco2, NIH3T3, and MCF7 cells. Lyral and lilial: (a) decreased the viability of HaCat cells with a 50% cell death at 100 and 60 nM respectively; (b) decreased significantly in a dose dependant manner the intracellular ATP level following 12-h of treatment; (c) inhibited complexes I and II of electron transport chain in liver sub-mitochondrial particles; and (d) increased reactive oxygen species generation that was reversed by N-acetyl cysteine and trolox and the natural antioxidant lipoic acid, without influencing the level of free and/or oxidized glutathione. Lipoic acid protected HaCat cells against the decrease in viability induced by either compound. Dehydrogenation of lyral and lilial produce α,β-unsaturated aldehydes, that reacts with lipoic acid requiring proteins resulting in their inhibition. We propose lyral and lilial as toxic to mitochondria that have a direct effect on electron transport chain, increase ROS production, derange mitochondrial membrane potential, and decrease cellular ATP level, leading thus to cell death. Copyright © 2012 Elsevier Ltd. All rights reserved.

  1. Promotion of initiated cells by radiation-induced cell inactivation.

    Science.gov (United States)

    Heidenreich, W F; Paretzke, H G

    2008-11-01

    Cells on the way to carcinogenesis can have a growth advantage relative to normal cells. It has been hypothesized that a radiation-induced growth advantage of these initiated cells might be induced by an increased cell replacement probability of initiated cells after inactivation of neighboring cells by radiation. Here Monte Carlo simulations extend this hypothesis for larger clones: The effective clonal expansion rate decreases with clone size. This effect is stronger for the two-dimensional than for the three-dimensional situation. The clones are irregular, far from a circular shape. An exposure-rate dependence of the effective clonal expansion rate could come in part from a minimal recovery time of the initiated cells for symmetric cell division.

  2. Acinar cell-specific knockout of the PTHrP gene decreases the proinflammatory and profibrotic responses in pancreatitis.

    Science.gov (United States)

    Bhatia, Vandanajay; Rastellini, Cristiana; Han, Song; Aronson, Judith F; Greeley, George H; Falzon, Miriam

    2014-09-01

    Pancreatitis is a necroinflammatory disease with acute and chronic manifestations. Accumulated damage incurred during repeated bouts of acute pancreatitis (AP) can lead to chronic pancreatitis (CP). Pancreatic parathyroid hormone-related protein (PTHrP) levels are elevated in a mouse model of cerulein-induced AP. Here, we show elevated PTHrP levels in mouse models of pancreatitis induced by chronic cerulein administration and pancreatic duct ligation. Because acinar cells play a major role in the pathophysiology of pancreatitis, mice with acinar cell-specific targeted disruption of the Pthrp gene (PTHrP(Δacinar)) were generated to assess the role of acinar cell-secreted PTHrP in pancreatitis. These mice were generated using Cre-LoxP technology and the acinar cell-specific elastase promoter. PTHrP(Δacinar) exerted protective effects in cerulein and pancreatic duct ligation models, evident as decreased edema, histological damage, amylase secretion, pancreatic stellate cell (PSC) activation, and extracellular matrix deposition. Treating acinar cells in vitro with cerulein increased IL-6 expression and NF-κB activity; these effects were attenuated in PTHrP(Δacinar) cells, as were the cerulein- and carbachol-induced elevations in amylase secretion. The cerulein-induced upregulation of procollagen I expression was lost in PSCs from PTHrP(Δacinar) mice. PTHrP immunostaining was elevated in human CP sections. The cerulein-induced upregulation of IL-6 and ICAM-1 (human acinar cells) and procollagen I (human PSCs) was suppressed by pretreatment with the PTH1R antagonist, PTHrP (7-34). These findings establish PTHrP as a novel mediator of inflammation and fibrosis associated with CP. Acinar cell-secreted PTHrP modulates acinar cell function via its effects on proinflammatory cytokine release and functions via a paracrine pathway to activate PSCs. Copyright © 2014 the American Physiological Society.

  3. Hypofractionated radiation induces a decrease in cell proliferation but no histological damage to organotypic multicellular spheroids of human glioblastomas

    International Nuclear Information System (INIS)

    Kaaijk, P.; Academic Medical Center, Amsterdam; Troost, D.; Leenstra, S.; Bosch, D.A.; Sminia, P.; Hulshof, M.C.C.M..; Kracht, A.H.W. van der

    1997-01-01

    The aim of this study was to examine the effect of radiation on glioblastoma, using an organotypic multicellular spheroid (OMS) model. Most glioblastoma cell lines are, in contrast to glioblastomas in vivo, relatively radiosensitive. This limits the value of using cell lines for studying the radiation effect of glioblastomas. The advantage of OMS is maintenance of the characteristics of the original tumour, which is lost in conventional cell cultures. OMS prepared from four glioblastomas were treated with hypofractionated radiation with a radiobiologically equivalent dose to standard radiation treatment for glioblastomas patients. After treatment, the histology as well as the cell proliferation of the OMS was examined. After radiation, a significant decrease in cell proliferation was found, although no histological damage to the OMS was observed. The modest effects of radiation on the OMS are in agreement with the limited therapeutic value of radiotherapy for glioblastoma patients. Therefore, OMS seems to be a good alternative for cell lines to study the radiobiological effect on glioblastomas. (author)

  4. Hypofractionated radiation induces a decrease in cell proliferation but no histological damage to organotypic multicellular spheroids of human glioblastomas

    Energy Technology Data Exchange (ETDEWEB)

    Kaaijk, P [Academic Medical Center, Amsterdam (Netherlands). Dept. of (Neuro) Pathology; [Academic Medical Center, Amsterdam (Netherlands). Dept. of Neurosurgery; Troost, D [Academic Medical Center, Amsterdam (Netherlands). Dept. of (Neuro) Pathology; Leenstra, S; Bosch, D A [Academic Medical Center, Amsterdam (Netherlands). Dept. of Neurosurgery; Sminia, P; Hulshof, M C.C.M.; Kracht, A.H.W. van der [Academic Medical Center, Amsterdam (Netherlands). Dept. of (Experimental) Radiotherapy

    1997-04-01

    The aim of this study was to examine the effect of radiation on glioblastoma, using an organotypic multicellular spheroid (OMS) model. Most glioblastoma cell lines are, in contrast to glioblastomas in vivo, relatively radiosensitive. This limits the value of using cell lines for studying the radiation effect of glioblastomas. The advantage of OMS is maintenance of the characteristics of the original tumour, which is lost in conventional cell cultures. OMS prepared from four glioblastomas were treated with hypofractionated radiation with a radiobiologically equivalent dose to standard radiation treatment for glioblastomas patients. After treatment, the histology as well as the cell proliferation of the OMS was examined. After radiation, a significant decrease in cell proliferation was found, although no histological damage to the OMS was observed. The modest effects of radiation on the OMS are in agreement with the limited therapeutic value of radiotherapy for glioblastoma patients. Therefore, OMS seems to be a good alternative for cell lines to study the radiobiological effect on glioblastomas. (author).

  5. Chromosome aberrations and cell survival in irradiated mammalian cells

    International Nuclear Information System (INIS)

    Tremp, J.

    1981-01-01

    A possible correlation between chromosome aberrations and reduced proliferation capacity or cell death was investigated. Synchronized Chinese hamster fibroblast cells were irradiated with 300 rad of x rays in early G 1 . Despite synchronization the cells reached the subsequent mitosis at different times. The frequency of chromosome aberrations was determined in the postirradiation division at 2-h intervals. The highest frequency occurred in cells with a first cell cycle of medium length. The colony-forming ability of mitotic cells was measured in parallel samples by following the progress of individual mitoses. The proportion of cells forming macrocolonies decreased with increasing cell cycle length, and the number of non-colony-forming cells increased. Irrespective of various first cell cycle lengths and different frequencies of chromosome aberrations, the number of cells forming microcolonies remained constant. A correlation was found between the absence of chromosome aberrations and the ability of cells to form macrocolonies. However, cells with a long first cell cycle formed fewer macrocolonies than expected

  6. Activin A, B and AB decrease progesterone production by down-regulating StAR in human granulosa cells.

    Science.gov (United States)

    Chang, Hsun-Ming; Cheng, Jung-Chien; Huang, He-Feng; Shi, Feng-Tao; Leung, Peter C K

    2015-09-05

    Activins are homo- or heterodimers of inhibin β subunits that play important roles in the reproductive system. Our previous work has shown that activins A (βAβA), B (βBβB) and AB (βAβB) induce aromatase/estradiol, but suppress StAR/progesterone production in human granulosa-lutein cells. However, the underlying molecular determinants of these effects have not been examined. In this continuing study, we used immortalized human granulosa cells (SVOG) to investigate the effects of activins in regulating StAR/progesterone and the potential mechanisms of action. In SVOG cells, activins A, B and AB produced comparable down-regulation of StAR expression and progesterone production. In addition, all three activin isoforms induced equivalent phosphorylation of both SMAD2 and SMAD3. Importantly, the activin-induced down-regulation of StAR, increase in SMAD2/3 phosphorylation, and decrease in progesterone were abolished by the TGF-β type I receptor inhibitor SB431542. Interestingly, the small interfering RNA-mediated knockdown of ALK4 but not ALK5 reversed the activin-induced suppression of StAR. Furthermore, the knockdown of SMAD4 or SMAD2 but not SMAD3 abolished the inhibitory effects of all three activin isoforms on StAR expression. These results provide evidence that activins A, B and AB down-regulate StAR expression and decrease progesterone production in human granulosa cells, likely via an ALK4-mediated SMAD2/SMAD4-dependent pathway. Our findings provide important insights into the molecular mechanisms underlying the regulatory effects of activins on human granulosa cell steroidogenesis. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  7. Pharmacologic suppression of target cell recognition by engineered T cells expressing chimeric T-cell receptors.

    Science.gov (United States)

    Alvarez-Vallina, L; Yañez, R; Blanco, B; Gil, M; Russell, S J

    2000-04-01

    Adoptive therapy with autologous T cells expressing chimeric T-cell receptors (chTCRs) is of potential interest for the treatment of malignancy. To limit possible T-cell-mediated damage to normal tissues that weakly express the targeted tumor antigen (Ag), we have tested a strategy for the suppression of target cell recognition by engineered T cells. Jurkat T cells were transduced with an anti-hapten chTCR tinder the control of a tetracycline-suppressible promoter and were shown to respond to Ag-positive (hapten-coated) but not to Ag-negative target cells. The engineered T cells were then reacted with hapten-coated target cells at different effector to target cell ratios before and after exposure to tetracycline. When the engineered T cells were treated with tetracycline, expression of the chTCR was greatly decreased and recognition of the hapten-coated target cells was completely suppressed. Tetracycline-mediated suppression of target cell recognition by engineered T cells may be a useful strategy to limit the toxicity of the approach to cancer gene therapy.

  8. Quercetin inhibits the invasion and mobility of murine melanoma B16-BL6 cells through inducing apoptosis via decreasing Bcl-2 expression.

    Science.gov (United States)

    Zhang, X; Xu, Q; Saiki, I

    2000-01-01

    Quercetin has been known to have anti-tumor and anti-oxidation activities. In the present study, we have investigated its in vitro anti-metastatic activity. Quercetin inhibited the invasion and mobility of murine melanoma B16-BL6 cells in a dose-dependent manner but did not affect their adhesion to either laminin, fibronectin, or type VI collagen. Moreover, quercetin significantly inhibited the proliferation of B16-BL6 cells only in the case of time incubation longer than 48 h. Quercetin dose-dependently decreased the cell rates in S and G2-M phases of cell cycle. The effect of quercetin to cause a remarkable apoptosis of B16-BL6 cells was also demonstrated by flow cytometric assay as well as DNA fragmentation with a typical 180-bp ladder band in agarose electrophoresis and a quantitative analysis. Furthermore, quercetin markedly inhibited the expression of anti-apoptotic protein Bcl-2 but hardly influenced Bcl-XL. These results suggest that the inhibition of quercetin on invasiveness and migration of B16-BL6 cells are closely associated with the arrest of cell cycle as well as the induction of apoptosis by decreasing the Bcl-2 expression.

  9. Arsenic trioxide decreases the amount and inhibits the function of regulatory T cells, which may contribute to its efficacy in the treatment of acute promyelocytic leukemia.

    Science.gov (United States)

    Xu, Wen; Li, Xiaoxia; Quan, Lina; Yao, Jiying; Mu, Guannan; Guo, Jingjie; Wang, Yitong

    2018-03-01

    Arsenic trioxide (ATO) exhibits substantial clinical efficacy in the treatment of acute promyelocytic leukemia (APL). Here, we investigated whether ATO exerts its efficacy by affecting regulatory T (Treg) cells. We determined whether ATO treatment influenced the amount and function of purified Treg cells. We also examined the effect of ATO treatment on Treg cells from APL patients. ATO treatment induced apoptosis in purified Treg cells and dampened the inhibition of effector T (Teff) cells proliferation and the secretion of cytokine by Treg cells. Treg cell levels in the peripheral blood and serum IL-10 levels were dramatically decreased in APL patients after single ATO treatment. In summary, our results show that ATO decreases the amount and inhibits the function of Treg cells, thereby enhancing Teff cell function and overall anti-tumor immunity.

  10. Leydig cell micronodules are a common finding in testicular biopsies from men with impaired spermatogenesis and are associated with decreased testosterone/LH ratio

    DEFF Research Database (Denmark)

    Holm, Mette; Rajpert-De Meyts, Ewa; Andersson, Anna-Maria

    2003-01-01

    To assess the biological significance of Leydig cell 'hyperplasia' in man, Leydig cell distribution, volume, and function were studied in patients with infertility or testicular cancer and in suddenly deceased controls. A total of 156 biopsies from 95 patients and 18 necropsies from 13 controls......), and were rare in testes from controls (median = 0, p = 0.02). The proportion of testicular tissue occupied by Leydig cells increased with decreasing spermatogenic capacity. In contrast, the total volume of Leydig cells per testis was roughly comparable irrespective of the histological pattern...... in the hyperstimulated testes, as reflected by an increased LH/testosterone ratio. In conclusion, Leydig cell micronodules were more frequent in biopsies with impaired spermatogenesis and associated with decreased ratios of testicular hormones to gonadotrophins. The presence of micronodules thus seems...

  11. Knockdown of AMPKα decreases ATM expression and increases radiosensitivity under hypoxia and nutrient starvation in an SV40-transformed human fibroblast cell line, LM217.

    Science.gov (United States)

    Murata, Yasuhiko; Hashimoto, Takuma; Urushihara, Yusuke; Shiga, Soichiro; Takeda, Kazuya; Jingu, Keiichi; Hosoi, Yoshio

    2018-01-22

    Presence of unperfused regions containing cells under hypoxia and nutrient starvation contributes to radioresistance in solid human tumors. It is well known that hypoxia causes cellular radioresistance, but little is known about the effects of nutrient starvation on radiosensitivity. We have reported that nutrient starvation induced decrease of mTORC1 activity and decrease of radiosensitivity in an SV40-transformed human fibroblast cell line, LM217, and that nutrient starvation induced increase of mTORC1 activity and increase of radiosensitivity in human liver cancer cell lines, HepG2 and HuH6 (Murata et al., BBRC 2015). Knockdown of mTOR using small interfering RNA (siRNA) for mTOR suppressed radiosensitivity under nutrient starvation alone in HepG2 cells, which suggests that mTORC1 pathway regulates radiosensitivity under nutrient starvation alone. In the present study, effects of hypoxia and nutrient starvation on radiosensitivity were investigated using the same cell lines. LM217 and HepG2 cells were used to examine the effects of hypoxia and nutrient starvation on cellular radiosensitivity, mTORC1 pathway including AMPK, ATM, and HIF-1α, which are known as regulators of mTORC1 activity, and glycogen storage, which is induced by HIF-1 and HIF-2 under hypoxia and promotes cell survival. Under hypoxia and nutrient starvation, AMPK activity and ATM expression were increased in LM217 cells and decreased in HepG2 cells compared with AMPK activity under nutrient starvation alone or ATM expression under hypoxia alone. Under hypoxia and nutrient starvation, radiosensitivity was decreased in LM217 cells and increased in HepG2 cells compared with radiosensitivity under hypoxia alone. Under hypoxia and nutrient starvation, knockdown of AMPK decreased ATM activity and increased radiation sensitivity in LM217 cells. In both cell lines, mTORC1 activity was decreased under hypoxia and nutrient starvation. Under hypoxia alone, knockdown of mTOR slightly increased ATM

  12. Inhibition of human melanoma cell growth by dietary flavonoid fisetin is associated with disruption of Wnt/β-catenin signaling and decreased Mitf levels

    Science.gov (United States)

    Syed, Deeba N.; Afaq, Farrukh; Maddodi, Nityanand; Johnson, Jeremy J.; Sarfaraz, Sami; Ahmad, Adeel; Setaluri, Vijayasaradhi; Mukhtar, Hasan

    2011-01-01

    The prognosis of advanced melanoma remains poor in spite of treatment advances, emphasizing the importance of additional preventive measures. Flavonoids, natural components of our diet are being investigated for their chemopreventive/therapeutic properties. Microphthalmia-associated transcription factor (Mitf), downstream of Wnt/β-catenin pathway has become an important prognostic marker of melanoma. Here, we show that treatment of 451Lu melanoma cells with the dietary flavonoid fisetin resulted in decreased cell viability with G1-phase arrest and disruption of Wnt/β-catenin signaling. This was accompanied with a decrease in expression of Wnt protein and its co-receptors and a parallel increase in the expression of endogenous Wnt inhibitors. Fisetin-treated cells showed increased cytosolic levels of Axin and β-TrCP and decreased phosphorylation of GSK3-β assocaited with decreased β-catenin stabilization. Fisetin-mediated interference with the functional cooperation between β-catenin and LEF/TCF-2 resulted in downregulation of positively regulated TCF targets such as c-myc, Brn-2 and Mitf. Flowcytometric analysis of Mitf overexpressing cells showed that fisetin repressed Mitf-induced cell proliferation. Finally, administration of fisetin to 451Lu xenografted nude mice resulted in inhibition of tumor development and decreased Mitf expression. Our data suggest that fisetin can be developed as an effective agent against melanoma due to its potential inhibitory effect on β-catenin/Mitf signaling. PMID:21346776

  13. Prolactin receptor attenuation induces zinc pool redistribution through ZnT2 and decreases invasion in MDA-MB-453 breast cancer cells

    International Nuclear Information System (INIS)

    Bostanci, Zeynep; Alam, Samina; Soybel, David I.; Kelleher, Shannon L.

    2014-01-01

    Prolactin receptor (PRL-R) activation regulates cell differentiation, proliferation, cell survival and motility of breast cells. Prolactin (PRL) and PRL-R over-expression are strongly implicated in breast cancer, particularly contributing to tumor growth and invasion in the more aggressive estrogen-receptor negative (ER−) disease. PRL-R antagonists have been suggested as potential therapeutic agents; however, mechanisms through which PRL-R antagonists exert their actions are not well-understood. Zinc (Zn) is a regulatory factor for over 10% of the proteome, regulating critical cell processes such as proliferation, cell signaling, transcription, apoptosis and autophagy. PRL-R signaling regulates Zn metabolism in breast cells. Herein we determined effects of PRL-R attenuation on cellular Zn metabolism and cell function in a model of ER-, PRL-R over-expressing breast cancer cells (MDA-MB-453). PRL-R attenuation post-transcriptionally increased ZnT2 abundance and redistributed intracellular Zn pools into lysosomes and mitochondria. ZnT2-mediated lysosomal Zn sequestration was associated with reduced matrix metalloproteinase 2 (MMP-2) activity and decreased invasion. ZnT2-mediated Zn accumulation in mitochondria was associated with increased mitochondrial oxidation. Our results suggest that PRL-R antagonism in PRL-R over-expressing breast cancer cells may reduce invasion through the redistribution of intracellular Zn pools critical for cellular function. - Highlights: • PRL-R attenuation increased ZnT2 expression. • PRL-R attenuation increased lysosomal and mitochondrial Zn accumulation. • PRL-R attenuation decreased MMP-2 and invasion. • PRL-R antagonists may modulate lysosomal and mitochondrial Zn pools

  14. Prolactin receptor attenuation induces zinc pool redistribution through ZnT2 and decreases invasion in MDA-MB-453 breast cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Bostanci, Zeynep, E-mail: zbostanci@hmc.psu.edu [The Pennsylvania State University, Department of Nutritional Sciences, 209 Chandlee Lab, University Park, PA 16802 (United States); The Pennsylvania State University Milton S. Hershey Medical Center, Department of Surgery, 500 University Dr., Hershey, PA 17033 (United States); Alam, Samina, E-mail: sra116@psu.edu [The Pennsylvania State University, Department of Nutritional Sciences, 209 Chandlee Lab, University Park, PA 16802 (United States); The Pennsylvania State University Milton S. Hershey Medical Center, Department of Surgery, 500 University Dr., Hershey, PA 17033 (United States); Soybel, David I., E-mail: dsoybel@hmc.psu.edu [The Pennsylvania State University, Department of Nutritional Sciences, 209 Chandlee Lab, University Park, PA 16802 (United States); The Pennsylvania State University Milton S. Hershey Medical Center, Department of Surgery, 500 University Dr., Hershey, PA 17033 (United States); The Pennsylvania State University College of Medicine, Department of Cell and Molecular Physiology, 500 University Dr., Hershey, PA 17033 (United States); Kelleher, Shannon L., E-mail: slk39@psu.edu [The Pennsylvania State University, Department of Nutritional Sciences, 209 Chandlee Lab, University Park, PA 16802 (United States); The Pennsylvania State University Milton S. Hershey Medical Center, Department of Surgery, 500 University Dr., Hershey, PA 17033 (United States); The Pennsylvania State University College of Medicine, Department of Cell and Molecular Physiology, 500 University Dr., Hershey, PA 17033 (United States)

    2014-02-15

    Prolactin receptor (PRL-R) activation regulates cell differentiation, proliferation, cell survival and motility of breast cells. Prolactin (PRL) and PRL-R over-expression are strongly implicated in breast cancer, particularly contributing to tumor growth and invasion in the more aggressive estrogen-receptor negative (ER−) disease. PRL-R antagonists have been suggested as potential therapeutic agents; however, mechanisms through which PRL-R antagonists exert their actions are not well-understood. Zinc (Zn) is a regulatory factor for over 10% of the proteome, regulating critical cell processes such as proliferation, cell signaling, transcription, apoptosis and autophagy. PRL-R signaling regulates Zn metabolism in breast cells. Herein we determined effects of PRL-R attenuation on cellular Zn metabolism and cell function in a model of ER-, PRL-R over-expressing breast cancer cells (MDA-MB-453). PRL-R attenuation post-transcriptionally increased ZnT2 abundance and redistributed intracellular Zn pools into lysosomes and mitochondria. ZnT2-mediated lysosomal Zn sequestration was associated with reduced matrix metalloproteinase 2 (MMP-2) activity and decreased invasion. ZnT2-mediated Zn accumulation in mitochondria was associated with increased mitochondrial oxidation. Our results suggest that PRL-R antagonism in PRL-R over-expressing breast cancer cells may reduce invasion through the redistribution of intracellular Zn pools critical for cellular function. - Highlights: • PRL-R attenuation increased ZnT2 expression. • PRL-R attenuation increased lysosomal and mitochondrial Zn accumulation. • PRL-R attenuation decreased MMP-2 and invasion. • PRL-R antagonists may modulate lysosomal and mitochondrial Zn pools.

  15. Stroke promotes survival of nearby transplanted neural stem cells by decreasing their activation of caspase 3 while not affecting their differentiation.

    Science.gov (United States)

    Kosi, Nina; Alić, Ivan; Salamon, Iva; Mitrečić, Dinko

    2018-02-14

    Although transplantation of stem cells improves recovery of the nervous tissue, little is known about the influence of different brain regions on transplanted cells. After we confirmed that cells with uniform differentiation potential can be generated in independent experiments, one million of neural stem cells isolated from B6.Cg-Tg(Thy1-YFP)16Jrs/J mouse embryos were transplanted into the brain 24 h after induction of stroke. The lateral ventricles, the corpus callosum and the striatum were tested. Two and four weeks after the transplantation, the cells transplanted in all three regions have been attracted to the ischemic core. The largest number of attracted cells has been observed after transplantation into the striatum. Their differentiation pattern and expression of neuroligin 1, SynCAM 1, postsynaptic density protein 95 and synapsin 1 followed the same pattern observed during in vitro cultivation and it did not differ among the tested regions. Differentiation pattern of the cells transplanted in the stroke-affected and healthy animals was the same. On the other hand, neural stem cells transplanted in the striatum of the animals affected by stroke exhibited significantly increased survival rates reaching 260 ± 19%, when compared to cells transplanted in their wild type controls. Surprisingly, improved survival two and four weeks after transplantation was not due to increased proliferation of the grafted cells and it was accompanied by decreased levels of activity of Casp3 (19.56 ± 3.1% in the stroke-affected vs. 30.14 ± 2.4% in healthy animals after four weeks). We assume that the decreased levels of Casp3 in cells transplanted near the ischemic region was linked to increased vasculogenesis, synaptogenesis, astrocytosis and axonogenesis detected in the host tissue affected by ischemia. Copyright © 2017 Elsevier B.V. All rights reserved.

  16. Bile acid receptor TGR5 overexpression is associated with decreased intestinal mucosal injury and epithelial cell proliferation in obstructive jaundice.

    Science.gov (United States)

    Ji, Chen-Guang; Xie, Xiao-Li; Yin, Jie; Qi, Wei; Chen, Lei; Bai, Yun; Wang, Na; Zhao, Dong-Qiang; Jiang, Xiao-Yu; Jiang, Hui-Qing

    2017-04-01

    Bile acids stimulate intestinal epithelial proliferation in vitro. We sought to investigate the role of the bile acid receptor TGR5 in the protection of intestinal epithelial proliferation in obstructive jaundice. Intestinal tissues and serum samples were obtained from patients with malignant obstructive jaundice and from bile duct ligation (BDL) rats. Intestinal permeability and morphological changes in the intestinal mucosa were observed. The functions of TGR5 in cell proliferation in intestinal epithelial injury were determined by overexpression or knockdown studies in Caco-2 and FHs 74 Int cells pretreated with lipopolysaccharide (LPS). Internal biliary drainage was superior to external biliary drainage in recovering intestinal permeability and mucosal histology in patients with obstructive jaundice. In BDL rats, feeding of chenodeoxycholic acid (CDCA) decreased intestinal mucosa injury. The levels of PCNA, a marker of proliferation, increased in response to CDCA feeding and were paralleled by elevated TGR5 expression. CDCA upregulated TGR5 expression and promoted proliferation in Caco-2 and FHs 74 Int cells pretreated with LPS. Overexpression of TGR5 resulted in increased PCNA, cell viability, EdU incorporation, and the proportion of cells in S phase, whereas knockdown of TGR5 had the opposite effect. Our data indicate that bile acids promote intestinal epithelial cell proliferation and decrease mucosal injury by upregulating TGR5 expression in obstructive jaundice. Copyright © 2016 Elsevier Inc. All rights reserved.

  17. Brazilian Red Propolis Induces Apoptosis-Like Cell Death and Decreases Migration Potential in Bladder Cancer Cells

    Directory of Open Access Journals (Sweden)

    Karine Rech Begnini

    2014-01-01

    Full Text Available Natural products continue to be an invaluable resource of anticancer drug discovery in recent years. Propolis is known for its biological activities such as antimicrobial and antitumor effects. This study assessed the effects of Brazilian red propolis (BRP on apoptosis and migration potential in human bladder cancer cells. The effect of BRP ethanolic extract (25, 50, and 100 μg/mL on 5637 cells was determined by MTT, LIVE/DEAD, and migration (scratch assay assays. Apoptosis induction was investigated through flow cytometry and gene expression profile was investigated by qRT-PCR. Results showed cytotoxicity on MTT and LIVE/DEAD assays, with IC50 values of 95 μg/mL in 24 h of treatment. Cellular migration of 5637 cells was significantly inhibited through lower doses of BRP ethanolic extract (25 and 50 μg/mL. Flow cytometry analyses showed that BRP induced cytotoxicity through apoptosis-like mechanisms in 5637 cells and qRT-PCR revealed increased levels of Bax/Bcl-2 ratio, p53, AIF, and antioxidant enzymes genes. Data suggest that BRP may be a potential source of drugs to bladder cancer treatment.

  18. Brazilian red propolis induces apoptosis-like cell death and decreases migration potential in bladder cancer cells.

    Science.gov (United States)

    Begnini, Karine Rech; Moura de Leon, Priscila Marques; Thurow, Helena; Schultze, Eduarda; Campos, Vinicius Farias; Martins Rodrigues, Fernanda; Borsuk, Sibele; Dellagostin, Odir Antônio; Savegnago, Lucielli; Roesch-Ely, Mariana; Moura, Sidnei; Padilha, Francine F; Collares, Tiago; Pêgas Henriques, João Antonio; Seixas, Fabiana Kömmling

    2014-01-01

    Natural products continue to be an invaluable resource of anticancer drug discovery in recent years. Propolis is known for its biological activities such as antimicrobial and antitumor effects. This study assessed the effects of Brazilian red propolis (BRP) on apoptosis and migration potential in human bladder cancer cells. The effect of BRP ethanolic extract (25, 50, and 100 μg/mL) on 5637 cells was determined by MTT, LIVE/DEAD, and migration (scratch assay) assays. Apoptosis induction was investigated through flow cytometry and gene expression profile was investigated by qRT-PCR. Results showed cytotoxicity on MTT and LIVE/DEAD assays, with IC50 values of 95 μg/mL in 24 h of treatment. Cellular migration of 5637 cells was significantly inhibited through lower doses of BRP ethanolic extract (25 and 50 μg/mL). Flow cytometry analyses showed that BRP induced cytotoxicity through apoptosis-like mechanisms in 5637 cells and qRT-PCR revealed increased levels of Bax/Bcl-2 ratio, p53, AIF, and antioxidant enzymes genes. Data suggest that BRP may be a potential source of drugs to bladder cancer treatment.

  19. Radiation-induced decrease of CD8+ dendritic cells contributes to Th1/Th2 shift.

    Science.gov (United States)

    Liu, Hu; Li, Bailong; Jia, Xiaojing; Ma, Yan; Gu, Yifeng; Zhang, Pei; Wei, Qun; Cai, Jianming; Cui, Jianguo; Gao, Fu; Yang, Yanyong

    2017-05-01

    Exposure to ionizing radiation (IR) often reduce the helper T (Th) 1 like function, resulting in a Th1/Th2 imbalance, which could affect the efficacy of cancer radiotherapy. As the most potent antigen presenting cells, dendritic cells (DC) can be divided into several subsets with specialized function. However, there is no literature covering the changes of DC subsets and their roles in immune regulation in response to IR. In the present study, we were aimed to investigate the changes of DC subsets after IR and its relationship with Th1/Th2 immunity. We found a significant decrease of BDCA3+DC in the blood of patients treated with radiotherapy. CD8+DC, a mouse equivalent of human BDCA3+DC, was also found decreased in mice spleen, peripheral blood and lymph node tissues after irradiation. As CD8+DC mainly induce Th1 immunity, we tested the changes of Th1/Th2 response and found that IR caused a repression of Th1 immunity, indicating a possible role of CD8+DC in radiation-induced Th1/Th2 imbalance. We also found that a CD8+DC-inducing cytokine, Fms-like tyrosine kinase 3 ligand (FLT3 ligand), restored CD8+DC and reversed Th1/Th2 shift. And then we found that bone marrow cells from irradiated mice differentiated into less CD8+DC, which was also protected by FLT3 ligand. In conclusion, our data showed that IR induced a decrease of CD8+DC and Th1/Th2 shift, which was reversed by Flt3 ligand treatment, suggesting a novel mechanism for radiation-induced immunosuppression. Copyright © 2017. Published by Elsevier B.V.

  20. Gestational lead exposure selectively decreases retinal dopamine amacrine cells and dopamine content in adult mice.

    Science.gov (United States)

    Fox, Donald A; Hamilton, W Ryan; Johnson, Jerry E; Xiao, Weimin; Chaney, Shawntay; Mukherjee, Shradha; Miller, Diane B; O'Callaghan, James P

    2011-11-01

    Gestational lead exposure (GLE) produces supernormal scotopic electroretinograms (ERG) in children, monkeys and rats, and a novel retinal phenotype characterized by an increased number of rod photoreceptors and bipolar cells in adult mice and rats. Since the loss of dopaminergic amacrine cells (DA ACs) in GLE monkeys and rats contributes to supernormal ERGs, the retinal DA system was analyzed in mice following GLE. C57BL/6 female mice were exposed to low (27 ppm), moderate (55 ppm) or high (109 ppm) lead throughout gestation and until postnatal day 10 (PN10). Blood [Pb] in control, low-, moderate- and high-dose GLE was ≤ 1, ≤ 10, ~25 and ~40 μg/dL, respectively, on PN10 and by PN30 all were ≤ 1 μg/dL. At PN60, confocal-stereology studies used vertical sections and wholemounts to characterize tyrosine hydroxylase (TH) expression and the number of DA and other ACs. GLE dose-dependently and selectively decreased the number of TH-immunoreactive (IR) DA ACs and their synaptic plexus without affecting GABAergic, glycinergic or cholinergic ACs. Immunoblots and confocal revealed dose-dependent decreases in retinal TH protein expression and content, although monoamine oxidase-A protein and gene expression were unchanged. High-pressure liquid chromatography showed that GLE dose-dependently decreased retinal DA content, its metabolites and DA utilization/release. The mechanism of DA selective vulnerability is unknown. However, a GLE-induced loss/dysfunction of DA ACs during development could increase the number of rods and bipolar cells since DA helps regulate neuronal proliferation, whereas during adulthood it could produce ERG supernormality as well as altered circadian rhythms, dark/light adaptation and spatial contrast sensitivity. Copyright © 2011 Elsevier Inc. All rights reserved.

  1. Natural killer cell lines preferentially kill clonogenic multiple myeloma cells and decrease myeloma engraftment in a bioluminescent xenograft mouse model.

    Science.gov (United States)

    Swift, Brenna E; Williams, Brent A; Kosaka, Yoko; Wang, Xing-Hua; Medin, Jeffrey A; Viswanathan, Sowmya; Martinez-Lopez, Joaquin; Keating, Armand

    2012-07-01

    Novel therapies capable of targeting drug resistant clonogenic MM cells are required for more effective treatment of multiple myeloma. This study investigates the cytotoxicity of natural killer cell lines against bulk and clonogenic multiple myeloma and evaluates the tumor burden after NK cell therapy in a bioluminescent xenograft mouse model. The cytotoxicity of natural killer cell lines was evaluated against bulk multiple myeloma cell lines using chromium release and flow cytometry cytotoxicity assays. Selected activating receptors on natural killer cells were blocked to determine their role in multiple myeloma recognition. Growth inhibition of clonogenic multiple myeloma cells was assessed in a methylcellulose clonogenic assay in combination with secondary replating to evaluate the self-renewal of residual progenitors after natural killer cell treatment. A bioluminescent mouse model was developed using the human U266 cell line transduced to express green fluorescent protein and luciferase (U266eGFPluc) to monitor disease progression in vivo and assess bone marrow engraftment after intravenous NK-92 cell therapy. Three multiple myeloma cell lines were sensitive to NK-92 and KHYG-1 cytotoxicity mediated by NKp30, NKp46, NKG2D and DNAM-1 activating receptors. NK-92 and KHYG-1 demonstrated 2- to 3-fold greater inhibition of clonogenic multiple myeloma growth, compared with killing of the bulk tumor population. In addition, the residual colonies after treatment formed significantly fewer colonies compared to the control in a secondary replating for a cumulative clonogenic inhibition of 89-99% at the 20:1 effector to target ratio. Multiple myeloma tumor burden was reduced by NK-92 in a xenograft mouse model as measured by bioluminescence imaging and reduction in bone marrow engraftment of U266eGFPluc cells by flow cytometry. This study demonstrates that NK-92 and KHYG-1 are capable of killing clonogenic and bulk multiple myeloma cells. In addition, multiple myeloma

  2. Dithiolethione modified valproate and diclofenac increase E-cadherin expression and decrease proliferation of non-small cell lung cancer cells.

    Science.gov (United States)

    Moody, Terry W; Switzer, Christopher; Santana-Flores, Wilmarie; Ridnour, Lisa A; Berna, Marc; Thill, Michelle; Jensen, Robert T; Sparatore, Anna; Del Soldato, Piero; Yeh, Grace C; Roberts, David D; Giaccone, Giuseppe; Wink, David A

    2010-05-01

    The effects of dithiolethione modified valproate, diclofenac and sulindac on non-small cell lung cancer (NSCLC) cells were investigated. Sulfur(S)-valproate and S-diclofenac at 1 microg/ml concentrations significantly reduced prostaglandin (PG)E(2) levels in NSCLC cell lines A549 and NCI-H1299 as did the COX-2 inhibitor DuP-697. In vitro, S-valproate, S-diclofenac and S-sulindac half-maximally inhibited the clonal growth of NCI-H1299 cells at 6, 6 and 15 microg/ml, respectively. Using the MTT assay, 10 microg/ml S-valproate, NO-aspirin and Cay10404, a selective COX-2 inhibitor, but not SC-560, a selective COX-1 inhibitor, inhibited the growth of A549 cells. In vivo, 18mg/kg i.p. of S-valproate and S-diclofenac, but not S-sulindac, significantly inhibited A549 or NCI-H1299 xenograft proliferation in nude mice, but had no effect on the nude mouse body weight. The mechanism by which S-valproate and S-diclofenac inhibited the growth of NSCLC cells was investigated. Nitric oxide-aspirin but not S-valproate caused apoptosis of NSCLC cells. By Western blot, S-valproate and S-diclofenac increased E-cadherin but reduced vimentin and ZEB1 (a transcriptional suppressor of E-cadherin) protein expression in NSCLC cells. Because S-valproate and S-diclofenac inhibit the growth of NSCLC cells and reduce PGE(2) levels, they may prove beneficial in the chemoprevention and/or therapy of NSCLC. Published by Elsevier Ireland Ltd.

  3. Decreased SAP expression in T cells from patients with SLE contributes to early signaling abnormalities and reduced IL-2 production

    Science.gov (United States)

    Karampetsou, Maria P.; Comte, Denis; Kis-Toth, Katalin; Terhorst, Cox; Kyttaris, Vasileios C.; Tsokos, George C.

    2016-01-01

    T cells from patients with systemic lupus erythematosus (SLE) display a number of functions including increased early signaling events following engagement of the T cell receptor (TCR). Signaling lymphocytic activation molecule family (SLAMF) cell surface receptors and the X-chromosome-defined signaling lymphocytic activation molecule-associated protein (SAP) adaptor are important in the development of several immunocyte lineages and modulating immune response. Here we present evidence that SAP protein levels are decreased in T cells and in their main subsets isolated from 32 women and 3 men with SLE independently of disease activity. In SLE T cells the SAP protein is also subject to increased degradation by a caspase-3. Forced expression of SAP in SLE T cells simultaneously heightened IL-2 production, calcium (Ca2+) responses and tyrosine phosphorylation of a number of proteins. Exposure of normal T cells to SLE serum IgG, known to contain anti-CD3/TCR antibodies, resulted in SAP downregulation. We conclude that SLE T cells display reduced levels of the adaptor protein SAP probably as a result of continuous T cell activation and degradation by caspase-3. Restoration of SAP levels in SLE T cells corrects the overexcitable lupus T cell phenotype. PMID:27183584

  4. Nanoparticle augmented radiation treatment decreases cancer cell proliferation.

    Science.gov (United States)

    Townley, Helen E; Rapa, Elizabeth; Wakefield, Gareth; Dobson, Peter J

    2012-05-01

    We report significant and controlled cell death using novel x-ray-activatable titania nanoparticles (NPs) doped with lanthanides. Preferential incorporation of such materials into tumor tissue can enhance the effect of radiation therapy. Herein, the incorporation of gadolinium into the NPs is designed to optimize localized energy absorption from a conventional medical x-ray. This result is further optimized by the addition of other rare earth elements. Upon irradiation, energy is transferred to the titania crystal structure, resulting in the generation of reactive oxygen species (ROS). The authors report significant and controlled cell death using x-ray-activated titania nanoparticles doped with lanthanides as enhancers. Upon irradiation X-ray energy is transferred to the titania crystal structure, resulting in the generation of reactive oxygen species. Copyright © 2012 Elsevier Inc. All rights reserved.

  5. Alginate gelation-induced cell death during laser-assisted cell printing

    International Nuclear Information System (INIS)

    Gudapati, Hemanth; Yan, Jingyuan; Huang, Yong; Chrisey, Douglas B

    2014-01-01

    Modified laser-induced forward transfer has emerged as a promising bioprinting technique. Depending on the operating conditions and cell properties, laser cell printing may cause cell injury and even death, which should be carefully elucidated for it to be a viable technology. This study has investigated the effects of alginate gelation, gelation time, alginate concentration, and laser fluence on the post-transfer cell viability of NIH 3T3 fibroblasts. Sodium alginate and calcium chloride are used as the gel precursor and gel reactant solution to form cell-laden alginate microspheres. It is found that the effects of gelation depend on the duration of gelation. Two-minute gelation is observed to increase the cell viability after 24 h incubation, mainly due to the protective cushion effect of the forming gel membrane during droplet landing. Despite the cushion effect from 10 min gelation, it is observed that the cell viability decreases after 24 h incubation because of the forming thick gel membrane that reduces nutrient and oxygen diffusion from the culture medium. In addition, the cell viability after 24 h incubation decreases as the laser fluence or alginate concentration increases. (paper)

  6. Cell damage evaluation of mammalian cells in cell manipulation by amplified femtosecond ytterbium laser

    Science.gov (United States)

    Hong, Z.-Y.; Iino, T.; Hagihara, H.; Maeno, T.; Okano, K.; Yasukuni, R.; Hosokawa, Y.

    2018-03-01

    A micrometer-scale explosion with cavitation bubble generation is induced by focusing a femtosecond laser in an aqueous solution. We have proposed to apply the explosion as an impulsive force to manipulate mammalian cells especially in microfluidic chip. Herein, we employed an amplified femtosecond ytterbium laser as an excitation source for the explosion and evaluated cell damage in the manipulation process to clarify the application potential. The damage of C2C12 myoblast cell prepared as a representative mammalian cell was investigated as a function of distance between cell and laser focal point. Although the cell received strong damage on the direct laser irradiation condition, the damage sharply decreased with increasing distance. Since the threshold distance, above which the cell had no damage, was consistent with radius of the cavitation bubble, impact of the cavitation bubble would be a critical factor for the cell damage. The damage had strong nonlinearity in the pulse energy dependence. On the other hand, cell position shift by the impact of the cavitation bubble was almost proportional to the pulse energy. In balance between the cell viability and the cell position shift, we elucidated controllability of the cell manipulation in microfluidic chip.

  7. Actinomycin D synergistically enhances the cytotoxicity of CDDP on KB cells by activating P53 via decreasing P53-MDM2 complex.

    Science.gov (United States)

    Wang, Lin; Pang, Xiao-Cong; Yu, Zi-Ru; Yang, Sheng-Qian; Liu, Ai-Lin; Wang, Jin-Hua; Du, Guan-Hua

    2017-06-01

    The aim of this study is to investigate the synergism of low dose of actinomycin D (LDActD) to the cytotoxicity of cisplatin (CDDP) on KB cells. The role of P53 reactivation by LDActD in the synergism and its mechanism were further studied. Cell viability was determined by MTT assay. Apoptosis was determined by AnnexinV-FITC/PI staining. Mitochondrial membrane potential (MMP) was detected by JC-1 staining. Expression of proteins was detected by Western blotting (WB) and/or immunofluorescence (IF). Molecular docking of actinomycin D (ACTD) to Mouse double minute 2 homolog (MDM2) and Mouse double minute 2 homolog X (MDMX). MDMX was analyzed by Discovery Studio. The content of P53-MDM2 complex was detected by ELISA assay. The cytotoxicity of CDDP was increased by the combination of LDActD in kinds of cancer cells. Molecular docking showed strong interaction between ACTD and MDM2/MDMX. Meanwhile, LDActD significantly decreased P53-MDM2 complex. Significant increase of the apoptotic activity by the combination therapy in KB cells is P53 upregulated modulator of apoptosis (PUMA) dependent. In addition to the decrease in MMP, LDActD increased P53 regulated protein and decreased BCL-XL in KB cells. LDActD efficiently enhanced the cytotoxicity of CDDP in cancer cells and induced P53-PUMA-dependent and mitochondria-mediated apoptosis in KB cells. The reactivation of P53 was probably achieved by disturbing the interaction of P53 and MDM2/MDMX.

  8. Intraportal injection of insulin-producing cells generated from human bone marrow mesenchymal stem cells decreases blood glucose level in diabetic rats.

    Science.gov (United States)

    Tsai, Pei-Jiun; Wang, Hwai-Shi; Lin, Chi-Hung; Weng, Zen-Chung; Chen, Tien-Hua; Shyu, Jia-Fwu

    2014-01-01

    We studied the process of trans-differentiation of human bone marrow mesenchymal stem cells (hBM-MSCs) into insulin-producing cells. Streptozotocin (STZ)-induced diabetic rat model was used to study the effect of portal vein transplantation of these insulin-producing cells on blood sugar levels. The BM-MSCs were differentiated into insulin-producing cells under defined conditions. Real-time PCR, immunocytochemistry and glucose challenge were used to evaluate in vitro differentiation. Flow cytometry showed that hBM-MSCs were strongly positive for CD44, CD105 and CD73 and negative for hematopoietic markers CD34, CD38 and CD45. Differentiated cells expressed C-peptide as well as β-cells specific genes and hormones. Glucose stimulation increased C-peptide secretion in these cells. The insulin-producing, differentiated cells were transplanted into the portal vein of STZ-induced diabetic rats using a Port-A catheter. The insulin-producing cells were localized in the liver of the recipient rat and expressed human C-peptide. Blood glucose levels were reduced in diabetic rats transplanted with insulin-producing cells. We concluded that hBM-MSCs could be trans-differentiated into insulin-producing cells in vitro. Portal vein transplantation of insulin-producing cells alleviated hyperglycemia in diabetic rats.

  9. Rice Hull Extract Suppresses Benign Prostate Hyperplasia by Decreasing Inflammation and Regulating Cell Proliferation in Rats.

    Science.gov (United States)

    Kim, Chae-Yun; Chung, Kyung-Sook; Cheon, Se-Yun; Lee, Jong-Hyun; Park, Youn-Bum; An, Hyo-Jin

    2016-08-01

    Even though rice hull has various physiological functions with high antioxidant potential, the molecular mechanism(s) underlying the effects of rice hull on benign prostatic hyperplasia (BPH) have not been evaluated. The aim of this study was to determine the protective effect of rice hull water extract (RHE) against BPH, which is a common disorder in elderly men and involves inflammation that induces an imbalance between cell proliferation and cell death. In this study, RHE-treated mice exhibited lower prostate weights and ratios of prostate weight to body weight compared to those for the BPH-induced group. In addition, RHE-treated mice had lower serum levels of dihydrotestosterone, mRNA expression of 5α-reductase2, and protein expressions of proliferating cell nuclear antigen (PCNA). Furthermore, RHE treatment significantly decreased cell proliferation by regulating the expression levels of inflammatory-related proteins (iNOS and COX-2) and apoptosis-associated proteins (Fas, FADD, procaspase-8, -3, and Bcl-2 family proteins). These results suggest that RHE could protect against the development of BPH through its anti-inflammatory and apoptotic properties and has good potential as a treatment for BPH.

  10. Nicotinamide induces differentiation of embryonic stem cells into insulin-secreting cells

    International Nuclear Information System (INIS)

    Vaca, Pilar; Berna, Genoveva; Araujo, Raquel; Carneiro, Everardo M.; Bedoya, Francisco J.; Soria, Bernat; Martin, Franz

    2008-01-01

    The poly(ADP-ribose) polymerase (PARP) inhibitor, nicotinamide, induces differentiation and maturation of fetal pancreatic cells. In addition, we have previously reported evidence that nicotinamide increases the insulin content of cells differentiated from embryonic stem (ES) cells, but the possibility of nicotinamide acting as a differentiating agent on its own has never been completely explored. Islet cell differentiation was studied by: (i) X-gal staining after neomycin selection; (ii) BrdU studies; (iii) single and double immunohistochemistry for insulin, C-peptide and Glut-2; (iv) insulin and C-peptide content and secretion assays; and (v) transplantation of differentiated cells, under the kidney capsule, into streptozotocin (STZ)-diabetic mice. Here we show that undifferentiated mouse ES cells treated with nicotinamide: (i) showed an 80% decrease in cell proliferation; (ii) co-expressed insulin, C-peptide and Glut-2; (iii) had values of insulin and C-peptide corresponding to 10% of normal mouse islets; (iv) released insulin and C-peptide in response to stimulatory glucose concentrations; and (v) after transplantation into diabetic mice, normalized blood glucose levels over 7 weeks. Our data indicate that nicotinamide decreases ES cell proliferation and induces differentiation into insulin-secreting cells. Both aspects are very important when thinking about cell therapy for the treatment of diabetes based on ES cells

  11. Long none coding RNA HOTTIP/HOXA13 act as synergistic role by decreasing cell migration and proliferation in Hirschsprung disease

    International Nuclear Information System (INIS)

    Xie, Hua; Zhu, Dongmei; Xu, Cao; Zhu, Hairong; Chen, Pingfa; Li, Hongxing; Liu, Xiang; Xia, Yankai; Tang, Weibing

    2015-01-01

    Long noncoding RNAs (lncRNAs) have been confirmed to be associated with various human diseases. However, whether they are associated with Hirschsprung disease (HSCR) progression remains unclear. In this study, we designed the experiment to explore the relationship between lncRNA HOTTIP and HOXA13, and their pathogenicity to HSCR. Quantitative real-time PCR and Western blot were performed to detect the levels of lncRNA, mRNAs, and proteins in colon tissues from 79 patients with HSCR and 79 controls. Small RNA interference transfection was used to study the function experiments in human 293T and SK-N-BE cell lines. The cell viability and activities were detected by the transwell assays, CCK8 assay, and flow cytometry, respectively. LncRNA HOTTIP and HOXA13 were significantly down-regulated in HSCR compared to the controls. Meanwhile, the declined extent of their expression levels makes sense between two main phenotype of HSCR. SiRNA-mediated knock-down of HOTTIP or HOXA13 correlated with decreased levels of each other and both reduced the cell migration and proliferation without affecting cell apoptosis or cell cycle. Our study demonstrates that aberrant reduction of HOTTIP and HOXA13, which have a bidirectional regulatory loop, may play an important role in the pathogenesis of HSCR. - Highlights: • LncRNA HOTTIP and HOXA13 are both down-regulated in HSCR. • HOTTIP and HOXA13 can regulate each other in 293T and SK-N-BE(2) cell lines. • Both HOTTIP and HOXA13 can decrease cell migration and proliferation

  12. Long none coding RNA HOTTIP/HOXA13 act as synergistic role by decreasing cell migration and proliferation in Hirschsprung disease

    Energy Technology Data Exchange (ETDEWEB)

    Xie, Hua; Zhu, Dongmei; Xu, Cao; Zhu, Hairong; Chen, Pingfa; Li, Hongxing [Department of Pediatric Surgery, State Key Laboratory of Reproductive Medicine, Nanjing Children' s Hospital Affiliated Nanjing Medical University, Nanjing 210008 (China); Key Laboratory of Modern Toxicology (Nanjing Medical University), Institute of Toxicology, School of Public Health, Nanjing Medical University, Ministry of Education, Nanjing 211166 (China); Liu, Xiang [Department of Pediatric Surgery, Anhui Provincial Children' s Hospital, Anhui 230000 (China); Xia, Yankai [Department of Pediatric Surgery, State Key Laboratory of Reproductive Medicine, Nanjing Children' s Hospital Affiliated Nanjing Medical University, Nanjing 210008 (China); Key Laboratory of Modern Toxicology (Nanjing Medical University), Institute of Toxicology, School of Public Health, Nanjing Medical University, Ministry of Education, Nanjing 211166 (China); Tang, Weibing, E-mail: twbcn@njmu.com [Department of Pediatric Surgery, State Key Laboratory of Reproductive Medicine, Nanjing Children' s Hospital Affiliated Nanjing Medical University, Nanjing 210008 (China); Key Laboratory of Modern Toxicology (Nanjing Medical University), Institute of Toxicology, School of Public Health, Nanjing Medical University, Ministry of Education, Nanjing 211166 (China)

    2015-08-07

    Long noncoding RNAs (lncRNAs) have been confirmed to be associated with various human diseases. However, whether they are associated with Hirschsprung disease (HSCR) progression remains unclear. In this study, we designed the experiment to explore the relationship between lncRNA HOTTIP and HOXA13, and their pathogenicity to HSCR. Quantitative real-time PCR and Western blot were performed to detect the levels of lncRNA, mRNAs, and proteins in colon tissues from 79 patients with HSCR and 79 controls. Small RNA interference transfection was used to study the function experiments in human 293T and SK-N-BE cell lines. The cell viability and activities were detected by the transwell assays, CCK8 assay, and flow cytometry, respectively. LncRNA HOTTIP and HOXA13 were significantly down-regulated in HSCR compared to the controls. Meanwhile, the declined extent of their expression levels makes sense between two main phenotype of HSCR. SiRNA-mediated knock-down of HOTTIP or HOXA13 correlated with decreased levels of each other and both reduced the cell migration and proliferation without affecting cell apoptosis or cell cycle. Our study demonstrates that aberrant reduction of HOTTIP and HOXA13, which have a bidirectional regulatory loop, may play an important role in the pathogenesis of HSCR. - Highlights: • LncRNA HOTTIP and HOXA13 are both down-regulated in HSCR. • HOTTIP and HOXA13 can regulate each other in 293T and SK-N-BE(2) cell lines. • Both HOTTIP and HOXA13 can decrease cell migration and proliferation.

  13. Role of BK channels in the apoptotic volume decrease in native eel intestinal cells

    DEFF Research Database (Denmark)

    Lionetto, Maria Giulia; Giordano, Maria Elena; Calisi, Antonio

    2010-01-01

    High conductance Ca(+)-activated K(+) channels (BK channels) have previously been demonstrated in the eel intestine. They are specifically activated following a hypotonic stress and sustain Regulatory Volume Decrease (RVD). The aim of the present work was to address the possible role...... enterocytes that BK channels, which are involved in RVD in these cells, plays also a crucial role in the AVD process and in the progression of apoptosis....

  14. MiR-133b is frequently decreased in gastric cancer and its overexpression reduces the metastatic potential of gastric cancer cells

    International Nuclear Information System (INIS)

    Zhao, Yu; Zhu, Zhenggang; Huang, Jie; Zhang, Li; Qu, Ying; Li, Jianfang; Yu, Beiqin; Yan, Min; Yu, Yingyan; Liu, Bingya

    2014-01-01

    Emerging evidence has shown that microRNAs are involved in gastric cancer development and progression. Here we examine the role of miR-133b in gastric cancer. Quantitative real-time PCR analysis was performed in 140 patient gastric cancer tissues and 8 gastric cancer cell lines. The effects of miR-133b in gastric cancer cells metastasis were examined by scratch assay, transwell migration and matrigel invasion. In vivo effects of miR-133b were examined in an intraperitoneal mouse tumor model. Targets of miR-133b were predicted by bioinformatics tools and validated by luciferase reporter analyses, western blot, and quantitative real-time PCR. MiR-133b was significantly downregulated in 70% (98/140) of gastric cancer patients. Expression of miR-133b was negatively correlated with lymph node metastasis of gastric cancer in patients. Similarly, the expression of miR-133b was significantly lower in seven tested gastric cancer cell lines than in the immortalized non-cancerous GES-1 gastric epithelial cells. Overexpression of miR-133b markedly inhibited metastasis of gastric cancer cells in vitro and in vivo. Moreover, the transcriptional factor Gli1 was identified as a direct target for miR-133b. Level of Gli1 protein but not mRNA was decreased by miR-133b. Activity of luciferase with Gli1 3′-untranslated region was markedly decreased by miR-133b in gastric cancer cells. Gli1 target genes, OPN and Zeb2, were also inhibited by miR133b. MiR-133b is frequently decreased in gastric cancer. Overexpression of miR-133b inhibits cell metastasis in vitro and in vivo partly by directly suppressing expression of Gli1 protein. These results suggested that miR-133b plays an important role in gastric cancer metastasis

  15. Dithiolethione modified valproate and diclofenac increase E-cadherin expression and decrease proliferation of non-small cell lung cancer cells

    OpenAIRE

    Moody, Terry W.; Switzer, Christopher; Santana-Flores, Wilmarie; Ridnour, Lisa A.; Berna, Marc; Thill, Michelle; Jensen, Robert T.; Sparatore, Anna; Del Soldato, Piero; Yeh, Grace C; Roberts, David D.; Giaccone, Giuseppe; Wink, David A.

    2009-01-01

    The effects of dithiolethione-modified valproate, diclofenac and sulindac on non-small cell lung cancer (NSCLC) cells were investigated. Sulfur(S)-valproate and S-diclofenac at 1 μg/ml concentrations significantly reduced prostaglandin (PG)E2 levels in NSCLC cell lines A549 and NCI-H1299 as did the COX-2 inhibitor DuP-697. In vitro, S-valproate, S-diclofenac and S-sulindac half-maximally inhibited the clonal growth of NCI-H1299 cells at 6, 6 and 15 μg/ml, respectively. Using the MTT assay, 10...

  16. The cell-free fetal DNA fraction in maternal blood decreases after physical activity

    DEFF Research Database (Denmark)

    Schlütter, Jacob Mørup; Hatt, Lotte; Bach, Cathrine

    2014-01-01

    of cycling with a pulse-rate of 150 beats per minute. The concentrations of cffDNA (DYS14) and cfDNA (RASSF1A) were assessed using quantitative real-time polymerase chain reaction. RESULTS: The fetal fraction decreased significantly in all participants after physical activity (p decrease varying......OBJECTIVE: If noninvasive prenatal testing using next generation sequencing is to be effective for pregnant women, a cell-free fetal DNA (cffDNA) fraction above 4% is essential unless the depth of sequencing is increased. This study's objective is to determine whether physical activity has...... from 1-17 percentage points. This was due to a significant increase in the concentration of cfDNA (p physical activity. CONCLUSION: When planning the timing of noninvasive...

  17. Schisandrin B protects PC12 cells by decreasing the expression of amyloid precursor protein and vacuolar protein sorting 35★

    Science.gov (United States)

    Yan, Mingmin; Mao, Shanping; Dong, Huimin; Liu, Baohui; Zhang, Qian; Pan, Gaofeng; Fu, Zhiping

    2012-01-01

    PC12 cell injury was induced using 20 μM amyloid β-protein 25–35 to establish a model of Alzheimer's disease. The cells were then treated with 5, 10, and 25 μM Schisandrin B. Methylthiazolyldiphenyl-tetrazolium bromide assays and Hoechst 33342 staining results showed that with increasing Schisandrin B concentration, the survival rate of PC12 cells injured by amyloid β-protein 25–35 gradually increased and the rate of apoptosis gradually decreased. Reverse transcription-PCR, immunocytochemical staining and western blot results showed that with increasing Schisandrin B concentration, the mRNA and protein expression of vacuolar protein sorting 35 and amyloid precursor protein were gradually decreased. Vacuolar protein sorting 35 and amyloid precursor protein showed a consistent trend for change. These findings suggest that 5, 10, and 25 μM Schisandrin B antagonizes the cellular injury induced by amyloid β-protein 25–35 in a dose-dependent manner. This may be caused by decreasing the expression of vacuolar protein sorting 35 and amyloid precursor protein. PMID:25745458

  18. 4-IBP, a σ1 Receptor Agonist, Decreases the Migration of Human Cancer Cells, Including Glioblastoma Cells, In Vitro and Sensitizes Them In Vitro and In Vivo to Cytotoxic Insults of Proapoptotic and Proautophagic Drugs

    Directory of Open Access Journals (Sweden)

    Veronique Mégalizzi

    2007-05-01

    Full Text Available Although the molecular function of cr receptors has not been fully defined and the natural ligand(s is still not known, there is increasing evidence that these receptors and their ligands might play a significant role in cancer biology. 4-(N-tibenzylpiperidin-4-yl-4iodobenzamide (4-IBP, a selective σ1, agonist, has been used to investigate whether this compound is able to modify: 1 in vitro the migration and proliferation of human cancer cells; 2 in vitro the sensitivity of human glioblastoma cells to cytotoxic drugs; and 3 in vivo in orthotopic glioblastoma and non-small cell lung carcinoma (NSCLC models the survival of mice coadministered cytotoxic agents. 4-IBP has revealed weak anti proliferative effects on human U373-MG glioblastoma and C32 melanoma cells but induced marked concentration-dependent decreases in the growth of human A549 NSCLC and PC3 prostate cancer cells. The compound was also significantly antimigratory in all four cancer cell lines. This may result, at least in U373-MG cells, from modifications to the actin cytoskeleton. 4-IBP modified the sensitivity of U373-MG cells in vitro to proapoptotic lomustin and proautophagic temozolomide, and markedly decreased the expression of two proteins involved in drug resistance: glucosylceramide synthase and Rho guanine nucleotide dissociation inhibitor. In vivo, 4-IBP increased the antitumor effects of temozolomide and irinotecan in immunodeficient mice that were orthotopically grafted with invasive cancer cells.

  19. Acrolein decreases endothelial cell migration and insulin sensitivity through induction of let-7a.

    Science.gov (United States)

    O'Toole, Timothy E; Abplanalp, Wesley; Li, Xiaohong; Cooper, Nigel; Conklin, Daniel J; Haberzettl, Petra; Bhatnagar, Aruni

    2014-08-01

    Acrolein is a major reactive component of vehicle exhaust, and cigarette and wood smoke. It is also present in several food substances and is generated endogenously during inflammation and lipid peroxidation. Although previous studies have shown that dietary or inhalation exposure to acrolein results in endothelial activation, platelet activation, and accelerated atherogenesis, the basis for these effects is unknown. Moreover, the effects of acrolein on microRNA (miRNA) have not been studied. Using AGILENT miRNA microarray high-throughput technology, we found that treatment of cultured human umbilical vein endothelial cells with acrolein led to a significant (>1.5-fold) upregulation of 12, and downregulation of 15, miRNAs. Among the miRNAs upregulated were members of the let-7 family and this upregulation was associated with decreased expression of their protein targets, β3 integrin, Cdc34, and K-Ras. Exposure to acrolein attenuated β3 integrin-dependent migration and reduced Akt phosphorylation in response to insulin. These effects of acrolein on endothelial cell migration and insulin signaling were reversed by expression of a let-7a inhibitor. Also, inhalation exposure of mice to acrolein (1 ppm x 6 h/day x 4 days) upregulated let-7a and led to a decrease in insulin-stimulated Akt phosphorylation in the aorta. These results suggest that acrolein exposure has broad effects on endothelial miRNA repertoire and that attenuation of endothelial cell migration and insulin signaling by acrolein is mediated in part by the upregulation of let-7a. This mechanism may be a significant feature of vascular injury caused by inflammation, oxidized lipids, and exposure to environmental pollutants. © The Author 2014. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For permissions, please email: journals.permissions@oup.com.

  20. Honokiol Increases CD4+ T Cell Activation and Decreases TNF but Fails to Improve Survival Following Sepsis.

    Science.gov (United States)

    Klingensmith, Nathan J; Chen, Ching-Wen; Liang, Zhe; Burd, Eileen M; Farris, Alton B; Arbiser, Jack L; Ford, Mandy L; Coopersmith, Craig M

    2017-10-11

    Honokiol is a biphenolic isolate extracted from the bark of the magnolia tree that has been used in traditional Chinese and Japanese medicine, and has more recently been investigated for its anti-inflammatory and anti-bacterial properties. Honokiol has previously been demonstrated to improve survival in sepsis models that have rapid 100% lethality. The purpose of this study was to determine the impact of Honokiol on the host response in a model of sepsis that more closely approximates human disease. Male and female C57BL/6 mice underwent cecal ligation and puncture (CLP) to induce polymicrobial intraabdominal sepsis. Mice were then randomized to receive an injection of either Honokiol (120 mg/kg/day) or vehicle and were sacrificed after 24 hours for functional studies or followed 7 days for survival. Honokiol treatment after sepsis increased the frequency of CD4 T cells and increased activation of CD4 T cells as measured by the activation marker CD69. Honokiol also increased splenic dendritic cells. Honokiol simultaneously decreased frequency and number of CD8 T cells. Honokiol decreased systemic TNF without impacting other systemic cytokines. Honokiol did not have a detectable effect on kidney function, lung physiology, liver function or intestinal integrity. In contrast to prior studies of Honokiol in a lethal model of sepsis, Honokiol did not alter survival at seven days (70% mortality for Honokiol vs. 60% mortality for vehicle). Honokiol is thus effective in modulating the host immune response and inflammation following a clinically relevant model of sepsis but is not sufficient to alter survival.

  1. Aging is associated with decreased maximal life span and accelerated senescence of bone marrow stromal cells

    DEFF Research Database (Denmark)

    Dokkedahl, Karin Stenderup; Justesen, Jeannette; Clausen, Christian

    2003-01-01

    Age-related decrease in bone formation is well described. However, the cellular causes are not known. Thus, we have established cultures of bone marrow stromal cells (MSC) from young (aged 18-29 years, n = 6) and old (aged 68-81 years, n = 5) donors. MSC were serially passaged until reaching maxi...

  2. Decreased neutrophil-associated miRNA and increased B-cell associated miRNA expression during tuberculosis.

    Science.gov (United States)

    van Rensburg, I C; du Toit, L; Walzl, G; du Plessis, N; Loxton, A G

    2018-05-20

    MicroRNAs are short non-coding RNAs that regulate gene expression by binding to, and suppressing the expression of genes. Research show that microRNAs have potential to be used as biomarkers for diagnosis, treatment response and can be used for therapeutic interventions. Furthermore, microRNA expression has effects on immune cell functions, which may lead to disease. Considering the important protective role of neutrophils and B-cells during M.tb infection, we evaluated the expression of microRNAs, known to alter function of these cells, in the context of human TB. We utilised real-time PCR to evaluate the levels of microRNA transcripts in the peripheral blood of TB cases and healthy controls. We found that neutrophil-associated miR-197-3p, miR-99b-5p and miR-191-5p transcript levels were significantly lower in TB cases. Additionally, B-cell-associated miR-320a, miR-204-5p, miR331-3p and other transcript levels were higher in TB cases. The miRNAs differentially expressed in neutrophils are predominantly implicated in signalling pathways leading to cytokine productions. Here, the decreased expression in TB cases may imply a lack of suppression on signalling pathways, which may lead to increased production of pro-inflammatory cytokines such as interferon-gamma. Furthermore, the miRNAs differentially expressed in B-cells are mostly involved in the induction/suppression of apoptosis. Further functional studies are however required to elucidate the significance and functional effects of changes in the expression of these microRNAs. Copyright © 2018 Elsevier B.V. All rights reserved.

  3. Impaired progenitor cell function in HIV-negative infants of HIV-positive mothers results in decreased thymic output and low CD4 counts

    DEFF Research Database (Denmark)

    Nielsen, S. D.; Jeppesen, D. L.; Kolte, L.

    2001-01-01

    and fetal thymic organ cultures (FTOCs). Lower naive CD4 counts (459.3 +/- 68.9 vs 1128.9 +/- 146.8 cells/microL, P mothers were found (frequency of CD4(+) cells with TRECs was 3.6% +/- 0.7% compared with 14.3% +/- 2.2% in controls, P ...). In combination with lower red blood cell counts in infants of HIV-positive mothers, this finding suggested impairment of progenitor cell function. Indeed, progenitors from infants of HIV-positive mothers had decreased cloning efficiency (15.7% +/- 2.6% vs 55.8% +/- 15.9%, P =.009) and seemed to generate fewer T...... cells in FTOCs. In conclusion, lower numbers of naive CD4(+) cells and reduced thymic output in HIV-negative infants of HIV-positive mothers may be due to impaired progenitor cell function....

  4. Inhibition of mutant IDH1 decreases D-2-HG levels without affecting tumorigenic properties of chondrosarcoma cell lines.

    Science.gov (United States)

    Suijker, Johnny; Oosting, Jan; Koornneef, Annemarie; Struys, Eduard A; Salomons, Gajja S; Schaap, Frank G; Waaijer, Cathelijn J F; Wijers-Koster, Pauline M; Briaire-de Bruijn, Inge H; Haazen, Lizette; Riester, Scott M; Dudakovic, Amel; Danen, Erik; Cleton-Jansen, Anne-Marie; van Wijnen, Andre J; Bovée, Judith V M G

    2015-05-20

    Mutations in isocitrate dehydrogenase 1 (IDH1) and IDH2 are found in a subset of benign and malignant cartilage tumors, gliomas and leukaemias. The mutant enzyme causes the production of D-2-hydroxyglutarate (D-2-HG), affecting CpG island and histone methylation. While mutations in IDH1/2 are early events in benign cartilage tumors, we evaluated whether these mutations play a role in malignant chondrosarcomas. Compared to IDH1/2 wildtype cell lines, chondrosarcoma cell lines harboring an endogenous IDH1 (n=3) or IDH2 mutation (n=2) showed up to a 100-fold increase in intracellular and extracellular D-2-HG levels. Specific inhibition of mutant IDH1 using AGI-5198 decreased levels of D-2-HG in a dose dependent manner. After 72 hours of treatment one out of three mutant IDH1 cell lines showed a moderate decrease in viability , while D-2-HG levels decreased >90%. Likewise, prolonged treatment (up to 20 passages) did not affect proliferation and migration. Furthermore, global gene expression, CpG island methylation as well as histone H3K4, -9, and -27 trimethylation levels remained unchanged. Thus, while IDH1/2 mutations cause enchondroma, malignant progression towards central chondrosarcoma renders chondrosarcoma growth independent of these mutations. Thus, monotherapy based on inhibition of mutant IDH1 appears insufficient for treatment of inoperable or metastasized chondrosarcoma patients.

  5. Real-time sensing of epithelial cell-cell and cell-substrate interactions by impedance spectroscopy on porous substrates

    Energy Technology Data Exchange (ETDEWEB)

    Mondal, D.; RoyChaudhuri, C., E-mail: chirosreepram@yahoo.com [Department of Electronics and Telecommunication Engineering, Indian Institute of Engineering Science and Technology, Shibpur, Howrah 711103 (India); Pal, D. [Department of Aerospace Engineering and Applied Mechanics, Indian Institute of Engineering Science and Technology, Shibpur, Howrah 711103 (India)

    2015-07-28

    Oxidized porous silicon (PS) is a common topographical biocompatible substrate that potentially provides a distinct in vitro environment for better understanding of in vivo behavior. But in the reported studies on oxidized PS, cell-cell and cell-substrate interactions have been detected only by fluorescent labeling. This paper is the first attempt to investigate real-time sensing of these interactions on HaCaT cells by label-free impedance spectroscopy on oxidized PS of two pore diameters (50 and 500 nm). One of the major requirements for successful impedance spectroscopy measurement is to restrict the channeling of electric field lines through the pores. To satisfy this criterion, we have designed the pore depths after analyzing the penetration of the medium by using computational fluid dynamics simulation. A distributed electrical model was also developed for estimating the various cellular attributes by considering a pseudorandom distribution of pores. It is observed from the impedance measurements and from the model that the proliferation rate increases for 50 nm pores but decreases for 500 nm pores compared to that for planar substrates. The rate of decrease in cell substrate separation (h) in the initial stage is more than the rate of increase in cell-cell junction resistance (R{sub b}) corresponding to the initial adhesion phase of cells. It is observed that R{sub b} and h are higher for 50 nm pores than those for planar substrates, corresponding to the fact that substrates more conducive toward cell adhesion encourage cell-cell interactions than direct cell-substrate interactions. Thus, the impedance spectroscopy coupled with the proposed theoretical framework for PS substrates can sense and quantify the cellular interactions.

  6. Real-time sensing of epithelial cell-cell and cell-substrate interactions by impedance spectroscopy on porous substrates

    International Nuclear Information System (INIS)

    Mondal, D.; RoyChaudhuri, C.; Pal, D.

    2015-01-01

    Oxidized porous silicon (PS) is a common topographical biocompatible substrate that potentially provides a distinct in vitro environment for better understanding of in vivo behavior. But in the reported studies on oxidized PS, cell-cell and cell-substrate interactions have been detected only by fluorescent labeling. This paper is the first attempt to investigate real-time sensing of these interactions on HaCaT cells by label-free impedance spectroscopy on oxidized PS of two pore diameters (50 and 500 nm). One of the major requirements for successful impedance spectroscopy measurement is to restrict the channeling of electric field lines through the pores. To satisfy this criterion, we have designed the pore depths after analyzing the penetration of the medium by using computational fluid dynamics simulation. A distributed electrical model was also developed for estimating the various cellular attributes by considering a pseudorandom distribution of pores. It is observed from the impedance measurements and from the model that the proliferation rate increases for 50 nm pores but decreases for 500 nm pores compared to that for planar substrates. The rate of decrease in cell substrate separation (h) in the initial stage is more than the rate of increase in cell-cell junction resistance (R b ) corresponding to the initial adhesion phase of cells. It is observed that R b and h are higher for 50 nm pores than those for planar substrates, corresponding to the fact that substrates more conducive toward cell adhesion encourage cell-cell interactions than direct cell-substrate interactions. Thus, the impedance spectroscopy coupled with the proposed theoretical framework for PS substrates can sense and quantify the cellular interactions

  7. The antitumor effect of tanshinone IIA on anti-proliferation and decreasing VEGF/VEGFR2 expression on the human non-small cell lung cancer A549 cell line

    Directory of Open Access Journals (Sweden)

    Jun Xie

    2015-11-01

    Full Text Available The effects of tanshinone IIA on the proliferation of the human non-small cell lung cancer cell line A549 and its possible mechanism on the VEGF/VEGFR signal pathway were investigated. The exploration of the interaction between tanshinone IIA and its target proteins provides a feasible platform for studying the anticancer mechanism of active components of herbs. The CCK-8 assay was used to evaluate the proliferative activity of A549 cells treated with tanshinone IIA (2.5−80 μmol/L for 24, 48 and 72 h, respectively. Flow cytometry was used for the detection of cell apoptosis and cell cycle perturbation. VEGF and VEGFR2 expression were studied by Western blotting. The binding mode of tanshinone IIA within the crystal structure of the VEGFR2 protein was evaluated with molecular docking analysis by use of the CDOCKER algorithm in Discovery Studio 2.1. The CCK-8 results showed that tanshinone IIA can significantly inhibit A549 cell proliferation in a dose- and time-dependent manner. Flow cytometry results showed that the apoptosis rate of tested group was higher than the vehicle control, and tanshinone IIA-treated cells accumulated at the S phase, which was higher than the vehicle control. Furthermore, the expression of VEGF and VEGFR2 was decreased in Western blot. Finally, molecular docking analysis revealed that tanshinone IIA could be stably docked into the kinase domain of VEGFR2 protein with its unique modes to form H-bonds with Cys917 and π–π stacking interactions with Val848. In conclusion, tanshinone IIA may suppress A549 proliferation, induce apoptosis and cell cycle arrest at the S phase. This drug may suppress angiogenesis by targeting the protein kinase domains of VEGF/VEGFR2.

  8. Inhibition of connective tissue growth factor (CTGF/CCN2) expression decreases the survival and myogenic differentiation of human rhabdomyosarcoma cells.

    Science.gov (United States)

    Croci, Stefania; Landuzzi, Lorena; Astolfi, Annalisa; Nicoletti, Giordano; Rosolen, Angelo; Sartori, Francesca; Follo, Matilde Y; Oliver, Noelynn; De Giovanni, Carla; Nanni, Patrizia; Lollini, Pier-Luigi

    2004-03-01

    Connective tissue growth factor (CTGF/CCN2), a cysteine-rich protein of the CCN (Cyr61, CTGF, Nov) family of genes, emerged from a microarray screen of genes expressed by human rhabdomyosarcoma cells. Rhabdomyosarcoma is a soft tissue sarcoma of childhood deriving from skeletal muscle cells. In this study, we investigated the role of CTGF in rhabdomyosarcoma. Human rhabdomyosarcoma cells of the embryonal (RD/12, RD/18, CCA) and the alveolar histotype (RMZ-RC2, SJ-RH4, SJ-RH30), rhabdomyosarcoma tumor specimens, and normal skeletal muscle cells expressed CTGF. To determine the function of CTGF, we treated rhabdomyosarcoma cells with a CTGF antisense oligonucleotide or with a CTGF small interfering RNA (siRNA). Both treatments inhibited rhabdomyosarcoma cell growth, suggesting the existence of a new autocrine loop based on CTGF. CTGF antisense oligonucleotide-mediated growth inhibition was specifically due to a significant increase in apoptosis, whereas cell proliferation was unchanged. CTGF antisense oligonucleotide induced a strong decrease in the level of myogenic differentiation of rhabdomyosarcoma cells, whereas the addition of recombinant CTGF significantly increased the proportion of myosin-positive cells. CTGF emerges as a survival and differentiation factor and could be a new therapeutic target in human rhabdomyosarcoma.

  9. Human neural progenitor cells decrease photoreceptor degeneration, normalize opsin distribution and support synapse structure in cultured porcine retina.

    Science.gov (United States)

    Mollick, Tanzina; Mohlin, Camilla; Johansson, Kjell

    2016-09-01

    Retinal neurodegenerative disorders like retinitis pigmentosa, age-related macular degeneration, diabetic retinopathy and retinal detachment decrease retinal functionality leading to visual impairment. The pathological events are characterized by photoreceptor degeneration, synaptic disassembly, remodeling of postsynaptic neurons and activation of glial cells. Despite intense research, no effective treatment has been found for these disorders. The current study explores the potential of human neural progenitor cell (hNPC) derived factors to slow the degenerative processes in adult porcine retinal explants. Retinas were cultured for 3 days with or without hNPCs as a feeder layer and investigated by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL), immunohistochemical, western blot and quantitative real time-polymerase chain reaction (qRT-PCR) techniques. TUNEL showed that hNPCs had the capacity to limit photoreceptor cell death. Among cone photoreceptors, hNPC coculture resulted in better maintenance of cone outer segments and reduced opsin mislocalization. Additionally, maintained synaptic structural integrity and preservation of second order calbindin positive horizontal cells was also observed. However, Müller cell gliosis only seemed to be alleviated in terms of reduced Müller cell density. Our observations indicate that at 3 days of coculture, hNPC derived factors had the capacity to protect photoreceptors, maintain synaptic integrity and support horizontal cell survival. Human neural progenitor cell applied treatment modalities may be an effective strategy to help maintain retinal functionality in neurodegenerative pathologies. Whether hNPCs can independently hinder Müller cell gliosis by utilizing higher concentrations or by combination with other pharmacological agents still needs to be determined. Copyright © 2016 Elsevier B.V. All rights reserved.

  10. Gallic acid reduces cell viability, proliferation, invasion and angiogenesis in human cervical cancer cells

    Science.gov (United States)

    ZHAO, BING; HU, MENGCAI

    2013-01-01

    Gallic acid is a trihydroxybenzoic acid, also known as 3,4,5-trihydroxybenzoic acid, which is present in plants worldwide, including Chinese medicinal herbs. Gallic acid has been shown to have cytotoxic effects in certain cancer cells, without damaging normal cells. The objective of the present study was to determine whether gallic acid is able to inhibit human cervical cancer cell viability, proliferation and invasion and suppress cervical cancer cell-mediated angiogenesis. Treatment of HeLa and HTB-35 human cancer cells with gallic acid decreased cell viability in a dose-dependent manner. BrdU proliferation and tube formation assays indicated that gallic acid significantly decreased human cervical cancer cell proliferation and tube formation in human umbilical vein endothelial cells, respectively. Additionally, gallic acid decreased HeLa and HTB-35 cell invasion in vitro. Western blot analysis demonstrated that the expression of ADAM17, EGFR, p-Akt and p-Erk was suppressed by gallic acid in the HeLa and HTB-35 cell lines. These data indicate that the suppression of ADAM17 and the downregulation of the EGFR, Akt/p-Akt and Erk/p-Erk signaling pathways may contribute to the suppression of cancer progression by Gallic acid. Gallic acid may be a valuable candidate for the treatment of cervical cancer. PMID:24843386

  11. Lipoic Acid Decreases the Viability of Breast Cancer Cells and Activity of PTP1B and SHP2.

    Science.gov (United States)

    Kuban-Jankowska, Alicja; Gorska-Ponikowska, Magdalena; Wozniak, Michal

    2017-06-01

    Protein tyrosine phosphatases PTP1B and SHP2 are potential targets for anticancer therapy, because of the essential role they play in the development of tumors. PTP1B and SHP2 are overexpressed in breast cancer cells, thus inhibition of their activity can be potentially effective in breast cancer therapy. Lipoic acid has been previously reported to inhibit the proliferation of colon, breast and thyroid cancer cells. We investigated the effect of alpha-lipoic acid (ALA) and its reduced form of dihydrolipoic acid (DHLA) on the viability of MCF-7 cancer cells and on the enzymatic activity of PTP1B and SHP2 phosphatases. ALA and DHLA decrease the activity of PTP1B and SHP2, and have inhibitory effects on the viability and proliferation of breast cancer cells. ALA and DHLA can be considered as potential agents for the adjunctive treatment of breast cancer. Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

  12. Male bovine GH transgenic mice have decreased adiposity with an adipose depot-specific increase in immune cell populations.

    Science.gov (United States)

    Benencia, Fabian; Harshman, Stephanie; Duran-Ortiz, Silvana; Lubbers, Ellen R; List, Edward O; Householder, Lara; Al-Naeeli, Mawadda; Liang, Xiaoyu; Welch, Lonnie; Kopchick, John J; Berryman, Darlene E

    2015-05-01

    White adipose tissue (WAT) is composed of mature adipocytes and a stromal vascular fraction (SVF), which contains a variety of cells, including immune cells that vary among the different WAT depots. Growth hormone (GH) impacts immune function and adiposity in an adipose depot-specific manner. However, its effects on WAT immune cell populations remain unstudied. Bovine GH transgenic (bGH) mice are commonly used to study the in vivo effects of GH. These giant mice have an excess of GH action, impaired glucose metabolism, decreased adiposity, increased lean mass, and a shortened lifespan. Therefore, the purpose of this study was to characterize the WAT depot-specific differences in immune cell populations in the presence of excess GH in vivo. Three WAT depots were assessed: inguinal (sc), epididymal (EPI), and mesenteric (MES). Subcutaneous and MES bGH WAT depots showed a significantly higher number of total SVF cells, yet only MES bGH WAT had higher leukocyte counts compared with control samples. By means of flow cytometry analysis of the SVF, we detected greater macrophage and regulatory T-cell infiltration in sc and MES bGH WAT depots compared with controls. However, no differences were observed in the EPI WAT depot. RNA-sequencing confirmed significant alterations in pathways related to T-cell infiltration and activation in the sc depot with fewer significant changes in the EPI bGH WAT depot. These findings collectively point to a previously unrecognized role for GH in influencing the distribution of WAT immune cell populations in a depot-specific manner.

  13. Cabergoline decreases alcohol drinking and seeking behaviors via glial cell line-derived neurotrophic factor.

    Science.gov (United States)

    Carnicella, Sebastien; Ahmadiantehrani, Somayeh; He, Dao-Yao; Nielsen, Carsten K; Bartlett, Selena E; Janak, Patricia H; Ron, Dorit

    2009-07-15

    Cabergoline is an ergotamine derivative that increases the expression of glial cell line-derived neurotrophic factor (GDNF) in vitro. We recently showed that GDNF in the ventral tegmental area (VTA) reduces the motivation to consume alcohol. We therefore set out to determine whether cabergoline administration decreases alcohol-drinking and -seeking behaviors via GDNF. Reverse transcription polymerase chain reaction (RT-PCR) and Enzyme-Linked ImmunoSorbent Assay (ELISA) were used to measure GDNF levels. Western blot analysis was used for phosphorylation experiments. Operant self-administration in rats and a two-bottle choice procedure in mice were used to assess alcohol-drinking behaviors. Instrumental performance tested during extinction was used to measure alcohol-seeking behavior. The [35S]GTPgammaS binding assay was used to assess the expression and function of the dopamine D2 receptor (D2R). We found that treatment of the dopaminergic-like cell line SH-SY5Y with cabergoline and systemic administration of cabergoline in rats resulted in an increase in GDNF level and in the activation of the GDNF pathway. Cabergoline treatment decreased alcohol-drinking and -seeking behaviors including relapse, and its action to reduce alcohol consumption was localized to the VTA. Finally, the increase in GDNF expression and the decrease in alcohol consumption by cabergoline were abolished in GDNF heterozygous knockout mice. Together, these findings suggest that cabergoline-mediated upregulation of the GDNF pathway attenuates alcohol-drinking behaviors and relapse. Alcohol abuse and addiction are devastating and costly problems worldwide. This study puts forward the possibility that cabergoline might be an effective treatment for these disorders.

  14. c-Myc-Dependent Cell Competition in Human Cancer Cells.

    Science.gov (United States)

    Patel, Manish S; Shah, Heta S; Shrivastava, Neeta

    2017-07-01

    Cell Competition is an interaction between cells for existence in heterogeneous cell populations of multicellular organisms. This phenomenon is involved in initiation and progression of cancer where heterogeneous cell populations compete directly or indirectly for the survival of the fittest based on differential gene expression. In Drosophila, cells having lower dMyc expression are eliminated by cell competition through apoptosis when present in the milieu of cells having higher dMyc expression. Thus, we designed a study to develop c-Myc (human homolog) dependent in vitro cell competition model of human cancer cells. Cells with higher c-Myc were transfected with c-myc shRNA to prepare cells with lower c-Myc and then co-cultured with the same type of cells having a higher c-Myc in equal ratio. Cells with lower c-Myc showed a significant decrease in numbers when compared with higher c-Myc cells, suggesting "loser" and "winner" status of cells, respectively. During microscopy, engulfment of loser cells by winner cells was observed with higher expression of JNK in loser cells. Furthermore, elimination of loser cells was prevented significantly, when co-cultured cells were treated with the JNK (apoptosis) inhibitor. Above results indicate elimination of loser cells in the presence of winner cells by c-Myc-dependent mechanisms of cell competition in human cancer cells. This could be an important mechanism in human tumors where normal cells are eliminated by c-Myc-overexpressed tumor cells. J. Cell. Biochem. 118: 1782-1791, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  15. The 2-oxoglutarate analog 3-oxoglutarate decreases normoxic hypoxia-inducible factor-1α in cancer cells, induces cell death, and reduces tumor xenograft growth

    Directory of Open Access Journals (Sweden)

    Koivunen P

    2016-03-01

    Full Text Available Peppi Koivunen,1 Stuart M Fell,2,3 Wenyun Lu,4 Joshua D Rabinowitz,4 Andrew L Kung,5,6 Susanne Schlisio,2,7 1Biocenter Oulu, Faculty of Biochemistry and Molecular Medicine, Oulu Center for Cell-Matrix Research, University of Oulu, Oulu, Finland; 2Ludwig Institute for Cancer Research Ltd, Stockholm, Sweden; 3Department of Cell and Molecular Biology, Karolinska Institutet, Stockholm, Sweden; 4Department of Chemistry and Integrative Genomics, Princeton University, Princeton, NJ, 5Department of Medical Oncology, Dana Farber Cancer Institute, Harvard Medical School, Boston, MA, 6Department of Pediatrics, Columbia University Medical Center, New York, NY, USA; 7Department of Microbiology and Tumor and Cell Biology, Karolinska Institutet, Stockholm, Sweden Abstract: The cellular response to hypoxia is primarily regulated by the hypoxia-inducible factors (HIFs. HIF-1α is also a major mediator of tumor physiology, and its abundance is correlated with therapeutic resistance in a broad range of cancers. Accumulation of HIF-1α under hypoxia is mainly controlled by the oxygen-sensing HIF prolyl 4-hydroxylases (EGLNs, also known as PHDs. Here, we identified a high level of normoxic HIF-1α protein in various cancer cell lines. EGLNs require oxygen and 2-oxoglutarate for enzymatic activity. We tested the ability of several cell-permeable 2-oxoglutarate analogs to regulate the abundance of HIF-1α protein. We identified 3-oxoglutarate as a potent regulator of HIF-1α in normoxic conditions. In contrast to 2-oxoglutarate, 3-oxoglutarate decreased the abundance of HIF-1α protein in several cancer cell lines in normoxia and diminished HIF-1α levels independent of EGLN enzymatic activity. Furthermore, we observed that 3-oxoglutarate was detrimental to cancer cell survival. We show that esterified 3-oxoglutarate, in combination with the cancer chemotherapeutic drug vincristine, induces apoptosis and inhibits tumor growth in vitro and in vivo. Our data

  16. Overexpression of human sperm protein 17 increases migration and decreases the chemosensitivity of human epithelial ovarian cancer cells

    International Nuclear Information System (INIS)

    Li, Fang-qiu; Han, Yan-ling; Liu, Qun; Wu, Bo; Huang, Wen-bin; Zeng, Su-yun

    2009-01-01

    Most deaths from ovarian cancer are due to metastases that are resistant to conventional therapies. But the factors that regulate the metastatic process and chemoresistance of ovarian cancer are poorly understood. In the current study, we investigated the aberrant expression of human sperm protein 17 (HSp17) in human epithelial ovarian cancer cells and tried to analyze its influences on the cell behaviors like migration and chemoresistance. Immunohistochemistry and immunocytochemistry were used to identify HSp17 in paraffin embedded ovarian malignant tumor specimens and peritoneal metastatic malignant cells. Then we examined the effect of HSp17 overexpression on the proliferation, migration, and chemoresistance of ovarian cancer cells to carboplatin and cisplatin in a human ovarian carcinoma cell line, HO8910. We found that HSp17 was aberrantly expressed in 43% (30/70) of the patients with primary epithelial ovarian carcinomas, and in all of the metastatic cancer cells of ascites from 8 patients. The Sp17 expression was also detected in the metastatic lesions the same as in ovarian lesions. None of the 7 non-epithelial tumors primarily developed in the ovaries was immunopositive for HSp17. Overexpression of HSp17 increased the migration but decreased the chemosensitivity of ovarian carcinoma cells to carboplatin and cisplatin. HSp17 is aberrantly expressed in a significant proportion of epithelial ovarian carcinomas. Our results strongly suggest that HSp17 plays a role in metastatic disease and resistance of epithelial ovarian carcinoma to chemotherapy

  17. Ketamine Metabolites Enantioselectively Decrease Intracellular D-Serine Concentrations in PC-12 Cells.

    Directory of Open Access Journals (Sweden)

    Nagendra S Singh

    Full Text Available D-Serine is an endogenous NMDA receptor co-agonist that activates synaptic NMDA receptors modulating neuronal networks in the cerebral cortex and plays a key role in long-term potentiation of synaptic transmission. D-serine is associated with NMDA receptor neurotoxicity and neurodegeneration and elevated D-serine concentrations have been associated with Alzheimer's and Parkinsons' diseases and amyotrophic lateral sclerosis. Previous studies have demonstrated that the ketamine metabolites (rac-dehydronorketamine and (2S,6S-hydroxynorketamine decrease intracellular D-serine concentrations in a concentration dependent manner in PC-12 cells. In the current study, PC-12 cells were incubated with a series of ketamine metabolites and the IC50 values associated with attenuated intracellular D-serine concentrations were determined. The results demonstrate that structural and stereochemical features of the studied compounds contribute to the magnitude of the inhibitory effect with (2S,6S-hydroxynorketamine and (2R,6R-hydroxynorketamine displaying the most potent inhibition with IC50 values of 0.18 ± 0.04 nM and 0.68 ± 0.09 nM. The data was utilized to construct a preliminary 3D-QSAR/pharmacophore model for use in the design of new and more efficient modulators of D-serine.

  18. Sensitization of gastric cancer cells to alkylating agents by glaucocalyxin B via cell cycle arrest and enhanced cell death.

    Science.gov (United States)

    Ur Rahman, Muhammad Saif; Zhang, Ling; Wu, Lingyan; Xie, Yuqiong; Li, Chunchun; Cao, Jiang

    2017-01-01

    Severe side effects are major problems with chemotherapy of gastric cancer (GC). These side effects can be reduced by using sensitizing agents in combination with therapeutic drugs. In this study, the low/nontoxic dosage of glaucocalyxin B (GLB) was used with other DNA linker agents mitomycin C (MMC), cisplatin (DDP), or cyclophosphamide (CTX) to treat GC cells. Combined effectiveness of GLB with drugs was determined by proliferation assay. The molecular mechanisms associated with cell proliferation, migration, invasion, cell cycle, DNA repair/replication, apoptosis, and autophagy were investigated by immunoblotting for key proteins involved. Cell cycle and apoptosis analysis were performed by flow cytometry. Reactive oxygen species level was also examined for identification of its role in apoptosis. Proliferation assay revealed that the addition of 5 µM GLB significantly sensitizes gastric cancer SGC-7901 cells to MMC, DDP, and CTX by decreasing half-maximal inhibitory concentration (IC 50 ) by up to 75.40%±5%, 45.10%±5%, and 52.10%±5%, respectively. GLB + drugs decreased the expression level of proteins involved in proliferation and migration, suggesting the anticancer potential of GLB + drugs. GLB + MMC, GLB + CTX, and GLB + DDP arrest the cells in G 0 /G 1 and G 1 /S phase, respectively, which may be the consequence of significant decrease in the level of enzymes responsible for DNA replication and telomerase shortening. Combined use of GLB with these drugs also induces DNA damage and apoptosis by activating caspase/PARP pathways and increased production of reactive oxygen species and increased autophagy in GC cells. GLB dosage sensitizes GC cells to the alkylating agents via arresting the cell cycle and enhancing cell death. This is of significant therapeutic importance in the reduction of side effects associated with these drugs.

  19. Adhesion defective BHK cell mutant has cell surface heparan sulfate proteoglycan of altered properties

    DEFF Research Database (Denmark)

    Couchman, J R; Austria, R; Woods, A

    1988-01-01

    In the light of accumulating data that implicate cell surface heparan sulfate proteoglycans (HSPGs) with a role in cell interactions with extracellular matrix molecules such as fibronectin, we have compared the properties of these molecules in wild-type BHK cells and an adhesion-defective ricin......-resistant mutant (RicR14). Our results showed that the mutant, unlike BHK cells, cannot form focal adhesions when adherent to planar substrates in the presence of serum. Furthermore, while both cell lines possess similar amounts of cell surface HSPG with hydrophobic properties, that of RicR14 cells had decreased...... sulfation, reduced affinity for fibronectin and decreased half-life on the cell surface when compared to the normal counterpart. Our conclusions based on this data are that these altered properties may, in part, account for the adhesion defect in the ricin-resistant mutant. Whether this results from...

  20. Induction of reactive oxygen species-stimulated distinctive autophagy by chelerythrine in non-small cell lung cancer cells.

    Science.gov (United States)

    Tang, Zheng-Hai; Cao, Wen-Xiang; Wang, Zhao-Yu; Lu, Jia-Hong; Liu, Bo; Chen, Xiuping; Lu, Jin-Jian

    2017-08-01

    Chelerythrine (CHE), a natural benzo[c]phenanthridine alkaloid, shows anti-cancer effect through a number of mechanisms. Herein, the effect and mechanism of the CHE-induced autophagy, a type II programmed cell death, in non-small cell lung cancer (NSCLC) cells were studied for the first time. CHE induced cell viability decrease, colony formation inhibition, and apoptosis in a concentration-dependent manner in NSCLC A549 and NCI-H1299 cells. In addition, CHE triggered the expression of phosphatidylethanolamine-modified microtubule-associated protein light-chain 3 (LC3-II). The CHE-induced expression of LC3-II was further increased in the combination treatment with chloroquine (CQ), an autophagy inhibitor, and large amounts of red-puncta were observed in the CHE-treated A549 cells with stable expression of mRFP-EGFP-LC3, indicating that CHE induces autophagy flux. Silence of beclin 1 reversed the CHE-induced expression of LC3-II. Inhibition of autophagy remarkably reversed the CHE-induced cell viability decrease and apoptosis in NCI-H1299 cells but not in A549 cells. Furthermore, CHE triggered reactive oxygen species (ROS) generation in both cell lines. A decreased level of ROS through pretreatment with N-acetyl-L-cysteine reversed the CHE-induced cell viability decrease, apoptosis, and autophagy. Taken together, CHE induced distinctive autophagy in A549 (accompanied autophagy) and NCI-H1299 (pro-death autophagy) cells and a decreased level of ROS reversed the effect of CHE in NSCLC cells in terms of cell viability, apoptosis, and autophagy. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

  1. Exogenous NAD(+) decreases oxidative stress and protects H2O2-treated RPE cells against necrotic death through the up-regulation of autophagy.

    Science.gov (United States)

    Zhu, Ying; Zhao, Ke-Ke; Tong, Yao; Zhou, Ya-Li; Wang, Yi-Xiao; Zhao, Pei-Quan; Wang, Zhao-Yang

    2016-05-31

    Increased oxidative stress, which can lead to the retinal pigment epithelium (RPE) cell death by inducing ATP depletion and DNA repair, is believed to be a prominent pathology in age-related macular degeneration (AMD). In the present study, we showed that and 0.1 mM nicotinamide adenine dinucleotide (NAD(+)) administration significantly blocked RPE cell death induced by 300 μM H2O2. Further investigation showed that H2O2 resulted in increased intracellular ROS level, activation of PARP-1 and subsequently necrotic death of RPE cells. Exogenous NAD(+) administration significantly decreased intracellular and intranuclear ROS levels in H2O2-treated RPE cells. In addition, NAD(+) administration to H2O2-treated RPE cells inhibited the activation of PARP-1 and protected the RPE cells against necrotic death. Moreover, exogenous NAD(+) administration up-regulated autophagy in the H2O2-treated RPE cells. Inhibition of autophagy by LY294002 blocked the decrease of intracellular and intranuclear ROS level. Besides, inhibition of autophagy by LY294002 abolished the protection of exogenous NAD(+) against H2O2-induced cell necrotic death. Taken together, our findings indicate that that exogenous NAD(+) administration suppresses H2O2-induced oxidative stress and protects RPE cells against PARP-1 mediated necrotic death through the up-regulation of autophagy. The results suggest that exogenous NAD(+) administration might be potential value for the treatment of AMD.

  2. Exogenous NAD+ decreases oxidative stress and protects H2O2-treated RPE cells against necrotic death through the up-regulation of autophagy

    Science.gov (United States)

    Zhu, Ying; Zhao, Ke-ke; Tong, Yao; Zhou, Ya-li; Wang, Yi-xiao; Zhao, Pei-quan; Wang, Zhao-yang

    2016-01-01

    Increased oxidative stress, which can lead to the retinal pigment epithelium (RPE) cell death by inducing ATP depletion and DNA repair, is believed to be a prominent pathology in age-related macular degeneration (AMD). In the present study, we showed that and 0.1 mM nicotinamide adenine dinucleotide (NAD+) administration significantly blocked RPE cell death induced by 300 μM H2O2. Further investigation showed that H2O2 resulted in increased intracellular ROS level, activation of PARP-1 and subsequently necrotic death of RPE cells. Exogenous NAD+ administration significantly decreased intracellular and intranuclear ROS levels in H2O2-treated RPE cells. In addition, NAD+ administration to H2O2-treated RPE cells inhibited the activation of PARP-1 and protected the RPE cells against necrotic death. Moreover, exogenous NAD+ administration up-regulated autophagy in the H2O2-treated RPE cells. Inhibition of autophagy by LY294002 blocked the decrease of intracellular and intranuclear ROS level. Besides, inhibition of autophagy by LY294002 abolished the protection of exogenous NAD+ against H2O2-induced cell necrotic death. Taken together, our findings indicate that that exogenous NAD+ administration suppresses H2O2-induced oxidative stress and protects RPE cells against PARP-1 mediated necrotic death through the up-regulation of autophagy. The results suggest that exogenous NAD+ administration might be potential value for the treatment of AMD. PMID:27240523

  3. Changes in T-cell subsets after radiation therapy

    International Nuclear Information System (INIS)

    Yang, S.J.; Rafla, S.; Youssef, E.; Selim, H.; Salloum, N.; Chuang, J.Y.

    1988-01-01

    The T-cell subsets of 129 patients with cancer were counted before and after radiation therapy. The cells were labeled with monoclonal antibodies that were specific for each type of T cell. Significant changes after therapy were decreases in the proportion of T-helper/inducer cells, pan-T cells, and in the ratio of T-helper/inducer to T-suppressor/cytotoxic cells. There was an increase in the percentage of T-suppressor/cytotoxic cells. When the site of the primary cancer was considered, genitourinary cancer and cancer of the head and neck both showed a decreased percentage of T-helper/inducer cells and a reduced ratio of T-helper/inducer to T-suppressor/cytotoxic cells. The percentage of pan-T cells in head and neck cancer and the ratio of T-helper/inducer to T-suppressor/cytotoxic cells in breast cancer were decreased. The percentage of T-helper cells was particularly decreased by radiation therapy in advanced stages of cancer, in higher grade tumors, and in larger tumors. The absolute numbers of various T-cell subsets were decreased in all groups

  4. Acrolein-induced cell death in PC12 cells: role of mitochondria-mediated oxidative stress.

    Science.gov (United States)

    Luo, Jian; Robinson, J Paul; Shi, Riyi

    2005-12-01

    Oxidative stress has been implicated in acrolein cytotoxicity in various cell types, including mammalian spinal cord tissue. In this study we report that acrolein also decreases PC12 cell viability in a reactive oxygen species (ROS)-dependent manner. Specifically, acrolein-induced cell death, mainly necrosis, is accompanied by the accumulation of cellular ROS. Elevating ROS scavengers can alleviate acrolein-induced cell death. Furthermore, we show that exposure to acrolein leads to mitochondrial dysfunction, denoted by the loss of mitochondrial transmembrane potential, reduction of cellular oxygen consumption, and decrease of ATP level. This raises the possibility that the cellular accumulation of ROS could result from the increased production of ROS in the mitochondria of PC12 cells as a result of exposure to acrolein. The acrolein-induced significant decrease of ATP production in mitochondria may also explain why necrosis, not apoptosis, is the dominant type of cell death. In conclusion, our data suggest that one possible mechanism of acrolein-induced cell death could be through mitochondria as its initial target. The subsequent increase of ROS then inflicts cell death and further worsens mitochondria function. Such mechanism may play an important role in CNS trauma and neurodegenerative diseases.

  5. Genistein inhibits cell invasion and motility by inducing cell differentiation in murine osteosarcoma cell line LM8.

    Science.gov (United States)

    Nakamura, Atsushi; Aizawa, Junichi; Sakayama, Kenshi; Kidani, Teruki; Takata, Tomoyo; Norimatsu, Yoshiaki; Miura, Hiromasa; Masuno, Hiroshi

    2012-09-26

    changes, markedly decreased the formation of multilayer masses of cells, and markedly increased the expression of osteocalcin mRNA. Genistein decreased invasive and motile potential by inducing cell differentiation in LM8 cells. Genistein may be useful as an anti-metastatic drug for osteosarcoma through its differentiation-inducing effects.

  6. Genistein inhibits cell invasion and motility by inducing cell differentiation in murine osteosarcoma cell line LM8

    Directory of Open Access Journals (Sweden)

    Nakamura Atsushi

    2012-09-01

    . Treatment of LM8 cells with genistein induced morphological changes, markedly decreased the formation of multilayer masses of cells, and markedly increased the expression of osteocalcin mRNA. Conclusions Genistein decreased invasive and motile potential by inducing cell differentiation in LM8 cells. Genistein may be useful as an anti-metastatic drug for osteosarcoma through its differentiation-inducing effects.

  7. UV-induced changes in cell cycle and gene expression within rabbit lens epithelial cells

    International Nuclear Information System (INIS)

    Sidjanin, D.; Grdina, D.; Woloschak, G.E.

    1996-01-01

    Damage to lens epithelial cells is a probable initiation process in cataract formation mediated by UV radiation. In these experiments, we investigated the effects of exposure to 254 nm radiation on cell cycle progression in the rabbit lens epithelial cell line N/N1003A. The RNA was harvested at various times following exposure to UV (254 nm) radiation and analyzed by dot-blot and northern blot hybridizations. These results revealed that during the first 6 h following exposure of the cells to UV, there was, associated with decreasing dose, a decrease in accumulation of transcripts specific for histones H3 and H4 and an increase in the mRNA encoding protein kinase C and β- and γ-actin. Using flow cytometry, we detected an accumulation of cells in G1/S phase of the cell cycle 1 h following exposure to 254 nm radiation. The observed changes in gene expression, especially the decreased accumulation of histone transcripts reported here, may play a role in UV-induced inhibition of cell cycle progression. (Author)

  8. Proliferative capacity of murine hematopoietic stem cells

    International Nuclear Information System (INIS)

    Hellman, S.; Botnick, L.E.; Hannon, E.C.; Vigneulle, R.M.

    1978-01-01

    The present study demonstrates a decrease in self-renewal capacity with serial transfer of murine hematopoietic stem cells. Production of differentiated cell progeny is maintained longer than stem cell self-renewal. In normal animals the capacity for self-renewal is not decreased with increasing donor age. The stem cell compartment in normal animals, both young and old, appears to be proliferatively quiescent. After apparent recovery from the alkylating agent busulfan, the probability of stem cell self-renewal is decreased, there is a permanent defect in the capacity of the bone marrow for serial transplantation, and the stem cells are proliferatively active. These findings support a model of the hematopoietic stem cell compartment as a continuum of cells with decreasing capacities for self-renewal, increasing likelihood for differentiation, and increasing proliferative activity. Cells progress in the continuum in one direction and such progression is not reversible

  9. Memantine Can Reduce Ethanol-Induced Caspase-3 Activity and Apoptosis in H4 Cells by Decreasing Intracellular Calcium.

    Science.gov (United States)

    Wang, Xiaolong; Chen, Jiajun; Wang, Hongbo; Yu, Hao; Wang, Changliang; You, Jiabin; Wang, Pengfei; Feng, Chunmei; Xu, Guohui; Wu, Xu; Zhao, Rui; Zhang, Guohua

    2017-08-01

    Caspase-3 activation and apoptosis are associated with various neurodegenerative disorders. Calcium activation is an important factor in promoting apoptosis. We, therefore, assessed the role of intracellular calcium in ethanol-induced activation of caspase-3 in H4 human neuroglioma cells and the protective effect of the NMDA receptor antagonist, memantine, on ethanol-induced apoptosis in H4 cells. H4 cells were treated with 100 mM EtOH (in culture medium) for 2 days. For interaction studies, cells were treated with memantine (4 μM), EDTA (1 mM), or BAPTA-AM (10 μM) before treatment with EtOH. Knockdown of the gene encoding the NR1 subunit of the NMDA receptor was performed using RNAi. Apoptosis was detected by Annexin V-FITC/PI staining and flow cytometry. Cell viability was detected using an MTS cell proliferation kit. Fluorescence dual wavelength spectrophotometry was used to determine the intracellular calcium concentration. The levels of NR1, caspase-3, IP3R1, and SERCA1 proteins were detected by western blotting. NR1, IP3R1, and SERCA1 mRNA levels were detected by qPCR. We observed increased expression of NR1, IP3R1, SERCA1, and increased intracellular levels of calcium ions in H4 cells exposed to ethanol. In addition, the calcium chelators, EDTA and BAPTA, and RNAi disruption of the NMDA receptor reduced ethanol-induced caspase-3 activation in H4 cells. Memantine treatment reduced the ethanol-induced increase of intracellular calcium, caspase-3 activation, apoptosis, and the ethanol-induced decrease in cell viability. Our results indicate that ethanol-induced caspase-3 activation and apoptosis are likely to be dependent on cytosolic calcium levels and that they can be reduced by memantine treatment.

  10. Reduced intracellular drug accumulation in drug-resistant leukemia cells is not solely due to MDR-mediated efflux but also to decreased uptake

    Directory of Open Access Journals (Sweden)

    Angela Oliveira Pisco

    2014-10-01

    Full Text Available Expression of ABC family transporter proteins that promote drug efflux from cancer cells is a widely observed mechanism of multi-drug resistance of cancer cells. Cell adaptation in long-term culture of HL60 leukemic cells in the presence of chemotherapy leads to induction and maintenance of the ABC transporters expression, preventing further accumulation of drugs. However, we found that decreased accumulation of drugs and fluorescent dyes was also contributed by a reduced uptake by the resistant cells. Confocal time-lapse microscopy and flow cytometry revealed that fluid-phase endocytosis was diminished in drug-resistant cells compared to drug-sensitive cells. Drug uptake was increased by insulin co-treatment when cells were grown in methylcellulose and monitored under the microscope, but not when cultured in suspension. We propose that multi-drug resistance is not solely achieved by enhanced efflux capacity but also by supressed intake of the drug offering an alternative target to overcome drug resistance or potentiate chemotherapy.

  11. Salt stress causes cell wall damage in yeast cells lacking mitochondrial DNA.

    Science.gov (United States)

    Gao, Qiuqiang; Liou, Liang-Chun; Ren, Qun; Bao, Xiaoming; Zhang, Zhaojie

    2014-03-03

    The yeast cell wall plays an important role in maintaining cell morphology, cell integrity and response to environmental stresses. Here, we report that salt stress causes cell wall damage in yeast cells lacking mitochondrial DNA (ρ 0 ). Upon salt treatment, the cell wall is thickened, broken and becomes more sensitive to the cell wall-perturbing agent sodium dodecyl sulfate (SDS). Also, SCW11 mRNA levels are elevated in ρ 0 cells. Deletion of SCW11 significantly decreases the sensitivity of ρ 0 cells to SDS after salt treatment, while overexpression of SCW11 results in higher sensitivity. In addition, salt stress in ρ 0 cells induces high levels of reactive oxygen species (ROS), which further damages the cell wall, causing cells to become more sensitive towards the cell wall-perturbing agent.

  12. The novel curcumin analog FLLL32 decreases STAT3 DNA binding activity and expression, and induces apoptosis in osteosarcoma cell lines

    International Nuclear Information System (INIS)

    Fossey, Stacey L; London, Cheryl A; Bear, Misty D; Lin, Jiayuh; Li, Chenglong; Schwartz, Eric B; Li, Pui-Kai; Fuchs, James R; Fenger, Joelle; Kisseberth, William C

    2011-01-01

    Curcumin is a naturally occurring phenolic compound shown to have a wide variety of antitumor activities; however, it does not attain sufficient blood levels to do so when ingested. Using structure-based design, a novel compound, FLLL32, was generated from curcumin. FLLL32 possesses superior biochemical properties and more specifically targets STAT3, a transcription factor important in tumor cell survival, proliferation, metastasis, and chemotherapy resistance. In our previous work, we found that several canine and human osteosarcoma (OSA) cell lines, but not normal osteoblasts, exhibit constitutive phosphorylation of STAT3. Compared to curcumin, we hypothesized that FLLL32 would be more efficient at inhibiting STAT3 function in OSA cells and that this would result in enhanced downregulation of STAT3 transcriptional targets and subsequent death of OSA cells. Human and canine OSA cells were treated with vehicle, curcumin, or FLLL32 and the effects on proliferation (CyQUANT ® ), apoptosis (SensoLyte ® Homogeneous AMC Caspase- 3/7 Assay kit, western blotting), STAT3 DNA binding (EMSA), and vascular endothelial growth factor (VEGF), survivin, and matrix metalloproteinase-2 (MMP2) expression (RT-PCR, western blotting) were measured. STAT3 expression was measured by RT-PCR, qRT- PCR, and western blotting. Our data showed that FLLL32 decreased STAT3 DNA binding by EMSA. FLLL32 promoted loss of cell proliferation at lower concentrations than curcumin leading to caspase-3- dependent apoptosis, as evidenced by PARP cleavage and increased caspase 3/7 activity; this could be inhibited by treatment with the pan-caspase inhibitor Z-VAD-FMK. Treatment of OSA cells with FLLL32 decreased expression of survivin, VEGF, and MMP2 at both mRNA and protein levels with concurrent decreases in phosphorylated and total STAT3; this loss of total STAT3 occurred, in part, via the ubiquitin-proteasome pathway. These data demonstrate that the novel curcumin analog FLLL32 has biologic activity

  13. The novel curcumin analog FLLL32 decreases STAT3 DNA binding activity and expression, and induces apoptosis in osteosarcoma cell lines

    Directory of Open Access Journals (Sweden)

    Li Pui-Kai

    2011-03-01

    Full Text Available Abstract Background Curcumin is a naturally occurring phenolic compound shown to have a wide variety of antitumor activities; however, it does not attain sufficient blood levels to do so when ingested. Using structure-based design, a novel compound, FLLL32, was generated from curcumin. FLLL32 possesses superior biochemical properties and more specifically targets STAT3, a transcription factor important in tumor cell survival, proliferation, metastasis, and chemotherapy resistance. In our previous work, we found that several canine and human osteosarcoma (OSA cell lines, but not normal osteoblasts, exhibit constitutive phosphorylation of STAT3. Compared to curcumin, we hypothesized that FLLL32 would be more efficient at inhibiting STAT3 function in OSA cells and that this would result in enhanced downregulation of STAT3 transcriptional targets and subsequent death of OSA cells. Methods Human and canine OSA cells were treated with vehicle, curcumin, or FLLL32 and the effects on proliferation (CyQUANT®, apoptosis (SensoLyte® Homogeneous AMC Caspase- 3/7 Assay kit, western blotting, STAT3 DNA binding (EMSA, and vascular endothelial growth factor (VEGF, survivin, and matrix metalloproteinase-2 (MMP2 expression (RT-PCR, western blotting were measured. STAT3 expression was measured by RT-PCR, qRT- PCR, and western blotting. Results Our data showed that FLLL32 decreased STAT3 DNA binding by EMSA. FLLL32 promoted loss of cell proliferation at lower concentrations than curcumin leading to caspase-3- dependent apoptosis, as evidenced by PARP cleavage and increased caspase 3/7 activity; this could be inhibited by treatment with the pan-caspase inhibitor Z-VAD-FMK. Treatment of OSA cells with FLLL32 decreased expression of survivin, VEGF, and MMP2 at both mRNA and protein levels with concurrent decreases in phosphorylated and total STAT3; this loss of total STAT3 occurred, in part, via the ubiquitin-proteasome pathway. Conclusions These data demonstrate

  14. Acidosis Decreases c-Myc Oncogene Expression in Human Lymphoma Cells: A Role for the Proton-Sensing G Protein-Coupled Receptor TDAG8

    Directory of Open Access Journals (Sweden)

    Zhigang Li

    2013-10-01

    Full Text Available Acidosis is a biochemical hallmark of the tumor microenvironment. Here, we report that acute acidosis decreases c-Myc oncogene expression in U937 human lymphoma cells. The level of c-Myc transcripts, but not mRNA or protein stability, contributes to c-Myc protein reduction under acidosis. The pH-sensing receptor TDAG8 (GPR65 is involved in acidosis-induced c-Myc downregulation. TDAG8 is expressed in U937 lymphoma cells, and the overexpression or knockdown of TDAG8 further decreases or partially rescues c-Myc expression, respectively. Acidic pH alone is insufficient to reduce c-Myc expression, as it does not decrease c-Myc in H1299 lung cancer cells expressing very low levels of pH-sensing G protein-coupled receptors (GPCRs. Instead, c-Myc is slightly increased by acidosis in H1299 cells, but this increase is completely inhibited by ectopic overexpression of TDAG8. Interestingly, TDAG8 expression is decreased by more than 50% in human lymphoma samples in comparison to non-tumorous lymph nodes and spleens, suggesting a potential tumor suppressor function of TDAG8 in lymphoma. Collectively, our results identify a novel mechanism of c-Myc regulation by acidosis in the tumor microenvironment and indicate that modulation of TDAG8 and related pH-sensing receptor pathways may be exploited as a new approach to inhibit Myc expression.

  15. Black cohosh inhibits 17β-estradiol-induced cell proliferation of endometrial adenocarcinoma cells.

    Science.gov (United States)

    Park, So Yun; Kim, Hee Ja; Lee, Sa Ra; Choi, Youn-Hee; Jeong, Kyungah; Chung, Hyewon

    2016-10-01

    This study was conducted to investigate the effect of black cohosh (BC) extract on the proliferation and apoptosis of Ishikawa cells. Ishikawa human endometrial adenocarcinoma cells were treated with or without BC (1, 5, 10 and 25 μM) and cell proliferation and cytotoxicity were measured by CCK-8 assays and flow cytometry analysis. Additionally, Ishikawa cells were treated with 17β-estradiol (E2), E2 + progesterone and E2 + BC (5 and 10 μM) and the effect of BC and progesterone on E2-induced cell proliferation was analyzed. BC decreased the proliferation of Ishikawa cells at a dose-dependent rate compared with the control group (p < 0.05). The proliferation of Ishikawa cells increased in the presence of E2, whereas the subsequent addition of progesterone or BC decreased proliferation to the level of the control group (p < 0.05). The inhibitory effect of BC on E2-induced cell proliferation was greater than the inhibitory effect of progesterone. In conclusion, BC induces apoptosis in Ishikawa cells and suppresses E2-induced cell proliferation in Ishikawa cells. BC could be considered a candidate co-treatment agent of estrogen-dependent tumors, especially those involving endometrial cells.

  16. Cell-cycle-dependent regulation of cell motility and determination of the role of Rac1

    DEFF Research Database (Denmark)

    Walmod, Peter S.; Hartmann-Petersen, Rasmus; Prag, S.

    2004-01-01

    comparable to those of control cells in G1. In contrast, transfection with dominant-negative Rac1 reduced cell speed and resulted in cellular displacements, which were identical in G1 and G2. These observations indicate that migration of cultured cells is regulated in a cell-cycle-dependent manner...... for calculation of three key parameters describing cell motility: speed, persistence time and rate of diffusion. All investigated cell lines demonstrated a lower cell displacement in the G2 phase than in the G1/S phases. This was caused by a decrease in speed and/or persistence time. The decrease in motility...... was accompanied by changes in morphology reflecting the larger volume of cells in G2 than in G1. Furthermore, L-cells and HeLa-cells appeared to be less adherent in the G2 phase. Transfection of L-cells with constitutively active Rac1 led to a general increase in the speed and rate of diffusion in G2 to levels...

  17. Effects of ionizing radiation on differentiation of murine bone marrow cells into mast cells

    International Nuclear Information System (INIS)

    Murakami, Sho; Yoshino, Hironori; Ishikawa, Junya; Yamaguchi, Masaru; Tsujiguchi, Takakiyo; Nishiyama, Ayaka; Yokoyama, Kouki; Kashiwakura, Ikuo

    2015-01-01

    Mast cells, immune effector cells produced from bone marrow cells, play a major role in immunoglobulin E–mediated allergic responses. Ionizing radiation affects the functions of mast cells, which are involved in radiation-induced tissue damage. However, whether ionizing radiation affects the differential induction of mast cells is unknown. Here we investigated whether bone marrow cells of X-irradiated mice differentiated into mast cells. To induce mast cells, bone marrow cells from X-irradiated and unirradiated mice were cultured in the presence of cytokines required for mast cell induction. Although irradiation at 0.5 Gy and 2 Gy decreased the number of bone marrow cells 1 day post-irradiation, the cultured bone marrow cells of X-irradiated and unirradiated mice both expressed mast cell–related cell-surface antigens. However, the percentage of mast cells in the irradiated group was lower than in the unirradiated group. Similar decreases in the percentage of mast cells induced in the presence of X-irradiation were observed 10 days post irradiation, although the number of bone marrow cells in irradiated mice had recovered by this time. Analysis of mast cell function showed that degranulation of mast cells after immunoglobulin E–mediated allergen recognition was significantly higher in the X-irradiated group compared with in the unirradiated group. In conclusion, bone marrow cells of X-irradiated mice differentiated into mast cells, but ionizing radiation affected the differentiation efficiency and function of mast cells. (author)

  18. Cuprizone decreases intermediate and late-stage progenitor cells in hippocampal neurogenesis of rats in a framework of 28-day oral dose toxicity study

    International Nuclear Information System (INIS)

    Abe, Hajime; Tanaka, Takeshi; Kimura, Masayuki; Mizukami, Sayaka; Saito, Fumiyo; Imatanaka, Nobuya; Akahori, Yumi; Yoshida, Toshinori; Shibutani, Makoto

    2015-01-01

    Developmental exposure to cuprizone (CPZ), a demyelinating agent, impairs intermediate-stage neurogenesis in the hippocampal dentate gyrus of rat offspring. To investigate the possibility of alterations in adult neurogenesis following postpubertal exposure to CPZ in a framework of general toxicity studies, CPZ was orally administered to 5-week-old male rats at 0, 120, or 600 mg/kg body weight/day for 28 days. In the subgranular zone (SGZ), 600 mg/kg CPZ increased the number of cleaved caspase-3 + apoptotic cells. At ≥ 120 mg/kg, the number of SGZ cells immunoreactive for TBR2, doublecortin, or PCNA was decreased, while that for SOX2 was increased. In the granule cell layer, CPZ at ≥ 120 mg/kg decreased the number of postmitotic granule cells immunoreactive for NEUN, CHRNA7, ARC or FOS. In the dentate hilus, CPZ at ≥ 120 mg/kg decreased phosphorylated TRKB + interneurons, although the number of reelin + interneurons was unchanged. At 600 mg/kg, mRNA levels of Bdnf and Chrna7 were decreased, while those of Casp4, Casp12 and Trib3 were increased in the dentate gyrus. These data suggest that CPZ in a scheme of 28-day toxicity study causes endoplasmic reticulum stress-mediated apoptosis of granule cell lineages, resulting in aberrations of intermediate neurogenesis and late-stage neurogenesis and following suppression of immediate early gene-mediated neuronal plasticity. Suppression of BDNF signals to interneurons caused by decreased cholinergic signaling may play a role in these effects of CPZ. The effects of postpubertal CPZ on neurogenesis were similar to those observed with developmental exposure, except for the lack of reelin response, which may contribute to a greater decrease in SGZ cells. - Highlights: • Effect of 28-day CPZ exposure on hippocampal neurogenesis was examined in rats. • CPZ suppressed intermediate neurogenesis and late-stage neurogenesis in the dentate gyrus. • CPZ suppressed BDNF signals to interneurons by decrease of cholinergic

  19. Cuprizone decreases intermediate and late-stage progenitor cells in hippocampal neurogenesis of rats in a framework of 28-day oral dose toxicity study

    Energy Technology Data Exchange (ETDEWEB)

    Abe, Hajime; Tanaka, Takeshi; Kimura, Masayuki; Mizukami, Sayaka [Laboratory of Veterinary Pathology, Tokyo University of Agriculture and Technology, 3-5-8 Saiwai-cho, Fuchu-shi, Tokyo 183-8509 (Japan); Pathogenetic Veterinary Science, United Graduate School of Veterinary Sciences, Gifu University, 1-1 Yanagido, Gifu-shi, Gifu 501-1193 (Japan); Saito, Fumiyo; Imatanaka, Nobuya; Akahori, Yumi [Chemicals Evaluation and Research Institute, Japan, 1-4-25 Koraku, Bunkyo-ku, Tokyo 112-0004 (Japan); Yoshida, Toshinori [Laboratory of Veterinary Pathology, Tokyo University of Agriculture and Technology, 3-5-8 Saiwai-cho, Fuchu-shi, Tokyo 183-8509 (Japan); Shibutani, Makoto, E-mail: mshibuta@cc.tuat.ac.jp [Laboratory of Veterinary Pathology, Tokyo University of Agriculture and Technology, 3-5-8 Saiwai-cho, Fuchu-shi, Tokyo 183-8509 (Japan)

    2015-09-15

    Developmental exposure to cuprizone (CPZ), a demyelinating agent, impairs intermediate-stage neurogenesis in the hippocampal dentate gyrus of rat offspring. To investigate the possibility of alterations in adult neurogenesis following postpubertal exposure to CPZ in a framework of general toxicity studies, CPZ was orally administered to 5-week-old male rats at 0, 120, or 600 mg/kg body weight/day for 28 days. In the subgranular zone (SGZ), 600 mg/kg CPZ increased the number of cleaved caspase-3{sup +} apoptotic cells. At ≥ 120 mg/kg, the number of SGZ cells immunoreactive for TBR2, doublecortin, or PCNA was decreased, while that for SOX2 was increased. In the granule cell layer, CPZ at ≥ 120 mg/kg decreased the number of postmitotic granule cells immunoreactive for NEUN, CHRNA7, ARC or FOS. In the dentate hilus, CPZ at ≥ 120 mg/kg decreased phosphorylated TRKB{sup +} interneurons, although the number of reelin{sup +} interneurons was unchanged. At 600 mg/kg, mRNA levels of Bdnf and Chrna7 were decreased, while those of Casp4, Casp12 and Trib3 were increased in the dentate gyrus. These data suggest that CPZ in a scheme of 28-day toxicity study causes endoplasmic reticulum stress-mediated apoptosis of granule cell lineages, resulting in aberrations of intermediate neurogenesis and late-stage neurogenesis and following suppression of immediate early gene-mediated neuronal plasticity. Suppression of BDNF signals to interneurons caused by decreased cholinergic signaling may play a role in these effects of CPZ. The effects of postpubertal CPZ on neurogenesis were similar to those observed with developmental exposure, except for the lack of reelin response, which may contribute to a greater decrease in SGZ cells. - Highlights: • Effect of 28-day CPZ exposure on hippocampal neurogenesis was examined in rats. • CPZ suppressed intermediate neurogenesis and late-stage neurogenesis in the dentate gyrus. • CPZ suppressed BDNF signals to interneurons by decrease of

  20. Surface conditioning with Escherichia coli cell wall components can reduce biofilm formation by decreasing initial adhesion

    Directory of Open Access Journals (Sweden)

    Luciana C. Gomes

    2017-07-01

    Full Text Available Bacterial adhesion and biofilm formation on food processing surfaces pose major risks to human health. Non-efficient cleaning of equipment surfaces and piping can act as a conditioning layer that affects the development of a new biofilm post-disinfection. We have previously shown that surface conditioning with cell extracts could reduce biofilm formation. In the present work, we hypothesized that E. coli cell wall components could be implicated in this phenomena and therefore mannose, myristic acid and palmitic acid were tested as conditioning agents. To evaluate the effect of surface conditioning and flow topology on biofilm formation, assays were performed in agitated 96-well microtiter plates and in a parallel plate flow chamber (PPFC, both operated at the same average wall shear stress (0.07 Pa as determined by computational fluid dynamics (CFD. It was observed that when the 96-well microtiter plate and the PPFC were used to form biofilms at the same shear stress, similar results were obtained. This shows that the referred hydrodynamic feature may be a good scale-up parameter from high-throughput platforms to larger scale flow cell systems as the PPFC used in this study. Mannose did not have any effect on E. coli biofilm formation, but myristic and palmitic acid inhibited biofilm development by decreasing cell adhesion (in about 50%. These results support the idea that in food processing equipment where biofilm formation is not critical below a certain threshold, bacterial lysis and adsorption of cell components to the surface may reduce biofilm buildup and extend the operational time.

  1. Cellular aging of mitochondrial DNA-depleted cells

    International Nuclear Information System (INIS)

    Park, Sun Young; Choi, Bongkun; Cheon, Hwanju; Pak, Youngmi Kim; Kulawiec, Mariola; Singh, Keshav K.; Lee, Myung-Shik

    2004-01-01

    We have reported that mitochondrial DNA-depleted ρ 0 cells are resistant to cell death. Because aged cells have frequent mitochondrial DNA mutations, the resistance of ρ 0 cells against cell death might be related to the apoptosis resistance of aged cells and frequent development of cancers in aged individuals. We studied if ρ 0 cells have features simulating aged cells. SK-Hep1 hepatoma ρ 0 cells showed typical morphology associated with aging such as increased size and elongated appearance. They had increased senescence-associated β-Gal activity, lipofuscin pigment, and plasminogen activator inhibitor-1 expression. Consistent with their decreased proliferation, the expression of mitotic cyclins was decreased and that of cdk inhibitors was increased. Rb hypophosphorylation and decreased telomerase activity were also noted. Features simulating aged cells were also observed in MDA-MB-435 ρ 0 cells. These results support the mitochondrial theory of aging, and suggest that ρ 0 cells could serve as an in vitro model for aged cells

  2. MicroRNA-101 inhibits cell proliferation, promotes cell apoptosis and increases sensitivity of breast cancer MDA-MB-231 cells to paclitaxel

    Directory of Open Access Journals (Sweden)

    Qiu-Lin Ke

    2016-02-01

    Full Text Available Objective: To explore the effect that miR-101 inhibits breast cancer MDA-MB-231 cell proliferation and increases the chemosensitivity of paclitaxel to breast cancer MDA-MB-231 cells and its influence on protein expression level of target gene Bcl2. Methods: miR-101 was artificially synthesized, it used liposome 3000 to transfect MDA-MB-231 cells, and experiment was divided into three groups: blank control group, negative control group and miR-101 group. MTT assay was used to detect the effect of miR-101 on MDA-MB-231 cell proliferation and chemosensitivity of paclitaxel-mediated MDA-MB-231 cells; flow cytometer was used to detect cell apoptosis. Real-time PCR and Western bloting were used to detect the changes of mRNA and protein expression levels of Bcl2. Results: After miR-101 transfected MDA-MB- 231 cells, cell proliferation ability significantly decreased compared with negative control group, and differences had statistical significance (P<0.01; after paclitaxel was used to process cells, IC50 of miR-101-processing group decreased by 2.45 times compared with blank control group, differences had statistical significance (P<0.05 and differences between blank control group and negative control group had no statistical significance; detection results by flow cytometer showed that both early-stage and late-stage apoptosis rates of MDA-MB-231 cells of miR-101 group were significantly higher than those of negative control group (P<0.05, and early-stage apoptosis rate was more significant (P<0.01; after transfection of miR-101, mRNA and protein levels of Bcl2 of MDA-MB-231 cells significantly decreased, and differences had statistical significance (P<0.05. Conclusion: miR-101 can inhibit breast cancer MDAMB- 231 cell proliferation through targeting and downregulating Bcl2, thereby increasing the chemosensitivity of breast cancer cells to paclitaxel and promoting cell apoptosis.

  3. Low White Blood Cell Count

    Science.gov (United States)

    Symptoms Low white blood cell count By Mayo Clinic Staff A low white blood cell count (leukopenia) is a decrease ... of white blood cell (neutrophil). The definition of low white blood cell count varies from one medical ...

  4. CXCR4 blockade decreases CD4+ T cell exhaustion and improves survival in a murine model of polymicrobial sepsis.

    Science.gov (United States)

    Ramonell, Kimberly M; Zhang, Wenxiao; Hadley, Annette; Chen, Ching-Wen; Fay, Katherine T; Lyons, John D; Klingensmith, Nathan J; McConnell, Kevin W; Coopersmith, Craig M; Ford, Mandy L

    2017-01-01

    Sepsis is a dysregulated systemic response to infection involving many inflammatory pathways and the induction of counter-regulatory anti-inflammatory processes that results in a state of immune incompetence and can lead to multi-organ failure. CXCR4 is a chemokine receptor that, following ligation by CXCL12, directs cells to bone marrow niches and also plays an important role in T cell cosignaling and formation of the immunological synapse. Here, we investigated the expression and function of CXCR4 in a murine model of polymicrobial sepsis. Results indicate that CXCR4 is selectively upregulated on naïve CD4+ and CD8+ T cells and CD4+ central memory T cells following the induction of sepsis, and that CXCR4 antagonism resulted in a significant decrease in sepsis-induced mortality. We probed the mechanistic basis for these findings and found that CXCR4 antagonism significantly increased the number of peripheral CD4+ and CD8+ T cells following sepsis. Moreover, mice treated with the CXCR4 antagonist contained fewer PD-1+ LAG-3+ 2B4+ cells, suggesting that blockade of CXCR4 mitigates CD4+ T cell exhaustion during sepsis. Taken together, these results characterize CXCR4 as an important pathway that modulates immune dysfunction and mortality following sepsis, which may hold promise as a target for future therapeutic intervention in septic patients.

  5. CXCR4 blockade decreases CD4+ T cell exhaustion and improves survival in a murine model of polymicrobial sepsis.

    Directory of Open Access Journals (Sweden)

    Kimberly M Ramonell

    Full Text Available Sepsis is a dysregulated systemic response to infection involving many inflammatory pathways and the induction of counter-regulatory anti-inflammatory processes that results in a state of immune incompetence and can lead to multi-organ failure. CXCR4 is a chemokine receptor that, following ligation by CXCL12, directs cells to bone marrow niches and also plays an important role in T cell cosignaling and formation of the immunological synapse. Here, we investigated the expression and function of CXCR4 in a murine model of polymicrobial sepsis. Results indicate that CXCR4 is selectively upregulated on naïve CD4+ and CD8+ T cells and CD4+ central memory T cells following the induction of sepsis, and that CXCR4 antagonism resulted in a significant decrease in sepsis-induced mortality. We probed the mechanistic basis for these findings and found that CXCR4 antagonism significantly increased the number of peripheral CD4+ and CD8+ T cells following sepsis. Moreover, mice treated with the CXCR4 antagonist contained fewer PD-1+ LAG-3+ 2B4+ cells, suggesting that blockade of CXCR4 mitigates CD4+ T cell exhaustion during sepsis. Taken together, these results characterize CXCR4 as an important pathway that modulates immune dysfunction and mortality following sepsis, which may hold promise as a target for future therapeutic intervention in septic patients.

  6. Perfusion cell culture decreases process and product heterogeneity in a head-to-head comparison with fed-batch.

    Science.gov (United States)

    Walther, Jason; Lu, Jiuyi; Hollenbach, Myles; Yu, Marcella; Hwang, Chris; McLarty, Jean; Brower, Kevin

    2018-05-30

    In this study, we compared the impacts of fed-batch and perfusion platforms on process and product attributes for IgG1- and IgG4-producing cell lines. A "plug-and-play" approach was applied to both platforms at bench scale, using commercially available basal and feed media, a standard feed strategy for fed-batch, and ATF filtration for perfusion. Product concentration in fed-batch was 2.5 times greater than perfusion, while average productivity in perfusion was 7.5 times greater than fed-batch. PCA revealed more variability in the cell environment and metabolism during the fed-batch run. LDH measurements showed that exposure of product to cell lysate was 7-10 times greater in fed-batch. Product analysis shows larger abundances of neutral species in perfusion, likely due to decreased bioreactor residence times and extracellular exposure. The IgG1 perfusion product also had higher purity and lower half-antibody. Glycosylation was similar across both culture modes. The first perfusion harvest slice for both product types showed different glycosylation than subsequent harvests, suggesting that product quality lags behind metabolism. In conclusion, process and product data indicate that intra-lot heterogeneity is decreased in perfusion cultures. Additional data and discussion is required to understand the developmental, clinical and commercial implications, and in what situations increased uniformity would be beneficial. This article is protected by copyright. All rights reserved.

  7. Quantifying cell behaviors in negative-pressure induced monolayer cell movement

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    Shu-Er Chow

    2016-02-01

    Conclusion: A quick membrane ruffling formation, an early cell–substratum separation, and an ensuing decrease in the cellular interaction occur in cells at NP. These specific monolayer cell behaviors at NP have been quantified and possibly accelerate wound healing.

  8. Quercetin inhibits the invasion of murine melanoma B16-BL6 cells by decreasing pro-MMP-9 via the PKC pathway.

    Science.gov (United States)

    Zhang, Xian-Ming; Huang, Shao-Peng; Xu, Qiang

    2004-01-01

    On the basis of the inhibitory effect of quercetin on the invasion of melanoma B16-BL6 cells previously reported by us, the mechanisms of quercetin-mediated inhibition of invasion were further investigated in the present study. The ability of B16-BL6 cells to invade and migrate was evaluated in terms of the numbers of cells penetrating a reconstituted basement membrane in the Transwell coculture system. The relative levels and activities of matrix metalloproteinase-9 (MMP-9) and MMP-2 were determined by gelatin zymography and quantified using LabWorks 4.0 software. The quercetin-mediated inhibition of invasion was partially blocked by phorbol-12,13-dibutyrate (PDB), a PKC (protein kinase C) activator, and by doxorubicin, a PKC inhibitor. Only the proforms of MMP-9 (92 kDa) and MMP-2 (72 kDa) were detected by gelatin zymography. Quercetin dose-dependently decreased the gelatinolytic activity of pro-MMP-9. Doxorubicin also markedly reversed the quercetin-induced decrease. Quercetin showed a dose-dependent antagonism of increases in gelatinolytic activity of pro-MMP-9 induced by PDB and free fatty acid (another PKC activator). Together with the report that quercetin directly reduces PKC activity, the results reported here suggest that quercetin may inhibit the invasion of B16-BL6 cells by decreasing pro-MMP-9 via the PKC pathway.

  9. IL-15 improves the cytotoxicity of cytokine-induced killer cells against leukemia cells by upregulating CD3+CD56+ cells and downregulating regulatory T cells as well as IL-35.

    Science.gov (United States)

    Tao, Qianshan; Chen, Tianping; Tao, Lili; Wang, Huiping; Pan, Ying; Xiong, Shudao; Zhai, Zhimin

    2013-01-01

    Cytokine-induced killer (CIK) cells are usually generated from peripheral blood mononuclear cells with the stimulation of IL-2 in vitro. Unlike the conventional IL-2-stimulated CIK cells (IL-2-CIK cells), we investigated the characteristics and potential mechanism of IL-15-stimulated CIK cells (IL-15-CIK cells) in this study. Compared with IL-2-CIK cells, the percentage of CD3CD56 cells was significantly increased in IL-15-CIK cells, but the expression of regulatory T (Treg) cells and IL-35 was significantly decreased in IL-15-CIK cells. Meanwhile, the in vitro cytotoxicity against human myeloid leukemia cells K562 of IL-15-CIK cells was significantly augmented compared with IL-2-CIK cells. These data suggest that IL-15 may improve the cytotoxicity of CIK cells against leukemia cells by upregulating CD3CD56 cells and downregulating Treg cells and IL-35.

  10. Cloning of a glutathione S-transferase decreasing during differentiation of HL60 cell line

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Jae Chul; Park, In Kyu; Lee, Kyu Bo; Sohn, Sang Kyun; Kim, Moo Kyu; Kim, Jung Chul [College of Medicine, Kyungpook National Univ., Taegu (Korea, Republic of)

    1999-06-01

    By sequencing the Expressed Sequence Tags of human dermal papilla cDNA library, we identified a clone named K872 of which the expression decreased during differentiation of HL60 cell line. K872 plasmid DNA was isolated according to QIA plasmid extraction kit (Qiagen GmbH, Germany). The nucleotide sequencing was performed by Sanger's method with K872 plasmid DNA. The most updated GenBank EMBL necleic acid banks were searched through the internet by using BLAST (Basic Local Alignment Search Tools) program. Northern bots were performed using RNA isolated from various human tissues and cancer cell lines. The gene expression of the fusion protein was achieved by His-Patch Thiofusion expression system and the protein product was identified on SDS-PAGE. K872 clone is 1006 nucleotides long, and has a coding region of 675 nucleotides and a 3' non-coding region of 280 nucleotides. The presumed open reading frame starting at the 5' terminus of K872 encodes 226 amino acids, including the initiation methionine residue. The amino acid sequence deduced from the open reading frame of K872 shares 70% identity with that of rat glutathione S-transferase kappa 1 (rGSTK1). The transcripts were expressed inh a variety of human tissues and cancer cells. The levels of transcript were relatively high in those tissues such as heart, skeletal muscle, and peripheral blood leukocyte. It is noteworthy that K872 was found to be abundantly expressed in colorectal cancer and melanoma cell lines. Homology search result suggests that K872 clone is the human homolog of the rGSTK1 which is known to be involved in the resistance of cytotoxic therapy. We propose that meticulous functional analysis should be followed to confirm that.

  11. Alternariol induce toxicity via cell death and mitochondrial damage on Caco-2 cells.

    Science.gov (United States)

    Fernández-Blanco, Celia; Juan-García, Ana; Juan, Cristina; Font, Guillermina; Ruiz, Maria-Jose

    2016-02-01

    Alternariol (AOH), a mycotoxin produced by Alternaria sp, appears as food contaminant in fruit, vegetables and cereal products. Its toxicity has been demonstrated, but the mechanisms involved have not been elucidated yet. In this study, the pathways triggered by AOH and degradation products generated on Caco-2 cells were evaluated. Cells were exposed to AOH sub-cytotoxic concentrations of 15, 30 and 60 μM. Cell cycle disruption, the induction of apoptosis/necrosis and changes in mitochondrial membrane potential (Δψm) after 24 and 48 h was asses by flow cytometry. Also, AOH and its degradation products were evaluated after 24 and 48 h by high-performance liquid chromatography with tandem mass spectrometric (LC-MS/MS) to detect and quantify its levels. Cell cycle was significantly decreased at G1 phase and increased at S and G2/M phase at the time of exposure. AOH induced necrosis, apoptosis/necrosis and loss of Δψm in a dose and time-dependent manner. The concentrations of AOH quantified in the culture media exposed to AOH decreased as the exposure time was increased. In conclusion, AOH caused cytotoxic effects supported by blocking cell cycle, decreasing cell proliferation and increasing apoptosis/necrosis cells. Copyright © 2015 Elsevier Ltd. All rights reserved.

  12. CD44+CD24+ subset of PANC-1 cells exhibits radiation resistance via decreased levels of reactive oxygen species.

    Science.gov (United States)

    Wang, Lei; Li, Pengping; Hu, Wei; Xia, Youyou; Hu, Chenxi; Liu, Liang; Jiang, Xiaodong

    2017-08-01

    Emerging evidence has suggested that pancreatic adenocarcinoma is sustained by pancreatic cancer stem cells. The present study aimed to investigate the expression patterns of the pancreatic cancer stem cell surface markers cluster of differentiation CD44 and CD24 in a pancreatic adenocarcinoma cell line, and to investigate the possible mechanisms for their radiation resistance. Flow cytometry was used to analyze the expression patterns of CD44 and CD24 in the pancreatic adenocarcinoma PANC-1 cell line. In addition, a multi-target click model was used to fit cell survival curves and determine the sensitizer enhancement ratio. The apoptosis and cycle distribution of the four cell subsets was determined using flow cytometry, and the level of reactive oxygen species (ROS) was determined using the 2',7'-dichlorofluorescin diacetate probe. The present results identified that the ratios of CD44 + and CD24 + in the sorted PANC-1 cell line were 92.0 and 4.7%, respectively. Prior to radiation, no statistically significant differences were observed among the four groups. Following treatment with 6 MV of X-rays, the rate of apoptosis was decreased in the CD44 + CD24 + group compared with other subsets. The percentage of G0/G1 cells was highest in the CD44 + CD24 + group compared with the three other groups, which exhibited increased radiosensitivity. In addition, the level of ROS in the CD44 + CD24 + group was reduced compared with the other groups. In summary, the results of the present study indicated that CD44 + CD24 + exhibited stem cell properties. The lower level of ROS and apoptosis in CD44 + CD24 + cells may contribute to their resistance to radiation in pancreatic adenocarcinoma.

  13. Effect of radiation dosage changes on the cell viability and the apoptosis induction on normal and tumorigenic cells

    International Nuclear Information System (INIS)

    Park, In Woo; Choi, Soon Chul; Lee, Sam Sun; Heo, Min Suk

    1999-01-01

    The study was aimed to detect the differences in the cell viability and the apoptosis induction after irradiation on normal and tumorigenic cells. The study, that was generated for two human normal cells (RHEK, HGF-1) and two human tumor cells (KB, HT-1080), was tested using MTT assay at 1 day and 3 day after irradiation and TUNEL assay under confocal laser scanning microscope at 1 day after irradiation. Single irradiation of 0.5, 1, 2, 4, and 8 Gy were applied to the cells. The two fractions of 1, 2, 4, and 8 Gy were separated with a 4 hour time interval. The irradiation was done with 5.38 Gy/min dose rate using Cs-137 irradiator at room temperature. 1. In 3-day group, the cell viability of HGF-1 cell was significantly decreased at 2, 4 and 8 Gy irradiation, the cell viability of KB cell was significantly decreased at 8 Gy irradiation and the cell viability of HT-1080 cell was significantly decreased at 4 and 8 Gy irradiation. 2. There was significant difference between RHEK and KB cell line in the cell viability of 3-day group at 8 Gy irradiation. There was significant difference between RHEK and HGF-1 cell line in the cell viability of 3-day group at 4 and 8 Gy irradiation. 3. There was a significantly decreased cell viability in 3-day group than those in 1-day group at 2, 4 and 8 Gy on HGF-1 cell, at 4 and 8 Gy on HT-1080 cell, at 8 Gy on KB cell.4. We could detect DNA fragmented cells only on KB cell. Number of apoptotic cells of KB cell was significantly increased at 4 and 8 Gy irradiation. However, there was no correlation between cell viability and apoptosis.5. On all 4 cell lines, there were no differences between single and split irradiation method in cell viability and apoptosis.

  14. 3-bromopyruvate ameliorate autoimmune arthritis by modulating Th17/Treg cell differentiation and suppressing dendritic cell activation.

    Science.gov (United States)

    Okano, Takaichi; Saegusa, Jun; Nishimura, Keisuke; Takahashi, Soshi; Sendo, Sho; Ueda, Yo; Morinobu, Akio

    2017-02-10

    Recent studies have shown that cellular metabolism plays an important role in regulating immune cell functions. In immune cell differentiation, both interleukin-17-producing T (Th17) cells and dendritic cells (DCs) exhibit increased glycolysis through the upregulation of glycolytic enzymes, such as hexokinase-2 (HK2). Blocking glycolysis with 2-deoxyglucose was recently shown to inhibit Th17 cell differentiation while promoting regulatory T (Treg) cell generation. However, 2-DG inhibits all isoforms of HK. Thus, it is unclear which isoform has a critical role in Th17 cell differentiation and in rheumatoid arthritis (RA) pathogenesis. Here we demonstrated that 3-bromopyruvate (BrPA), a specific HK2 inhibitor, significantly decreased the arthritis scores and the histological scores in SKG mice, with a significant increase in Treg cells, decrease in Th17 cells, and decrease in activated DCs in the spleen. In vitro, BrPA facilitated the differentiation of Treg cells, suppressed Th17 cells, and inhibited the activation of DCs. These results suggested that BrPA may be a therapeutic target of murine arthritis. Although the role of IL-17 is not clarified in the treatment of RA, targeting cell metabolism to alter the immune cell functions might lead to a new therapeutic strategy for RA.

  15. Short-lived, transitory cell-cell interactions foster migration-dependent aggregation.

    Directory of Open Access Journals (Sweden)

    Melissa D Pope

    Full Text Available During embryonic development, motile cells aggregate into cohesive groups, which give rise to tissues and organs. The role of cell migration in regulating aggregation is unclear. The current paradigm for aggregation is based on an equilibrium model of differential cell adhesivity to neighboring cells versus the underlying substratum. In many biological contexts, however, dynamics is critical. Here, we provide evidence that multicellular aggregation dynamics involves both local adhesive interactions and transport by cell migration. Using time-lapse video microscopy, we quantified the duration of cell-cell contacts among migrating cells that collided and adhered to another cell. This lifetime of cell-cell interactions exhibited a monotonic decreasing dependence on substratum adhesivity. Parallel quantitative measurements of cell migration speed revealed that across the tested range of adhesive substrata, the mean time needed for cells to migrate and encounter another cell was greater than the mean adhesion lifetime, suggesting that aggregation dynamics may depend on cell motility instead of the local differential adhesivity of cells. Consistent with this hypothesis, aggregate size exhibited a biphasic dependence on substratum adhesivity, matching the trend we observed for cell migration speed. Our findings suggest a new role for cell motility, alongside differential adhesion, in regulating developmental aggregation events and motivate new design principles for tuning aggregation dynamics in tissue engineering applications.

  16. Inhibition of myeloperoxidase decreases vascular oxidative stress and increases vasodilatation in sickle cell disease mice1[S

    Science.gov (United States)

    Zhang, Hao; Xu, Hao; Weihrauch, Dorothee; Jones, Deron W.; Jing, Xigang; Shi, Yang; Gourlay, David; Oldham, Keith T.; Hillery, Cheryl A.; Pritchard, Kirkwood A.

    2013-01-01

    Activated leukocytes and polymorphonuclear neutrophils (PMN) release myeloperoxidase (MPO), which binds to endothelial cells (EC), is translocated, and generates oxidants that scavenge nitric oxide (NO) and impair EC function. To determine whether MPO impairs EC function in sickle cell disease (SCD), control (AA) and SCD mice were treated with N-acetyl-lysyltyrosylcysteine-amide (KYC). SCD humans and mice have high plasma MPO and soluble L-selectin (sL-selectin). KYC had no effect on MPO but decreased plasma sL-selectin and malondialdehyde in SCD mice. MPO and 3-chlorotyrosine (3-ClTyr) were increased in SCD aortas. KYC decreased MPO and 3-ClTyr in SCD aortas to the levels in AA aortas. Vasodilatation in SCD mice was impaired. KYC increased vasodilatation in SCD mice more than 2-fold, to ∼60% of levels in AA mice. KYC inhibited MPO-dependent 3-ClTyr formation in EC proteins. SCD mice had high plasma alanine transaminase (ALT), which tended to decrease in KYC-treated SCD mice (P = 0.07). KYC increased MPO and XO/XDH and decreased 3-ClTyr and 3-nitrotyrosine (3-NO2Tyr) in SCD livers. These data support the hypothesis that SCD increases release of MPO, which generates oxidants that impair EC function and injure livers. Inhibiting MPO is an effective strategy for decreasing oxidative stress and liver injury and restoring EC function in SCD. PMID:23956444

  17. Baicalein induces G1 arrest in oral cancer cells by enhancing the degradation of cyclin D1 and activating AhR to decrease Rb phosphorylation

    Energy Technology Data Exchange (ETDEWEB)

    Cheng, Ya-Hsin, E-mail: yhcheng@mail.cmu.edu.tw [Department of Physiology, School of Medicine, China Medical University, Taichung 40402, Taiwan, ROC (China); Li, Lih-Ann; Lin, Pinpin; Cheng, Li-Chuan [Division of Environmental Health and Occupational Medicine, National Health Research Institutes, Zhunan, Miaoli 35053, Taiwan, ROC (China); Hung, Chein-Hui [Graduate Institute of Clinical Medicine Sciences, Chang Gung University, Puizi City, Chiayi 613, Taiwan, ROC (China); Chang, Nai Wen [Department of Biochemistry, School of Medicine, China Medical University, Taichung, Taiwan, ROC (China); Lin, Chingju [Department of Physiology, School of Medicine, China Medical University, Taichung 40402, Taiwan, ROC (China)

    2012-09-15

    Baicalein is a flavonoid, known to have anti-inflammatory and anti-cancer effects. As an aryl hydrocarbon receptor (AhR) ligand, baicalein at high concentrations blocks AhR-mediated dioxin toxicity. Because AhR had been reported to play a role in regulating the cell cycle, we suspected that the anti-cancer effect of baicalein is associated with AhR. This study investigated the molecular mechanism involved in the anti-cancer effect of baicalein in oral cancer cells HSC-3, including whether such effect would be AhR-mediated. Results revealed that baicalein inhibited cell proliferation and increased AhR activity in a dose-dependent manner. Cell cycle was arrested at the G1 phase and the expression of CDK4, cyclin D1, and phosphorylated retinoblastoma (pRb) was decreased. When the AhR was suppressed by siRNA, the reduction of pRb was partially reversed, accompanied by a decrease of cell population at G1 phase and an increase at S phase, while the reduction of cyclin D1 and CDK4 did not change. This finding suggests that the baicalein activation of AhR is indeed associated with the reduction of pRb, but is independent of the reduction of cyclin D1 and CDK4. When cells were pre-treated with LiCl, the inhibitor of GSK-3β, the decrease of cyclin D1 was blocked and the reduction of pRb was recovered. The data indicates that in HSC-3 the reduction of pRb is both mediated by baicalein through activation of AhR and facilitation of cyclin D1 degradation, which causes cell cycle arrest at the G1 phase, and results in the inhibition of cell proliferation. -- Highlights: ► Baicalein causes the G1 phase arrest by decreasing Rb phosphorylation. ► Baicalein modulates AhR-mediated cell proliferation. ► Both AhR activation and cyclin D1 degradation results in hypophosphorylation of Rb. ► Baicalein facilitates cyclin D1 degradation by signalling the GSK-3β pathway.

  18. Celecoxib and tauro-ursodeoxycholic acid co-treatment inhibits cell growth in familial adenomatous polyposis derived LT97 colon adenoma cells

    Energy Technology Data Exchange (ETDEWEB)

    Heumen, Bjorn W.H. van, E-mail: b.vanheumen@mdl.umcn.nl [Department of Gastroenterology and Hepatology, Radboud University Nijmegen Medical Centre, Nijmegen (Netherlands); Roelofs, Hennie M.J.; Morsche, Rene H.M. te [Department of Gastroenterology and Hepatology, Radboud University Nijmegen Medical Centre, Nijmegen (Netherlands); Marian, Brigitte [Institute of Cancer Research, Wien University, Vienna (Austria); Nagengast, Fokko M.; Peters, Wilbert H.M. [Department of Gastroenterology and Hepatology, Radboud University Nijmegen Medical Centre, Nijmegen (Netherlands)

    2012-04-15

    Chemoprevention would be a desirable strategy to avoid duodenectomy in patients with familial adenomatous polyposis (FAP) suffering from duodenal adenomatosis. We investigated the in vitro effects on cell proliferation, apoptosis, and COX-2 expression of the potential chemopreventives celecoxib and tauro-ursodeoxycholic acid (UDCA). HT-29 colon cancer cells and LT97 colorectal micro-adenoma cells derived from a patient with FAP, were exposed to low dose celecoxib and UDCA alone or in combination with tauro-cholic acid (CA) and tauro-chenodeoxycholic acid (CDCA), mimicking bile of FAP patients treated with UDCA. In HT-29 cells, co-treatment with low dose celecoxib and UDCA resulted in a decreased cell growth (14-17%, p < 0.01). A more pronounced decrease (23-27%, p < 0.01) was observed in LT97 cells. Cell growth of HT-29 cells exposed to 'artificial bile' enriched with UDCA, was decreased (p < 0.001), either in the absence or presence of celecoxib. In LT97 cells incubated with 'artificial bile' enriched with UDCA, cell growth was decreased only in the presence of celecoxib (p < 0.05). No clear evidence was found for involvement of proliferating cell nuclear antigen, caspase-3, or COX-2 in the cellular processes leading to the observed changes in cell growth. In conclusion, co-treatment with low dose celecoxib and UDCA has growth inhibitory effects on colorectal adenoma cells derived from a patient with FAP, and further research on this combination as promising chemopreventive strategy is desired. -- Highlights: Black-Right-Pointing-Pointer Celecoxib and UDCA acid co-treatment decreases cell growth in colon tumor cells. Black-Right-Pointing-Pointer UDCA enriched 'artificial bile' decreases LT-97 cell growth only in presence of celecoxib. Black-Right-Pointing-Pointer PCNA, caspase-3, nor COX-2 seem to be involved in the observed changes in cell growth.

  19. Celecoxib and tauro-ursodeoxycholic acid co-treatment inhibits cell growth in familial adenomatous polyposis derived LT97 colon adenoma cells

    International Nuclear Information System (INIS)

    Heumen, Bjorn W.H. van; Roelofs, Hennie M.J.; Morsche, René H.M. te; Marian, Brigitte; Nagengast, Fokko M.; Peters, Wilbert H.M.

    2012-01-01

    Chemoprevention would be a desirable strategy to avoid duodenectomy in patients with familial adenomatous polyposis (FAP) suffering from duodenal adenomatosis. We investigated the in vitro effects on cell proliferation, apoptosis, and COX-2 expression of the potential chemopreventives celecoxib and tauro-ursodeoxycholic acid (UDCA). HT-29 colon cancer cells and LT97 colorectal micro-adenoma cells derived from a patient with FAP, were exposed to low dose celecoxib and UDCA alone or in combination with tauro-cholic acid (CA) and tauro-chenodeoxycholic acid (CDCA), mimicking bile of FAP patients treated with UDCA. In HT-29 cells, co-treatment with low dose celecoxib and UDCA resulted in a decreased cell growth (14–17%, p < 0.01). A more pronounced decrease (23–27%, p < 0.01) was observed in LT97 cells. Cell growth of HT-29 cells exposed to ‘artificial bile’ enriched with UDCA, was decreased (p < 0.001), either in the absence or presence of celecoxib. In LT97 cells incubated with ‘artificial bile’ enriched with UDCA, cell growth was decreased only in the presence of celecoxib (p < 0.05). No clear evidence was found for involvement of proliferating cell nuclear antigen, caspase-3, or COX-2 in the cellular processes leading to the observed changes in cell growth. In conclusion, co-treatment with low dose celecoxib and UDCA has growth inhibitory effects on colorectal adenoma cells derived from a patient with FAP, and further research on this combination as promising chemopreventive strategy is desired. -- Highlights: ► Celecoxib and UDCA acid co-treatment decreases cell growth in colon tumor cells. ► UDCA enriched ‘artificial bile’ decreases LT-97 cell growth only in presence of celecoxib. ► PCNA, caspase-3, nor COX-2 seem to be involved in the observed changes in cell growth.

  20. Pro-inflammatory activated Kupffer cells by lipids induce hepatic NKT cells deficiency through activation-induced cell death.

    Directory of Open Access Journals (Sweden)

    Tongfang Tang

    Full Text Available BACKGROUND: Dietary lipids play an important role in the progression of non-alcoholic fatty liver disease (NAFLD through alternation of liver innate immune response. AIMS: The present study was to investigate the effect of lipid on Kupffer cells phenotype and function in vivo and in vitro. And further to investigate the impact of lipid on ability of Kupffer cell lipid antigen presentation to activate NKT cells. METHODS: Wild type male C57BL/6 mice were fed either normal or high-fat diet. Hepatic steatosis, Kupffer cell abundance, NKT cell number and cytokine gene expression were evaluated. Antigen presentation assay was performed with Kupffer cells treated with certain fatty acids in vitro and co-cultured with NKT cells. RESULTS: High-fat diet induced hepatosteatosis, significantly increased Kupffer cells and decreased hepatic NKT cells. Lipid treatment in vivo or in vitro induced increase of pro-inflammatory cytokines gene expression and toll-like receptor 4 (TLR4 expression in Kupffer cells. Kupffer cells expressed high levels of CD1d on cell surface and only presented exogenous lipid antigen to activate NKT cells. Ability of Kupffer cells to present antigen and activate NKT cells was enhanced after lipid treatment. In addition, pro-inflammatory activated Kupffer cells by lipid treatment induced hepatic NKT cells activation-induced apoptosis and necrosis. CONCLUSION: High-fat diet increase Kupffer cells number and induce their pro-inflammatory status. Pro-inflammatory activated Kupfffer cells by lipid promote hepatic NKT cell over-activation and cell death, which lead to further hepatic NKT cell deficiency in the development of NAFLD.

  1. Pro-inflammatory activated Kupffer cells by lipids induce hepatic NKT cells deficiency through activation-induced cell death.

    Science.gov (United States)

    Tang, Tongfang; Sui, Yongheng; Lian, Min; Li, Zhiping; Hua, Jing

    2013-01-01

    Dietary lipids play an important role in the progression of non-alcoholic fatty liver disease (NAFLD) through alternation of liver innate immune response. The present study was to investigate the effect of lipid on Kupffer cells phenotype and function in vivo and in vitro. And further to investigate the impact of lipid on ability of Kupffer cell lipid antigen presentation to activate NKT cells. Wild type male C57BL/6 mice were fed either normal or high-fat diet. Hepatic steatosis, Kupffer cell abundance, NKT cell number and cytokine gene expression were evaluated. Antigen presentation assay was performed with Kupffer cells treated with certain fatty acids in vitro and co-cultured with NKT cells. High-fat diet induced hepatosteatosis, significantly increased Kupffer cells and decreased hepatic NKT cells. Lipid treatment in vivo or in vitro induced increase of pro-inflammatory cytokines gene expression and toll-like receptor 4 (TLR4) expression in Kupffer cells. Kupffer cells expressed high levels of CD1d on cell surface and only presented exogenous lipid antigen to activate NKT cells. Ability of Kupffer cells to present antigen and activate NKT cells was enhanced after lipid treatment. In addition, pro-inflammatory activated Kupffer cells by lipid treatment induced hepatic NKT cells activation-induced apoptosis and necrosis. High-fat diet increase Kupffer cells number and induce their pro-inflammatory status. Pro-inflammatory activated Kupfffer cells by lipid promote hepatic NKT cell over-activation and cell death, which lead to further hepatic NKT cell deficiency in the development of NAFLD.

  2. Bisphenol A Disrupts Transcription and Decreases Viability in Aging Vascular Endothelial Cells

    Science.gov (United States)

    Ribeiro-Varandas, Edna; Pereira, H. Sofia; Monteiro, Sara; Neves, Elsa; Brito, Luísa; Boavida Ferreira, Ricardo; Viegas, Wanda; Delgado, Margarida

    2014-01-01

    Bisphenol A (BPA) is a widely utilized endocrine disruptor capable of mimicking endogenous hormones, employed in the manufacture of numerous consumer products, thereby interfering with physiological cellular functions. Recent research has shown that BPA alters epigenetic cellular mechanisms in mammals and may be correlated to enhanced cellular senescence. Here, the effects of BPA at 10 ng/mL and 1 µg/mL, concentrations found in human samples, were analyzed on HT29 human colon adenocarcinona cell line and Human Umbilical Vein Endothelial Cells (HUVEC). Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) transcriptional analysis of the Long Interspersed Element-1 (LINE-1) retroelement showed that BPA induces global transcription deregulation in both cell lines, although with more pronounced effects in HUVEC cells. Whereas there was an increase in global transcription in HT29 exclusively after 24 h of exposure, this chemical had prolonged effects on HUVEC. Immunoblotting revealed that this was not accompanied by alterations in the overall content of H3K9me2 and H3K4me3 epigenetic marks. Importantly, cell viability assays and transcriptional analysis indicated that prolonged BPA exposure affects aging processes in senescent HUVEC. To our knowledge this is the first report that BPA interferes with senescence in primary vascular endothelial cells, therefore, suggesting its association to the etiology of age-related human pathologies, such as atherosclerosis. PMID:25207595

  3. Experimental study of cell reversal of a high temperature polymer electrolyte membrane fuel cell caused by H2 starvation

    DEFF Research Database (Denmark)

    Zhou, Fan; Andreasen, Søren Juhl; Kær, Søren Knudsen

    2015-01-01

    Operation under fuel starvation has been proved to be harmful to the fuel cell by causing severe and irreversible degradation. To characterize the behaviors of the high temperature PEM fuel cell under fuel starvation conditions, the cell voltage and local current density is measured simultaneously...... under different H2 stoichiometries below 1.0 and at different current loads. The experimental results show that the cell voltage decreases promptly when the H2 stoichiometry decreases to below 1.0. Negative cell voltage can be observed which indicates cell reversal. The local current density starts...... to diverge when the cell voltage decreases. In the H2 upstream regions the current densities show an increasing trend, while those in the H2 downstream regions show a decreasing trend. Consequently, the current density distribution becomes very uneven. The current density is the highest in the upstream...

  4. Cell cycle arrest and cell survival induce reverse trends of cardiolipin remodeling.

    Directory of Open Access Journals (Sweden)

    Yu-Jen Chao

    Full Text Available Cell survival from the arrested state can be a cause of the cancer recurrence. Transition from the arrest state to the growth state is highly regulated by mitochondrial activity, which is related to the lipid compositions of the mitochondrial membrane. Cardiolipin is a critical phospholipid for the mitochondrial integrity and functions. We examined the changes of cardiolipin species by LC-MS in the transition between cell cycle arrest and cell reviving in HT1080 fibrosarcoma cells. We have identified 41 cardiolipin species by MS/MS and semi-quantitated them to analyze the detailed changes of cardiolipin species. The mass spectra of cardiolipin with the same carbon number form an envelope, and the C64, C66, C68, C70 C72 and C74 envelopes in HT1080 cells show a normal distribution in the full scan mass spectrum. The cardiolipin quantity in a cell decreases while entering the cell cycle arrest, but maintains at a similar level through cell survival. While cells awakening from the arrested state and preparing itself for replication, the groups with short acyl chains, such as C64, C66 and C68 show a decrease of cardiolipin percentage, but the groups with long acyl chains, such as C70 and C72 display an increase of cardiolipin percentage. Interestingly, the trends of the cardiolipin species changes during the arresting state are completely opposite to cell growing state. Our results indicate that the cardiolipin species shift from the short chain to long chain cardiolipin during the transition from cell cycle arrest to cell progression.

  5. Nuclear orphan receptor TLX affects gene expression, proliferation and cell apoptosis in beta cells

    International Nuclear Information System (INIS)

    Shi, Xiaoli; Xiong, Xiaokan; Dai, Zhe; Deng, Haohua; Sun, Li; Hu, Xuemei; Zhou, Feng; Xu, Yancheng

    2015-01-01

    Nuclear orphan receptor TLX is an essential regulator of the growth of neural stem cells. However, its exact function in pancreatic islet cells is still unknown. In the present study, gene expression profiling analysis revealed that overexpression of TLX in beta cell line MIN6 causes suppression of 176 genes and upregulation of 49 genes, including a cadre of cell cycle, cell proliferation and cell death control genes, such as Btg2, Ddit3 and Gadd45a. We next examined the effects of TLX overexpression on proliferation, apoptosis and insulin secretion in MIN6 cells. Proliferation analysis using EdU assay showed that overexpression of TLX increased percentage of EdU-positive cells. Cell cycle and apoptosis analysis revealed that overexpression of TLX in MIN6 cells resulted in higher percentage of cells exiting G1 into S-phase, and a 58.8% decrease of cell apoptosis induced by 0.5 mM palmitate. Moreover, TLX overexpression did not cause impairment of insulin secretion. Together, we conclude that TLX is among factors capable of controlling beta cell proliferation and survival, which may serve as a target for the development of novel therapies for diabetes. - Highlights: • TLX overexpression in MIN6 cell causes significant expression changes of 225 genes. • TLX overexpression promotes MIN6 cell proliferation and decreases cell apoptosis. • TLX overexpression does not cause impairment of insulin secretion.

  6. Nuclear orphan receptor TLX affects gene expression, proliferation and cell apoptosis in beta cells

    Energy Technology Data Exchange (ETDEWEB)

    Shi, Xiaoli; Xiong, Xiaokan; Dai, Zhe; Deng, Haohua; Sun, Li; Hu, Xuemei; Zhou, Feng; Xu, Yancheng, E-mail: oxyccc@163.com

    2015-12-04

    Nuclear orphan receptor TLX is an essential regulator of the growth of neural stem cells. However, its exact function in pancreatic islet cells is still unknown. In the present study, gene expression profiling analysis revealed that overexpression of TLX in beta cell line MIN6 causes suppression of 176 genes and upregulation of 49 genes, including a cadre of cell cycle, cell proliferation and cell death control genes, such as Btg2, Ddit3 and Gadd45a. We next examined the effects of TLX overexpression on proliferation, apoptosis and insulin secretion in MIN6 cells. Proliferation analysis using EdU assay showed that overexpression of TLX increased percentage of EdU-positive cells. Cell cycle and apoptosis analysis revealed that overexpression of TLX in MIN6 cells resulted in higher percentage of cells exiting G1 into S-phase, and a 58.8% decrease of cell apoptosis induced by 0.5 mM palmitate. Moreover, TLX overexpression did not cause impairment of insulin secretion. Together, we conclude that TLX is among factors capable of controlling beta cell proliferation and survival, which may serve as a target for the development of novel therapies for diabetes. - Highlights: • TLX overexpression in MIN6 cell causes significant expression changes of 225 genes. • TLX overexpression promotes MIN6 cell proliferation and decreases cell apoptosis. • TLX overexpression does not cause impairment of insulin secretion.

  7. Biomek Cell Workstation: A Variable System for Automated Cell Cultivation.

    Science.gov (United States)

    Lehmann, R; Severitt, J C; Roddelkopf, T; Junginger, S; Thurow, K

    2016-06-01

    Automated cell cultivation is an important tool for simplifying routine laboratory work. Automated methods are independent of skill levels and daily constitution of laboratory staff in combination with a constant quality and performance of the methods. The Biomek Cell Workstation was configured as a flexible and compatible system. The modified Biomek Cell Workstation enables the cultivation of adherent and suspension cells. Until now, no commercially available systems enabled the automated handling of both types of cells in one system. In particular, the automated cultivation of suspension cells in this form has not been published. The cell counts and viabilities were nonsignificantly decreased for cells cultivated in AutoFlasks in automated handling. The proliferation of manual and automated bioscreening by the WST-1 assay showed a nonsignificant lower proliferation of automatically disseminated cells associated with a mostly lower standard error. The disseminated suspension cell lines showed different pronounced proliferations in descending order, starting with Jurkat cells followed by SEM, Molt4, and RS4 cells having the lowest proliferation. In this respect, we successfully disseminated and screened suspension cells in an automated way. The automated cultivation and dissemination of a variety of suspension cells can replace the manual method. © 2015 Society for Laboratory Automation and Screening.

  8. Cytotoxicity of ophthalmic solutions with and without preservatives to human corneal endothelial cells, epithelial cells and conjunctival epithelial cells.

    Science.gov (United States)

    Ayaki, Masahiko; Yaguchi, Shigeo; Iwasawa, Atsuo; Koide, Ryohei

    2008-08-01

    The cytotoxicity of a range of commercial ophthalmic solutions in the presence and absence of preservatives was assessed in human corneal endothelial cells (HCECs), corneal epithelia and conjunctival epithelia using in vitro techniques. Cell survival was measured using the WST-1 assay for endothelial cells and the MTT assay for epithelial cells. Commercially available timolol, carteolol, cromoglicate, diclofenac, bromfenac and hyaluronic acid ophthalmic solutions were assessed for cytotoxicity in the presence and absence of preservatives. The preservatives benzalkonium, chlorobutanol and polysorbate were also tested. The survival of cells exposed to test ophthalmic solutions was expressed as a percentage of cell survival in the control solution (distilled water added to media) after 48 h exposure. HCEC survival was 20-30% in ophthalmic solutions diluted 10-fold. The survival of HCEC was significantly greater in all solutions in the absence of preservative than in the presence of preservative. The survival of corneal and conjunctival epithelia was consistent with that of HCECs for all test ophthalmic solutions. The preservatives polysorbate and benzalkonium were highly cytotoxic with cell survival decreasing to 20% at the concentration estimated in commercial ophthalmic solutions. By comparison, the survival of cells exposed to chlorobutanol was 80% or greater. The cytotoxicity of ophthalmic solutions to HCEC, corneal epithelia and conjunctival epithelia decreased in the absence of preservative.

  9. Acacetin enhances the therapeutic efficacy of doxorubicin in non-small-cell lung carcinoma cells.

    Directory of Open Access Journals (Sweden)

    Reenu Punia

    Full Text Available Anthracyclines are efficient and potent agents to treat broad range of cancers but cytotoxicity induced by them limits their use in therapeutics. Use of plant-derived agents help to prevent or delay the process of cancer progression and their combination increases the anti-cancer potential of mainstream compound. However, multidrug resistance is major cause of treatment failure in cancer patients.In this study, combination treatments of fisetin or acacetin with doxorubicin were explored for their potential synergistic effect on non-small-cell lung carcinoma (NSCLC cells.During this study, NSCLC model cell lines A549 and H1299 were used to determine the combinatorial effect of phytochemicals namly acacetin and fisetin with doxorubicin.The effects of individual compounds and their combination on cell viability, clonogenic potential and cell cycle progression were studied. Efflux of doxorubicin was measured by spectrofluorophotometer, whereas accumulation inside the cells was analyzed by flow cytometry and confocal microscopy. Expression of MDR1 was checked by semi-quantitative PCR.The results showed that the cell viability of A549 and H1299 cells were significantly decreased in time- and dose-dependent manner, although A549 cells showed more sensitivity toward doxorubicin than H1299 cells. Mostly, combination of doxorubicin showed good synergy with acacetin in both the cell lines whereas, fisetin exerted synergistic effect only at 72 h of treatment in H1299 cells. Acacetin with doxorubicin caused G2/M arrest by downregulating CDK-cyclin complex in A549 cells. Acacetin-doxorubicin combination decreased the clonogenic potential of A549 and H1299 cells upto 82% and 59%, respectively, as compared to control. Acacetin also decreased efflux of doxorubicin by 59% after 30 mins of exposure to A549 cells and further increased accumulation of doxorubicin inside the cells upto 55% in 2 h. The modulatory effect of acacetin-doxorubicin combination on

  10. Over-expression of CD8+ T-cell activation is associated with decreased CD4+ cells in patients seeking treatment of Alcohol Use Disorder.

    Science.gov (United States)

    Zuluaga, Paola; Sanvisens, Arantza; Martínez-Cáceres, Eva; Teniente, Aina; Tor, Jordi; Muga, Robert

    2017-11-01

    Harmful alcohol consumption may have an impact on the adaptive immune system through an imbalance in T cell subpopulations and changes in cell activation. We aimed to analyze profiles of CD4 and CD8T cell activation in patients with alcohol use disorder (AUD). We used a cross-sectional study with patients seeking treatment of the disorder. Blood samples for immunophenotyping were obtained at admission. Profiles of T cell activation were defined: (I) CD38 + /HLA-DR + , (II) CD38 + /HLA-DR - , (III) CD38 - /HLA-DR + , (IV) CD38 - /HLA-DR - and compared with healthy controls. We calculated a CD8 + T cell activation indicator (AI) that was defined as the quotient of non-activated cells (CD38 - /HLA-DR - ) and activated cells (CD38 + /HLA-DR + ). 60 patients were eligible (83%M); median age was 49 years [IQR: 44-54] and alcohol consumption was 145g/day [IQR: 90-205]. Mean±SD of CD38 + /HLA-DR - was 50.3±50.6 cells/μL in patients and 33.5±24.5 cells/μL in controls (p=0.03), for the CD38 - /HLA-DR + it was 61±62.2 cells/μL in patients and 21.2±17.3 cells/μL in controls (pcells/μL in patients and 10.8±10.3 cells/μL in controls (pcells, and the percentage of CD38 + /HLA-DR + CD8 + T cells (r=0.37, p=0.003; r=0.2, p=0.086, respectively). Patients with AUD have an increased expression of immune activation with respect to healthy individuals. This excess of activated CD8 + T cells correlates with the absolute CD4 + T cells. Copyright © 2017 Elsevier B.V. All rights reserved.

  11. PTEN-induction in U251 glioma cells decreases the expression of insulin-like growth factor binding protein-2

    International Nuclear Information System (INIS)

    Levitt, Randy J.; Georgescu, Maria-Magdalena; Pollak, Michael

    2005-01-01

    PTEN is a tumor suppressor gene whose loss of function is observed in ∼40-50% of human cancers. Although insulin-like growth factor binding protein-2 (IGFBP-2) was classically described as a growth inhibitor, multiple recent reports have shown an association of overexpression and/or high serum levels of IGFBP-2 with poor prognosis of several malignancies, including gliomas. Using an inducible PTEN expression system in the PTEN-null glioma cell line U251, we demonstrate that PTEN-induction is associated with reduced proliferation, increased apoptosis, and a substantial reduction of the high levels of IGFBP-2 expression. The PTEN-induced decrease in IGFBP-2 expression could be mimicked with the PI3-kinase inhibitor LY294002, indicating that the lipid phosphatase activity of PTEN is responsible for the observed effect. However, the rapamycin analog CCI-779 did not affect IGFBP-2 expression, suggesting that the PTEN-induced decrease in IGFBP-2 expression is not attributable to decreased mTOR signalling. Recombinant human IGFBP-2 was unable to rescue U251-PTEN cells from the antiproliferative effects of PTEN, and IGFBP-2 siRNA did not affect the IGF-dependent or -independent growth of this cell line. These results suggest that the clinical data linking IGFBP-2 expression to poor prognosis may arise, at least in part, because high levels of IGFBP-2 expression correlate with loss of function of PTEN, which is well known to lead to aggressive behavior of gliomas. Our results motivate translational research regarding the relationship between IGFBP-2 expression and loss of function of PTEN

  12. 7-ketocholesterol inhibits Na,K-ATPase activity by decreasing expression of its α1-subunit and membrane fluidity in human endothelial cells.

    Science.gov (United States)

    Duran, M J; Pierre, S V; Lesnik, P; Pieroni, G; Bourdeaux, M; Dignat-Georges, F; Sampol, J; Maixent, J M

    2010-11-09

    As cholesterol, oxysterols, can insert the cell membrane and thereby modify the functions of membrane-bound proteins. The Na,K-ATPase is very sensitive to its lipid environment, seems to be involved in important endothelial functions as the regulation of nitric oxide (NO) release. The effects of 7-ketocholesterol , an oxysterol present in oxidized LDL, was investigated on Na,K-ATPase in isolated human endothelial cells. Cells were incubated 24h with lecithin-, cholesterol- or 7-ketocholesterol liposomes (6 μg/ml). K+-stimulated paranitrophenyl phosphatase activity, reflecting Na,K-ATPase activity, was evaluated as well as cell viability and lipoperoxidation. The expression of Na,K-ATPase subunits mRNAs and membrane fluidity were also investigated. As Na,K-ATPase and nitric oxide seem to be related, we determined the production of NO and the expression of endothelial NO synthase mRNAs. Na,K-ATPase activity was strongly decreased by 7-ketocholesterol. This decrease, not related to lipoperoxidation, was correlated with a decreased expression of the Na,K-ATPase α1-subunit messengers and with rigidity of plasma membranes. Cholesterol induced similar effects but was less potent than 7-ketocholesterol. Basal NO production and expression of endothelial NO synthase mRNAs were not modified by 7-ketocholesterol. Our new findings demonstrate that 7-ketocholesterol, used at non toxic doses, was very potent to disrupt the transport of ions by Na,K-ATPase and perturb membrane structure. These data demonstrate that 7-ketocholesterol induces endothelial dysfunction without cell death that may contribute to early events in atherosclerosis.

  13. Iodine-131 treatment of thyroid cancer cells leads to suppression of cell proliferation followed by induction of cell apoptosis and cell cycle arrest by regulation of B-cell translocation gene 2-mediated JNK/NF-κB pathways

    Energy Technology Data Exchange (ETDEWEB)

    Zhao, L.M.; Pang, A.X., E-mail: zhaoliming515@126.com [Department of Nuclear Medicine, Linyi People' s Hospital, Linyi (China); Department of Urology, Linyi People' s Hospital, Linyi (China)

    2017-10-01

    Iodine-131 ({sup 131}I) is widely used for the treatment of thyroid-related diseases. This study aimed to investigate the expression of p53 and BTG2 genes following {sup 131}I therapy in thyroid cancer cell line SW579 and the possible underlying mechanism. SW579 human thyroid squamous carcinoma cells were cultured and treated with {sup 131}I. They were then assessed for {sup 131}I uptake, cell viability, apoptosis, cell cycle arrest, p53 expression, and BTG2 gene expression. SW579 cells were transfected with BTG2 siRNA, p53 siRNA and siNC and were then examined for the same aforementioned parameters. When treated with a JNK inhibitor of SP600125 and {sup 131}I or with a NF-kB inhibitor of BMS-345541 and {sup 131}I, non-transfected SW579 cells were assessed in JNK/NFkB pathways. It was observed that {sup 131}I significantly inhibited cell proliferation, promoted cell apoptosis and cell cycle arrest. Both BTG2 and p53 expression were enhanced in a dose-dependent manner. An increase in cell viability by up-regulation in Bcl2 gene, a decrease in apoptosis by enhanced CDK2 gene expression and a decrease in cell cycle arrest at G{sub 0}/G{sub 1} phase were also observed in SW579 cell lines transfected with silenced BTG2 gene. When treated with SP600125 and {sup 131}I, the non transfected SW579 cell lines significantly inhibited JNK pathway, NF-kB pathway and the expression of BTG2. However, when treated with BMS-345541 and {sup 131}I, only the NF-kB pathway was suppressed. {sup 131}I suppressed cell proliferation, induced cell apoptosis, and promoted cell cycle arrest of thyroid cancer cells by up-regulating B-cell translocation gene 2-mediated activation of JNK/NF--κB pathways. (author)

  14. Filaggrin 2 deficiency results in abnormal cell-cell adhesion in the cornified cell layers and causes peeling skin syndrome type A.

    Science.gov (United States)

    Mohamad, Janan; Sarig, Ofer; Godsel, Lisa M; Peled, Alon; Malchin, Natalia; Bochner, Ron; Vodo, Dan; Rabinowitz, Tom; Pavlovsky, Mor; Taiber, Shahar; Fried, Maya; Eskin-Schwartz, Marina; Assi, Siwar; Shomron, Noam; Uitto, Jouni; Koetsier, Jennifer L; Bergman, Reuven; Green, Kathleen J; Sprecher, Eli

    2018-05-11

    Peeling skin syndromes form a large and heterogeneous group of inherited disorders characterized by superficial detachment of the epidermal cornified cell layers, often associated with inflammatory features. Here we report on a consanguineous family featuring non-inflammatory peeling of the skin exacerbated by exposure to heat and mechanical stress. Whole exome sequencing revealed a homozygous nonsense mutation in FLG2, encoding filaggrin 2, which co-segregated with the disease phenotype in the family. The mutation was found to result in decreased FLG2 RNA levels as well almost total absence of filaggrin 2 in the patient epidermis. Filaggrin 2 was found to be expressed throughout the cornified cell layers and to co-localize with corneodesmosin which plays a crucial role in maintaining cell-cell adhesion in this region of the epidermis. Absence of filaggrin 2 in the patient skin was associated with markedly decreased corneodesmosin expression, which may contribute to the peeling phenotype displayed by the patients. Accordingly, using the dispase dissociation assay, we showed that FLG2 down-regulation interferes with keratinocyte cell-cell adhesion. Of particular interest, this effect was aggravated by temperature elevation, consistent with the clinical phenotype. Restoration of CDSN levels by ectopic expression rescued cell-cell adhesion.Taken together, the present data suggest that filaggrin 2 is essential for normal cell-cell adhesion in the cornified cell layers. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

  15. Nuclear receptor TLX regulates cell cycle progression in neural stem cells of the developing brain.

    Science.gov (United States)

    Li, Wenwu; Sun, Guoqiang; Yang, Su; Qu, Qiuhao; Nakashima, Kinichi; Shi, Yanhong

    2008-01-01

    TLX is an orphan nuclear receptor that is expressed exclusively in vertebrate forebrains. Although TLX is known to be expressed in embryonic brains, the mechanism by which it influences neural development remains largely unknown. We show here that TLX is expressed specifically in periventricular neural stem cells in embryonic brains. Significant thinning of neocortex was observed in embryonic d 14.5 TLX-null brains with reduced nestin labeling and decreased cell proliferation in the germinal zone. Cell cycle analysis revealed both prolonged cell cycles and increased cell cycle exit in TLX-null embryonic brains. Increased expression of a cyclin-dependent kinase inhibitor p21 and decreased expression of cyclin D1 provide a molecular basis for the deficiency of cell cycle progression in embryonic brains of TLX-null mice. Furthermore, transient knockdown of TLX by in utero electroporation led to precocious cell cycle exit and differentiation of neural stem cells followed by outward migration. Together these results indicate that TLX plays an important role in neural development by regulating cell cycle progression and exit of neural stem cells in the developing brain.

  16. Keratin 5/14‑mediated cell differentiation and transformation are regulated by TAp63 and Notch‑1 in oral squamous cell carcinoma‑derived cells.

    Science.gov (United States)

    Srivastava, Saumya S; Alam, Hunain; Patil, Sonam J; Shrinivasan, Rashmi; Raikundalia, Sweta; Chaudhari, Pratik Rajeev; Vaidya, Milind M

    2018-05-01

    Keratins 5/14 (K5/14) are intermediate filament proteins expressed in the basal layer of stratified epithelial cells and are known targets of p63. Previous research in our laboratory showed that upon K5/14 downregulation in oral squamous cell carcinoma (OSCC)‑derived cells, there was an increase in intracellular Notch‑1 levels and differentiation markers such as involucrin, keratin 1 and a decrease in tumorigenic potential in vivo. However, the molecules involved in the K14 regulated cell differentiation and transformation are not known to date. In order to understand the possible role of TAp63, we downregulated TAp63 in a K14‑knockdown background. We observed that there was a decrease in the expression of Notch‑1. Expression levels of differentiation markers such as involucrin, K1, loricrin and filaggrin were also decreased. Furthermore, TAp63 downregulation led to an increase in invasion, migration and in vivo tumorigenic potential of these cells. We observed a decrease in β‑catenin signaling in K14‑downregulated cells. Notably, when TAp63 was downregulated in K14‑knockdown cells, there was increase in non‑phospho β‑catenin levels. Hence, this study indicates that TAp63 plays an important role in K14‑downregulated cells possibly by regulating the Notch‑1 expression. K14 regulates the expression of TAp63 which in turn regulates expression of Notch‑1. The present study is a step forward in our quest to understand the functional significance of molecules that regulate the process of differentiation and tumorigenesis in stratified epithelial cells.

  17. Interference with PSMB4 Expression Exerts an Anti-Tumor Effect by Decreasing the Invasion and Proliferation of Human Glioblastoma Cells

    Directory of Open Access Journals (Sweden)

    Yu-Chen Cheng

    2018-01-01

    Full Text Available Background/Aims: Glioblastoma (GBM is a malignant brain tumor with a poor prognosis. Proteasome subunit beta type-4 (PSMB4 is an essential subunit that contributes to the assembly of the 20S proteasome complex. However, the role of PSMB4 in glioblastomas remains to be clarified. The aim of this study was to investigate the role of PSMB4 in GBM tumor progression. Methods: We first analyzed the PSMB4 protein and mRNA expression in 80 clinical brain specimens and 77 datasets from the National Center for Biotechnology Information (NCBI Gene Expression Omnibus (GEO database. Next, we inhibited the PSMB4 expression by siRNA in cellular and animal models to explore PSMB4’s underlying mechanisms. The cell survival after siPSMB4 transfection was assayed by MTT assay. Annexin V and propidium iodide staining was used to monitor the apoptosis by flow cytometric analysis. Moreover, the migration and invasion were evaluated by wound healing and Transwell assays. The expression of migration-related and invasion-related proteins after PSMB4 inhibition was detected by Western blotting. In addition, an orthotropic xenograft mouse model was used to assay the effect of PSMB4 knockdown in the in vivo study. Results: Basis on the results of bioinformatics study, glioma patients with higher PSMB4 expression had a shorter survival time than those with lower PSMB4 expression. The staining of clinical brain tissues showed elevated PSMB4 expression in GBM tissues compared with normal brain tissues. The PSMB4 inhibition decreased proliferation, migration and invasion abilities in human GBM cells. Downregulated PSMB4 resulted in cell cycle arrest and apoptosis in vitro. In an orthotropic xenograft mouse model, the glioma tumors progression was reduced when PSMB4 was down-regulated. The decreased PSMB4 enhanced the anti-tumor effect of temozolomide (TMZ on tumor growth. In addition, the absence of PSMB4 decreased the expression of phosphorylated focal adhesion kinase and

  18. Mast cells infiltration and decreased E-cadherin expression in ketamine-induced cystitis

    Directory of Open Access Journals (Sweden)

    Mengqiang Li

    2015-01-01

    Conclusions: Increased mast cells in bladder wall and downregulated expression of E-cadherin junction protein in epithelial cells were probably associated with interstitial inflammation and fissures in mucosa. It implied that ketamine induced an interstitial cystitis.

  19. Decreased SAP Expression in T Cells from Patients with Systemic Lupus Erythematosus Contributes to Early Signaling Abnormalities and Reduced IL-2 Production.

    Science.gov (United States)

    Karampetsou, Maria P; Comte, Denis; Kis-Toth, Katalin; Terhorst, Cox; Kyttaris, Vasileios C; Tsokos, George C

    2016-06-15

    T cells from patients with systemic lupus erythematosus (SLE) display a number of abnormalities, including increased early signaling events following engagement of the TCR. Signaling lymphocytic activation molecule family cell surface receptors and the X-chromosome-defined signaling lymphocytic activation molecule-associated protein (SAP) adaptor are important in the development of several immunocyte lineages and modulating the immune response. We present evidence that SAP protein levels are decreased in T cells and in their main subsets isolated from 32 women and three men with SLE, independent of disease activity. In SLE T cells, SAP protein is also subject to increased degradation by caspase-3. Forced expression of SAP in SLE T cells normalized IL-2 production, calcium (Ca(2+)) responses, and tyrosine phosphorylation of a number of proteins. Exposure of normal T cells to SLE serum IgG, known to contain anti-CD3/TCR Abs, resulted in SAP downregulation. We conclude that SLE T cells display reduced levels of the adaptor protein SAP, probably as a result of continuous T cell activation and degradation by caspase-3. Restoration of SAP levels in SLE T cells corrects the overexcitable lupus T cell phenotype. Copyright © 2016 by The American Association of Immunologists, Inc.

  20. Rho GTPase activity modulates paramyxovirus fusion protein-mediated cell-cell fusion

    International Nuclear Information System (INIS)

    Schowalter, Rachel M.; Wurth, Mark A.; Aguilar, Hector C.; Lee, Benhur; Moncman, Carole L.; McCann, Richard O.; Dutch, Rebecca Ellis

    2006-01-01

    The paramyxovirus fusion protein (F) promotes fusion of the viral envelope with the plasma membrane of target cells as well as cell-cell fusion. The plasma membrane is closely associated with the actin cytoskeleton, but the role of actin dynamics in paramyxovirus F-mediated membrane fusion is unclear. We examined cell-cell fusion promoted by two different paramyxovirus F proteins in three cell types in the presence of constitutively active Rho family GTPases, major cellular coordinators of actin dynamics. Reporter gene and syncytia assays demonstrated that expression of either Rac1 V12 or Cdc42 V12 could increase cell-cell fusion promoted by the Hendra or SV5 glycoproteins, though the effect was dependent on the cell type expressing the viral glycoproteins. In contrast, RhoA L63 decreased cell-cell fusion promoted by Hendra glycoproteins but had little affect on SV5 F-mediated fusion. Also, data suggested that GTPase activation in the viral glycoprotein-containing cell was primarily responsible for changes in fusion. Additionally, we found that activated Cdc42 promoted nuclear rearrangement in syncytia

  1. Role of Cell-Cell bond for the viability and the function of vascular smooth muscle cells

    Directory of Open Access Journals (Sweden)

    M. Mura

    2010-01-01

    Full Text Available Vascular smooth muscle cell (VSMC viability and homeostasis is regulated by cell-matrix and cell-cell contact: disruption of these interactions are responsible of a switch from a mature to a high proliferative phenotype. VSMCs migration, rate of growth and apoptosis, and the extent of their extracellular matrix (ECM deposition can be also modulated by proatherogenic peptides. Among them, ATII induces the transactivation of IGF I R, which, together with the binding protein IGFBP3, represents a determinant of cell survival, growth and proliferation. Aim of our in vitro study was to verify the role of elective cell-cell bond in moulating the response to ATII. Thus, we evaluated viability, proliferation, IGFIR, IGFBP3 expression and the long term survival and production of ECM in a provisional tissue. A7r5 cell-line was used in adherent cultures or incubated in agarose-coated culture plates to inhibit cell-matrix interactions. Cells, treated or not with ATII 100 nM, were evaluated for apoptosis rate, cell cycle, IGFIR and IGFBP3 protei expression. Fibrin provisional tissue was developed polymerizing a fibrin solution. cantaining A7r5 cells with thrombin. Histological stainings for ECM components were performed on sections of prvisional tissue. An exclusive cell-cell contact resulted to monolayer cell cultures. ATII did not affect the cell survival in both culture conditions, but promoted a 10% decrease in "S" phase and an increases IGFIR expression only in adherent cells. while suspended cell aggregates were resistant to ATII administration; IGFBP3 was reduced both in ATII treated adherent cells and in floating clustered cells, irrespective of the treatmentn. VSMC conditioning in agarose-coated plates before seeding in fibrin provisional matrix reduced, but not abolished, the cell ability to colonize the clot and to produce ECM. This study demonstrates that the elective cell-cell contact induces a quiescent status in cells lacking of cell

  2. HYDROXYUREA TREATMENT DECREASES GLOMERULAR HYPERFILTRATION IN CHILDREN WITH SICKLE CELL ANEMIA

    Science.gov (United States)

    Aygun, Banu; Mortier, Nicole A.; Smeltzer, Matthew P.; Shulkin, Barry L.; Hankins, Jane S.; Ware, Russell E.

    2015-01-01

    Background Glomerular hyperfiltration and microalbuminuria/proteinuria are early manifestations of sickle nephropathy. The effects of hydroxyurea therapy on these renal manifestations of sickle cell anemia (SCA) are not well defined. Objective To investigate the effects of hydroxyurea on glomerular filtration rate (GFR) measured by 99mTc-DTPA clearance, and on microalbuminuria/proteinuria in children with SCA. Study Design Hydroxyurea Study of Long-Term Effects (HUSTLE) is a prospective study (NCT00305175) with the goal of describing the long-term cellular, molecular, and clinical effects of hydroxyurea therapy in SCA. Glomerular filtration rate, urine microalbumin, and serum cystatin C were measured before initiating hydroxyurea therapy and then repeated after 3 years. Baseline and Year 3 values for HUSTLE subjects were compared using the Wilcoxon Signed Rank test. Associations between continuous variables were evaluated using Spearman correlation coefficient. Results Twenty-three children with SCA (median age 7.5 years, range 2.5–14.0 years) received hydroxyurea at maximum tolerated dose (MTD, 24.4 ± 4.5 mg/kg/day, range 15.3–30.6 mg/kg/day). After three years of treatment, GFR measured by 99mTc-DTPA decreased significantly from 167 ± 46 mL/min/1.73m2 to 145 ± 27 mL/min/1.73m2 (p=0.016). This decrease in GFR was significantly associated with increase in fetal hemoglobin (p= 0.042) and decrease in lactate dehydrogenase levels (p=0.035). Urine microalbumin and cystatin C levels did not change significantly. Conclusions Hydroxyurea at MTD is associated with a decrease in hyperfiltration in young children with SCA. PMID:23255310

  3. Hemidesmosomal linker proteins regulate cell motility, invasion and tumorigenicity in oral squamous cell carcinoma derived cells.

    Science.gov (United States)

    Chaudhari, Pratik Rajeev; Charles, Silvania Emlit; D'Souza, Zinia Charlotte; Vaidya, Milind Murlidhar

    2017-11-15

    BPAG1e and Plectin are hemidesmosomal linker proteins which anchor intermediate filament proteins to the cell surface through β4 integrin. Recent reports indicate that these proteins play a role in various cellular processes apart from their known anchoring function. However, the available literature is inconsistent. Further, the previous study from our laboratory suggested that Keratin8/18 pair promotes cell motility and tumor progression by deregulating β4 integrin signaling in oral squamous cell carcinoma (OSCC) derived cells. Based on these findings, we hypothesized that linker proteins may have a role in neoplastic progression of OSCC. Downregulation of hemidesmosomal linker proteins in OSCC derived cells resulted in reduced cell migration accompanied by alterations in actin organization. Further, decreased MMP9 activity led to reduced cell invasion in linker proteins knockdown cells. Moreover, loss of these proteins resulted in reduced tumorigenic potential. SWATH analysis demonstrated upregulation of N-Myc downstream regulated gene 1 (NDRG1) in linker proteins downregulated cells as compared to vector control cells. Further, the defects in phenotype upon linker proteins ablation were rescued upon loss of NDRG1 in linker proteins knockdown background. These data together indicate that hemidesmosomal linker proteins regulate cell motility, invasion and tumorigenicity possibly through NDRG1 in OSCC derived cells. Copyright © 2017 Elsevier Inc. All rights reserved.

  4. SHP1-mediated cell cycle redistribution inhibits radiosensitivity of non-small cell lung cancer

    International Nuclear Information System (INIS)

    Cao, Rubo; Ding, Qian; Li, Pindong; Xue, Jun; Zou, Zhenwei; Huang, Jing; Peng, Gang

    2013-01-01

    Radioresistance is the common cause for radiotherapy failure in non-small cell lung cancer (NSCLC), and the degree of radiosensitivity of tumor cells is different during different cell cycle phases. The objective of the present study was to investigate the effects of cell cycle redistribution in the establishment of radioresistance in NSCLC, as well as the signaling pathway of SH2 containing Tyrosine Phosphatase (SHP1). A NSCLC subtype cell line, radioresistant A549 (A549S1), was induced by high-dose hypofractionated ionizing radiations. Radiosensitivity-related parameters, cell cycle distribution and expression of cell cycle-related proteins and SHP1 were investigated. siRNA was designed to down-regulate SHP1expression. Compared with native A549 cells, the proportion of cells in the S phase was increased, and cells in the G0/G1 phase were consequently decreased, however, the proportion of cells in the G2/M phase did not change in A549S1 cells. Moreover, the expression of SHP1, CDK4 and CylinD1 were significantly increased, while p16 was significantly down-regulated in A549S1 cells compared with native A549 cells. Furthermore, inhibition of SHP1 by siRNA increased the radiosensitivity of A549S1 cells, induced a G0/G1 phase arrest, down-regulated CDK4 and CylinD1expressions, and up-regulated p16 expression. SHP1 decreases the radiosensitivity of NSCLC cells through affecting cell cycle distribution. This finding could unravel the molecular mechanism involved in NSCLC radioresistance

  5. Zinc oxide nanoparticles decrease the expression and activity of plasma membrane calcium ATPase, disrupt the intracellular calcium homeostasis in rat retinal ganglion cells.

    Science.gov (United States)

    Guo, Dadong; Bi, Hongsheng; Wang, Daoguang; Wu, Qiuxin

    2013-08-01

    Zinc oxide nanoparticle is one of the most important materials with diverse applications. However, it has been reported that zinc oxide nanoparticles are toxic to organisms, and that oxidative stress is often hypothesized to be an important factor in cytotoxicity mediated by zinc oxide nanoparticles. Nevertheless, the mechanism of toxicity of zinc oxide nanoparticles has not been completely understood. In this study, we investigated the cytotoxic effect of zinc oxide nanoparticles and the possible molecular mechanism involved in calcium homeostasis mediated by plasma membrane calcium ATPase in rat retinal ganglion cells. Real-time cell electronic sensing assay showed that zinc oxide nanoparticles could exert cytotoxic effect on rat retinal ganglion cells in a concentration-dependent manner; flow cytometric analysis indicated that zinc oxide nanoparticles could lead to cell damage by inducing the overproduction of reactive oxygen species. Furthermore, zinc oxide nanoparticles could also apparently decrease the expression level and their activity of plasma membrane calcium ATPase, which finally disrupt the intracellular calcium homeostasis and result in cell death. Taken together, zinc oxide nanoparticles could apparently decrease the plasma membrane calcium ATPase expression, inhibit their activity, cause the elevated intracellular calcium ion level and disrupt the intracellular calcium homeostasis. Further, the disrupted calcium homeostasis will trigger mitochondrial dysfunction, generate excessive reactive oxygen species, and finally initiate cell death. Thus, the disrupted calcium homeostasis is involved in the zinc oxide nanoparticle-induced rat retinal ganglion cell death. Copyright © 2013 Elsevier Ltd. All rights reserved.

  6. Prostate stromal cells express the progesterone receptor to control cancer cell mobility.

    Science.gov (United States)

    Yu, Yue; Lee, Jennifer Suehyun; Xie, Ning; Li, Estelle; Hurtado-Coll, Antonio; Fazli, Ladan; Cox, Michael; Plymate, Stephen; Gleave, Martin; Dong, Xuesen

    2014-01-01

    Reciprocal interactions between epithelium and stroma play vital roles for prostate cancer development and progression. Enhanced secretions of cytokines and growth factors by cancer associated fibroblasts in prostate tumors create a favorable microenvironment for cancer cells to grow and metastasize. Our previous work showed that the progesterone receptor (PR) was expressed specifically in prostate stromal fibroblasts and smooth muscle cells. However, the expression levels of PR and its impact to tumor microenvironment in prostate tumors are poorly understood. Immunohistochemistry assays are applied to human prostate tissue biopsies. Cell migration, invasion and proliferation assays are performed using human prostate cells. Real-time PCR and ELISA are applied to measure gene expression at molecular levels. Immunohistochemistry assays showed that PR protein levels were decreased in cancer associated stroma when compared with paired normal prostate stroma. Using in vitro prostate stromal cell models, we showed that conditioned media collected from PR positive stromal cells inhibited prostate cancer cell migration and invasion, but had minor suppressive impacts on cancer cell proliferation. PR suppressed the secretion of stromal derived factor-1 (SDF-1) and interlukin-6 (IL-6) by stromal cells independent to PR ligands. Blocking PR expression by siRNA or supplementation of exogenous SDF-1 or IL-6 to conditioned media from PR positive stromal cells counteracted the inhibitory effects of PR to cancer cell migration and invasion. Decreased expression of the PR in cancer associated stroma may contribute to the elevated SDF-1 and IL-6 levels in prostate tumors and enhance prostate tumor progression.

  7. Prostate stromal cells express the progesterone receptor to control cancer cell mobility.

    Directory of Open Access Journals (Sweden)

    Yue Yu

    Full Text Available Reciprocal interactions between epithelium and stroma play vital roles for prostate cancer development and progression. Enhanced secretions of cytokines and growth factors by cancer associated fibroblasts in prostate tumors create a favorable microenvironment for cancer cells to grow and metastasize. Our previous work showed that the progesterone receptor (PR was expressed specifically in prostate stromal fibroblasts and smooth muscle cells. However, the expression levels of PR and its impact to tumor microenvironment in prostate tumors are poorly understood.Immunohistochemistry assays are applied to human prostate tissue biopsies. Cell migration, invasion and proliferation assays are performed using human prostate cells. Real-time PCR and ELISA are applied to measure gene expression at molecular levels.Immunohistochemistry assays showed that PR protein levels were decreased in cancer associated stroma when compared with paired normal prostate stroma. Using in vitro prostate stromal cell models, we showed that conditioned media collected from PR positive stromal cells inhibited prostate cancer cell migration and invasion, but had minor suppressive impacts on cancer cell proliferation. PR suppressed the secretion of stromal derived factor-1 (SDF-1 and interlukin-6 (IL-6 by stromal cells independent to PR ligands. Blocking PR expression by siRNA or supplementation of exogenous SDF-1 or IL-6 to conditioned media from PR positive stromal cells counteracted the inhibitory effects of PR to cancer cell migration and invasion.Decreased expression of the PR in cancer associated stroma may contribute to the elevated SDF-1 and IL-6 levels in prostate tumors and enhance prostate tumor progression.

  8. Cell-kinetics of the human uterine cervical carcinoma cells during radium irradiation

    International Nuclear Information System (INIS)

    Iwata, Masaharu; Sasaki, Hiroshi

    1983-01-01

    HeLa cells grown in a monolayer culture and in nude mice were exposed to graded dose rates (37, 55 or 200 rad/hour) and doses (500-2,000 rad) of radiation and analyzed in terms of their cell cycle distribution using flow-microfluorometry. In the case of the cultured HeLa cells, dose-survival curves were constructed using colony formation as the end-point. The HeLa cells, both in vitro and in vivo, accumulated in G2-M phases after both acute and chronic irradiation. The dose rate of 37 rad/hour proved to be the most effective in producing G2-M accumulation, which is the sensitive phase of the cell cycle. In comparing the G2-M accumulation to the irradiation time, 55 and 37 rad/hour proved to be similarly efficient in producing G2-M accumulation, both in vitro and in vivo. When survival of HeLa cells in vitro was studied, the radiation-induced changes in cell distribution correlated with cell survival and accounted for the change in the dose rate effect above 1,000 rads. In the case of in vivo HeLa cells, the decrease in the number of G0+G1 stage cells was demonstrated during chronic irradiation (37 and 55 rad/hour). The two low dose rates were equally efficient in producing a decrease in the number of G0+G1 cells. These data indicate that chronic irradiation induces redistribution and recruitment more effectively than acute irradiation. (author)

  9. Murine Lung Cancer Increases CD4+ T Cell Apoptosis and Decreases Gut Proliferative Capacity in Sepsis.

    Science.gov (United States)

    Lyons, John D; Mittal, Rohit; Fay, Katherine T; Chen, Ching-Wen; Liang, Zhe; Margoles, Lindsay M; Burd, Eileen M; Farris, Alton B; Ford, Mandy L; Coopersmith, Craig M

    2016-01-01

    Mortality is significantly higher in septic patients with cancer than in septic patients without a history of cancer. We have previously described a model of pancreatic cancer followed by sepsis from Pseudomonas aeruginosa pneumonia in which cancer septic mice have higher mortality than previously healthy septic mice, associated with increased gut epithelial apoptosis and decreased T cell apoptosis. The purpose of this study was to determine whether this represents a common host response by creating a new model in which both the type of cancer and the model of sepsis are altered. C57Bl/6 mice received an injection of 250,000 cells of the lung cancer line LLC-1 into their right thigh and were followed three weeks for development of palpable tumors. Mice with cancer and mice without cancer were then subjected to cecal ligation and puncture and sacrificed 24 hours after the onset of sepsis or followed 7 days for survival. Cancer septic mice had a higher mortality than previously healthy septic mice (60% vs. 18%, p = 0.003). Cancer septic mice had decreased number and frequency of splenic CD4+ lymphocytes secondary to increased apoptosis without changes in splenic CD8+ numbers. Intestinal proliferation was also decreased in cancer septic mice. Cancer septic mice had a higher bacterial burden in the peritoneal cavity, but this was not associated with alterations in local cytokine, neutrophil or dendritic cell responses. Cancer septic mice had biochemical evidence of worsened renal function, but there was no histologic evidence of renal injury. Animals with cancer have a significantly higher mortality than previously healthy animals following sepsis. The potential mechanisms associated with this elevated mortality differ significantly based upon the model of cancer and sepsis utilized. While lymphocyte apoptosis and intestinal integrity are both altered by the combination of cancer and sepsis, the patterns of these alterations vary greatly depending on the models used.

  10. Murine Lung Cancer Increases CD4+ T Cell Apoptosis and Decreases Gut Proliferative Capacity in Sepsis.

    Directory of Open Access Journals (Sweden)

    John D Lyons

    Full Text Available Mortality is significantly higher in septic patients with cancer than in septic patients without a history of cancer. We have previously described a model of pancreatic cancer followed by sepsis from Pseudomonas aeruginosa pneumonia in which cancer septic mice have higher mortality than previously healthy septic mice, associated with increased gut epithelial apoptosis and decreased T cell apoptosis. The purpose of this study was to determine whether this represents a common host response by creating a new model in which both the type of cancer and the model of sepsis are altered.C57Bl/6 mice received an injection of 250,000 cells of the lung cancer line LLC-1 into their right thigh and were followed three weeks for development of palpable tumors. Mice with cancer and mice without cancer were then subjected to cecal ligation and puncture and sacrificed 24 hours after the onset of sepsis or followed 7 days for survival.Cancer septic mice had a higher mortality than previously healthy septic mice (60% vs. 18%, p = 0.003. Cancer septic mice had decreased number and frequency of splenic CD4+ lymphocytes secondary to increased apoptosis without changes in splenic CD8+ numbers. Intestinal proliferation was also decreased in cancer septic mice. Cancer septic mice had a higher bacterial burden in the peritoneal cavity, but this was not associated with alterations in local cytokine, neutrophil or dendritic cell responses. Cancer septic mice had biochemical evidence of worsened renal function, but there was no histologic evidence of renal injury.Animals with cancer have a significantly higher mortality than previously healthy animals following sepsis. The potential mechanisms associated with this elevated mortality differ significantly based upon the model of cancer and sepsis utilized. While lymphocyte apoptosis and intestinal integrity are both altered by the combination of cancer and sepsis, the patterns of these alterations vary greatly depending on

  11. Delayed luminescence to monitor programmed cell death induced by berberine on thyroid cancer cells

    Science.gov (United States)

    Scordino, Agata; Campisi, Agata; Grasso, Rosaria; Bonfanti, Roberta; Gulino, Marisa; Iauk, Liliana; Parenti, Rosalba; Musumeci, Francesco

    2014-11-01

    Correlation between apoptosis and UVA-induced ultraweak photon emission delayed luminescence (DL) from tumor thyroid cell lines was investigated. In particular, the effects of berberine, an alkaloid that has been reported to have anticancer activities, on two cancer cell lines were studied. The FTC-133 and 8305C cell lines, as representative of follicular and anaplastic thyroid human cancer, respectively, were chosen. The results show that berberine is able to arrest cell cycle and activate apoptotic pathway as shown in both cell lines by deoxyribonucleic acid fragmentation, caspase-3 cleavage, p53 and p27 protein overexpression. In parallel, changes in DL spectral components after berberine treatment support the hypothesis that DL from human cells originates mainly from mitochondria, since berberine acts especially at the mitochondrial level. The decrease of DL blue component for both cell lines could be related to the decrease of intra-mitochondrial nicotinamide adenine dinucleotide and may be a hallmark of induced apoptosis. In contrast, the response in the red spectral range is different for the two cell lines and may be ascribed to a different iron homeostasis.

  12. Decreased miR-106a inhibits glioma cell glucose uptake and proliferation by targeting SLC2A3 in GBM.

    Science.gov (United States)

    Dai, Dong-Wei; Lu, Qiong; Wang, Lai-Xing; Zhao, Wen-Yuan; Cao, Yi-Qun; Li, Ya-Nan; Han, Guo-Sheng; Liu, Jian-Min; Yue, Zhi-Jian

    2013-10-14

    MiR-106a is frequently down-regulated in various types of human cancer. However the underlying mechanism of miR-106a involved in glioma remains elusive. The association of miR-106a with glioma grade and patient survival was analyzed. The biological function and target of miR-106a were determined by bioinformatic analysis and cell experiments (Western blot, luciferase reporter, cell cycle, ntracellular ATP production and glucose uptake assay). Finally, rescue expression of its target SLC2A3 was used to test the role of SLC2A3 in miR-106a-mediated cell glycolysis and proliferation. Here we showed that miR-106a was a tumor suppressor miRNA was involved in GBM cell glucose uptake and proliferation. Decreased miR-106a in GBM tissues and conferred a poor survival of GBM patients. SLC2A3 was identified as a core target of miR-106a in GBM cells. Inhibition of SLC2A3 by miR-106a attenuated cell proliferation and inhibited glucose uptake. In addition, for each biological process we identified ontology-associated transcripts that significantly correlated with SLC2A3 expression. Finally, the expression of SLC2A3 largely abrogated miR-106a-mediated cell proliferation and glucose uptake in GBM cells. Taken together, miR-106a and SLC2A3 could be potential therapeutic approaches for GBM.

  13. Aging increases cell-to-cell transcriptional variability upon immune stimulation.

    Science.gov (United States)

    Martinez-Jimenez, Celia Pilar; Eling, Nils; Chen, Hung-Chang; Vallejos, Catalina A; Kolodziejczyk, Aleksandra A; Connor, Frances; Stojic, Lovorka; Rayner, Timothy F; Stubbington, Michael J T; Teichmann, Sarah A; de la Roche, Maike; Marioni, John C; Odom, Duncan T

    2017-03-31

    Aging is characterized by progressive loss of physiological and cellular functions, but the molecular basis of this decline remains unclear. We explored how aging affects transcriptional dynamics using single-cell RNA sequencing of unstimulated and stimulated naïve and effector memory CD4 + T cells from young and old mice from two divergent species. In young animals, immunological activation drives a conserved transcriptomic switch, resulting in tightly controlled gene expression characterized by a strong up-regulation of a core activation program, coupled with a decrease in cell-to-cell variability. Aging perturbed the activation of this core program and increased expression heterogeneity across populations of cells in both species. These discoveries suggest that increased cell-to-cell transcriptional variability will be a hallmark feature of aging across most, if not all, mammalian tissues. Copyright © 2017, American Association for the Advancement of Science.

  14. Investigation of the response of low-dose irradiated cells. Pt. 2. Radio-adaptive response of human embryonic cells is related to cell-to-cell communication

    International Nuclear Information System (INIS)

    Ishii, Keiichiro; Watanabe, Masami.

    1994-01-01

    To clarify the radio-adaptive response of normal cells to low-dose radiation, we irradiated human embryonic cells and HeLa cells with low-dose X-ray and examined the changes in sensitivity to subsequent high-dose X-irradiation. The results obtained were as follows; (1) When HE cells were irradiated by a high-dose of 200 cGy, the growth ratio of the living cells five days after the irradiation decreased to 37% of that of the cells which received no X-irradiation. When the cells received a preliminary irradiation of 10 to 20 cGy four hours before the irradiation of 200 cGy, the relative growth ratios increased significantly to 45-53%. (2) This preliminary irradiation effect was not observed in HeLa cells, being cancer cells. (3) When the HE cells suspended in a Ca 2+ iron-free medium or TPA added medium while receiving the preliminary irradiation of 13 cGy, the effect of the preliminary irradiation in increasing the relative growth ratio of living cells was not observed. (4) This indicates that normal cells shows an adaptive response to low-dose radiation and become more radioresistant. This phenomenon is considered to involve cell-to-cell communication maintained in normal cells and intracellular signal transduction in which Ca 2+ ion plays a role. (author)

  15. RNA interference-mediated silencing of speckle-type POZ protein promotes apoptosis of renal cell cancer cells.

    Science.gov (United States)

    Liu, Xiaoxia; Sun, Guiling; Sun, Xiuju

    2016-01-01

    This study aimed to investigate the effects of silencing the speckle-type POZ protein (SPOP) gene on renal cell cancer (RCC) cells and to explore its possible mechanism. The A498 and ACHN RCC cells were transfected with small interference RNA (siRNA)-SPOP by lipofection methods. The silencing efficiency was monitored by quantitative real-time polymerase chain reaction and Western blot. The effects of SPOP silencing on cell apoptosis, cell viability, colony formation ability, cell migration ability, and chemosensitivity to Sorafenib were assessed by flow cytometry, an MTT assay, a colony formation assay, a trans-well migration assay, and a CCK-8 assay, respectively. Its effects on the expression of several cytokines were determined by a protein microarray. Relevant signaling pathways were also analyzed. Compared with the control group, the cell apoptosis rate was significantly higher; the cell viability, the colony formation, and migration ability were significantly decreased in the siRNA-SPOP group. The protein microarray screening showed that the expression of vascular endothelial growth factor receptor, matrix metallopeptidase-9, vascular cell adhesion molecule-1, and stromal cell-derived factor-1 in the siRNA group was significantly decreased and that the expression of granulocyte-macrophage colony-stimulating factor and E-cadherin was significantly increased (Pmatrix organization signal pathway. SPOP gene silencing induced cell apoptosis, decreased cell viability, colony formation, and migration ability, and elevated the drug sensitivity in the RCC cells. A possible mechanism is that silencing SPOP induces the differential expression of E-cadherin, vascular endothelial growth factor receptor, matrix metallopeptidase-9, and vascular cell adhesion molecule, which are related to the integrin-mediated cell surface interactions and extracellular matrix organization signaling pathway.

  16. Splenectomy reduces packed red cell transfusion requirement in children with sickle cell disease.

    Science.gov (United States)

    Haricharan, Ramanath N; Roberts, Jared M; Morgan, Traci L; Aprahamian, Charles J; Hardin, William D; Hilliard, Lee M; Georgeson, Keith E; Barnhart, Douglas C

    2008-06-01

    The purpose of the study was to measure the effect of splenectomy on packed-cell transfusion requirement in children with sickle cell disease. Thirty-seven sickle cell children who underwent splenectomies between January 2000 and May 2006 at a children's hospital were reviewed. Data were collected 6 months preoperatively to 12 months postsplenectomy. Paired t test, analysis of variance, and multivariable regression analyses were performed. Of 37 children with median age 11 years (range, 2-18 years), 34 (21 males) had data that allowed analyses. Twenty-six had Hgb-SS, 5 had Hgb-SC, and 3 had Hgb S-Thal. Laparoscopic splenectomy was attempted in 36 and completed successfully in 34 (94% success). The number of units transfused decreased by 38% for 0 to 6 months and by 45% for 6 to 12 months postsplenectomy. Postoperatively, hematocrit levels increased and reticulocytes concurrently decreased with a reduction in transfusion clinic visits. The decrease in transfusion was not influenced by spleen weight, age, or hemoglobin type. Two children had acute chest syndrome (6%), and 1 had severe pneumonia (3%). Laparoscopic splenectomy can be successfully completed in sickle cell children. Splenectomy significantly reduces the packed red cell transfusion requirement and frequency of clinic visits, in sickle cell children for at least 12 months postoperatively.

  17. Cell-to-cell communication and cellular environment alter the somatostatin status of delta cells

    Energy Technology Data Exchange (ETDEWEB)

    Kelly, Catriona, E-mail: catriona.kelly@qub.ac.uk [SAAD Centre for Pharmacy and Diabetes, School of Biomedical Sciences, University of Ulster, Coleraine (United Kingdom); Flatt, Peter R.; McClenaghan, Neville H. [SAAD Centre for Pharmacy and Diabetes, School of Biomedical Sciences, University of Ulster, Coleraine (United Kingdom)

    2010-08-20

    Research highlights: {yields} TGP52 cells display enhanced functionality in pseudoislet form. {yields} Somatostatin content was reduced, but secretion increased in high glucose conditions. {yields} Cellular interactions and environment alter the somatostatin status of TGP52 cells. -- Abstract: Introduction: Somatostatin, released from pancreatic delta cells, is a potent paracrine inhibitor of insulin and glucagon secretion. Islet cellular interactions and glucose homeostasis are essential to maintain normal patterns of insulin secretion. However, the importance of cell-to-cell communication and cellular environment in the regulation of somatostatin release remains unclear. Methods: This study employed the somatostatin-secreting TGP52 cell line maintained in DMEM:F12 (17.5 mM glucose) or DMEM (25 mM glucose) culture media. The effect of pseudoislet formation and culture medium on somatostatin content and release in response to a variety of stimuli was measured by somatostatin EIA. In addition, the effect of pseudoislet formation on cellular viability (MTT and LDH assays) and proliferation (BrdU ELISA) was determined. Results: TGP52 cells readily formed pseudoislets and showed enhanced functionality in three-dimensional form with increased E-cadherin expression irrespective of the culture environment used. However, culture in DMEM decreased cellular somatostatin content (P < 0.01) and increased somatostatin secretion in response to a variety of stimuli including arginine, calcium and PMA (P < 0.001) when compared with cells grown in DMEM:F12. Configuration of TGP52 cells as pseudoislets reduced the proliferative rate and increased cellular cytotoxicity irrespective of culture medium used. Conclusions: Somatostatin secretion is greatly facilitated by cell-to-cell interactions and E-cadherin expression. Cellular environment and extracellular glucose also significantly influence the function of delta cells.

  18. Cell-to-cell communication and cellular environment alter the somatostatin status of delta cells

    International Nuclear Information System (INIS)

    Kelly, Catriona; Flatt, Peter R.; McClenaghan, Neville H.

    2010-01-01

    Research highlights: → TGP52 cells display enhanced functionality in pseudoislet form. → Somatostatin content was reduced, but secretion increased in high glucose conditions. → Cellular interactions and environment alter the somatostatin status of TGP52 cells. -- Abstract: Introduction: Somatostatin, released from pancreatic delta cells, is a potent paracrine inhibitor of insulin and glucagon secretion. Islet cellular interactions and glucose homeostasis are essential to maintain normal patterns of insulin secretion. However, the importance of cell-to-cell communication and cellular environment in the regulation of somatostatin release remains unclear. Methods: This study employed the somatostatin-secreting TGP52 cell line maintained in DMEM:F12 (17.5 mM glucose) or DMEM (25 mM glucose) culture media. The effect of pseudoislet formation and culture medium on somatostatin content and release in response to a variety of stimuli was measured by somatostatin EIA. In addition, the effect of pseudoislet formation on cellular viability (MTT and LDH assays) and proliferation (BrdU ELISA) was determined. Results: TGP52 cells readily formed pseudoislets and showed enhanced functionality in three-dimensional form with increased E-cadherin expression irrespective of the culture environment used. However, culture in DMEM decreased cellular somatostatin content (P < 0.01) and increased somatostatin secretion in response to a variety of stimuli including arginine, calcium and PMA (P < 0.001) when compared with cells grown in DMEM:F12. Configuration of TGP52 cells as pseudoislets reduced the proliferative rate and increased cellular cytotoxicity irrespective of culture medium used. Conclusions: Somatostatin secretion is greatly facilitated by cell-to-cell interactions and E-cadherin expression. Cellular environment and extracellular glucose also significantly influence the function of delta cells.

  19. miR-122 targets NOD2 to decrease intestinal epithelial cell injury in Crohn’s disease

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Yu; Wang, Chengxiao; Liu, Ying; Tang, Liwei; Zheng, Mingxia [Department of Pediatrics, Jiangwan Hospital of Shanghai, Shanghai 200434 (China); Xu, Chundi [Department of Pediatrics, Ruijin affiliated to Shanghai Jiaotong University School of Medicine, Shanghai, 200025 (China); Song, Jian, E-mail: jiansongkxy@126.com [Department of Gastroenterology, Jiangwan Hospital of Shanghai, Shanghai 200434 (China); Meng, Xiaochun [Department of Pediatrics, Jiangwan Hospital of Shanghai, Shanghai 200434 (China)

    2013-08-16

    Highlights: •NOD2 is a target gene of miR-122. •miR-122 inhibits LPS-induced apoptosis by suppressing NOD2 in HT-29 cells. •miR-122 reduces the expression of pro-inflammatory cytokines (TNF-α and IFN-γ). •miR-122 promotes the release of anti-inflammatory cytokines (IL-4 and IL-10). •NF-κB signaling pathway is involved in inflammatory response induced by LPS. -- Abstract: Crohn’s disease (CD) is one of the two major types of inflammatory bowel disease (IBD) thought to be caused by genetic and environmental factors. Recently, miR-122 was found to be deregulated in association with CD progression. However, the underlying molecular mechanisms remain unclear. In the present study, the gene nucleotide-binding oligomerization domain 2 (NOD2/CARD15), which is strongly associated with susceptibility to CD, was identified as a functional target of miR-122. MiR-122 inhibited LPS-induced apoptosis by suppressing NOD2 in HT-29 cells. NOD2 interaction with LPS initiates signal transduction mechanisms resulting in the activation of nuclear factor κB (NF-κB) and the stimulation of downstream pro-inflammatory events. The activation of NF-κB was inhibited in LPS-stimulated HT-29 cells pretreated with miR-122 precursor or NOD2 shRNA. The expression of the pro-inflammatory cytokines TNF-α and IFN-γ was significantly decreased, whereas therelease of the anti-inflammatory cytokines IL-4 and IL-10 was increased in LPS-stimulated HT-29 cells pretreated with miR-122 precursor, NOD2 shRNA or the NF-κB inhibitor QNZ. Taken together, these results indicate that miR-122 and its target gene NOD2 may play an important role in the injury of intestinal epithelial cells induced by LPS.

  20. Gigantol Suppresses Cancer Stem Cell-Like Phenotypes in Lung Cancer Cells

    Directory of Open Access Journals (Sweden)

    Narumol Bhummaphan

    2015-01-01

    Full Text Available As cancer stem cells (CSCs contribute to malignancy, metastasis, and relapse of cancers, potential of compound in inhibition of CSCs has garnered most attention in the cancer research as well as drug development fields recently. Herein, we have demonstrated for the first time that gigantol, a pure compound isolated from Dendrobium draconis, dramatically suppressed stem-like phenotypes of human lung cancer cells. Gigantol at nontoxic concentrations significantly reduced anchorage-independent growth and survival of the cancer cells. Importantly, gigantol significantly reduced the ability of the cancer cells to form tumor spheroids, a critical hallmark of CSCs. Concomitantly, the treatment of the compound was shown to reduce well-known lung CSCs markers, including CD133 and ALDH1A1. Moreover, we revealed that gigantol decreased stemness in the cancer cells by suppressing the activation of protein kinase B (Akt signal which in turn decreased the cellular levels of pluripotency and self-renewal factors Oct4 and Nanog. In conclusion, gigantol possesses CSCs suppressing activity which may facilitate the development of this compound for therapeutic approaches by targeting CSCs.

  1. Rheological characteristics of cell suspension and cell culture of Perilla frutescens.

    Science.gov (United States)

    Zhong, J J; Seki, T; Kinoshita, S; Yoshida, T

    1992-12-05

    Physical properties such as viscosity, fluid dynamic behavior of cell suspension, and size distribution of cell aggregates of a plant, Perilla frustescens, cultured in a liquid medium were studied. As a result of investigations using cells harvester after 12 days of cultivation in a flask, it was found that the apparent viscosity of the cell suspension did not change with any variation of cell concentration below 5 g dry cell/L but markedly increased when the cell concentration increased over 12.8 g dry cell/L. The cell suspension exhibited the characteristics of a Bingham plastic fluid with a small yield stress. The size of cell aggregates in the range 74 to 500 mum did not influence the rheological characteristics of the cell suspension. The rheological characteristics of cultivation mixtures of P. frutescens cultivated in a flask and in a bioreactor were also investigated. The results showed that the flow characteristics of the cell culture could be described by a Bingham plastic model. At the later stage of cultivation, the apparent viscosity increased steadily, even though the biomass concentration (by dry weight) decreased, due to the increase of individual cell size. (c) 1992 John Wiley & Sons, Inc.

  2. Decreased Expression of T-Cell Costimulatory Molecule CD28 on CD4 and CD8 T Cells of Mexican Patients with Pulmonary Tuberculosis

    Directory of Open Access Journals (Sweden)

    German Bernal-Fernandez

    2010-01-01

    Full Text Available Patients with tuberculosis frequently develop anergy, a state of T-cell hyporesponsiveness in which defective T-cell costimulation could be a factor. To know if the expression of T-cell costimulatory molecules was altered in tuberculosis, we analyzed the peripheral blood T-cell phenotype of 23 Mexican patients with pulmonary tuberculosis. There was severe CD4 (P<.001 and CD8 (P<.01 lymphopenia and upregulation of costimulatory molecule CD30 on CD4 and CD8 T cells (P<.05; this increase was higher in relapsing tuberculosis. The main finding was severe downregulation of the major costimulatory molecule CD28 on both CD8 and CD4 T cells (P<.001. Depletion of the CD4/CD28 subset, a hitherto undescribed finding, is relevant because CD4 T cells constitute the main arm of the cell-mediated antimycobacterial immune response.

  3. Planar Cell Polarity Controls Pancreatic Beta Cell Differentiation and Glucose Homeostasis

    Directory of Open Access Journals (Sweden)

    Cedric Cortijo

    2012-12-01

    Full Text Available Planar cell polarity (PCP refers to the collective orientation of cells within the epithelial plane. We show that progenitor cells forming the ducts of the embryonic pancreas express PCP proteins and exhibit an active PCP pathway. Planar polarity proteins are acquired at embryonic day 11.5 synchronously to apicobasal polarization of pancreas progenitors. Loss of function of the two PCP core components Celsr2 and Celsr3 shows that they control the differentiation of endocrine cells from polarized progenitors, with a prevalent effect on insulin-producing beta cells. This results in a decreased glucose clearance. Loss of Celsr2 and 3 leads to a reduction of Jun phosphorylation in progenitors, which, in turn, reduces beta cell differentiation from endocrine progenitors. These results highlight the importance of the PCP pathway in cell differentiation in vertebrates. In addition, they reveal that tridimensional organization and collective communication of cells are needed in the pancreatic epithelium in order to generate appropriate numbers of endocrine cells.

  4. Sphere-forming cell subpopulations with cancer stem cell properties in human hepatoma cell lines

    Directory of Open Access Journals (Sweden)

    Chen Lei

    2011-06-01

    Full Text Available Abstract Background Cancer stem cells (CSCs are regarded as the cause of tumor formation and recurrence. The isolation and identification of CSCs could help to develop novel therapeutic strategies specifically targeting CSCs. Methods Human hepatoma cell lines were plated in stem cell conditioned culture system allowed for sphere forming. To evaluate the stemness characteristics of spheres, the self-renewal, proliferation, chemoresistance, tumorigenicity of the PLC/PRF/5 sphere-forming cells, and the expression levels of stem cell related proteins in the PLC/PRF/5 sphere-forming cells were assessed, comparing with the parental cells. The stem cell RT-PCR array was performed to further explore the biological properties of liver CSCs. Results The PLC/PRF/5, MHCC97H and HepG2 cells could form clonal nonadherent 3-D spheres and be serially passaged. The PLC/PRF/5 sphere-forming cells possessed a key criteria that define CSCs: persistent self-renewal, extensive proliferation, drug resistance, overexpression of liver CSCs related proteins (Oct3/4, OV6, EpCAM, CD133 and CD44. Even 500 sphere-forming cells were able to form tumors in NOD/SCID mice, and the tumor initiating capability was not decreased when spheres were passaged. Besides, downstream proteins DTX1 and Ep300 of the CSL (CBF1 in humans, Suppressor of hairless in Drosophila and LAG1 in C. elegans -independent Notch signaling pathway were highly expressed in the spheres, and a gamma-secretase inhibitor MRK003 could significantly inhibit the sphere formation ability. Conclusions Nonadherent tumor spheres from hepatoma cell lines cultured in stem cell conditioned medium possess liver CSC properties, and the CSL-independent Notch signaling pathway may play a role in liver CSCs.

  5. Control of density-dependent, cell state-specific signal transduction by the cell adhesion molecule CEACAM1, and its influence on cell cycle regulation

    International Nuclear Information System (INIS)

    Scheffrahn, Inka; Singer, Bernhard B.; Sigmundsson, Kristmundur; Lucka, Lothar; Oebrink, Bjoern

    2005-01-01

    Growth factor receptors, extracellular matrix receptors, and cell-cell adhesion molecules co-operate in regulating the activities of intracellular signaling pathways. Here, we demonstrate that the cell adhesion molecule CEACAM1 co-regulates growth-factor-induced DNA synthesis in NBT-II epithelial cells in a cell-density-dependent manner. CEACAM1 exerted its effects by regulating the activity of the Erk 1/2 MAP kinase pathway and the expression levels of the cyclin-dependent kinase inhibitor p27 Kip1 . Interestingly, both inhibitory and stimulatory effects were observed. Confluent cells continuously exposed to fetal calf serum showed little Erk activity and DNA synthesis compared with sparse cells. Under these conditions, anti-CEACAM1 antibodies strongly stimulated Erk activation, decreased p27 expression, and induced DNA synthesis. In serum-starved confluent cells, re-addition of 10% fetal calf serum activated the Erk pathway, decreased p27 expression, and stimulated DNA synthesis to the same levels as in sparse cells. Under these conditions anti-CEACAM1 antibodies de-activated Erk, restored the level of p27, and inhibited DNA synthesis. These data indicate that CEACAM1 mediates contact inhibition of proliferation in cells that are constantly exposed to growth factors, but co-activates growth-factor-induced proliferation in cells that have been starved for growth factors; exposure to extracellular CEACAM1 ligands reverts these responses

  6. Planar Cell Polarity Controls Pancreatic Beta Cell Differentiation and Glucose Homeostasis

    DEFF Research Database (Denmark)

    Cortijo, Cedric; Gouzi, Mathieu; Tissir, Fadel

    2012-01-01

    glucose clearance. Loss of Celsr2 and 3 leads to a reduction of Jun phosphorylation in progenitors, which, in turn, reduces beta cell differentiation from endocrine progenitors. These results highlight the importance of the PCP pathway in cell differentiation in vertebrates. In addition, they reveal.......5 synchronously to apicobasal polarization of pancreas progenitors. Loss of function of the two PCP core components Celsr2 and Celsr3 shows that they control the differentiation of endocrine cells from polarized progenitors, with a prevalent effect on insulin-producing beta cells. This results in a decreased...

  7. SATB2 expression increased anchorage-independent growth and cell migration in human bronchial epithelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Wu, Feng; Jordan, Ashley; Kluz, Thomas [Department of Environmental Medicine, New York University School of Medicine, 57 Old Forge Road, Tuxedo, NY 10987 (United States); Shen, Steven [Center for Health Informatics and Bioinformatics, New York University Langone Medical Center, New York, NY 10016 (United States); Sun, Hong; Cartularo, Laura A. [Department of Environmental Medicine, New York University School of Medicine, 57 Old Forge Road, Tuxedo, NY 10987 (United States); Costa, Max, E-mail: Max.Costa@nyumc.org [Department of Environmental Medicine, New York University School of Medicine, 57 Old Forge Road, Tuxedo, NY 10987 (United States)

    2016-02-15

    The special AT-rich sequence-binding protein 2 (SATB2) is a protein that binds to the nuclear matrix attachment region of the cell and regulates gene expression by altering chromatin structure. In our previous study, we reported that SATB2 gene expression was induced in human bronchial epithelial BEAS-2B cells transformed by arsenic, chromium, nickel and vanadium. In this study, we show that ectopic expression of SATB2 in the normal human bronchial epithelial cell-line BEAS-2B increased anchorage-independent growth and cell migration, meanwhile, shRNA-mediated knockdown of SATB2 significantly decreased anchorage-independent growth in Ni transformed BEAS-2B cells. RNA sequencing analyses of SATB2 regulated genes revealed the enrichment of those involved in cytoskeleton, cell adhesion and cell-movement pathways. Our evidence supports the hypothesis that SATB2 plays an important role in BEAS-2B cell transformation. - Highlights: • We performed SATB2 overexpression in the BEAS-2B cell line. • We performed SATB2 knockdown in a Ni transformed BEAS-2B cell line. • SATB2 induced anchorage-independent growth and increased cell migration. • SATB2 knockdown significantly decreased anchorage-independent growth. • We identified alterations in gene involved in cytoskeleton, cell adhesion.

  8. SATB2 expression increased anchorage-independent growth and cell migration in human bronchial epithelial cells

    International Nuclear Information System (INIS)

    Wu, Feng; Jordan, Ashley; Kluz, Thomas; Shen, Steven; Sun, Hong; Cartularo, Laura A.; Costa, Max

    2016-01-01

    The special AT-rich sequence-binding protein 2 (SATB2) is a protein that binds to the nuclear matrix attachment region of the cell and regulates gene expression by altering chromatin structure. In our previous study, we reported that SATB2 gene expression was induced in human bronchial epithelial BEAS-2B cells transformed by arsenic, chromium, nickel and vanadium. In this study, we show that ectopic expression of SATB2 in the normal human bronchial epithelial cell-line BEAS-2B increased anchorage-independent growth and cell migration, meanwhile, shRNA-mediated knockdown of SATB2 significantly decreased anchorage-independent growth in Ni transformed BEAS-2B cells. RNA sequencing analyses of SATB2 regulated genes revealed the enrichment of those involved in cytoskeleton, cell adhesion and cell-movement pathways. Our evidence supports the hypothesis that SATB2 plays an important role in BEAS-2B cell transformation. - Highlights: • We performed SATB2 overexpression in the BEAS-2B cell line. • We performed SATB2 knockdown in a Ni transformed BEAS-2B cell line. • SATB2 induced anchorage-independent growth and increased cell migration. • SATB2 knockdown significantly decreased anchorage-independent growth. • We identified alterations in gene involved in cytoskeleton, cell adhesion.

  9. Identification of glycan structure alterations on cell membrane proteins in desoxyepothilone B resistant leukemia cells.

    Science.gov (United States)

    Nakano, Miyako; Saldanha, Rohit; Göbel, Anja; Kavallaris, Maria; Packer, Nicolle H

    2011-11-01

    Resistance to tubulin-binding agents used in cancer is often multifactorial and can include changes in drug accumulation and modified expression of tubulin isotypes. Glycans on cell membrane proteins play important roles in many cellular processes such as recognition and apoptosis, and this study investigated whether changes to the glycan structures on cell membrane proteins occur when cells become resistant to drugs. Specifically, we investigated the alteration of glycan structures on the cell membrane proteins of human T-cell acute lymphoblastic leukemia (CEM) cells that were selected for resistance to desoxyepothilone B (CEM/dEpoB). The glycan profile of the cell membrane glycoproteins was obtained by sequential release of N- and O-glycans from cell membrane fraction dotted onto polyvinylidene difluoride membrane with PNGase F and β-elimination respectively. The released glycan alditols were analyzed by liquid chromatography (graphitized carbon)-electrospray ionization tandem MS. The major N-glycan on CEM cell was the core fucosylated α2-6 monosialo-biantennary structure. Resistant CEM/dEpoB cells had a significant decrease of α2-6 linked sialic acid on N-glycans. The lower α2-6 sialylation was caused by a decrease in activity of β-galactoside α2-6 sialyltransferase (ST6Gal), and decreased expression of the mRNA. It is clear that the membrane glycosylation of leukemia cells changes during acquired resistance to dEpoB drugs and that this change occurs globally on all cell membrane glycoproteins. This is the first identification of a specific glycan modification on the surface of drug resistant cells and the mechanism of this downstream effect on microtubule targeting drugs may offer a route to new interventions to overcome drug resistance.

  10. Coculture with BJ fibroblast cells inhibits the adipogenesis and lipogenesis in 3T3-L1 cells

    International Nuclear Information System (INIS)

    Jeong, Hyun Jeong; Park, Sahng Wook; Kim, Hojeong; Park, Sang-Kyu; Yoon, Dojun

    2010-01-01

    Mouse or human fibroblasts are commonly used as feeder cells to prevent differentiation in stem or primary cell culture. In the present study, we addressed whether fibroblasts can affect the differentiation of adipocytes. We found that the differentiation of 3T3-L1 preadipocytes was strongly suppressed when the cells were cocultured with human fibroblast (BJ) cells. BrdU incorporation analysis indicated that mitotic clonal expansion, an early event required for 3T3-L1 cell adipogenesis, was not affected by BJ cells. The 3T3-L1 cell expression levels of peroxisome proliferator-activated receptor γ2, CCAAT/enhancer-binding protein alpha (C/EBPα), sterol regulatory element binding protein-1c, and Krueppel-like factor 15, but not those of C/EBPβ or C/EBPδ, were decreased by coculture with BJ cells. When mature 3T3-L1 adipocytes were cocultured with BJ cells, their lipid contents were significantly reduced, with decreased fatty acid synthase expression and increased phosphorylated form of acetyl-CoA carboxylase 1. Our data indicate that coculture with BJ fibroblast cells inhibits the adipogenesis of 3T3-L1 preadipocytes and decreases the lipogenesis of mature 3T3-L1 adipocytes.

  11. B Cells Promote Th1- Skewed NKT Cell Response by CD1d-TCR Interaction.

    Science.gov (United States)

    Shin, Jung Hoon; Park, Se-Ho

    2013-10-01

    CD1d expressing dendritic cells (DCs) are good glyco-lipid antigen presenting cells for NKT cells. However, resting B cells are very weak stimulators for NKT cells. Although α-galactosylceramide (α-GalCer) loaded B cells can activate NKT cells, it is not well defined whether B cells interfere NKT cell stimulating activity of DCs. Unexpectedly, we found in this study that B cells can promote Th1-skewed NKT cell response, which means a increased level of IFN-γ by NKT cells, concomitant with a decreased level of IL-4, in the circumstance of co-culture of DCs and B Cells. Remarkably, the response promoted by B cells was dependent on CD1d expression of B cells.

  12. Sensitization of gastric cancer cells to alkylating agents by glaucocalyxin B via cell cycle arrest and enhanced cell death

    Directory of Open Access Journals (Sweden)

    Ur Rahman MS

    2017-08-01

    Full Text Available Muhammad Saif Ur Rahman,1 Ling Zhang,2 Lingyan Wu,1 Yuqiong Xie,1 Chunchun Li,1 Jiang Cao1 1Clinical Research Center, 2Cardiovascular Key Laboratory of Zhejiang Province, The Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang Province, People’s Republic of China Abstract: Severe side effects are major problems with chemotherapy of gastric cancer (GC. These side effects can be reduced by using sensitizing agents in combination with therapeutic drugs. In this study, the low/nontoxic dosage of glaucocalyxin B (GLB was used with other DNA linker agents mitomycin C (MMC, cisplatin (DDP, or cyclophosphamide (CTX to treat GC cells. Combined effectiveness of GLB with drugs was determined by proliferation assay. The molecular mechanisms associated with cell proliferation, migration, invasion, cell cycle, DNA repair/replication, apoptosis, and autophagy were investigated by immunoblotting for key proteins involved. Cell cycle and apoptosis analysis were performed by flow cytometry. Reactive oxygen species level was also examined for identification of its role in apoptosis. Proliferation assay revealed that the addition of 5 µM GLB significantly sensitizes gastric cancer SGC-7901 cells to MMC, DDP, and CTX by decreasing half-maximal inhibitory concentration (IC50 by up to 75.40%±5%, 45.10%±5%, and 52.10%±5%, respectively. GLB + drugs decreased the expression level of proteins involved in proliferation and migration, suggesting the anticancer potential of GLB + drugs. GLB + MMC, GLB + CTX, and GLB + DDP arrest the cells in G0/G1 and G1/S phase, respectively, which may be the consequence of significant decrease in the level of enzymes responsible for DNA replication and telomerase shortening. Combined use of GLB with these drugs also induces DNA damage and apoptosis by activating caspase/PARP pathways and increased production of reactive oxygen species and increased autophagy in GC cells. GLB dosage sensitizes GC

  13. Suppression of Glut1 and Glucose Metabolism by Decreased Akt/mTORC1 Signaling Drives T Cell Impairment in B Cell Leukemia

    NARCIS (Netherlands)

    Siska, Peter J.; van der Windt, Gerritje J. W.; Kishton, Rigel J.; Cohen, Sivan; Eisner, William; MacIver, Nancie J.; Kater, Arnon P.; Weinberg, J. Brice; Rathmell, Jeffrey C.

    2016-01-01

    Leukemia can promote T cell dysfunction and exhaustion that contributes to increased susceptibility to infection and mortality. The treatment-independent mechanisms that mediate leukemia-associated T cell impairments are poorly understood, but metabolism tightly regulates T cell function and may

  14. Blocking CD147 induces cell death in cancer cells through impairment of glycolytic energy metabolism

    International Nuclear Information System (INIS)

    Baba, Miyako; Inoue, Masahiro; Itoh, Kazuyuki; Nishizawa, Yasuko

    2008-01-01

    CD147 is a multifunctional transmembrane protein and promotes cancer progression. We found that the anti-human CD147 mouse monoclonal antibody MEM-M6/1 strongly induces necrosis-like cell death in LoVo, HT-29, WiDr, and SW620 colon cancer cells and A2058 melanoma cells, but not in WI-38 and TIG-113 normal fibroblasts. Silencing or overexpression of CD147 in LoVo cells enhanced or decreased the MEM-M6/1 induced cell death, respectively. CD147 is known to form complex with proton-linked monocarboxylate transporters (MCTs), which is critical for lactate transport and intracellular pH (pHi) homeostasis. In LoVo cells, CD147 and MCT-1 co-localized on the cell surface, and MEM-M6/1 inhibited the association of these molecules. MEM-M6/1 inhibited lactate uptake, lactate release, and reduced pHi. Further, the induction of acidification was parallel to the decrease of the glycolytic flux and intracellular ATP levels. These effects were not found in the normal fibroblasts. As cancer cells depend on glycolysis for their energy production, CD147 inhibition might induce cell death specific to cancer cells

  15. Curcumin decreases the expression of Pokemon by suppressing the binding activity of the Sp1 protein in human lung cancer cells.

    Science.gov (United States)

    Cui, Jiajun; Meng, Xianfeng; Gao, Xudong; Tan, Guangxuan

    2010-03-01

    Pokemon, which stands for POK erythroid myeloid ontogenic factor, can regulate expression of many genes and plays an important role in tumorigenesis. Curcumin, a natural and non-toxic yellow compound, has capacity for antioxidant, free radical scavenger, anti-inflammatory properties. Recent studies shows it is a potential inhibitor of cell proliferation in a variety of tumour cells. To investigate whether curcumin can regulate the expression of Pokemon, a series of experiments were carried out. Transient transfection experiments demonstrated that curcumin could decrease the activity of the Pokemon promoter. Western blot analysis suggested that curcumin could significantly decrease the expression of the Pokemon. Overexpression of Sp1 could enhance the activity of the Pokemon promoter, whereas knockdown of Sp1 could decrease its activity. More important, we also found that curcumin could decrease the expression of the Pokemon by suppressing the stimulation of the Sp1 protein. Therefore, curcumin is a potential reagent for tumour therapy which may target Pokemon.

  16. Inhibition of Cholesterol Synthesis in HepG2 Cells by GINST-Decreasing HMG-CoA Reductase Expression Via AMP-Activated Protein Kinase.

    Science.gov (United States)

    Han, Joon-Seung; Sung, Jong Hwan; Lee, Seung Kwon

    2017-11-01

    GINST, a hydrolyzed ginseng extract, has been reported to have antidiabetic effects and to reduce hyperglycemia and hyperlipidemia. Hypercholesterolemia is caused by diet or genetic factors and can lead to atherosclerosis and coronary heart disease. Thus, the purpose of this study is to determine whether GINST and the ginsenoside metabolite, IH-901 (compound K), reduce cholesterol synthesis in HepG2 cells and the signal transduction pathways involved. Concentrations of cholesterol were measured by using an enzymatic method. Expression levels of sterol regulatory element-binding protein 2 (SREBP2), HMG-CoA reductase (HMGCR), peroxisome proliferators-activated receptor γ (PPARγ), CCAAT/enhancer-binding proteins α (C/EBPα), GAPDH, and phosphorylation of AMP-activated protein kinase α (AMPKα), protein kinase B (PKB, also known as Akt), and mechanistic target of rapamycin complex 1 (mTORC1) were measured using western blot. Total cholesterol concentration decreased after GINST treatment for 24 and 48 h. Expression of HMGCR decreased more with GINST than with the inhibitors, U18666A and atorvastatin, after 48 h in a dose-dependent manner. Phosphorylation of AMPKα increased 2.5x by GINST after 360 min of treatment, and phosphorylation of Akt decreased after 120 and 360 min. We separated compound K from GINST extracts flash chromatography. Compound K decreased cholesterol synthesis in HepG2 cells at 24 and 48 h. Therefore, we conclude that GINST inhibits cholesterol synthesis in HepG2 cells by decreasing HMGCR expression via AMPKα activation. GINST, a hydrolyzed ginseng extract, can inhibit cholesterol synthesis in liver cells via activation of AMPKα. IH-901 (compound K), which is the main component with bioactivity in GINST, also has anticholesterol effects. Thus, we suggest that GINST can be used to reduce hypercholesterolemia. © 2017 Institute of Food Technologists®.

  17. Impaired Telomere Maintenance and Decreased Canonical WNT Signaling but Normal Ribosome Biogenesis in Induced Pluripotent Stem Cells from X-Linked Dyskeratosis Congenita Patients.

    Directory of Open Access Journals (Sweden)

    Bai-Wei Gu

    Full Text Available Dyskeratosis congenita (DC is an inherited bone marrow failure syndrome characterized by the presence of short telomeres at presentation. Mutations in ten different genes, whose products are involved in the telomere maintenance pathway, have been shown to cause DC. The X-linked form is the most common form of the disease and is caused by mutations in the gene DKC1, encoding the protein dyskerin. Dyskerin is required for the assembly and stability of telomerase and is also involved in ribosomal RNA (rRNA processing where it converts specific uridines to pseudouridine. DC is thought to result from failure to maintain tissues, like blood, that are renewed by stem cell activity, but research into pathogenic mechanisms has been hampered by the difficulty of obtaining stem cells from patients. We reasoned that induced pluripotent stem (iPS cells from X-linked DC patients may provide information about the mechanisms involved. Here we describe the production of iPS cells from DC patients with DKC1 mutations Q31E, A353V and ΔL37. In addition we constructed "corrected" lines with a copy of the wild type dyskerin cDNA expressed from the AAVS1 safe harbor locus. We show that in iPS cells with DKC1 mutations telomere maintenance is compromised with short telomere lengths and decreased telomerase activity. The degree to which telomere lengths are affected by expression of telomerase during reprograming, or with ectopic expression of wild type dyskerin, is variable. The recurrent mutation A353V shows the most severe effect on telomere maintenance. A353V cells but not Q31E or ΔL37 cells, are refractory to correction by expression of wild type DKC1 cDNA. Because dyskerin is involved in both telomere maintenance and ribosome biogenesis it has been postulated that defective ribosome biogenesis and translation may contribute to the disease phenotype. Evidence from mouse and zebra fish models has supported the involvement of ribosome biogenesis but primary cells

  18. Impaired Telomere Maintenance and Decreased Canonical WNT Signaling but Normal Ribosome Biogenesis in Induced Pluripotent Stem Cells from X-Linked Dyskeratosis Congenita Patients.

    Science.gov (United States)

    Gu, Bai-Wei; Apicella, Marisa; Mills, Jason; Fan, Jian-Meng; Reeves, Dara A; French, Deborah; Podsakoff, Gregory M; Bessler, Monica; Mason, Philip J

    2015-01-01

    Dyskeratosis congenita (DC) is an inherited bone marrow failure syndrome characterized by the presence of short telomeres at presentation. Mutations in ten different genes, whose products are involved in the telomere maintenance pathway, have been shown to cause DC. The X-linked form is the most common form of the disease and is caused by mutations in the gene DKC1, encoding the protein dyskerin. Dyskerin is required for the assembly and stability of telomerase and is also involved in ribosomal RNA (rRNA) processing where it converts specific uridines to pseudouridine. DC is thought to result from failure to maintain tissues, like blood, that are renewed by stem cell activity, but research into pathogenic mechanisms has been hampered by the difficulty of obtaining stem cells from patients. We reasoned that induced pluripotent stem (iPS) cells from X-linked DC patients may provide information about the mechanisms involved. Here we describe the production of iPS cells from DC patients with DKC1 mutations Q31E, A353V and ΔL37. In addition we constructed "corrected" lines with a copy of the wild type dyskerin cDNA expressed from the AAVS1 safe harbor locus. We show that in iPS cells with DKC1 mutations telomere maintenance is compromised with short telomere lengths and decreased telomerase activity. The degree to which telomere lengths are affected by expression of telomerase during reprograming, or with ectopic expression of wild type dyskerin, is variable. The recurrent mutation A353V shows the most severe effect on telomere maintenance. A353V cells but not Q31E or ΔL37 cells, are refractory to correction by expression of wild type DKC1 cDNA. Because dyskerin is involved in both telomere maintenance and ribosome biogenesis it has been postulated that defective ribosome biogenesis and translation may contribute to the disease phenotype. Evidence from mouse and zebra fish models has supported the involvement of ribosome biogenesis but primary cells from human

  19. EphA2 Is a Potential Player of Malignant Cellular Behavior in Non-Metastatic Renal Cell Carcinoma Cells but Not in Metastatic Renal Cell Carcinoma Cells.

    Science.gov (United States)

    Cho, Min Chul; Cho, Sung Yong; Yoon, Cheol Yong; Lee, Seung Bae; Kwak, Cheol; Kim, Hyeon Hoe; Jeong, Hyeon

    2015-01-01

    To investigate the role of EphA2 in malignant cellular behavior in renal cell carcinoma (RCC) cells and whether FAK/RhoA signaling can act as downstream effectors of EphA2 on RCC cells. Expression of EphA2 protein in non-metastatic RCC (Caki-2 and A498), metastatic RCC cells (Caki-1 and ACHN), HEK-293 cells and prostate cancer cells (PC-3 and DU-145; positive controls of EphA2 expression) was evaluated by Western blot. Changes in mRNA or protein expression of EphA2, FAK or membrane-bound RhoA following EphA2, FAK or RhoA small interfering RNA (siRNA) transfection were determined by reverse transcription polymerase chain reaction or Western blot. The effect of siRNA treatment on cellular viability, apoptosis and invasion was analyzed by cell counting kit-8, Annexin-V and modified Matrigel-Boyden assays, respectively. In all RCC cell lines, the expression of EphA2 protein was detectable at variable levels; however, in HEK-293 cells, EphA2 expression was very low. Treatment with EphA2 siRNA significantly reduced the expression of EphA2 mRNA and protein in all RCC cell lines. For non-metastatic RCC cells (Caki-2 and A498) but not metastatic RCC cells (Caki-1 and ACHN), cellular viability, invasiveness, resistance to apoptosis, expression of membrane-bound RhoA protein and FAK phosphorylation were significantly decreased in EphA2 siRNA-treated cells compared to the control. In non-metastatic RCC cells, FAK siRNA significantly attenuated the invasiveness, resistance to apoptosis, as well as expression of membrane-bound RhoA protein without changing protein expression of EphA2. RhoA siRNA significantly decreased the malignant cellular behavior and expression of membrane-bound RhoA protein without changing EphA2 protein expression or FAK phosphorylation. Our data provide the first functional evidence that the EphA2/FAK/RhoA signaling pathway plays a critical role in the malignant cellular behavior of RCC and appears to be functional particularly in the early stage of

  20. Relative Contributions of B Cells and Dendritic Cells from Lupus-Prone Mice to CD4+ T Cell Polarization.

    Science.gov (United States)

    Choi, Seung-Chul; Xu, Zhiwei; Li, Wei; Yang, Hong; Roopenian, Derry C; Morse, Herbert C; Morel, Laurence

    2018-05-01

    Mouse models of lupus have shown that multiple immune cell types contribute to autoimmune disease. This study sought to investigate the involvement of B cells and dendritic cells in supporting the expansion of inflammatory and regulatory CD4 + T cells that are critical for lupus pathogenesis. We used lupus-prone B6.NZM2410.Sle1.Sle2.Sle3 (TC) and congenic C57BL/6J (B6) control mice to investigate how the genetic predisposition of these two cell types controls the activity of normal B6 T cells. Using an allogeneic in vitro assay, we showed that TC B1-a and conventional B cells expanded Th17 cells significantly more than their B6 counterparts. This expansion was dependent on CD86 and IL-6 expression and mapped to the Sle1 lupus-susceptibility locus. In vivo, TC B cells promoted greater differentiation of CD4 + T cells into Th1 and follicular helper T cells than did B6 B cells, but they limited the expansion of Foxp3 regulatory CD4 + T cells to a greater extent than did B6 B cells. Finally, when normal B6 CD4 + T cells were introduced into Rag1 -/- mice, TC myeloid/stromal cells caused their heightened activation, decreased Foxp3 regulatory CD4 + T cell differentiation, and increased renal infiltration of Th1 and Th17 cells in comparison with B6 myeloid/stromal cells. The results show that B cells from lupus mice amplify inflammatory CD4 + T cells in a nonredundant manner with myeloid/stromal cells. Copyright © 2018 by The American Association of Immunologists, Inc.

  1. Canthin-6-one induces cell death, cell cycle arrest and differentiation in human myeloid leukemia cells.

    Science.gov (United States)

    Vieira Torquato, Heron F; Ribeiro-Filho, Antonio C; Buri, Marcus V; Araújo Júnior, Roberto T; Pimenta, Renata; de Oliveira, José Salvador R; Filho, Valdir C; Macho, Antonio; Paredes-Gamero, Edgar J; de Oliveira Martins, Domingos T

    2017-04-01

    Canthin-6-one is a natural product isolated from various plant genera and from fungi with potential antitumor activity. In the present study, we evaluate the antitumor effects of canthin-6-one in human myeloid leukemia lineages. Kasumi-1 lineage was used as a model for acute myeloid leukemia. Cells were treated with canthin-6-one and cell death, cell cycle and differentiation were evaluated in both total cells (Lin + ) and leukemia stem cell population (CD34 + CD38 - Lin -/low ). Among the human lineages tested, Kasumi-1 was the most sensitive to canthin-6-one. Canthin-6-one induced cell death with apoptotic (caspase activation, decrease of mitochondrial potential) and necrotic (lysosomal permeabilization, double labeling of annexin V/propidium iodide) characteristics. Moreover, canthin-6-one induced cell cycle arrest at G 0 /G 1 (7μM) and G 2 (45μM) evidenced by DNA content, BrdU incorporation and cyclin B1/histone 3 quantification. Canthin-6-one also promoted differentiation of Kasumi-1, evidenced by an increase in the expression of myeloid markers (CD11b and CD15) and the transcription factor PU.1. Furthermore, a reduction of the leukemic stem cell population and clonogenic capability of stem cells were observed. These results show that canthin-6-one can affect Kasumi-1 cells by promoting cell death, cell cycle arrest and cell differentiation depending on concentration used. Canthin-6-one presents an interesting cytotoxic activity against leukemic cells and represents a promising scaffold for the development of molecules for anti-leukemic applications, especially by its anti-leukemic stem cell activity. Copyright © 2017 Elsevier B.V. All rights reserved.

  2. Treatment with a JNK inhibitor increases, whereas treatment with a p38 inhibitor decreases, H2O2-induced calf pulmonary arterial endothelial cell death.

    Science.gov (United States)

    Park, Woo Hyun

    2017-08-01

    Oxidative stress induces apoptosis in endothelial cells (ECs). Reactive oxygen species (ROS) promote cell death by regulating the activity of various mitogen-activated protein kinases (MAPKs) in ECs. The present study investigated the effects of MAPK inhibitors on cell survival and glutathione (GSH) levels upon H 2 O 2 treatment in calf pulmonary artery ECs (CPAECs). H 2 O 2 treatment inhibited the growth and induced the death of CPAECs, as well as causing GSH depletion and the loss of mitochondrial membrane potential (MMP). While treatment with the MEK or JNK inhibitor impaired the growth of H 2 O 2 -treated CPAECs, treatment with the p38 inhibitor attenuated this inhibition of growth. Additionally, JNK inhibitor treatment increased the proportion of sub-G 1 phase cells in H 2 O 2 -treated CPAECs and further decreased the MMP. However, treatment with a p38 inhibitor reversed the effects of H 2 O 2 treatment on cell growth and the MMP. Similarly, JNK inhibitor treatment further increased, whereas p38 inhibitor treatment decreased, the proportion of GSH-depleted cells in H 2 O 2 -treated CPAECs. Each of the MAPK inhibitors affected cell survival, and ROS or GSH levels differently in H 2 O 2 -untreated, control CPAECs. The data suggest that the exposure of CPAECs to H 2 O 2 caused the cell growth inhibition and cell death through GSH depletion. Furthermore, JNK inhibitor treatment further enhanced, whereas p38 inhibitors attenuated, these effects. Thus, the results of the present study suggest a specific protective role for the p38 inhibitor, and not the JNK inhibitor, against H 2 O 2 -induced cell growth inhibition and cell death.

  3. Decreased neural precursor cell pool in NADPH oxidase 2-deficiency: From mouse brain to neural differentiation of patient derived iPSC

    Directory of Open Access Journals (Sweden)

    Zeynab Nayernia

    2017-10-01

    Full Text Available There is emerging evidence for the involvement of reactive oxygen species (ROS in the regulation of stem cells and cellular differentiation. Absence of the ROS-generating NADPH oxidase NOX2 in chronic granulomatous disease (CGD patients, predominantly manifests as immune deficiency, but has also been associated with decreased cognition. Here, we investigate the role of NOX enzymes in neuronal homeostasis in adult mouse brain and in neural cells derived from human induced pluripotent stem cells (iPSC. High levels of NOX2 were found in mouse adult neurogenic regions. In NOX2-deficient mice, neurogenic regions showed diminished redox modifications, as well as decrease in neuroprecursor numbers and in expression of genes involved in neural differentiation including NES, BDNF and OTX2. iPSC from healthy subjects and patients with CGD were used to study the role of NOX2 in human in vitro neuronal development. Expression of NOX2 was low in undifferentiated iPSC, upregulated upon neural induction, and disappeared during neuronal differentiation. In human neurospheres, NOX2 protein and ROS generation were polarized within the inner cell layer of rosette structures. NOX2 deficiency in CGD-iPSCs resulted in an abnormal neural induction in vitro, as revealed by a reduced expression of neuroprogenitor markers (NES, BDNF, OTX2, NRSF/REST, and a decreased generation of mature neurons. Vector-mediated NOX2 expression in NOX2-deficient iPSCs rescued neurogenesis. Taken together, our study provides novel evidence for a regulatory role of NOX2 during early stages of neurogenesis in mouse and human.

  4. Nanomolar Caffeic Acid Decreases Glucose Uptake and the Effects of High Glucose in Endothelial Cells.

    Directory of Open Access Journals (Sweden)

    Lucia Natarelli

    Full Text Available Epidemiological studies suggest that moderate and prolonged consumption of coffee is associated with a reduced risk of developing type 2 diabetes but the molecular mechanisms underlying this effect are not known. In this study, we report the effects of physiological concentrations of caffeic acid, easily achievable by normal dietary habits, in endothelial cells cultured in 25 mM of glucose (high glucose, HG. In HG, the presence of 10 nM caffeic acid was associated with a decrease of glucose uptake but not to changes of GLUT-1 membrane localization or mRNA levels. Moreover, caffeic acid countered HG-induced loss of barrier integrity, reducing actin rearrangement and FITC-dextran passage. The decreased flux of glucose associated to caffeic acid affected HG induced apoptosis by down-regulating the expression of initiator (caspase 8 and 9 and effector caspases (caspase 7 and 3 and by increasing the levels of phosphorylated Bcl-2. We also observed that caffeic acid in HG condition was associated to a reduction of p65 subunit nuclear levels with respect to HG alone. NF-κB activation has been shown to lead to apoptosis in HG treated cells and the analysis of the expression of a panel of about 90 genes related to NF-κB signaling pathway revealed that caffeic acid significantly influenced gene expression changes induced by HG. In conclusion, our results suggest that caffeic acid, decreasing the metabolic stress induced by HG, allows the activation of survival mechanisms mediated by a different modulation of NF-κB-related signaling pathways and to the activation of anti-apoptotic proteins.

  5. Ochratoxim A alters cell adhesion and gap junction intercellular communication in MDCK cells

    International Nuclear Information System (INIS)

    Mally, Angela; Decker, Martina; Bekteshi, Michaela; Dekant, Wolfgang

    2006-01-01

    Ochratoxin A (OTA) is one of the most potent renal carcinogens studied to date, but the mechanism of tumor formation by ochratoxin A remains largely unknown. Cell adhesion and cell-cell communication participate in the regulation of signaling pathways involved in cell proliferation and growth control and it is therefore not surprising that modulation of cell-cell signaling has been implicated in cancer development. Several nephrotoxicants and renal carcinogens have been shown to alter cell-cell signaling by interference with gap junction intercell communication (GJIC) and/or cell adhesion, and the aim of this study was to determine if disruption of cell-cell interactions occurs in kidney epithelial cells in response to OTA treatment. MDCK cells were treated with OTA (0-50 μM) for up to 24 h and gap junction function was analyzed using the scrape-load/dye transfer assay. In addition, expression and intracellular localization of Cx43, E-cadherin and β-catenin were determined by immunoblot and immunofluorescence analysis. A clear decrease in the distance of dye transfer was evident following treatment with OTA at concentrations/incubation times which did not affect cell viability. Consistent with the functional inhibition of GJIC, treatment with OTA resulted in a dose-dependent decrease in Cx43 expression. In contrast to Cx43, OTA did not alter total amount of the adherens junction proteins E-cadherin and β-catenin. Moreover, Western blot analysis of Triton X-100 soluble and insoluble protein fractions did not indicate translocation of cell adhesion molecules from the membrane to the cytoplasm. However, a ∼78 kDa fragment of β-catenin was detected in the detergent soluble fraction, indicating proteolytic cleavage of β-catenin. Immunofluorescence analysis also revealed changes in the pattern of both β-catenin and E-cadherin labeling, suggesting that OTA may alter cell-adhesion. Taken together, these data support the hypothesis that disruption of cell-cell

  6. Treatment with a Small Molecule Mutant IDH1 Inhibitor Suppresses Tumorigenic Activity and Decreases Production of the Oncometabolite 2-Hydroxyglutarate in Human Chondrosarcoma Cells

    Science.gov (United States)

    Li, Luyuan; Paz, Ana C.; Wilky, Breelyn A.; Johnson, Britt; Galoian, Karina; Rosenberg, Andrew; Hu, Guozhi; Tinoco, Gabriel; Bodamer, Olaf; Trent, Jonathan C.

    2015-01-01

    Chondrosarcomas are malignant bone tumors that produce cartilaginous matrix. Mutations in isocitrate dehydrogenase enzymes (IDH1/2) were recently described in several cancers including chondrosarcomas. The IDH1 inhibitor AGI-5198 abrogates the ability of mutant IDH1 to produce the oncometabolite D-2 hydroxyglutarate (D-2HG) in gliomas. We sought to determine if treatment with AGI-5198 would similarly inhibit tumorigenic activity and D-2HG production in IDH1-mutant human chondrosarcoma cells. Two human chondrosarcoma cell lines, JJ012 and HT1080 with endogenous IDH1 mutations and a human chondrocyte cell line C28 with wild type IDH1 were employed in our study. Mutation analysis of IDH was performed by PCR-based DNA sequencing, and D-2HG was detected using tandem mass spectrometry. We confirmed that JJ012 and HT1080 harbor IDH1 R132G and R132C mutation, respectively, while C28 has no mutation. D-2HG was detectable in cell pellets and media of JJ012 and HT1080 cells, as well as plasma and urine from an IDH-mutant chondrosarcoma patient, which decreased after tumor resection. AGI-5198 treatment decreased D-2HG levels in JJ012 and HT1080 cells in a dose-dependent manner, and dramatically inhibited colony formation and migration, interrupted cell cycling, and induced apoptosis. In conclusion, our study demonstrates anti-tumor activity of a mutant IDH1 inhibitor in human chondrosarcoma cell lines, and suggests that D-2HG is a potential biomarker for IDH mutations in chondrosarcoma cells. Thus, clinical trials of mutant IDH inhibitors are warranted for patients with IDH-mutant chondrosarcomas. PMID:26368816

  7. Treatment with a Small Molecule Mutant IDH1 Inhibitor Suppresses Tumorigenic Activity and Decreases Production of the Oncometabolite 2-Hydroxyglutarate in Human Chondrosarcoma Cells.

    Directory of Open Access Journals (Sweden)

    Luyuan Li

    Full Text Available Chondrosarcomas are malignant bone tumors that produce cartilaginous matrix. Mutations in isocitrate dehydrogenase enzymes (IDH1/2 were recently described in several cancers including chondrosarcomas. The IDH1 inhibitor AGI-5198 abrogates the ability of mutant IDH1 to produce the oncometabolite D-2 hydroxyglutarate (D-2HG in gliomas. We sought to determine if treatment with AGI-5198 would similarly inhibit tumorigenic activity and D-2HG production in IDH1-mutant human chondrosarcoma cells. Two human chondrosarcoma cell lines, JJ012 and HT1080 with endogenous IDH1 mutations and a human chondrocyte cell line C28 with wild type IDH1 were employed in our study. Mutation analysis of IDH was performed by PCR-based DNA sequencing, and D-2HG was detected using tandem mass spectrometry. We confirmed that JJ012 and HT1080 harbor IDH1 R132G and R132C mutation, respectively, while C28 has no mutation. D-2HG was detectable in cell pellets and media of JJ012 and HT1080 cells, as well as plasma and urine from an IDH-mutant chondrosarcoma patient, which decreased after tumor resection. AGI-5198 treatment decreased D-2HG levels in JJ012 and HT1080 cells in a dose-dependent manner, and dramatically inhibited colony formation and migration, interrupted cell cycling, and induced apoptosis. In conclusion, our study demonstrates anti-tumor activity of a mutant IDH1 inhibitor in human chondrosarcoma cell lines, and suggests that D-2HG is a potential biomarker for IDH mutations in chondrosarcoma cells. Thus, clinical trials of mutant IDH inhibitors are warranted for patients with IDH-mutant chondrosarcomas.

  8. Cell elasticity with altered cytoskeletal architectures across multiple cell types.

    Science.gov (United States)

    Grady, Martha E; Composto, Russell J; Eckmann, David M

    2016-08-01

    The cytoskeleton is primarily responsible for providing structural support, localization and transport of organelles, and intracellular trafficking. The structural support is supplied by actin filaments, microtubules, and intermediate filaments, which contribute to overall cell elasticity to varying degrees. We evaluate cell elasticity in five different cell types with drug-induced cytoskeletal derangements to probe how actin filaments and microtubules contribute to cell elasticity and whether it is conserved across cell type. Specifically, we measure elastic stiffness in primary chondrocytes, fibroblasts, endothelial cells (HUVEC), hepatocellular carcinoma cells (HUH-7), and fibrosarcoma cells (HT 1080) subjected to two cytoskeletal destabilizers: cytochalasin D and nocodazole, which disrupt actin and microtubule polymerization, respectively. Elastic stiffness is measured by atomic force microscopy (AFM) and the disruption of the cytoskeleton is confirmed using fluorescence microscopy. The two cancer cell lines showed significantly reduced elastic moduli values (~0.5kPa) when compared to the three healthy cell lines (~2kPa). Non-cancer cells whose actin filaments were disrupted using cytochalasin D showed a decrease of 60-80% in moduli values compared to untreated cells of the same origin, whereas the nocodazole-treated cells showed no change in elasticity. Overall, we demonstrate actin filaments contribute more to elastic stiffness than microtubules but this result is cell type dependent. Cancer cells behaved differently, exhibiting increased stiffness as well as stiffness variability when subjected to nocodazole. We show that disruption of microtubule dynamics affects cancer cell elasticity, suggesting therapeutic drugs targeting microtubules be monitored for significant elastic changes. Copyright © 2016 Elsevier Ltd. All rights reserved.

  9. Regulation of beta cell replication

    DEFF Research Database (Denmark)

    Lee, Ying C; Nielsen, Jens Høiriis

    2008-01-01

    Beta cell mass, at any given time, is governed by cell differentiation, neogenesis, increased or decreased cell size (cell hypertrophy or atrophy), cell death (apoptosis), and beta cell proliferation. Nutrients, hormones and growth factors coupled with their signalling intermediates have been...... suggested to play a role in beta cell mass regulation. In addition, genetic mouse model studies have indicated that cyclins and cyclin-dependent kinases that determine cell cycle progression are involved in beta cell replication, and more recently, menin in association with cyclin-dependent kinase...... inhibitors has been demonstrated to be important in beta cell growth. In this review, we consider and highlight some aspects of cell cycle regulation in relation to beta cell replication. The role of cell cycle regulation in beta cell replication is mostly from studies in rodent models, but whether...

  10. Strenuous exercise decreases the percentage of type 1 T cells in the circulation

    DEFF Research Database (Denmark)

    Steensberg, A; Toft, A D; Bruunsgaard, H

    2001-01-01

    -gamma and interleukin (IL)-2, and type 2 (Th2 and Tc2) cells, which produce IL-4. The question addressed in the present study was whether exercise affected the relative balance between the circulating levels of these cytokine-producing T cells. Nine male runners performed treadmill running for 2.5 h at 75% of maximal...... oxygen consumption. The intracellular expression of cytokines was detected following stimulation with ionomycin and phorbol 12-myristate 13-acetate in blood obtained before, during, and after exercise. The percentage of type 1 T cells in the circulation was suppressed at the end of exercise and 2 h after......Prolonged strenuous exercise is followed by a temporary functional immune impairment. Low numbers of CD4+ T helper (Th) and CD8+ T cytotoxic (Tc) cells are found in the circulation. These cells can be divided according to their cytokine profile into type 1 (Th1 and Tc1), which produce interferon...

  11. Targeting survivin with prodigiosin isolated from cell wall of Serratia marcescens induces apoptosis in hepatocellular carcinoma cells.

    Science.gov (United States)

    Yenkejeh, R A; Sam, M R; Esmaeillou, M

    2017-04-01

    Abnormal activation of the Wnt/β-catenin signaling pathway increases survivin expression that is involved in hepatocarcinogenesis. Therefore, downregulation of survivin may provide an attractive strategy for treatment of hepatocellular carcinoma. In this regard, little is known about the anticancer effects of prodigiosin isolated from cell wall of Serratia marcescens on the survivin expression and induction of apoptosis in hepatocellular carcinoma cells. Human hepatocellular carcinoma (HepG2) cells were treated with 100-, 200-, 400-, and 600-nM prodigiosin after which morphology of cells, cell number, growth inhibition, survivin expression, caspase-3 activation, and apoptotic rate were evaluated by inverted microscope, hemocytometer, MTT assay, RT-PCR, fluorometric immunosorbent enzyme assay, and flow cytometric analysis, respectively. Prodigiosin changed morphology of cells to apoptotic forms and disrupted cell connections. This compound significantly increased growth inhibition rate and decreased metabolic activity of HepG2 cells in a dose- and time-dependent manner. After 24-, 48-, and 72-h treatments with prodigiosin at concentrations ranging from 100 nM to 600 nM, growth inhibition rates were measured to be 1.5-10%, 24-47.5%, and 55.5-72.5%, respectively, compared to untreated cells. At the same conditions, metabolic activities were measured to be 91-83%, 74-53%, and 47-31% for indicated concentrations of prodigiosin, respectively, compared to untreated cells. We also found that treatment of HepG2 cells for 48 h decreased significantly cell number and survivin expression and increased caspase-3 activation in a dose-dependent manner. Specifically, treatment with 600-nM prodigiosin resulted in 77% decrease in cell number, 88.5% decrease in survivin messenger RNA level, and 330% increase in caspase-3 activation level compared to untreated cells. An increase in the number of apoptotic cells (late apoptosis) ranging from 36.9% to 97.4% was observed with increasing

  12. CD 4 + CD 25 + T cells maintain homeostasis by promoting TER - 119 cell development and inhibiting T cell activation

    Directory of Open Access Journals (Sweden)

    Muhaimin Rifa’i

    2014-05-01

    Full Text Available CD4+ CD25+ regulatory T cells involved in the regulation of self- tolerance and normality of homeostasis. CD122 deficient mice are model animals that have an abnormal immune system characteristically have a high number of activated T cells and TER-119 cell decreased. Here we showed evidence that the transfer of CD4+ CD25+ regulatory T cells derived from normal mice to CD122- defficient neonates prevent the development of activated memory T cells and elicit TER-119 differentiation. Bone marrow reconstitution derived from CD122-/- mice to normal mice resulting tolerance to individual that genetically different. Importantly, CD4+ CD25+ regulatory T cells derived from normal mice can replace CD4+ CD25+ cells derived from CD122-/- mice. The results of this experiment suggest that regulatory T cells from normal mice exert a critical role in maintaining peripheral tolerance and controlling hematopoietic disorder.

  13. The influence of continuous γ-irradiation at decreasing dose-rate on the survival rote and induction of gene mutations in cultured Chinese hamster cells

    International Nuclear Information System (INIS)

    Feoktistova, T.P.; Elisova, E.V.; Stavrakova, N.M.

    1991-01-01

    Continuous γ-irradiation at decreasing dose-rate was shown to be less effective than acute exposure with regard to the lethal effect and frequency of mutations of resistance to 6-thioguanine in cultured Chinese hamster cells. The cell population subjected to continuons irradiation was d more radioresistant than the intact one. Lethal and genetic effects of continuous irradiation at decreasing dose-rate were mainly determined by the contribution of the radiation dose received during the first 24 h of exposure

  14. Suppression of topoisomerase IIα expression and function in human cells decreases chromosomal radiosensitivity

    International Nuclear Information System (INIS)

    Terry, Samantha Y.A.; Riches, Andrew C.; Bryant, Peter E.

    2009-01-01

    The mechanism behind chromatid break formation is as yet unclear, although it is known that DNA double-strand breaks (DSBs) are the initiating lesions. Chromatid breaks formed in cells in the G2-phase of the cell-cycle disappear ('rejoin') as a function of time between radiation exposure and cell fixation. However, the kinetics of disappearance of chromatid breaks does not correspond to those of DSB rejoining, leading us to seek alternative models. We have proposed that chromatid breaks could be formed indirectly from DSB and that the mechanism involves topoisomerase IIα. In support of this hypothesis we have recently shown that frequencies of radiation-induced chromatid breaks are lower in two variant human promyelocytic leukaemic cell lines with reduced topoisomerase IIα expression. Here we report that suppression of topoisomerase IIα in human hTERT-RPE1 cells, either by its abrogation using specific siRNA or by inhibition of its catalytic activity with the inhibitor ICRF-193, causes a reduction in frequency of chromatid breaks in radiation-exposed cells. The findings support our hypothesis for the involvement of topoisomerase IIα in the formation of radiation-induced chromatid breaks, and could help explain inter-individual variation in human chromosomal radiosensitivity; elevation of which has been linked with cancer susceptibility.

  15. Regulation of nonsmall-cell lung cancer stem cell like cells by neurotransmitters and opioid peptides.

    Science.gov (United States)

    Banerjee, Jheelam; Papu John, Arokya M S; Schuller, Hildegard M

    2015-12-15

    Nonsmall-cell lung cancer (NSCLC) is the leading type of lung cancer and has a poor prognosis. We have shown that chronic stress promoted NSCLC xenografts in mice via stress neurotransmitter-activated cAMP signaling downstream of beta-adrenergic receptors and incidental beta-blocker therapy was reported to improve clinical outcomes in NSCLC patients. These findings suggest that psychological stress promotes NSCLC whereas pharmacologically or psychologically induced decreases in cAMP may inhibit NSCLC. Cancer stem cells are thought to drive the development, progression and resistance to therapy of NSCLC. However, their potential regulation by stress neurotransmitters has not been investigated. In the current study, epinephrine increased the number of cancer stem cell like cells (CSCs) from three NSCLC cell lines in spheroid formation assays while enhancing intracellular cAMP and the stem cell markers sonic hedgehog (SHH), aldehyde dehydrogenase-1 (ALDH-1) and Gli1, effects reversed by GABA or dynorphin B via Gαi -mediated inhibition of cAMP formation. The growth of NSCLC xenografts in a mouse model of stress reduction was significantly reduced as compared with mice maintained under standard conditions. Stress reduction reduced serum levels of corticosterone, norepinephrine and epinephrine while the inhibitory neurotransmitter γ-aminobutyric acid (GABA) and opioid peptides increased. Stress reduction significantly reduced cAMP, VEGF, p-ERK, p-AKT, p-CREB, p-SRc, SHH, ALDH-1 and Gli1 in xenograft tissues whereas cleaved caspase-3 and p53 were induced. We conclude that stress neurotransmitters activate CSCs in NSCLC via multiple cAMP-mediated pathways and that pharmacologically or psychologically induced decreases in cAMP signaling may improve clinical outcomes in NSCLC patients. © 2015 UICC.

  16. Age-related decrease in rod bipolar cell density of the human retina ...

    Indian Academy of Sciences (India)

    PRAKASH

    Retinal bipolar cells situated in the inner nuclear layer are predominantly involved in mediating the transfer of signals from the photoreceptors to the ganglion cells. Their synapses are located both at the level of the outer and inner plexiform layers. In the former, their dendritic processes synapse with photoreceptors in the ...

  17. Decreased naive and increased memory CD4(+ T cells are associated with subclinical atherosclerosis: the multi-ethnic study of atherosclerosis.

    Directory of Open Access Journals (Sweden)

    Nels C Olson

    Full Text Available Adaptive immunity has been implicated in atherosclerosis in animal models and small clinical studies. Whether chronic immune activation is associated with atherosclerosis in otherwise healthy individuals remains underexplored. We hypothesized that activation of adaptive immune responses, as reflected by higher proportions of circulating CD4(+ memory cells and lower proportions of naive cells, would be associated with subclinical atherosclerosis.We examined cross-sectional relationships of circulating CD4(+ naive and memory T cells with biomarkers of inflammation, serologies, and subclinical atherosclerosis in 912 participants of the Multi-Ethnic Study of Atherosclerosis (MESA. Circulating CD4(+ naive cells were higher in women than men and decreased with age (all p-values <0.0001. European-Americans had higher levels of naive cells and lower levels of memory cells compared with African-Americans and Hispanic-Americans (all p-values ≤0.0005. Lower naive/higher memory cells were associated with interleukin-6 levels. In multivariate models, cytomegalovirus (CMV and H. Pylori titers were strongly associated with higher memory and lower naive cells (all p-values <0.05. Higher memory cells were associated with coronary artery calcification (CAC level in the overall population [β-Coefficient (95% confidence interval (CI  = 0.20 (0.03, 0.37]. Memory and naive (inversely cells were associated with common carotid artery intimal media thickness (CC IMT in European-Americans [memory: β =  0.02 (0.006, 0.04; naive: β = -0.02 (-0.004, -0.03].These results demonstrate that the degree of chronic adaptive immune activation is associated with both CAC and CC IMT in otherwise healthy individuals, consistent with the known role of CD4(+ T cells, and with innate immunity (inflammation, in atherosclerosis. These data are also consistent with the hypothesis that immunosenescence accelerates chronic diseases by putting a greater burden on the innate

  18. Cell-cycle distributions and radiation responses of Chinese hamster cells cultured continuously under hypoxic conditions

    International Nuclear Information System (INIS)

    Tokita, N.; Carpenter, S.G.; Raju, M.R.

    1984-01-01

    Cell-cycle distributions were measured by flow cytometry for Chinese hamster (CHO) cells cultured continuously under hypoxic conditions. DNA histograms showed an accumulation of cells in the early S phase followed by a traverse delay through the S phase, and a G 2 block. During hypoxic culturing, cell viability decreased rapidly to less than 0.1% at 120 h. Radiation responses for cells cultured under these conditions showed an extreme radioresistance at 72 h. Results suggest that hypoxia induces a condition similar to cell synchrony which itself changes the radioresistance of hypoxic cells. (author)

  19. Practical cell labeling with magnetite cationic liposomes for cell manipulation.

    Science.gov (United States)

    Ito, Hiroshi; Nonogaki, Yurika; Kato, Ryuji; Honda, Hiroyuki

    2010-07-01

    Personalization of the cell culture process for cell therapy is an ideal strategy to obtain maximum treatment effects. In a previous report, we proposed a strategy using a magnetic manipulation device that combined a palm-top size device and a cell-labeling method using magnetite cationic liposomes (MCLs) to enable feasible personalized cell processing. In the present study, we focused on optimizing the MCL-labeling technique with respect to cell manipulation in small devices. From detailed analysis with different cell types, 4 pg/cell of MCL-label was found to be obtained immediately after mixing with MCLs, which was sufficient for magnetic cell manipulation. The amount of label increased within 24 h depending on cell type, although in all cases it decreased along with cell doubling, indicating that the labeling potential of MCLs was limited. The role of free MCLs not involved in labeling was also investigated; MCLs' role was found to be a supportive one that maximized the manipulation performance up to 100%. We also determined optimum conditions to manipulate adherent cells by MCL labeling using the MCL dispersed in trypsin solution. Considering labeling feasibility and practical performance with 10(3)-10(5) cells for personalized cell processing, we determined that 10 microg/ml of label without incubation time (0 h incubation) was the universal MCL-labeling condition. We propose the optimum specifications for a device to be combined with this method. 2010. Published by Elsevier B.V.

  20. RON kinase isoforms demonstrate variable cell motility in normal cells.

    Science.gov (United States)

    Greenbaum, Alissa; Rajput, Ashwani; Wan, Guanghua

    2016-09-01

    Aberrant RON (Recepteur d'Origine Nantais) tyrosine kinase activation causes the epithelial cell to evade normal growth pathways, resulting in unregulated cell proliferation, increased cell motility and decreased apoptosis. Wildtype (wt) RON has been shown to play a role in metastasis of epithelial malignancies. It presents an important potential therapeutic target for colorectal, breast, gastric and pancreatic cancer. Little is known about functional differences amongst RON isoforms RON155, RON160 and RON165. The purpose of this study was to determine the effect of various RON kinase isoforms on cell motility. Cell lines with stable expression of wtRON were generated by inserting the coding region of RON in pTagRFP (tagged red fluorescence protein plasmid). The expression constructs of RON variants (RON155, RON160 and RON165) were generated by creating a mutagenesis-based wtRON-pTag RFP plasmid and stably transfected into HEK 293 cells. The wound closure scratch assay was used to investigate the effect on cell migratory capacity of wild type RON and its variants. RON transfected cells demonstrated increased cell motility compared to HEK293 control cells. RON165 cell motility was significantly increased compared to RON160 (mean percentage of wound covered 37.37% vs. 32.40%; p = 0.03). RON tyrosine kinase isoforms have variable cell motility. This may reflect a difference in the behavior of malignant epithelial cells and their capacity for metastasis.

  1. Modification of cell growth rate by irradiation

    International Nuclear Information System (INIS)

    Itoh, Hisao; Takemasa, Kazuhiko; Nishiguchi, Iku; Ka, Wei-Jei; Kutsuki, Shoji; Hashimoto, Shozo

    1993-01-01

    The effect of irradiation on the proliferation kinetics of the monolayer cells has been studied. Two human cell lines with different doubling times (HeLa-P and RMUG) and two clones that have the same radiosensitivity but different doubling times (HeLa-R and HeLa-S) were irradiated with a daily dose of 2 Gy for 6 days. The number of the clonogenic cells/dish was calculated by multiplying the number of total cell/dish by the survival fraction. In the rapidly growing cells (HeLa-P, HeLa-R), the number of the clonogenic cells was not decreased by the first two fractionated irradiations, but decreased thereafter at a similar rate as by single-dose fractionation, whereas the clonogenic cell number decreased from the first fractionated irradiation in the slowly growing cells (RMUG, HeLa-S). When the proliferation of clonogenic cell number increased along with a similar growth rates that was seen in all other types of cells. Further, no correlation was seen between the growth rates of cells without irradiation and cells that received irradiation. This latter result suggests that the slow growth rate of non-irradiated cells may not be the predictive factor of the tumor cure and the interruption of radiotherapy may reduce the beneficial effect of this treatment even in slow growing tumors. (author)

  2. Quercetin decrease somatic cells count in mastitis of dairy cows.

    Science.gov (United States)

    Burmańczuk, Artur; Hola, Piotr; Milczak, Andrzej; Piech, Tomasz; Kowalski, Cezary; Wojciechowska, Beata; Grabowski, Tomasz

    2018-04-01

    Quercetin is a dietary flavonoid which has an effect on inflammation, angiogenesis and vascular inflammation. In several other flavonoids (e.g. kaempferol, astragalin, alpinetin, baicalein, indirubin), anti-inflammatory mechanism was proven by using mice mastitis model. The aim of the current study was pilot analysis of quercetin tolerability and its impact on somatic cells count (SCC) after multiple intramammary treatment on dairy cows with clinical mastitis. Based on SCC and clinical investigation, 9 dairy cows with clinical mastitis of one quarter were selected for the pilot study. Baseline analysis (hematology, TNFα, SCC) was performed every 24h among all cows three days before the first dose (B1-B3). After the baseline monitoring (B1-B3) eight days treatment (D1-D8) was performed with a high and low dose. Selected blood parameters were analyzed. Starting from D1 to D8, a decrease of SCC in relation to baseline was characterized by declining trend. The presented results allowed the confirmation of the significant influence of quercetin on the reduction of SCC in mastitis in dairy cows after 8days of therapy. Copyright © 2018. Published by Elsevier Ltd.

  3. Phytol isolated from watermelon (Citrullus lanatus) sprouts induces cell death in human T-lymphoid cell line Jurkat cells via S-phase cell cycle arrest.

    Science.gov (United States)

    Itoh, Tomohiro; Ono, Akito; Kawaguchi, Kaori; Teraoka, Sayaka; Harada, Mayo; Sumi, Keitaro; Ando, Masashi; Tsukamasa, Yasuyuki; Ninomiya, Masayuki; Koketsu, Mamoru; Hashizume, Toshiharu

    2018-05-01

    The phytol isolated from watermelon (Citrullus lanatus) sprouts inhibited the growth of a human T-cell leukemia line Jurkat cell and suppressed tumor progression in a xenograft model of human lung adenocarcinoma epithelial cell line A549 in nude mice. To elucidate the mechanisms underlying the phytol-induced cell death in the present study, we examined the changes in cell morphology, DNA fragmentation, and intracellular reactive oxygen species (ROS) levels and performed flow cytometric analysis to evaluate cell cycle stage. There were no significant changes in apoptosis, autophagy, and necrosis marker in cells treated with the phytol. But, we found, for the first time, that phytol remarkably induced S-phase cell cycle arrest accompanied with intracellular ROS production. Western blot analyses showed that phytolinduced S-phase cell cycle arrest was mediated through the decreased expression of cyclins A and D and the downregulations of MAPK and PI3K/Akt. The tumor volume levels in mice treated with phytol were lower than those of non-treatment groups, and it showed very similar suppression compared with those of mice treated with cyclophosphamide. Based on the data of in vitro and in vivo studies and previous studies, we suggest phytol as a potential therapeutic compound for cancer. Copyright © 2018 Elsevier Ltd. All rights reserved.

  4. Radotinib Induces Apoptosis of CD11b+ Cells Differentiated from Acute Myeloid Leukemia Cells.

    Directory of Open Access Journals (Sweden)

    Sook-Kyoung Heo

    Full Text Available Radotinib, developed as a BCR/ABL tyrosine kinase inhibitor (TKI, is approved for the second-line treatment of chronic myeloid leukemia (CML in South Korea. However, therapeutic effects of radotinib in acute myeloid leukemia (AML are unknown. In the present study, we demonstrate that radotinib significantly decreases the viability of AML cells in a dose-dependent manner. Kasumi-1 cells were more sensitive to radotinib than NB4, HL60, or THP-1 cell lines. Furthermore, radotinib induced CD11b expression in NB4, THP-1, and Kasumi-1 cells either in presence or absence of all trans-retinoic acid (ATRA. We found that radotinib promoted differentiation and induced CD11b expression in AML cells by downregulating LYN. However, CD11b expression induced by ATRA in HL60 cells was decreased by radotinib through upregulation of LYN. Furthermore, radotinib mainly induced apoptosis of CD11b+ cells in the total population of AML cells. Radotinib also increased apoptosis of CD11b+ HL60 cells when they were differentiated by ATRA/dasatinib treatment. We show that radotinib induced apoptosis via caspase-3 activation and the loss of mitochondrial membrane potential (ΔΨm in CD11b+ cells differentiated from AML cells. Our results suggest that radotinib may be used as a candidate drug in AML or a chemosensitizer for treatment of AML by other therapeutics.

  5. Salinomycin induces cell death and differentiation in head and neck squamous cell carcinoma stem cells despite activation of epithelial-mesenchymal transition and Akt

    International Nuclear Information System (INIS)

    Kuo, Selena Z; Blair, Katherine J; Rahimy, Elham; Kiang, Alan; Abhold, Eric; Fan, Jian-Bing; Wang-Rodriguez, Jessica; Altuna, Xabier; Ongkeko, Weg M

    2012-01-01

    Cancer stem cells (CSC) are believed to play a crucial role in cancer recurrence due to their resistance to conventional chemotherapy and capacity for self-renewal. Recent studies have reported that salinomycin, a livestock antibiotic, selectively targets breast cancer stem cells 100-fold more effectively than paclitaxel. In our study we sought to determine the effects of salinomycin on head and neck squamous cell carcinoma (HNSCC) stem cells. MTS and TUNEL assays were used to study cell proliferation and apoptosis as a function of salinomycin exposure in JLO-1, a putative HNSCC stem cell culture. MTS and trypan blue dye exclusion assays were performed to investigate potential drug interactions between salinomycin and cisplatin or paclitaxel. Stem cell-like phenotype was measured by mRNA expression of stem cell markers, sphere-forming capacity, and matrigel invasion assays. Immunoblotting was also used to determine expression of epithelial-mesenchymal transition (EMT) markers and Akt phosphorylation. Arrays by Illumina, Inc. were used to profile microRNA expression as a function of salinomycin dose. In putative HNSCC stem cells, salinomycin was found to significantly inhibit cell viability, induce a 71.5% increase in levels of apoptosis, elevate the Bax/Bcl-2 ratio, and work synergistically with cisplatin and paclitaxel in inducing cell death. It was observed that salinomycin significantly inhibited sphere forming-capability and repressed the expression of CD44 and BMI-1 by 3.2-fold and 6.2-fold, respectively. Furthermore, salinomycin reduced invasion of HNSCC stem cells by 2.1 fold. Contrary to expectations, salinomycin induced the expression of EMT markers Snail, vimentin, and Zeb-1, decreased expression of E-cadherin, and also induced phosphorylation of Akt and its downstream targets GSK3-β and mTOR. These results demonstrate that in HNSCC cancer stem cells, salinomycin can cause cell death and decrease stem cell properties despite activation of both EMT and

  6. RNA interference-mediated silencing of speckle-type POZ protein promotes apoptosis of renal cell cancer cells

    Directory of Open Access Journals (Sweden)

    Liu X

    2016-04-01

    Full Text Available Xiaoxia Liu, Guiling Sun, Xiuju Sun Department of Nephrology, Affiliated Hospital of Weifang Medical University, Weifang, People’s Republic of China Abstract: This study aimed to investigate the effects of silencing the speckle-type POZ protein (SPOP gene on renal cell cancer (RCC cells and to explore its possible mechanism. The A498 and ACHN RCC cells were transfected with small interference RNA (siRNA-SPOP by lipofection methods. The silencing efficiency was monitored by quantitative real-time polymerase chain reaction and Western blot. The effects of SPOP silencing on cell apoptosis, cell viability, colony formation ability, cell migration ability, and chemosensitivity to Sorafenib were assessed by flow cytometry, an MTT assay, a colony formation assay, a trans-well migration assay, and a CCK-8 assay, respectively. Its effects on the expression of several cytokines were determined by a protein microarray. Relevant signaling pathways were also analyzed. Compared with the control group, the cell apoptosis rate was significantly higher; the cell viability, the colony formation, and migration ability were significantly decreased in the siRNA-SPOP group. The protein microarray screening showed that the expression of vascular endothelial growth factor receptor, matrix metallopeptidase-9, vascular cell adhesion molecule-1, and stromal cell-derived factor-1 in the siRNA group was significantly decreased and that the expression of granulocyte–macrophage colony-stimulating factor and E-cadherin was significantly increased (P<0.05. The relevant signaling pathways were the integrin-mediated cell surface interactions pathway and extracellular matrix organization signal pathway. SPOP gene silencing induced cell apoptosis, decreased cell viability, colony formation, and migration ability, and elevated the drug sensitivity in the RCC cells. A possible mechanism is that silencing SPOP induces the differential expression of E-cadherin, vascular endothelial

  7. Protective Effects of Mouse Bone Marrow Mesenchymal Stem Cell Soup on Staurosporine Induced Cell Death in PC12 and U87 Cell Lines

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    Hossein Zhaleh

    2016-11-01

    Full Text Available Mouse bone marrow mesenchymal stem cells (mBMSCs soup is promising tool for the treatment of neurodegenerative diseases. mBMSCs soup is easily obtained and is capable of transplantation without rejection. We investigated the effects of mBMSC soup on staurosporine-induced cell death in PC12 and U87 cells lines. The percentage of cell viability, cell death, NO concentration, total neurite length (TNL and fraction of cell differentiation (f% were assessed. Viability assay showed that mBM soup (24 and 48h in time dependent were increased cell viability (p<0.05 and also cell death assay showed that cell death in time dependent were decreased, respectively (p<0.05. TNL and fraction of cell differentiation significantly were increased compared with treatment1 (p<0.05. Our data showed that mBM Soup protects cells, increases cell viability, suppresses cell death and improvement the neurite elongation. We concluded that Mouse bone marrow mesenchymal stem cell soup plays an important protective role in staurosporine-induced cell death in PC12 and U87 cell lines.

  8. ArtinM Mediates Murine T Cell Activation and Induces Cell Death in Jurkat Human Leukemic T Cells

    Science.gov (United States)

    Oliveira-Brito, Patrícia Kellen Martins; Gonçalves, Thiago Eleutério; Vendruscolo, Patrícia Edivânia; Roque-Barreira, Maria Cristina

    2017-01-01

    The recognition of cell surface glycans by lectins may be critical for the innate and adaptive immune responses. ArtinM, a d-mannose-binding lectin from Artocarpus heterophyllus, activates antigen-presenting cells by recognizing TLR2 N-glycans and induces Th1 immunity. We recently demonstrated that ArtinM stimulated CD4+ T cells to produce proinflammatory cytokines. Here, we further studied the effects of ArtinM on adaptive immune cells. We showed that ArtinM activates murine CD4+ and CD8+ T cells, augmenting their positivity for CD25, CD69, and CD95 and showed higher interleukin (IL)-2 and interferon (IFN)-γ production. The CD4+ T cells exhibited increased T-bet expression in response to ArtinM, and IL-2 production by CD4+ and CD8+ T cells depended on the recognition of CD3εγ-chain glycans by ArtinM. The ArtinM effect on aberrantly-glycosylated neoplastic lymphocytes was studied in Jurkat T cells, in which ArtinM induced IL-2, IFN-γ, and IL-1β production, but decreased cell viability and growth. A higher frequency of AnnexinV- and propidium iodide-stained cells demonstrated the induction of Jurkat T cells apoptosis by ArtinM, and this apoptotic response was reduced by caspases and protein tyrosine kinase inhibitors. The ArtinM effects on murine T cells corroborated with the immunomodulatory property of lectin, whereas the promotion of Jurkat T cells apoptosis may reflect a potential applicability of ArtinM in novel strategies for treating lymphocytic leukemia. PMID:28665310

  9. Planar cell polarity gene expression correlates with tumor cell viability and prognostic outcome in neuroblastoma

    International Nuclear Information System (INIS)

    Dyberg, Cecilia; Papachristou, Panagiotis; Haug, Bjørn Helge; Lagercrantz, Hugo; Kogner, Per; Ringstedt, Thomas; Wickström, Malin; Johnsen, John Inge

    2016-01-01

    The non-canonical Wnt/Planar cell polarity (PCP) signaling pathway is a major player in cell migration during embryonal development and has recently been implicated in tumorigenesis. Transfections with cDNA plasmids or siRNA were used to increase and suppress Prickle1 and Vangl2 expression in neuroblastoma cells and in non-tumorigenic cells. Cell viability was measured by trypan blue exclusion and protein expression was determined with western blotting. Transcriptional activity was studied with luciferase reporter assay and mRNA expression with real-time RT-PCR. Immunofluorescence stainings were used to study the effects of Vangl2 overexpression in non-tumorigenic embryonic cells. Statistical significance was tested with t-test or one-way ANOVA. Here we show that high expression of the PCP core genes Prickle1 and Vangl2 is associated with low-risk neuroblastoma, suppression of neuroblastoma cell growth and decreased Wnt/β-catenin signaling. Inhibition of Rho-associated kinases (ROCKs) that are important in mediating non-canonical Wnt signaling resulted in increased expression of Prickle1 and inhibition of β-catenin activity in neuroblastoma cells. In contrast, overexpression of Vangl2 in MYC immortalized neural stem cells induced accumulation of active β-catenin and decreased the neural differentiation marker Tuj1. Similarly, genetically modified mice with forced overexpression of Vangl2 in nestin-positive cells showed decreased Tuj1 differentiation marker during embryonal development. Our experimental data demonstrate that high expression of Prickle1 and Vangl2 reduce the growth of neuroblastoma cells and indicate different roles of PCP proteins in tumorigenic cells compared to normal cells. These results suggest that the activity of the non-canonical Wnt/PCP signaling pathway is important for neuroblastoma development and that manipulation of the Wnt/PCP pathway provides a possible therapy for neuroblastoma. The online version of this article (doi:10.1186/s

  10. Decreased hypertrophic differentiation accompanies enhanced matrix formation in co-cultures of outer meniscus cells with bone marrow mesenchymal stromal cells

    Science.gov (United States)

    2012-01-01

    Introduction The main objective of this study was to determine whether meniscus cells from the outer (MCO) and inner (MCI) regions of the meniscus interact similarly to or differently with mesenchymal stromal stem cells (MSCs). Previous study had shown that co-culture of meniscus cells with bone marrow-derived MSCs result in enhanced matrix formation relative to mono-cultures of meniscus cells and MSCs. However, the study did not examine if cells from the different regions of the meniscus interacted similarly to or differently with MSCs. Methods Human menisci were harvested from four patients undergoing total knee replacements. Tissue from the outer and inner regions represented pieces taken from one third and two thirds of the radial distance of the meniscus, respectively. Meniscus cells were released from the menisci after collagenase treatment. Bone marrow MSCs were obtained from the iliac crest of two patients after plastic adherence and in vitro culture until passage 2. Primary meniscus cells from the outer (MCO) or inner (MCI) regions of the meniscus were co-cultured with MSCs in three-dimensional (3D) pellet cultures at 1:3 ratio, respectively, for 3 weeks in the presence of serum-free chondrogenic medium containing TGF-β1. Mono-cultures of MCO, MCI and MSCs served as experimental control groups. The tissue formed after 3 weeks was assessed biochemically, histochemically and by quantitative RT-PCR. Results Co-culture of inner (MCI) or outer (MCO) meniscus cells with MSCs resulted in neo-tissue with increased (up to 2.2-fold) proteoglycan (GAG) matrix content relative to tissues formed from mono-cultures of MSCs, MCI and MCO. Co-cultures of MCI or MCO with MSCs produced the same amount of matrix in the tissue formed. However, the expression level of aggrecan was highest in mono-cultures of MSCs but similar in the other four groups. The DNA content of the tissues from co-cultured cells was not statistically different from tissues formed from mono-cultures of

  11. Good Cell Culture Practice for stem cells and stem-cell-derived models.

    Science.gov (United States)

    Pamies, David; Bal-Price, Anna; Simeonov, Anton; Tagle, Danilo; Allen, Dave; Gerhold, David; Yin, Dezhong; Pistollato, Francesca; Inutsuka, Takashi; Sullivan, Kristie; Stacey, Glyn; Salem, Harry; Leist, Marcel; Daneshian, Mardas; Vemuri, Mohan C; McFarland, Richard; Coecke, Sandra; Fitzpatrick, Suzanne C; Lakshmipathy, Uma; Mack, Amanda; Wang, Wen Bo; Yamazaki, Daiju; Sekino, Yuko; Kanda, Yasunari; Smirnova, Lena; Hartung, Thomas

    2017-01-01

    The first guidance on Good Cell Culture Practice (GCCP) dates back to 2005. This document expands this to include aspects of quality assurance for in vitro cell culture focusing on the increasingly diverse cell types and culture formats used in research, product development, testing and manufacture of biotechnology products and cell-based medicines. It provides a set of basic principles of best practice that can be used in training new personnel, reviewing and improving local procedures, and helping to assure standard practices and conditions for the comparison of data between laboratories and experimentation performed at different times. This includes recommendations for the documentation and reporting of culture conditions. It is intended as guidance to facilitate the generation of reliable data from cell culture systems, and is not intended to conflict with local or higher level legislation or regulatory requirements. It may not be possible to meet all recommendations in this guidance for practical, legal or other reasons. However, when it is necessary to divert from the principles of GCCP, the risk of decreasing the quality of work and the safety of laboratory staff should be addressed and any conclusions or alternative approaches justified. This workshop report is considered a first step toward a revised GCCP 2.0.

  12. Berberine Suppresses Cell Motility Through Downregulation of TGF-β1 in Triple Negative Breast Cancer Cells

    Directory of Open Access Journals (Sweden)

    Sangmin Kim

    2018-01-01

    Full Text Available Background/Aims: Transforming growth factor-beta proteins (TGF-βs are multifunctional growth factors and powerful modulators of the epithelial-mesenchymal transition (EMT in a variety of cancer types including breast and lung cancer cells. Here, we demonstrated the inhibitory effect of berberine (BBR on tumor growth and metastasis of triple negative breast cancer (TNBC cells via suppression of TGF-β1 expression. Methods: The levels of mRNA expression were analyzed by real-time PCR. The levels of MMP-2, MMP-9 and TGF-β1 protein expression were analyzed by zymography and confocal microscopy, respectively. Cell migration was analyzed by wound healing assay. Tumorigenicity of TNBC cells such as tumor growth and metastasis was analyzed using xenograft models. Results: In a clinical data set, aberrant TGF-β1 expression was associated with poor prognosis of breast cancer patients. Our in vitro results using TNBC cells showed that the expression levels of matrix metalloproteinase (MMP-2 and MMP-9 and the capacity for cell migration were increased by TGF-β1 treatment. In contrast, basal levels of MMP-2 and MMP-9 were suppressed by a specific TGF-β receptor I inhibitor, SB431542. In addition, TGF-β1–induced MMP-2 and MMP-9 expression and cell migration were decreased by SB431542. Interestingly, we showed for the first time that BBR decreased the level of TGF-β1, but not TGF-β2, in TNBC cells. Furthermore, BBR significantly decreased the level of MMP-2 expression as well as the capacity for cell migration in TNBC cells. Finally, we examined the effect of BBR on in vivo tumor growth and lung metastasis in MDA-MB231 and 4T1 breast cancer xenograft models and showed that both were significantly decreased following BBR treatment. Conclusion: BBR suppresses tumorigenicity of TNBC cells through inhibition of TGF-β1 expression. Therefore, we demonstrate that BBR could be a promising drug for treatment of TNBC.

  13. The Cytotoxic Role of Intermittent High Glucose on Apoptosis and Cell Viability in Pancreatic Beta Cells

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    Zhen Zhang

    2014-01-01

    Full Text Available Objectives. Glucose fluctuations are both strong predictor of diabetic complications and crucial factor for beta cell damages. Here we investigated the effect of intermittent high glucose (IHG on both cell apoptosis and proliferation activity in INS-1 cells and the potential mechanisms. Methods. Cells were treated with normal glucose (5.5 mmol/L, constant high glucose (CHG (25 mmol/L, and IHG (rotation per 24 h in 11.1 or 25 mmol/L for 7 days. Reactive oxygen species (ROS, xanthine oxidase (XOD level, apoptosis, cell viability, cell cycle, and expression of cyclinD1, p21, p27, and Skp2 were determined. Results. We found that IHG induced more significant apoptosis than CHG and normal glucose; intracellular ROS and XOD levels were more markedly increased in cells exposed to IHG. Cells treated with IHG showed significant decreased cell viability and increased cell proportion in G0/G1 phase. Cell cycle related proteins such as cyclinD1 and Skp2 were decreased significantly, but expressions of p27 and p21 were increased markedly. Conclusions. This study suggested that IHG plays a more toxic effect including both apoptosis-inducing and antiproliferative effects on INS-1 cells. Excessive activation of cellular stress and regulation of cyclins might be potential mechanism of impairment in INS-1 cells induced by IHG.

  14. Changes in radiosensitivity of V-79 cells accompanying growth and cell division

    International Nuclear Information System (INIS)

    Lehnert, S.

    1975-01-01

    The X-ray survival curve for asynchronous Chinese hamster V-79 cells at 17 to 20 hr after plating when cells are irradiated as microclones of two to four cells differs from the survival curve seen at short times after plating, when single cells are irradiated, in having higher D 0 (300 rad vs 160 rad) and negligible extrapolation number. As a consequence of the difference in D 0 the difference in survival between single cells and clones increases with increasing dose. Transient cyclic changes in survival occur at early times after plating and are probably related to partial synchronization induced by trypsinization. In addition there is a progressive increase in survival which develops with increasing time after plating, as the number of cells in the clones increases. Decrease in radiosensitivity with increasing number of cells irradiated is also observed for synchronous cells when cells at corresponding points in the cell cycle are irradiated. Accumulation and repair of sublethal damage is demonstrable in cells irradiated at short times after plating, but cannot be shown at 20 hr after plating when cells are irradiated as microclones. (U.S.)

  15. Expression of heparanase in basal cell carcinoma and squamous cell carcinoma.

    Science.gov (United States)

    Pinhal, Maria Aparecida Silva; Almeida, Maria Carolina Leal; Costa, Alessandra Scorse; Theodoro, Thérèse Rachell; Serrano, Rodrigo Lorenzetti; Machado, Carlos D'Apparecida Santos

    2016-01-01

    Heparanase is an enzyme that cleaves heparan sulfate chains. Oligosaccharides generated by heparanase induce tumor progression. Basal cell carcinoma and squamous cell carcinoma comprise types of nonmelanoma skin cancer. Evaluate the glycosaminoglycans profile and expression of heparanase in two human cell lines established in culture, immortalized skin keratinocyte (HaCaT) and squamous cell carcinoma (A431) and also investigate the expression of heparanase in basal cell carcinoma, squamous cell carcinoma and eyelid skin of individuals not affected by the disease (control). Glycosaminoglycans were quantified by electrophoresis and indirect ELISA method. The heparanase expression was analyzed by quantitative RT-PCR (qRTPCR). The A431 strain showed significant increase in the sulfated glycosaminoglycans, increased heparanase expression and decreased hyaluronic acid, comparing to the HaCaT lineage. The mRNA expression of heparanase was significantly higher in Basal cell carcinoma and squamous cell carcinoma compared with control skin samples. It was also observed increased heparanase expression in squamous cell carcinoma compared to the Basal cell carcinoma. The glycosaminoglycans profile, as well as heparanase expression are different between HaCaT and A431 cell lines. The increased expression of heparanase in Basal cell carcinoma and squamous cell carcinoma suggests that this enzyme could be a marker for the diagnosis of such types of non-melanoma cancers, and may be useful as a target molecule for future alternative treatment.

  16. Basics elements for modelling the dynamics of cell migration in cell culture

    International Nuclear Information System (INIS)

    FarIas, Ro; Vidal, Cs; Rapacioli, M; Flores, V

    2007-01-01

    This paper introduces some basic elements for modelling the dynamics of cell migration activity over a bi-dimensional substratum. A square matrix, representing the substratum, is implemented in order to generate virtual cells with an initial random uniform distribution, with the ability to freely move within the matrix and to interact with each others by mean of adhesive forces. Two different conditions were examined: A) cells can freely move and after contacting with another cell they both completely inhibit their migration; B) cells that come into contact have the ability to rotate respect to each other without losing their contacts and retaining the ability to move together but at a slower rate, being the decrease in the rate of movement proportional to the number of contacting cells. The dynamics of the migration process in these two conditions was evaluated by recording the evolution of several parameters as a function of time. Minor modifications in some parameters (mobility, intensity of cell-cell and cell-substratum adhesiveness) significantly change the dynamics and the final result of the virtual migrating cells

  17. Complex cells decrease errors for the Müller-Lyer illusion in a model of the visual ventral stream

    Directory of Open Access Journals (Sweden)

    Astrid eZeman

    2014-09-01

    Full Text Available To improve robustness in object recognition, many artificial visual systems imitate the way in which the human visual cortex encodes object information as a hierarchical set of features. These systems are usually evaluated in terms of their ability to accurately categorize well-defined, unambiguous objects and scenes. In the real world, however, not all objects and scenes are presented clearly, with well-defined labels and interpretations. Visual illusions demonstrate a disparity between perception and objective reality, allowing psychophysicists to methodically manipulate stimuli and study our interpretation of the environment. One prominent effect, the Müller-Lyer illusion, is demonstrated when the perceived length of a line is contracted (or expanded by the addition of arrowheads (or arrow-tails to its ends. HMAX, a benchmark object recognition system, consistently produces a bias when classifying Müller-Lyer images. HMAX is a hierarchical, artificial neural network that imitates the ‘simple’ and ‘complex’ cell layers found in the visual ventral stream. In this study, we perform two experiments to explore the Müller-Lyer illusion in HMAX, asking: 1 How do simple versus complex cell operations within HMAX affect illusory bias and precision? 2 How does varying the position of the figures in the input image affect classification using HMAX? In our first experiment, we assessed classification after traversing each layer of HMAX and found that in general, kernel operations performed by simple cells increase bias and uncertainty while max-pooling operations executed by complex cells decrease bias and uncertainty. In our second experiment, we increased variation in the positions of figures in the input that reduced bias and uncertainty in HMAX. Our findings suggest that the Müller-Lyer illusion is exacerbated by the vulnerability of simple cell operations to positional fluctuations, but ameliorated by the robustness of complex cell

  18. Complex cells decrease errors for the Müller-Lyer illusion in a model of the visual ventral stream.

    Science.gov (United States)

    Zeman, Astrid; Obst, Oliver; Brooks, Kevin R

    2014-01-01

    To improve robustness in object recognition, many artificial visual systems imitate the way in which the human visual cortex encodes object information as a hierarchical set of features. These systems are usually evaluated in terms of their ability to accurately categorize well-defined, unambiguous objects and scenes. In the real world, however, not all objects and scenes are presented clearly, with well-defined labels and interpretations. Visual illusions demonstrate a disparity between perception and objective reality, allowing psychophysicists to methodically manipulate stimuli and study our interpretation of the environment. One prominent effect, the Müller-Lyer illusion, is demonstrated when the perceived length of a line is contracted (or expanded) by the addition of arrowheads (or arrow-tails) to its ends. HMAX, a benchmark object recognition system, consistently produces a bias when classifying Müller-Lyer images. HMAX is a hierarchical, artificial neural network that imitates the "simple" and "complex" cell layers found in the visual ventral stream. In this study, we perform two experiments to explore the Müller-Lyer illusion in HMAX, asking: (1) How do simple vs. complex cell operations within HMAX affect illusory bias and precision? (2) How does varying the position of the figures in the input image affect classification using HMAX? In our first experiment, we assessed classification after traversing each layer of HMAX and found that in general, kernel operations performed by simple cells increase bias and uncertainty while max-pooling operations executed by complex cells decrease bias and uncertainty. In our second experiment, we increased variation in the positions of figures in the input images that reduced bias and uncertainty in HMAX. Our findings suggest that the Müller-Lyer illusion is exacerbated by the vulnerability of simple cell operations to positional fluctuations, but ameliorated by the robustness of complex cell responses to such

  19. Impact of cladribine therapy on changes in circulating dendritic cell subsets, T cells and B cells in patients with multiple sclerosis.

    Science.gov (United States)

    Mitosek-Szewczyk, Krystyna; Tabarkiewicz, Jacek; Wilczynska, Barbara; Lobejko, Katarzyna; Berbecki, Jerzy; Nastaj, Marcin; Dworzanska, Ewa; Kolodziejczyk, Beata; Stelmasiak, Zbigniew; Rolinski, Jacek

    2013-09-15

    Cladribine causes sustained reduction in peripheral T and B cell populations while sparing other immune cells. We determined two populations of dendritic cells (DCs): namely CD1c(+)/CD19(-) (myeloid DCs) and CD303(+)/CD123(+) (plasmacytoid DCs), CD19(+) B lymphocytes, CD3(+) T lymphocytes and CD4(+) or CD8(+) subpopulations in patients with multiple sclerosis after cladribine therapy. We examined 50 patients with secondary progressive multiple sclerosis (SP MS) according to McDonalds et al.'s criteria, 2001 [15]. Blood samples were collected before the initiation of cladribine therapy and after 1st, 2nd, 3th, 4th and 5th courses of treatment. DC subsets, T and B cells were analyzed by flow cytometry. During cladribine treatment the myeloid DCs CD1c(+)/CD19(-) did not change (p=0.73175), and the plasmacytoid DCs CD303(+)/CD123(+) significantly increased (p=0.00034) which resulted in significant changes in the ratio of myeloid DCs to plasmacytoid DCs (p=0.00273). During therapy, B lymphocyte CD19(+) significantly decreased (p=0.00005) and significant changes in CD4(+) cells (p=0.00191), changes in CD8(+) cells (p=0.05760) and significant changes in CD3(+) (p=0.01822) were found. We noticed significant trend to increase the CD303(+) circulating the dendritic cells. This population produces large amounts of IFN-alfa. We found significant and rapid decrease in B cells and CD4(+) Th cells. Our results suggest two possible ways of beneficial cladribine influence on immune system in MS. Induction of IFN-alfa producing cells and their predominance over BDCA-1(+) DCs, which are associated with cytotoxic response. Additionally, cladribine could influence two populations of lymphocytes: B cells and Th lymphocytes responsible for induction of immune response against myelin antigens. Copyright © 2013 Elsevier B.V. All rights reserved.

  20. Fluoxetine Decreases the Proliferation and Adipogenic Differentiation of Human Adipose-Derived Stem Cells

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    Bo Kyung Sun

    2015-07-01

    Full Text Available Fluoxetine was originally developed as an antidepressant, but it has also been used to treat obesity. Although the anti-appetite effect of fluoxetine is well-documented, its potential effects on human adipose-derived stem cells (ASCs or mature adipocytes have not been investigated. Therefore, we investigated the mechanisms underlying the inhibitory effects of fluoxetine on the proliferation of ASCs. We also investigated its inhibitory effect on adipogenic differentiation. Fluoxetine significantly decreased ASC proliferation, and signal transduction PCR array analysis showed that it increased expression of autophagy-related genes. In addition, fluoxetine up-regulated SQSTM1 and LC3B protein expression as detected by western blotting and immunofluorescence. The autophagy inhibitor, 3-methyladenine (3-MA, significantly attenuated fluoxetine-mediated effects on ASC proliferation and SQSTM1/LC3B expression. In addition, 3-MA decreased the mRNA expression of two autophagy-related genes, beclin-1 and Atg7, in ASCs. Fluoxetine also significantly inhibited lipid accumulation and down-regulated the levels of PPAR-γ and C/EBP-α in ASCs. Collectively, these results indicate that fluoxetine decreases ASC proliferation and adipogenic differentiation. This is the first in vitro evidence that fluoxetine can reduce fat accumulation by inhibiting ASC proliferation and differentiation.

  1. Fluoxetine Decreases the Proliferation and Adipogenic Differentiation of Human Adipose-Derived Stem Cells

    Science.gov (United States)

    Sun, Bo Kyung; Kim, Ji Hye; Choi, Joon-Seok; Hwang, Sung-Joo; Sung, Jong-Hyuk

    2015-01-01

    Fluoxetine was originally developed as an antidepressant, but it has also been used to treat obesity. Although the anti-appetite effect of fluoxetine is well-documented, its potential effects on human adipose-derived stem cells (ASCs) or mature adipocytes have not been investigated. Therefore, we investigated the mechanisms underlying the inhibitory effects of fluoxetine on the proliferation of ASCs. We also investigated its inhibitory effect on adipogenic differentiation. Fluoxetine significantly decreased ASC proliferation, and signal transduction PCR array analysis showed that it increased expression of autophagy-related genes. In addition, fluoxetine up-regulated SQSTM1 and LC3B protein expression as detected by western blotting and immunofluorescence. The autophagy inhibitor, 3-methyladenine (3-MA), significantly attenuated fluoxetine-mediated effects on ASC proliferation and SQSTM1/LC3B expression. In addition, 3-MA decreased the mRNA expression of two autophagy-related genes, beclin-1 and Atg7, in ASCs. Fluoxetine also significantly inhibited lipid accumulation and down-regulated the levels of PPAR-γ and C/EBP-α in ASCs. Collectively, these results indicate that fluoxetine decreases ASC proliferation and adipogenic differentiation. This is the first in vitro evidence that fluoxetine can reduce fat accumulation by inhibiting ASC proliferation and differentiation. PMID:26204837

  2. Decrease in the red cell cofactor 2,3-diphosphoglycerate increases hemoglobin oxygen affinity in the hibernating brown bear Ursus arctos.

    Science.gov (United States)

    Revsbech, Inge G; Malte, Hans; Fröbert, Ole; Evans, Alina; Blanc, Stéphane; Josefsson, Johan; Fago, Angela

    2013-01-01

    During winter hibernation, brown bears (Ursus arctos) reduce basal O(2) consumption rate to ∼25% compared with the active state, while body temperature decreases moderately (to ∼30°C), suggesting a temperature-independent component in their metabolic depression. To establish whether changes in O(2) consumption during hibernation correlate with changes in blood O(2) affinity, we took blood samples from the same six individuals of hibernating and nonhibernating free-ranging brown bears during winter and summer, respectively. A single hemoglobin (Hb) component was detected in all samples, indicating no switch in Hb synthesis. O(2) binding curves measured on red blood cell lysates at 30°C and 37°C showed a less temperature-sensitive O(2) affinity than in other vertebrates. Furthermore, hemolysates from hibernating bears consistently showed lower cooperativity and higher O(2) affinity than their summer counterparts, regardless of the temperature. We found that this increase in O(2) affinity was associated with a significant decrease in the red cell Hb-cofactor 2,3-diphosphoglycerate (DPG) during hibernation to approximately half of the summer value. Experiments performed on purified Hb, to which DPG had been added to match summer and winter levels, confirmed that the low DPG content was the cause of the left shift in the Hb-O(2) equilibrium curve during hibernation. Levels of plasma lactate indicated that glycolysis is not upregulated during hibernation and that metabolism is essentially aerobic. Calculations show that the increase in Hb-O(2) affinity and decrease in cooperativity resulting from decreased red cell DPG may be crucial in maintaining a fairly constant tissue oxygen tension during hibernation in vivo.

  3. Immunocytochemical characterization of the cell walls of bean cell suspensions during habituation and dehabituation to dichlobenil

    DEFF Research Database (Denmark)

    Garcia-Angulo, P.; Willats, W. G. T.; Encina, A. E.

    2006-01-01

    The effects of the cellulose inhibitor dichlobenil on the cell wall composition and structure during the habituation/dehabituation process of suspension-cultured bean cells were assessed. A range of techniques were used including cell wall fractionation, sugar analysis, immunofluorescence...... and fluorochrome labelling of resin-embedded sections, and immunodot assays (IDAs) of cell wall fractions. The cell walls from bean cell suspensions with initial levels of habituation to dichlobenil had decreased levels of cellulose, but this effect lessened with increasing numbers of subcultures. All cell walls...

  4. Cell killing and radiosensitization by caffeic acid phenethyl ester (CAPE) in lung cancer cells

    International Nuclear Information System (INIS)

    Chen, Miao-Fen; Chen, Wen-Cheng; Wu, Chun-Te; King, P.C.

    2004-01-01

    Caffeic acid phenethyl ester (CAPE) is a biologically active ingredient of honeybee propoplis. The cytotoxicity and radiation sensitization effects of CAPE were evaluated in human lung cancer A549 cells and normal lung fibroblast WI-38 cells. A549 cells treated with 6 μg/ml CAPE showed marked growth inhibition (60%) at 48 hr after treatments. During the same time, the number of viable cells decreased to 46% of the control value. In contrast, WI-38 cells showed 20% growth inhibition with no change in the number of viable cells under the same treatment conditions. At 72 hr after CAPE treatment (6 μg/ml), the percentage of apoptotic cells in A549 cultures increased significantly to 67% and an S/G2 arrest was also detected in the culture. Furthermore, there was a significant decrease in the level of intracellular glutathione and hydrogen peroxide contents within one hr after CAPE treatment, and the expression of cyclin B 1 was reduced 6 hr after treatment. The radiation sensitization effect of CAPE on A549 cells was determined from the clonogenic survival curves, and the results showed a small but significant difference in radiation survival between cells treated with or without CAPE. Taken together, our results suggest that the effects of CAPE on differential cytotoxicity, apoptosis, and radiosensitization are associated with glutathione depletion that occurred shortly after treatments. (author)

  5. Hakai reduces cell-substratum adhesion and increases epithelial cell invasion

    International Nuclear Information System (INIS)

    Rodríguez-Rigueiro, Teresa; Valladares-Ayerbes, Manuel; Haz-Conde, Mar; Aparicio, Luis A; Figueroa, Angélica

    2011-01-01

    expression of Paxillin was found to be regulated by a proteasome-independent mechanism, possibly due to the decreased abundance of E-cadherin. Taken together, these results suggest that Hakai may be involved in two hallmark aspects of tumour progression, the lowering cell-substratum adhesion and the enhancement of cell invasion

  6. Substantial decrease in cell wall α-1,3-glucan caused by disruption of the kexB gene encoding a subtilisin-like processing protease in Aspergillus oryzae.

    Science.gov (United States)

    Mizutani, Osamu; Shiina, Matsuko; Yoshimi, Akira; Sano, Motoaki; Watanabe, Takeshi; Yamagata, Youhei; Nakajima, Tasuku; Gomi, Katsuya; Abe, Keietsu

    2016-09-01

    Disruption of the kexB encoding a subtilisin-like processing protease in Aspergillus oryzae (ΔkexB) leads to substantial morphological defects when the cells are grown on Czapek-Dox agar plates. We previously found that the disruption of kexB causes a constitutive activation of the cell wall integrity pathway. To understand how the disruption of the kexB affects cell wall organization and components, we analyzed the cell wall of ΔkexB grown on the plates. The results revealed that both total N-acetylglucosamine content, which constitutes chitin, and chitin synthase activities were increased. Whereas total glucose content, which constitutes β-1,3-glucan and α-1,3-glucan, was decreased; this decrease was attributed to a remarkable decrease in α-1,3-glucan. Additionally, the β-1,3-glucan in the alkali-insoluble fraction of the ΔkexB showed a high degree of polymerization. These results suggested that the loss of α-1,3-glucan in the ΔkexB was compensated by increases in the chitin content and the average degree of β-1,3-glucan polymerization.

  7. Granulocyte colony-stimulating factor decreases the Th1/Th2 ratio in peripheral blood mononuclear cells from patients with chronic immune thrombocytopenic purpura in vitro.

    Science.gov (United States)

    Ge, Fei; Zhang, Zhuo; Hou, Jinxiao; Cao, Fenglin; Zhang, Yingmei; Wang, Ping; Wei, Hong; Zhou, Jin

    2016-12-01

    Chronic immune thrombocytopenia purpura (ITP) is an autoimmune disease that exhibits an abnormally high Th1/Th2 ratio. Granulocyte colony-stimulating factor (G-CSF) has been shown to decrease the Th1/Th2 ratio in healthy donors. In this study, we investigated the effects of G-CSF treatment on the Th1/Th2 cells and the underlying mechanisms in patients with ITP in vitro. Peripheral blood mononuclear cells (PBMCs) isolated from patients with ITP and healthy controls were treated with G-CSF. Expression levels of interferon (IFN)-γ, interleukin (IL)-2, IL-4, and IL-13 in supernatants were measured by enzyme-linked immunosorbent assays. The expression of IFN-γ, IL-4, and G-CSF receptor (G-CSFR) on Th1 and Th2 cells were examined by flow cytometry and confocal microscopy. The mRNA expression of IFN-γ, IL-2, IL-4, IL-13, and T-box expressed in T cells (T-bet) and GATA-binding protein 3 (GATA-3) in PBMCs was evaluated by reverse transcription polymerase chain reaction. The results showed that G-CSF could significantly reduce the Th1/Th2 ratio in PBMCs from patients with ITP in vitro. As the concentration of G-CSF increased, Th1/Th2 ([IFN-γ+IL-2]/[IL-4+IL-13]) cytokine ratios and T-bet/GATA-3 mRNA ratios decreased in a concentration-dependent manner. Th1 cells and Th2 cells both expressed G-CSFR. These results suggest that G-CSF could decrease the Th1/Th2 ratio in the context of ITP, and elucidate the direct and indirect immunomodulatory mechanisms underlying G-CSF functions in Th1/Th2 cells, thus supporting the therapeutic potential of G-CSF in the treatment of patients with ITP. Copyright © 2016 Elsevier Ltd. All rights reserved.

  8. Inhibitory Activity of (+-Usnic Acid against Non-Small Cell Lung Cancer Cell Motility.

    Directory of Open Access Journals (Sweden)

    Yi Yang

    Full Text Available Lichens are symbiotic organisms that produce various unique chemicals that can be used for pharmaceutical purposes. With the aim of screening new anti-cancer agents that inhibit cancer cell motility, we tested the inhibitory activity of seven lichen species collected from the Romanian Carpathian Mountains against migration and invasion of human lung cancer cells and further investigated the molecular mechanisms underlying their anti-metastatic activity. Among them, Alectoria samentosa, Flavocetraria nivalis, Alectoria ochroleuca, and Usnea florida showed significant inhibitory activity against motility of human lung cancer cells. HPLC results showed that usnic acid is the main compound in these lichens, and (+-usnic acid showed similar inhibitory activity that crude extract have. Mechanistically, β-catenin-mediated TOPFLASH activity and KITENIN-mediated AP-1 activity were decreased by (+-usnic acid treatment in a dose-dependent manner. The quantitative real-time PCR data showed that (+-usnic acid decreased the mRNA level of CD44, Cyclin D1 and c-myc, which are the downstream target genes of both β-catenin/LEF and c-jun/AP-1. Also, Rac1 and RhoA activities were decreased by treatment with (+-usnic acid. Interestingly, higher inhibitory activity for cell invasion was observed when cells were treated with (+-usnic acid and cetuximab. These results implied that (+-usnic acid might have potential activity in inhibition of cancer cell metastasis, and (+-usnic acid could be used for anti-cancer therapy with a distinct mechanisms of action.

  9. Inhibitory Activity of (+)-Usnic Acid against Non-Small Cell Lung Cancer Cell Motility

    Science.gov (United States)

    Yang, Yi; Nguyen, Thanh Thi; Jeong, Min-Hye; Crişan, Florin; Yu, Young Hyun; Ha, Hyung-Ho; Choi, Kyung Hee; Jeong, Hye Gwang; Jeong, Tae Cheon; Lee, Kwang Youl; Kim, Kyung Keun; Hur, Jae-Seoun; Kim, Hangun

    2016-01-01

    Lichens are symbiotic organisms that produce various unique chemicals that can be used for pharmaceutical purposes. With the aim of screening new anti-cancer agents that inhibit cancer cell motility, we tested the inhibitory activity of seven lichen species collected from the Romanian Carpathian Mountains against migration and invasion of human lung cancer cells and further investigated the molecular mechanisms underlying their anti-metastatic activity. Among them, Alectoria samentosa, Flavocetraria nivalis, Alectoria ochroleuca, and Usnea florida showed significant inhibitory activity against motility of human lung cancer cells. HPLC results showed that usnic acid is the main compound in these lichens, and (+)-usnic acid showed similar inhibitory activity that crude extract have. Mechanistically, β-catenin-mediated TOPFLASH activity and KITENIN-mediated AP-1 activity were decreased by (+)-usnic acid treatment in a dose-dependent manner. The quantitative real-time PCR data showed that (+)-usnic acid decreased the mRNA level of CD44, Cyclin D1 and c-myc, which are the downstream target genes of both β-catenin/LEF and c-jun/AP-1. Also, Rac1 and RhoA activities were decreased by treatment with (+)-usnic acid. Interestingly, higher inhibitory activity for cell invasion was observed when cells were treated with (+)-usnic acid and cetuximab. These results implied that (+)-usnic acid might have potential activity in inhibition of cancer cell metastasis, and (+)-usnic acid could be used for anti-cancer therapy with a distinct mechanisms of action. PMID:26751081

  10. Single-Cell RNA-Sequencing Reveals a Continuous Spectrum of Differentiation in Hematopoietic Cells

    Directory of Open Access Journals (Sweden)

    Iain C. Macaulay

    2016-02-01

    Full Text Available The transcriptional programs that govern hematopoiesis have been investigated primarily by population-level analysis of hematopoietic stem and progenitor cells, which cannot reveal the continuous nature of the differentiation process. Here we applied single-cell RNA-sequencing to a population of hematopoietic cells in zebrafish as they undergo thrombocyte lineage commitment. By reconstructing their developmental chronology computationally, we were able to place each cell along a continuum from stem cell to mature cell, refining the traditional lineage tree. The progression of cells along this continuum is characterized by a highly coordinated transcriptional program, displaying simultaneous suppression of genes involved in cell proliferation and ribosomal biogenesis as the expression of lineage specific genes increases. Within this program, there is substantial heterogeneity in the expression of the key lineage regulators. Overall, the total number of genes expressed, as well as the total mRNA content of the cell, decreases as the cells undergo lineage commitment.

  11. CDK2 differentially controls normal cell senescence and cancer cell proliferation upon exposure to reactive oxygen species

    International Nuclear Information System (INIS)

    Hwang, Chae Young; Lee, Seung-Min; Park, Sung Sup; Kwon, Ki-Sun

    2012-01-01

    Highlights: ► H 2 O 2 differently adjusted senescence and proliferation in normal and cancer cells. ► H 2 O 2 exposure transiently decreased PCNA levels in normal cells. ► H 2 O 2 exposure transiently increased CDK2 activity in cancer cells. ► p21 Cip1 is likely dispensable when H 2 O 2 induces senescence in normal cells. ► Suggestively, CDK2 and PCNA play critical roles in H 2 O 2 -induced cell fate decision. -- Abstract: Reactive oxygen species modulate cell fate in a context-dependent manner. Sublethal doses of H 2 O 2 decreased the level of proliferating cell nuclear antigen (PCNA) in normal cells (including primary human dermal fibroblasts and IMR-90 cells) without affecting cyclin-dependent kinase 2 (CDK2) activity, leading to cell cycle arrest and subsequent senescence. In contrast, exposure of cancer cells (such as HeLa and MCF7 cells) to H 2 O 2 increased CDK2 activity with no accompanying change in the PCNA level, leading to cell proliferation. A CDK2 inhibitor, CVT-313, prevented H 2 O 2 -induced cancer cell proliferation. These results support the notion that the cyclin/CDK2/p21 Cip1 /PCNA complex plays an important role as a regulator of cell fate decisions.

  12. Decreased levels of irisin, a skeletal muscle cell-derived myokine, are related to emphysema associated with chronic obstructive pulmonary disease.

    Science.gov (United States)

    Sugiyama, Yukari; Asai, Kazuhisa; Yamada, Kazuhiro; Kureya, Yuko; Ijiri, Naoki; Watanabe, Tetsuya; Kanazawa, Hiroshi; Hirata, Kazuto

    2017-01-01

    Cigarette smoking-induced oxidant-antioxidant imbalance is a factor that contributes to the pathogenesis of COPD through epithelial cell apoptosis. Irisin is a skeletal muscle cell-derived myokine associated with physical activity. Irisin is also known to decrease oxidant-induced apoptosis in patients with diabetes mellitus. However, the correlation between irisin and emphysema in COPD and its role in epithelial cell apoptosis remains unknown. Forty patients with COPD were enrolled in this study. Pulmonary function tests and measurements of the percentage of low-attenuation area on high-resolution computed tomography images were performed, and the results were evaluated for correlation with serum irisin levels. The effect of irisin on cigarette-smoke extract-induced A549 cell apoptosis and the expression of Nrf2, a transcription factor for antioxidants, was also examined in vitro. Serum irisin levels were significantly correlated with lung diffusing capacity for carbon monoxide divided by alveolar volume ( r =0.56, P emphysema in patients with COPD and involved in epithelial apoptosis, resulting in emphysema. Irisin could be a novel treatment for emphysema in patients with COPD.

  13. Fisetin and luteolin protect human retinal pigment epithelial cells from oxidative stress-induced cell death and regulate inflammation

    Science.gov (United States)

    Hytti, Maria; Piippo, Niina; Korhonen, Eveliina; Honkakoski, Paavo; Kaarniranta, Kai; Kauppinen, Anu

    2015-01-01

    Degeneration of retinal pigment epithelial (RPE) cells is a clinical hallmark of age-related macular degeneration (AMD), the leading cause of blindness among aged people in the Western world. Both inflammation and oxidative stress are known to play vital roles in the development of this disease. Here, we assess the ability of fisetin and luteolin, to protect ARPE-19 cells from oxidative stress-induced cell death and to decrease intracellular inflammation. We also compare the growth and reactivity of human ARPE-19 cells in serum-free and serum-containing conditions. The absence of serum in the culture medium did not prevent ARPE-19 cells from reaching full confluency but caused an increased sensitivity to oxidative stress-induced cell death. Both fisetin and luteolin protected ARPE-19 cells from oxidative stress-induced cell death. They also significantly decreased the release of pro-inflammatory cytokines into the culture medium. The decrease in inflammation was associated with reduced activation of MAPKs and CREB, but was not linked to NF- κB or SIRT1. The ability of fisetin and luteolin to protect and repair stressed RPE cells even after the oxidative insult make them attractive in the search for treatments for AMD. PMID:26619957

  14. Osimertinib induces autophagy and apoptosis via reactive oxygen species generation in non-small cell lung cancer cells

    International Nuclear Information System (INIS)

    Tang, Zheng-Hai; Cao, Wen-Xiang; Su, Min-Xia; Chen, Xiuping; Lu, Jin-Jian

    2017-01-01

    Osimertinib (OSI), also known as AZD9291, is a third-generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor that has been approved for the treatment of non-small cell lung cancer (NSCLC) patients harboring EGFR T790M mutation. Herein, we indicated for the first time that OSI increased the accumulations of cytoplasmic vacuoles, the expression of phosphatidylethanolamine-modified microtubule-associated protein light-chain 3 (LC3-II), and the formation of GFP-LC3 puncta in various cancer cells. The OSI-induced expression of LC3-II was further increased when combined treatment with chloroquine (CQ), an autophagy inhibitor, and the mRFP-EGFP-LC3 plasmid-transfected cells exposed to OSI led to the production of more red-fluorescent puncta than green-fluorescent puncta, indicating OSI induced autophagic flux in the NSCLC cells. Knockdown of EGFR showed no effect on the OSI-induced expression of LC3-II in NCI-H1975 cells. In addition, OSI increased reactive oxygen species (ROS) generation and scavenge of ROS via pretreatment with N-acetyl-L-cysteine (NAC), catalase (CAT), or vitamin E (Vita E) significantly inhibited OSI-induced the accumulations of cytoplasmic vacuoles, the expression of LC3-II, as well as the formation of GFP-LC3 puncta. Combinative treatment with CQ could not remarkably change the OSI-induced cell viability decrease, whereas the OSI-induced cell viability decrease and apoptosis could be reversed through pretreatment with NAC, CAT, and Vita E, respectively. Taken together, this is the first report that OSI induces an accompanied autophagy and the generation of ROS is critical for the OSI-induced autophagy, cell viability decrease, and apoptosis in NSCLC cells. - Highlights: • Osimertinib induced the expressions of cytoplasmic vacuoles and autophagic markers in different cancer cells. • Osimertinib induced autophagic flux in NSCLC NCI-H1975 and HCC827 cell lines. • ROS generation contributed to osimertinib-induced cytoplasmic

  15. Osimertinib induces autophagy and apoptosis via reactive oxygen species generation in non-small cell lung cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Tang, Zheng-Hai; Cao, Wen-Xiang; Su, Min-Xia; Chen, Xiuping; Lu, Jin-Jian, E-mail: jinjianlu@umac.mo

    2017-04-15

    Osimertinib (OSI), also known as AZD9291, is a third-generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor that has been approved for the treatment of non-small cell lung cancer (NSCLC) patients harboring EGFR T790M mutation. Herein, we indicated for the first time that OSI increased the accumulations of cytoplasmic vacuoles, the expression of phosphatidylethanolamine-modified microtubule-associated protein light-chain 3 (LC3-II), and the formation of GFP-LC3 puncta in various cancer cells. The OSI-induced expression of LC3-II was further increased when combined treatment with chloroquine (CQ), an autophagy inhibitor, and the mRFP-EGFP-LC3 plasmid-transfected cells exposed to OSI led to the production of more red-fluorescent puncta than green-fluorescent puncta, indicating OSI induced autophagic flux in the NSCLC cells. Knockdown of EGFR showed no effect on the OSI-induced expression of LC3-II in NCI-H1975 cells. In addition, OSI increased reactive oxygen species (ROS) generation and scavenge of ROS via pretreatment with N-acetyl-L-cysteine (NAC), catalase (CAT), or vitamin E (Vita E) significantly inhibited OSI-induced the accumulations of cytoplasmic vacuoles, the expression of LC3-II, as well as the formation of GFP-LC3 puncta. Combinative treatment with CQ could not remarkably change the OSI-induced cell viability decrease, whereas the OSI-induced cell viability decrease and apoptosis could be reversed through pretreatment with NAC, CAT, and Vita E, respectively. Taken together, this is the first report that OSI induces an accompanied autophagy and the generation of ROS is critical for the OSI-induced autophagy, cell viability decrease, and apoptosis in NSCLC cells. - Highlights: • Osimertinib induced the expressions of cytoplasmic vacuoles and autophagic markers in different cancer cells. • Osimertinib induced autophagic flux in NSCLC NCI-H1975 and HCC827 cell lines. • ROS generation contributed to osimertinib-induced cytoplasmic

  16. Keratin23 (KRT23) knockdown decreases proliferation and affects the DNA damage response of colon cancer cells

    DEFF Research Database (Denmark)

    Birkenkamp-Demtröder, Karin; Hahn, Stephan; Mansilla, Francisco

    2013-01-01

    correlated with absent expression, while increased KRT23 expression in tumor samples correlated with promoter hypomethylation, as confirmed by bisulfite sequencing. Demethylation induced KRT23 expression in vitro. Expression profiling of shRNA mediated stable KRT23 knockdown in colon cancer cell lines showed...... response, mainly molecules of the double strand break repair homologous recombination pathway. KRT23 knockdown decreased the transcript and protein expression of key molecules as e.g. MRE11A, E2F1, RAD51 and BRCA1. Knockdown of KRT23 rendered colon cancer cells more sensitive to irradiation and reduced......Keratin 23 (KRT23) is strongly expressed in colon adenocarcinomas but absent in normal colon mucosa. Array based methylation profiling of 40 colon samples showed that the promoter of KRT23 was methylated in normal colon mucosa, while hypomethylated in most adenocarcinomas. Promoter methylation...

  17. CCL22-specific T Cells

    DEFF Research Database (Denmark)

    Martinenaite, Evelina; Munir Ahmad, Shamaila; Hansen, Morten

    2016-01-01

    Tumor cells and tumor-infiltrating macrophages produce the chemokine CCL22, which attracts regulatory T cells (Tregs) into the tumor microenvironment, decreasing anticancer immunity. Here, we investigated the possibility of targeting CCL22-expressing cells by activating specific T cells. We...... analyzed the CCL22 protein signal sequence, identifying a human leukocyte antigen A2- (HLA-A2-) restricted peptide epitope, which we then used to stimulate peripheral blood mononuclear cells (PMBCs) to expand populations of CCL22-specific T cells in vitro. T cells recognizing an epitope derived from...... the signal-peptide of CCL22 will recognize CCL22-expressing cells even though CCL22 is secreted out of the cell. CCL22-specific T cells recognized and killed CCL22-expressing cancer cells. Furthermore, CCL22-specific T cells lysed acute monocytic leukemia cells in a CCL22 expression-dependent manner. Using...

  18. Diethylstilbestrol alters positive and negative selection of T cells in the thymus and modulates T-cell repertoire in the periphery

    International Nuclear Information System (INIS)

    Brown, Nicole; Nagarkatti, Mitzi; Nagarkatti, Prakash S.

    2006-01-01

    Prenatal exposure to diethylstilbestrol (DES) is known to cause altered immune functions and increased susceptibility to autoimmune disease in humans. In the current study, we investigated the effects of DES on T-cell differentiation in the thymus using the HY-TCR transgenic (Tg) mouse model in which the female mice exhibit positive selection of T cells bearing the Tg TCR, while the male mice show negative selection of such T cells. In female HY-TCR-Tg mice, exposure to DES showed more pronounced decrease in thymic cellularity when compared to male mice. Additionally, female mice also showed a significant decrease in the proportion of double-positive (DP) T cells in the thymus and HY-TCR-specific CD8 + T cells in the periphery. Male mice exhibiting negative selection also showed decreased thymic cellularity following DES exposure. Moreover, the male mice showed increased proportion of double-negative (DN) T cells in the thymus and decreased proportion of CD8 + T cells. The density of expression of HY-TCR on CD8 + cells was increased following DES exposure in both females and males. Finally, the proliferative response of thymocytes to mitogens and peripheral lymph node T cells to male H-Y antigen was significantly altered in female and male mice following DES treatment. Taken together, these data suggest that DES alters T-cell differentiation in the thymus by interfering with positive and negative selection processes, which in turn modulates the T-cell repertoire in the periphery

  19. Diethylstilbestrol alters positive and negative selection of T cells in the thymus and modulates T-cell repertoire in the periphery

    Energy Technology Data Exchange (ETDEWEB)

    Brown, Nicole [Department of Microbiology and Immunology, Medical College of Virginia Campus, Virginia Commonwealth University, Richmond, VA 23298 (United States); Nagarkatti, Mitzi [Department of Microbiology and Immunology, Medical College of Virginia Campus, Virginia Commonwealth University, Richmond, VA 23298 (United States); Nagarkatti, Prakash S [Department of Pharmacology and Toxicology, PO Box 980613, Virginia Commonwealth University Medical Center, Richmond, VA 23298-0613 (United States)

    2006-04-15

    Prenatal exposure to diethylstilbestrol (DES) is known to cause altered immune functions and increased susceptibility to autoimmune disease in humans. In the current study, we investigated the effects of DES on T-cell differentiation in the thymus using the HY-TCR transgenic (Tg) mouse model in which the female mice exhibit positive selection of T cells bearing the Tg TCR, while the male mice show negative selection of such T cells. In female HY-TCR-Tg mice, exposure to DES showed more pronounced decrease in thymic cellularity when compared to male mice. Additionally, female mice also showed a significant decrease in the proportion of double-positive (DP) T cells in the thymus and HY-TCR-specific CD8{sup +} T cells in the periphery. Male mice exhibiting negative selection also showed decreased thymic cellularity following DES exposure. Moreover, the male mice showed increased proportion of double-negative (DN) T cells in the thymus and decreased proportion of CD8{sup +} T cells. The density of expression of HY-TCR on CD8{sup +} cells was increased following DES exposure in both females and males. Finally, the proliferative response of thymocytes to mitogens and peripheral lymph node T cells to male H-Y antigen was significantly altered in female and male mice following DES treatment. Taken together, these data suggest that DES alters T-cell differentiation in the thymus by interfering with positive and negative selection processes, which in turn modulates the T-cell repertoire in the periphery.

  20. Mesenchymal Stem Cells from Patients with Rheumatoid Arthritis Display Impaired Function in Inhibiting Th17 Cells

    Directory of Open Access Journals (Sweden)

    Yue Sun

    2015-01-01

    Full Text Available Mesenchymal stem cells (MSCs possess multipotent and immunomodulatory properties and are suggested to be involved in the pathogenesis of immune-related diseases. This study explored the function of bone marrow MSCs from rheumatoid arthritis (RA patients, focusing on immunomodulatory effects. RA MSCs showed decreased proliferative activity and aberrant migration capacity. No significant differences were observed in cytokine profiles between RA and control MSCs. The effects of RA MSCs on proliferation of peripheral blood mononuclear cells (PBMCs and distribution of specific CD4+ T cell subtypes (Th17, Treg, and Tfh cells were investigated. RA MSCs appeared to be indistinguishable from controls in suppressing PBMC proliferation, decreasing the proportion of Tfh cells, and inducing the polarization of Treg cells. However, the capacity to inhibit Th17 cell polarization was impaired in RA MSCs, which was related to the low expression of CCL2 in RA MSCs after coculture with CD4+ T cells. These findings indicated that RA MSCs display defects in several important biological activities, especially the capacity to inhibit Th17 cell polarization. These functionally impaired MSCs may contribute to the development of RA disease.

  1. Collective cell migration without proliferation: density determines cell velocity and wave velocity

    Science.gov (United States)

    Tlili, Sham; Gauquelin, Estelle; Li, Brigitte; Cardoso, Olivier; Ladoux, Benoît; Delanoë-Ayari, Hélène; Graner, François

    2018-05-01

    Collective cell migration contributes to embryogenesis, wound healing and tumour metastasis. Cell monolayer migration experiments help in understanding what determines the movement of cells far from the leading edge. Inhibiting cell proliferation limits cell density increase and prevents jamming; we observe long-duration migration and quantify space-time characteristics of the velocity profile over large length scales and time scales. Velocity waves propagate backwards and their frequency depends only on cell density at the moving front. Both cell average velocity and wave velocity increase linearly with the cell effective radius regardless of the distance to the front. Inhibiting lamellipodia decreases cell velocity while waves either disappear or have a lower frequency. Our model combines conservation laws, monolayer mechanical properties and a phenomenological coupling between strain and polarity: advancing cells pull on their followers, which then become polarized. With reasonable values of parameters, this model agrees with several of our experimental observations. Together, our experiments and model disantangle the respective contributions of active velocity and of proliferation in monolayer migration, explain how cells maintain their polarity far from the moving front, and highlight the importance of strain-polarity coupling and density in long-range information propagation.

  2. The cell death factor, cell wall elicitor of rice blast fungus (Magnaporthe grisea) causes metabolic alterations including GABA shunt in rice cultured cells

    OpenAIRE

    Takahashi, Hideyuki; Matsumura, Hideo; Kawai-Yamada, Maki; Uchimiya, Hirofumi

    2008-01-01

    An elicitor derived from the cell wall of rice blast fungus (Magnaporthe grisea) causes cell death in suspension cultured cells of rice (Oryza sativa L.). To elucidate the role of M. grisea elicitor on metabolic pathway of rice cells, we performed metabolite profiling using capillary electrophoresis-mass spectrometry (CE/MS). Treatment with M. grisea elicitor increased the amounts of antioxidants and free amino acids and decreased the amount of metabolites in the tricarboxylic acid (TCA) cycl...

  3. Arsenic trioxide inhibits cell proliferation and human papillomavirus oncogene expression in cervical cancer cells

    International Nuclear Information System (INIS)

    Wang, Hongtao; Gao, Peng; Zheng, Jie

    2014-01-01

    Highlights: • As 2 O 3 inhibits growth of cervical cancer cells and expression of HPV oncogenes in these cells. • HPV-negative cervical cancer cells are more sensitive to As 2 O 3 than HPV-positive cervical cancer cells. • HPV-18 positive cervical cancer cells are more sensitive to As 2 O 3 than HPV-16 positive cancer cells. • Down-regulation of HPV oncogenes by As 2 O 3 is partially due to the diminished AP-1 binding. - Abstract: Arsenic trioxide (As 2 O 3 ) has shown therapeutic effects in some leukemias and solid cancers. However, the molecular mechanisms of its anticancer efficacy have not been clearly elucidated, particularly in solid cancers. Our previous data showed that As 2 O 3 induced apoptosis of human papillomavirus (HPV) 16 DNA-immortalized human cervical epithelial cells and cervical cancer cells and inhibited the expression of HPV oncogenes in these cells. In the present study, we systemically examined the effects of As 2 O 3 on five human cervical cancer cell lines and explored the possible molecular mechanisms. MTT assay showed that HPV-negative C33A cells were more sensitive to growth inhibition induced by As 2 O 3 than HPV-positive cervical cancer cells, and HPV 18-positive HeLa and C4-I cells were more sensitive to As 2 O 3 than HPV 16-positive CaSki and SiHa cells. After As 2 O 3 treatment, both mRNA and protein levels of HPV E6 and E7 obviously decreased in all HPV positive cell lines. In contrast, p53 and Rb protein levels increased in all tested cell lines. Transcription factor AP-1 protein expression decreased significantly in HeLa, CaSki and C33A cells with ELISA method. These results suggest that As 2 O 3 is a potential anticancer drug for cervical cancer

  4. Arsenic trioxide inhibits cell proliferation and human papillomavirus oncogene expression in cervical cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Hongtao [Department of Pathology, School of Medicine, Southeast University, Nanjing 210009 (China); Gao, Peng [Department of Internal Medicine, University of Iowa, Iowa City, IA 52242 (United States); Zheng, Jie, E-mail: jiezheng54@126.com [Department of Pathology, School of Medicine, Southeast University, Nanjing 210009 (China)

    2014-09-05

    Highlights: • As{sub 2}O{sub 3} inhibits growth of cervical cancer cells and expression of HPV oncogenes in these cells. • HPV-negative cervical cancer cells are more sensitive to As{sub 2}O{sub 3} than HPV-positive cervical cancer cells. • HPV-18 positive cervical cancer cells are more sensitive to As{sub 2}O{sub 3} than HPV-16 positive cancer cells. • Down-regulation of HPV oncogenes by As{sub 2}O{sub 3} is partially due to the diminished AP-1 binding. - Abstract: Arsenic trioxide (As{sub 2}O{sub 3}) has shown therapeutic effects in some leukemias and solid cancers. However, the molecular mechanisms of its anticancer efficacy have not been clearly elucidated, particularly in solid cancers. Our previous data showed that As{sub 2}O{sub 3} induced apoptosis of human papillomavirus (HPV) 16 DNA-immortalized human cervical epithelial cells and cervical cancer cells and inhibited the expression of HPV oncogenes in these cells. In the present study, we systemically examined the effects of As{sub 2}O{sub 3} on five human cervical cancer cell lines and explored the possible molecular mechanisms. MTT assay showed that HPV-negative C33A cells were more sensitive to growth inhibition induced by As{sub 2}O{sub 3} than HPV-positive cervical cancer cells, and HPV 18-positive HeLa and C4-I cells were more sensitive to As{sub 2}O{sub 3} than HPV 16-positive CaSki and SiHa cells. After As{sub 2}O{sub 3} treatment, both mRNA and protein levels of HPV E6 and E7 obviously decreased in all HPV positive cell lines. In contrast, p53 and Rb protein levels increased in all tested cell lines. Transcription factor AP-1 protein expression decreased significantly in HeLa, CaSki and C33A cells with ELISA method. These results suggest that As{sub 2}O{sub 3} is a potential anticancer drug for cervical cancer.

  5. Ubiquitin-specific protease 2 decreases p53-dependent apoptosis in cutaneous T-cell lymphoma

    DEFF Research Database (Denmark)

    Wei, Tianling; Biskup, Edyta; Gjerdrum, Lise Mette Rahbek

    2016-01-01

    expression is 50% lower in CTCL cell lines (MyLa2000, SeAx and Hut-78) than in normal T-cells. USP2 is expressed in neoplastic cells in early, plaque-stage mycosis fungoides (MF) and is downregulated in advanced tumor stages. Upon treatment with psoralen with UVA (PUVA) or a p53 activator, nutlin3a, USP2...

  6. Biochemical and microscopic evidence for the internalization and degradation of heparin-containing mast cell granules by bovine endothelial cells

    International Nuclear Information System (INIS)

    Atkins, F.M.; Friedman, M.M.; Metcalfe, D.D.

    1985-01-01

    Incubation of [ 35 S]heparin-containing mast cell granules with cultured bovine endothelial cells was followed by the appearance of 35 S-granule-associated radioactivity within the endothelial cells and a decrease in radioactivity in the extracellular fluid. These changes occurred during the first 24 hours of incubation and suggested ingestion of the mast cell granules by the endothelial cells. Periodic electron microscopic examination of the monolayers confirmed this hypothesis by demonstrating apposition of the granules to the plasmalemma of endothelial cells, which was followed by the engulfment of the granules by cytoplasmic projections. Under light microscopic examination, mast cell granules within endothelial cells then appeared to undergo degradation. The degradation of [ 35 S]heparin in mast cell granules was demonstrated by a decrease in the amount of intracellular [ 35 S]heparin proteoglycan after 24 hours and the appearance of free [ 35 S]sulfate in the extracellular compartment. Intact endothelial cells were more efficient at degrading [ 35 S]heparin than were cell lysates or cell supernatants. These data provide evidence of the ability of endothelial cells to ingest mast cell granules and degrade native heparin that is presented as a part of the mast cell granule

  7. Triiodothyronine regulates cell growth and survival in renal cell cancer.

    Science.gov (United States)

    Czarnecka, Anna M; Matak, Damian; Szymanski, Lukasz; Czarnecka, Karolina H; Lewicki, Slawomir; Zdanowski, Robert; Brzezianska-Lasota, Ewa; Szczylik, Cezary

    2016-10-01

    Triiodothyronine plays an important role in the regulation of kidney cell growth, differentiation and metabolism. Patients with renal cell cancer who develop hypothyreosis during tyrosine kinase inhibitor (TKI) treatment have statistically longer survival. In this study, we developed cell based model of triiodothyronine (T3) analysis in RCC and we show the different effects of T3 on renal cell cancer (RCC) cell growth response and expression of the thyroid hormone receptor in human renal cell cancer cell lines from primary and metastatic tumors along with human kidney cancer stem cells. Wild-type thyroid hormone receptor is ubiquitously expressed in human renal cancer cell lines, but normalized against healthy renal proximal tube cell expression its level is upregulated in Caki-2, RCC6, SKRC-42, SKRC-45 cell lines. On the contrary the mRNA level in the 769-P, ACHN, HKCSC, and HEK293 cells is significantly decreased. The TRβ protein was abundant in the cytoplasm of the 786-O, Caki-2, RCC6, and SKRC-45 cells and in the nucleus of SKRC-42, ACHN, 769-P and cancer stem cells. T3 has promoting effect on the cell proliferation of HKCSC, Caki-2, ASE, ACHN, SK-RC-42, SMKT-R2, Caki-1, 786-0, and SK-RC-45 cells. Tyrosine kinase inhibitor, sunitinib, directly inhibits proliferation of RCC cells, while thyroid hormone receptor antagonist 1-850 (CAS 251310‑57-3) has less significant inhibitory impact. T3 stimulation does not abrogate inhibitory effect of sunitinib. Renal cancer tumor cells hypostimulated with T3 may be more responsive to tyrosine kinase inhibition. Moreover, some tumors may be considered as T3-independent and present aggressive phenotype with thyroid hormone receptor activated independently from the ligand. On the contrary proliferation induced by deregulated VHL and or c-Met pathways may transgress normal T3 mediated regulation of the cell cycle.

  8. miRNA-720 controls stem cell phenotype, proliferation and differentiation of human dental pulp cells.

    Directory of Open Access Journals (Sweden)

    Emilio Satoshi Hara

    Full Text Available Dental pulp cells (DPCs are known to be enriched in stem/progenitor cells but not well characterized yet. Small non-coding microRNAs (miRNAs have been identified to control protein translation, mRNA stability and transcription, and have been reported to play important roles in stem cell biology, related to cell reprogramming, maintenance of stemness and regulation of cell differentiation. In order to characterize dental pulp stem/progenitor cells and its mechanism of differentiation, we herein sorted stem-cell-enriched side population (SP cells from human DPCs and periodontal ligament cells (PDLCs, and performed a locked nucleic acid (LNA-based miRNA array. As a result, miR-720 was highly expressed in the differentiated main population (MP cells compared to that in SP cells. In silico analysis and a reporter assay showed that miR-720 targets the stem cell marker NANOG, indicating that miR-720 could promote differentiation of dental pulp stem/progenitor cells by repressing NANOG. Indeed, gain-and loss-of-function analyses showed that miR-720 controls NANOG transcript and protein levels. Moreover, transfection of miR-720 significantly decreased the number of cells positive for the early stem cell marker SSEA-4. Concomitantly, mRNA levels of DNA methyltransferases (DNMTs, which are known to play crucial factors during stem cell differentiation, were also increased by miR-720 through unknown mechanism. Finally, miR-720 decreased DPC proliferation as determined by immunocytochemical analysis against ki-67, and promoted odontogenic differentiation as demonstrated by alizarin red staining, as well as alkaline phosphatase and osteopontin mRNA levels. Our findings identify miR-720 as a novel miRNA regulating the differentiation of DPCs.

  9. UV-induced changes in cell cycle and gene expression within rabbit lens epithelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Sidjanin, D. [Northern Illinois Univ., De Kalb, IL (United States). Dept. of Biological Sciences; Grdina, D. [Argonne National Lab., IL (United States); Woloschak, G.E. [Northern Illinois Univ., De Kalb, IL (United States). Dept. of Biological Sciences

    1994-11-01

    Damage to lens epithelial cells is a probable initiation process in cataract formation induced by ultraviolet radiation. These experiments investigated the ability of 254 nm radiation on cell cycle progression and gene expression in rabbit lens epithelial cell line N/N1003A. No changes in expression of c-fos, c-jun, alpha- tubulin, or vimentin was observed following UV exposure. Using flow cytometry, an accumulation of cells in G1/S phase of the cell cycle 1 hr following exposure. The observed changes in gene expression, especially the decreased histone transcripts reported here may play a role in UV induced inhibition of cell cycle progression.

  10. Uterine-derived progenitor cells are immunoprivileged and effectively improve cardiac regeneration when used for cell therapy.

    Science.gov (United States)

    Ludke, Ana; Wu, Jun; Nazari, Mansoreh; Hatta, Kota; Shao, Zhengbo; Li, Shu-Hong; Song, Huifang; Ni, Nathan C; Weisel, Richard D; Li, Ren-Ke

    2015-07-01

    Cell therapy to prevent cardiac dysfunction after myocardial infarction (MI) is less effective in aged patients because aged cells have decreased regenerative capacity. Allogeneic transplanted stem cells (SCs) from young donors are usually rejected. Maintaining transplanted SC immunoprivilege may dramatically improve regenerative outcomes. The uterus has distinct immune characteristics, and we showed that reparative uterine SCs home to the myocardium post-MI. Here, we identify immunoprivileged uterine SCs and assess their effects on cardiac regeneration after allogeneic transplantation. We found more than 20% of cells in the mouse uterus have undetectable MHC I expression by flow cytometry. Uterine MHC I((neg)) and MHC I((pos)) cells were separated by magnetic cell sorting. The MHC I((neg)) population expressed the SC markers CD34, Sca-1 and CD90, but did not express MHC II or c-kit. In vitro, MHC I((neg)) and ((pos)) SCs show colony formation and endothelial differentiation capacity. In mixed leukocyte co-culture, MHC I((neg)) cells showed reduced cell death and leukocyte proliferation compared to MHC I((pos)) cells. MHC I((neg)) and ((pos)) cells had significantly greater angiogenic capacity than mesenchymal stem cells. The benefits of intramyocardial injection of allogeneic MHC I((neg)) cells after MI were comparable to syngeneic bone marrow cell transplantation, with engraftment in cardiac tissue and limited recruitment of CD4 and CD8 cells up to 21 days post-MI. MHC I((neg)) cells preserved cardiac function, decreased infarct size and improved regeneration post-MI. This new source of immunoprivileged cells can induce neovascularization and could be used as allogeneic cell therapy for regenerative medicine. Copyright © 2015 Elsevier Ltd. All rights reserved.

  11. Effect on pancreatic beta cells and nerve cells by low let x-ray

    Energy Technology Data Exchange (ETDEWEB)

    Park, Kwang Hun [Dept. of Nuclear Medicine, Kyungbuk National University Hospital, Daegu (Korea, Republic of); Kim, Kgu Hwan [Dept. of Radiological Technology, Daegu health College, Daegu (Korea, Republic of)

    2014-03-15

    Cultured pancreatic beta cells and nerve cells, it is given normal condition of 10% FBS (fetal bovine serum), 11.1 mM glucose and hyperglycemia condition of 1% FBS, 30 mM glucose. For low LET X-ray irradiated with 0.5 Gy/hr dose-rate(total dose: 0.5 to 5 Gy). Survival rates were measured by MTT assay. When non irradiated, differentiated in the pancreatic beta cells experiment is hyperglycemia conditions survival rate compared to normal conditions survival rate seemed a small reduction. However increasing the total dose of X-ray, the survival rate of normal conditions decreased slightly compared to the survival rate of hyperglycemia conditions, the synergistic effect was drastically reduced. When non irradiated, undifferentiated in the nerve cells experiment is hyperglycemia conditions survival rate compared to normal conditions survival rate seemed a large reduction. As the cumulative dose of X-ray normal conditions and hyperglycemia were all relatively rapid cell death. But the rate of decreased survivals by almost parallel to the reduction proceed and it didn't show synergistic effect.

  12. Chorein Sensitivity of Actin Polymerization, Cell Shape and Mechanical Stiffness of Vascular Endothelial Cells

    Directory of Open Access Journals (Sweden)

    Ioana Alesutan

    2013-09-01

    Full Text Available Background/Aims: Endothelial cell stiffness plays a key role in endothelium-dependent control of vascular tone and arterial blood pressure. Actin polymerization and distribution of microfilaments is essential for mechanical cell stiffness. Chorein, a protein encoded by the VPS13A gene, defective in chorea-acanthocytosis (ChAc, is involved in neuronal cell survival as well as cortical actin polymerization of erythrocytes and blood platelets. Chorein is expressed in a wide variety of further cells, yet nothing is known about the impact of chorein on cells other than neurons, erythrocytes and platelets. The present study explored whether chorein is expressed in human umbilical vein endothelial cells (HUVECs and addressed the putative role of chorein in the regulation of cytoskeletal architecture, stiffness and survival of those cells. Methods: In HUVECs with or without silencing of the VPS13A gene, VPS13A mRNA expression was determined utilizing quantitative RT-PCR, cytoskeletal organization visualized by confocal microscopy, G/F actin ratio and phosphorylation status of focal adhesion kinase quantified by western blotting, cell death determined by flow cytometry, mechanical properties studied by atomic force microscopy (AFM and cell morphology analysed by scanning ion conductance microscopy (SICM. Results: VPS13A mRNA expression was detectable in HUVECs. Silencing of the VPS13A gene attenuated the filamentous actin network, decreased the ratio of soluble G-actin over filamentous F-actin, reduced cell stiffness and changed cell morphology as compared to HUVECs silenced with negative control siRNA. These effects were paralleled by a significant decrease in FAK phosphorylation following VPS13A silencing. Moreover, silencing of the VPS13A gene increased caspase 3 activity and induced necrosis in HUVECs. Conclusions: Chorein is a novel regulator of cytoskeletal architecture, cell shape, mechanical stiffness and survival of vascular endothelial cells.

  13. Protein Kinase G facilitates EGFR-mediated cell death in MDA-MB-468 cells

    Energy Technology Data Exchange (ETDEWEB)

    Jackson, Nicole M.; Ceresa, Brian P., E-mail: brian.ceresa@louisville.edu

    2016-08-15

    The Epidermal Growth Factor Receptor (EGFR) is a transmembrane receptor tyrosine kinase with critical implications in cell proliferation, migration, wound healing and the regulation of apoptosis. However, the EGFR has been shown to be hyper-expressed in a number of human malignancies. The MDA-MB-468 metastatic breast cell line is one example of this. This particular cell line hyper-expresses the EGFR and undergoes EGFR-mediated apoptosis in response to EGF ligand. The goal of this study was to identify the kinases that could be potential intermediates for the EGFR-mediated induction of apoptosis intracellularly. After identifying Cyclic GMP-dependent Protein Kinase G (PKG) as a plausible intermediate, we wanted to determine the temporal relationship of these two proteins in the induction of apoptosis. We observed a dose-dependent decrease in MDA-MB-468 cell viability, which was co-incident with increased PKG activity as measured by VASPSer239 phosphorylation. In addition, we observed a dose dependent decrease in cell viability, as well as an increase in apoptosis, in response to two different PKG agonists, 8-Bromo-cGMP and 8-pCPT-cGMP. MDA-MB-468 cells with reduced PKG activity had attenuated EGFR-mediated apoptosis. These findings indicate that PKG does not induce cell death via transphosphorylation of the EGFR. Instead, PKG activity occurs following EGFR activation. Together, these data indicate PKG as an intermediary in EGFR-mediated cell death, likely via apoptotic pathway.

  14. Fibronectin Modulates Cell Adhesion and Signaling to Promote Single Cell Migration of Highly Invasive Oral Squamous Cell Carcinoma

    Science.gov (United States)

    Ramos, Grasieli de Oliveira; Bernardi, Lisiane; Lauxen, Isabel; Sant’Ana Filho, Manoel; Horwitz, Alan Rick; Lamers, Marcelo Lazzaron

    2016-01-01

    Cell migration is regulated by adhesion to the extracellular matrix (ECM) through integrins and activation of small RhoGTPases, such as RhoA and Rac1, resulting in changes to actomyosin organization. During invasion, epithelial-derived tumor cells switch from laminin-enriched basal membrane to collagen and fibronectin-enriched connective tissue. How this switch affects the tumor migration is still unclear. We tested the hypothesis that ECM dictates the invasiveness of Oral Squamous Cell Carcinoma (OSCC). We analyzed the migratory properties of two OSCC lines, a low invasive cell line with high e-cadherin levels (Linv/HE-cad) or a highly invasive cell line with low e-cadherin levels (Hinv/LE-cad), plated on different ECM components. Compared to laminin, fibronectin induced non-directional collective migration and decreased RhoA activity in Linv/HE-cad OSCC. For Hinv/LE-cad OSCC, fibronectin increased Rac1 activity and induced smaller adhesions, resulting in a fast single cell migration in both 2D and 3D environments. Consistent with these observations, human OSCC biopsies exhibited similar changes in cell-ECM adhesion distribution at the invasive front of the tumor, where cells encounter fibronectin. Our results indicate that ECM composition might induce a switch from collective to single cell migration according to tumor invasiveness due to changes in cell-ECM adhesion and the resulting signaling pathways that alter actomyosin organization. PMID:26978651

  15. Fibronectin Modulates Cell Adhesion and Signaling to Promote Single Cell Migration of Highly Invasive Oral Squamous Cell Carcinoma.

    Directory of Open Access Journals (Sweden)

    Grasieli de Oliveira Ramos

    Full Text Available Cell migration is regulated by adhesion to the extracellular matrix (ECM through integrins and activation of small RhoGTPases, such as RhoA and Rac1, resulting in changes to actomyosin organization. During invasion, epithelial-derived tumor cells switch from laminin-enriched basal membrane to collagen and fibronectin-enriched connective tissue. How this switch affects the tumor migration is still unclear. We tested the hypothesis that ECM dictates the invasiveness of Oral Squamous Cell Carcinoma (OSCC. We analyzed the migratory properties of two OSCC lines, a low invasive cell line with high e-cadherin levels (Linv/HE-cad or a highly invasive cell line with low e-cadherin levels (Hinv/LE-cad, plated on different ECM components. Compared to laminin, fibronectin induced non-directional collective migration and decreased RhoA activity in Linv/HE-cad OSCC. For Hinv/LE-cad OSCC, fibronectin increased Rac1 activity and induced smaller adhesions, resulting in a fast single cell migration in both 2D and 3D environments. Consistent with these observations, human OSCC biopsies exhibited similar changes in cell-ECM adhesion distribution at the invasive front of the tumor, where cells encounter fibronectin. Our results indicate that ECM composition might induce a switch from collective to single cell migration according to tumor invasiveness due to changes in cell-ECM adhesion and the resulting signaling pathways that alter actomyosin organization.

  16. Surface receptor Toso controls B cell-mediated regulation of T cell immunity.

    Science.gov (United States)

    Yu, Jinbo; Duong, Vu Huy Hoang; Westphal, Katrin; Westphal, Andreas; Suwandi, Abdulhadi; Grassl, Guntram A; Brand, Korbinian; Chan, Andrew C; Föger, Niko; Lee, Kyeong-Hee

    2018-05-01

    The immune system is tightly controlled by regulatory processes that allow for the elimination of invading pathogens, while limiting immunopathological damage to the host. In the present study, we found that conditional deletion of the cell surface receptor Toso on B cells unexpectedly resulted in impaired proinflammatory T cell responses, which led to impaired immune protection in an acute viral infection model and was associated with reduced immunopathological tissue damage in a chronic inflammatory context. Toso exhibited its B cell-inherent immunoregulatory function by negatively controlling the pool of IL-10-competent B1 and B2 B cells, which were characterized by a high degree of self-reactivity and were shown to mediate immunosuppressive activity on inflammatory T cell responses in vivo. Our results indicate that Toso is involved in the differentiation/maintenance of regulatory B cells by fine-tuning B cell receptor activation thresholds. Furthermore, we showed that during influenza A-induced pulmonary inflammation, the application of Toso-specific antibodies selectively induced IL-10-competent B cells at the site of inflammation and resulted in decreased proinflammatory cytokine production by lung T cells. These findings suggest that Toso may serve as a novel therapeutic target to dampen pathogenic T cell responses via the modulation of IL-10-competent regulatory B cells.

  17. Edaravone Decreases Paraquat Toxicity in A549 Cells and Lung Isolated Mitochondria

    OpenAIRE

    Shokrzadeh, Mohammad; Shaki, Fatemeh; Mohammadi, Ebrahim; Rezagholizadeh, Neda; Ebrahimi, Fatemeh

    2014-01-01

    Edaravone, an antioxidant and radical scavenger, showed protective effects against oxidative stress-like condition. Paraquat (PQ) is toxic herbicide considerable evidence suggests that oxidative stress and mitochondrial dysfunction contribute to PQ toxicity. In this study, protective effect of edaravone against PQ induced toxicity and reactive oxygen species (ROS) generation in A549 cells and lung isolated mitochondria were evaluated. A549 cells and lung isolated mitochondria were divided int...

  18. TGF-β1 downregulates StAR expression and decreases progesterone production through Smad3 and ERK1/2 signaling pathways in human granulosa cells.

    Science.gov (United States)

    Fang, Lanlan; Chang, Hsun-Ming; Cheng, Jung-Chien; Leung, Peter C K; Sun, Ying-Pu

    2014-11-01

    Regulation of progesterone production in granulosa cells is important for normal reproductive functions. Steroidogenic acute regulatory protein (StAR) is recognized as the key regulatory protein involved in the rate-limiting step of steroidogenesis. TGF-β1 protein is detected in human follicular fluid, and TGF-β1 and its receptors are expressed in human granulosa cells. However, the functional role of TGF-β1 in the regulation of StAR expression and progesterone production in human granulosa cells remains unknown. Our objective was to investigate the effects of TGF-β1 on StAR expression and progesterone production in human granulosa cells. SVOG cells are human granulosa cells that were obtained from women undergoing in vitro fertilization and immortalized with SV40 large T antigen. SVOG cells were used to investigate the effects of TGF-β1 on StAR expression and progesterone production at an academic research center. Levels of mRNA and protein were examined by RT-qPCR and western blotting, respectively. The accumulation levels of progesterone were measured by enzyme-linked immunosorbent assay (ELISA). TGF-β1 treatment downregulated StAR expression and decreased progesterone production. The suppressive effects of TGF-β1 on StAR expression and progesterone production were abolished by the inhibition of TGF-β type I receptor. In addition, treatment with TGF-β1 activated the Smad2/3 and ERK1/2 signaling pathways. The inhibition of the Smad3 and ERK1/2 signaling pathways attenuated the TGF-β1-induced downregulation of StAR expression and progesterone production. TGF-β1 downregulated StAR expression and decreased progesterone production by activating the Smad3 and ERK1/2 signaling pathways in human granulosa cells.

  19. Radiation cell survival and growth delay studies in multicellular spheroids of small-cell lung carcinoma

    International Nuclear Information System (INIS)

    Duchesne, G.M.; Peacock, J.H.

    1987-01-01

    The radiation sensitivity of two small-cell lung carcinoma cell lines growing as multicellular spheroids in static culture was determined using clonogenic cell survival and growth delay as endpoints. Growth delay determination suggested that clonogenic cell kill was less than was obtained by direct assay of cell survival. Recovery from potentially lethal damage was assayed in one line (HC12) but was not demonstrable, and clonogenic cell survival decreased with time in treated spheroids with diameters greater than 300 μm which contained a hypoxic cell population. Microscopic examination of the treated spheroids showed the emergence of an abnormal giant-cell population, and the progressive clonogenic cell loss that occurred after treatment was thought to be due to oxygen and nutrient deprivation of the remaining viable cells by this doomed cell population. Correction of the growth delay measurements for changes in cell size and clonogenic cell population allowed correlation of the growth delay and cell survival data. (author)

  20. Self-renewal and chemotherapy resistance of p75NTR positive cells in esophageal squamous cell carcinomas

    International Nuclear Information System (INIS)

    Huang, Sheng-Dong; Yuan, Yang; Liu, Xiao-Hong; Gong, De-Jun; Bai, Chen-Guang; Wang, Feng; Luo, Jun-Hui; Xu, Zhi-Yun

    2009-01-01

    p75 NTR has been used to isolate esophageal and corneal epithelial stem cells. In the present study, we investigated the expression of p75 NTR in esophageal squamous cell carcinoma (ESCC) and explored the biological properties of p75 NTR+ cells. p75 NTR expression in ESCC was assessed by immunohistochemistry. p75 NTR+ and p75 NTR- cells of 4 ESCC cell lines were separated by fluorescence-activated cell sorting. Differentially expressed genes between p75 NTR+ and p75 NTR- cells were determined by real-time quantitative reverse transcription-PCR. Sphere formation assay, DDP sensitivity assay, 64 copper accumulation assay and tumorigenicity analysis were performed to determine the capacity of self-renewal, chemotherapy resistance and tumorigenicity of p75 NTR+ cells. In ESCC specimens, p75 NTR was found mainly confined to immature cells and absent in cells undergoing terminal differentiation. The percentage of p75 NTR+ cells was 1.6%–3.7% in Eca109 and 3 newly established ESCC cell lines. The expression of Bmi-1, which is associated with self-renewal of stem cells, was significantly higher in p75 NTR+ cells. p63, a marker identified in keratinocyte stem cells, was confined mainly to p75 NTR+ cells. The expression of CTR1, which is associated with cisplatin (DDP)-resistance, was significantly decreased in p75 NTR+ cells. Expression levels of differentiation markers, such as involucrin, cytokeratin 13, β1-integrin and β4-integrin, were lower in p75 NTR+ cells. In addition, p75 NTR+ cells generated both p75 NTR+ and p75 NTR- cells, and formed nonadherent spherical clusters in serum-free medium supplemented with growth factors. Furthermore, p75 NTR+ cells were found to be more resistant to DDP and exhibited lower 64 copper accumulation than p75 NTR- cells. Our results demonstrated that p75 NTR+ cells possess some characteristics of CSCs, namely, self-renewal and chemotherapy resistance. Chemotherapy resistance of p75 NTR+ cells may probably be attributable to

  1. Exosomes decrease sensitivity of breast cancer cells to adriamycin by delivering microRNAs.

    Science.gov (United States)

    Mao, Ling; Li, Jian; Chen, Wei-Xian; Cai, Yan-Qin; Yu, Dan-Dan; Zhong, Shan-Liang; Zhao, Jian-Hua; Zhou, Jian-Wei; Tang, Jin-Hai

    2016-04-01

    While adriamycin (adr) offers improvement in survival for breast cancer (BCa) patients, unfortunately, drug resistance is almost inevitable. Mounting evidence suggests that exosomes act as a vehicle for genetic cargo and constantly shuttle biologically active molecules including microRNAs (miRNAs) between heterogeneous populations of tumor cells, engendering a resistance-promoting niche for cancer progression. Our recent study showed that exosomes from docetaxel-resistance BCa cells could modulate chemosensitivity by delivering miRNAs. Herein, we expand on our previous finding and explore the relevance of exosome-mediated miRNA delivery in resistance transmission of adr-resistant BCa sublines. We now demonstrated the selective packing of miRNAs within the exosomes (A/exo) derived from adr-resistant BCa cells. The highly expressed miRNAs in A/exo were significantly increased in recipient fluorescent sensitive cells (GFP-S) after A/exo incorporation. Gene ontology analysis of predicted targets showed that the top 30 most abundant miRNAs in A/exo were involved in crucial biological processes. Moreover, A/exo not only loaded miRNAs for its production and release but also carried miRNAs associated with Wnt signaling pathway. Furthermore, A/exo co-culture assays indicated that miRNA-containing A/exo was able to increase the overall resistance of GFP-S to adr exposure and regulate gene levels in GFP-S. Our results reinforce our earlier reports that adr-resistant BCa cells could manipulate a more deleterious microenvironment and transmit resistance capacity through altering gene expressions in sensitive cells by transferring specific miRNAs contained within exosomes.

  2. Differential concentration-specific effects of caffeine on cell viability, oxidative stress, and cell cycle in pulmonary oxygen toxicity in vitro

    International Nuclear Information System (INIS)

    Tiwari, Kirti Kumar; Chu, Chun; Couroucli, Xanthi; Moorthy, Bhagavatula; Lingappan, Krithika

    2014-01-01

    Highlights: • Caffeine at 0.05 mM decreases oxidative stress in hyperoxia. • Caffeine at 1 mM decreases cell viability, increases oxidative stress in hyperoxia. • Caffeine at 1 but not 0.05 mM, abrogates hyperoxia-induced G2/M arrest. - Abstract: Caffeine is used to prevent bronchopulmonary dysplasia (BPD) in premature neonates. Hyperoxia contributes to the development of BPD, inhibits cell proliferation and decreases cell survival. The mechanisms responsible for the protective effect of caffeine in pulmonary oxygen toxicity remain largely unknown. A549 and MLE 12 pulmonary epithelial cells were exposed to hyperoxia or maintained in room air, in the presence of different concentrations (0, 0.05, 0.1 and 1 mM) of caffeine. Caffeine had a differential concentration-specific effect on cell cycle progression, oxidative stress and viability, with 1 mM concentration being deleterious and 0.05 mM being protective. Reactive oxygen species (ROS) generation during hyperoxia was modulated by caffeine in a similar concentration-specific manner. Caffeine at 1 mM, but not at the 0.05 mM concentration decreased the G2 arrest in these cells. Taken together this study shows the novel funding that caffeine has a concentration-specific effect on cell cycle regulation, ROS generation, and cell survival in hyperoxic conditions

  3. Differential concentration-specific effects of caffeine on cell viability, oxidative stress, and cell cycle in pulmonary oxygen toxicity in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Tiwari, Kirti Kumar; Chu, Chun; Couroucli, Xanthi; Moorthy, Bhagavatula; Lingappan, Krithika, E-mail: lingappa@bcm.edu

    2014-08-08

    Highlights: • Caffeine at 0.05 mM decreases oxidative stress in hyperoxia. • Caffeine at 1 mM decreases cell viability, increases oxidative stress in hyperoxia. • Caffeine at 1 but not 0.05 mM, abrogates hyperoxia-induced G2/M arrest. - Abstract: Caffeine is used to prevent bronchopulmonary dysplasia (BPD) in premature neonates. Hyperoxia contributes to the development of BPD, inhibits cell proliferation and decreases cell survival. The mechanisms responsible for the protective effect of caffeine in pulmonary oxygen toxicity remain largely unknown. A549 and MLE 12 pulmonary epithelial cells were exposed to hyperoxia or maintained in room air, in the presence of different concentrations (0, 0.05, 0.1 and 1 mM) of caffeine. Caffeine had a differential concentration-specific effect on cell cycle progression, oxidative stress and viability, with 1 mM concentration being deleterious and 0.05 mM being protective. Reactive oxygen species (ROS) generation during hyperoxia was modulated by caffeine in a similar concentration-specific manner. Caffeine at 1 mM, but not at the 0.05 mM concentration decreased the G2 arrest in these cells. Taken together this study shows the novel funding that caffeine has a concentration-specific effect on cell cycle regulation, ROS generation, and cell survival in hyperoxic conditions.

  4. Effects of γ-radiation on cell growth, cell cycle and promoter methylation of 22 cell cycle genes in the 1321NI astrocytoma cell line.

    Science.gov (United States)

    Alghamian, Yaman; Abou Alchamat, Ghalia; Murad, Hossam; Madania, Ammar

    2017-09-01

    DNA damage caused by radiation initiates biological responses affecting cell fate. DNA methylation regulates gene expression and modulates DNA damage pathways. Alterations in the methylation profiles of cell cycle regulating genes may control cell response to radiation. In this study we investigated the effect of ionizing radiation on the methylation levels of 22 cell cycle regulating genes in correlation with gene expression in 1321NI astrocytoma cell line. 1321NI cells were irradiated with 2, 5 or 10Gy doses then analyzed after 24, 48 and 72h for cell viability using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliu bromide) assay. Flow cytometry were used to study the effect of 10Gy irradiation on cell cycle. EpiTect Methyl II PCR Array was used to identify differentially methylated genes in irradiated cells. Changes in gene expression was determined by qPCR. Azacytidine treatment was used to determine whether DNA methylation affectes gene expression. Our results showed that irradiation decreased cell viability and caused cell cycle arrest at G2/M. Out of 22 genes tested, only CCNF and RAD9A showed some increase in DNA methylation (3.59% and 3.62%, respectively) after 10Gy irradiation, and this increase coincided with downregulation of both genes (by 4 and 2 fold, respectively). with azacytidine confirmed that expression of CCNF and RAD9A genes was regulated by methylation. 1321NI cell line is highly radioresistant and that irradiation of these cells with a 10Gy dose increases DNA methylation of CCNF and RAD9A genes. This dose down-regulates these genes, favoring G2/M arrest. Copyright © 2017 Medical University of Bialystok. Published by Elsevier B.V. All rights reserved.

  5. Anti-sense suppression of epidermal growth factor receptor expression alters cellular proliferation, cell-adhesion and tumorigenicity in ovarian cancer cells.

    Science.gov (United States)

    Alper, O; De Santis, M L; Stromberg, K; Hacker, N F; Cho-Chung, Y S; Salomon, D S

    2000-11-15

    Over-expression of epidermal growth factor receptor (EGFR) in ovarian cancer has been well documented. Human NIH:OVCAR-8 ovarian carcinoma cells were transfected with an expression vector containing the anti-sense orientation of truncated human EGFR cDNA. EGFR anti-sense over-expression resulted in decreased EGFR protein and mRNA expression, cell proliferation and tumor formation in nude mice. In accordance with the reduced levels of EGFR in EGFR anti-sense-expressing cells, tyrosine phosphorylation of EGFR was decreased compared to untransfected parental cells treated with EGF. In EGFR anti-sense-transfected cells, expression of erbB-3, but not erbB-2, was increased. In addition, basal and heregulin-beta 1-stimulated tyrosine phosphorylation of erbB-3 was higher in EGFR anti-sense vector-transfected cells. A morphological alteration in EGFR anti-sense gene-expressing cells was correlated with a decrease in the expression of E-cadherin, alpha-catenin and, to a lesser extent, beta-catenin. Changes in the expression of these proteins were associated with a reduction in complex formation among E-cadherin, beta-catenin and alpha-catenin and between beta-catenin and EGFR in EGFR anti-sense-expressing cells compared to sense-transfected control cells. These results demonstrate that EGFR expression in ovarian carcinoma cells regulates expression of cell adhesion proteins that may enhance cell growth and invasiveness. Copyright 2000 Wiley-Liss, Inc.

  6. CXCL10 Decreases GP73 Expression in Hepatoma Cells at the Early Stage of Hepatitis C Virus (HCV Infection

    Directory of Open Access Journals (Sweden)

    Yuan Liu

    2013-12-01

    Full Text Available Golgi protein 73 (GP73, which is up-regulated in hepatocellular carcinoma (HCC, has recently been identified as a novel serum marker for HCC diagnosis. Several reports also noted the increased levels of GP73 expression in chronic liver disease in patients with acute hepatitis of various etiologies, chronic Hepatitis C virus (HCV infection and alcoholic liver disease. The molecular mechanisms of GP73 expression in HCV related liver disease still need to be determined. In this study, we aimed to evaluate the effect of HCV infection on GP73 expression. GP73 was highly expressed in Huh7, Hep3B, 293T and HUVEC cells, and was low-expressed in HepG2 cells. HCV infection led to down-regulation of GP73 in Huh7 and HepG2/CD81 cells at the early stage of infection. CXCL10 decreased GP73 expression in Huh7 and HepG2 cells. Up-regulation of GP73 was noted in hepatocytes with cytopathic effect at advanced stage of HCV infection, and further research is needed to determine the unknown factors affecting GP73 expression. In conclusion, our study provided additional evidence for the roles of GP73 in liver disease.

  7. Tributyltin (TBT) and Dibutyltin (DBT) Alter Secretion of Tumor Necrosis Factor Alpha (TNFα) from Human Natural Killer (NK) Cells and a Mixture of T cells and NK Cells

    Science.gov (United States)

    Hurt, Kelsi; Hurd-Brown, Tasia; Whalen, Margaret

    2012-01-01

    Butyltins (BTs) have been in widespread use. Tributyltin (TBT) has been used as a biocide in a variety of applications and is found in human blood samples. Dibutyltin (DBT) has been used as a stabilizer in polyvinyl chloride plastics and as a de-worming agent in poultry. DBT, like TBT, is found in human blood. Human natural killer (NK) cells are the earliest defense against tumors and viral infections and secrete the cytokine tumor necrosis factor (TNF) alpha (α). TNFα is an important regulator of adaptive and innate immune responses. TNFα promotes inflammation and an association between malignant transformation and inflammation has been established. Previously, we have shown that TBT and DBT were able to interfere with the ability of NK cells to lyse tumor target cells. Here we show that BTs alter cytokine secretion by NK cells as well as a mixture of T and NK lymphocytes (T/NK cells). We examined 24 h, 48 h, and 6 day exposures to TBT (200- 2.5 nM) and DBT (5- 0.05 µM) on TNFα secretion by highly enriched human NK cells and T/NK cells. The results indicate that TBT (200 - 2.5 nM) decreased TNFα secretion from NK cells. In the T/NK cells 200 nM TBT decreased secretion while 100-5 nM TBT increased secretion of TNFα. NK cells or T/NK cells exposed to higher concentrations of DBT showed decreased TNFα secretion while lower concentrations showed increased secretion. The effects of BTs on TNFα secretion are seen at concentrations present in human blood. PMID:23047847

  8. Resveratrol and Estradiol Exert Disparate Effects on Cell Migration, Cell Surface Actin Structures, and Focal Adhesion Assembly in MDA-MB-231 Human Breast Cancer Cells

    Directory of Open Access Journals (Sweden)

    Nicolas G. Azios

    2005-02-01

    Full Text Available Resveratrol, a grape polyphenol, is thought to be a cancer preventive, yet its effects on metastatic breast cancer are relatively unknown. Since cancer cell invasion is dependent on cell migration, the chemotactic response of MDA-MB-231 metastatic human breast cancer cells to resveratrol, estradiol (E2, or epidermal growth factor (EGF was investigated. Resveratrol decreased while E2 and EGF increased directed cell migration. Resveratrol may inhibit cell migration by altering the cytoskeleton. Resveratrol induced a rapid global array of filopodia and decreased focal adhesions and focal adhesion kinase (FAK activity. E2 or EGF treatment did not affect filopodia extension but increased lamellipodia and associated focal adhesions that are integral for cell migration. Combined resveratrol and E2 treatment resulted in a filopodia and focal adhesion response similar to resveratrol alone. Combined resveratrol and EGF resulted in a lamellipodia and focal adhesion response similar to EGF alone. E2 and to a lesser extent resveratrol increased EGFR activity. The cytoskeletal changes and EGFR activity in response to E2 were blocked by EGFR1 inhibitor indicating that E2 may increase cell migration via crosstalk with EGFR signaling. These data suggest a promotional role for E2 in breast cancer cell migration but an antiestrogenic, preventative role for resveratrol.

  9. Generation and Characterisation of Cisplatin-Resistant Non-Small Cell Lung Cancer Cell Lines Displaying a Stem-Like Signature

    Science.gov (United States)

    Barr, Martin P.; Gray, Steven G.; Hoffmann, Andreas C.; Hilger, Ralf A.; Thomale, Juergen; O’Flaherty, John D.; Fennell, Dean A.; Richard, Derek; O’Leary, John J.; O’Byrne, Kenneth J.

    2013-01-01

    Introduction Inherent and acquired cisplatin resistance reduces the effectiveness of this agent in the management of non-small cell lung cancer (NSCLC). Understanding the molecular mechanisms underlying this process may result in the development of novel agents to enhance the sensitivity of cisplatin. Methods An isogenic model of cisplatin resistance was generated in a panel of NSCLC cell lines (A549, SKMES-1, MOR, H460). Over a period of twelve months, cisplatin resistant (CisR) cell lines were derived from original, age-matched parent cells (PT) and subsequently characterized. Proliferation (MTT) and clonogenic survival assays (crystal violet) were carried out between PT and CisR cells. Cellular response to cisplatin-induced apoptosis and cell cycle distribution were examined by FACS analysis. A panel of cancer stem cell and pluripotent markers was examined in addition to the EMT proteins, c-Met and β-catenin. Cisplatin-DNA adduct formation, DNA damage (γH2AX) and cellular platinum uptake (ICP-MS) was also assessed. Results Characterisation studies demonstrated a decreased proliferative capacity of lung tumour cells in response to cisplatin, increased resistance to cisplatin-induced cell death, accumulation of resistant cells in the G0/G1 phase of the cell cycle and enhanced clonogenic survival ability. Moreover, resistant cells displayed a putative stem-like signature with increased expression of CD133+/CD44+cells and increased ALDH activity relative to their corresponding parental cells. The stem cell markers, Nanog, Oct-4 and SOX-2, were significantly upregulated as were the EMT markers, c-Met and β-catenin. While resistant sublines demonstrated decreased uptake of cisplatin in response to treatment, reduced cisplatin-GpG DNA adduct formation and significantly decreased γH2AX foci were observed compared to parental cell lines. Conclusion Our results identified cisplatin resistant subpopulations of NSCLC cells with a putative stem-like signature, providing

  10. Generation and characterisation of cisplatin-resistant non-small cell lung cancer cell lines displaying a stem-like signature.

    Directory of Open Access Journals (Sweden)

    Martin P Barr

    Full Text Available Inherent and acquired cisplatin resistance reduces the effectiveness of this agent in the management of non-small cell lung cancer (NSCLC. Understanding the molecular mechanisms underlying this process may result in the development of novel agents to enhance the sensitivity of cisplatin.An isogenic model of cisplatin resistance was generated in a panel of NSCLC cell lines (A549, SKMES-1, MOR, H460. Over a period of twelve months, cisplatin resistant (CisR cell lines were derived from original, age-matched parent cells (PT and subsequently characterized. Proliferation (MTT and clonogenic survival assays (crystal violet were carried out between PT and CisR cells. Cellular response to cisplatin-induced apoptosis and cell cycle distribution were examined by FACS analysis. A panel of cancer stem cell and pluripotent markers was examined in addition to the EMT proteins, c-Met and β-catenin. Cisplatin-DNA adduct formation, DNA damage (γH2AX and cellular platinum uptake (ICP-MS was also assessed.Characterisation studies demonstrated a decreased proliferative capacity of lung tumour cells in response to cisplatin, increased resistance to cisplatin-induced cell death, accumulation of resistant cells in the G0/G1 phase of the cell cycle and enhanced clonogenic survival ability. Moreover, resistant cells displayed a putative stem-like signature with increased expression of CD133+/CD44+cells and increased ALDH activity relative to their corresponding parental cells. The stem cell markers, Nanog, Oct-4 and SOX-2, were significantly upregulated as were the EMT markers, c-Met and β-catenin. While resistant sublines demonstrated decreased uptake of cisplatin in response to treatment, reduced cisplatin-GpG DNA adduct formation and significantly decreased γH2AX foci were observed compared to parental cell lines.Our results identified cisplatin resistant subpopulations of NSCLC cells with a putative stem-like signature, providing a further understanding of the

  11. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) induces Fas-dependent activation-induced cell death in superantigen-primed T cells

    Energy Technology Data Exchange (ETDEWEB)

    Camacho, Iris A; Nagarkatti, Mitzi [Department of Microbiology and Immunology, Medical College of Virginia Campus, Virginia Commonwealth University, Richmond, VA 23298 (United States); Nagarkatti, Prakash S [Department of Pharmacology and Toxicology, PO Box 980613, Medical College of Virginia Campus, Virginia Commonwealth University, Richmond, VA 23298-0613 (United States)

    2002-10-01

    Immune response against a foreign antigen is characterized by a growth phase, in which antigen-specific T cells clonally expand, followed by a decline phase in which the activated T cells undergo apoptosis, a process termed activation-induced cell death (AICD). In the current study, we have investigated the phase at which 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) acts to downregulate the antigen-specific T cell response. To this end, C57BL/6 +/+ mice were injected with staphylococcal enterotoxin A (SEA) into the footpads (10 {mu}g/footpad), and simultaneously treated with TCDD (10 or 50 {mu}g/kg intraperitoneally). At various time points, the draining lymph node (LN) cells were analyzed for SEA-activated T cells. The data demonstrated that in C57BL/6 +/+ mice, TCDD treatment did not alter the growth phase but facilitated the decline phase of SEA-reactive T cells. TCDD caused a significant decrease in the percentage and absolute numbers of CD4{sup +} and CD8{sup +} SEA-responsive T cells expressing V{beta}3{sup +} and V{beta}11{sup +} but did not affect SEA-nonresponsive V{beta}8{sup +} T cells. Upon in vitro culture, TCDD-exposed SEA-immunized LN cells exhibited increased levels of apoptosis when compared with the vehicle controls. When Fas-deficient (C57BL/6 lpr/lpr) or Fas ligand defective (C57BL/6 gld/gld) mice were treated with TCDD, they failed to exhibit a decrease in percentage and cellularity of SEA-reactive T cells, thereby suggesting a role of Fas-Fas ligand interactions in the TCDD-induced downregulation of SEA-reactive T cell response. The resistance to TCDD-induced decrease in T cell responsiveness to SEA seen in Fas- and FasL-mutant mice was neither due to decreased aryl hydrocabon receptor (AhR) expression nor to altered T cell responsiveness to SEA. The current study demonstrates that TCDD does not prevent T cell activation, but prematurely induces Fas-based AICD, which may contribute to the deletion of antigen-primed T cells. (orig.)

  12. The Hippo pathway controls border cell migration through distinct mechanisms in outer border cells and polar cells of the Drosophila ovary.

    Science.gov (United States)

    Lin, Tzu-Huai; Yeh, Tsung-Han; Wang, Tsu-Wei; Yu, Jenn-Yah

    2014-11-01

    The Hippo pathway is a key signaling cascade in controlling organ size. The core components of this pathway are two kinases, Hippo (Hpo) and Warts (Wts), and a transcriptional coactivator, Yorkie (Yki). Yes-associated protein (YAP, a Yki homolog in mammals) promotes epithelial-mesenchymal transition and cell migration in vitro. Here, we use border cells in the Drosophila ovary as a model to study Hippo pathway functions in cell migration in vivo. During oogenesis, polar cells secrete Unpaired (Upd), which activates JAK/STAT signaling of neighboring cells and specifies them into outer border cells. The outer border cells form a cluster with polar cells and undergo migration. We find that hpo and wts are required for migration of the border cell cluster. In outer border cells, overexpression of hpo disrupts polarization of the actin cytoskeleton and attenuates migration. In polar cells, knockdown of hpo and wts or overexpression of yki impairs border cell induction and disrupts migration. These manipulations in polar cells reduce JAK/STAT activity in outer border cells. Expression of upd-lacZ is increased and decreased in yki and hpo mutant polar cells, respectively. Furthermore, forced expression of upd in polar cells rescues defects of border cell induction and migration caused by wts knockdown. These results suggest that Yki negatively regulates border cell induction by inhibiting JAK/STAT signaling. Together, our data elucidate two distinct mechanisms of the Hippo pathway in controlling border cell migration: (1) in outer border cells, it regulates polarized distribution of the actin cytoskeleton; (2) in polar cells, it regulates upd expression to control border cell induction and migration. Copyright © 2014 by the Genetics Society of America.

  13. Anisomycin-induced GATA-6 degradation accompanying a decrease of proliferation of colorectal cancer cell

    Energy Technology Data Exchange (ETDEWEB)

    Ushijima, Hironori; Horyozaki, Akiko; Maeda, Masatomo, E-mail: mmaeda@nupals.ac.jp

    2016-09-09

    Transcription factor GATA-6 plays a key role in normal cell differentiation of the mesoderm and endoderm. On the other hand, GATA-6 is abnormally overexpressed in many clinical gastrointestinal cancer tissue samples, and accelerates cell proliferation or an anti-apoptotic response in cancerous tissues. We previously showed that activation of the JNK signaling cascade causes proteolysis of GATA-6. In this study, we demonstrated that anisomycin, a JNK activator, stimulates nuclear export of GATA-6 in a colorectal cancer cell line, DLD-1. Concomitantly, anisomycin remarkably inhibits the proliferation of DLD-1 cells via G2/M arrest in a plate culture. However, it did not induce apoptosis under growth arrest conditions. Furthermore, the growth of DLD-1 cells in a spheroid culture was suppressed by anisomycin. Although 5-FU showed only a slight inhibitory effect on 3D spheroid cultures, the same concentration of 5-FU together with a low concentration of anisomycin exhibited strong growth inhibition. These results suggest that the induction of GATA-6 dysfunction may be more effective for chemotherapy for colorectal cancer, although the mechanism underlying the synergistic effect of 5-FU and anisomycin remains unknown. - Highlights: • Anisomycin induces proteolysis of GATA-6 in DLD-1 cells. • Anisomycin remarkably inhibits the proliferation of DLD-1 cells via G2/M arrest. • Anisomycin suppresses the growth of spheroids of DLD-1, and enhances the effect of 5-FU.

  14. Decreased number of CD4+ and CD8+ T cells that express the interleukin-7 receptor in blood and tissues of SIV-infected macaques

    International Nuclear Information System (INIS)

    Moniuszko, Marcin; Edghill-Smith, Yvette; Venzon, David; Stevceva, Liljana; Nacsa, Janos; Tryniszewska, Elzbieta; Tsai, Wen-Po; Franchini, Genoveffa

    2006-01-01

    Acute HIV/SIV (human/simian immunodeficiency virus) infection results in severe CD4 + T cell depletion in lymphoid compartments. During the chronic phase of infection, CD4 + T cell numbers rebound in blood but remain low in the gut-associated lymphoid tissue (GALT), even when viral replication is suppressed by antiretroviral therapy (ART). Thus, strategies to repopulate lymphoid compartments may ameliorate the clinical outcome of HIV/SIV infection. Interleukin (IL)-7 is a key cytokine for the maintenance of homeostatic proliferation of T cells. In HIV/SIV infection, IL-7 expression is increased, likely to compensate for T cell loss, suggesting that supraphysiological administration of IL-7 could provide additional benefit. However, the ability of T cells to respond to IL-7 is dependent on the level of expression of the IL-7 receptor (IL-7R) in T cells in various body compartments. In here, we investigated the proportion of IL-7R + T cells in blood, spleen, gut, and genitourinary tract of healthy and SIV-infected macaques with various degrees of CD4 + T cell depletion. We found that the percentage of T cells expressing IL-7R was significantly lower in both CD4 + and CD8 + T cell subsets in SIV-infected macaques than in healthy animals and this decrease directly correlated with the CD4 + T cell number. Importantly, the proportion of CD4 + and CD8 + T cells expressing IL-7R in blood paralleled that found in tissues. IL-7R + T cells within the SIV-specific CD8 + T cells varied and were lowest in most tissues of viremic macaques, likely reflecting continuous antigen stimulation of effector cells

  15. NCAM regulates cell motility

    DEFF Research Database (Denmark)

    Prag, Søren; Lepekhin, Eugene A; Kolkova, Kateryna

    2002-01-01

    Cell migration is required during development of the nervous system. The regulatory mechanisms for this process, however, are poorly elucidated. We show here that expression of or exposure to the neural cell adhesion molecule (NCAM) strongly affected the motile behaviour of glioma cells...... independently of homophilic NCAM interactions. Expression of the transmembrane 140 kDa isoform of NCAM (NCAM-140) caused a significant reduction in cellular motility, probably through interference with factors regulating cellular attachment, as NCAM-140-expressing cells exhibited a decreased attachment...... to a fibronectin substratum compared with NCAM-negative cells. Ectopic expression of the cytoplasmic part of NCAM-140 also inhibited cell motility, presumably via the non-receptor tyrosine kinase p59(fyn) with which NCAM-140 interacts. Furthermore, we showed that the extracellular part of NCAM acted as a paracrine...

  16. Cell cycle inhibitor, p19INK4d, promotes cell survival and decreases chromosomal aberrations after genotoxic insult due to enhanced DNA repair.

    Science.gov (United States)

    Scassa, María E; Marazita, Mariela C; Ceruti, Julieta M; Carcagno, Abel L; Sirkin, Pablo F; González-Cid, Marcela; Pignataro, Omar P; Cánepa, Eduardo T

    2007-05-01

    Genome integrity and cell proliferation and survival are regulated by an intricate network of pathways that includes cell cycle checkpoints, DNA repair and recombination, and programmed cell death. It makes sense that there should be a coordinated regulation of these different processes, but the components of such mechanisms remain unknown. In this report, we demonstrate that p19INK4d expression enhances cell survival under genotoxic conditions. By using p19INK4d-overexpressing clones, we demonstrated that p19INK4d expression correlates with the cellular resistance to UV treatment with increased DNA repair activity against UV-induced lesions. On the contrary, cells transfected with p19INK4d antisense cDNA show reduced ability to repair DNA damage and increased sensitivity to genotoxic insult when compared with their p19INK4d-overexpressing counterparts. Consistent with these findings, our studies also show that p19INK4d-overexpressing cells present not only a minor accumulation of UV-induced chromosomal aberrations but a lower frequency of spontaneous chromosome abnormalities than p19INK4d-antisense cells. Lastly, we suggest that p19INK4d effects are dissociated from its role as CDK4/6 inhibitor. The results presented herein support a crucial role for p19INK4d in regulating genomic stability and overall cell viability under conditions of genotoxic stress. We propose that p19INK4d would belong to a protein network that would integrate DNA repair, apoptotic and checkpoint mechanisms in order to maintain the genomic integrity.

  17. Inhibition of macrophage migration inhibitory factor decreases proliferation and cytokine expression in bladder cancer cells

    International Nuclear Information System (INIS)

    Meyer-Siegler, Katherine L; Leifheit, Erica C; Vera, Pedro L

    2004-01-01

    The importance of various inflammatory cytokines in maintaining tumor cell growth and viability is well established. Increased expression of the proinflammatory cytokine macrophage migration inhibitory factor (MIF) has previously been associated with various types of adenocarcinoma. MIF IHC was used to localize MIF in human bladder tissue. ELISA and Western blot analysis determined the synthesis and secretion of MIF by human bladder transitional cell carcinoma cells. The effects of MIF inhibitors (high molecular weight hyaluronate (HA), anti-MIF antibody or MIF anti-sense) on cell growth and cytokine expression were analyzed. Human bladder cancer cells (HT-1376) secrete detectable amounts of MIF protein. Treatment with HA, anti-MIF antibody and MIF anti-sense reduced HT-1376 cell proliferation, MIF protein secretion, MIF gene expression and secreted inflammatory cytokines. Our evidence suggests MIF interacts with the invariant chain, CD74 and the major cell surface receptor for HA, CD44. This study is the first to report MIF expression in the human bladder and these findings support a role for MIF in tumor cell proliferation. Since MIF participates in the inflammatory response and bladder cancer is associated with chronic inflammatory conditions, these new findings suggest that neutralizing bladder tumor MIF may serve as a novel therapeutic treatment for bladder carcinoma

  18. Shear stress-induced mitochondrial biogenesis decreases the release of microparticles from endothelial cells

    OpenAIRE

    Kim, Ji-Seok; Kim, Boa; Lee, Hojun; Thakkar, Sunny; Babbitt, Dianne M.; Eguchi, Satoru; Brown, Michael D.; Park, Joon-Young

    2015-01-01

    This study assesses effects of aerobic exercise training on the release of microparticles from endothelial cells and corroborates these findings using an in vitro experimental exercise stimulant, laminar shear stress. Furthermore, this study demonstrated that shear stress-induced mitochondrial biogenesis mediates these effects against endothelial cell activation and injury.

  19. Expression of HSF2 decreases in mitosis to enable stress-inducible transcription and cell survival

    Science.gov (United States)

    Elsing, Alexandra N.; Aspelin, Camilla; Björk, Johanna K.; Bergman, Heidi A.; Himanen, Samu V.; Kallio, Marko J.; Roos-Mattjus, Pia

    2014-01-01

    Unless mitigated, external and physiological stresses are detrimental for cells, especially in mitosis, resulting in chromosomal missegregation, aneuploidy, or apoptosis. Heat shock proteins (Hsps) maintain protein homeostasis and promote cell survival. Hsps are transcriptionally regulated by heat shock factors (HSFs). Of these, HSF1 is the master regulator and HSF2 modulates Hsp expression by interacting with HSF1. Due to global inhibition of transcription in mitosis, including HSF1-mediated expression of Hsps, mitotic cells are highly vulnerable to stress. Here, we show that cells can counteract transcriptional silencing and protect themselves against proteotoxicity in mitosis. We found that the condensed chromatin of HSF2-deficient cells is accessible for HSF1 and RNA polymerase II, allowing stress-inducible Hsp expression. Consequently, HSF2-deficient cells exposed to acute stress display diminished mitotic errors and have a survival advantage. We also show that HSF2 expression declines during mitosis in several but not all human cell lines, which corresponds to the Hsp70 induction and protection against stress-induced mitotic abnormalities and apoptosis. PMID:25202032

  20. Cell cycle progression in irradiated endothelial cells cultured from bovine aorta

    International Nuclear Information System (INIS)

    Rubin, D.B.; Drab, E.A.; Ward, W.F.; Bauer, K.D.

    1988-01-01

    Logarithmically growing endothelial cells from bovine aortas were exposed to single doses of 0-10 Gy of 60Co gamma rays, and cell cycle phase distribution and progression were examined by flow cytometry and autoradiography. In some experiments, cells were synchronized in the cell cycle with hydroxyurea (1 mM). Cell number in sham-irradiated control cultures doubled in approximately 24 h. Estimated cycle stage times for control cells were 14.4 h for G1 phase, 7.2 h for S phase, and 2.4 h for G2 + M phase. Irradiated cells demonstrated a reduced distribution at the G1/S phase border at 4 h, and an increased distribution in G2 + M phase at 24 h postirradiation. Autoradiographs of irradiated cells after continuous [3H]thymidine labeling indicated a block in G1 phase or at the G1/S-phase border. The duration of the block was dose dependent (2-3 min/cGy). Progression of the endothelial cells through S phase after removal of the hydroxyurea block also was retarded by irradiation, as demonstrated by increased distribution in early S phase and decreased distribution in late S phase. These results indicate that progression of asynchronous cultured bovine aortic endothelial cells through the DNA synthetic cycle is susceptible to radiation inhibition at specific sites in the cycle, resulting in redistribution and partial synchronization of the population. Thus aortic endothelial cells, diploid cells from a normal tissue, resemble many immortal cell types that have been examined in this regard in vitro

  1. Ataxia telangiectasia derived iPS cells show preserved x-ray sensitivity and decreased chromosomal instability

    OpenAIRE

    Fukawatase, Yoshihiro; Toyoda, Masashi; Okamura, Kohji; Nakamura, Ken-ichi; Nakabayashi, Kazuhiko; Takada, Shuji; Yamazaki-Inoue, Mayu; Masuda, Akira; Nasu, Michiyo; Hata, Kenichiro; Hanaoka, Kazunori; Higuchi, Akon; Takubo, Kaiyo; Umezawa, Akihiro

    2014-01-01

    Ataxia telangiectasia is a neurodegenerative inherited disease with chromosomal instability and hypersensitivity to ionizing radiation. iPS cells lacking ATM (AT-iPS cells) exhibited hypersensitivity to X-ray irradiation, one of the characteristics of the disease. While parental ataxia telangiectasia cells exhibited significant chromosomal abnormalities, AT-iPS cells did not show any chromosomal instability in vitro for at least 80 passages (560 days). Whole exome analysis also showed a compa...

  2. Defective TFH Cell Function and Increased TFR Cells Contribute to Defective Antibody Production in Aging.

    Science.gov (United States)

    Sage, Peter T; Tan, Catherine L; Freeman, Gordon J; Haigis, Marcia; Sharpe, Arlene H

    2015-07-14

    Defective antibody production in aging is broadly attributed to immunosenescence. However, the precise immunological mechanisms remain unclear. Here, we demonstrate an increase in the ratio of inhibitory T follicular regulatory (TFR) cells to stimulatory T follicular helper (TFH) cells in aged mice. Aged TFH and TFR cells are phenotypically distinct from those in young mice, exhibiting increased programmed cell death protein-1 expression but decreased ICOS expression. Aged TFH cells exhibit defective antigen-specific responses, and programmed cell death protein-ligand 1 blockade can partially rescue TFH cell function. In contrast, young and aged TFR cells have similar suppressive capacity on a per-cell basis in vitro and in vivo. Together, these studies reveal mechanisms contributing to defective humoral immunity in aging: an increase in suppressive TFR cells combined with impaired function of aged TFH cells results in reduced T-cell-dependent antibody responses in aged mice. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

  3. Murraya koenigii leaf extract inhibits proteasome activity and induces cell death in breast cancer cells.

    Science.gov (United States)

    Noolu, Bindu; Ajumeera, Rajanna; Chauhan, Anitha; Nagalla, Balakrishna; Manchala, Raghunath; Ismail, Ayesha

    2013-01-09

    Inhibition of the proteolytic activity of 26S proteasome, the protein-degrading machine, is now considered a novel and promising approach for cancer therapy. Interestingly, proteasome inhibitors have been demonstrated to selectively kill cancer cells and also enhance the sensitivity of tumor cells to chemotherapeutic agents. Recently, polyphenols/flavonoids have been reported to inhibit proteasome activity. Murraya koenigii Spreng, a medicinally important herb of Indian origin, has been used for centuries in the Ayurvedic system of medicine. Here we show that Murraya koenigii leaves (curry leaves), a rich source of polyphenols, inhibit the proteolytic activity of the cancer cell proteasome, and cause cell death. Hydro-methanolic extract of curry leaves (CLE) was prepared and its total phenolic content [TPC] determined by, the Folin-Ciocalteau's method. Two human breast carcinoma cell lines: MCF-7 and MDA-MB-231 and a normal human lung fibroblast cell line, WI-38 were used for the studies. Cytotoxicity of the CLE was assessed by the MTT assay. We studied the effect of CLE on growth kinetics using colony formation assay. Growth arrest was assessed by cell cycle analysis and apoptosis by Annexin-V binding using flow cytometry. Inhibition of the endogenous 26S proteasome was studied in intact cells and cell extracts using substrates specific to 20S proteasomal enzymes. CLE decreased cell viability and altered the growth kinetics in both the breast cancer cell lines in a dose-dependent manner. It showed a significant arrest of cells in the S phase albeit in cancer cells only. Annexin V binding data suggests that cell death was via the apoptotic pathway in both the cancer cell lines. CLE treatment significantly decreased the activity of the 26S proteasome in the cancer but not normal cells. Our study suggests M. koenigii leaves to be a potent source of proteasome inhibitors that lead to cancer cell death. Therefore, identification of active component(s) from the leaf

  4. Trehalose improves cell proliferation and dehydration tolerance of human HaCaT cells

    Directory of Open Access Journals (Sweden)

    Lee Kyung Eun

    2015-01-01

    Full Text Available Trehalose is a disaccharide molecule that serves as a natural osmotic regulator in halophilic microorganisms and plants but not in mammals. We observed that human HaCaT cells supplied with trehalose improved cell proliferation and extended viability under dehydration. In HaCaT cells, in response to increasing concentrations of exogenous trehalose, the levels of heat shock protein (HSP 70 increased and matrix metalloproteinase (MMP 1 decreased. Proteome analysis of trehalose-treated HaCaT cells revealed remarkable increases in the levels of proteins involved in cell signaling and the cell cycle, including p21 activated kinase I, Sec I family domain protein and elongation factor G. Moreover, the proteins for cell stress resistance, tryptophan hydroxylase, serine/cysteine proteinase inhibitors and vitamin D receptors were also increased. In addition, the proteins responsible for the maintenance of the cytoskeleton and cellular structures including procollagen-lysine dioxygenase, vinculin and ezrin were increased. Proteomic data revealed that trehalose affected HaCaT cells by inducing the proteins involved in cell proliferation. These results suggest that trehalose improves the proliferation and dehydration tolerance of HaCaT cells by inducing proteins involved in cell growth and dehydration protection.

  5. Ataxia-telangiectasia mutated (ATM) deficiency decreases reprogramming efficiency and leads to genomic instability in iPS cells

    Energy Technology Data Exchange (ETDEWEB)

    Kinoshita, Taisuke [Department of Cell Differentiation, The Sakaguchi Laboratory, School of Medicine, Keio University, Tokyo 160-8582 (Japan); Nagamatsu, Go, E-mail: gonag@sc.itc.keio.ac.jp [Department of Cell Differentiation, The Sakaguchi Laboratory, School of Medicine, Keio University, Tokyo 160-8582 (Japan); Precursory Research for Embryonic Science and Technology, Japan Science and Technology Agency, Kawaguchi, Saitama 332-0012 (Japan); Kosaka, Takeo [Department of Urology, School of Medicine, Keio University, Tokyo 160-8582 (Japan); Takubo, Keiyo [Department of Cell Differentiation, The Sakaguchi Laboratory, School of Medicine, Keio University, Tokyo 160-8582 (Japan); Hotta, Akitsu [Precursory Research for Embryonic Science and Technology, Japan Science and Technology Agency, Kawaguchi, Saitama 332-0012 (Japan); Department of Reprogramming Science, Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto (Japan); Ellis, James [Ontario Human iPS Cell Facility, Molecular Genetics, University of Toronto, Developmental and Stem Cell Biology, SickKids, Toronto, Canada MG1L7 (Canada); Suda, Toshio, E-mail: sudato@sc.itc.keio.ac.jp [Department of Cell Differentiation, The Sakaguchi Laboratory, School of Medicine, Keio University, Tokyo 160-8582 (Japan)

    2011-04-08

    Highlights: {yields} iPS cells were induced with a fluorescence monitoring system. {yields} ATM-deficient tail-tip fibroblasts exhibited quite a low reprogramming efficiency. {yields} iPS cells obtained from ATM-deficient cells had pluripotent cell characteristics. {yields} ATM-deficient iPS cells had abnormal chromosomes, which were accumulated in culture. -- Abstract: During cell division, one of the major features of somatic cell reprogramming by defined factors, cells are potentially exposed to DNA damage. Inactivation of the tumor suppressor gene p53 raised reprogramming efficiency but resulted in an increased number of abnormal chromosomes in established iPS cells. Ataxia-telangiectasia mutated (ATM), which is critical in the cellular response to DNA double-strand breaks, may also play an important role during reprogramming. To clarify the function of ATM in somatic cell reprogramming, we investigated reprogramming in ATM-deficient (ATM-KO) tail-tip fibroblasts (TTFs). Although reprogramming efficiency was greatly reduced in ATM-KO TTFs, ATM-KO iPS cells were successfully generated and showed the same proliferation activity as WT iPS cells. ATM-KO iPS cells had a gene expression profile similar to ES cells and WT iPS cells, and had the capacity to differentiate into all three germ layers. On the other hand, ATM-KO iPS cells accumulated abnormal genome structures upon continuous passages. Even with the abnormal karyotype, ATM-KO iPS cells retained pluripotent cell characteristics for at least 20 passages. These data indicate that ATM does participate in the reprogramming process, although its role is not essential.

  6. Ataxia-telangiectasia mutated (ATM) deficiency decreases reprogramming efficiency and leads to genomic instability in iPS cells

    International Nuclear Information System (INIS)

    Kinoshita, Taisuke; Nagamatsu, Go; Kosaka, Takeo; Takubo, Keiyo; Hotta, Akitsu; Ellis, James; Suda, Toshio

    2011-01-01

    Highlights: → iPS cells were induced with a fluorescence monitoring system. → ATM-deficient tail-tip fibroblasts exhibited quite a low reprogramming efficiency. → iPS cells obtained from ATM-deficient cells had pluripotent cell characteristics. → ATM-deficient iPS cells had abnormal chromosomes, which were accumulated in culture. -- Abstract: During cell division, one of the major features of somatic cell reprogramming by defined factors, cells are potentially exposed to DNA damage. Inactivation of the tumor suppressor gene p53 raised reprogramming efficiency but resulted in an increased number of abnormal chromosomes in established iPS cells. Ataxia-telangiectasia mutated (ATM), which is critical in the cellular response to DNA double-strand breaks, may also play an important role during reprogramming. To clarify the function of ATM in somatic cell reprogramming, we investigated reprogramming in ATM-deficient (ATM-KO) tail-tip fibroblasts (TTFs). Although reprogramming efficiency was greatly reduced in ATM-KO TTFs, ATM-KO iPS cells were successfully generated and showed the same proliferation activity as WT iPS cells. ATM-KO iPS cells had a gene expression profile similar to ES cells and WT iPS cells, and had the capacity to differentiate into all three germ layers. On the other hand, ATM-KO iPS cells accumulated abnormal genome structures upon continuous passages. Even with the abnormal karyotype, ATM-KO iPS cells retained pluripotent cell characteristics for at least 20 passages. These data indicate that ATM does participate in the reprogramming process, although its role is not essential.

  7. Endothelial induced EMT in breast epithelial cells with stem cell properties.

    Directory of Open Access Journals (Sweden)

    Valgardur Sigurdsson

    Full Text Available Epithelial to mesenchymal transition (EMT is a critical event in cancer progression and is closely linked to the breast epithelial cancer stem cell phenotype. Given the close interaction between the vascular endothelium and cancer cells, especially at the invasive front, we asked whether endothelial cells might play a role in EMT. Using a 3D culture model we demonstrate that endothelial cells are potent inducers of EMT in D492 an immortalized breast epithelial cell line with stem cell properties. Endothelial induced mesenchymal-like cells (D492M derived from D492, show reduced expression of keratins, a switch from E-Cadherin (E-Cad to N-Cadherin (N-Cad and enhanced migration. Acquisition of cancer stem cell associated characteristics like increased CD44(high/CD24(low ratio, resistance to apoptosis and anchorage independent growth was also seen in D492M cells. Endothelial induced EMT in D492 was partially blocked by inhibition of HGF signaling. Basal-like breast cancer, a vascular rich cancer with stem cell properties and adverse prognosis has been linked with EMT. We immunostained several basal-like breast cancer samples for endothelial and EMT markers. Cancer cells close to the vascular rich areas show no or decreased expression of E-Cad and increased N-Cad expression suggesting EMT. Collectively, we have shown in a 3D culture model that endothelial cells are potent inducers of EMT in breast epithelial cells with stem cell properties. Furthermore, we demonstrate that basal-like breast cancer contains cells with an EMT phenotype, most prominently close to vascular rich areas of these tumors. We conclude that endothelial cells are potent inducers of EMT and may play a role in progression of basal-like breast cancer.

  8. Endothelial induced EMT in breast epithelial cells with stem cell properties.

    Science.gov (United States)

    Sigurdsson, Valgardur; Hilmarsdottir, Bylgja; Sigmundsdottir, Hekla; Fridriksdottir, Agla J R; Ringnér, Markus; Villadsen, Rene; Borg, Ake; Agnarsson, Bjarni A; Petersen, Ole William; Magnusson, Magnus K; Gudjonsson, Thorarinn

    2011-01-01

    Epithelial to mesenchymal transition (EMT) is a critical event in cancer progression and is closely linked to the breast epithelial cancer stem cell phenotype. Given the close interaction between the vascular endothelium and cancer cells, especially at the invasive front, we asked whether endothelial cells might play a role in EMT. Using a 3D culture model we demonstrate that endothelial cells are potent inducers of EMT in D492 an immortalized breast epithelial cell line with stem cell properties. Endothelial induced mesenchymal-like cells (D492M) derived from D492, show reduced expression of keratins, a switch from E-Cadherin (E-Cad) to N-Cadherin (N-Cad) and enhanced migration. Acquisition of cancer stem cell associated characteristics like increased CD44(high)/CD24(low) ratio, resistance to apoptosis and anchorage independent growth was also seen in D492M cells. Endothelial induced EMT in D492 was partially blocked by inhibition of HGF signaling. Basal-like breast cancer, a vascular rich cancer with stem cell properties and adverse prognosis has been linked with EMT. We immunostained several basal-like breast cancer samples for endothelial and EMT markers. Cancer cells close to the vascular rich areas show no or decreased expression of E-Cad and increased N-Cad expression suggesting EMT. Collectively, we have shown in a 3D culture model that endothelial cells are potent inducers of EMT in breast epithelial cells with stem cell properties. Furthermore, we demonstrate that basal-like breast cancer contains cells with an EMT phenotype, most prominently close to vascular rich areas of these tumors. We conclude that endothelial cells are potent inducers of EMT and may play a role in progression of basal-like breast cancer.

  9. Expression of WNT genes in cervical cancer-derived cells: Implication of WNT7A in cell proliferation and migration

    International Nuclear Information System (INIS)

    Ramos-Solano, Moisés; Meza-Canales, Ivan D.; Torres-Reyes, Luis A.; Alvarez-Zavala, Monserrat

    2015-01-01

    According to the multifactorial model of cervical cancer (CC) causation, it is now recognized that other modifications, in addition to Human papillomavirus (HPV) infection, are necessary for the development of this neoplasia. Among these, it has been proposed that a dysregulation of the WNT pathway might favor malignant progression of HPV-immortalized keratinocytes. The aim of this study was to identify components of the WNT pathway differentially expressed in CC vs. non-tumorigenic, but immortalized human keratinocytes. Interestingly, WNT7A expression was found strongly downregulated in cell lines and biopsies derived from CC. Restoration of WNT7A in CC-derived cell lines using a lentiviral gene delivery system or after adding a recombinant human protein decreases cell proliferation. Likewise, WNT7A silencing in non-tumorigenic cells markedly accelerates proliferation. Decreased WNT7A expression was due to hypermethylation at particular CpG sites. To our knowledge, this is the first study reporting reduced WNT7A levels in CC-derived cells and that ectopic WNT7A restoration negatively affects cell proliferation and migration. - Highlights: • WNT7A is expressed in normal keratinocytes or cervical cells without lesion. • WNT7A is significantly reduced in cervical cancer-derived cells. • Restoration of WNT7A expression in HeLa decreases proliferation and cell migration. • Silencing of WNT7A in HaCaT induces an increased proliferation and migration rate. • Decreased WNT7A expression in this model is due to hypermethylation

  10. Expression of WNT genes in cervical cancer-derived cells: Implication of WNT7A in cell proliferation and migration

    Energy Technology Data Exchange (ETDEWEB)

    Ramos-Solano, Moisés, E-mail: mrsolano84@gmail.com [División de Inmunología, Centro de Investigación Biomédica de Occidente (CIBO)-Instituto Mexicano del Seguro Social (IMSS), Guadalajara, Jalisco (Mexico); Programa de Doctorado en Ciencias Biomédica, Centro Universitario de Ciencias de la Salud (CUCS), Universidad de Guadalajara, Guadalajara, Jalisco (Mexico); Meza-Canales, Ivan D., E-mail: imezacanales@ice.mpg.de [Department of Molecular Ecology, Max Planck Institute for Chemical Ecology, 07745 Jena (Germany); Torres-Reyes, Luis A., E-mail: torres_reyes_88@hotmail.com [División de Inmunología, Centro de Investigación Biomédica de Occidente (CIBO)-Instituto Mexicano del Seguro Social (IMSS), Guadalajara, Jalisco (Mexico); Programa de Doctorado en Ciencias Biomédica, Centro Universitario de Ciencias de la Salud (CUCS), Universidad de Guadalajara, Guadalajara, Jalisco (Mexico); Alvarez-Zavala, Monserrat, E-mail: monse_belan@hotmail.com [División de Inmunología, Centro de Investigación Biomédica de Occidente (CIBO)-Instituto Mexicano del Seguro Social (IMSS), Guadalajara, Jalisco (Mexico); Programa de Doctorado en Ciencias Biomédica, Centro Universitario de Ciencias de la Salud (CUCS), Universidad de Guadalajara, Guadalajara, Jalisco (Mexico); and others

    2015-07-01

    According to the multifactorial model of cervical cancer (CC) causation, it is now recognized that other modifications, in addition to Human papillomavirus (HPV) infection, are necessary for the development of this neoplasia. Among these, it has been proposed that a dysregulation of the WNT pathway might favor malignant progression of HPV-immortalized keratinocytes. The aim of this study was to identify components of the WNT pathway differentially expressed in CC vs. non-tumorigenic, but immortalized human keratinocytes. Interestingly, WNT7A expression was found strongly downregulated in cell lines and biopsies derived from CC. Restoration of WNT7A in CC-derived cell lines using a lentiviral gene delivery system or after adding a recombinant human protein decreases cell proliferation. Likewise, WNT7A silencing in non-tumorigenic cells markedly accelerates proliferation. Decreased WNT7A expression was due to hypermethylation at particular CpG sites. To our knowledge, this is the first study reporting reduced WNT7A levels in CC-derived cells and that ectopic WNT7A restoration negatively affects cell proliferation and migration. - Highlights: • WNT7A is expressed in normal keratinocytes or cervical cells without lesion. • WNT7A is significantly reduced in cervical cancer-derived cells. • Restoration of WNT7A expression in HeLa decreases proliferation and cell migration. • Silencing of WNT7A in HaCaT induces an increased proliferation and migration rate. • Decreased WNT7A expression in this model is due to hypermethylation.

  11. Mitigating the Effects of Xuebijing Injection on Hematopoietic Cell Injury Induced by Total Body Irradiation with γ rays by Decreasing Reactive Oxygen Species Levels

    Directory of Open Access Journals (Sweden)

    Deguan Li

    2014-06-01

    Full Text Available Hematopoietic injury is the most common side effect of radiotherapy. However, the methods available for the mitigating of radiation injury remain limited. Xuebijing injection (XBJ is a traditional Chinese medicine used to treat sepsis in the clinic. In this study, we investigated the effects of XBJ on the survival rate in mice with hematopoietic injury induced by γ ray ionizing radiation (IR. Mice were intraperitoneally injected with XBJ daily for seven days after total body irradiation (TBI. Our results showed that XBJ (0.4 mL/kg significantly increased 30-day survival rates in mice exposed to 7.5 Gy TBI. This effect may be attributable to improved preservation of white blood cells (WBCs and hematopoietic cells, given that bone marrow (BM cells from XBJ-treated mice produced more granulocyte-macrophage colony forming units (CFU-GM than that in the 2 Gy/TBI group. XBJ also decreased the levels of reactive oxygen species (ROS by increasing glutathione (GSH and superoxide dismutase (SOD levels in serum and attenuated the increased BM cell apoptosis caused by 2 Gy/TBI. In conclusion, these findings suggest that XBJ enhances the survival rate of irradiated mice and attenuates the effects of radiation on hematopoietic injury by decreasing ROS production in BM cells, indicating that XBJ may be a promising therapeutic candidate for reducing hematopoietic radiation injury.

  12. Balancing Cell Migration with Matrix Degradation Enhances Gene Delivery to Cells Cultured Three-Dimensionally Within Hydrogels

    Science.gov (United States)

    Shepard, Jaclyn A.; Huang, Alyssa; Shikanova, Ariella; Shea, Lonnie D.

    2010-01-01

    In regenerative medicine, hydrogels are employed to fill defects and support the infiltration of cells that can ultimately regenerate tissue. Gene delivery within hydrogels targeting infiltrating cells has the potential to promote tissue formation, but the delivery efficiency of nonviral vectors within hydrogels is low hindering their applicability in tissue regeneration. To improve their functionality, we have conducted a mechanistic study to investigate the contribution of cell migration and matrix degradation on gene delivery. In this report, lipoplexes were entrapped within hydrogels based on poly(ethylene glycol) (PEG) crosslinked with peptides containing matrix metalloproteinase degradable sequences. The mesh size of these hydrogels is substantially less than the size of the entrapped lipoplexes, which can function to retain vectors. Cell migration and transfection were simultaneously measured within hydrogels with varying density of cell adhesion sites (Arg-Gly-Asp peptides) and solids content. Increasing RGD density increased expression levels up to 100-fold, while greater solids content sustained expression levels for 16 days. Increasing RGD density and decreasing solids content increased cell migration, which indicates expression levels increase with increased cell migration. Initially exposing cells to vector resulted in transient expression that declined after 2 days, verifying the requirement of migration to sustain expression. Transfected cells were predominantly located within the population of migrating cells for hydrogels that supported cell migration. Although the small mesh size retained at least 70% of the lipoplexes in the absence of cells after 32 days, the presence of cells decreased retention to 10% after 16 days. These results indicate that vectors retained within hydrogels contact migrating cells, and that persistent cell migration can maintain elevated expression levels. Thus matrix degradation and cell migration are fundamental design

  13. Local differentiation of cell wall matrix polysaccharides in sinuous pavement cells: its possible involvement in the flexibility of cell shape.

    Science.gov (United States)

    Sotiriou, P; Giannoutsou, E; Panteris, E; Galatis, B; Apostolakos, P

    2018-03-01

    The distribution of homogalacturonans (HGAs) displaying different degrees of esterification as well as of callose was examined in cell walls of mature pavement cells in two angiosperm and two fern species. We investigated whether local cell wall matrix differentiation may enable pavement cells to respond to mechanical tension forces by transiently altering their shape. HGA epitopes, identified with 2F4, JIM5 and JIM7 antibodies, and callose were immunolocalised in hand-made or semithin leaf sections. Callose was also stained with aniline blue. The structure of pavement cells was studied with light and transmission electron microscopy (TEM). In all species examined, pavement cells displayed wavy anticlinal cell walls, but the waviness pattern differed between angiosperms and ferns. The angiosperm pavement cells were tightly interconnected throughout their whole depth, while in ferns they were interconnected only close to the external periclinal cell wall and intercellular spaces were developed between them close to the mesophyll. Although the HGA epitopes examined were located along the whole cell wall surface, the 2F4- and JIM5- epitopes were especially localised at cell lobe tips. In fern pavement cells, the contact sites were impregnated with callose and JIM5-HGA epitopes. When tension forces were applied on leaf regions, the pavement cells elongated along the stretching axis, due to a decrease in waviness of anticlinal cell walls. After removal of tension forces, the original cell shape was resumed. The presented data support that HGA epitopes make the anticlinal pavement cell walls flexible, in order to reversibly alter their shape. Furthermore, callose seems to offer stability to cell contacts between pavement cells, as already suggested in photosynthetic mesophyll cells. © 2017 German Society for Plant Sciences and The Royal Botanical Society of the Netherlands.

  14. Adrenaline promotes cell proliferation and increases chemoresistance in colon cancer HT29 cells through induction of miR-155

    International Nuclear Information System (INIS)

    Pu, Jun; Bai, Danna; Yang, Xia; Lu, Xiaozhao; Xu, Lijuan; Lu, Jianguo

    2012-01-01

    Highlights: ► Adrenaline increases colon cancer cell proliferation and its resistance to cisplatin. ► Adrenaline activates NFκB in a dose dependent manner. ► NFκB–miR-155 pathway contributes to cell proliferation and resistance to cisplatin. -- Abstract: Recently, catecholamines have been described as being involved in the regulation of cancer genesis and progression. Here, we reported that adrenaline increased the cell proliferation and decreased the cisplatin induced apoptosis in HT29 cells. Further study found that adrenaline increased miR-155 expression in an NFκB dependent manner. HT29 cells overexpressing miR-155 had a higher cell growth rate and more resistance to cisplatin induced apoptosis. In contrast, HT29 cells overexpressing miR-155 inhibitor displayed decreased cell proliferation and sensitivity to cisplatin induced cell death. In summary, our study here revealed that adrenaline–NFκB–miR-155 pathway at least partially contributes to the psychological stress induced proliferation and chemoresistance in HT29 cells, shedding light on increasing the therapeutic strategies of cancer chemotherapy.

  15. Opioid management strategy decreases admissions in high-utilizing adults with sickle cell disease.

    Science.gov (United States)

    Mager, Amy; Pelot, Kristin; Koch, Kathryn; Miller, Lawrence; Hubler, Collin; Ndifor, Anisah; Coan, Canice; Leonard, Cynthia; Field, Joshua J

    A subset of adults with sickle cell disease (SCD) heavily utilizes the emergency department (ED) and hospital. The objective of our study was to determine the efficacy of a multidisciplinary strategy to address unmet needs in highly utilizing adults with SCD. In a prospective study, adults with SCD with ≥10 admissions per year were assessed by a multidisciplinary team for gaps in medical, social, and psychological care. Thereafter, the team decided upon the subject's predominant domain that drove admissions and instituted an interventional plan. All plans included an opioid management strategy. Preintervention and postintervention admission rate, as well as opioid use, was compared. Twelve subjects were enrolled. Median rate of ED and hospital admissions preintervention was 25 per year. The predominant domains identified were social needs (n = 6), psychological disorder (n = 1), and substance use disorder (n = 5). Multifaceted interventional plans were developed to address a wide range of gaps in care, but an opioid management strategy was the only intervention successfully completed. Even so, when the preintervention versus postintervention admission rate was compared, regardless of the domain, there was a 40 percent decline in hospital admissions (p = 0.03). Consistent with the successful implementation of an opioid management plan, the decrease in admissions was accompanied by a 37 percent decrease in intravenous opioid use (p = 0.02) and 10 percent decrease in oral opioid use (p = 0.04). An opioid management strategy, as part of a larger effort to improve care for high-utilizing adults with SCD, decreased rate of admissions and opioid use.

  16. Sorting of cells of the same size, shape, and cell cycle stage for a single cell level assay without staining

    Directory of Open Access Journals (Sweden)

    Yomo Tetsuya

    2006-06-01

    cell viability but decreases the proliferative ability of the PC12 cells. Conclusion We demonstrated a pretreatment method to collect well-characterized, viable, single cells without using fluorescent labels and without significant damage to the cells. This method is quantitative, rapid, single-step, and yields cells of high purity, making it applicable for a variety of single-cell level analyses.

  17. MS4a4B, a CD20 homologue in T cells, inhibits T cell propagation by modulation of cell cycle.

    Directory of Open Access Journals (Sweden)

    Hui Xu

    2010-11-01

    Full Text Available MS4a4B, a CD20 homologue in T cells, is a novel member of the MS4A gene family in mice. The MS4A family includes CD20, FcεRIβ, HTm4 and at least 26 novel members that are characterized by their structural features: with four membrane-spanning domains, two extracellular domains and two cytoplasmic regions. CD20, FcεRIβ and HTm4 have been found to function in B cells, mast cells and hematopoietic cells respectively. However, little is known about the function of MS4a4B in T cell regulation. We demonstrate here that MS4a4B negatively regulates mouse T cell proliferation. MS4a4B is highly expressed in primary T cells, natural killer cells (NK and some T cell lines. But its expression in all malignant T cells, including thymoma and T hybridoma tested, was silenced. Interestingly, its expression was regulated during T cell activation. Viral vector-driven overexpression of MS4a4B in primary T cells and EL4 thymoma cells reduced cell proliferation. In contrast, knockdown of MS4a4B accelerated T cell proliferation. Cell cycle analysis showed that MS4a4B regulated T cell proliferation by inhibiting entry of the cells into S-G2/M phase. MS4a4B-mediated inhibition of cell cycle was correlated with upregulation of Cdk inhibitory proteins and decreased levels of Cdk2 activity, subsequently leading to inhibition of cell cycle progression. Our data indicate that MS4a4B negatively regulates T cell proliferation. MS4a4B, therefore, may serve as a modulator in the negative-feedback regulatory loop of activated T cells.

  18. MS4a4B, a CD20 homologue in T cells, inhibits T cell propagation by modulation of cell cycle.

    Science.gov (United States)

    Xu, Hui; Yan, Yaping; Williams, Mark S; Carey, Gregory B; Yang, Jingxian; Li, Hongmei; Zhang, Guang-Xian; Rostami, Abdolmohamad

    2010-11-01

    MS4a4B, a CD20 homologue in T cells, is a novel member of the MS4A gene family in mice. The MS4A family includes CD20, FcεRIβ, HTm4 and at least 26 novel members that are characterized by their structural features: with four membrane-spanning domains, two extracellular domains and two cytoplasmic regions. CD20, FcεRIβ and HTm4 have been found to function in B cells, mast cells and hematopoietic cells respectively. However, little is known about the function of MS4a4B in T cell regulation. We demonstrate here that MS4a4B negatively regulates mouse T cell proliferation. MS4a4B is highly expressed in primary T cells, natural killer cells (NK) and some T cell lines. But its expression in all malignant T cells, including thymoma and T hybridoma tested, was silenced. Interestingly, its expression was regulated during T cell activation. Viral vector-driven overexpression of MS4a4B in primary T cells and EL4 thymoma cells reduced cell proliferation. In contrast, knockdown of MS4a4B accelerated T cell proliferation. Cell cycle analysis showed that MS4a4B regulated T cell proliferation by inhibiting entry of the cells into S-G2/M phase. MS4a4B-mediated inhibition of cell cycle was correlated with upregulation of Cdk inhibitory proteins and decreased levels of Cdk2 activity, subsequently leading to inhibition of cell cycle progression. Our data indicate that MS4a4B negatively regulates T cell proliferation. MS4a4B, therefore, may serve as a modulator in the negative-feedback regulatory loop of activated T cells.

  19. Inhibition of macrophage migration inhibitory factor decreases proliferation and cytokine expression in bladder cancer cells

    Directory of Open Access Journals (Sweden)

    Leifheit Erica C

    2004-07-01

    Full Text Available Abstract Background The importance of various inflammatory cytokines in maintaining tumor cell growth and viability is well established. Increased expression of the proinflammatory cytokine macrophage migration inhibitory factor (MIF has previously been associated with various types of adenocarcinoma. Methods MIF IHC was used to localize MIF in human bladder tissue. ELISA and Western blot analysis determined the synthesis and secretion of MIF by human bladder transitional cell carcinoma cells. The effects of MIF inhibitors (high molecular weight hyaluronate (HA, anti-MIF antibody or MIF anti-sense on cell growth and cytokine expression were analyzed. Results Human bladder cancer cells (HT-1376 secrete detectable amounts of MIF protein. Treatment with HA, anti-MIF antibody and MIF anti-sense reduced HT-1376 cell proliferation, MIF protein secretion, MIF gene expression and secreted inflammatory cytokines. Our evidence suggests MIF interacts with the invariant chain, CD74 and the major cell surface receptor for HA, CD44. Conclusions This study is the first to report MIF expression in the human bladder and these findings support a role for MIF in tumor cell proliferation. Since MIF participates in the inflammatory response and bladder cancer is associated with chronic inflammatory conditions, these new findings suggest that neutralizing bladder tumor MIF may serve as a novel therapeutic treatment for bladder carcinoma.

  20. Thioredoxin reductase deficiency potentiates oxidative stress, mitochondrial dysfunction and cell death in dopaminergic cells.

    Directory of Open Access Journals (Sweden)

    Pamela Lopert

    Full Text Available Mitochondria are considered major generators of cellular reactive oxygen species (ROS which are implicated in the pathogenesis of neurodegenerative diseases such as Parkinson's disease (PD. We have recently shown that isolated mitochondria consume hydrogen peroxide (H₂O₂ in a substrate- and respiration-dependent manner predominantly via the thioredoxin/peroxiredoxin (Trx/Prx system. The goal of this study was to determine the role of Trx/Prx system in dopaminergic cell death. We asked if pharmacological and lentiviral inhibition of the Trx/Prx system sensitized dopaminergic cells to mitochondrial dysfunction, increased steady-state H₂O₂ levels and death in response to toxicants implicated in PD. Incubation of N27 dopaminergic cells or primary rat mesencephalic cultures with the Trx reductase (TrxR inhibitor auranofin in the presence of sub-toxic concentrations of parkinsonian toxicants paraquat; PQ or 6-hydroxydopamine; 6OHDA (for N27 cells resulted in a synergistic increase in H₂O₂ levels and subsequent cell death. shRNA targeting the mitochondrial thioredoxin reductase (TrxR2 in N27 cells confirmed the effects of pharmacological inhibition. A synergistic decrease in maximal and reserve respiratory capacity was observed in auranofin treated cells and TrxR2 deficient cells following incubation with PQ or 6OHDA. Additionally, TrxR2 deficient cells showed decreased basal mitochondrial oxygen consumption rates. These data demonstrate that inhibition of the mitochondrial Trx/Prx system sensitizes dopaminergic cells to mitochondrial dysfunction, increased steady-state H₂O₂, and cell death. Therefore, in addition to their role in the production of cellular H₂O₂ the mitochondrial Trx/Prx system serve as a major sink for cellular H₂O₂ and its disruption may contribute to dopaminergic pathology associated with PD.

  1. TNF-α Regulates Mast Cell Functions by Inhibiting Cell Degranulation

    Directory of Open Access Journals (Sweden)

    Yuwei Gao

    2017-11-01

    Full Text Available Background/Aims: The aim of this study was to investigate the involvement of inducible co-stimulatory ligand (ICOSL expression in stimulation of mast cells (MCs by TNF-α and the ability of TNF-α stimulation of MCs to influence CD4+ T cell differentiation and function. The mechanisms underlying TNF-α stimulation of MCs were also explored. Methods: Mast cells and CD4+ T cells were prepared from C57BL/6 mice (aged 6–8 weeks. ICOSL expression by MCs was measured by real-time PCR and flow cytometry, and levels of IL-4, IL-10 and IFN-γ were measured by ELISA. Results: ICOSL expression by MCs was increased by TNF-α stimulation, and resulted in interaction with CD4+ T cells. The IL-4 and IL-10 levels in the co-culture system increased, while IFN-γ levels decreased. Furthermore, CD4+CD25+Foxp3+ T cell proliferation was induced by co-culture with TNF-α-stimulated MCs. The mechanism by which TNF-α stimulated MCs was dependent on the activation of the MAPK signaling pathway. Conclusion: TNF-α upregulated the expression of ICOSL on mast cells via a mechanism that is dependent on MAPK phosphorylation. TNF-α-treated MCs promoted the differentiation of regulatory T cells and induced a shift in cytokine expression from a Th1 to a Th2 profile by up-regulation ICOSL expression and inhibition of MC degranulation. Our study reveals a novel mechanism by which mast cells regulate T cell function.

  2. Ion Channels Involved in Cell Volume Regulation

    DEFF Research Database (Denmark)

    Hoffmann, Else Kay

    2011-01-01

    regulatory ion channels involved, and the mechanisms (cellular signalling pathways) that regulate these channels. Finally, I shall also briefly review current investigations in these two cell lines that focuses on how changes in cell volume can regulate cell functions such as cell migration, proliferation......This mini review outlines studies of cell volume regulation in two closely related mammalian cell lines: nonadherent Ehrlich ascites tumour cells (EATC) and adherent Ehrlich Lettre ascites (ELA) cells. Focus is on the regulatory volume decrease (RVD) that occurs after cell swelling, the volume...

  3. Glucose capped silver nanoparticles induce cell cycle arrest in HeLa cells.

    Science.gov (United States)

    Panzarini, Elisa; Mariano, Stefania; Vergallo, Cristian; Carata, Elisabetta; Fimia, Gian Maria; Mura, Francesco; Rossi, Marco; Vergaro, Viviana; Ciccarella, Giuseppe; Corazzari, Marco; Dini, Luciana

    2017-06-01

    This study aims to determine the interaction (uptake and biological effects on cell viability and cell cycle progression) of glucose capped silver nanoparticles (AgNPs-G) on human epithelioid cervix carcinoma (HeLa) cells, in relation to amount, 2×10 3 or 2×10 4 NPs/cell, and exposure time, up to 48h. The spherical and well dispersed AgNPs (30±5nm) were obtained by using glucose as reducing agent in a green synthesis method that ensures to stabilize AgNPs avoiding cytotoxic soluble silver ions Ag + release. HeLa cells take up abundantly and rapidly AgNPs-G resulting toxic to cells in amount and incubation time dependent manner. HeLa cells were arrested at S and G2/M phases of the cell cycle and subG1 population increased when incubated with 2×10 4 AgNPs-G/cell. Mitotic index decreased accordingly. The dissolution experiments demonstrated that the observed effects were due only to AgNPs-G since glucose capping prevents Ag + release. The AgNPs-G influence on HeLa cells viability and cell cycle progression suggest that AgNPs-G, alone or in combination with chemotherapeutics, may be exploited for the development of novel antiproliferative treatment in cancer therapy. However, the possible influence of the cell cycle on cellular uptake of AgNPs-G and the mechanism of AgNPs entry in cells need further investigation. Copyright © 2017 Elsevier B.V. All rights reserved.

  4. Nutrient Shielding in Clusters of Cells

    Science.gov (United States)

    Lavrentovich, Maxim O.; Koschwanez, John H.; Nelson, David R.

    2014-01-01

    Cellular nutrient consumption is influenced by both the nutrient uptake kinetics of an individual cell and the cells’ spatial arrangement. Large cell clusters or colonies have inhibited growth at the cluster's center due to the shielding of nutrients by the cells closer to the surface. We develop an effective medium theory that predicts a thickness ℓ of the outer shell of cells in the cluster that receives enough nutrient to grow. The cells are treated as partially absorbing identical spherical nutrient sinks, and we identify a dimensionless parameter ν that characterizes the absorption strength of each cell. The parameter ν can vary over many orders of magnitude between different cell types, ranging from bacteria and yeast to human tissue. The thickness ℓ decreases with increasing ν, increasing cell volume fraction ϕ, and decreasing ambient nutrient concentration ψ∞. The theoretical results are compared with numerical simulations and experiments. In the latter studies, colonies of budding yeast, Saccharomyces cerevisiae, are grown on glucose media and imaged under a confocal microscope. We measure the growth inside the colonies via a fluorescent protein reporter and compare the experimental and theoretical results for the thickness ℓ. PMID:23848711

  5. Increased extracellular matrix density decreases MCF10A breast cell acinus formation in 3D culture conditions.

    Science.gov (United States)

    Lance, Amanda; Yang, Chih-Chao; Swamydas, Muthulekha; Dean, Delphine; Deitch, Sandy; Burg, Karen J L; Dréau, Didier

    2016-01-01

    The extracellular matrix (ECM) contributes to the generation and dynamic of normal breast tissue, in particular to the generation of polarized acinar and ductal structures. In vitro 3D culture conditions, including variations in the composition of the ECM, have been shown to directly influence the formation and organization of acinus-like and duct-like structures. Furthermore, the density of the ECM appears to also play a role in the normal mammary tissue and tumour formation. Here we show that the density of the ECM directly influences the number, organization and function of breast acini. Briefly, non-malignant human breast MCF10A cells were incubated in increasing densities of a Matrigel®-collagen I matrix. Elastic moduli near and distant to the acinus structures were measured by atomic force microscopy, and the number of acinus structures was determined. Immunochemistry was used to investigate the expression levels of E-cadherin, laminin, matrix metalloproteinase-14 and ß-casein in MCF10A cells. The modulus of the ECM was significantly increased near the acinus structures and the number of acinus structures decreased with the increase in Matrigel-collagen I density. As evaluated by the expression of laminin, the organization of the acinus structures present was altered as the density of the ECM increased. Increases in both E-cadherin and MMP14 expression by MCF10A cells as ECM density increased were also observed. In contrast, MCF10A cells expressed lower ß-casein levels as the ECM density increased. Taken together, these observations highlight the key role of ECM density in modulating the number, organization and function of breast acini. Copyright © 2013 John Wiley & Sons, Ltd.

  6. Inhibition of IGF-1-Mediated Cellular Migration and Invasion by Migracin A in Ovarian Clear Cell Carcinoma Cells.

    Science.gov (United States)

    Ukaji, Tamami; Lin, Yinzhi; Banno, Kouji; Okada, Shoshiro; Umezawa, Kazuo

    2015-01-01

    Previously we isolated migracin A from a Streptomyces culture filtrate as an inhibitor of cancer cell migration. In the present research, we found that migracin A inhibited migration and invasion of ovarian clear cell carcinoma ES-2 cells. In the course of our mechanistic study, migracin A was shown to enhance vasohibin-1 expression in an angiogenesis array. We also confirmed that it increased the mRNA expression of this protein. Moreover, overexpression of vasohibin-1 lowered the migration but not the invasion of ES-2 cells. Then, we looked for another target protein employing a motility array, and found that migracin A lowered the IGF-1 expression. Knockdown of IGF-1 by siRNA decreased the migration and invasion of ES-2 cells. Migracin A also decreased Akt phosphorylation involved in the downstream signaling. Crosstalk analysis indicated that overexpression of vasohibin-1 decreased the IGF-1 expression. On the other hand, it showed no direct anticancer activity in terms of the ES-2 growth in agar. Migracin A inhibited the migration and IGF-1 expression in not only ES-2 but also another ovarian clear cell carcinoma JHOC-5 cells. In addition, it also inhibited capillary tube formation of human umbilical vein endothelial cells. Since its cytotoxicity is very low, migracin A may be a candidate for an anti-metastasis agent not exhibiting prominent toxicity.

  7. Increased circulating follicular helper T cells with decreased programmed death-1 in chronic renal allograft rejection

    OpenAIRE

    Shi, Jian; Luo, Fengbao; Shi, Qianqian; Xu, Xianlin; He, Xiaozhou; Xia, Ying

    2015-01-01

    Background Chronic antibody-mediated rejection is a major issue that affects long-term renal allograft survival. Since follicular helper T (Tfh) cells promote the development of antigen-specific B cells in alloimmune responses, we investigated the potential roles of Tfh cells, B cells and their alloimmune-regulating molecules in the pathogenesis of chronic renal allograft rejection in this study. Methods The frequency of Tfh, B cells and the levels of their alloimmune-regulating molecules inc...

  8. Differentiation of Dental Pulp Stem Cells into Neuron-Like Cells in Serum-Free Medium

    Directory of Open Access Journals (Sweden)

    Shahrul Hisham Zainal Ariffin

    2013-01-01

    Full Text Available Dental pulp tissue contains dental pulp stem cells (DPSCs. Dental pulp cells (also known as dental pulp-derived mesenchymal stem cells are capable of differentiating into multilineage cells including neuron-like cells. The aim of this study was to examine the capability of DPSCs to differentiate into neuron-like cells without using any reagents or growth factors. DPSCs were isolated from teeth extracted from 6- to 8-week-old mice and maintained in complete medium. The cells from the fourth passage were induced to differentiate by culturing in medium without serum or growth factors. RT-PCR molecular analysis showed characteristics of Cd146+, Cd166+, and Cd31− in DPSCs, indicating that these cells are mesenchymal stem cells rather than hematopoietic stem cells. After 5 days of neuronal differentiation, the cells showed neuron-like morphological changes and expressed MAP2 protein. The activation of Nestin was observed at low level prior to differentiation and increased after 5 days of culture in differentiation medium, whereas Tub3 was activated only after 5 days of neuronal differentiation. The proliferation of the differentiated cells decreased in comparison to that of the control cells. Dental pulp stem cells are induced to differentiate into neuron-like cells when cultured in serum- and growth factor-free medium.

  9. Zebularine exerts its antiproliferative activity through S phase delay and cell death in human malignant mesothelioma cells.

    Science.gov (United States)

    Takemura, Yukitoshi; Satoh, Motohiko; Hatanaka, Kenichi; Kubota, Shunichiro

    2018-04-24

    Malignant mesothelioma is an asbestos-related aggressive tumor and current therapy remains ineffective. Zebularine as a DNA methyltransferase (DNMT) inhibitor has an anti-tumor effect in several human cancer cells. The aim of the present study was to investigate whether zebularine could induce antiproliferative effect in human malignant mesothelioma cells. Zebularine induced cell growth inhibition in a dose-dependent manner. In addition, zebularine dose-dependently decreased expression of DNMT1 in all malignant mesothelioma cells tested. Cell cycle analysis indicated that zebularine induced S phase delay. Zebularine also induced cell death in malignant mesothelioma cells. In contrast, zebularine did not induce cell growth inhibition and cell death in human normal fibroblast cells. These results suggest that zebularine has a potential for the treatment of malignant mesothelioma by inhibiting cell growth and inducing cell death.

  10. Epithelial Cell Cultures

    Directory of Open Access Journals (Sweden)

    Imran S. Chaudhry

    2011-01-01

    Full Text Available The biological effects of only a finite number of tobacco toxins have been studied. Here, we describe exposure of cultures of human bronchial epithelial cells to low concentrations of tobacco carcinogens: nickel sulphate, benzo(bfluoranthene, N-nitrosodiethylamine, and 4-(methylnitrosamino-1-(3-pyridyl-1-butanone (NNK. After a 24-hour exposure, EGFR was expressed in cell membrane and cytoplasm, BCL-2 was expressed only in the irregular nuclei of large atypical cells, MKI67 was expressed in nuclei with no staining in larger cells, cytoplasmic BIRC5 with stronger nuclear staining was seen in large atypical cells, and nuclear TP53 was strongly expressed in all cells. After only a 24-hour exposure, cells exhibited atypical nuclear and cytoplasmic features. After a 48-hour exposure, EGFR staining was localized to the nucleus, BCL-2 was slightly decreased in intensity, BIRC5 was localized to the cytoplasm, and TP53 staining was increased in small and large cells. BCL2L1 was expressed in both the cytoplasm and nuclei of cells at 24- and 48-hour exposures. We illustrate that short-termexposure of a bronchial epithelial cell line to smoking-equivalent concentrations of tobacco carcinogens alters the expression of key proliferation regulatory genes, EGFR, BCL-2, BCL2L1, BIRC5, TP53, and MKI67, similar to that reported in biopsy specimens of pulmonary epithelium described to be preneoplastic lesions.

  11. Bioactive compounds from crocodile (Crocodylus siamensis) white blood cells induced apoptotic cell death in hela cells.

    Science.gov (United States)

    Patathananone, Supawadee; Thammasirirak, Sompong; Daduang, Jureerut; Chung, Jing Gung; Temsiripong, Yosapong; Daduang, Sakda

    2016-08-01

    Crocodile (Crocodylus siamensis) white blood cell extracts (WBCex) were examined for anticancer activity in HeLa cell lines using the MTT assay. The percentage viability of HeLa cells significantly deceased after treatment with WBCex in a dose- and time-dependent manner. The IC50 dose was suggested to be approximately 225 μg/mL protein. Apoptotic cell death occurred in a time-dependent manner based on investigation by flow cytometry using annexin V-FITC and PI staining. DAPI nucleic acid staining indicated increased chromatin condensation. Caspase-3, -8 and -9 activities also increased, suggesting the induction of the caspase-dependent apoptotic pathway. Furthermore, the mitochondrial membrane potential (ΔΨm ) of HeLa cells was lost as a result of increasing levels of Bax and reduced levels of Bcl-2, Bcl-XL, Bcl-Xs, and XIAP. The decreased ΔΨm led to the release of cytochrome c and the activation of caspase-9 and -3. Apoptosis-inducing factor translocated into the nuclei, and endonuclease G (Endo G) was released from the mitochondria. These results suggest that anticancer agents in WBCex can induce apoptosis in HeLa cells via both caspase-dependent and -independent pathways. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 986-997, 2016. © 2015 Wiley Periodicals, Inc.

  12. IL13Rα2 siRNA inhibited cell proliferation, induced cell apoptosis, and suppressed cell invasion in papillary thyroid carcinoma cells

    Directory of Open Access Journals (Sweden)

    Gu MJ

    2018-03-01

    Full Text Available Mingjun Gu Department of Endocrinology, Shanghai Gongli Hospital, The Second Military Medical University, Shanghai, People’s Republic of China Aim: Papillary thyroid carcinoma (PTC is the most common type of thyroid cancer. Infiltrative growth and metastasis are the two most intractable characteristics of PTC. Interleukin-13 receptor α2 (IL13Rα2 with high affinity for Th2-derived cytokine IL-13 has been reported to be overexpressed in several tumors. In this study, an analysis of IL13Rα2 expression in PTC and matched paracancerous tissues was undertaken, and its biologic functions in PTC were assessed. Methods: IL13Rα2 and vascular endothelial growth factor (VEGF expression were detected by using real-time polymerase chain reaction and immunohistochemistry analyses. Cell proliferation, invasion, apoptosis, and caspase activity were measured with the Cell Counting Kit-8, Transwell, flow cytometry analyses, and biochemistry assay, respectively. Results: Upregulation of IL13Rα2 and VEGF was observed in PTC tissues compared with matched paracancerous tissues. Pearson’s correlation analysis indicated that IL13Rα2 mRNA level in the tested PTC tissues was positively correlated with VEGF mRNA level. Besides, inhibited cell proliferation, induced cell apoptosis, and suppressed cell invasion were detected in IL13Rα2-silenced TPC-1 cells. Increased activity of Caspase 3 and Caspase 9, along with elevated cleaved Caspase 3 and poly (ADP-ribose polymerase indicated the signal pathway of cell apoptosis induced by IL13Rα2 siRNA. In addition, downregulated metastasis- and angiogenesis-related proteins VEGF, VEGFR2, MMP2, and MMP9 indicated the decreased number of invading cells after knockdown of IL13Rα2. Conclusion: The results demonstrate that IL13Rα2 plays an important role in the progress of PTC. IL13Rα2 knockdown in PTC cells inhibited cell proliferation, induced cell apoptosis, and suppressed cell invasion. These data suggest that IL13Rα2

  13. Tofacitinib induces G1 cell-cycle arrest and inhibits tumor growth in Epstein-Barr virus-associated T and natural killer cell lymphoma cells.

    Science.gov (United States)

    Ando, Shotaro; Kawada, Jun-Ichi; Watanabe, Takahiro; Suzuki, Michio; Sato, Yoshitaka; Torii, Yuka; Asai, Masato; Goshima, Fumi; Murata, Takayuki; Shimizu, Norio; Ito, Yoshinori; Kimura, Hiroshi

    2016-11-22

    Epstein-Barr virus (EBV) infects not only B cells, but also T cells and natural killer (NK) cells, and is associated with T or NK cell lymphoma. These lymphoid malignancies are refractory to conventional chemotherapy. We examined the activation of the JAK3/STAT5 pathway in EBV-positive and -negative B, T and NK cell lines and in cell samples from patients with EBV-associated T cell lymphoma. We then evaluated the antitumor effects of the selective JAK3 inhibitor, tofacitinib, against these cell lines in vitro and in a murine xenograft model. We found that all EBV-positive T and NK cell lines and patient samples tested displayed activation of the JAK3/STAT5 pathway. Treatment of these cell lines with tofacitinib reduced the levels of phospho-STAT5, suppressed proliferation, induced G1 cell-cycle arrest and decreased EBV LMP1 and EBNA1 expression. An EBV-negative NK cell line was also sensitive to tofacitinib, whereas an EBV-infected NK cell line was more sensitive to tofacitinib than its parental line. Tofacitinib significantly inhibited the growth of established tumors in NOG mice. These findings suggest that tofacitinib may represent a useful therapeutic agent for patients with EBV-associated T and NK cell lymphoma.

  14. p53 inactivation decreases dependence on estrogen/ERK signalling for proliferation but promotes EMT and susceptility to 3-bromopyruvate in ERα+ breast cancer MCF-7 cells.

    Science.gov (United States)

    Rieber, Manuel; Strasberg-Rieber, Mary

    2014-03-15

    Most breast cancers express the estrogen receptor alpha (ERα(+)), harbor wt TP53, depend on estrogen/ERK signalling for proliferation, and respond to anti-estrogens. However, concomittant activation of the epidermal growth factor receptor (EGFR)/MEK pathway promotes resistance by decreasing estrogen dependence. Previously, we showed that retroviral transduction of mutant p53 R175H into wt TP53 ERα(+) MCF-7 cells induces epidermal growth factor (EGF)-independent proliferation, activation of the EGF receptor (p-EGFR) and some characteristics of epithelial-mesenchymal transition (EMT). To investigate whether p53 inactivation augments ERα(+) cell proliferation in response to restrictive estradiol, chemical MEK inhibition or metabolic inhibitors. Introduction of mutant p53 R175H lowered expression of p53-dependent PUMA and p21WAF1, decreased E-cadherin and cytokeratin 18 associated with EMT, but increased the % of proliferating ERα(+)/Ki67 cells, diminishing estrogen dependence. These cells also exhibited higher proliferation in the presence of MEK-inhibitor UO126, reciprocally correlating with preferential susceptibility to the pyruvate analog 3-bromopyruvate (3-BrPA) without a comparable response to 2-deoxyglucose. p53 siRNA silencing by electroporation in wt TP53 MCF-7 cells also decreased estrogen dependence and response to MEK inhibition, while also conferring susceptibility to 3-BrPA. (a) ERα(+) breast cancer cells dysfunctional for TP53 which proliferate irrespective of low estrogen and chemical MEK inhibition are likely to increase metabolic consumption becoming increasingly susceptible to 3-BrPA; (b) targeting the pyruvate pathway may improve response to endocrine therapy in ERα(+) breast cancer with p53 dysfunction. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

  15. Barium inhibits arsenic-mediated apoptotic cell death in human squamous cell carcinoma cells.

    Science.gov (United States)

    Yajima, Ichiro; Uemura, Noriyuki; Nizam, Saika; Khalequzzaman, Md; Thang, Nguyen D; Kumasaka, Mayuko Y; Akhand, Anwarul A; Shekhar, Hossain U; Nakajima, Tamie; Kato, Masashi

    2012-06-01

    Our fieldwork showed more than 1 μM (145.1 μg/L) barium in about 3 μM (210.7 μg/L) arsenic-polluted drinking well water (n = 72) in cancer-prone areas in Bangladesh, while the mean concentrations of nine other elements in the water were less than 3 μg/L. The types of cancer include squamous cell carcinomas (SCC). We hypothesized that barium modulates arsenic-mediated biological effects, and we examined the effect of barium (1 μM) on arsenic (3 μM)-mediated apoptotic cell death of human HSC-5 and A431 SCC cells in vitro. Arsenic promoted SCC apoptosis with increased reactive oxygen species (ROS) production and JNK1/2 and caspase-3 activation (apoptotic pathway). In contrast, arsenic also inhibited SCC apoptosis with increased NF-κB activity and X-linked inhibitor of apoptosis protein (XIAP) expression level and decreased JNK activity (antiapoptotic pathway). These results suggest that arsenic bidirectionally promotes apoptotic and antiapoptotic pathways in SCC cells. Interestingly, barium in the presence of arsenic increased NF-κB activity and XIAP expression and decreased JNK activity without affecting ROS production, resulting in the inhibition of the arsenic-mediated apoptotic pathway. Since the anticancer effect of arsenic is mainly dependent on cancer apoptosis, barium-mediated inhibition of arsenic-induced apoptosis may promote progression of SCC in patients in Bangladesh who keep drinking barium and arsenic-polluted water after the development of cancer. Thus, we newly showed that barium in the presence of arsenic might inhibit arsenic-mediated cancer apoptosis with the modulation of the balance between arsenic-mediated promotive and suppressive apoptotic pathways.

  16. Altered ganglioside GD3 in HeLa cells might influence the cytotoxic abilities of NK cells

    OpenAIRE

    Lee, Wen-Chi; Lee, Wen-Ling; Shyong, Wen-Yuann; Yang, Lin-Wei; Ko, Min-Chun; Yeh, Chang-Ching; Edmond Hsieh, Shie-Liang; Wang, Peng-Hui

    2012-01-01

    Objective: Previously, we found that altered sialidases in HeLa cells in a natural killer-HeLa (NK-HeLa) coculture system contributed to the decreased cytotoxic ability of NK cells. However, changes that occur in the glycosylation of the HeLa cells in the NK-HeLa coculture system remain unknown. Materials and Methods: An NK-HeLa coculture system was used to examine the changes that occur in the gangliosides of HeLa cells. Results: GD3 expression in HeLa cells was significantly increased...

  17. Unexpected features of exponentially growing Tobacco Bright Yellow-2 cell suspension culture in relation to excreted extracellular polysaccharides and cell wall composition.

    Science.gov (United States)

    Issawi, Mohammad; Muhieddine, Mohammad; Girard, Celine; Sol, Vincent; Riou, Catherine

    2017-10-01

    This article presents a new insight about TBY-2 cells; from extracellular polysaccharides secretion to cell wall composition during cell suspension culture. In the medium of cells taken 2 days after dilution (end of lag phase), a two unit pH decrease from 5.38 to 3.45 was observed and linked to a high uronic acid (UA) amount secretion (47.8%) while, in 4 and 7 day-old spent media, pH increased and UA amounts decreased 35.6 and 42.3% UA, respectively. To attain deeper knowledge of the putative link between extracellular polysaccharide excretion and cell wall composition, we determined cell wall UA and neutral sugar composition of cells from D2 to D12 cultures. While cell walls from D2 and D3 cells contained a large amount of uronic acid (twice as much as the other analysed cell walls), similar amounts of neutral sugar were detected in cells from lag to end of exponential phase cells suggesting an enriched pectin network in young cultures. Indeed, monosaccharide composition analysis leads to an estimated percentage of pectins of 56% for D3 cell wall against 45% D7 cell walls indicating that the cells at the mid-exponential growth phase re-organized their cell wall linked to a decrease in secreted UA that finally led to a stabilization of the spent medium pH to 5.4. In conclusion, TBY-2 cell suspension from lag to stationary phase showed cell wall remodeling that could be of interest in drug interaction and internalization study.

  18. Cell survival studies using ultrasoft x rays

    International Nuclear Information System (INIS)

    Schillaci, M.E.; Raju, M.R.; Carpenter, S.; Cornforth, M.; Wilder, M.

    1987-01-01

    Cell survival was studied for V79 hamster, 10T1/2 mouse, and human skin fibroblast cell lines, using carbon K (0.28 keV), copper K (8.0 keV), and 250 kVp x rays. Because of the rapid attenuation of the carbon x rays, cellular dimensions at the time of exposure were measured using optical and electron microscopy, and frequency distributions of mean dose absorbed by the cell nucleus were obtained. The results indicate that the differences in cell killing between ultra-soft and hard x rays may depend on the nuclear thickness of the cells. Studies of the effects of hypoxia on V79 and 10T1/2 cells using carbon K, aluminum K (1.5 keV), and copper K x rays show decreasing OER values with decreasing x-ray energy and no difference between the two cell lines. Age response studies with V79 cells show similar cell-cycle variation of survival for carbon K and aluminum K x rays as for hard x rays

  19. Gallic acid modulates phenotypic behavior and gene expression in oral squamous cell carcinoma cells by interfering with leptin pathway.

    Science.gov (United States)

    Santos, Eliane Macedo Sobrinho; da Rocha, Rogério Gonçalves; Santos, Hércules Otacílio; Guimarães, Talita Antunes; de Carvalho Fraga, Carlos Alberto; da Silveira, Luiz Henrique; Batista, Paulo Ricardo; de Oliveira, Paulo Sérgio Lopes; Melo, Geraldo Aclécio; Santos, Sérgio Henrique; de Paula, Alfredo Maurício Batista; Guimarães, André Luiz Sena; Farias, Lucyana Conceição

    2018-01-01

    Gallic acid is a polyphenolic compost appointed to interfere with neoplastic cells behavior. Evidence suggests an important role of leptin in carcinogenesis pathways, inducing a proliferative phenotype. We investigated the potential of gallic acid to modulate leptin-induced cell proliferation and migration of oral squamous cell carcinoma cell lines. The gallic acid effect on leptin secretion by oral squamous cell carcinoma cells, as well as the underlying molecular mechanisms, was also assessed. For this, we performed proliferation, migration, immunocytochemical and qPCR assays. The expression levels of cell migration-related genes (MMP2, MMP9, Col1A1, and E-cadherin), angiogenesis (HIF-1α, mir210), leptin signaling (LepR, p44/42 MAPK), apoptosis (casp-3), and secreted leptin levels by oral squamous cell carcinoma cells were also measured. Gallic acid decreased proliferation and migration of leptin-treated oral squamous cell carcinoma cells, and reduced mRNA expression of MMP2, MMP9, Col1A1, mir210, but did not change HIF-1α. Gallic acid decreased levels of leptin secreted by oral squamous cell carcinoma cells, accordingly with downregulation of p44/42 MAPK expression. Thus, gallic acid appears to break down neoplastic phenotype of oral squamous cell carcinoma cells by interfering with leptin pathway. Copyright © 2017 Elsevier GmbH. All rights reserved.

  20. Influence of Cell Detachment on the Respiration Rate of Tumor and Endothelial Cells

    Science.gov (United States)

    Danhier, Pierre; Copetti, Tamara; De Preter, Géraldine; Leveque, Philippe; Feron, Olivier; Jordan, Bénédicte F.; Sonveaux, Pierre; Gallez, Bernard

    2013-01-01

    Cell detachment is a procedure routinely performed in cell culture and a necessary step in many biochemical assays including the determination of oxygen consumption rates (OCR) in vitro. In vivo, cell detachment has been shown to exert profound metabolic influences notably in cancer but also in other pathologies, such as retinal detachment for example. In the present study, we developed and validated a new technique combining electron paramagnetic resonance (EPR) oximetry and the use of cytodex 1 and collagen-coated cytodex 3 dextran microbeads, which allowed the unprecedented comparison of the OCR of adherent and detached cells with high sensitivity. Hence, we demonstrated that both B16F10 melanoma cells and human umbilical vein endothelial cells (HUVEC) experience strong OCR decrease upon trypsin or collagenase treatments. The reduction of cell oxygen consumption was more pronounced with a trypsin compared to a collagenase treatment. Cells remaining in suspension also encounter a marked intracellular ATP depletion and an increase in the lactate production/glucose uptake ratio. These findings highlight the important influence exerted by cell adhesion/detachment on cell respiration, which can be probed with the unprecedented experimental assay that was developed and validated in this study. PMID:23382841

  1. Influence of cell detachment on the respiration rate of tumor and endothelial cells.

    Science.gov (United States)

    Danhier, Pierre; Copetti, Tamara; De Preter, Géraldine; Leveque, Philippe; Feron, Olivier; Jordan, Bénédicte F; Sonveaux, Pierre; Gallez, Bernard

    2013-01-01

    Cell detachment is a procedure routinely performed in cell culture and a necessary step in many biochemical assays including the determination of oxygen consumption rates (OCR) in vitro. In vivo, cell detachment has been shown to exert profound metabolic influences notably in cancer but also in other pathologies, such as retinal detachment for example. In the present study, we developed and validated a new technique combining electron paramagnetic resonance (EPR) oximetry and the use of cytodex 1 and collagen-coated cytodex 3 dextran microbeads, which allowed the unprecedented comparison of the OCR of adherent and detached cells with high sensitivity. Hence, we demonstrated that both B16F10 melanoma cells and human umbilical vein endothelial cells (HUVEC) experience strong OCR decrease upon trypsin or collagenase treatments. The reduction of cell oxygen consumption was more pronounced with a trypsin compared to a collagenase treatment. Cells remaining in suspension also encounter a marked intracellular ATP depletion and an increase in the lactate production/glucose uptake ratio. These findings highlight the important influence exerted by cell adhesion/detachment on cell respiration, which can be probed with the unprecedented experimental assay that was developed and validated in this study.

  2. Tumor budding cells, cancer stem cells and epithelial-mesenchymal transition-type cells in pancreatic cancer

    International Nuclear Information System (INIS)

    Karamitopoulou, Eva

    2013-01-01

    Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal cancers with a 5-year survival rate of less than 5%. Moreover, PDAC escapes early detection and resists treatment. Multiple combinations of genetic alterations are known to occur in PDAC including mutational activation of KRAS, inactivation of p16/CDKN2A and SMAD4 (DPC4) and dysregulation of PTEN/PI3K/AKT signaling. Through their interaction with Wingless-INT pathway, the downstream molecules of these pathways have been implicated in the promotion of epithelial–mesenchymal transition (EMT). Emerging evidence has demonstrated that cancer stem cells (CSCs), small populations of which have been identified in PDAC, and EMT-type cells play critical roles in drug resistance, invasion, and metastasis in pancreatic cancer. EMT may be histologically represented by the presence of tumor budding which is described as the occurrence of single tumor cells or small clusters (<5) of dedifferentiated cells at the invasive front of gastrointestinal (including colorectal, oesophageal, gastric, and ampullary) carcinomas and is linked to poor prognosis. Tumor budding has recently been shown to occur frequently in PDAC and to be associated with adverse clinicopathological features and decreased disease-free and overall survival. The aim of this review is to present a short overview on the morphological and molecular aspects that underline the relationship between tumor budding cells, CSCs, and EMT-type cells in PDAC.

  3. Tumor budding cells, cancer stem cells and epithelial-mesenchymal transition-type cells in pancreatic cancer.

    Science.gov (United States)

    Karamitopoulou, Eva

    2012-01-01

    Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal cancers with a 5-year survival rate of less than 5%. Moreover, PDAC escapes early detection and resists treatment. Multiple combinations of genetic alterations are known to occur in PDAC including mutational activation of KRAS, inactivation of p16/CDKN2A and SMAD4 (DPC4) and dysregulation of PTEN/PI3K/AKT signaling. Through their interaction with Wingless-INT pathway, the downstream molecules of these pathways have been implicated in the promotion of epithelial-mesenchymal transition (EMT). Emerging evidence has demonstrated that cancer stem cells (CSCs), small populations of which have been identified in PDAC, and EMT-type cells play critical roles in drug resistance, invasion, and metastasis in pancreatic cancer. EMT may be histologically represented by the presence of tumor budding which is described as the occurrence of single tumor cells or small clusters (<5) of dedifferentiated cells at the invasive front of gastrointestinal (including colorectal, oesophageal, gastric, and ampullary) carcinomas and is linked to poor prognosis. Tumor budding has recently been shown to occur frequently in PDAC and to be associated with adverse clinicopathological features and decreased disease-free and overall survival. The aim of this review is to present a short overview on the morphological and molecular aspects that underline the relationship between tumor budding cells, CSCs, and EMT-type cells in PDAC.

  4. Tumor Budding Cells, Cancer Stem Cells and Epithelial-Mesenchymal Transition-type Cells in Pancreatic Cancer

    Directory of Open Access Journals (Sweden)

    Eva eKaramitopoulou

    2013-01-01

    Full Text Available Pancreatic ductal adenocarcinoma (PDAC is one of the most lethal cancers with a 5-year survival rate of less than 5%. Moreover, PDAC escapes early detection and resists treatment. Multiple combinations of genetic alterations are known to occur in PDAC including mutational activation of KRAS, inactivation of p16/CDKN2A and SMAD4 (DPC4 and dysregulation of PTEN/PI3K/AKT signaling. Through their interaction with WNT pathway, the downstream molecules of these pathways have been implicated in the promotion of epithelial-mesenchymal transition (EMT. Emerging evidence has demonstrated that cancer stem cells (CSCs, small populations of which have been identified in PDAC, and EMT-type cells play critical roles in drug resistance, invasion and metastasis in pancreatic cancer. EMT may be histologically represented by the presence of tumor budding which is described as the occurrence of single tumor cells or small clusters (<5 of dedifferentiated cells at the invasive front of gastrointestinal (including colorectal, oesophageal, gastric and ampullary carcinomas and is linked to poor prognosis. Tumor budding has recently been shown to occur frequently in PDAC and to be associated with adverse clinicopathological features and decreased disease-free and overall survival. The aim of this review is to present a short overview on the morphological and molecular aspects that underline the relationship between tumor budding cells, CSCs and EMT-type cells in PDAC.

  5. Survivin Modulates Squamous Cell Carcinoma-Derived Stem-Like Cell Proliferation, Viability and Tumor Formation in Vivo

    Directory of Open Access Journals (Sweden)

    Roberta Lotti

    2016-01-01

    Full Text Available Squamous Cell Carcinoma-derived Stem-like Cells (SCC-SC originate from alterations in keratinocyte stem cells (KSC gene expression and sustain tumor development, invasion and recurrence. Since survivin, a KSC marker, is highly expressed in SCC-SC, we evaluate its role in SCC-SC cell growth and SCC models. Survivin silencing by siRNA decreases clonal growth of SCC keratinocytes and viability of total, rapidly adhering (RAD and non-RAD (NRAD cells from primary SCC. Similarly, survivin silencing reduces the expression of stem cell markers (OCT4, NOTCH1, CD133, β1-integrin, while it increases the level of differentiation markers (K10, involucrin. Moreover, survivin silencing improves the malignant phenotype of SCC 3D-reconstruct, as demonstrated by reduced epidermal thickness, lower Ki-67 positive cell number, and decreased expression of MMP9 and psoriasin. Furthermore, survivin depletion by siRNA in RasG12V-IκBα-derived tumors leads to smaller tumor formation characterized by lower mitotic index and reduced expression of the tumor-associated marker HIF1α, VEGF and CD51. Therefore, our results indicate survivin as a key gene in regulating SCC cancer stem cell formation and cSCC development.

  6. Pigment Cell Differentiation in Sea Urchin Blastula-Derived Primary Cell Cultures

    Science.gov (United States)

    Ageenko, Natalya V.; Kiselev, Konstantin V.; Dmitrenok, Pavel S.; Odintsova, Nelly A.

    2014-01-01

    The quinone pigments of sea urchins, specifically echinochrome and spinochromes, are known for their effective antioxidant, antibacterial, antifungal, and antitumor activities. We developed in vitro technology for inducing pigment differentiation in cell culture. The intensification of the pigment differentiation was accompanied by a simultaneous decrease in cell proliferation. The number of pigment cells was two-fold higher in the cells cultivated in the coelomic fluids of injured sea urchins than in those intact. The possible roles of the specific components of the coelomic fluids in the pigment differentiation process and the quantitative measurement of the production of naphthoquinone pigments during cultivation were examined by MALDI and electrospray ionization mass spectrometry. Echinochrome A and spinochrome E were produced by the cultivated cells of the sand dollar Scaphechinus mirabilis in all tested media, while only spinochromes were found in the cultivated cells of another sea urchin, Strongylocentrotus intermedius. The expression of genes associated with the induction of pigment differentiation was increased in cells cultivated in the presence of shikimic acid, a precursor of naphthoquinone pigments. Our results should contribute to the development of new techniques in marine biotechnology, including the generation of cell cultures producing complex bioactive compounds with therapeutic potential. PMID:24979272

  7. Activation of mammalian target of rapamycin signaling promotes cell cycle progression and protects cells from apoptosis in mantle cell lymphoma.

    Science.gov (United States)

    Peponi, Evangelia; Drakos, Elias; Reyes, Guadalupe; Leventaki, Vasiliki; Rassidakis, George Z; Medeiros, L Jeffrey

    2006-12-01

    Mantle cell lymphoma (MCL) is characterized by the t(11;14) and cyclin D1 overexpression. However, additional molecular events are most likely required for oncogenesis, possibly through cell cycle and apoptosis deregulation. We hypothesized that mammalian target of rapamycin (mTOR) is activated in MCL and contributes to tumor proliferation and survival. In MCL cell lines, pharmacological inhibition of the phosphoinositide 3-kinase/AKT pathway was associated with decreased phosphorylation (activation) of mTOR and its downstream targets phosphorylated (p)-4E-BP1, p-p70S6 kinase, and p-ribosomal protein S6, resulting in apoptosis and cell cycle arrest. These changes were associated with down-regulation of cyclin D1 and the anti-apoptotic proteins cFLIP, BCL-XL, and MCL-1. Furthermore, silencing of mTOR expression using mTOR-specific short interfering RNA decreased phosphorylation of mTOR signaling proteins and induced cell cycle arrest and apoptosis. Silencing of eukaryotic initiation factor (eIF4E), a downstream effector of mTOR, recapitulated these results. We also assessed mTOR signaling in MCL tumors using immunohistochemical methods and a tissue microarray: 10 of 30 (33%) expressed Ser473p-AKT, 13 of 21 (62%) Ser2448p-mTOR, 22 of 22 (100%) p-p70S6K, and 5 of 20 (25%) p-ribosomal protein S6. Total eIF4E binding protein 1 and eukaryotic initiation factor 4E were expressed in 13 of 14 (93%) and 16 of 29 (55%) MCL tumors, respectively. These findings suggest that the mTOR signaling pathway is activated and may contribute to cell cycle progression and tumor cell survival in MCL.

  8. Radioresistant cell strain of human fibrosarcoma cells obtained after long-term exposure to X-rays

    International Nuclear Information System (INIS)

    Wei, K.; Kodym, R.

    1998-01-01

    A radioresistant cell strain from human fibrosarcoma HT1080 has been obtained after prolonged exposure to x-rays for 7 months (2 Gy per day, 5 days per week). This new strain, HT1080R, differs from HT1080 in a significantly increased ability of clonogenical survival, with coefficient α decreasing from 0.161 to 0.123 Gy -1 and coefficient β decreasing from 0.0950 to 0.0565 Gy -2 . Furthermore, the radioresistance of HT1080R proved to be stable in long-term passaged cultures as well as in frozen samples. Differences between the two cell lines are also observed in the G-banded karyotype; the new cell line shows monosomy of chromosome 17 and loss of 5p + and 11q + present in the parental cells. These data suggest that the radioresistance may have been caused by radiation-induced cell mutation and that the resistant cells may have been selected by repeated irradiations. In order to characterize this new strain, the ability of the cells to rejoin DNA double-strand breaks, the cell cycle distribution and the amount of apoptosis after irradiation have been estimated; however, no differences are observed between these two cell strains. Although the mechanism of the elevated radioresistance remains unknown, this pair of cell strains can provide a new model system for further investigations with regard to the mechanisms of cellular radioresistance. The results also show that any type of irradiation similar to the schedules used in radiotherapy can lead to the formation and selection of more radioresistant cell clones in vitro, a phenomenon with possible implications for radiotherapy. (orig.)

  9. Cell-free culture supernatant of Bifidobacterium breve CNCM I-4035 decreases pro-inflammatory cytokines in human dendritic cells challenged with Salmonella typhi through TLR activation.

    Science.gov (United States)

    Bermudez-Brito, Miriam; Muñoz-Quezada, Sergio; Gomez-Llorente, Carolina; Matencio, Esther; Bernal, Maria J; Romero, Fernando; Gil, Angel

    2013-01-01

    Dendritic cells (DCs) constitute the first point of contact between gut commensals and our immune system. Despite growing evidence of the immunomodulatory effects of probiotics, the interactions between the cells of the intestinal immune system and bacteria remain largely unknown. Indeed,, the aim of this work was to determine whether the probiotic Bifidobacterium breve CNCM I-4035 and its cell-free culture supernatant (CFS) have immunomodulatory effects in human intestinal-like dendritic cells (DCs) and how they respond to the pathogenic bacterium Salmonella enterica serovar Typhi, and also to elucidate the molecular mechanisms involved in these interactions. Human DCs were directly challenged with B. breve/CFS, S. typhi or a combination of these stimuli for 4 h. The expression pattern of genes involved in Toll-like receptor (TLR) signaling pathway and cytokine secretion was analyzed. CFS decreased pro-inflammatory cytokines and chemokines in human intestinal DCs challenged with S. typhi. In contrast, the B. breve CNCM I-4035 probiotic strain was a potent inducer of the pro-inflammatory cytokines and chemokines tested, i.e., TNF-α, IL-8 and RANTES, as well as anti-inflammatory cytokines including IL-10. CFS restored TGF-β levels in the presence of Salmonella. Live B.breve and its supernatant enhanced innate immune responses by the activation of TLR signaling pathway. These treatments upregulated TLR9 gene transcription. In addition, CFS was a more potent inducer of TLR9 expression than the probiotic bacteria in the presence of S. typhi. Expression levels of CASP8 and IRAK4 were also increased by CFS, and both treatments induced TOLLIP gene expression. Our results indicate that the probiotic strain B. breve CNCM I-4035 affects the intestinal immune response, whereas its supernatant exerts anti-inflammatory effects mediated by DCs. This supernatant may protect immune system from highly infectious agents such as Salmonella typhi and can down-regulate pro

  10. Cell-free culture supernatant of Bifidobacterium breve CNCM I-4035 decreases pro-inflammatory cytokines in human dendritic cells challenged with Salmonella typhi through TLR activation.

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    Miriam Bermudez-Brito

    Full Text Available Dendritic cells (DCs constitute the first point of contact between gut commensals and our immune system. Despite growing evidence of the immunomodulatory effects of probiotics, the interactions between the cells of the intestinal immune system and bacteria remain largely unknown. Indeed,, the aim of this work was to determine whether the probiotic Bifidobacterium breve CNCM I-4035 and its cell-free culture supernatant (CFS have immunomodulatory effects in human intestinal-like dendritic cells (DCs and how they respond to the pathogenic bacterium Salmonella enterica serovar Typhi, and also to elucidate the molecular mechanisms involved in these interactions. Human DCs were directly challenged with B. breve/CFS, S. typhi or a combination of these stimuli for 4 h. The expression pattern of genes involved in Toll-like receptor (TLR signaling pathway and cytokine secretion was analyzed. CFS decreased pro-inflammatory cytokines and chemokines in human intestinal DCs challenged with S. typhi. In contrast, the B. breve CNCM I-4035 probiotic strain was a potent inducer of the pro-inflammatory cytokines and chemokines tested, i.e., TNF-α, IL-8 and RANTES, as well as anti-inflammatory cytokines including IL-10. CFS restored TGF-β levels in the presence of Salmonella. Live B.breve and its supernatant enhanced innate immune responses by the activation of TLR signaling pathway. These treatments upregulated TLR9 gene transcription. In addition, CFS was a more potent inducer of TLR9 expression than the probiotic bacteria in the presence of S. typhi. Expression levels of CASP8 and IRAK4 were also increased by CFS, and both treatments induced TOLLIP gene expression. Our results indicate that the probiotic strain B. breve CNCM I-4035 affects the intestinal immune response, whereas its supernatant exerts anti-inflammatory effects mediated by DCs. This supernatant may protect immune system from highly infectious agents such as Salmonella typhi and can down

  11. The roles of Sertoli cells in fate determinations of spermatogonial stem cells

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    Maryam Khanehzad

    2016-03-01

    Full Text Available Background: Spermatogenesis is a complex and highly organized process of proliferation and differentiation of spermatogonial stem cells. Spermatogonial stem cells (SSCs as a unique stem cell have the potential to self-renewal, differentiation and transmit genetic information to the next generation and play a vital role in maintaining fertility. Sertoli cells as the only somatic cells within the seminiferous epithelium play central roles in the formation of niche and balance between self-renewal and differentiation by secrete many growth factors. Given the importance and widespread use of SSCs, particularly in the treatment of infertility, the aim of this study was to create an optimal environment for the proliferation of SSCs. So we decided to study of undifferentiated (ID4 and differentiated (c-Kit gene expression in SSCs followed by co-culture with Sertoli cells for a one-month. Methods: This experimental study was conducted from November 2013 to December 2014 in Department of Anatomy, School of Medicine, Tehran University of Medical Sciences, on immature NMRI mouse (6-3 days old. Initially, Sertoli cells and SSCs were isolated from neonates mouse testes during the two-step enzymatic digestion characteristics Sertoli cells with vimentin marker and SSCs with promyelocytic leukemia zinc-finger (PLZF marker were confirmed. Then SSCs were cultured in two groups: co-culture with Sertoli and without co-culture (control. Undifferentiated (ID4 and differentiation (c-Kit gene expression were evaluated by Real-time PCR technique. Results: Spermatogonial stem cells purity was obtained 66.91% by flow cytometry. The relative expression levels of gene ID4 in co-culture group at the end of each week, compared to the control group showed a significant increase (P<0.05. While the expression of this gene significantly decreased in each group over time (P<0.05. The results of the comparison of the relative expression of c-Kit gene in co-culture group are

  12. The effect of wool hydrolysates on squamous cell carcinoma cells in vitro. Possible implications for cancer treatment.

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    Tatsiana Damps

    Full Text Available Squamous cell carcinoma of the skin is the second most common cutaneous malignancy. Despite various available treatment methods and advances in noninvasive diagnostic techniques, the incidence of metastatic cutaneous squamous cell carcinoma is rising. Deficiency in effective preventive or treatment methods of transformed keratinocytes leads to necessity of searching for new anticancer agents. The present study aims to evaluate the possibility of using wool hydrolysates as such agents. Commercially available compounds such as 5-fluorouracil, ingenol mebutate, diclofenac sodium salt were also used in this study. The process of wool degradation was based on chemical pre-activation and enzymatic digestion of wool. The effect of mentioned compounds on cell viability of squamous carcinoma cell line and healthy keratinocytes was evaluated. The obtained data show a significantly stronger effect of selected wool hydrolysates compared to commercial compounds (p<0.05 on viability of cells. The wool hydrolysates decreased squamous cell carcinoma cells viability by up to 67% comparing to untreated cells. These results indicate bioactive properties of wool hydrolysates, which affect the viability of squamous carcinoma cells and decrease their number. We hypothesize that these agents may be used topically for treatment of transformed keratinocytes in actinic keratosis and invasive squamous skin cancer in humans.

  13. Endothelial Cells Control Pancreatic Cell Fate at Defined Stages through EGFL7 Signaling

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    Der-I Kao

    2015-02-01

    Full Text Available Although endothelial cells have been shown to affect mouse pancreatic development, their precise function in human development remains unclear. Using a coculture system containing human embryonic stem cell (hESC-derived progenitors and endothelial cells, we found that endothelial cells play a stage-dependent role in pancreatic development, in which they maintain pancreatic progenitor (PP self-renewal and impair further differentiation into hormone-expressing cells. The mechanistic studies suggest that the endothelial cells act through the secretion of EGFL7. Consistently, endothelial overexpression of EGFL7 in vivo using a transgenic mouse model resulted in an increase of PP proliferation rate and a decrease of differentiation toward endocrine cells. These studies not only identified the role of EGFL7 as the molecular handle involved in the crosstalk between endothelium and pancreatic epithelium, but also provide a paradigm for using hESC stepwise differentiation to dissect the stage-dependent roles of signals controlling organogenesis.

  14. Alterations in the regulatory volume decrease (RVD) and swelling-activated Cl- current associated with neuroendocrine differentiation of prostate cancer epithelial cells.

    NARCIS (Netherlands)

    Lemonnier, L.; Lazarenko, R.; Shuba, Y.; Thebault, S.C.; Roudbaraki, M.; Lepage, G.; Prevarskaya, N.; Skryma, R.

    2005-01-01

    Neuroendocrine (NE) differentiation of prostate epithelial/basal cells is a hallmark of advanced, androgen-independent prostate cancer, for which there is no successful therapy. Here we report for the first time on alterations in regulatory volume decrease (RVD) and its key determinant,

  15. Increased numbers of preexisting memory CD8 T cells and decreased T-bet expression can restrain terminal differentiation of secondary effector and memory CD8 T cells.

    Science.gov (United States)

    Joshi, Nikhil S; Cui, Weiguo; Dominguez, Claudia X; Chen, Jonathan H; Hand, Timothy W; Kaech, Susan M

    2011-10-15

    Memory CD8 T cells acquire effector memory cell properties after reinfection and may reach terminally differentiated, senescent states ("Hayflick limit") after multiple infections. The signals controlling this process are not well understood, but we found that the degree of secondary effector and memory CD8 T cell differentiation was intimately linked to the amount of T-bet expressed upon reactivation and preexisting memory CD8 T cell number (i.e., primary memory CD8 T cell precursor frequency) present during secondary infection. Compared with naive cells, memory CD8 T cells were predisposed toward terminal effector (TE) cell differentiation because they could immediately respond to IL-12 and induce T-bet, even in the absence of Ag. TE cell formation after secondary (2°) or tertiary infections was dependent on increased T-bet expression because T-bet(+/-) cells were resistant to these phenotypic changes. Larger numbers of preexisting memory CD8 T cells limited the duration of 2° infection and the amount of IL-12 produced, and consequently, this reduced T-bet expression and the proportion of 2° TE CD8 T cells that formed. Together, these data show that over repeated infections, memory CD8 T cell quality and proliferative fitness is not strictly determined by the number of serial encounters with Ag or cell divisions, but is a function of the CD8 T cell differentiation state, which is genetically controlled in a T-bet-dependent manner. This differentiation state can be modulated by preexisting memory CD8 T cell number and the intensity of inflammation during reinfection. These results have important implications for vaccinations involving prime-boost strategies.

  16. Changes of lymphocyte subsets after local irradiation for early stage breast cancer and seminoma testis: long-term increase of activated (HLA-DR+) T-cells and decrease of ''naive'' (CD4-CD45R) T lymphocytes

    International Nuclear Information System (INIS)

    De Ruysscher, D.; Aerts, R.; Vantongelen, K.; Schueren, E. van der; Waer, M.; Vandeputte, M.

    1992-01-01

    Blood lymphocyte subsets of early breast cancer patients and of men with stage I seminoma of the testis were studied up to 6 years after radiotherapy. Similar results were obtained in the two patient groups. After a temporary decrease, the CD4-w29 or ''memory'' T cells recovered completely, while the CD4-45R or ''naive'' T cells remained decreased up to 6 years after irradiation. The number of CD8 T lymphocytes did not change during or after treatment. Because of the decrease of a subset of CD4 cells, and the unchanged values of CD8 cells, the CD4/CD8 ratio decreased significantly after irradiation, and remained lower than before treatment up to 5-6 years after radiotherapy. The number of both HLA-DR positive CD4 and HLA-DR positive CD8 T cells (''activated'' T cells) increased significantly after irradiation. The natural killer (NK) cells were not affected by treatment. The authors propose that recovery of the CD4 cells is limited to the CD4-w29 (''memory'') population because of thymic dysfunction in older humans. (Author)

  17. Inhibition of neutral sphingomyelinase decreases elevated levels of inducible nitric oxide synthase and apoptotic cell death in ocular hypertensive rats

    Energy Technology Data Exchange (ETDEWEB)

    Aslan, Mutay, E-mail: mutayaslan@akdeniz.edu.tr [Department of Medical Biochemistry, Akdeniz University Faculty of Medicine, Antalya (Turkey); Basaranlar, Goksun [Department of Biophysics, Akdeniz University Faculty of Medicine, Antalya (Turkey); Unal, Mustafa [Department of Ophthalmology, Akdeniz University Faculty of Medicine, Antalya (Turkey); Ciftcioglu, Akif [Department of Pathology, Akdeniz University Faculty of Medicine, Antalya (Turkey); Derin, Narin [Department of Biophysics, Akdeniz University Faculty of Medicine, Antalya (Turkey); Mutus, Bulent [Department of Chemistry and Biochemistry, University of Windsor, Windsor, Ontario (Canada)

    2014-11-01

    Endoplasmic reticulum (ER) stress and excessive nitric oxide production via induction of inducible nitric oxide synthase (NOS2) have been implicated in the pathogenesis of neuronal retinal cell death in ocular hypertension. Neutral sphingomyelinase (N-SMase)/ceramide pathway can regulate NOS2 expression, hence this study determined the role of selective neutral sphingomyelinase (N-SMase) inhibition on retinal NOS2 levels, ER stress, apoptosis and visual evoked potentials (VEPs) in a rat model of elevated intraocular pressure (EIOP). NOS2 expression and retinal protein nitration were significantly greater in EIOP and significantly decreased with N-SMase inhibition. A significant increase was observed in retinal ER stress markers pPERK, CHOP and GRP78 in EIOP, which were not significantly altered by N-SMase inhibition. Retinal TUNEL staining showed increased apoptosis in all EIOP groups; however N-SMase inhibition significantly decreased the percent of apoptotic cells in EIOP. Caspase-3, -8 and -9 activities were significantly increased in EIOP and returned to baseline levels following N-SMase inhibition. Latencies of all VEP components were significantly prolonged in EIOP and shortened following N-SMase inhibition. Data confirm the role of nitrative injury in EIOP and highlight the protective effect of N-SMase inhibition in EIOP via down-regulation of NOS2 levels and nitrative stress. - Highlights: • Inhibition of N-SMase decreases NOS2 levels in ocular hypertension. • Inhibition of N-SMase decreases protein nitration in ocular hypertension. • Inhibition of N-SMase decreases caspase activation in ocular hypertension. • Inhibition of N-SMase decreases apoptosis in ocular hypertension.

  18. Muscle satellite cell heterogeneity and self-renewal

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    Motohashi, Norio; Asakura, Atsushi

    2014-01-01

    Adult skeletal muscle possesses extraordinary regeneration capacities. After muscle injury or exercise, large numbers of newly formed muscle fibers are generated within a week as a result of expansion and differentiation of a self-renewing pool of muscle stem cells termed muscle satellite cells. Normally, satellite cells are mitotically quiescent and reside beneath the basal lamina of muscle fibers. Upon regeneration, satellite cells are activated, and give rise to daughter myogenic precursor cells. After several rounds of proliferation, these myogenic precursor cells contribute to the formation of new muscle fibers. During cell division, a minor population of myogenic precursor cells returns to quiescent satellite cells as a self-renewal process. Currently, accumulating evidence has revealed the essential roles of satellite cells in muscle regeneration and the regulatory mechanisms, while it still remains to be elucidated how satellite cell self-renewal is molecularly regulated and how satellite cells are important in aging and diseased muscle. The number of satellite cells is decreased due to the changing niche during ageing, resulting in attenuation of muscle regeneration capacity. Additionally, in Duchenne muscular dystrophy (DMD) patients, the loss of satellite cell regenerative capacity and decreased satellite cell number due to continuous needs for satellite cells lead to progressive muscle weakness with chronic degeneration. Thus, it is necessary to replenish muscle satellite cells continuously. This review outlines recent findings regarding satellite cell heterogeneity, asymmetric division and molecular mechanisms in satellite cell self-renewal which is crucial for maintenance of satellite cells as a muscle stem cell pool throughout life. In addition, we discuss roles in the stem cell niche for satellite cell maintenance, as well as related cell therapies for approaching treatment of DMD. PMID:25364710

  19. Muscle Satellite Cell Heterogeneity and Self-Renewal

    Directory of Open Access Journals (Sweden)

    Norio eMotohashi

    2014-01-01

    Full Text Available Adult skeletal muscle possesses extraordinary regeneration capacities. After muscle injury or exercise, large numbers of newly formed muscle fibers are generated within a week as a result of expansion and differentiation of a self-renewing pool of muscle stem cells termed muscle satellite cells. Normally, satellite cells are mitotically quiescent and reside beneath the basal lamina of muscle fibers. Upon regeneration, satellite cells are activated, and give rise to daughter myogenic precursor cells. After several rounds of proliferation, these myogenic precursor cells contribute to the formation of new muscle fibers. During cell division, a minor population of myogenic precursor cells returns to quiescent satellite cells as a self-renewal process. Currently, accumulating evidence has revealed the essential roles of satellite cells in muscle regeneration and the regulatory mechanisms, while it still remains to be elucidated how satellite cell self-renewal is molecularly regulated and how satellite cells are important in aging and diseased muscle. The number of satellite cells is decreased due to the changing niche during ageing, resulting in attenuation of muscle regeneration capacity. Additionally, in Duchenne muscular dystrophy (DMD patients, the loss of satellite cell regenerative capacity and decreased satellite cell number due to continuous needs for satellite cells lead to progressive muscle weakness with chronic degeneration. Thus, it is necessary to replenish muscle satellite cells continuously. This review outlines recent findings regarding satellite cell heterogeneity, asymmetric division and molecular mechanisms in satellite cell self-renewal which is crucial for maintenance of satellite cells as a muscle stem cell pool throughout life. In addition, we discuss roles in the stem cell niche for satellite cell maintenance, as well as related cell therapies for approaching treatment of DMD.

  20. Curcumin affects cell survival and cell volume regulation in human renal and intestinal cells

    International Nuclear Information System (INIS)

    Kössler, Sonja; Nofziger, Charity; Jakab, Martin; Dossena, Silvia; Paulmichl, Markus

    2012-01-01

    Curcumin (1,7-bis(4-hydroxy-3-methoxyphenyl)-1E,6E-heptadiene-3,5-dione or diferuloyl methane) is a polyphenol derived from the Curcuma longa plant, commonly known as turmeric. This substance has been used extensively in Ayurvedic medicine for centuries for its anti-oxidant, analgesic, anti-inflammatory and antiseptic activity. More recently curcumin has been found to possess anti-cancer properties linked to its pro-apoptotic and anti-proliferative actions. The underlying mechanisms of these diverse effects are complex, not fully elucidated and subject of intense scientific debate. Despite increasing evidence indicating that different cation channels can be a molecular target for curcumin, very little is known about the effect of curcumin on chloride channels. Since, (i) the molecular structure of curcumin indicates that the substance could potentially interact with chloride channels, (ii) chloride channels play a role during the apoptotic process and regulation of the cell volume, and (iii) apoptosis is a well known effect of curcumin, we set out to investigate whether or not curcumin could (i) exert a modulatory effect (direct or indirect) on the swelling activated chloride current ICl swell in a human cell system, therefore (ii) affect cell volume regulation and (iii) ultimately modulate cell survival. The ICl swell channels, which are essential for regulating the cell volume after swelling, are also known to be activated under isotonic conditions as an early event in the apoptotic process. Here we show that long-term exposure of a human kidney cell line to extracellular 0.1–10 μM curcumin modulates ICl swell in a dose-dependent manner (0.1 μM curcumin is ineffective, 0.5–5.0 μM curcumin increase, while 10 μM curcumin decrease the current), and short-term exposure to micromolar concentrations of curcumin does not affect ICl swell neither if applied from the extracellular nor from the intracellular side – therefore, a direct effect of curcumin on ICl