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  1. Red blood cell decreases of microgravity

    Science.gov (United States)

    Johnson, P. C.

    1985-01-01

    Postflight decreases in red blood cell mass (RBCM) have regularly been recorded after exposure to microgravity. These 5-25 percent decreases do not relate to the mission duration, workload, caloric intake or to the type of spacecraft used. The decrease is accompanied by normal red cell survivals, increased ferritin levels, normal radioactive iron studies, and increases in mean red blood cell volume. Comparable decreases in red blood cell mass are not found after bed rest, a commonly used simulation of the microgravity state. Inhibited bone marrow erythropoiesis has not been proven to date, although reticulocyte numbers in the peripheral circulation are decreased about 50 percent. To date, the cause of the microgravity induced decreases in RBCM is unknown. Increased splenic trapping of circulating red blood cells seem the most logical way to explain the results obtained.

  2. 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.

  3. Glucocorticoids decrease Treg cell numbers in lungs of allergic mice.

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    Olsen, P C; Kitoko, J Z; Ferreira, T P; de-Azevedo, C T; Arantes, A C; Martins, Μ A

    2015-01-15

    Glucocorticoids have been the hallmark anti-inflammatory drug used to treat asthma. It has been shown that glucocorticoids ameliorate asthma by increasing numbers and activity of Tregs, in contrast recent data show that glucocorticoid might have an opposite effect on Treg cells from normal mice. Since Tregs are target cells that act on the resolution of asthma, the aim of this study was to elucidate the effect of glucocorticoid treatment on lung Tregs in mouse models of asthma. Allergen challenged mice were treated with either oral dexamethasone or nebulized budesonide. Broncoalveolar lavage and airway hyperresponsiveness were evaluated after allergenic challenge. Lung, thymic and lymph node cells were phenotyped on Treg through flow cytometry. Lung cytokine secretion was detected by ELISA. Although dexamethasone inhibited airway inflammation and hyperresponsiveness, improving resolution, we have found that both dexamethasone and budesonide induce a reduction of Treg numbers on lungs and lymphoid organs of allergen challenged mice. The reduction of lung Treg levels was independent of mice strain or type of allergen challenge. Our study also indicates that both glucocorticoids do not increase Treg activity through production of IL-10. Glucocorticoid systemic or localized treatment induced thymic atrophy. Taken together, our results demonstrate that glucocorticoids decrease Treg numbers and activity in different asthma mouse models, probably by reducing thymic production of T cells. Therefore, it is possible that glucocorticoids do not have beneficial effects on lung populations of Treg cells from asthmatic patients.

  4. The insecticide DDT decreases membrane potential and cell input resistance of cultured human liver cells.

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    Schefczik, K; Buff, K

    1984-10-03

    The resting membrane potential, Em, and the cell input resistance, Rinp, of cultured human Chang liver cells were measured using the single electrode 'double-pulse' current clamp technique, following exposure of the cells to the insecticide DDT (20 microM). In control (unexposed) cells, the mean Em was -24 mV, and the mean Rinp was 30 M omega. Neither parameter was significantly impaired after 1 h of cell exposure to DDT. But after 7 and 48 h, the Em was depolarized by 15 and 25 mV, respectively, in parallel with a decrease of the cell input resistance. The strongly time-delayed effect of DDT on Chang liver cell membranes may indicate a mode of interaction different from excitable membranes.

  5. Stem cells decreased neuronal cell death after hypoxic stress in primary fetal rat neurons in vitro.

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    Sakai, Tetsuro; Xu, Yan

    2012-01-01

    To explore stem cell-mediated neuronal protection through extracellular signaling pathways by transplanted stem cells, we sought to identify potential candidate molecules responsible for neuronal protection using an in vitro coculture system. Primary fetal rat hippocampal neurons underwent hypoxia (≤1% oxygen) for 96 h nad then were returned to a normoxic condition. The study group then received rat umbilical cord matrix-derived stem cells, while the control group received fresh media only. The experimental group showed decreased neuronal apoptosis compared to the control group [44.5 ± 1.6% vs. 71.0 ± 4.2% (mean ± SD, p = 0.0005) on day 5] and higher neuronal survival (4.9 ± 1.2 cells/100× field vs. 2.2 ± 0.3, p = 0.02 on day 5). Among 90 proteins evaluated using a protein array, stem cell coculture media showed increased protein secretion of TIMP-1 (5.61-fold), TIMP-2 (4.88), CNTF-Rα (3.42), activin A (2.20), fractalkine (2.04), CCR4 (2.02), and decreased secretion in MIP-2 (0.30-fold), AMPK α1 (0.43), TROY (0.48), and TIMP-3 (0.50). This study demonstrated that coculturing stem cells with primary neurons in vitro decreased neuronal cell death after hypoxia with significantly altered protein secretion. The results suggest that stem cells may offer neuronal protection through extracellular signaling.

  6. 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

  7. Metronidazole decreases viability of DLD-1 colorectal cancer cell line.

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    Sadowska, Anna; Krętowski, Rafał; Szynaka, Beata; Cechowska-Pasko, Marzanna; Car, Halina

    2013-10-01

    The aim of our study was to evaluate the impact of metronidazole (MTZ) on DLD-1 colorectal cancer cell (CRC) line. Toxicity of MTZ was determined by MTT test. Cells were incubated with MTZ used in different concentrations for 24, 48, and 72 hours. The effect of MTZ on DNA synthesis was measured as [3H]-thymidine incorporation. The morphological changes in human DLD-1 cell line were defined by transmission electron microscope OPTON 900. The influence of MTZ on the apoptosis of DLD-1 cell lines was detected by flow cytometry and fluorescence microscopy, while cell concentration, volume, and diameter were displayed by Scepter Cell Counter from Millipore. Our results show that cell viability was diminished in all experimental groups in comparison with the control, and the differences were statistically significant. We did not find any significant differences in [3H]-thymidine incorporation in all experimental groups and times of observation. Cytofluorimetric assays demonstrated a statistically significant increase of apoptotic rate in MTZ concentrations 10 and 50 μg/mL after 24 hours; 0.1, 10, 50, and 250 μg/mL after 48 hours; and in all concentrations after 72 hours compared with control groups. In the ultrastructural studies, necrotic or apoptotic cells were occasionally seen. In conclusion, MTZ affects human CRC cell line viability. The reduction of cell viability was consistent with the apoptotic test.

  8. Contrast Adaptation Decreases Complexity in Retinal Ganglion Cell Spike Train

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    WANG Guang-Li; HUANG Shi-Yong; ZHANG Ying-Ying; LIANG Pei-Ji

    2007-01-01

    @@ The difference in temporal structures of retinal ganglion cell spike trains between spontaneous activity and firing activity after contrast adaptation is investigated. The Lempel-Ziv complexity analysis reveals that the complexity of the neural spike train decreases after contrast adaptation. This implies that the behaviour of the neuron becomes ordered, which may carry relevant information about the external stimulus. Thus, during the neuron activity after contrast adaptation, external information could be encoded in forms of some certain patterns in the temporal structure of spike train that is significantly different, compared to that of the spike train during spontaneous activity, although the firing rates in spontaneous activity and firing activity after contrast adaptation are sometime similar.

  9. Decreased natural killer cell activity is associated with atherosclerosis in elderly humans

    DEFF Research Database (Denmark)

    Bruunsgaard, H.; Pedersen, Agnes Nadelmann; Schroll, M.;

    2001-01-01

    of elderly humans. It was tested if the potential of natural cytotoxicity in the blood (evaluated as an index including cytotoxicity per NK cell and the number of circulating NK cells) was preserved in 174 81-yearold humans versus 91 young controls and if NK cell mediated immunity was associated with age......-related inflammatory diseases such as atherosclerosis. Elderly people had decreased cytotoxicity per NK cell in short-term but not in long-term assays. Ca2+ independent cytotoxicity was unaltered, and NK cells maintained their cytotoxic responses to interleukin-2 and interferon-alpha signals. The decreased...... cytotoxicity per NK cell was not completely counteracted by increased circulating numbers of NK cells in the blood. Elderly people with severe medical disorders had low numbers of circulating NK cells. Furthermore, elderly people with atherosclerosis had low cytotoxicity per NK cell and a high number...

  10. Decreased CD8+ T cell response to Epstein-Barr virus infected B cells in multiple sclerosis is not due to decreased HLA class I expression on B cells or monocytes

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    Csurhes Peter A

    2011-08-01

    Full Text Available Abstract Background Patients with multiple sclerosis (MS have a decreased frequency of CD8+ T cells reactive to their own Epstein-Barr virus (EBV infected B cells. We have proposed that this might predispose to the development of MS by allowing EBV-infected autoreactive B cells to accumulate in the central nervous system. The decreased CD8+ T cell response to EBV results from a general CD8+ T cell deficiency and also a decreased proportion of EBV-specific T cells within the total CD8+ T cell population. Because decreased HLA class I expression on monocytes and B cells has been reported in MS and could influence the generation and effector function of EBV-specific CD8+ T cells, the present study was undertaken to measure the expression of HLA molecules on B cells and monocytes in patients with MS. Methods We used flow cytometry to determine the proportions of T cells, natural killer cells, B cells and monocytes in peripheral blood mononuclear cells (PBMC and to quantify the expression of HLA molecules on T cells, B cells and monocytes of 59 healthy subjects and 62 patients with MS who had not received corticosteroids or immunomodulatory therapy in the previous 3 months. Results The levels of HLA class I and class II molecules expressed on T cells, B cells and monocytes were normal in patients with MS, with the exception of two patients with secondary progressive MS with very low class II expression on B cells. In confirmation of previous studies we also found that the percentage of CD8+ T cells was significantly decreased whereas the percentage of CD4+ T cells and the CD4:CD8 ratio were significantly increased in patients with MS compared to healthy subjects. Conclusions The decreased CD8+ T cell response to EBV-infected B cells in MS patients is not due to decreased HLA class I expression on monocytes or B cells. In a small proportion of patients decreased HLA class II expression on B cells might impair the CD8+ T cell response to EBV by

  11. Autophagy Alleviates Melamine-Induced Cell Death in PC12 Cells Via Decreasing ROS Level.

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    Wang, Hui; Gao, Na; Li, Zhigui; Yang, Zhuo; Zhang, Tao

    2016-04-01

    Since melamine was illegally added to raw milk for increasing the apparent protein content, such a scandal has not been quite blown out. Previous studies showed that melamine induced apoptosis and oxidative damage in both in vivo and in vitro experiments. It is well known that autophagy is closely related to oxidative stress. In the present study, we examined whether autophagy played an important role in protecting PC12 cells, which were damaged by melamine. Immunofluorescence assay showed that melamine enhanced the number of punctuate dot, indicating the increase of autophagosomes. Western blot assay presented that melamine significantly elevated the expression level of autophagy markers including LC3-II/LC3-I ratio, beclin-1, and Atg 7. Rapamycin further enhanced the effect, whereas 3-methyadenine (3-MA) inhibited it. MTT assay exhibited that rapamycin significantly enhanced the cell viability (P melamine-treated PC12 cells (P melamine-treated PC12 cells (P melamine-induced cell death via inhibiting the excessive generation of ROS. Regulating autophagy may become a new targeted therapy to relieve the damage induced by melamine.

  12. Decreased PD-1 positive blood follicular helper T cells in patients with psoriasis.

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    Shin, Dongyun; Kim, Dae Suk; Kim, Sung Hee; Je, Jung Hwan; Kim, Hee Ju; Young Kim, Do; Kim, Soo Min; Lee, Min-Geol

    2016-10-01

    Follicular helper T (Tfh) cells are recently characterized subset of helper T cells, which are initially found in the germinal centers of B cell follicles. The major role of Tfh cells is helping B cell activation and antibody production during humoral immunity. Recently, blood Tfh cells were shown to be associated with autoimmune diseases, such as systemic lupus erythematosus, rheumatoid arthritis, bullous pemphigoid and psoriasis. There is only one study which investigated Tfh cells in psoriasis patients. Therefore, in this study, we evaluated and analyzed blood Tfh cells in Korean patients with psoriasis. A total of 28 psoriasis patients and 16 healthy controls were enrolled. The frequency and absolute number of CXCR5(+)PD-1(+) Tfh cells were decreased in patients with psoriasis compared to healthy controls. CD4(+)CXCR5(+) T cells and CXCR5(+)ICOS(+) Tfh cells did not show differences. The frequency and absolute number of CXCR5(+)PD-1(+) Tfh cells in psoriasis patients negatively correlated with erythrocyte sedimentation rate and positively correlated with disease duration. The absolute number of CXCR5(+)ICOS(+) Tfh cells also showed positive correlation with disease duration. However, the subpopulations of Tfh cells did not correlate with Psoriasis Area and Severity Index. Serum interleukin-21 level was significantly increased in psoriasis patients compared to healthy controls, however, its level did not correlate with clinical and experimental parameters of psoriasis patients. These findings suggest the decreased function of Tfh cells in psoriasis, which could result in attenuated B cell immune responses in the pathogenesis of psoriasis. However, further investigations are necessary to confirm the function of Tfh cells in psoriasis vulgaris.

  13. Exposure of isoflurane-treated cells to hyperoxia decreases cell viability and activates the mitochondrial apoptotic pathway.

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    Kim, Gunn Hee; Lee, Jeong Jin; Lee, Sang Hyun; Chung, Yang Hoon; Cho, Hyun Sung; Kim, Jie Ae; Kim, Min Kyung

    2016-04-01

    Isoflurane has either neuroprotective or neurotoxic effects. High-dose oxygen is frequently used throughout the perioperative period. We hypothesized that hyperoxia will affect cell viability of rat pheochromocytoma (PC12) cells that were exposed to isoflurane and reactive oxygen species (ROS) may be involved. PC12 cells were exposed to 1.2% or 2.4% isoflurane for 6 or 24h respectively, and cell viability was evaluated. To investigate the effects of hyperoxia, PC12 cells were treated with 21%, 50%, or 95% oxygen and 2.4% isoflurane for 6h, and cell viability, TUNEL staining, ROS production, and expression of B-cell lymphoma 2 (BCL-2), BCL2-associated X protein (BAX), caspase-3 and beta-site APP cleaving enzyme (BACE) were measured. ROS involvement was evaluated using the ROS scavenger 2-mercaptopropiopylglycine (MPG). The viability of cells exposed to 2.4% isoflurane was lower than that of cells exposed to 1.2% isoflurane. Prolonged exposure (6h vs. 24h) to 2.4% isoflurane resulted in a profound reduction in cell viability. Treatment with 95% (but not 50%) oxygen enhanced the decrease in cell viability induced by 2.4% isoflurane alone. Levels of ROS, Bax, caspase-3 and BACE were increased, whereas expression of Bcl-2 was decreased, in cells treated with 95% oxygen plus 2.4% isoflurane compared with the control and 2.4% isoflurane plus air groups. MPG attenuated the effects of oxygen and isoflurane. In conclusion, isoflurane affects cell viability in a dose- and time-dependent manner. This effect is augmented by hyperoxia and may involve ROS, the mitochondrial apoptotic signaling pathway, and β-amyloid protein.

  14. Bioactive dietary polyphenols decrease heme iron absorption by decreasing basolateral iron release in human intestinal Caco-2 cells.

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    Ma, Qianyi; Kim, Eun-Young; Han, Okhee

    2010-06-01

    Because dietary polyphenolic compounds have a wide range of effects in vivo and vitro, including chelation of metals such as iron, it is prudent to test whether the regular consumption of dietary bioactive polyphenols impair the utilization of dietary iron. Because our previous study showed the inhibitory effect of (-) -epigallocatechin-3-gallate (EGCG) and grape seed extract (GSE) on nonheme iron absorption, we investigated whether EGCG and GSE also affect iron absorption from heme. The fully differentiated intestinal Caco-2 cells grown on microporous membrane inserts were incubated with heme (55)Fe in uptake buffer containing EGCG or GSE in the apical compartment for 7 h. Both EGCG and GSE decreased (P heme-derived iron. However, apical heme iron uptake was increased (P heme (55)Fe, the transfer of iron across the intestinal basolateral membrane was extremely low, indicating that basolateral export was impaired by GSE. In contrast, EGCG moderately decreased the cellular assimilation of heme (55)Fe, but the basolateral iron transfer was extremely low, suggesting that the basolateral efflux of heme iron was also inhibited by EGCG. Expression of heme oxygenase, ferroportin, and hephaestin protein was not changed by EGCG and GSE. The apical uptake of heme iron was temperature dependent and saturable in fully differentiated Caco-2 cells. Our data show that bioactive dietary polyphenols inhibit heme iron absorption mainly by reducing basolateral iron exit rather than decreasing apical heme iron uptake in intestinal cells.

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

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    Silva, Patricio; Soto, Nicolás [Institute for Research in Dental Sciences, Faculty of Dentistry, Universidad de Chile, Santiago (Chile); Díaz, Jorge [Institute for Research in Dental Sciences, Faculty of Dentistry, Universidad de Chile, Santiago (Chile); Center for Molecular Studies of the Cell, Institute of Biomedical Sciences (ICBM), Faculty of Medicine, Universidad de Chile, Santiago (Chile); Mendoza, Pablo [Institute for Research in Dental Sciences, Faculty of Dentistry, Universidad de Chile, Santiago (Chile); Díaz, Natalia [Institute for Research in Dental Sciences, Faculty of Dentistry, Universidad de Chile, Santiago (Chile); Center for Molecular Studies of the Cell, Institute of Biomedical Sciences (ICBM), Faculty of Medicine, Universidad de Chile, Santiago (Chile); Quest, Andrew F.G. [Center for Molecular Studies of the Cell, Institute of Biomedical Sciences (ICBM), Faculty of Medicine, Universidad de Chile, Santiago (Chile); Advanced Center for Chronic Diseases (ACCDiS), Faculty of Medicine, Universidad de Chile, Santiago (Chile); Torres, Vicente A., E-mail: vatorres@med.uchile.cl [Institute for Research in Dental Sciences, Faculty of Dentistry, Universidad de Chile, Santiago (Chile); Advanced Center for Chronic Diseases (ACCDiS), Faculty of Medicine, Universidad de Chile, Santiago (Chile)

    2015-08-21

    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.

  16. Phosphatidylinositol 3-kinase mediates the ability of retinol to decrease colorectal cancer cell invasion.

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    Lengyel, Jennifer N Griffin; Park, Eun Young; Brunson, Anna R; Pinali, Daniel; Lane, Michelle A

    2014-01-01

    Previously, we showed that retinol (vitamin A) decreased both colorectal cancer cell invasion and phosphatidylinositol 3-kinase (PI3K) activity through a retinoic acid receptor-independent mechanism. Here, we determined if these phenomena were related by using parental HCT-116 cells that harbor 1 allele of wild-type PI3K and 1 allele of constitutively active (ca) PI3K and 2 mutant HCT-116 cell lines homozygous for caPI3K. In vitro, treatment of parental HCT-116 cells with 10 μM retinol reduced cell invasion whereas treatment of mutant HCT-116 cell lines with retinol did not. Treatment with 10 μM retinol also decreased the activity of matrixmetalloproteinase-9 and increased tissue inhibitor of matrixmetalloproteinase-I levels in parental, but not mutant, HCT-116 cells. Finally, parental or mutant cells were intrasplenically injected into athymic mice consuming diets with or without supplemental vitamin A. As expected, vitamin A supplementation tended (P = 0.18) to reduce the incidence of metastases in mice injected with the parental cell line and consuming the supplemented diet. In contrast, metastatic incidence was not affected (P = 1.00) by vitamin A supplementation in mice injected with mutant cells. These data indicate that the capacity of retinol to inhibit PI3K activity confers its ability to decrease colorectal cancer metastasis.

  17. Decreases in CD31 and CD47 levels on the cell surface during etoposide-induced Jurkat cell apoptosis.

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    Azuma, Yutaro; Nakagawa, Hideaki; Dote, Kanae; Higai, Koji; Matsumoto, Kojiro

    2011-01-01

    Engulfment of apoptotic cells is regulated by 'eat me' and 'don't eat me' signals on the cell surface. Alterations to the 'eat me' signals have been well described; however, very little is known about the 'don't eat me' signals on the cell surface during apoptosis. In the present study, apoptosis of Jurkat cells was induced by treatment with topoisomerase II inhibitor etoposide, and then the CD31 and CD47 levels on the apoptotic cell surface and in microparticles were estimated by flow cytometry and immunoblotting methods in the presence of caspase, metalloproteinase, and Rho-associated coiled-coil containing protein kinase 1 (ROCK1) inhibitors. The CD31 and CD47 levels on the cell surface of apoptotic Jurkat cells had decreased after treatment with etoposide. These decreases in CD31 and CD47 levels on the apoptotic cell surface were almost completely suppressed by the caspase 3 inhibitor, Ac-DEVD-CHO, and partially suppressed by caspase 8 (Ac-IETD-CHO) and caspase 9 (Ac-LEHE-CHO) inhibitors but not by the metalloproteinase inhibitors GM6001 and TAPI-0. Microparticle counts in culture supernatants were higher during etoposide-induced apoptosis. The ROCK1 inhibitor, Y27632, suppressed blebbing formation and microparticle release. Moreover, flow cytometry and immunoblotting revealed CD31 and CD47 in the microparticles. These results indicate that CD31 and CD47 were released by the apoptotic Jurkat cells into the culture supernatant in microparticles, but not in soluble forms, resulting in decreased levels on the apoptotic cell surface.

  18. MiR-30a Decreases Multidrug Resistance (MDR) of Gastric Cancer Cells

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    Li, Chunying; Zou, Jinhai; Zheng, Guoqi; Chu, Jiankun

    2016-01-01

    Background The effectiveness of chemotherapy for gastric cancer is largely limited by either intrinsic or acquired drug resistance. In this study, we aimed to explore the association between miR-30a dysregulation and multidrug resistance (MDR) in gastric cancer cells. Material/Methods We recruited 20 patients with advanced gastric cancer. Chemosensitivity was assessed after completion of the chemotherapy. SGC-7901 and SGC-7901/DDP cells were transfected for miR-30a overexpression or knockdown. Then, MTT assay was performed to assess the IC50 of DPP and 5-FU in SGC-7901 and SGC-7901/DDP cells. Flow cytometry analysis was used to detect DPP- and 5-FU-induced cell apoptosis. Western blot analysis and immunofluorescence staining were used to assess EMT of the cells. Results MiR-30a was significantly downregulated in the chemoresistant tissues. In both SGC-7901 and SGC-7901/DDP cells, miR-30a overexpression decreased the expression of P-gp, a MDR-related protein. MTT assay and flow cytometry analysis showed that miR-30a inhibition increased chemoresistance, while miR-30a overexpression decreased chemoresistance in gastric cancer cells. Both Western blot analysis and immunofluorescence staining confirmed that miR-30a inhibition decreased E-cadherin but increased N-cadherin in SGC-7901 cells, while miR-30a overexpression increased E-cadherin but decreased N-cadherin in SGC-7901 cells. Conclusions MiR-30a can decrease multidrug resistance (MDR) of gastric cancer cells. It is also an important miRNA modulating EMT of the cancer cells.

  19. Involvement of regulatory volume decrease in the migration of nasopharyngeal carcinoma cells

    Institute of Scientific and Technical Information of China (English)

    Jian Wen MAO; Li Xin CHEN; Li Wei WANG; Tim JACOB; Xue Rong SUN; Hui LI; Lin Yan ZHU; Pan LI; Ping ZHONG; Si Huai NIE

    2005-01-01

    The transwell chamber migration assay and CCD digital camera imaging techniques were used to investigate the relationship between regulatory volume decrease (RVD) and cell migration in nasopharyngeal carcinoma cells (CNE-2Z cells). Both migrated and non-migrated CNE-2Z cells, when swollen by 47% hypotonic solution, exhibited RVD which was inhibited by extracellular application of chloride channel blockers adenosine 5'-triphosphate (ATP), 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB) and tamoxifen. However, RVD rate in migrated CNE-2Z cells was bigger than that of non-migrated cells and the sensitivity of migrated cells to NPPB and tamoxifen was higher than that of nonmigrated cells. ATP, NPPB and tamoxifen also inhibited migration of CNE-2Z cells. The inhibition of migration was positively correlated to the blockage of RVD, with a correlation coefficient (r) = 0.99, suggesting a functional relationship between RVD and cell migration. We conclude that RVD is involved in cell migration and RVD may play an important role in migratory process in CNE-2Z cells.

  20. 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.

  1. Tetrathiomolybdate inhibits head and neck cancer metastasis by decreasing tumor cell motility, invasiveness and by promoting tumor cell anoikis

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    Merajver Sofia D

    2010-08-01

    Full Text Available Abstract Background The metastatic spread of solid tumors is directly or indirectly responsible for most cancer-related deaths. Tumor metastasis is very complex and this process requires a tumor cell to acquire enhanced motility, invasiveness and anoikis resistance to successfully establish a tumor at a distal site. Metastatic potential of tumor cells is directly correlated with the expression levels of several angiogenic cytokines. Copper is a mandatory cofactor for the function of many of these angiogenic mediators as well as other proteins that play an important role in tumor cell motility and invasiveness. We have previously shown that tetrathiomolybdate (TM is a potent chelator of copper and it mediates its anti-tumor effects by suppressing tumor angiogenesis. However, very little is known about the effect of TM on tumor cell function and tumor metastasis. In this study, we explored the mechanisms underlying TM-mediated inhibition of tumor metastasis. Results We used two in vivo models to examine the effects of TM on tumor metastasis. Animals treated with TM showed a significant decrease in lung metastasis in both in vivo models as compared to the control group. In addition, tumor cells from the lungs of TM treated animals developed significantly smaller colonies and these colonies had significantly fewer tumor cells. TM treatment significantly decreased tumor cell motility and invasiveness by inhibiting lysyl oxidase (LOX activity, FAK activation and MMP2 levels. Furthermore, TM treatment significantly enhanced tumor cell anoikis by activating p38 MAPK cell death pathway and by downregulating XIAP survival protein expression. Conclusions Taken together, these results suggest that TM is a potent suppressor of head and neck tumor metastasis by modulating key regulators of tumor cell motility, invasiveness and anoikis resistance.

  2. Metformin inhibits growth and decreases resistance to anoikis in medullary thyroid cancer cells.

    Science.gov (United States)

    Klubo-Gwiezdzinska, Joanna; Jensen, Kirk; Costello, John; Patel, Aneeta; Hoperia, Victoria; Bauer, Andrew; Burman, Kenneth D; Wartofsky, Leonard; Vasko, Vasyl

    2012-06-01

    Medullary thyroid cancer (MTC) is associated with activation of mammalian target of rapamycin (mTOR) signaling pathways. Recent studies showed that the antidiabetic agent metformin decreases proliferation of cancer cells through 5'-AMP-activated protein kinase (AMPK)-dependent inhibition of mTOR. In the current study, we assessed the effect of metformin on MTC cells. For this purpose, we determined growth, viability, migration, and resistance to anoikis assays using two MTC-derived cell lines (TT and MZ-CRC-1). Expressions of molecular targets of metformin were examined in MTC cell lines and in 14 human MTC tissue samples. We found that metformin inhibited growth and decreased expression of cyclin D1 in MTC cells. Treatment with metformin was associated with inhibition of mTOR/p70S6K/pS6 signaling and downregulation of pERK in both TT and MZ-CRC-1 cells. Metformin had no significant effects on pAKT in the cell lines examined. Metformin-inducible AMPK activation was noted only in TT cells. Treatment with AMPK inhibitor (compound C) or AMPK silencing did not prevent growth inhibitory effects of metformin in TT cells. Metformin had no effect on MTC cell migration but reduced the ability of cells to form multicellular spheroids in nonadherent conditions. Immunostaining of human MTC showed over-expression of cyclin D1 in all tumors compared with corresponding normal tissue. Activation of mTOR/p70S6K was detected in 8/14 (57.1%) examined tumors. Together, these findings indicate that growth inhibitory effects in MTC cells are associated with downregulation of both mTOR/6SK and pERK signaling pathways. Expression of metformin's molecular targets in human MTC cells suggests its potential utility for the treatment of MTC in patients.

  3. Physical exercise decreases the number of fetal cells in maternal blood

    DEFF Research Database (Denmark)

    Schlütter, Jacob Mørup; Kirkegaard, Ida; Christensen, Connie Britta;

    liability company). Fetal cells in the blood, bound to fetal cell specific markers, were initially isolated by magnetic cell sorting, then stained with a cocktail of intracellular antibodies, identified and counted. Information about 6 variables reflecting the physical activity of the participants......Physical exercise decreases the number of fetal cells in maternal blood J. M. Schlütter1, I. Kirkegaard1, B. Christensen2, S. Kølvraa3, N. Uldbjerg1 1. Department of Gynecology and Obstetrics, Aarhus University Hospital, Skejby, Aarhus N, Denmark. 2. FCMB ApS, Vejle, Denmark. 3. Department...... of Clinical Genetics, Vejle Hospital, Vejle, Denmark Objectives We have established a robust method to specifically identify and isolate a subgroup of fetal cells in maternal blood (fcmb) at a gestational age of 12 weeks. The concentration of these cells, however, varies considerably among pregnant women...

  4. Levels of CD105(+) cells increase and cell proliferation decreases during S-phase arrest of amniotic fluid cells in long-term culture.

    Science.gov (United States)

    Wang, Ding; Chen, Rui; Zhong, Xuan; Fan, Yong; Lai, Weiqiang; Sun, Xiaofang

    2014-11-01

    The present study aimed to improve the characterization of amniotic fluid cells (AFCs) in order to optimize their use in chromosomal prenatal diagnosis and as seed or stem cells for tissue engineering. The AFCs used in the current study were obtained from three females in their second trimester of pregnancy. The cells were cultured independently and characterized by cell morphology, cell markers, cell cycle distribution and chromosome Giemsa banding in an early- and late-passage. The AFCs remained homogeneous in culture and expressed mesenchymal markers, but not endothelial markers along the culture process. In addition, compared with the early-passage cells, the late-passage cells exhibit an increase in CD105 expression, a decrease in cell division and a delay in the cell cycle, and a number of cells underwent cell cycle arrest. However, the cells retained a normal karyotype. Therefore, the current study characterized AFCs in a clinical culture and confirmed that AFCs are mesenchymal precursors. The results obtained may be useful for the application of AFCs in prenatal diagnosis.

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

    Science.gov (United States)

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

    2014-01-01

    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.

  6. Cell-free supernatants from probiotic Lactobacillus casei and Lactobacillus rhamnosus GG decrease colon cancer cell invasion in vitro.

    Science.gov (United States)

    Escamilla, Juanita; Lane, Michelle A; Maitin, Vatsala

    2012-08-01

    Probiotics have been shown to have a preventative role in colorectal carcinogenesis but research concerning their prophylactic potential in the later stages of colorectal cancer, specifically metastasis is limited. This study explored the potential of cell-free supernatants (CFS) from 2 probiotic Lactobacillus sp., Lactobacillus casei and Lactobacillus rhamnosus GG, to inhibit colon cancer cell invasion by influencing matrix metalloproteinase-9 (MMP-9) activity and levels of the tight junction protein zona occludens-1 (ZO-1) in cultured metastatic human colorectal carcinoma cells. HCT-116 cells were treated with CFS from L. casei, L. rhamnosus, or Bacteroides thetaiotaomicron (a gut commensal); or with uninoculated bacterial growth media. Treatment with CFS from both Lactobacillus sp. decreased colorectal cell invasion but treatment with CFS from B. thetaiotaomicron did not. CFS from both Lactobacillus sp. decreased MMP-9 and increased ZO-1 protein levels. L. rhamnosus CFS also lowered MMP-9 activity. To begin elucidating the secreted bacterial factor conveying these responses, Lactobacillus sp. CFS were fractionated into defined molecular weight ranges and cell invasion assessed. Fractionation revealed that the inhibitory activity was contained primarily in the >100 kDa and 50-100 kDa fractions, suggesting the inhibitory compound may be a macromolecule such as a protein, nucleic acid, or a polysaccharide.

  7. 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.

  8. β-Cyclodextrins Decrease Cholesterol Release and ABC-Associated Transporter Expression in Smooth Muscle Cells and Aortic Endothelial Cells

    Science.gov (United States)

    Coisne, Caroline; Hallier-Vanuxeem, Dorothée; Boucau, Marie-Christine; Hachani, Johan; Tilloy, Sébastien; Bricout, Hervé; Monflier, Eric; Wils, Daniel; Serpelloni, Michel; Parissaux, Xavier; Fenart, Laurence; Gosselet, Fabien

    2016-01-01

    Atherosclerosis is an inflammatory disease that leads to an aberrant accumulation of cholesterol in vessel walls forming atherosclerotic plaques. During this process, the mechanism regulating complex cellular cholesterol pools defined as the reverse cholesterol transport (RCT) is altered as well as expression and functionality of transporters involved in this process, namely ABCA1, ABCG1, and SR-BI. Macrophages, arterial endothelial and smooth muscle cells (SMCs) have been involved in the atherosclerotic plaque formation. As macrophages are widely described as the major cell type forming the foam cells by accumulating intracellular cholesterol, RCT alterations have been poorly studied at the arterial endothelial cell and SMC levels. Amongst the therapeutics tested to actively counteract cellular cholesterol accumulation, the methylated β-cyclodextrin, KLEPTOSE® CRYSMEβ, has recently shown promising effects on decreasing the atherosclerotic plaque size in atherosclerotic mouse models. Therefore we investigated in vitro the RCT process occurring in SMCs and in arterial endothelial cells (ABAE) as well as the ability of some modified β-CDs with different methylation degree to modify RCT in these cells. To this aim, cells were incubated in the presence of different methylated β-CDs, including KLEPTOSE® CRYSMEβ. Both cell types were shown to express basal levels of ABCA1 and SR-BI whereas ABCG1 was solely found in ABAE. Upon CD treatments, the percentage of membrane-extracted cholesterol correlated to the methylation degree of the CDs independently of the lipid composition of the cell membranes. Decreasing the cellular cholesterol content with CDs led to reduce the expression levels of ABCA1 and ABCG1. In addition, the cholesterol efflux to ApoA-I and HDL particles was significantly decreased suggesting that cells forming the blood vessel wall are able to counteract the CD-induced loss of cholesterol. Taken together, our observations suggest that methylated

  9. 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

    donors were able to form similar amounts of mineralized matrix in vitro and of normal lamellar bone in vivo. In adipogenic medium similar numbers of adipocytes formed in cultures of young and old donors. In conclusion, aging is associated with decreased proliferative capacity of osteoprogenitor cells......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......, suggesting that decreased osteoblastic cell number, and not function, leads to age-related decrease in bone formation....

  10. Formaldehyde induces apoptosis through decreased Prx 2 via p38 MAPK in lung epithelial cells.

    Science.gov (United States)

    Lim, Seul Ki; Kim, Jong Chun; Moon, Chang Jong; Kim, Gye Yeop; Han, Ho Jae; Park, Soo Hyun

    2010-05-27

    Formaldehyde (FA) is an important substance that induces sick house syndrome and diseases, such as asthma and allergies. Oxidative stress is involved in the development of respiratory disease, and diverse antioxidants may protect respiratory tract cells from apoptosis. Peroxiredoxin is a pivotal endogenous antioxidant. In the present study, FA induced death in A549 cells, a lung epithelial cell line, in a dose-dependent manner. FA also increased lipid peroxide formation (LPO) in A549 cells, suggesting a role for oxidative stress. Additionally, FA decreased peroxiredoxin 2 (Prx 2) protein levels after a 24 or 48h exposure to FA. We also examined whether the FA-induced decrease in Prx 2 was associated with apoptosis. Prx 2 overexpression protected against FA-induced cell apoptosis but not necrosis. Prx 2 overexpression blocked FA-induced increase in Bax, a pro-apoptotic molecule, and a decrease in Bcl-2, an anti-apoptotic molecule. Prx 2 overexpression also protected against FA-induced activation of some special apoptosis-associated proteins [caspase-3, caspase-9, and polypeptide poly (ADP-ribose) polymerase (PARP)]. Furthermore, we examined the signaling molecules involved in the FA-induced decrease in Prx 2 expression. The FA-induced decrease in Prx 2 and increase in cell apoptosis was restored by treatment with SB203580 [a p38 mitogen activated protein kinase (MAPK) inhibitor], but not by SP600125 [a c-jun-N-terminal kinase (JNK) inhibitor]. Also, FA-induced events were blocked by treatment with p38 siRNA, but not by scrambled siRNA. Indeed, FA increased p38 MAPK activation, suggesting a role for p38 MAPK in FA action. In conclusion, FA mediated apoptosis in lung epithelial cells by decreasing Prx 2 via p38 MAPK.

  11. Human Adipose-Derived Mesenchymal Stem Cells Cryopreservation and Thawing Decrease α4-Integrin Expression

    Directory of Open Access Journals (Sweden)

    Ana Carolina Irioda

    2016-01-01

    Full Text Available Aim. The effects of cryopreservation on adipose tissue-derived mesenchymal stem cells are not clearly documented, as there is a growing body of evidence about the importance of adipose-derived mesenchymal stem cells for regenerative therapies. The aim of this study was to analyze human adipose tissue-derived mesenchymal stem cells phenotypic expression (CD34, CD45, CD73, CD90, CD105, and CD49d, colony forming unit ability, viability, and differentiation potential before and after cryopreservation. Materials and Methods. 12 samples of the adipose tissue were collected from a healthy donor using the liposuction technique. The cell isolation was performed by enzymatic digestion and then the cells were cultured up to passage 2. Before and after cryopreservation the immunophenotype, cellular viability analysis by flow cytometer, colony forming units ability, differentiation potential into adipocytes and osteoblasts as demonstrated by Oil Red O and Alizarin Red staining, respectively. Results. The immunophenotypic markers expression was largely preserved, and their multipotency was maintained. However, after cryopreservation, the cells decreased α4-integrin expression (CD49d, cell viability, and number of colony forming units. Conclusions. These findings suggest that ADMSC transplanted after cryopreservation might compromise the retention of transplanted cells in the host tissue. Therefore, further studies are warranted to standardize protocols related to cryopreservation to attain full benefits of stem cell therapy.

  12. Wuweizisu C from Schisandra chinensis decreases membrane potential in C6 glioma cells

    Institute of Scientific and Technical Information of China (English)

    Young-whan CHOI; Kyeok KIM; Ji-yeong JO; Hyo-lim KIM; You-jin LEE; Woo-jung SHIN; Santosh J SACKET; Mijin HAN; Dong-soon IM

    2008-01-01

    Aim:To study the effects of dibenzocyclooctadiene lignans isolated from Schi-sandra chinensis, such as wuweizisu C, gomisin N, gomisin A, and schisandrin, on the membrane potential in C6 glioma cells. Methods: The membrane po-tential was estimated by measuring the fluorescence change in DiBAC-loaded glioma cells. Results: Wuweizisu C decreased the membrane potential in a concentration-dependent manner. Gomisin N and gomisin A, however, showed differential modulation and no change was induced by schisandrin or dimethyl-4,4'-dimethoxy-5,6,5',6'-dimethylene dioxybiphenyl-2,2'-dicarboxylate, a syn-thetic drug derived from dibenzocyclooctadiene lignans. We found no involve-ment of Gi/o proteins, phospholipase C, and extracellular Na+ on the wuweizisu C-indueed decrease of the membrane potential. Wuweizisu C by itself did not change the intracellular Ca2+ [Ca2+]I concentration, but decreased the ATP-indu-ted Ca2+ increase in C6 glioma cells. The 4 lignans at all concentrations used in this study did not induce any effect on cell viability. Furthermore, we found a similar decrease of the membrane potential by wuweizisu C in PC12 neuronal cells. Conclusion: Our results suggest that the decrease in the membrane poten-tial and the modulation of [Ca2+]I concentration by wuweizisu C could be impor-tant action mechanisms ofwuweizisu C.

  13. Histone deacetylase inhibitors inducing human cervical cancer cell apoptosis by decreasing DNA-methyltransferase 3B

    Institute of Scientific and Technical Information of China (English)

    LIU Ning; ZHAO Li-jun; LI Xiao-ping; WANG Jian-liu; CHAI Guo-lin; WEI Li-hui

    2012-01-01

    Background Histone deacetylase (HDAC) inhibitors are a group of small chemical molecules that inhibit histone deacetylase.At cell level,HDAC inhibitors have multiple biological effects such as cell cycle arrest,apoptosis,cell differentiation and auotophagy.At molecular level,HDAC inhibitors cause histone and nonhistone acetylation and induce gene expression.HDAC inhibitors are widely used in cancer therapy because of its function of inducing apoptosis.However,the mechanisms of apoptosis effect are not fully understood.TSA is a classical HDAC inhibitor and widely used in epigenetic and anti-cancer research.In this study,we selected Trichostatin A (TSA) to investigate the mechanisms of HDAC inhibitors apoptotic effect on cancer cells.Methods Cervical cancer cell lines such as Hela,Caski and normal human keratinocyte line HaCaT were treated with various concentrations of TSA.Crystal violent assay and 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay were performed to determine cell number.PARP cleavage and FITC-AnexinV were performed to determine apoptosis.DNA-methyltransferase (DNMT)1,DNMT3A and DNMT3B were determined by regular PCR,qPCR and Western Blotting.Small interfering RNA (SiRNAi) was used to knock down DNMT3B.Results HDAC inhibitors only induce cervical cancer cell apoptosis.At 1 μmol/L of TSA,86% of Hela cell and 76% of Caski went apoptosis.For normal cells,HDAC inhibitors have no cytotoxic effect at therapeutic dosage,(90.0±8.4)% of normal cell survive after treated with 1 μmol/L of TSA.We compared 1 μmol/L group with untreated control with t-test.There was no significance between 1 μmol/L group and untreated control for normal cell (P >0.05).HDAC inhibitors decreased DNMT3B in cancer cell but not in normal cell.Manually knock-down of DNMT3B induced Hela and Caski cell apoptosis.More than 99% of Hela and Caski cell went apoptosis after deprived of DNMT3B.Conclusions DNMT3B was essential to cervical cancer cell survival

  14. Inhibition of focal adhesion kinase (FAK) signaling in focal adhesions decreases cell motility and proliferation.

    OpenAIRE

    1996-01-01

    It has been proposed that the focal adhesion kinase (FAK) mediates focal adhesion formation through tyrosine phosphorylation during cell adhesion. We investigated the role of FAK in focal adhesion structure and function. Loading cells with a glutathione-S-transferase fusion protein (GST-Cterm) containing the FAK focal adhesion targeting sequence, but not the kinase domain, decreased the association of endogenous FAK with focal adhesions. This displacement of endogenous FAK in both BALB/c 3T3 ...

  15. Ubiquitin-specific protease 2 decreases p53-dependent apoptosis in cutaneous T-cell lymphoma.

    Science.gov (United States)

    Wei, Tianling; Biskup, Edyta; Gjerdrum, Lise Mette Rahbek; Niazi, Omid; Ødum, Niels; Gniadecki, Robert

    2016-07-26

    Treatment of advanced cutaneous T-cell lymphomas (CTCL) is challenging because they are resistant to conventional chemotherapy. USP2 has been shown to promote resistance to chemotherapeutic agents in several cancer models.We show here USP2 is expressed in quiescent and activated T-cells and its 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 expression is significantly increased in MyLa2000 (p53wt/wt), but not in SeAx (p53mut) or Hut-78 (p53-/-). USP2 knockdown decreases MyLa2000 cell viability after PUVA by 50% but not Hut-78, suggesting that the function of USP2 in CTCL cells is p53-dependent. Furthermore, USP2 knockdown results in a decreased Mdm2 expression and upregulation of p53. Taken together, our findings suggest that USP2 stabilizes Mdm2 which antagonizes pro-apoptotic activity of p53 and possibly contributes to therapeutic resistance in CTCL.

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

    Directory of Open Access Journals (Sweden)

    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.

  17. Sudden collapse of vacuoles in Saintpaulia sp. palisade cells induced by a rapid temperature decrease.

    Directory of Open Access Journals (Sweden)

    Noriaki Kadohama

    Full Text Available It is well known that saintpaulia leaf is damaged by the rapid temperature decrease when cold water is irrigated onto the leaf surface. We investigated this temperature sensitivity and the mechanisms of leaf damage in saintpaulia (Saintpaulia sp. cv. 'Iceberg' and other Gesneriaceae plants. Saintpaulia leaves were damaged and discolored when subjected to a rapid decrease in temperature, but not when the temperature was decreased gradually. Sensitivity to rapid temperature decrease increased within 10 to 20 min during pre-incubation at higher temperature. Injury was restricted to the palisade mesophyll cells, where there was an obvious change in the color of the chloroplasts. During a rapid temperature decrease, chlorophyll fluorescence monitored by a pulse amplitude modulated fluorometer diminished and did not recover even after rewarming to the initial temperature. Isolated chloroplasts were not directly affected by the rapid temperature decrease. Intracellular pH was monitored with a pH-dependent fluorescent dye. In palisade mesophyll cells damaged by rapid temperature decrease, the cytosolic pH decreased and the vacuolar membrane collapsed soon after a temperature decrease. In isolated chloroplasts, chlorophyll fluorescence declined when the pH of the medium was lowered. These results suggest that a rapid temperature decrease directly or indirectly affects the vacuolar membrane, resulting in a pH change in the cytosol that subsequently affects the chloroplasts in palisade mesophyll cells. We further confirmed that the same physiological damage occurs in other Gesneriaceae plants. These results strongly suggested that the vacuoles of palisade mesophyll cells collapsed during the initial phase of leaf injury.

  18. Sudden collapse of vacuoles in Saintpaulia sp. palisade cells induced by a rapid temperature decrease.

    Science.gov (United States)

    Kadohama, Noriaki; Goh, Tatsuaki; Ohnishi, Miwa; Fukaki, Hidehiro; Mimura, Tetsuro; Suzuki, Yoshihiro

    2013-01-01

    It is well known that saintpaulia leaf is damaged by the rapid temperature decrease when cold water is irrigated onto the leaf surface. We investigated this temperature sensitivity and the mechanisms of leaf damage in saintpaulia (Saintpaulia sp. cv. 'Iceberg') and other Gesneriaceae plants. Saintpaulia leaves were damaged and discolored when subjected to a rapid decrease in temperature, but not when the temperature was decreased gradually. Sensitivity to rapid temperature decrease increased within 10 to 20 min during pre-incubation at higher temperature. Injury was restricted to the palisade mesophyll cells, where there was an obvious change in the color of the chloroplasts. During a rapid temperature decrease, chlorophyll fluorescence monitored by a pulse amplitude modulated fluorometer diminished and did not recover even after rewarming to the initial temperature. Isolated chloroplasts were not directly affected by the rapid temperature decrease. Intracellular pH was monitored with a pH-dependent fluorescent dye. In palisade mesophyll cells damaged by rapid temperature decrease, the cytosolic pH decreased and the vacuolar membrane collapsed soon after a temperature decrease. In isolated chloroplasts, chlorophyll fluorescence declined when the pH of the medium was lowered. These results suggest that a rapid temperature decrease directly or indirectly affects the vacuolar membrane, resulting in a pH change in the cytosol that subsequently affects the chloroplasts in palisade mesophyll cells. We further confirmed that the same physiological damage occurs in other Gesneriaceae plants. These results strongly suggested that the vacuoles of palisade mesophyll cells collapsed during the initial phase of leaf injury.

  19. Decreased differentiation of erythroid cells exacerbates ineffective erythropoiesis in beta-thalassemia.

    Science.gov (United States)

    Libani, Ilaria V; Guy, Ella C; Melchiori, Luca; Schiro, Raffaella; Ramos, Pedro; Breda, Laura; Scholzen, Thomas; Chadburn, Amy; Liu, YiFang; Kernbach, Margrit; Baron-Lühr, Bettina; Porotto, Matteo; de Sousa, Maria; Rachmilewitz, Eliezer A; Hood, John D; Cappellini, M Domenica; Giardina, Patricia J; Grady, Robert W; Gerdes, Johannes; Rivella, Stefano

    2008-08-01

    In beta-thalassemia, the mechanism driving ineffective erythropoiesis (IE) is insufficiently understood. We analyzed mice affected by beta-thalassemia and observed, unexpectedly, a relatively small increase in apoptosis of their erythroid cells compared with healthy mice. Therefore, we sought to determine whether IE could also be characterized by limited erythroid cell differentiation. In thalassemic mice, we observed that a greater than normal percentage of erythroid cells was in S-phase, exhibiting an erythroblast-like morphology. Thalassemic cells were associated with expression of cell cycle-promoting genes such as EpoR, Jak2, Cyclin-A, Cdk2, and Ki-67 and the antiapoptotic protein Bcl-X(L). The cells also differentiated less than normal erythroid ones in vitro. To investigate whether Jak2 could be responsible for the limited cell differentiation, we administered a Jak2 inhibitor, TG101209, to healthy and thalassemic mice. Exposure to TG101209 dramatically decreased the spleen size but also affected anemia. Although our data do not exclude a role for apoptosis in IE, we propose that expansion of the erythroid pool followed by limited cell differentiation exacerbates IE in thalassemia. In addition, these results suggest that use of Jak2 inhibitors has the potential to profoundly change the management of this disorder.

  20. Urate Oxidase Knockdown Decreases Oxidative Stress in a Murine Hepatic Cell Line

    Directory of Open Access Journals (Sweden)

    Beth M. Cleveland

    2009-01-01

    Full Text Available Humans, birds, and some primates do not express the uric acid degrading enzyme urate oxidase (UOX and, as a result, have plasma uric acid concentrations higher than UOX expressing animals. Although high uric acid concentrations are suggested to increase the antioxidant defense system and provide a health advantage to animals without UOX, knockout mice lacking UOX develop pathological complications including gout and kidney failure. As an alternative to the knockout model, RNA interference was used to decrease UOX expression using stable transfection in a mouse hepatic cell line (ATCC, FL83B. Urate oxidase mRNA was reduced 66% (p < 0.05 compared to wild type, as measured by real time RT-PCR. To determine if UOX knockdown resulted in enhanced protection against oxidative stress, cells were challenged with hexavalent chromium (Cr(VI or 3-morpholinosydnonimine hydrochloride (SIN-1. Compared to wild type, cells with UOX knockdown exhibited a 37.2 ± 3.5% reduction (p < 0.05 in the electron spin resonance (ESR signal after being exposed to Cr(VI and displayed less DNA fragmentation (p < 0.05 following SIN-1 treatment. Cell viability decreased in wild type cells (p < 0.05, but not cells with UOX knockdown, after treatment with SIN-1. These results are consistent with an increased intracellular uric acid concentration and an increased defense against oxidative stress.

  1. 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.

  2. Cortisol level decreases natural killer cell activity among women with aircraft noise

    Directory of Open Access Journals (Sweden)

    Hartono Hartono

    2016-02-01

    Full Text Available One of the impacts of exposure to noise is stress. Natural killer (NK cells are one of the leukocyte subsets that are responsive to physiological and psychological stress. The objective of the present research was to determine the relationship between cortisol levels and NK cell activity among women with aircraft noise stress in the area of Adi Sumarmo Airport, Solo. This study was an analytical survey with a cross sectional design. The number of subjects was 39, who were divided into 3 groups of 13 subjects each. Groups 1 to 3 were exposed to noise levels of 92.29 dB, 71.79 dB and 52.17 dB, respectively. The sample was taken using simple random sampling. The data were analyzed by Pearson correlation test and Anova followed by post hoc test using LSD test. The Anova test showed that there were significant differences in circulating cortisol levels among all groups (p = 0.018. The Pearson correlation test showed that there was a positive association between circulating cortisol levels and the number of NK cells (r = 0.547; p< 0.05 and a negative association between circulating cortisol levels and NK cell activity (r = - 0.578; p < 0.05. This study indicated that cortisol levels decreased NK cell activity among women with exposure to aircraft noise. Women who experienced aircraft noise stress showed increased cortisol levels and decreased NK cells activity.

  3. The pesticide methoxychlor decreases myotube formation in cell culture by slowing myoblast proliferation.

    Science.gov (United States)

    Steffens, Bradley W; Batia, Lyn M; Baarson, Chad J; Choi, Chang-Kun Charles; Grow, Wade A

    2007-08-01

    We studied the effect of the estrogenic pesticide methoxychlor (MXC) on skeletal muscle development using C2C12 cell culture. Myoblast cultures were exposed to various concentrations of MXC at various times during the process of myoblast fusion into myotubes. We observed that MXC exposure decreased myotube formation. In addition, we observed myoblasts with cytoplasmic vacuoles in cultures exposed to MXC. Because cytoplasmic vacuoles can be characteristic of cell death, apoptosis assays and trypan blue exclusion assays were performed. We found no difference in the frequency of apoptosis or in the frequency of cell death for cultures exposed to MXC and untreated cultures. Collectively, these results indicate that MXC exposure decreases myotube formation without causing cell death. In contrast, when cell proliferation was assessed, untreated cultures had a myoblast proliferation rate 50% greater than cultures exposed to MXC. We conclude that MXC decreases myotube formation at least in part by slowing myoblast proliferation. Furthermore, we suggest that direct exposure to MXC could affect skeletal muscle development in animals or humans, in addition to the defects in reproductive development that have previously been reported.

  4. 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.

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

    Science.gov (United States)

    Suemori, Shin-ichiro; Wada, Hideho; Nakanishi, Hidekazu; Tsujioka, Takayuki; Sugihara, Takashi; Tohyama, Kaoru

    2015-01-01

    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.

  6. Low-dose testosterone treatment decreases oxidative damage in TM3 Leydig cells

    Institute of Scientific and Technical Information of China (English)

    Thomas IS Hwang; Tien-Ling Liao; Ji-Fan Lin; Yi-Chia Lin; Shu-Yu Lee; Yen-Chun Lai; Shu-Huei Kao

    2011-01-01

    Testosterone replacement therapy has benefits for aging men and those with hypogonadism. However, the effects of exogenous testosterone on Leydig cells are still unclear and need to be clarified. In this report, we demonstrate that testosterone supplementation can reduce oxidative damage in Leydig cells. The TM3 Leydig cell line was used as an in vitro cell model in this study. Cytoprotective effects were identified with 100-nmol l-1 testosterone treatment, but cytotoxic effects were found with ≥ 500-nmol l-1 testosterone supplementation. Significantly reduced reactive oxygen species (ROS) generation, lipid peroxide contents and hypoxia induction factor (HIF)-1α stabilization and activation were found with 100-nmol l-1 testosterone treatment. There was a 1.72-fold increase in ROS generation in the 500-nmol l-1 compared to the 100-nmol l-1 testosterone treatment. A 1.58-fold increase in steroidogenic acute regulatory protein (StAR) expression was found in 50-nmol l-1 testosterone-treated cells (P<0.01). Chemically induced hypoxia was attenuated by testosterone supplementation. Leydig cells treated with low-dose testosterone supplementation showed cytoprotection by decreasing ROS and lipid peroxides, increasing StAR expression and relieving hypoxia stress as demonstrated by HIF-1α stabilization. Increased oxidative damage was found with ≥ 500-nmol l-1 testosterone manipulation. The mechanism governing the differential dose effects of testosterone on Leydig cells needs further investigation in order to shed light on testosterone replacement therapy.

  7. Trans-differentiation of prostatic stromal cells leads to decreased glycoprotein hormone alpha production.

    Science.gov (United States)

    Rumpold, Holger; Mascher, Katarina; Untergasser, Gerold; Plas, Eugen; Hermann, Martin; Berger, Peter

    2002-11-01

    Age-related development of benign prostatic hyperplasia is an important health issue in developed countries. The histopathogenetic hallmark of this disease is the increase in relative and absolute numbers of smooth muscle cells (SMC). Glycoprotein hormone alpha-subunit (GPHalpha) is expressed in the human prostate, and, because of its structural similarities to other cystine knot growth factors, it has been considered to have growth regulatory functions of its own. Primary cell cultures allowing for selective cultivation of prostatic epithelial cells, fibroblasts, and SMC were established. Directed trans-differentiation and cellular homogeneity was pursued by confocal scanning laser microscopy with cell type-specific markers. GPHalpha production by these cells was assessed by immunofluorimetric assays. Its predominant source was young fibroblasts, whereas replicative senescent fibroblasts, SMC, and control fibroblasts from foreskin did not produce significant amounts. Functionally, GPHalpha reduced growth of stromal cells at concentrations of 10 and 100 nmol/liter as shown by cell viability assays. It is concluded that histogenetic reorganization over the adult lifetime, guided by endocrine factors like steroid hormones together with senescence of fibroblasts, leads to a decreased production of growth inhibitors, such as GPHalpha, favoring proliferation and the development of benign prostatic hyperplasia.

  8. 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.

  9. Decrease in spermidine content during logarithmic phase of cell growth delays spore formation of Bacillus subtilis.

    Science.gov (United States)

    Ishii, I; Takada, H; Terao, K; Kakegawa, T; Igarashi, K; Hirose, S

    1994-11-01

    Bacillus subtilis 168M contained a large amount of spermidine during the logarithmic phase of growth, but the amount decreased drastically during the stationary phase. The extracts, prepared from B. subtilis cells harvested in the logarithmic phase, contained activity of arginine decarboxylase (ADC) rather than the activity of ornithine decarboxylase. In the presence of alpha-difluoromethylarginine (DFMA), a specific and irreversible inhibitor of ADC, the amount of spermidine in B. subtilis during the logarithmic phase decreased to about 25% of the control cells. Under these conditions, spore formation of B. subtilis 168M delayed greatly without significant inhibition of cell growth. The decrease in spermidine content in the logarithmic phase rather than in the stationary phase was involved in the delay of sporulation. Electron microscopy of cells at 24 hrs. of culture confirmed the delay of spore formation by the decrease of spermidine content. Furthermore, the delay of sporulation was negated by the addition of spermidine. These data suggest that a large amount of spermidine existing during the logarithmic phase plays an important role in the sporulation of B. subtilis.

  10. 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

    of these channels in the Apoptotic Volume Decrease (AVD) of isolated eel enterocytes, and the possible interaction between BK channels and the progression of apoptosis. The detection of apoptosis was performed by confocal microscopy and annexin V and propidium iodide labelling; cell volume changes were monitored...

  11. Decreased cell proliferation and higher oxidative stress in fibroblasts from Down Syndrome fetuses. Preliminary study.

    Science.gov (United States)

    Gimeno, Amparo; García-Giménez, José Luis; Audí, Laura; Toran, Nuria; Andaluz, Pilar; Dasí, Francisco; Viña, José; Pallardó, Federico V

    2014-01-01

    Down Syndrome is the most common chromosomal disease and is also known for its decreased incidence of solid tumors and its progeroid phenotype. Cellular and systemic oxidative stress has been considered as one of the Down Syndrome phenotype causes. We correlated, in a preliminary study, the fibroblast proliferation rate and different cell proliferation key regulators, like Rcan1 and the telomere length from Down Syndrome fetuses, with their oxidative stress profile and the Ribonucleic acid and protein expression of the main antioxidant enzymes together with their activity. Increased oxidized glutathione/glutathione ratio and high peroxide production were found in our cell model. These results correlated with a distorted antioxidant shield. The messenger RNA (SOD1) and protein levels of copper/zinc superoxide dismutase were increased together with a decreased mRNA expression and protein levels of glutathione peroxidase (GPx). As a consequence the [Cu/ZnSOD/(catalase+GPx)] activity ratio increases which explains the oxidative stress generated in the cell model. In addition, the expression of thioredoxin 1 and glutaredoxin 1 is decreased. The results obtained show a decreased antioxidant phenotype that correlates with increased levels of Regulator of calcineurin 1 and attrition of telomeres, both related to oxidative stress and cell cycle impairment. Our preliminary results may explain the proneness to a progeroid phenotype.

  12. Cells

    Directory of Open Access Journals (Sweden)

    Zhao-Hui Jin

    2012-11-01

    Full Text Available As cancer stem cells (CSCs are postulated to play critical roles in cancer development, including metastasis and recurrence, CSC imaging would provide valuable information for cancer treatment and lead to CSC-targeted therapy. To assess the possibility of in vivo CSC targeting, we conducted basic studies on radioimmunotargeting of cancer cells positive for CD133, a CSC marker recognized in various cancers. Antibodies against CD133 were labeled with 125I, and their in vitro cell binding properties were tested. Using the same isotype IgG as a control, in vivo biodistribution of the labeled antibody retaining immunoreactivity was examined in mice bearing an HCT116 xenograft in which a population of the cancer cells expressed CD133. Intratumoral distribution of the labeled antibody was examined and compared to the CD133 expression pattern. The 125I-labeled anti-CD133 antibody showed a modest but significantly higher accumulation in the HCT116 xenograft compared to the control IgG. The intratumoral distribution of the labeled antibody mostly overlapped with the CD133 expression, whereas the control IgG was found in the area close to the necrotic tumor center. Our results indicate that noninvasive in vivo targeting of CSCs could be possible with radiolabeled antibodies against cell membrane markers.

  13. N-cadherin knock-down decreases invasiveness of esophageal squamous cell carcinoma in vitro

    Institute of Scientific and Technical Information of China (English)

    Ke Li; Wei He; Na Lin; Xin Wang; Qing-Xia Fan

    2009-01-01

    AIM: To examine the expressions of N-cadherin and E-cadherin in specimens of 62 normal esophageal epithela, 31 adjacent atypical hyperplastic epithelia and 62 esophageal squamous cell carcinomas (ESCCs), and to investigate the roles of N-cadherin in the invasiveness of ESCC cell line EC9706 transfected by N-cadherin shRNA. METHODS: PV immunohistochemistry was used to detect the expression pattern of N-cadherin and E-cadherin in specimens of 62 normal esophageal epithelia, 31 adjacent atypical hyperplastic epithelia and 62 ESCCs. The invasiveness of ESCC line EC9706 was determined by transwell assay after EC9706 was transfected by N-cadherin shRNA. RESULTS: The positive rates of N-cadherin decreased in the carcinoma, adjacent atypical hyperplastic and normal esophageal tissues (75.8%, 61.3% and 29.0%, P < 0.05), respectively, while those of E-cadherin increased (40.3%, 71.0% and 95.2%, P < 0.05). The increased expression of N-cadherin and decreased expression of E-cadherin were related to invasion, differentiation, and lymph node metastasis ( P < 0.05). The expression level of N-cadherin decreased in the N-cadherin knocked down cells, and the invasiveness of those cells decreased significantly as well. The number of cells which crossed the basement membrane filter 0.05). CONCLUSION: E-cadherin and N-cadherin expression is correlated with the invasion and aggravation of ESCC. The down-regulation of N-cadherin lowers the invasiveness of EC9706 cell line.

  14. Thyroid Autoimmunity is Associated with Decreased Cytotoxicity T Cells in Women with Repeated Implantation Failure

    Directory of Open Access Journals (Sweden)

    Chunyu Huang

    2015-08-01

    Full Text Available Thyroid autoimmunity (TAI, which is defined as the presence of autoantibodies against thyroid peroxidase (TPO and/or thyroglobulin (TG, is related to repeated implantation failure (RIF. It is reported that TAI was involved in reproductive failure not only through leading thyroid function abnormality, but it can also be accompanied with immune imbalance. Therefore, this study was designed to investigate the association of thyroid function, immune status and TAI in women with RIF. Blood samples were drawn from 72 women with RIF to evaluate the prevalence of TAI, the thyroid function, the absolute numbers and percentages of lymphocytes. The prevalence of thyroid function abnormality in RIF women with TAI was not significantly different from that in RIF women without TAI (c2 = 0.484, p > 0.05. The absolute number and percentage of T cells, T helper (Th cells, B cells and natural killer (NK cells were not significantly different in RIF women with TAI compared to those without TAI (all p > 0.05. The percentage of T cytotoxicity (Tc cells was significantly decreased in RIF women with TAI compared to those without TAI (p < 0.05. Meanwhile, Th/Tc ratio was significantly increased (p < 0.05. These results indicated that the decreased Tc percentage and increased Th/Tc ratio may be another influential factor of adverse pregnancy outcomes in RIF women with TAI.

  15. 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...... expression is significantly increased in MyLa2000 (p53wt/wt), but not in SeAx (p53mut) or Hut-78 (p53-/-). USP2 knockdown decreases MyLa2000 cell viability after PUVA by 50% but not Hut-78, suggesting that the function of USP2 in CTCL cells is p53-dependent. Furthermore, USP2 knockdown results in a decreased...... Mdm2 expression and upregulation of p53. Taken together, our findings suggest that USP2 stabilizes Mdm2 which antagonizes pro-apoptotic activity of p53 and possibly contributes to therapeutic resistance in CTCL....

  16. Increased apoptosis and decreased density of medial smooth muscle cells in human abdominal aortic aneurysms

    Institute of Scientific and Technical Information of China (English)

    ZHANG Jian张健; Jan Schmidt; Eduard Ryschich; Hardy Schumacher; Jens R Allenberg

    2003-01-01

    Objective To determine the increase of apoptosis and the decrease of smooth muscle cells (SMCs) density in human abdominal aortic aneurysms (AAA). Methods In situ terminal transferase-mediated dUTP nick end labeling (TUNEL) was employed to detect apoptosis of SMCs in patients with AAA (n=25) and normal abdominal aortae (n=10). Positive cells were identified by specific cell marker in combination with immunohistochemistry. Meanwhile SMC counting was performed by anti-α-actin immunohistostaining to compare the SMC density. Results TUNEL staining revealed that there was significantly increased apoptosis in AAAs (average 8.6%) compared with normal abdominal aortae (average 0.95%, P<0.01). Double staining showed that most of these cells were SMCs. Counting of α-actin positive SMCs revealed that medial SMC density of AAAs (37.5±7.6 SMCs /HPF) was reduced by 79.1% in comparison with that of normal abdominal aortae (179.2±16.1 SMCs /HPF, P<0.01). Conclusions Significantly increased SMCs of AAA bear apoptotic markers initiating cell death. Elevated apoptosis may result in a decreased density of SMCs in AAA, which may profoundly influence the development of AAA.

  17. 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.

  18. miR-17 overexpression in cystic fibrosis airway epithelial cells decreases interleukin-8 production.

    Science.gov (United States)

    Oglesby, Irene K; Vencken, Sebastian F; Agrawal, Raman; Gaughan, Kevin; Molloy, Kevin; Higgins, Gerard; McNally, Paul; McElvaney, Noel G; Mall, Marcus A; Greene, Catherine M

    2015-11-01

    Interleukin (IL)-8 levels are higher than normal in cystic fibrosis (CF) airways, causing neutrophil infiltration and non-resolving inflammation. Overexpression of microRNAs that target IL-8 expression in airway epithelial cells may represent a therapeutic strategy for cystic fibrosis. IL-8 protein and mRNA were measured in cystic fibrosis and non-cystic fibrosis bronchoalveolar lavage fluid and bronchial brushings (n=20 per group). miRNAs decreased in the cystic fibrosis lung and predicted to target IL-8 mRNA were quantified in βENaC-transgenic, cystic fibrosis transmembrane conductance regulator (Cftr)-/- and wild-type mice, primary cystic fibrosis and non-cystic fibrosis bronchial epithelial cells and a range of cystic fibrosis versus non-cystic fibrosis airway epithelial cell lines or cells stimulated with lipopolysaccharide, Pseudomonas-conditioned medium or cystic fibrosis bronchoalveolar lavage fluid. The effect of miRNA overexpression on IL-8 protein production was measured. miR-17 regulates IL-8 and its expression was decreased in adult cystic fibrosis bronchial brushings, βENaC-transgenic mice and bronchial epithelial cells chronically stimulated with Pseudomonas-conditioned medium. Overexpression of miR-17 inhibited basal and agonist-induced IL-8 protein production in F508del-CFTR homozygous CFTE29o(-) tracheal, CFBE41o(-) and/or IB3 bronchial epithelial cells. These results implicate defective CFTR, inflammation, neutrophilia and mucus overproduction in regulation of miR-17. Modulating miR-17 expression in cystic fibrosis bronchial epithelial cells may be a novel anti-inflammatory strategy for cystic fibrosis and other chronic inflammatory airway diseases.

  19. Cell Volume Regulation and Apoptotic Volume Decrease in Rat Distal Colon Superficial Enterocytes

    Directory of Open Access Journals (Sweden)

    Stefania Antico

    2013-12-01

    Full Text Available Background: The colon epithelium is physiologically exposed to osmotic stress, and the activation of cell volume regulation mechanisms is essential in colonocyte physiology. Moreover, colon is characterized by a high apoptotic rate of mature cells balancing the high division rate of stem cells. Aim: The aim of the present work was to investigate the main cell volume regulation mechanisms in rat colon surface colonocytes and their role in apoptosis. Methods: Cell volume changes were measured by light microscopy and video imaging on colon explants; apoptosis sign appearance was monitored by confocal microscopy on annexin V/propidium iodide labeled explants. Results: Superficial colonocytes showed a dynamic regulation of their cell volume during anisosmotic conditions with a Regulatory Volume Increase (RVI response following hypertonic shrinkage and Regulatory Volume Decrease (RVD response following hypotonic swelling. RVI was completely inhibited by bumetanide, while RVD was completely abolished by high K+ or iberiotoxin treatment and by extracellular Ca2+ removal. DIDS incubation was also able to affect the RVD response. When colon explants were exposed to H2O2 as apoptotic inducer, colonocytes underwent an isotonic volume decrease ascribable to Apoptotic Volume Decrease (AVD within about four hours of exposure. AVD was shown to precede annexin V positivity. It was also inhibited by high K+ or iberiotoxin treatment. Interestingly, treatment with iberiotoxin significantly inhibited apoptosis progression. Conclusions: In rat superficial colonocytes K+ efflux through high conductance Ca2+-activated K+ channels (BK channels was demonstrated to be the main mechanism of RVD and to plays also a crucial role in the AVD process and in the progression of apoptosis.

  20. Age-related decrease in rod bipolar cell density of the human retina: an immunohistochemical study

    Indian Academy of Sciences (India)

    P Aggarwal; T C Nag; S Wadhwa

    2007-03-01

    During normal ageing, the rods (and other neurones) undergo a significant decrease in density in the human retina from the fourth decade of life onward. Since the rods synapse with the rod bipolar cells in the outer plexiform layer, a decline in rod density (mainly due to death) may ultimately cause an associated decline of the neurones which, like the rod bipolar cells, are connected to them. The rod bipolar cells are selectively stained with antibodies to protein kinase C-. This study examined if rod bipolar cell density changes with ageing of the retina, utilizing donor human eyes (age: 6–91 years). The retinas were fixed and their temporal parts from the macula to the mid-periphery sectioned and processed for protein kinase C- immunohistochemistry. The density of the immunopositive rod bipolar cells was estimated in the mid-peripheral retina (eccentricity: 3–5 mm) along the horizontal temporal axis. The results show that while there is little change in the density of the rod bipolar cells from 6 to 35 years (2.2%), the decline during the period from 35 to 62 years is about 21% and between seventh and tenth decades, it is approximately 27%.

  1. Cardamonin Inhibits Metastasis of Lewis Lung Carcinoma Cells by Decreasing mTOR Activity.

    Directory of Open Access Journals (Sweden)

    Pei-Guang Niu

    Full Text Available The mammalian target of rapamycin (mTOR regulates the motility and invasion of cancer cells. Cardamonin is a chalcone that exhibits anti-tumor activity. The previous study had proved that the anti-tumor effect of cardamonin was associated with mTOR inhibition. In the present study, the anti-metastatic effect of cardamonin and its underlying molecule mechanisms were investigated on the highly metastatic Lewis lung carcinoma (LLC cells. The proliferation, invasion and migration of LLC cells were measured by MTT, transwell and wound healing assays, respectively. The expression and activation of mTOR- and adhesion-related proteins were assessed by Western blotting. The in vivo effect of cardamonin on the metastasis of the LLC cells was investigated by a mouse model. Treated with cardamonin, the proliferation, invasion and migration of LLC cells were significantly inhibited. The expression of Snail was decreased by cardamonin, while that of E-cadherin was increased. In addition, cardamonin inhibited the activation of mTOR and its downstream target ribosomal S6 kinase 1 (S6K1. Furthermore, the tumor growth and its lung metastasis were inhibited by cardamonin in C57BL/6 mice. It indicated that cardamonin inhibited the invasion and metastasis of LLC cells through inhibiting mTOR. The metastasis inhibitory effect of cardamonin was correlated with down-regulation of Snail and up-regulation of E-cadherin.

  2. 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.

  3. Stable expression of sialyl-Tn antigen in T47-D cells induces a decrease of cell adhesion and an increase of cell migration.

    Science.gov (United States)

    Julien, Sylvain; Lagadec, Chann; Krzewinski-Recchi, Marie-Ange; Courtand, Gilles; Le Bourhis, Xuefen; Delannoy, Philippe

    2005-03-01

    Sialyl-Tn is a carbohydrate antigen overexpressed in several epithelial cancers including breast cancer, and usually associated with poor prognosis. Sialyl-Tn is synthesized by a CMP-Neu5Ac: GalNAc alpha2,6-sialyltransferase: ST6GalNAc I, which catalyzes the transfer of a sialic acid residue in alpha2,6-linkage to the GalNAcalpha1-O-Ser/Thr structure. The resulting disaccharide (Neu5Acalpha2-6GalNAcalpha1-O-Ser/Thr) cannot be further elongated and sialyl-Tn expression results therefore in a shortening of the O-glycan chains. However, usual breast cancer cell lines express neither ST6GalNAc I nor sialyl-Tn antigen. We have previously shown that stable transfection of MDA-MB-231 cells with the hST6GalNAc I cDNA induces the sialyl-Tn antigen expression at the cell surface and leads to a decreased cell growth and an increased cell migration. We describe herein the generation of new T47-D clones expressing sialyl-Tn antigen after hST6GalNAc I cDNA stable transfection. sialyl-Tn antigen is carried by several high molecular weight membrane bound O-glycoproteins, including MUC1. We show that sialyl-Tn expression induces a decrease of cell growth and adhesion, and an increase of cell migration in sialyl-Tn positive clones compared to mock transfected cells. These observations show that the alteration of the O-glycans pattern is sufficient to modify the biological features of cancer cells. These T47-D sialyl-Tn expressing clones might allow further in vivo investigation to determine precisely the impact of such O-glycosylation modifications on breast cancer development.

  4. 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.

  5. Caspase 3 siRNA decreases apoptosis in cultured neuronal cells

    Institute of Scientific and Technical Information of China (English)

    Chunting Ye; Yaoxiong Huang; Xiaohong Yang; Honghui Chen

    2009-01-01

    BACKGROUND: Lentiviral vectors, a type of retroviral vector, are able to infect cells at all phases of cell cycle. They are able to express exogenous target genes in vivo over long periods of time with limited immunological reaction. OBJECTIVE: To inhibit neuronal apoptosis by blocking the apoptotic cascade reaction, gene silencing of Caspase 3, and transfection of Caspase 3 short hairpin ribonucleic acid (shRNA) into Neuro 2a cells using a lentiviral vector. DESIGN, TIME AND SETTING: An observational, genetic engineering cellular biology experiment was performed in Guangzhou Red Cross Hospital and Guangzhou institute of Traumatic Surgery between March 2007 and June 2008.MATERIALS: PLL3.7, PCMV-VSV-G, and PH'8.9△PR plasmids were provided by the CBR institute for Biomedical Research, Harvard Medical School, USA. Staurosporine was purchased from Sigma, USA. METHODS: Caspase 3 siRNA was synthesized and cloned into the PLL3.7 plasmid. The Caspase 3 shRNA-PLL3.7 lentivirus was generated in 293T cells using a calcium phosphate transfection kit. After the lentivirus was transfected into Neuro 2a cells, apoptosis was induced in both the transfected and untransfected cells by staurosporine. Cell apoptosis was assessed by flow cytometry. MAIN OUTCOME MEASURES: Caspase 3 mRNA expression was measured by RT-PCR and Caspase protein expression was assessed by Western blot. Cellular apoptosis was determined by flow cytometry using Annevin V-PE/7aad-Cy7.RESULTS: The transfection rate of caspase 3 shRNA was>98% using the ientiviral vector. RT-PCR and Western blot results demonstrated that significantly reduced Caspase 3 mRNA and protein expression in the transfected Neuro 2a. The control group exhibited 38.7% Annexin V/7aad-positive cells, which suggested apoptotic anaphase, while only 5.0% cells in the Caspase 3 gene silencing group entered apoptotic anaphase. CONCLUSION: Caspase 3 shRNA inhibited Caspase 3 expression in Neuro 2a cells and decreased drug-induced apoptosis of

  6. 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.

  7. 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.

  8. 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

  9. Increased Mesenchymal Stem Cell Response and Decreased Staphylococcus aureus Adhesion on Titania Nanotubes without Pharmaceuticals

    Science.gov (United States)

    Xu, Zhiqiang; Lai, Yingzhen; Wu, Dong; Huang, Wenxiu; Huang, Sijia; Zhou, Lin; Chen, Jiang

    2015-01-01

    Titanium (Ti) implants with enhanced biocompatibility and antibacterial property are highly desirable and characterized by improved success rates. In this study, titania nanotubes (TNTs) with various tube diameters were fabricated on Ti surfaces through electrochemical anodization at 10, 30, and 60 V (denoted as NT10, NT30, and NT60, resp.). Ti was also investigated and used as a control. NT10 with a diameter of 30 nm could promote the adhesion and proliferation of bone marrow mesenchymal stem cells (BMSCs) without noticeable differentiation. NT30 with a diameter of 100 nm could support the adhesion and proliferation of BMSCs and induce osteogenesis. NT60 with a diameter of 200 nm demonstrated the best ability to promote cell spreading and osteogenic differentiation; however, it clearly impaired cell adhesion and proliferation. As the tube diameter increased, bacterial adhesion on the TNTs decreased and reached the lowest value on NT60. Therefore, NT30 without pharmaceuticals could be used to increase mesenchymal stem cell response and decrease Staphylococcus aureus adhesion and thus should be further studied for improving the efficacy of Ti-based orthopedic implants. PMID:26640782

  10. Decrease in Circulating Dendritic Cell Precursors in Patients with Peripheral Artery Disease

    Directory of Open Access Journals (Sweden)

    Daniel Kretzschmar

    2015-01-01

    Full Text Available Peripheral artery disease (PAD is a common manifestation of atherosclerosis. Inflammation is important for initiation and progression of the disease. Dendritic cells (DCs as antigen-presenting cells play an important role in the immune system. Therefore, we hypothesize that, in patients with PAD, DCPs might be reduced in blood due to their recruitment into the vascular wall and induce a proinflammatory response. The numbers of myeloid DCPs, plasmacytoid DCPs, and total DCPs were analyzed by flow cytometry in blood of patients with PAD (n=52 compared to controls (n=60. Femoralis plaques (n=12 of patients who underwent surgery were immunostained for CD209 and CD83 (mDCs as well as CD304, CD123 (pDCs, and HLA-DR. In patients with PAD, a significant decrease in mDCPs, pDCPs, and tDCPs was observed. In immunostaining, markers indicative for mDCs (CD209: 16 versus 8 cells/0.1 mm2, P=0.02; CD83: 19 versus 5 cells/0.1 mm2, P=0.03 were significantly elevated in femoralis plaques compared to control vessels. We show for the first time that mDCPs, pDCPs, and tDCPs are significantly reduced in patients with PAD. Immunohistochemical analysis unraveled that the decrease in DCPs might be due to their recruitment into atherosclerotic plaques.

  11. 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...

  12. Iron increases HMOX1 and decreases hepatitis C viral expression in HCV-expressing cells

    Institute of Scientific and Technical Information of China (English)

    Wei-Hong Hou; Lisa Rossi; Ying Shan; Jian-Yu Zheng; Richard W Lambrecht; Herbert L Bonkovsky

    2009-01-01

    AIM: To investigate effects of iron on oxidative stress,heme oxygenase-1 (HMOX1) and hepatitis C viral (HCV) expression in human hepatoma cells stably expressing HCV proteins.METHODS: Effects of iron on oxidative stress, HMOX1,and HCV expression were assessed in CON1 cells.Measurements included mRNA by quantitative reverse transcription-polymerase chain reaction, and protein levels by Western blots.RESULTS: Iron, in the form of ferric nitrilotriacetate,increased oxidative stress and up-regulated HMOX1 gene expression. Iron did not affect mRNA or protein levels of Bach1, a repressor of HMOX1. Silencing the up-regulation of HMOX1 nuclear factor-erythroid 2-related factor 2 (Nrf2) by Nrf2-siRNA decreased FeNTA-mediated up-regulation of HMOX1 mRNA levels. These iron effects were completely blocked by deferoxamine (DFO). Iron also significantly decreased levels of HCV core mRNA and protein by 80%-90%,nonstructural 5A mRNA by 90% and protein by about 50% in the Con1 full length HCV replicon cells,whereas DFO increased them.CONCLUSION: Excess iron up-regulates HMOX1 and down-regulates HCV gene expression in hepatoma cells. This probably mitigates liver injury caused by combined iron overload and HCV infection.

  13. Decreased Polysaccharide Feruloylation Compromises Plant Cell Wall Integrity and Increases Susceptibility to Necrotrophic Fungal Pathogens.

    Science.gov (United States)

    Reem, Nathan T; Pogorelko, Gennady; Lionetti, Vincenzo; Chambers, Lauran; Held, Michael A; Bellincampi, Daniela; Zabotina, Olga A

    2016-01-01

    The complexity of cell wall composition and structure determines the strength, flexibility, and function of the primary cell wall in plants. However, the contribution of the various components to cell wall integrity (CWI) and function remains unclear. Modifications of cell wall composition can induce plant responses known as CWI control. In this study, we used transgenic expression of the fungal feruloyl esterase AnFAE to examine the effect of post-synthetic modification of Arabidopsis and Brachypodium cell walls. Transgenic Arabidopsis plants expressing AnFAE showed a significant reduction of monomeric ferulic acid, decreased amounts of wall-associated extensins, and increased susceptibility to Botrytis cinerea, compared with wild type. Transgenic Brachypodium showed reductions in monomeric and dimeric ferulic acids and increased susceptibility to Bipolaris sorokiniana. Upon infection, transgenic Arabidopsis and Brachypodium plants also showed increased expression of several defense-related genes compared with wild type. These results demonstrate a role, in both monocot and dicot plants, of polysaccharide feruloylation in plant CWI, which contributes to plant resistance to necrotrophic pathogens.

  14. Decreased hydrocortisone sensitivity of T cell function in multiple sclerosis-associated major depression.

    Science.gov (United States)

    Fischer, Anja; Otte, Christian; Krieger, Thorsten; Nicholls, Robert A; Krüger, Schulamith; Ziegler, Kristin J; Schulz, Karl-Heinz; Heesen, Christoph; Gold, Stefan M

    2012-10-01

    Multiple sclerosis (MS) is an inflammatory, demyelinating disease of the CNS with a high prevalence of depression. Both MS and depression have been linked to elevated cortisol levels and inflammation, indicating disturbed endocrine-immune regulation. An imbalance in mineralocorticoid versus glucocorticoid signaling in the CNS has been proposed as a pathogenetic mechanism of depression. Intriguingly, both receptors are also expressed in lymphocytes, but their role for 'escape' of the immune system from endocrine control is unknown. Using steroid sensitivity of T cell function as a read-out system, we here investigate a potential role of mineralocorticoid receptor (MR) versus glucocorticoid receptor (GR) regulation in the immune system as a biological mechanism underlying MS-associated major depression. Twelve female MS patients meeting diagnostic criteria for current major depressive disorder (MDD) were compared to twelve carefully matched MS patients without depression. We performed lymphocyte phenotyping by flow cytometry. In addition, steroid sensitivity of T cell proliferation was tested using hydrocortisone as well as MR (aldosterone) and GR (dexamethasone) agonists. Sensitivity to hydrocortisone was decreased in T cells from depressed MS patients. Experiments with agonists suggested disturbed MR regulation, but intact GR function. Importantly, there were no differences in lymphocyte composition and frequency of T cell subsets, indicating that the differences in steroid sensitivity are unlikely to be secondary to shifts in the immune compartment. To our knowledge, this study provides first evidence for altered steroid sensitivity of T cells from MS patients with comorbid MDD possibly due to MR dysregulation.

  15. Electroporation transiently decreases GJB2 (connexin 26) expression in B16/BL6 melanoma cell line.

    Science.gov (United States)

    Rangel, Marcelo Monte Mór; Chaible, Lucas Martins; Nagamine, Marcia Kazumi; Mennecier, Gregory; Cogliati, Bruno; de Oliveira, Krishna Duro; Fukumasu, Heidge; Sinhorini, Idércio Luiz; Mir, Lluis Maria; Dagli, Maria Lúcia Zaidan

    2015-02-01

    Connexins are proteins that form gap junctions. Perturbations in the cell membrane reportedly promote changes in the expression profile of connexins. Electroporation promotes destabilization by applying electrical pulses, and this procedure is used in electrochemotherapy and gene therapy, among others. This in vitro work aimed to study the interference of electroporation on the expression profile of GJB2 (Cx26 gene) and Connexin 26 in melanoma cell line B16/BL6. The techniques of immunocytochemistry, Western blot, and real-time PCR were used. After electroporation, cells showed a transient decrease in GJB2 mRNA. The immunostaining of Cx26 showed no noticeable change after electroporation at different time points. However, Western blot showed a significant reduction in Cx26 30 min after electroporation. Our results showed that electroporation interferes transiently in the expression of Connexin 26 in melanoma and are consistent with the idea that electroporation is a process of intense stress that promotes cell homeostatic imbalance and results in disruption of cell physiological processes such as transcription and translation.

  16. Decreased frequencies and impaired functions of the CD31(+) subpopulation in Treg cells associated with decreased FoxP3 expression and enhanced Treg cell defects in patients with coronary heart disease.

    Science.gov (United States)

    Huang, L; Zheng, Y; Yuan, X; Ma, Y; Xie, G; Wang, W; Chen, H; Shen, L

    2017-03-01

    Coronary heart disease (CHD) is one of the most common types of organ lesions caused by atherosclerosis, in which CD4(+) CD25(+) forkhead box protein 3 (FoxP3(+) ) regulatory T cells (Treg ) play an atheroprotective role. However, Treg cell numbers are decreased and their functions are impaired in atherosclerosis; the underlying mechanisms remain unclear. CD31 plays an important part in T cell response and contributes to maintaining T cell tolerance. The immunomodulatory effects of CD31 are also implicated in atherosclerosis. In this study, we found that decreased frequencies of the CD31(+) subpopulation in Treg cells (CD31(+) Tr cells) correlated positively with decreased FoxP3 expression in CHD patients. Cell culture in vitro demonstrated CD31(+) Tr cells maintaining stable FoxP3 expression after activation and exhibited enhanced proliferation and immunosuppression compared with the CD31(-) subpopulation in Treg cells (CD31(-) Tr cells). We also confirmed impaired secretion of transforming growth factor (TGF)-β1 and interleukin (IL)-10 in CD31(+) Tr cells of CHD patients. Further analysis revealed reduced phospho-SHP2 (associated with CD31 activation) and phospho-signal transducer and activator of transcription-5 (STAT-5) (associated with FoxP3 transcription) levels in CD31(+) Tr cells of CHD patients, suggesting that decreased FoxP3 expression in CD31(+) Tr cells might be because of attenuated SHP2 and STAT-5 activation. These data indicate that decreased frequencies and impaired functions of the CD31(+) Tr subpopulation associated with decreased FoxP3 expression give rise, at least in part, to Treg cell defects in CHD patients. Our findings emphasize the important role of the CD31(+) Tr subpopulation in maintaining Treg cell normal function and may provide a novel explanation for impaired immunoregulation of Treg cells in CHD.

  17. 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

    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...

  18. Saccharomyces cerevisiae Live Cells Decreased In vitro Methane Production in Intestinal Content of Pigs.

    Science.gov (United States)

    Gong, Y L; Liao, X D; Liang, J B; Jahromi, M F; Wang, H; Cao, Z; Wu, Y B

    2013-06-01

    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 1.8×10(10) 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 (pyeast. 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 (pyeast supplementation. Quantity of methanogenic archaea per milliliter of inoculum decreased (pyeast 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 bacteria, both of which serve as a competitive pathway for the available H2 resulting in the reduction of methanogenic archaea.

  19. Monoclonal B-cell lymphocytosis (MBL) with normal lymphocyte counts is associated with decreased numbers of normal circulating B-cell subsets.

    Science.gov (United States)

    Hauswirth, Alexander W; Almeida, Julia; Nieto, Wendy G; Teodosio, Cristina; Rodriguez-Caballero, Arancha; Romero, Alfonso; López, Antonio; Fernandez-Navarro, Paulino; Vega, Tomas; Perez-Andres, Martin; Valent, Peter; Jäger, Ulrich; Orfao, Alberto

    2012-07-01

    Monoclonal B-cell lymphocytosis (MBL) with normal lymphocyte counts is associated with decreased numbers of normal circulating B-cell subsets.Little is known about the distribution of normal lymphoid cells and their subsets in the peripheral blood (PB) of subjects with monoclonal B-cell lymphocytosis (MBL). In our study, we compared the absolute number of PB lymphoid cells and their subpopulations in 95 MBL cases with normal lymphocyte counts vs. 617 age-/sex-matched non-MBL healthy subjects (controls), using highly sensitive flow cytometry. MBL cases showed significantly reduced numbers of normal circulating B-cells, at the expense of immature and naive B-cells; in addition, CD4+CD8+ double-positive T-cells and CD8+ T-cells were significantly lower and higher vs. controls, respectively. Moreover, most normal B-cell subsets were significantly decreased in PB at >1% MBL-counts, vs. "low-count" MBL cases, and lower amounts of immature/naive B-cells were detected in biclonal (particularly in cases with coexisting CLL-like- and non-CLL-like B-cell clones) vs. monoclonal MBL subjects. In summary, our results show imbalanced (reduced) absolute numbers of recently produced normal circulating B-cells (e.g., immature and naıve B-cells) in MBL, which becomes more pronounced as the MBL cell count increases.

  20. Red blood cell aquaporin-1 expression is decreased in hereditary spherocytosis.

    Science.gov (United States)

    Crisp, Renée L; Maltaneri, Romina E; Vittori, Daniela C; Solari, Liliana; Gammella, Daniel; Schvartzman, Gabriel; García, Eliana; Rapetti, María C; Donato, Hugo; Nesse, Alcira

    2016-10-01

    Aquaporin-1 (AQP1) is the membrane water channel responsible for changes in erythrocyte volume in response to the tonicity of the medium. As the aberrant distribution of proteins in hereditary spherocytosis (HS) generates deficiencies of proteins other than those codified by the mutated gene, we postulated that AQP1 expression might be impaired in spherocytes. AQP1 expression was evaluated through flow cytometry in 5 normal controls, 1 autoimmune hemolytic anemia, 10 HS (2 mild, 3 moderate, 2 severe, and 3 splenectomized), and 3 silent carriers. The effect of AQP1 inhibitors was evaluated through water flow-based tests: osmotic fragility and hypertonic cryohemolysis. Serum osmolality was measured in 20 normal controls and 13 HS. The effect of erythropoietin (Epo) on AQP1 expression was determined in cultures of erythroleukemia UT-7 cells, dependent on Epo to survive. Independent of erythrocyte size, HS patients showed a lower content of AQP1 in erythrocyte membranes which correlated with the severity of the disease. Accordingly, red blood cells from HS subjects were less sensitive to cryohemolysis than normal erythrocytes after inhibition of the AQP1 water channel. A lower serum osmolality in HS with respect to normal controls suggests alterations during reticulocyte remodeling. The decreased AQP1 expression could contribute to explain variable degrees of anemia in hereditary spherocytosis. The finding of AQP1 expression induced by Epo in a model of erythroid cells may be interpreted as a mechanism to restore the balance of red cell water fluxes.

  1. Bone Marrow Homing Enriches Stem Cells Responsible for Neogenesis of Insulin-Producing Cells, While Radiation Decreases Homing Efficiency.

    Science.gov (United States)

    Goldenberg-Cohen, Nitza; Iskovich, Svetlana; Askenasy, Nadir

    2015-10-01

    Small-sized adult bone marrow cells isolated by counterflow centrifugal elutriation and depleted of lineage markers (Fr25lin(-)) have the capacity to differentiate into insulin-producing cells and stabilize glycemic control. This study assessed competitive migration of syngeneic stem cells to the bone marrow and islets in a murine model of chemical diabetes. VLA-4 is expressed in ∼ 25% of these cells, whereas CXCR4 is not detected, however, it is transcriptionally upregulated (6-fold). The possibility to enrich stem cells by a bone marrow homing (BM-H) functional assay was assessed in sequential transplants. Fr25lin(-) cells labeled with PKH26 were grafted into primary myeloablated recipients, and mitotically quiescent Fr25lin(-)PKH(bright) cells were sorted from the bone marrow after 2 days. The contribution of bone marrow-homed stem cells was remarkably higher in secondary recipients compared to freshly elutriated cells. The therapeutic efficacy was further increased by omission of irradiation in the secondary recipients, showing a 25-fold enrichment of islet-reconstituting cells by the bone marrow homing assay. Donor cells identified by the green fluorescent protein (GFP) and a genomic marker in sex-mismatched transplants upregulated PDX-1 and produced proinsulin, affirming the capacity of BM-H cells to convert in the injured islets. There was no evidence of transcriptional priming of freshly elutriated subsets to express PDX-1, insulin, and other markers of endocrine progenitors, indicating that the bone marrow harbors stem cells with versatile differentiation capacity. Affinity to the bone marrow can be used to enrich stem cells for pancreatic regeneration, and reciprocally, conditioning reduces the competitive incorporation in the injured islets.

  2. 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.

  3. 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.'

  4. Decreased RORC-dependent silencing of prostaglandin receptor EP2 induces autoimmune Th17 cells.

    Science.gov (United States)

    Kofler, David M; Marson, Alexander; Dominguez-Villar, Margarita; Xiao, Sheng; Kuchroo, Vijay K; Hafler, David A

    2014-06-01

    Prostaglandin E2 (PGE2) promotes Th17 expansion while otherwise inhibiting other CD4+ T cell subsets. Here, we identified a PGE2-dependent pathway that induces pathogenic Th17 cells in autoimmune disease and is regulated by the transcription factor RORC. Compared with other CD4+ cell types from healthy subjects, there is a surprising lack of the prostaglandin receptor EP2 on Th17 cells; therefore, we examined the hypothesis that RORγt, which is highly expressed in Th17 cells, mediates EP2 downregulation. Chromatin immunoprecipitation followed by DNA sequencing revealed that RORγt binds directly to Ptger2 (the gene encoding EP2 receptor) in Th17 cells isolated from WT mice. In Th17 cells isolated from humans, RORC repressed EP2 by directly silencing PTGER2 transcription, and knock down of RORC restored EP2 expression in Th17 cells. Compared with Th17 cells from healthy individuals, Th17 cells from patients with MS exhibited reduced RORC binding to the PTGER2 promoter region, resulting in higher EP2 levels and increased expression of IFN-γ and GM-CSF. Finally, overexpression of EP2 in Th17 cells from healthy individuals induced a specific program of inflammatory gene transcription that produced a pathogenic Th17 cell phenotype. These findings reveal that RORC directly regulates the effects of PGE2 on Th17 cells, and dysfunction of this pathway induces a pathogenic Th17 cell phenotype.

  5. 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

  6. Acrylamide decreased dopamine levels and increased 3-nitrotyrosine (3-NT) levels in PC 12 cells.

    Science.gov (United States)

    Tareke, Eden; Lyn-Cook, Beverly D; Duhart, Helen; Newport, Glenn; Ali, Syed

    2009-07-17

    Acrylamide is a chemical known to produce neurotoxicity in animals, as well as in humans. The mechanism of acrylamide-induced neurotoxicity is not fully known. However, recent studies have revealed that acrylamide affects the dopaminergic system. Therefore, the aim of this study was to investigate the effect of acrylamide on dopamine (DA) and the metabolites, 3,4-dihydroxy phenylacetic acid (DOPAC) and homovanillicacid (HVA), levels in Pheochromocytoma (PC 12) cells. In addition, the generation of peroxynitrite (ONOO(-)), measured by 3-nitrotyrosine (3-NT), was investigated as a possible mechanism in acrylamide-induced neurotoxicity. HPLC-coupled to electrochemical detection (ECD) was used to determine DA, DOPAC, HVA and 3-NT levels. Acrylamide (0.01-5mM) exposure produced a dose- and time (1-42h)-dependent decrease in DA levels. The decrease (P<0.05) in DA levels was noted at 24h after exposure to acrylamide. The study also revealed that 3-NT levels in PC 12 increased as a result of treatment with acrylamide. Thus, these data suggest that acrylamide-induced decrease in DA levels in PC 12 cells may be associated with peroxynitrite formation, measured as 3-NT levels.

  7. 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.

  8. Decreased IL-27 Negatively Correlated with Th17 Cells in Non-Small-Cell Lung Cancer Patients.

    Science.gov (United States)

    Duan, Minchao; Ning, Zhengqing; Fu, Zhijun; Zhang, Jianquan; Liu, Guangnan; Wei, Qiu; Zheng, Xiaoyu

    2015-01-01

    The presence of Th17 cells and IL-27 is observed in a variety of inflammatory associated cancers. However, there are some data on the role of Th17 cells and IL-27 in the regulation of immune reactions in non-small-cell lung cancer (NSCLC). The aim of this study is to assess the variation of Th17 cells and IL-27 in the peripheral blood (PB) of patients with NSCLC. The proportion of Th17 cells in peripheral blood mononuclear cells (PBMCs) was evaluated by flow cytometry. The serum concentrations of IL-27 and IL-17 were measured by enzyme-linked immunosorbent assay (ELISA). The mRNA expression of RORγt and IL-27 in the peripheral blood was examined by real-time quantitative polymerase chain reaction (QPCR). Expression of IL-27 was lower in NSCLC patients compared with normal controls. The frequency of Th17 cells was increased in NSCLC patients, accompanied by the upregulation of IL-17 and RORγt. IL-27 negatively correlated with the number of Th17 cells and the RORγt mRNA. Our results indicate that IL-27 might inhibit Th17 differentiation in NSCLC patients and better understanding of the regulatory effects of IL-27 on Th17 cells may shed light on potential new targets in cancer prevention and therapy.

  9. Autistic children exhibit decreased levels of essential Fatty acids in red blood cells.

    Science.gov (United States)

    Brigandi, Sarah A; Shao, Hong; Qian, Steven Y; Shen, Yiping; Wu, Bai-Lin; Kang, Jing X

    2015-05-04

    Omega-6 (n-6) and omega-3 (n-3) polyunsaturated fatty acids (PUFA) are essential nutrients for brain development and function. However, whether or not the levels of these fatty acids are altered in individuals with autism remains debatable. In this study, we compared the fatty acid contents between 121 autistic patients and 110 non-autistic, non-developmentally delayed controls, aged 3-17. Analysis of the fatty acid composition of red blood cell (RBC) membrane phospholipids showed that the percentage of total PUFA was lower in autistic patients than in controls; levels of n-6 arachidonic acid (AA) and n-3 docosahexaenoic acid (DHA) were particularly decreased (pautism.

  10. 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.

  11. 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.

  12. Decreased Number of Circulating Endothelial Progenitor Cells (CD133+/KDR+) in Patients with Psoriatic Arthritis.

    Science.gov (United States)

    Batycka-Baran, Aleksandra; Paprocka, Maria; Baran, Wojciech; Szepietowski, Jacek C

    2016-08-23

    Cardiovascular diseases are a major cause of mortality in patients with psoriatic arthritis (PsA), but the precise mechanism of increased cardiovascular risk is unknown. Endothelial dysfunction plays a crucial role in the development of atherosclerosis. Circulating endothelial progenitor cells (CEPCs) contribute to endothelial regeneration and their level may be affected by chronic inflammation. The aim of this study was to evaluate the number of CEPCs in patients with PsA (n = 24) compared with controls (n = 26). CEPCs were identified as CD133+/ KDR+ cells in peripheral blood, using flow cytometry. A significantly decreased number of CEPCs was observed in patients with PsA (p number of these cells was significantly, inversely correlated with the severity of skin and joint involvement (Psoriasis Area and Severity Index (PASI), DAS28) and the level of C-reactive protein. We hypothesize that the reduced number of CEPCs may indicate and contribute to the increased cardiovascular risk in patients with PsA.

  13. Src activity increases and Yes activity decreases during mitosis of human colon carcinoma cells.

    OpenAIRE

    Park, J.; Cartwright, C A

    1995-01-01

    Src and Yes protein-tyrosine kinase activities are elevated in malignant and premalignant tumors of the colon. To determine whether Src activity is elevated throughout the human colon carcinoma cell cycle as it is in polyomavirus middle T antigen- or F527 Src-transformed cells, and whether Yes activity, which is lower than that of Src in the carcinoma cells, is regulated differently, we measured their activities in cycling cells. We observed that the activities of both kinases were higher thr...

  14. 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.

  15. Gaucher Disease-Induced Pluripotent Stem Cells Display Decreased Erythroid Potential and Aberrant Myelopoiesis.

    Science.gov (United States)

    Sgambato, Judi A; Park, Tea Soon; Miller, Diana; Panicker, Leelamma M; Sidransky, Ellen; Lun, Yu; Awad, Ola; Bentzen, Søren M; Zambidis, Elias T; Feldman, Ricardo A

    2015-08-01

    Gaucher disease (GD) is the most common lysosomal storage disease resulting from mutations in the lysosomal enzyme glucocerebrosidase (GCase). The hematopoietic abnormalities in GD include the presence of characteristic Gaucher macrophages that infiltrate patient tissues and cytopenias. At present, it is not clear whether these cytopenias are secondary to the pathological activity of Gaucher cells or a direct effect of GCase deficiency on hematopoietic development. To address this question, we differentiated induced pluripotent stem cells (iPSCs) derived from patients with types 1, 2, and 3 GD to CD34(+)/CD45(+)/CD43(+)/CD143(+) hematopoietic progenitor cells (HPCs) and examined their developmental potential. The formation of GD-HPCs was unaffected. However, these progenitors demonstrated a skewed lineage commitment, with increased myeloid differentiation and decreased erythroid differentiation and maturation. Interestingly, myeloid colony-formation assays revealed that GD-HPCs, but not control-HPCs, gave rise to adherent, macrophage-like cells, another indication of abnormal myelopoiesis. The extent of these hematologic abnormalities correlated with the severity of the GCase mutations. All the phenotypic abnormalities of GD-HPCs observed were reversed by incubation with recombinant GCase, indicating that these developmental defects were caused by the mutated GCase. Our results show that GCase deficiency directly impairs hematopoietic development. Additionally, our results suggest that aberrant myelopoiesis might contribute to the pathological properties of Gaucher macrophages, which are central to GD manifestations. The hematopoietic developmental defects we observed reflect hematologic abnormalities in patients with GD, demonstrating the utility of GD-iPSCs for modeling this disease.

  16. Overexpression of forkhead box J2 can decrease the migration of breast cancer cells.

    Science.gov (United States)

    Wang, Yingying; Yang, Shuyun; Ni, Qichao; He, Song; Zhao, Yunhong; Yuan, Qin; Li, Chunmiao; Chen, Hongwei; Zhang, Li; Zou, Lin; Shen, Aiguo; Cheng, Chun

    2012-08-01

    The prognosis of breast cancer patients with metastases is generally poor, so it is essential to elucidate related molecules mechanisms. Forkhead Box J2 (FOXJ2) is a member of Forkhead Box transcription factors, many of which have been reported to participate in tumor migration and invasion. In this study, we showed the expression of FOXJ2 was higher in primary breast cancer tissues without lymph nodes metastases than those with, and there was statistical significance between the expression of FXOJ2 and the clinical factors. Hence, we identified a novel function of metastasis, which was not previously known for FOXJ2. Overexpression of FOXJ2 decreased the motility property of highly migrative MDA-MB-231 cells in vitro by wound healing assays and trans-well migration assays, and it was concurrent with the increased expression of epithelial marker E-cadherin and the decreased expression of mesenchymal marker vimentin by Western blot analysis, reverse transcription PCR analysis, and immunofluorescence analysis. Consistent with these observations, the repression of FOXJ2 in weakly metastatic MCF-7 cells remarkably promoted cellular motility. Our study demonstrates that FOXJ2 can inhibit the metastasis of human breast cancer by regulating the EMT key markers E-cadherin and vimentin.

  17. High IP-10 levels decrease T cell function in HIV-1-infected individuals on ART

    Science.gov (United States)

    Ramirez, L. A.; Arango, T. A.; Thompson, E.; Naji, M.; Tebas, P.; Boyer, J. D.

    2014-01-01

    HIV-1-infected subjects, despite control of viral replication with ART, have an altered immune cytokine/chemokine milieu. Changes in systemic cytokines and chemokines can alter immune responses. IP-10, in particular, has been associated with pathogenesis in a number of conditions, and we found that IP-10 is increased in serum in subjects who are HIV-1 infected and on stable ART compared with HIV-1-uninfected individuals. In a series of in vitro studies, we found that PBMCs exposed to IP-10 showed a significant decrease in the number of cells capable of secreting IFN-γ, as well as other cytokines, when stimulated with recall antigens. Furthermore, treatment with IP-10 led to decreased antigen-specific calcium signaling and MAPK38 phosphorylation. Importantly, the cytokines, as well as proliferative responses, could be enhanced with an IP-10 Nab. Our findings suggest that IP-10-modulating drugs may potentially enhance T cell responses to vaccination and HIV-1 in HIV+ subjects on ART. PMID:25157027

  18. Reduced Number of Transitional and Naive B Cells in Addition to Decreased BAFF Levels in Response to the T Cell Independent Immunogen Pneumovax®23.

    Directory of Open Access Journals (Sweden)

    Alena Roth

    Full Text Available Protective immunity against T cell independent (TI antigens such as Streptococcus pneumoniae is characterized by antibody production of B cells induced by the combined activation of T cell independent type 1 and type 2 antigens in the absence of direct T cell help. In mice, the main players in TI immune responses have been well defined as marginal zone (MZ B cells and B-1 cells. However, the existence of human equivalents to these B cell subsets and the nature of the human B cell compartment involved in the immune reaction remain elusive. We therefore analyzed the effect of a TI antigen on the B cell compartment through immunization of healthy individuals with the pneumococcal polysaccharide (PnPS-based vaccine Pneumovax®23, and subsequent characterization of B cell subpopulations. Our data demonstrates a transient decrease of transitional and naïve B cells, with a concomitant increase of IgA+ but not IgM+ or IgG+ memory B cells and a predominant generation of PnPS-specific IgA+ producing plasma cells. No alterations could be detected in T cells, or proposed human B-1 and MZ B cell equivalents. Consistent with the idea of a TI immune response, antigen-specific memory responses could not be observed. Finally, BAFF, which is supposed to drive class switching to IgA, was unexpectedly found to be decreased in serum in response to Pneumovax®23. Our results demonstrate that a characteristic TI response induced by Pneumovax®23 is associated with distinct phenotypical and functional changes within the B cell compartment. Those modulations occur in the absence of any modulations of T cells and without the development of a specific memory response.

  19. Reduced Number of Transitional and Naive B Cells in Addition to Decreased BAFF Levels in Response to the T Cell Independent Immunogen Pneumovax®23.

    Science.gov (United States)

    Roth, Alena; Glaesener, Stephanie; Schütz, Katharina; Meyer-Bahlburg, Almut

    2016-01-01

    Protective immunity against T cell independent (TI) antigens such as Streptococcus pneumoniae is characterized by antibody production of B cells induced by the combined activation of T cell independent type 1 and type 2 antigens in the absence of direct T cell help. In mice, the main players in TI immune responses have been well defined as marginal zone (MZ) B cells and B-1 cells. However, the existence of human equivalents to these B cell subsets and the nature of the human B cell compartment involved in the immune reaction remain elusive. We therefore analyzed the effect of a TI antigen on the B cell compartment through immunization of healthy individuals with the pneumococcal polysaccharide (PnPS)-based vaccine Pneumovax®23, and subsequent characterization of B cell subpopulations. Our data demonstrates a transient decrease of transitional and naïve B cells, with a concomitant increase of IgA+ but not IgM+ or IgG+ memory B cells and a predominant generation of PnPS-specific IgA+ producing plasma cells. No alterations could be detected in T cells, or proposed human B-1 and MZ B cell equivalents. Consistent with the idea of a TI immune response, antigen-specific memory responses could not be observed. Finally, BAFF, which is supposed to drive class switching to IgA, was unexpectedly found to be decreased in serum in response to Pneumovax®23. Our results demonstrate that a characteristic TI response induced by Pneumovax®23 is associated with distinct phenotypical and functional changes within the B cell compartment. Those modulations occur in the absence of any modulations of T cells and without the development of a specific memory response.

  20. 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.

  1. Prenatal infection decreases calbindin, decreases Purkinje cell volume and density and produces long-term motor deficits in Sprague-Dawley rats.

    Science.gov (United States)

    Wallace, K; Veerisetty, S; Paul, I; May, W; Miguel-Hidalgo, J J; Bennett, W

    2010-01-01

    The cerebellum is involved in the control of motor functions with Purkinje cells serving as the only output from the cerebellum. Purkinje cells are important targets for toxic substances and are vulnerable to prenatal insults. Intrauterine infection (IUI) has been shown to selectively target the developing cerebral white matter through lesioning, necrosis and inflammatory cytokine activation. Developmental and cognitive delays have been associated with animal models of IUI. The aim of this study was to determine if IUI leads to damage to Purkinje cells in the developing cerebellum and if any damage is associated with decreases in calbindin and motor behaviors in surviving pups. Pregnant rats were injected with Escherichia coli (1 × 10⁵ colony-forming units) or sterile saline at gestational day 17. Beginning at postnatal day (PND) 2, the pups were subjected to a series of developmental tests to examine developmental milestones. At PND 16, some pups were sacrificed and their brains extracted and processed for histology or protein studies. Hematoxylin and eosin (HE) staining was done to examine the general morphology of the Purkinje cells and to examine Purkinje cell density, area and volume. Calbindin expression was examined in the cerebellum via immunohistochemistry and Western blot techniques. The remaining rat pups were used to examine motor coordination and balance on a rotating rotarod at the prepubertal and adult ages. Prenatal E. coli injection did not significantly change birth weight or delivery time, but did delay surface righting and negative geotaxis in pups. Pups in the E. coli group also had a decrease in the number of Purkinje cells, as well as a decrease in Purkinje cell density and volume. HE staining demonstrated a change in Purkinje cell morphology. Calbindin expression was decreased in rats from the E. coli group as well. Locomotor tests indicated that while there were no significant changes in gross motor activity, motor coordination and

  2. 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.

  3. Fluoxetine Decreases the Proliferation and Adipogenic Differentiation of Human Adipose-Derived Stem Cells

    Directory of Open Access Journals (Sweden)

    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.

  4. 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

    2015-07-22

    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.

  5. Adipose-Derived Mesenchymal Stem Cells from Ventral Hernia Repair Patients Demonstrate Decreased Vasculogenesis

    Directory of Open Access Journals (Sweden)

    Jeffrey Lisiecki

    2014-01-01

    Full Text Available Introduction. In adipose tissue healing, angiogenesis is stimulated by adipose-derived stromal stem cells (ASCs. Ventral hernia repair (VHR patients are at high risk for wound infections. We hypothesize that ASCs from VHR patients are less vasculogenic than ASCs from healthy controls. Methods. ASCs were harvested from the subcutaneous fat of patients undergoing VHR by the component separation technique and from matched abdominoplasty patients. RNA and protein were harvested on culture days 0 and 3. Both groups of ASCs were subjected to hypoxic conditions for 12 and 24 hours. RNA was analyzed using qRT-PCR, and protein was used for western blotting. ASCs were also grown in Matrigel under hypoxic conditions and assayed for tubule formation after 24 hours. Results. Hernia patient ASCs demonstrated decreased levels of VEGF-A protein and vasculogenic RNA at 3 days of growth in differentiation media. There were also decreases in VEGF-A protein and vasculogenic RNA after growth in hypoxic conditions compared to control ASCs. After 24 hours in hypoxia, VHR ASCs formed fewer tubules in Matrigel than in control patient ASCs. Conclusion. ASCs derived from VHR patients appear to express fewer vasculogenic markers and form fewer tubules in Matrigel than ASCs from abdominoplasty patients, suggesting decreased vasculogenic activity.

  6. Function of Treg Cells Decreased in Patients With Systemic Lupus Erythematosus Due To the Effect of Prolactin.

    Science.gov (United States)

    Legorreta-Haquet, María Victoria; Chávez-Rueda, Karina; Chávez-Sánchez, Luis; Cervera-Castillo, Hernando; Zenteno-Galindo, Edgar; Barile-Fabris, Leonor; Burgos-Vargas, Rubén; Álvarez-Hernández, Everardo; Blanco-Favela, Francisco

    2016-02-01

    Prolactin has different functions, including cytokine secretion and inhibition of the suppressor effect of regulatory T (Treg) cells in healthy individuals. Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by defects in the functions of B, T, and Treg cells. Prolactin plays an important role in the physiopathology of SLE. Our objective was to establish the participation of prolactin in the regulation of the immune response mediated by Treg cells from patients with SLE. CD4CD25CD127 cells were purified using magnetic beads and the relative expression of prolactin receptor was measured. The functional activity was evaluated by proliferation assay and cytokine secretion in activated cells, in the presence and absence of prolactin. We found that both percentage and function of Treg cells decrease in SLE patients compared to healthy individuals with statistical significance. The prolactin receptor is constitutively expressed on Treg and effector T (Teff) cells in SLE patients, and this expression is higher than in healthy individuals. The expression of this receptor differs in inactive and active patients: in the former, the expression is higher in Treg cells than in Teff cells, similar to healthy individuals, whereas there is no difference in the expression between Treg and Teff cells from active patients. In Treg:Teff cell cocultures, addition of prolactin decreases the suppressor effect exerted by Treg cells and increases IFNγ secretion. Our results suggest that prolactin plays an important role in the activation of the disease in inactive patients by decreasing the suppressor function exerted by Treg cells over Teff cells, thereby favoring an inflammatory microenvironment.

  7. Inhibition of miR-15b decreases cell migration and metastasis in colorectal cancer.

    Science.gov (United States)

    Li, Jian; Chen, Yuxiang; Guo, Xiong; Zhou, Ling; Jia, Zeming; Tang, Yaping; Lin, Ling; Liu, Weidong; Ren, Caiping

    2016-07-01

    Colorectal cancer (CRC) has a high prevalence and mortality rate. Biomarkers for predicting the recurrence of CRC are not clinically available. This study investigated the role of circulating miR-15b in the prediction of CRC recurrence and the associated mechanism. miR-15b levels in plasma and tissues were measured by real-time PCR. Metastasis suppressor-1 (MTSS1) and Klotho protein expression were detected by Western blot and immunohistochemistry. Invasion and migration of CRC tumor cells were measured by transwell plates. Liver metastasis was established by intraspleen injection of HCT116 cells. Plasma miR-15b levels were significantly higher in CRC patients than in healthy controls, in CRC patients with metastasis than in CRC patients without metastasis, and in CRC patients with recurrence than in CRC patients without recurrence in the 5-year follow-up. miR-15b level in CRC tumors was significantly higher than that in peritumoral tissues. High plasma miR-15b level and negative MTSS1 and Klotho expression in tumor tissues significantly correlated with poor survival. Inhibition of miR-15b activity by adenovirus carrying antimiR-15b sequence significantly increased MTSS1 and Klotho protein expression and subsequently decreased colony formation ability, invasion, and migration of HCT116 cells in vitro and liver metastasis of HCT116 tumors in vivo. In conclusion, high abundance of circulating miR-15b correlated with tumor metastasis, recurrence, and poor patient prognosis through downregulation of MTSS1 and Klotho protein expression.

  8. Mitochondrial Fission Increases Apoptosis and Decreases Autophagy in Renal Proximal Tubular Epithelial Cells Treated with High Glucose.

    Science.gov (United States)

    Lee, Wen-Chin; Chiu, Chien-Hua; Chen, Jin-Bor; Chen, Chiu-Hua; Chang, Hsueh-Wei

    2016-11-01

    The aim of this study was to examine the effect of mitochondrial morphogenesis changes on apoptosis and autophagy of high-glucose-treated proximal tubular epithelial cells (HK2). Cell viability, apoptosis, and mitochondrial morphogenesis were examined using crystal violet, terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL), and mitotracker staining, respectively. High glucose inhibited cell viability and induced mitochondrial fission in HK2 cells. After depleting mitofusin 1 (MFN1), the MFN1(-) HK2 cells (fission type) became more susceptible to high-glucose-induced apoptosis and mitochondrial fragmentation observed by TUNEL and mitotracker assays. In siMFN2 HK2 cells (fission type), mitochondria were highly fragmented (>80% fission rate) with or without high-glucose treatment; however, siFIS1 (mitochondrial fission protein 1) HK2 cells (fusion type) exhibited little fragmentation (High-glucose treatment induced autophagy, characterized by the formation of autophagosome and microtubule-associated protein light chain 3 (LC3) B-II, as observed by transmission electron microscopy and western blotting, respectively. LC3B-II levels decreased in both MFN1(-) and siMFN2 HK2 cells, but increased in siFIS1 HK2 cells. Moreover, autophagy displays a protective role against high-glucose-induced cell death based on cotreatment with autophagy inhibitors (3-methyladenine and chloroquine). Mitochondrial fission may increase apoptosis and decrease autophagy of high-glucose-treated HK2 cells.

  9. 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.

  10. Decreased proliferation ability and differentiation potential of mesenchymal stem cells of osteoporosis rat

    Institute of Scientific and Technical Information of China (English)

    Qiang Wang; Bing Zhao; Chao Li; Jie-Sheng Rong; Shu-Qing Tao; Tian-Zun Tao

    2014-01-01

    Objective:To explore decreased proliferation ability and differentiation potential of mesenchymal stem cells(MSCs) of osteoporosis rat.Methods:MSCs were obtained from osteoporosis rat, and proliferation potency and impaired osteogenic differentiation potential were determined. Results:The result showed a significant downregulation ofMSCs pluripotency related gene(Oct 4) and osteogenic genes(BSP,OCN) expression inOVXMSCs compared withShamMSCs(P<0.05). Conclusions:These data suggest thatMSCs are aging in osteoporosis body, and autologous OVXMSCs transplantation is not appropriate to treat osteoporosis if necessary.There will be a possibility in establishing a new clinical application ofMSCs autologous transplantation to treat osteoporosis, ifOVXMSCs have stronger proliferation and differentiation.

  11. Regulatory T cells reduce acute lung injury fibroproliferation by decreasing fibrocyte recruitment.

    Science.gov (United States)

    Garibaldi, Brian T; D'Alessio, Franco R; Mock, Jason R; Files, D Clark; Chau, Eric; Eto, Yoshiki; Drummond, M Bradley; Aggarwal, Neil R; Sidhaye, Venkataramana; King, Landon S

    2013-01-01

    Acute lung injury (ALI) causes significant morbidity and mortality. Fibroproliferation in ALI results in worse outcomes, but the mechanisms governing fibroproliferation remain poorly understood. Regulatory T cells (Tregs) are important in lung injury resolution. Their role in fibroproliferation is unknown. We sought to identify the role of Tregs in ALI fibroproliferation, using a murine model of lung injury. Wild-type (WT) and lymphocyte-deficient Rag-1(-/-) mice received intratracheal LPS. Fibroproliferation was characterized by histology and the measurement of lung collagen. Lung fibrocytes were measured by flow cytometry. To dissect the role of Tregs in fibroproliferation, Rag-1(-/-) mice received CD4(+)CD25(+) (Tregs) or CD4(+)CD25(-) Tcells (non-Tregs) at the time of LPS injury. To define the role of the chemokine (C-X-C motif) ligand 12 (CXCL12)-CXCR4 pathway in ALI fibroproliferation, Rag-1(-/-) mice were treated with the CXCR4 antagonist AMD3100 to block fibrocyte recruitment. WT and Rag-1(-/-) mice demonstrated significant collagen deposition on Day 3 after LPS. WT mice exhibited the clearance of collagen, but Rag-1(-/-) mice developed persistent fibrosis. This fibrosis was mediated by the sustained epithelial expression of CXCL12 (or stromal cell-derived factor 1 [SDF-1]) that led to increased fibrocyte recruitment. The adoptive transfer of Tregs resolved fibroproliferation by decreasing CXCL12 expression and subsequent fibrocyte recruitment. Blockade of the CXCL12-CXCR4 axis with AMD3100 also decreased lung fibrocytes and fibroproliferation. These results indicate a central role for Tregs in the resolution of ALI fibroproliferation by reducing fibrocyte recruitment along the CXCL12-CXCR4 axis. A dissection of the role of Tregs in ALI fibroproliferation may inform the design of new therapeutic tools for patients with ALI.

  12. High expression of osteoglycin decreases the metastatic capability of mouse hepatocarcinoma Hca-F cells to lymph nodes

    Institute of Scientific and Technical Information of China (English)

    Xiaonan Cui; Bo Song; Li Hou; Zhiyi Wei; Jianwu Tang

    2008-01-01

    Osteoglycin, one of the matrix molecules, belongs to the small leucine-rich proteoglycan gene family and might play important roles in cell growth and differentiation and in pathological processes such as fibrosis and cancer growth.In this study, a eukaryotic expression plasmid pIRESpuro3 osteoglycin(+) was constructed and transfected into mouse hepatocarcinoma Hca-F cells to evaluate the contribution of osteoglycin to the malignant behavior of Hca-F. It was foundthat Hca-F cells transfected with pIRESpuro3 osteoglycin(+) showed significantly decreased potential for both migration and invasion. Furthermore, Hca-F cells transfected with osteoglycin showed decreased metastatic potential to peripheral lymph nodes. However, proliferation potential and adhesive capacity of Hca-F cells to different protein substrates were not influenced by osteoglycin transfection. In summary,high expression of osteoglycin decreases the metastatic capability of Hca-F to lymph nodes.

  13. Decreased prolactin-inducible protein expression exhibits inhibitory effects on the metastatic potency of breast cancer cells

    Institute of Scientific and Technical Information of China (English)

    Zhendong Zheng; Xiaodong Xie

    2013-01-01

    Objective: The aim of the research was to study the effects of prolactin-inducible protein (PIP) downregulation on metastatic abilities of human breast cancer MDA-MB-453 cells. Methods: PIP-siRNA was transfected into human breast cancer MDA-MB-453 cells through liposome. Reverse transcription PCR and immunocytochemistry were employed to detect the downregulated expression of PIP. Cell migration, adhesion and invasion assays were performed to assess the impacts of PIP downregulation on cell migration, adhesion and invasion respectively. Results: Knockdown of PIP obviously inhibited cell migration, the migrated cells were decreased by 83.1% compared with the negative control group. Cell adhesion was also reduced, the adhesion rates at 30 min and 60 min were decreased by 42.6% and 48.5% respectively compared with the negative control group. Moreover, PIP downregulation resulted in decreased invasion rate by 73.9%. Conclusion: Reduced PIP expression in MDA-MB-453 cells can inhibit the abilities of migration, adhesion and invasion, which suggests that PIP plays an important role in the metastatic potency of breast cancer cells.

  14. Silibinin decreases prostate-specific antigen with cell growth inhibition via G1 arrest, leading to differentiation of prostate carcinoma cells: Implications for prostate cancer intervention

    OpenAIRE

    Zi, Xiaolin; Agarwal, Rajesh

    1999-01-01

    Reduction in serum prostate-specific antigen (PSA) levels has been proposed as an endpoint biomarker for hormone-refractory human prostate cancer intervention. We examined whether a flavonoid antioxidant silibinin (an active constituent of milk thistle) decreases PSA levels in hormone-refractory human prostate carcinoma LNCaP cells and whether this effect has biological relevance. Silibinin treatment of cells grown in serum resulted in a significant decrease in both intracellular and secreted...

  15. Transmethylation inhibitors decrease chemotactic sensitivity and delay cell aggregation in Dictyostelium discoideum

    NARCIS (Netherlands)

    van Waarde, A; van Haastert, P J

    1984-01-01

    In Dictyostelium discoideum, extracellular cyclic AMP (cAMP) induces chemotaxis and cell aggregation. Suspensions of cAMP-sensitive cells respond to a cAMP pulse with a rapid, transient increase of protein carboxyl methylation. The transmethylation inhibitors cycloleucine, L-homocysteine thiolactone

  16. Contact sensitizers decrease 33D1 expression on mature Langerhans cells.

    Science.gov (United States)

    Herouet, C; Cottin, M; Galanaud, P; Leclaire, J; Rousset, F

    1999-01-01

    Langerhans cells play a critical role in allergic contact hypersensitivity. In vivo, these cells capture xenobiotics that penetrate the skin and transport them through the lymphatic vessels into regional lymph nodes for presentation to T cells. During this migration step, Langerhans cells become mature dendritic cells according to their phenotype and their high immunostimulatory capacity. In vitro, when isolated from the skin and cultured for 3 days, Langerhans cells undergo similar phenotypic and functional maturation. In this study, the capacity of sensitizers, irritants and neutral chemicals to modulate the surface marker expression and morphology of pure mature murine Langerhans cells in vitro was examined. Contact with 4 sensitizers (2,4-dinitrobenzenesulfate, 4-ethoxymethylene-2-phenyl-2-oxazolin-5-one, p-phenylenediamine, mercaptobenzo-thiazole) resulted in a rapid, specific, marked fall in 33D1 expression, a murine specific dendritic cell marker. No effect was observed with 2 neutral chemicals (sodium chloride, methyl nicotinate) or 2 irritants (dimethyl sulfoxide, benzalkonium chloride). Nevertheless, sodium lauryl sulfate, a very irritant detergent, altered morphology and down-regulated all membrane markers. These preliminary data suggest that in vitro modulation of 33D1 expression by strong sensitizers may be an approach to the development of an in vitro model for the identification of chemicals that have the potential to cause skin sensitization and to distinguish them as far as possible from irritants.

  17. R-(+)-perillyl alcohol-induced cell cycle changes, altered actin cytoskeleton, and decreased ras and p34(cdc2) expression in colonic adenocarcinoma SW480 cells.

    Science.gov (United States)

    Cerda, S R; Wilkinson, J; Thorgeirsdottir, S; Broitman, S A

    1999-01-01

    Monoterpenes as S-(-)-perillyl alcohol (PA) have been shown to inhibit the isoprenylation of such growth regulatory proteins as ras. In this study, we investigated the effects of the R-(+) enantiomer of PA on cell cycle, signaling, and cytoskeletal control in the colonic adenocarcinoma cell line SW480, which carries a K-ras mutation. Cell cycle analysis by flow cytometry of SW480 cells treated with 1 mM PA for 24 hours demonstrated an increase in the number of cells in G0/G1 with a decrease in S phase, compared with untreated control cells. These cell cycle changes correlated with an inhibition of protein isoprenylation from (14)C-mevalonate and decreased expression of the cell cycle regulatory kinase p34(cdc2). Additionally, PA-treated cells acquired a flattened morphology with a condensation of cytoskeletal actin spikes to the periphery. This was in contrast to treatment with 15 microM mevinolin (MVN), a direct mevalonate synthesis inhibitor, which imparted to SW480 cells a more rounded and spindly morphology, associated with the depolymerization of actin microfilaments. Together, these data suggest that fluctuations in mevalonate and isoprenoid pools may involve different morphologic phenomenon. Because ras mediated signaling is related to the organization of the actin cytoskeleton, we investigated the effects of PA on the isoprenylation of ras. Although MVN treatment inhibited ras farnesylation, PA treatment decreased the expression of total ras protein. In summary, R-(+)-PA-induced cell signaling events correlated with alterations in the organization of cytoskeletal actin and decreased protein expression of growth regulatory proteins, such as ras and cdc2 kinase. These effects may contribute to the growth inhibitory activity of R-(+)-PA.

  18. 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.

  19. 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.

  20. Simvastatin decreases steroid production in the H295R cell line and decreases steroids and FSH in female rats

    DEFF Research Database (Denmark)

    Jensen, Anna Guldvang; Hansen, Cecilie Hurup; Weisser, Johan J;

    2015-01-01

    .3 (L), 5.0 (M), and 20.0 (H)mg SV/kg bw/day for 14 days. 10 Steroids were investigated in H295R growth media, and in tissues and plasma from rats using GC-MS/MS. Plasma LH and FSH were quantified by ELISA. In the H295R assay, SV and SVA particularly decreased progestagens with IC50-values from 0...

  1. SHP-1 overexpression increases the radioresistance of NPC cells by enhancing DSB repair, increasing S phase arrest and decreasing cell apoptosis.

    Science.gov (United States)

    Pan, Xiaofen; Mou, Jingjing; Liu, Sha; Sun, Ziyi; Meng, Rui; Zhou, Zhenwei; Wu, Gang; Peng, Gang

    2015-06-01

    The present study aimed to investigate the influence of SHP-1 on the radioresistance of the nasopharyngeal carcinoma (NPC) cell line CNE-2 and the relevant underlying mechanisms. The human NPC cell line CNE-2 was transfected with a lentivirus that contained the SHP-1 gene or a nonsense sequence (referred to as LP-H1802Lv201 and LP-NegLv201 cells, respectively). Cells were irradiated with different ionizing radiation (IR) doses. Cell survival, DNA double-strand breaks (DSBs), apoptosis, cell cycle distribution, and the expression of related proteins were assessed using colony formation assay, immunofluorescent assays (IFAs), flow cytometry (FCM) and western blot analyses, respectively. Compared with the control (CNE-2 cells) and LP-NegLv201 cells, LP-H1802Lv201 cells were more resistant to IR. IFAs showed that IR caused less histone H2AX phosphorylation (γH2AX) and RAD51 foci in the LP-H1802Lv201 cells. Compared with the control and LP-NegLv201 cells, LP-H1802Lv201 cells showed increased S phase arrest. After IR, the apoptotic rate of the LP-H1802Lv201 cells was lower in contrast to the control and LP-NegLv201 cells. Western blot analyses showed that IR increased the phosphorylation of ataxia telangiectasia mutated (ATM) kinase, checkpoint kinase 2 (CHK2), ataxia telangiectasia and Rad3-related (ATR) protein, checkpoint kinase 1 (CHK1) and p53. In LP-H1802Lv201 cells, the phosphorylation levels of ATM and CHK2 were significantly increased while the p53 phosphorylation level was decreased compared to these levels in the control and LP-NegLv201 cells. Phosphorylation of ATR and CHK1 did not show significant differences in the three cell groups. Overexpression of SHP-1 in the CNE-2 cells led to radioresistance and the radioresistance was related to enhanced DNA DSB repair, increased S phase arrest and decreased cell apoptosis.

  2. Trichostatin A Induced Bcl-2 Protein Level Decrease Mediated A549/CDDP Cells Apoptosis by Mitochondria Pathway

    Directory of Open Access Journals (Sweden)

    Jun WU

    2009-11-01

    Full Text Available Background and objective The use of platinum-based combination chemotherpy remains the standard treatment for non-small cell lung cancer. However, the resistance to platinum limits further treatment clinically. Trichostatin A (TSA is one of histone deacetylase (HDAC inhibitors. It inhibits tumor cell proliferation and acts as a chemosensitizer. The aim of this study is to investigate the action mechanism of TSA on cisplatin-resistant human lung adenocarcinoma cell line A549/CDDP. Methods Cytotoxicity and cell viability was assayed by Neutral Red method. Morphologic assessment of apoptosis was determined by fluorescence microscope; cell cycle and mitochondrial membrane potential were detected by flow cytometry. In addition, A549/CDDP cells were transfected with Bcl-2 expression Vector and siRNA-bcl-2. Results A549/CDDP cells treated with TSA showed apparently cytotoxicity, IC50 of TSA was (446.59±27.32 nmol/L. The growth curve showed the ratio of growth decreased with the increase of concentration of TSA. The apoptosis appeared 24 hours after treated by (125-500 nmol/L TSA, morphologic changes including nuclear chromatin condensation. Fluorescence strength was observed with fluorescence microscope. Treated by TSA, mitochondrial membrane potential was decreased and cells were arrested at S phase. Western blotting analyses showed that the levels of Bcl-2 decreased, while expression of Bax increased. Simultaneously caspase-3 was activated. Over expression of Bcl-2 can inhibit TSA-induced A549/CDDP cell apoptosis, while the decrease of Bcl-2 enhanced the sensitivity of A549/CDDP cell to TSA. Conclusion TSA induce A549/CDDP cell apoptosis by mitochondria pathway.

  3. Circulating natural killer and γδ T cells decrease soon after infection of rhesus macaques with lymphocytic choriomeningitis virus

    Directory of Open Access Journals (Sweden)

    Juan D Rodas

    2009-07-01

    Full Text Available Rhesus macaques infected with the WE strain of lymphocytic choriomeningitis virus (LCMV-WE serve as a model for human infection with Lassa fever virus. To identify the earliest events of acute infection, rhesus macaques were monitored immediately after lethal infection for changes in peripheral blood mononuclear cells (PBMCs. Changes in CD3, CD4, CD8 and CD20 subsets did not vary outside the normal fluctuations of these blood cell populations; however, natural killer (NK and γδ T cells increased slightly on day 1 and then decreased significantly after two days. The NK subsets responsible for the decrease were primarily CD3-CD8+ or CD3-CD16+ and not the NKT (primarily CD3+CD56+ subset. Macaques infected with a non-virulent arenavirus, LCMV-Armstrong, showed a similar drop in circulating NK and γδ T cells, indicating that this is not a pathogenic event. V³9 T cells, representing the majority of circulating γδ T cells in rhesus macaques, displayed significant apoptosis when incubated with LCMV in cell culture; however, the low amount of cell death for virus-co-cultured NK cells was insufficient to account for the observed disappearance of this subset. Our observations in primates are similar to those seen in LCMV-infected mice, where decreased circulating NK cells were attributed to margination and cell death. Thus, the disappearance of these cells during acute hemorrhagic fever in rhesus macaques may be a cytokine-induced lymphopenia common to many virus infections.

  4. Downregulation of β-catenin decreases the tumorigenicity, but promotes epithelial-mesenchymal transition in breast cancer cells

    Directory of Open Access Journals (Sweden)

    Kai Cai

    2014-01-01

    Full Text Available Background: Wnt/β-catenin signaling pathway plays a key role in human breast cancer progression. In this study, we down regulated β-catenin expression in human breast cancer MDA-MB-231 cells and investigated the effect of β-catenin knockdown on the cell biological characteristics. Materials and Methods: The recombinant plasmids of pSUPER-enhancement green fluorescent protein 1 (EGFP1-scrabble-β-catenin-short hairpin ribonucleic acid (shRNA and pSUPER-EGFP1-β-catenin-shRNA-1 were transfected into MDA-MB-231 cells, respectively, and the stably transfected cells were isolated from G418 selected clones. The β-catenin gene silenced efficiency was measured by quantitative reverse transcriptase polymerase chain reaction (QRT-PCR and Western blot. The biological characteristics of MDA-MB-231 cells with down regulated β-catenin were evaluated by analyzing cell proliferation, clonogenicity, cell mobility and tumorigenicity. The expression of E-cadherin and Vimentin was concurrently detected by QRT-PCR. Results: The β-catenin-shRNA-1 stably transfected MDA-MB-231 cells significantly decreased β-catenin expression, cell proliferation, clonogenicity, and tumorigenicity in Balb/c nude mice compared with the MDA-MB-231 cells transfected with pSUPER-EGFP1-scrabble-β-catenin-shRNA. Interestingly, knockdown of β-catenin led to the reduction of epithelial E-cadherin expression, the increase of cell mobility and mesenchymal vimentin expression in MDA-MB-231 cells, indicating an epithelial to mesenchymal transition. Conclusion: Knockdown of β-catenin expression in human breast cancer MDA-MB-231 cells inhibits cell tumorigenicity in mice, but promotes cell epithelial-mesenchymal transition.

  5. Decrease in Ins(+)Glut2(LO)β-cells with advancing age in mouse and human pancreas.

    Science.gov (United States)

    Beamish, Christine A; Mehta, Sofia; Strutt, Brenda J; Chakrabarti, Subrata; Hara, Manami; Hill, David J

    2017-03-27

    The presence and location of resident pancreatic β-cell progenitors is controversial. A subpopulation of insulin-expressing but glucose transporter-2-low (Ins+Glut2LO) cells may represent multipotent pancreatic progenitors in adult mouse and human islets, and in mice are enriched in small, extra-islet β-cell clusters (in mouse and human pancreata throughout life. Mouse pancreata were collected at postnatal days (d) 7, 14, 21, 28, and at 3, 6, 12, and 18 months of age, and in the first 28 days after β-cell mass depletion following Streptozotocin (STZ) administration. Human pancreas samples were examined during fetal life (22-30 weeks gestation), infancy (0-1 year), childhood (2-9), adolescence (10-17), and adulthood (18-80). Tissues were analyzed by immunohistochemistry for the expression and location of insulin, glut2, and ki67. The proportion of β-cells within clusters relative to islets was higher in human pancreas than mouse at all ages examined, and decreased significantly at adolescence. In mice, the total number of Ins+Glut2LO cells decreased after 7 d, concurrent with the proportion of clusters. These cells were more abundant in clusters than islets in both species. A positive association existed between the appearance of new β-cells after STZ treatment of young mice, particularly in clusters and smaller islets, and an increased proportional presence of Ins+Glut2LO cells during early β-cell regeneration. These data suggest that Ins+Glut2LO cells are preferentially located within β-cell clusters throughout life in mouse and human pancreas, and may represent a source of β-cell plasticity.

  6. Decreased Polysaccharide Feruloylation Compromises Plant Cell Wall Integrity and Increases Susceptibility to Necrotrophic Fungal Pathogens

    Directory of Open Access Journals (Sweden)

    Nathan T Reem

    2016-05-01

    Full Text Available The complexity of cell wall composition and structure determines the strength, flexibility, and function of the primary cell wall in plants. However, the contribution of the various components to cell wall integrity and function remains unclear. Modifications of cell wall composition can induce plant responses known as Cell Wall Integrity control. In this study, we used transgenic expression of the fungal feruloyl esterase AnFAE to examine the effect of post-synthetic modification of Arabidopsis and Brachypodium cell walls. Transgenic Arabidopsis plants expressing AnFAE showed a significant reduction of monomeric ferulic acid, increased amounts of wall-associated extensins, and increased susceptibility to Botrytis cinerea, compared with wild type. Transgenic Brachypodium showed reductions in monomeric and dimeric ferulic acids and increased susceptibility to Bipolaris sorokiniana. Upon infection, transgenic Arabidopsis and Brachypodium plants also showed increased expression of several defense-related genes compared with wild type. These results demonstrate a role, in both monocot and dicot plants, of polysaccharide feruloylation in plant cell wall integrity, which contributes to plant resistance to necrotrophic pathogens.

  7. 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; Sistonen, Lea

    2014-09-15

    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.

  8. Rutin inhibits proliferation, attenuates superoxide production and decreases adhesion and migration of human cancerous cells.

    Science.gov (United States)

    Ben Sghaier, Mohamed; Pagano, Alessandra; Mousslim, Mohamed; Ammari, Youssef; Kovacic, Hervé; Luis, José

    2016-12-01

    Lung and colorectal cancer are the principal causes of death in the world. Rutin, an active flavonoid compound, is known for possessing a wide range of biological activities. In this study, we examined the effect of rutin on the viability, superoxide anion production, adhesion and migration of human lung (A549) and colon (HT29 and Caco-2) cancer cell lines. In order to control the harmlessness of the tested concentrations of rutin, the viability of cancer cell lines was assessed using a 3-(4,5-dimethylthiazol- 2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay. ROS generation was measured by lucigenin chemiluminescence detecting superoxide ions. To investigate the effect of rutin on the behavior of human lung and colon cancer cell lines, we performed adhesion assays, using various purified extracellular matrix (ECM) proteins. Finally, in vitro cell migration assays were explored using modified Boyden chambers. The viability of cancerous cells was inhibited by rutin. It also significantly attenuated the superoxide production in HT29 cells. In addition, rutin affected adhesion and migration of A549 and HT29 cell. These findings indicate that rutin, a natural molecule, might have potential as anticancer agent against lung and colorectal carcinogenesis.

  9. Extracellular magnesium decreases the secretory response of rat peritoneal mast cells to compound 48/80 in vitro

    DEFF Research Database (Denmark)

    Bertelsen, Niels Haldor; Johansen, Torben

    1991-01-01

    Exposure of rat peritoneal mast cells to magnesium in the absence of extracellular calcium resulted in a time- and dose-dependent decrease in the secretory response induced by compound 48/80. The decrease was prevented by a low extracellular concentration of calcium. Furthermore, the decreased...... and the secretory stimulus. A dose-dependent decrease in antigen induced histamine secretion that was reversed by calcium was also observed. Exposure of the mast cells to magnesium for 15 min resulted in a parallel decrease in histamine secretion and in the cellular content of 45Ca2+. These observations suggest...... secretory responsiveness was dose-dependently restored by the addition of calcium to the cells simultaneously with compound 48/80. Preincubation with magnesium also inhibited antigen-induced histamine secretion in a dose-dependent manner. This was reversed by the simultaneous addition of calcium...

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

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    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.

  11. Lithium chloride decreases proliferation and migration of C6 glioma cells harboring isocitrate dehydrogenase 2 mutant via GSK-3β.

    Science.gov (United States)

    Fu, Yuejun; Zheng, Yali; Chan, Kok-Gan; Liang, Aihua; Hu, Fengyun

    2014-06-01

    The gene encoding isocitrate dehydrogenase (IDH) is somatically mutated predominantly in secondary glioblastoma multiforme. Mutations of IDH1 and IDH2 lead to simultaneous loss and gain of activities in the production of α-ketoglutarate and 2-hydroxyglutarate, respectively. Lithium chloride was recently proved efficient in inhibiting glioma cell migration. The mechanism of lithium chloride on C6 glioma cells harboring IDH2 mutation has not been studied. Here, we found lithium chloride induced inhibitive effects on cell proliferation of both C6 glioma cells with and without IDH2 mutation, although IDH2 mutation increased the stability of HIF-1α. GSK-3β could be phosphorylated at Ser9 and its activity was inhibited when C6 glioma cells were treated by lithium chloride. The degree of phosphorylation in IDH2(R172G) treatment group was lower than that as compared to the control and IDH2 treatment groups. At the same time, the accumulation of β-catenin in C6 cell nucleus was decreased. Moreover, although the β-catenin and HIF-1α increased the secretion of metalloproteinase-2,-9 in C6 glioma cells harboring IDH2 mutation, the migration potential of lithium chloride-treated C6 glioma cells harboring the IDH2 and its mutant was uniform. These results indicated lithium chloride could decrease the proliferation and migration potential of C6 glioma cells harboring IDH2 mutation.

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

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    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. Fractalkine restores the decreased expression of StAR and progesterone in granulosa cells from patients with polycystic ovary syndrome.

    Science.gov (United States)

    Huang, Shuo; Pang, Yanli; Yan, Jie; Lin, Shengli; Zhao, Yue; Lei, Li; Yan, Liying; Li, Rong; Ma, Caihong; Qiao, Jie

    2016-07-08

    Low progesterone levels are associated with luteal phase deficiency in women with polycystic ovary syndrome (PCOS). The mechanisms regulating progesterone biosynthesis in the granulosa cells from women with PCOS is largely unknown. Fractalkine is expressed in human ovaries, and is reported to regulate progesterone production in granulosa cells of healthy women. In the current study, we aimed to examine the role of fractalkine in women with PCOS. Reduced fractalkine levels were found in follicular fluid and granulosa cells, accompanied by decreased progesterone production and reduced steroidogenic acute regulatory protein (StAR) expression in the granulosa cells of patients with PCOS. Administration of fractalkine reversed the inhibition of progesterone and StAR expression. The mechanism mediating these effects may be associated with the inhibition of ERK activity in the granulosa cells from women with PCOS. Our findings revealed that fractalkine regulated steroidogenesis in follicular granulosa cells of women with PCOS.

  14. Fractalkine restores the decreased expression of StAR and progesterone in granulosa cells from patients with polycystic ovary syndrome

    Science.gov (United States)

    Huang, Shuo; Pang, Yanli; Yan, Jie; Lin, Shengli; Zhao, Yue; Lei, Li; Yan, Liying; Li, Rong; Ma, Caihong; Qiao, Jie

    2016-01-01

    Low progesterone levels are associated with luteal phase deficiency in women with polycystic ovary syndrome (PCOS). The mechanisms regulating progesterone biosynthesis in the granulosa cells from women with PCOS is largely unknown. Fractalkine is expressed in human ovaries, and is reported to regulate progesterone production in granulosa cells of healthy women. In the current study, we aimed to examine the role of fractalkine in women with PCOS. Reduced fractalkine levels were found in follicular fluid and granulosa cells, accompanied by decreased progesterone production and reduced steroidogenic acute regulatory protein (StAR) expression in the granulosa cells of patients with PCOS. Administration of fractalkine reversed the inhibition of progesterone and StAR expression. The mechanism mediating these effects may be associated with the inhibition of ERK activity in the granulosa cells from women with PCOS. Our findings revealed that fractalkine regulated steroidogenesis in follicular granulosa cells of women with PCOS. PMID:27386819

  15. 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...... 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...

  16. Decreased autocrine EGFR signaling in metastatic breast cancer cells inhibits tumor growth in bone and mammary fat pad.

    Science.gov (United States)

    Nickerson, Nicole K; Mohammad, Khalid S; Gilmore, Jennifer L; Crismore, Erin; Bruzzaniti, Angela; Guise, Theresa A; Foley, John

    2012-01-01

    Breast cancer metastasis to bone triggers a vicious cycle of tumor growth linked to osteolysis. Breast cancer cells and osteoblasts express the epidermal growth factor receptor (EGFR) and produce ErbB family ligands, suggesting participation of these growth factors in autocrine and paracrine signaling within the bone microenvironment. EGFR ligand expression was profiled in the bone metastatic MDA-MB-231 cells (MDA-231), and agonist-induced signaling was examined in both breast cancer and osteoblast-like cells. Both paracrine and autocrine EGFR signaling were inhibited with a neutralizing amphiregulin antibody, PAR34, whereas shRNA to the EGFR was used to specifically block autocrine signaling in MDA-231 cells. The impact of these was evaluated with proliferation, migration and gene expression assays. Breast cancer metastasis to bone was modeled in female athymic nude mice with intratibial inoculation of MDA-231 cells, and cancer cell-bone marrow co-cultures. EGFR knockdown, but not PAR34 treatment, decreased osteoclasts formed in vitro (p<0.01), reduced osteolytic lesion tumor volume (p<0.01), increased survivorship in vivo (p<0.001), and resulted in decreased MDA-231 growth in the fat pad (p<0.01). Fat pad shEGFR-MDA-231 tumors produced in nude mice had increased necrotic areas and decreased CD31-positive vasculature. shEGFR-MDA-231 cells also produced decreased levels of the proangiogenic molecules macrophage colony stimulating factor-1 (MCSF-1) and matrix metalloproteinase 9 (MMP9), both of which were decreased by EGFR inhibitors in a panel of EGFR-positive breast cancer cells. Thus, inhibiting autocrine EGFR signaling in breast cancer cells may provide a means for reducing paracrine factor production that facilitates microenvironment support in the bone and mammary gland.

  17. Decreased autocrine EGFR signaling in metastatic breast cancer cells inhibits tumor growth in bone and mammary fat pad.

    Directory of Open Access Journals (Sweden)

    Nicole K Nickerson

    Full Text Available Breast cancer metastasis to bone triggers a vicious cycle of tumor growth linked to osteolysis. Breast cancer cells and osteoblasts express the epidermal growth factor receptor (EGFR and produce ErbB family ligands, suggesting participation of these growth factors in autocrine and paracrine signaling within the bone microenvironment. EGFR ligand expression was profiled in the bone metastatic MDA-MB-231 cells (MDA-231, and agonist-induced signaling was examined in both breast cancer and osteoblast-like cells. Both paracrine and autocrine EGFR signaling were inhibited with a neutralizing amphiregulin antibody, PAR34, whereas shRNA to the EGFR was used to specifically block autocrine signaling in MDA-231 cells. The impact of these was evaluated with proliferation, migration and gene expression assays. Breast cancer metastasis to bone was modeled in female athymic nude mice with intratibial inoculation of MDA-231 cells, and cancer cell-bone marrow co-cultures. EGFR knockdown, but not PAR34 treatment, decreased osteoclasts formed in vitro (p<0.01, reduced osteolytic lesion tumor volume (p<0.01, increased survivorship in vivo (p<0.001, and resulted in decreased MDA-231 growth in the fat pad (p<0.01. Fat pad shEGFR-MDA-231 tumors produced in nude mice had increased necrotic areas and decreased CD31-positive vasculature. shEGFR-MDA-231 cells also produced decreased levels of the proangiogenic molecules macrophage colony stimulating factor-1 (MCSF-1 and matrix metalloproteinase 9 (MMP9, both of which were decreased by EGFR inhibitors in a panel of EGFR-positive breast cancer cells. Thus, inhibiting autocrine EGFR signaling in breast cancer cells may provide a means for reducing paracrine factor production that facilitates microenvironment support in the bone and mammary gland.

  18. Decreased peripheral natural killer cells activity in the immune activated stage of chronic hepatitis B.

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    Yuan Li

    Full Text Available BACKGROUND & AIMS: The natural course of chronic hepatitis B virus (HBV infection is characterized by different immune responses, ranging from immune tolerant (IT to immune activated (IA stages. In our study, we investigated the natural killer (NK cells activity in patients at different immunological stages of chronic HBV infection. METHODS: Blood samples obtained from 57 HBeAg positive patients with chronic hepatitis B (CHB, including 15 patients in the immune tolerant (IT stage, 42 patients in the immune activated (IA stage, and 18 healthy individuals (HI. The analyses included flow cytometry to detect NK cells, the determination of cytokine levels as well as of surface receptor expression and cytotoxicity. RESULTS: NK cells in peripheral blood were significantly lower in patients in the IA stage of CHB compared to HI (p<0.05. Patients in the IA stage of CHB had lower levels of NK cells activating receptor NKp30 and NKG2D expression, cytokine interferon-γ (IFN-γ and tumor necrosis factor-α (TNF-α production, as compared to patients in the IT stage and HI, respectively (p<0.05. Cytotoxicity of NK cells was lower in patients in the IA stage of CHB compared to patients in the IT stage and HI, respectively (p<0.05. The level of IFN-γ but not level of TNF-α and cytotoxicity of NK cells was inversely correlated with serum HBV load in patients with CHB. Peripheral NK cells activity did not correlate with ALT level. CONCLUSION: NK cells activity was lower in CHB patients, especially in those in the IA stage.

  19. Gradient sensing by a bistable regulatory motif enhances signal amplification but decreases accuracy in individual cells

    Science.gov (United States)

    Sharma, Rati; Roberts, Elijah

    2016-06-01

    Many vital eukaryotic cellular functions require the cell to respond to a directional gradient of a signaling molecule. The first two steps in any eukaryotic chemotactic/chemotropic pathway are gradient detection and cell polarization. Like many processes, such chemotactic and chemotropic decisions are made using a relatively small number of molecules and are thus susceptible to internal and external fluctuations during signal transduction. Large cell-to-cell variations in the magnitude and direction of a response are therefore possible and do, in fact, occur in natural systems. In this work we use three-dimensional probabilistic modeling of a simple gradient sensing pathway to study the capacity for individual cells to accurately determine the direction of a gradient, despite fluctuations. We include a stochastic external gradient in our simulations using a novel gradient boundary condition modeling a point emitter a short distance away. We compare and contrast three different variants of the pathway, one monostable and two bistable. The simulation data show that an architecture combining bistability with spatial positive feedback permits the cell to both accurately detect and internally amplify an external gradient. We observe strong polarization in all individual cells, but in a distribution of directions centered on the gradient. Polarization accuracy in our study was strongly dependent upon a spatial positive feedback term that allows the pathway to trade accuracy for polarization strength. Finally, we show that additional feedback links providing information about the gradient to multiple levels in the pathway can help the cell to refine initial inaccuracy in the polarization direction.

  20. A new method for decreasing cell-load variation in dynamic cellular manufacturing systems

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    Aidin Delgoshaei

    2016-01-01

    Full Text Available Cell load variation is considered a significant shortcoming in scheduling of cellular manufacturing systems. In this article, a new method is proposed for scheduling dynamic cellular manufacturing systems in the presence of bottleneck and parallel machines. The aim of this method is to control cell load variation during the process of determining the best trading off values between in-house manufacturing and outsourcing. A genetic algorithm (GA is developed because of the high potential of trapping in the local optima, and results are compared with the results of LINGO® 12.0 software. The Taguchi method (an L_9 orthogonal optimization is used to estimate parameters of GA in order to solve experiments derived from literature. An in-depth analysis is conducted on the results in consideration of various factors, and control charts are used on machine-load variation. Our findings indicate that the dynamic condition of product demands affects the routing of product parts and may induce machine-load variations that yield to cell-load diversity. An increase in product uncertainty level causes the loading level of each cell to vary, which in turn results in the development of “complex dummy sub-cells”. The effect of the complex sub-cells is measured using another mathematical index. The results showed that the proposed GA can provide solutions with limited cell-load variations.

  1. NKT cell activation by local α-galactosylceramide administration decreases susceptibility to HSV-2 infection

    DEFF Research Database (Denmark)

    Iversen, Marie Beck; Jensen, Simon Kok; Hansen, Anne Louise;

    2015-01-01

    NKT cells are a subgroup of T cells, which express a restricted TCR repertoire and are critical for the innate immune responses to viral infections. Activation of NKT cells depends on the major histocompatibility complex-related molecule CD1d, which presents bioactive lipids to NKT cells....... The marine sponge derived lipid αGalCer has recently been demonstrated as a specific agonist for activation of human and murine NKT cells. In the present study we investigated the applicability of αGalCer pre-treatment for immune protection against intra-vaginal HSV-2 infection. We found that C57BL/6 WT mice...... that received local pre-treatment with αGalCer prior to intra-vaginal HSV-2 infection had a lower mean disease score, mortality and viral load in the vagina following infection, compared to mice that did not receive αGalCer pre-treatment. Further, we found increased numbers of CD45 and NK1.1 positive cells...

  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. Activation of P2X7 receptors decreases the proliferation of murine luteal cells.

    Science.gov (United States)

    Wang, Jing; Liu, Shuangmei; Nie, Yijun; Wu, Bing; Wu, Qin; Song, Miaomiao; Tang, Min; Xiao, Li; Xu, Ping; Tan, Ximin; Zhang, Luyin; Li, Gang; Liang, Shangdong; Zhang, Chunping

    2015-11-01

    Extracellular ATP regulates cellular function in an autocrine or paracrine manner through activating purinergic signalling. Studies have shown that purinergic receptors were expressed in mammalian ovaries and they have been proposed as an intra-ovarian regulatory mechanism. P2X7 was expressed in porcine ovarian theca cells and murine and human ovarian surface epithelium and is involved in ATP-induced apoptotic cell death. However, the role of P2X7 in corpus luteum is still unclear. The aim of this study was to investigate the role of ATP signalling in murine luteal cells and the possible mechanism(s) involved. We found that P2X7 was highly expressed in murine small luteal cells. The agonists of P2X7, ATP and BzATP, inhibited the proliferation of luteal cells. P2X7 antagonist BBG reversed the inhibition induced by ATP and BzATP. Further studies showed that ATP and BzATP inhibited the expression of cell cycle regulators cyclinD2 and cyclinE2. ATP and BzATP also inhibited the p38-mitogen-activated protein kinase (MAPK) signalling pathway. These results reveal that P2X7 receptor activation is involved in corpus luteum formation and function.

  4. Glucosamine inhibits decidualization of human endometrial stromal cells and decreases litter sizes in mice.

    Science.gov (United States)

    Tsai, Jui-He; Schulte, Maureen; O'Neill, Kathleen; Chi, Maggie M-Y; Frolova, Antonina I; Moley, Kelle H

    2013-07-01

    Embryo implantation in the uterus depends on decidualization of the endometrial stromal cells (ESCs), and glucose utilization via the pentose phosphate pathway is critical in this process. We hypothesized that the amino sugar glucosamine may block the pentose phosphate pathway via inhibition of the rate-limiting enzyme glucose-6-phosphate dehydrogenase in ESCs and therefore impair decidualization and embryo implantation, thus preventing pregnancy. Both human primary and immortalized ESCs were decidualized in vitro in the presence of 0, 2.5, or 5 mM glucosamine for 9 days. Viability assays demonstrated that glucosamine was well tolerated by human ESCs. Exposure of human ESCs to glucosamine resulted in significant decreases in the activity and expression of glucose-6-phosphate dehydrogenase and in the mRNA expression of the decidual markers prolactin, somatostatin, interleukin-15, and left-right determination factor 2. In mouse ESCs, expression of the decidual marker Prp decreased upon addition of glucosamine. In comparison with control mice, glucosamine-treated mice showed weak artificial deciduoma formation along the stimulated uterine horn. In a complementary in vivo experiment, a 60-day-release glucosamine (15, 150, or 1500 μg) or placebo pellet was implanted in a single uterine horn of mice. Mice with a glucosamine pellet delivered fewer live pups per litter than those with a control pellet, and pup number returned to normal after the end of the pellet-active period. In conclusion, glucosamine is a nonhormonal inhibitor of decidualization of both human and mouse ESCs and of pregnancy in mice. Our data indicate the potential for development of glucosamine as a novel, reversible, nonhormonal contraceptive.

  5. Histone deacetylase 3 depletion in osteo/chondroprogenitor cells decreases bone density and increases marrow fat.

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    David F Razidlo

    Full Text Available Histone deacetylase (Hdac3 is a nuclear enzyme that contributes to epigenetic programming and is required for embryonic development. To determine the role of Hdac3 in bone formation, we crossed mice harboring loxP sites around exon 7 of Hdac3 with mice expressing Cre recombinase under the control of the osterix promoter. The resulting Hdac3 conditional knockout (CKO mice were runted and had severe deficits in intramembranous and endochondral bone formation. Calvarial bones were significantly thinner and trabecular bone volume in the distal femur was decreased 75% in the Hdac3 CKO mice due to a substantial reduction in trabecular number. Hdac3-CKO mice had fewer osteoblasts and more bone marrow adipocytes as a proportion of tissue area than their wildtype or heterozygous littermates. Bone formation rates were depressed in both the cortical and trabecular regions of Hdac3 CKO femurs. Microarray analyses revealed that numerous developmental signaling pathways were affected by Hdac3-deficiency. Thus, Hdac3 depletion in osterix-expressing progenitor cells interferes with bone formation and promotes bone marrow adipocyte differentiation. These results demonstrate that Hdac3 inhibition is detrimental to skeletal health.

  6. Autistic Children Exhibit Decreased Levels of Essential Fatty Acids in Red Blood Cells

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    Sarah A. Brigandi

    2015-05-01

    Full Text Available Omega-6 (n-6 and omega-3 (n-3 polyunsaturated fatty acids (PUFA are essential nutrients for brain development and function. However, whether or not the levels of these fatty acids are altered in individuals with autism remains debatable. In this study, we compared the fatty acid contents between 121 autistic patients and 110 non-autistic, non-developmentally delayed controls, aged 3–17. Analysis of the fatty acid composition of red blood cell (RBC membrane phospholipids showed that the percentage of total PUFA was lower in autistic patients than in controls; levels of n-6 arachidonic acid (AA and n-3 docosahexaenoic acid (DHA were particularly decreased (p < 0.001. In addition, plasma levels of the pro-inflammatory AA metabolite prostaglandin E2 (PGE2 were higher in a subset of the autistic participants (n = 20 compared to controls. Our study demonstrates an alteration in the PUFA profile and increased production of a PUFA-derived metabolite in autistic patients, supporting the hypothesis that abnormal lipid metabolism is implicated in autism.

  7. Fasciola hepatica Kunitz type molecule decreases dendritic cell activation and their ability to induce inflammatory responses.

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    Cristian R Falcón

    Full Text Available The complete repertoire of proteins with immunomodulatory activity in Fasciola hepatica (Fh has not yet been fully described. Here, we demonstrated that Fh total extract (TE reduced LPS-induced DC maturation, and the DC ability to induce allogeneic responses. After TE fractionating, a fraction lower than 10 kDa (F<10 kDa was able to maintain the TE properties to modulate the DC pro- and anti-inflammatory cytokine production induced by LPS. In addition, TE or F<10 kDa treatment decreased the ability of immature DC to stimulate the allogeneic responses and induced a novo allogeneic CD4+CD25+Foxp3+ T cells. In contrast, treatment of DC with T/L or F<10 kDa plus LPS (F<10/L induced a regulatory IL-27 dependent mechanism that diminished the proliferative and Th1 and Th17 allogeneic responses. Finally, we showed that a Kunitz type molecule (Fh-KTM, present in F<10 kDa, was responsible for suppressing pro-inflammatory cytokine production in LPS-activated DC, by printing tolerogenic features on DC that impaired their ability to induce inflammatory responses. These results suggest a modulatory role for this protein, which may be involved in the immune evasion mechanisms of the parasite.

  8. Decreased frequencies of circulating follicular helper T cell counterparts and plasmablasts in ankylosing spondylitis patients Naive for TNF blockers.

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    María-Belén Bautista-Caro

    Full Text Available Follicular helper T cells (Tfh, localized in lymphoid organs, promote B cell differentiation and function. Circulating CD4 T cells expressing CXCR5, ICOS and/or PD-1 are counterparts of Tfh. Three subpopulations of circulating CD4+CXCR5+ cells have been described: CXCR3+CCR6- (Tfh-Th1, CXCR3-CCR6+ (Tfh-Th17, and CXCR3-CCR6- (Tfh-Th2. Only Tfh-Th17 and Tfh-Th2 function as B cell helpers. Our objective was to study the frequencies of circulating Tfh (cTfh, cTfh subsets and plasmablasts (CD19+CD20-CD27+CD38high cells, and the function of cTfh cells, in patients with Ankylosing Spondylitis (AS. To this end, peripheral blood was drawn from healthy controls (HC (n = 50, AS patients naïve for TNF blockers (AS/nb (n = 25 and AS patients treated with TNF blockers (AS/b (n = 25. The frequencies of cTfh and plasmablasts were determined by flow cytometry. Cocultures of magnetically sorted CD4+CXCR5+ T cells with autologous CD19+CD27- naïve B cells were established from 3 AS/nb patients and 3 HC, and concentrations of IgG, A and M were measured in supernatants. We obseved that AS/nb but not AS/b patients, demonstrated decreased frequencies of circulating CD4+CXCR5+ICOS+PD-1+ cells and plasmablasts, together with a decreased (Tfh-Th17+Tfh-Th2/Tfh-Th1 ratio. The amounts of IgG and IgA produced in cocultures of CD4+CXCR5+ T cells with CD19+CD27- B cells of AS/nb patients were significantly lower than observed in cocultures established from HC. In summary, AS/nb but not AS/b patients, demonstrate a decreased frequency of cTfh and plasmablasts, and an underrepresentation of cTfh subsets bearing a B helper phenotype. In addition, peripheral blood CD4+CXCR5+ T cells of AS/nb patients showed a decreased capacity to help B cells ex vivo.

  9. Lipoxin A₄ modulates adaptive immunity by decreasing memory B-cell responses via an ALX/FPR2-dependent mechanism.

    Science.gov (United States)

    Ramon, Sesquile; Bancos, Simona; Serhan, Charles N; Phipps, Richard P

    2014-02-01

    Specialized proresolving mediators are endogenous bioactive lipid molecules that play a fundamental role in the regulation of inflammation and its resolution. Lipoxins and other specialized proresolving mediators have been identified in important immunological tissues including bone marrow, spleen, and blood. Lipoxins regulate functions of the innate immune system including the promotion of monocyte recruitment and increase macrophage phagocytosis of apoptotic neutrophils. A major knowledge gap is whether lipoxins influence adaptive immune cells. Here, we analyzed the actions of lipoxin A₄ (LXA₄) and its receptor ALX/FPR2 on human and mouse B cells. LXA₄ decreased IgM and IgG production on activated human B cells through ALX/FPR2-dependent signaling, which downregulated NF-κB p65 nuclear translocation. LXA₄ also inhibited human memory B-cell antibody production and proliferation, but not naïve B-cell function. Lastly, LXA₄ decreased antigen-specific antibody production in an OVA immunization mouse model. To our knowledge, this is the first description of the actions of lipoxins on human B cells, demonstrating a link between resolution signals and adaptive immunity. Regulating antibody production is crucial to prevent unwanted inflammation. Harnessing the ability of lipoxins to decrease memory B-cell antibody production can be beneficial to threat inflammatory and autoimmune disorders.

  10. A rat tail temporary static compression model reproduces different stages of intervertebral disc degeneration with decreased notochordal cell phenotype.

    Science.gov (United States)

    Hirata, Hiroaki; Yurube, Takashi; Kakutani, Kenichiro; Maeno, Koichiro; Takada, Toru; Yamamoto, Junya; Kurakawa, Takuto; Akisue, Toshihiro; Kuroda, Ryosuke; Kurosaka, Masahiro; Nishida, Kotaro

    2014-03-01

    The intervertebral disc nucleus pulposus (NP) has two phenotypically distinct cell types-notochordal cells (NCs) and non-notochordal chondrocyte-like cells. In human discs, NCs are lost during adolescence, which is also when discs begin to show degenerative signs. However, little evidence exists regarding the link between NC disappearance and the pathogenesis of disc degeneration. To clarify this, a rat tail disc degeneration model induced by static compression at 1.3 MPa for 0, 1, or 7 days was designed and assessed for up to 56 postoperative days. Radiography, MRI, and histomorphology showed degenerative disc findings in response to the compression period. Immunofluorescence displayed that the number of DAPI-positive NP cells decreased with compression; particularly, the decrease was notable in larger, vacuolated, cytokeratin-8- and galectin-3-co-positive cells, identified as NCs. The proportion of TUNEL-positive cells, which predominantly comprised non-NCs, increased with compression. Quantitative PCR demonstrated isolated mRNA up-regulation of ADAMTS-5 in the 1-day loaded group and MMP-3 in the 7-day loaded group. Aggrecan-1 and collagen type 2α-1 mRNA levels were down-regulated in both groups. This rat tail temporary static compression model, which exhibits decreased NC phenotype, increased apoptotic cell death, and imbalanced catabolic and anabolic gene expression, reproduces different stages of intervertebral disc degeneration.

  11. Increased Adipogenic and Decreased Chondrogenic Differentiation of Adipose Derived Stem Cells on Nanowire Surfaces

    Directory of Open Access Journals (Sweden)

    Nathan A. Trujillo

    2014-03-01

    Full Text Available Despite many advances in tissue engineering, there are still significant challenges associated with restructuring, repairing, or replacing damaged tissue in the body. Currently, a major obstacle has been trying to develop a scaffold for cartilage tissue engineering that provides the correct mechanical properties to endure the loads associated with articular joints as well as promote cell-scaffold interactions to aid in extracellular matrix deposition. In addition, adipogenic tissue engineering is widely growing due to an increased need for more innovative reconstructive therapies following adipose tissue traumas and cosmetic surgeries. Recently, lipoaspirate tissue has been identified as a viable alternative source for mesenchymal stem cells because it contains a supportive stroma that can easily be isolated. Adipose derived stem cells (ADSCs can differentiate into a variety of mesodermal lineages including the adipogenic and chondrogenic phenotypes. Biodegradable polymeric scaffolds have been shown to be a promising alternative and stem cells have been widely used to evaluate the compatibility, viability, and bioactivity of these materials. Polycaprolactone is a bioresorbable polymer, which has been widely used for biomedical and tissue engineering applications. The fundamental concept behind successful synthetic tissue-engineered scaffolds is to promote progenitor cell migration, adhesion, proliferation, and induce differentiation, extracellular matrix synthesis, and finally integration with host tissue. In this study, we investigated the adhesion, proliferation, and chondrogenic and adipogenic differentiation of ADSCs on nanowire surfaces. A solvent-free gravimetric template technique was used to fabricate polycaprolactone nanowires surfaces. The results indicated that during the growth period i.e., initial 7 days of culture, the nanowire surfaces (NW supported adhesion and proliferation of the cells that had elongated morphologies. However

  12. MDSC-decreasing chemotherapy increases the efficacy of cytokine-induced killer cell immunotherapy in metastatic renal cell carcinoma and pancreatic cancer.

    Science.gov (United States)

    Wang, Zibing; Liu, Yuqing; Zhang, Yong; Shang, Yiman; Gao, Quanli

    2016-01-26

    Adoptive immunotherapy using cytokine-induced killer (CIK) cells is a promising cancer treatment, but its efficacy is restricted by various factors, including the accumulation of myeloid-derived suppressor cells (MDSCs). In this study, we determine whether chemotherapeutic drugs that reduce MDSC levels enhance the efficacy of CIK cell therapy in the treatment of solid tumors. Fifty-three patients were included in this study; 17 were diagnosed with metastatic renal cell carcinoma (MRCC), 10 with advanced pancreatic cancer (PC), and 26 with metastatic melanoma (MM). These patients were divided into two groups: CIK cell therapy alone and CIK cell therapy combined with chemotherapy. Combining CIK cell therapy and chemotherapy increased 1-year survival rates and median survival times in MRCC and PC patients, but not in MM patients. The disease control rate did not differ between treatment groups for MRCC or MM patients, but was higher in PC patients receiving combined treatment than CIK cell treatment alone. These data suggest that addition of MDSC-decreasing chemotherapy to CIK cell therapy improves survival in MRCC and PC patients.

  13. Targeting sphingosine kinase 1 in carcinoma cells decreases proliferation and survival by compromising PKC activity and cytokinesis.

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    Nataliya Kotelevets

    Full Text Available Sphingosine kinases (SK catalyze the phosphorylation of proapoptotic sphingosine to the prosurvival factor sphingosine 1-phosphate (S1P, thereby promoting oncogenic processes. Breast (MDA-MB-231, lung (NCI-H358, and colon (HCT 116 carcinoma cells were transduced with shRNA to downregulate SK-1 expression or treated with a pharmacologic SK-1 inhibitor. The effects of SK-1 targeting were investigated by measuring the level of intracellular sphingosine, the activity of protein kinase C (PKC and cell cycle regulators, and the mitotic index. Functional assays included measurement of cell proliferation, colony formation, apoptosis, and cell cycle analysis. Downregulation of SK-1 or its pharmacologic inhibition increased intracellular sphingosine and decreased PKC activity as shown by reduced phosphorylation of PKC substrates. In MDA-MB-231 cells this effect was most pronounced and reduced cell proliferation and colony formation, which could be mimicked using exogenous sphingosine or the PKC inhibitor RO 31-8220. SK-1 downregulation in MDA-MB-231 cells increased the number of cells with 4N and 8N DNA content, and similar effects were observed upon treatment with sphingosine or inhibitors of SK-1 or PKC. Examination of cell cycle regulators unveiled decreased cdc2 activity and expression of Chk1, which may compromise spindle checkpoint function and cytokinesis. Indeed, SK-1 kd cells entered mitosis but failed to divide, and in the presence of taxol also failed to sustain mitotic arrest, resulting in further increased endoreduplication and apoptosis. Our findings delineate an intriguing link between SK-1, PKC and components of the cell cycle machinery, which underlines the significance of SK-1 as a target for cancer therapy.

  14. Hydrostatic pressure decreases membrane fluidity and lipid desaturase expression in chondrocyte progenitor cells.

    Science.gov (United States)

    Montagne, Kevin; Uchiyama, Hiroki; Furukawa, Katsuko S; Ushida, Takashi

    2014-01-22

    Membrane biomechanical properties are critical in modulating nutrient and metabolite exchange as well as signal transduction. Biological membranes are predominantly composed of lipids, cholesterol and proteins, and their fluidity is tightly regulated by cholesterol and lipid desaturases. To determine whether such membrane fluidity regulation occurred in mammalian cells under pressure, we investigated the effects of pressure on membrane lipid order of mouse chondrogenic ATDC5 cells and desaturase gene expression. Hydrostatic pressure linearly increased membrane lipid packing and simultaneously repressed lipid desaturase gene expression. We also showed that cholesterol mimicked and cholesterol depletion reversed those effects, suggesting that desaturase gene expression was controlled by the membrane physical state itself. This study demonstrates a new effect of hydrostatic pressure on mammalian cells and may help to identify the molecular mechanisms involved in hydrostatic pressure sensing in chondrocytes.

  15. Erythropoietin reduces storage lesions and decreases apoptosis indices in blood bank red blood cells

    Science.gov (United States)

    Penuela, Oscar Andrés; Palomino, Fernando; Gómez, Lina Andrea

    2015-01-01

    Background Recent evidence shows a selective destruction of the youngest circulating red blood cells (neocytolysis) trigged by a drop in erythropoietin levels. Objective The aim of this study was to evaluate the effect of recombinant human erythropoietin beta on the red blood cell storage lesion and apoptosis indices under blood bank conditions. Methods Each one of ten red blood cell units preserved in additive solution 5 was divided in two volumes of 100 mL and assigned to one of two groups: erythropoietin (addition of 665 IU of recombinant human erythropoietin) and control (isotonic buffer solution was added). The pharmacokinetic parameters of erythropoietin were estimated and the following parameters were measured weekly, for six weeks: Immunoreactive erythropoietin, hemolysis, percentage of non-discocytes, adenosine triphosphate, glucose, lactate, lactate dehydrogenase, and annexin-V/esterase activity. The t-test or Wilcoxon's test was used for statistical analysis with significance being set for a p-value 6 weeks under blood bank conditions, with persistent supernatant concentrations of erythropoietin during the entire storage period. Adenosine triphosphate was higher in the Erythropoietin Group in Week 6 (4.19 ± 0.05 μmol/L vs. 3.53 ± 0.02 μmol/L; p-value = 0.009). The number of viable cells in the Erythropoietin Group was higher than in the Control Group (77% ± 3.8% vs. 71% ± 2.3%; p-value <0.05), while the number of apoptotic cells was lower (9.4% ± 0.3% vs. 22% ± 0.8%; p-value <0.05). Conclusions Under standard blood bank conditions, an important proportion of red blood cells satisfy the criteria of apoptosis. Recombinant human erythropoietin beta seems to improve storage lesion parameters and mitigate apoptosis. PMID:26969770

  16. 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

    Treatment of advanced cutaneous T-cell lymphomas (CTCL) is challenging because they are resistant to conventional chemotherapy. USP2 has been shown to promote resistance to chemotherapeutic agents in several cancer models.We show here USP2 is expressed in quiescent and activated T-cells and its e...... Mdm2 expression and upregulation of p53. Taken together, our findings suggest that USP2 stabilizes Mdm2 which antagonizes pro-apoptotic activity of p53 and possibly contributes to therapeutic resistance in CTCL....

  17. Effect of decreasing electrical resistance in Characeae cell membranes caused by the flow of alternating current

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    Edward Śpiewla

    2014-02-01

    Full Text Available By means of the techniques of external electrodes and microelectrodes, it was found that evanescent flow of an alternating current through plasmalemma of Characeae cells neutralises oscillatory change in their electrical resistance and reversibly diminishes its value. This effect is particularly significant in the case of "high resistance cells", but it weakens with increasing temperature. The value of the estimated activation energy indicates that, after flow of the alternating current through the membrane, a rapid increase in the conductivity may be caused by an increase in conductivity of potassium channels. This result seems to support the hypothesis of electroconformational feedback.

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

    OpenAIRE

    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...

  19. Flavone inhibits migration through DLC1/RhoA pathway by decreasing ROS generation in breast cancer cells.

    Science.gov (United States)

    Zhu, Wenzhen; Ma, Long; Yang, Bingwu; Zheng, Zhaodi; Chai, Rongfei; Liu, Tingting; Liu, Zhaojun; Song, Taiyu; Li, Fenglin; Li, Guorong

    2016-05-01

    Tumor suppressor protein deleted in liver cancer 1 (DLC1) is a RhoGTPase-activating protein (RhoGAP) and inhibits cancer cell migration by inactivating downstream target protein RhoA. A few studies have reported the regulations of reactive oxygen species (ROS) on RhoGAP. In this study, we investigated flavone (the core structure of flavonoids)-induced regulation on ROS generation and DLC1/RhoA pathway in MCF-7 and MDA-MB-231 breast cancer cells and explored whether flavone-induced upregulation of DLC1 is mediated by ROS. Our results showed that flavone decreased ROS production and inhibited cell migration through DLC1/RhoA pathway. To further investigate the role of ROS in flavone-induced regulation on DLC1/RhoA pathway, hydrogen peroxide was added to restore the ROS levels. Flavone-induced upregulation of DLC1 expression, downregulation of RhoA activity, and inhibition of cell migration were all restrained by hydrogen peroxide. We also found that flavone increased DLC1 stability by inhibiting DLC1 protein degradation in breast cancer cells. In summary, our study demonstrated that flavone inhibited cell migration through DLC1/RhoA pathway by decreasing ROS generation and suppressed DLC1 degradation in MCF-7 and MDA-MB-231 breast cancer cells.

  20. Decreased proportion of cytomegalovirus specific CD8 T-cells but no signs of general immunosenescence in Alzheimer's disease.

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    Gabriel Westman

    Full Text Available Cytomegalovirus (CMV has been suggested as a contributing force behind the impaired immune responsiveness in the elderly, with decreased numbers of naïve T-cells and an increased proportion of effector T-cells. Immunological impairment is also implicated as a part of the pathogenesis in Alzheimer's disease (AD. The aim of this study was to investigate whether AD patients present with a different CMV-specific CD8 immune profile compared to non-demented controls. Blood samples from 50 AD patients and 50 age-matched controls were analysed for HLA-type, CMV serostatus and systemic inflammatory biomarkers. Using multi-colour flow cytometry, lymphocytes from peripheral blood mononuclear cells were analysed for CMV-specific CD8 immunity with MHC-I tetramers A01, A02, A24, B07, B08 and B35 and further classified using CD27, CD28, CD45RA and CCR7 antibodies. Among CMV seropositive subjects, patients with AD had significantly lower proportions of CMV-specific CD8 T-cells compared to controls, 1.16 % vs. 4.13 % (p=0.0057. Regardless of dementia status, CMV seropositive subjects presented with a lower proportion of naïve CD8 cells and a higher proportion of effector CD8 cells compared to seronegative subjects. Interestingly, patients with AD showed a decreased proportion of CMV-specific CD8 cells but no difference in general CD8 differentiation.

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

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    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.

  2. Mechanical Anisotropy of Rat Aortic Smooth Muscle Cells Decreases with Their Contraction

    Science.gov (United States)

    Nagayama, Kazuaki; Matsumoto, Takeo

    Tensile properties of smooth muscle cells freshly isolated from rat thoracic aortas (FSMCs) in their major and minor axes were measured using a laboratory-made micro tensile tester. The relationship between the tension applied to a cell and its elongation was obtained in untreated cells and those treated with 10-5M serotonin to induce contraction. An initial stiffness of untreated FSMCs, normalized by their initial cross-sectional area perpendicular to the stretch direction, was significantly higher in the major axis (14.8±4.3kPa, mean±SEM, n=5) than the minor axis (2.8±1.0kPa, n=5). The stiffness increased significantly in response to the contraction, but the increase was much higher in the minor axis (59.0±9.4kPa, n=4) than in the major (88.1±13.3kPa, n=4). The difference between the two directions was insignificant in the contracted state. Observations of the morphology of actin filaments with a confocal laser scanning microscope in untreated FSMCs revealed that they were long fibers running almost parallel to the major axis, while those in contracted cells showed an aggregated structure without a preferential direction. These results may indicate that anisotropy in untreated FSMCs is caused by the anisotropic alignment of their actin filaments, and that such anisotropy disappears in response to actin filament reorganization caused by the contraction.

  3. Ratiometric fluorescence imaging of cellular polarity: decrease in mitochondrial polarity in cancer cells.

    Science.gov (United States)

    Jiang, Na; Fan, Jiangli; Xu, Feng; Peng, Xiaojun; Mu, Huiying; Wang, Jingyun; Xiong, Xiaoqing

    2015-02-16

    Mitochondrial polarity strongly influences the intracellular transportation of proteins and interactions between biomacromolecules. The first fluorescent probe capable of the ratiometric imaging of mitochondrial polarity is reported. The probe, termed BOB, has two absorption maxima (λabs = 426 and 561 nm) and two emission maxima--a strong green emission (λem = 467 nm) and a weak red emission (642 nm in methanol)--when excited at 405 nm. However, only the green emission is markedly sensitive to polarity changes, thus providing a ratiometric fluorescence response with a good linear relationship in both extensive and narrow ranges of solution polarity. BOB possesses high specificity to mitochondria (Rr =0.96) that is independent of the mitochondrial membrane potential. The mitochondrial polarity in cancer cells was found to be lower than that of normal cells by ratiometric fluorescence imaging with BOB. The difference in mitochondrial polarity might be used to distinguish cancer cells from normal cells.

  4. Decreased pCO(2) accumulation by eliminating bicarbonate addition to high cell-density cultures.

    Science.gov (United States)

    Goudar, Chetan T; Matanguihan, Ricaredo; Long, Edward; Cruz, Christopher; Zhang, Chun; Piret, James M; Konstantinov, Konstantin B

    2007-04-15

    High-density perfusion cultivation of mammalian cells can result in elevated bioreactor CO(2) partial pressure (pCO(2)), a condition that can negatively influence growth, metabolism, productivity, and protein glycosylation. For BHK cells in a perfusion culture at 20 x 10(6) cells/mL, the bioreactor pCO(2) exceeded 225 mm Hg with approximate contributions of 25% from cellular respiration, 35% from medium NaHCO(3), and 40% from NaHCO(3) added for pH control. Recognizing the limitations to the practicality of gas sparging for CO(2) removal in perfusion systems, a strategy based on CO(2) reduction at the source was investigated. The NaHCO(3) in the medium was replaced with a MOPS-Histidine buffer, while Na(2)CO(3) replaced NaHCO(3) for pH control. These changes resulted in 63-70% pCO(2) reductions in multiple 15 L perfusion bioreactors, and were reproducible at the manufacturing-scale. Bioreactor pCO(2) values after these modifications were in the 68-85 mm Hg range, pCO(2) reductions consistent with those theoretically expected. Low bioreactor pCO(2) was accompanied by both 68-123% increased growth rates and 58-92% increased specific productivity. Bioreactor pCO(2) reduction and the resulting positive implications for cell growth and productivity were brought about by process changes that were readily implemented and robust. This philosophy of pCO(2) reduction at the source through medium and base modification should be readily applicable to large-scale fed-batch cultivation of mammalian cells.

  5. Platelet-Activating Factor Antagonists Decrease Follicular Dendritic-Cell Stimulation of Human B Lymphocytes

    Directory of Open Access Journals (Sweden)

    Halickman Isaac

    2005-06-01

    Full Text Available Abstract Both B-lymphoblastoid cell lines and tonsillar B lymphocytes express receptors for platelet-activating factor (PAF. In lymph node germinal centres, B lymphocytes interact with follicular dendritic cells (FDCs, which present antigen-containing immune complexes to B lymphocytes. FDCs have phenotypic features that are similar to those of stromal cells and monocytes and may therefore be a source of lipid mediators. In this study, we evaluated the effects of the PAF antagonist WEB 2170 on the activation of tonsillar B lymphocytes by FDCs. FDCs were isolated from tonsils by Bovine Serum Albumin (BSA gradient centrifugation. After being cultured for 6 to 10 days, they were incubated with freshly isolated B cells in the presence or absence of the specific PAF receptor antagonist WEB 2170. B-lymphocyte proliferation was assessed by [3H]-thymidine incorporation, and immunoglobulin (Ig G and IgM secretion was assessed by enzyme-linked immunosorbent assay (ELISA. WEB 2170 (10-6 to 10-8 M inhibited [3H]-thymidine incorporation by up to 35% ± 3%. Moreover, the secretion of IgG and IgM was inhibited by up to 50% by WEB 2170 concentrations ranging from 10-6 to 10-8 M. There was no evidence of toxicity by trypan blue staining, and the addition of WEB 2170 to B cells in the absence of FDCs did not inhibit the spontaneous production of IgG or IgM. The effect of the PAF antagonist is primarily on B lymphocytes, as reverse transcription polymerase chain reaction detected little PAF receptor messenger ribonucleic acid (mRNA from FDCs. These data suggest that endogenous production of PAF may be important in the interaction of B lymphocytes with FDCs.

  6. Helional induces Ca2+ decrease and serotonin secretion of QGP-1 cells via a PKG-mediated pathway.

    Science.gov (United States)

    Kalbe, Benjamin; Schlimm, Marian; Mohrhardt, Julia; Scholz, Paul; Jansen, Fabian; Hatt, Hanns; Osterloh, Sabrina

    2016-10-01

    The secretion, motility and transport by intestinal tissues are regulated among others by specialized neuroendocrine cells, the so-called enterochromaffin (EC) cells. These cells detect different luminal stimuli, such as mechanical stimuli, fatty acids, glucose and distinct chemosensory substances. The EC cells react to the changes in their environment through the release of transmitter molecules, most importantly serotonin, to mediate the corresponding physiological response. However, little is known about the molecular targets of the chemical stimuli delivered from consumed food, spices and cosmetics within EC cells. In this study, we evaluated the expression of the olfactory receptor (OR) 2J3 in the human pancreatic EC cell line QGP-1 at the mRNA and protein levels. Using ratiofluorometric Ca(2+) imaging experiments, we demonstrated that the OR2J3-specific agonist helional induces a transient dose-dependent decrease in the intracellular Ca(2+) levels. This Ca(2+) decrease is mediated by protein kinase G (PKG) on the basis that the specific pharmacological inhibition of PKG with Rp-8-pCPT-cGMPS abolished the helional-induced Ca(2+) response. Furthermore, stimulation of QGP-1 cells with helional caused a dose-dependent release of serotonin that was comparable with the release induced by the application of a direct PKG activator (8-bromo-cGMP). Taken together, our results demonstrate that luminal odorants can be detected by specific ORs in QGP-1 cells and thus cause the directed release of serotonin and a PKG-dependent decrease in intracellular Ca(2.)

  7. Activation of D4 dopamine receptor decreases angiotensin II type 1 receptor expression in rat renal proximal tubule cells.

    Science.gov (United States)

    Chen, Ken; Deng, Kun; Wang, Xiaoyan; Wang, Zhen; Zheng, Shuo; Ren, Hongmei; He, Duofen; Han, Yu; Asico, Laureano D; Jose, Pedro A; Zeng, Chunyu

    2015-01-01

    The dopaminergic and renin-angiotensin systems interact to regulate blood pressure. Disruption of the D4 dopamine receptor gene in mice produces hypertension that is associated with increased renal angiotensin type 1 (AT1) receptor expression. We hypothesize that the D4 receptor can inhibit AT1 receptor expression and function in renal proximal tubule cells from Wistar-Kyoto (WKY) rats, but the D4 receptor regulation of AT1 receptor is aberrant in renal proximal tubule cells from spontaneously hypertensive rats (SHRs). The D4 receptor agonist, PD168077, decreased AT1 receptor protein expression in a time- and concentration-dependent manner in WKY cells. By contrast, in SHR cells, PD168077 increased AT1 receptor protein expression. The inhibitory effect of D4 receptor on AT1 receptor expression in WKY cells was blocked by a calcium channel blocker, nicardipine, or calcium-free medium, indicating that calcium is involved in the D4 receptor-mediated signaling pathway. Angiotensin II increased Na(+)-K(+) ATPase activity in WKY cells. Pretreatment with PD168077 decreased the stimulatory effect of angiotensin II on Na(+)-K(+) ATPase activity in WKY cells. In SHR cells, the inhibitory effect of D4 receptor on angiotensin II-mediated stimulation of Na(+)-K(+) ATPase activity was aberrant; pretreatment with PD168077 augmented the stimulatory effect of AT1 receptor on Na(+)-K(+) ATPase activity in SHR cells. This was confirmed in vivo; pretreatment with PD128077 for 1 week augmented the antihypertensive and natriuretic effect of losartan in SHRs but not in WKY rats. We suggest that an aberrant interaction between D4 and AT1 receptors may play a role in the abnormal regulation of sodium excretion in hypertension.

  8. Decreased β-Cell Function Is Associated with Reduced Skeletal Muscle Mass in Japanese Subjects without Diabetes

    Science.gov (United States)

    Sakai, Satoshi; Tanimoto, Keiji; Imbe, Ayumi; Inaba, Yuiko; Shishikura, Kanako; Tanimoto, Yoshimi; Ushiroyama, Takahisa; Terasaki, Jungo; Hanafusa, Toshiaki

    2016-01-01

    Background Decreased insulin secretion has a great impact on the incidence of type 2 diabetes in Japanese subjects. It is not clear whether β-cell function is related to muscle mass in subjects without diabetes. We investigated the relationship between β-cell function and skeletal muscle mass in Japanese subjects without diabetes. Methods The study included 1098 subjects (538 men and 560 women) aged 40 to 79 years, without diabetes (fasting glucose lower than 126 mg/dL and glycosylated hemoglobin lower than 6.5%), who consulted Osaka Medical College Health Science Clinic for a medical examination. Appendicular muscle mass was measured by bioelectrical impedance analysis. Appendicular muscle mass index was calculated as appendicular muscle mass divided by height squared (kg/m2). The homeostatic model assessment of β-cell function was used to assess β-cell function. The homeostatic model assessment of insulin resistance was used as a measure of insulin resistance. The association between appendicular muscle mass index and clinical parameters of β-cell function and insulin resistance was examined. Results Log-transformed homeostatic model assessment of β-cell function and Log-transformed homeostatic model assessment of insulin resistance showed a normal distribution. In both men and women, there was a significant positive correlation between appendicular muscle mass index and clinical parameters of β-cell function and insulin resistance. Tertile analysis, following stratification according to appendicular muscle mass index, found that low appendicular muscle mass index was significantly associated with the Log homeostatic model assessment of β-cell function and Log-transformed homeostatic model assessment of insulin resistance. Conclusion This study shows that decreased β cell function is associated with reduced skeletal muscle mass in Japanese subjects without diabetes. PMID:27612202

  9. Vasculoprotective Effects of Combined Endothelial Progenitor Cells and Mesenchymal Stem Cells in Diabetic Wound Care: Their Potential Role in Decreasing Wound-Oxidative Stress

    Directory of Open Access Journals (Sweden)

    Supakanda Sukpat

    2013-01-01

    Full Text Available To investigate whether the combined endothelial progenitor cells (EPCs and mesenchymal stem cells (MSCs could enhance angiogenesis and wound healing in diabetic mice. Balb/c nude mice were divided into five groups, including a control group, diabetic group (DM, DM injected with 1 × 106  cells MSCs, DM injected with 1 × 106  cells EPCs, and DM injected with combined 0.5 × 106  cells MSCs and 0.5 × 106  cells EPCs. After seven weeks, the mice were anesthetized, and bilateral full-thickness excision skin wounds were made on the dorsorostral back. The percentage of wound closure in DM group decreased significantly than in control and all other treated groups on day 7 and day 14 (P<0.005. On day 14, the percentage of capillary vascularity in combine-treated group was significantly higher than in DM (P<0.005. In the present study, we have demonstrated that the combined EPCs and MSCs can increase vascular endothelial growth factor (VEGF level and angiogenesis which resulted in reduced neutrophil infiltration, decreased malondialdehyde (MDA levels, and enhanced wound healing in diabetic mice model.

  10. Vasculoprotective effects of combined endothelial progenitor cells and mesenchymal stem cells in diabetic wound care: their potential role in decreasing wound-oxidative stress.

    Science.gov (United States)

    Sukpat, Supakanda; Isarasena, Nipan; Wongphoom, Jutamas; Patumraj, Suthiluk

    2013-01-01

    To investigate whether the combined endothelial progenitor cells (EPCs) and mesenchymal stem cells (MSCs) could enhance angiogenesis and wound healing in diabetic mice. Balb/c nude mice were divided into five groups, including a control group, diabetic group (DM), DM injected with 1 × 10(6) cells MSCs, DM injected with 1 × 10(6) cells EPCs, and DM injected with combined 0.5 × 10(6) cells MSCs and 0.5 × 10(6) cells EPCs. After seven weeks, the mice were anesthetized, and bilateral full-thickness excision skin wounds were made on the dorsorostral back. The percentage of wound closure in DM group decreased significantly than in control and all other treated groups on day 7 and day 14 (P < 0.005). On day 14, the percentage of capillary vascularity in combine-treated group was significantly higher than in DM (P < 0.005). In the present study, we have demonstrated that the combined EPCs and MSCs can increase vascular endothelial growth factor (VEGF) level and angiogenesis which resulted in reduced neutrophil infiltration, decreased malondialdehyde (MDA) levels, and enhanced wound healing in diabetic mice model.

  11. Decreased lung carcinoma cell density on select polymer nanometer surface features for lung replacement therapies

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    Lijuan Zhang

    2010-04-01

    Full Text Available Lijuan Zhang1, Young Wook Chun2, Thomas J Webster21Department of Chemistry and 2Division of Engineering, Brown University, Providence, RI USAAbstract: Poly(lactic-co-glycolic acid (PLGA has been widely used as a biomaterial in regenerative medicine because of its biocompatibility and biodegradability properties. Previous studies have shown that cells (such as bladder smooth muscle cells, chondrocytes, and osteoblasts respond differently to nanostructured PLGA surfaces compared with nanosmooth surfaces. The purpose of the present in vitro research was to prepare PLGA films with various nanometer surface features and determine whether lung cancer epithelial cells respond differently to such topographies. To create nanosurface features on PLGA, different sized (190 nm, 300 nm, 400 nm, and 530 nm diameter polystyrene beads were used to cast polydimethylsiloxane (PDMS molds which were used as templates to create nanofeatured PLGA films. Atomic force microscopy (AFM images and root mean square roughness (RMS values indicated that the intended spherical surface nanotopographies on PLGA with RMS values of 2.23, 5.03, 5.42, and 36.90 nm were formed by employing 190, 300, 400, and 530 nm beads. A solution evaporation method was also utilized to modify PLGA surface features by using 8 wt% (to obtain an AFM RMS value of 0.62 nm and 4 wt% (to obtain an AFM RMS value of 2.23 nm PLGA in chloroform solutions. Most importantly, lung cancer epithelial cells adhered less on the PLGA surfaces with RMS values of 0.62, 2.23, and 5.42 nm after four hours of culture compared with any other PLGA surface created here. After three days, PLGA surfaces with an RMS value of 0.62 nm had much lower cell density than any other sample. In this manner, PLGA with specific nanometer surface features may inhibit lung cancer cell density which may provide an important biomaterial for the treatment of lung cancer (from drug delivery to regenerative medicine.Keywords: nanotechnology

  12. Sox10 directs neural stem cells toward the oligodendrocyte lineage by decreasing Suppressor of Fused expression

    Science.gov (United States)

    Pozniak, Christine D.; Langseth, Abraham J.; Dijkgraaf, Gerrit J. P.; Choe, Youngshik; Werb, Zena; Pleasure, Samuel J.

    2010-01-01

    Oligodendrocyte precursor cells (OPCs) are lineage-restricted progenitors generally limited in vivo to producing oligodendrocytes. Mechanisms controlling genesis of OPCs are of interest because of their importance in myelin development and their potential for regenerative therapies in multiple sclerosis and dysmyelinating syndromes. We show here that the SoxE transcription factors (comprising Sox8, 9, and 10) induce multipotent neural precursor cells (NPCs) from the early postnatal subventricular zone (SVZ) to become OPCs in an autonomous manner. We performed a chromatin immunoprecipitation-based bioinformatic screen and identified Suppressor of Fused (Sufu) as a direct target of repression by Sox10. In vitro, overexpression of Sufu blocked OPC production, whereas RNAi-mediated inhibition augmented OPC production. Furthermore, mice heterozygous for Sufu have increased numbers of OPCs in the telencephalon during development. We conclude that Sox10 acts to restrict the potential of NPCs toward the oligodendrocyte lineage in part by regulating the expression of Sufu. PMID:21098272

  13. Gomisin N Decreases Inflammatory Cytokine Production in Human Periodontal Ligament Cells.

    Science.gov (United States)

    Hosokawa, Yoshitaka; Hosokawa, Ikuko; Shindo, Satoru; Ozaki, Kazumi; Matsuo, Takashi

    2017-04-01

    Gomisin N, which is a lignan isolated from Schisandra chinensis, has some pharmacological effects. However, the anti-inflammatory effects of gomisin N on periodontal disease are uncertain. The aim of this study was to examine the effect of gomisin N on inflammatory mediator production in tumor necrosis factor (TNF)-α-stimulated human periodontal ligament cells (HPDLC). Gomisin N inhibited interleukin (IL)-6, IL-8, CC chemokine ligand (CCL) 2, and CCL20 production in TNF-α-stimulated HPDLC in a dose-dependent manner. Moreover, we revealed that gomisin N could suppress extracellular signal-regulated kinase (ERK) and c-Jun N terminal kinase (JNK) phosphorylation in TNF-α-stimulated HPDLC though protein kinase B (Akt) phosphorylation was not suppressed by gomisin N treatment. In summary, gomisin N might exert anti-inflammatory effects by attenuating cytokine production in periodontal ligament cells via inhibiting the TNF-α-stimulated ERK and JNK pathways activation.

  14. Rapamycin decreases DNA damage accumulation and enhances cell growth of WRN-deficient human fibroblasts.

    Science.gov (United States)

    Saha, Bidisha; Cypro, Alexander; Martin, George M; Oshima, Junko

    2014-06-01

    Werner syndrome (WS), caused by mutations at the WRN helicase gene, is a progeroid syndrome characterized by multiple features consistent with accelerated aging. Aberrant double-strand DNA damage repair leads to genomic instability and reduced replicative lifespan of somatic cells. We observed increased autophagy in WRN knockdown cells; this was further increased by short-term rapamycin treatment. Long-term rapamycin treatment resulted in improved growth rate, reduced accumulation of DNA damage foci and improved nuclear morphology; autophagy markers were reduced to near-normal levels, possibly due to clearance of damaged proteins. These data suggest that protein aggregation plays a role in the development of WS phenotypes and that the mammalian target of rapamycin complex 1 pathway is a potential therapeutic target of WS.

  15. Axonal outgrowth is associated with increased ERK 1/2 activation but decreased caspase 3 linked cell death in Schwann cells after immediate nerve repair in rats

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    Kanje Martin

    2011-01-01

    Full Text Available Abstract Background Extracellular-signal regulated kinase (ERK1/2 is activated by nerve damage and its activation precedes survival and proliferation of Schwann cells. In contrast, activation of caspase 3, a cysteine protease, is considered as a marker for apoptosis in Schwann cells. In the present study, axonal outgrowth, activation of ERK1/2 by phosphorylation (p-ERK 1/2 and immunoreactivity of cleaved caspase 3 were examined after immediate, delayed, or no repair of transected rat sciatic nerves. Results Axonal outgrowth, detected by neurofilament staining, was longer after immediate repair than after either the delayed or no repair conditions. Immediate repair also showed a higher expression of p-ERK 1/2 and a lower number of cleaved caspase 3 stained Schwann cells than after delayed nerve repair. If the transected nerve was not repaired a lower level of p-ERK 1/2 was found than in either the immediate or delayed repair conditions. Axonal outgrowth correlated to p-ERK 1/2, but not clearly with cleaved caspase 3. Contact with regenerating axons affected Schwann cells with respect to p-ERK 1/2 and cleaved caspase 3 after immediate nerve repair only. Conclusion The decreased regenerative capacity that has historically been observed after delayed nerve repair may be related to impaired activation of Schwann cells and increased Schwann cell death. Outgrowing axons influence ERK 1/2 activation and apoptosis of Schwann cells.

  16. The foetal distress decreases the number of stem cells in umbilical cord blood

    OpenAIRE

    Pafumi, Carlo; PALUMBO, M A; LEANZA, V; TEODORO, M C; COCO, L; RISOLETI, E VI; VIZZINI S; Belvedere, G.; ZARBO, G

    2010-01-01

    The authors evaluated the blood volume of foetal blood remaining in the placenta after giving birth with the foetal distress and after a physiological delivery While the amount of blood collected did non differ between groups, the number of CD34 cells was grater in the physiological may be the foetal distress during labour leads to a shift of blood from the placenta to the foetal circulation compartment.

  17. ST6GALNAC5 Expression Decreases the Interactions between Breast Cancer Cells and the Human Blood-Brain Barrier

    Science.gov (United States)

    Drolez, Aurore; Vandenhaute, Elodie; Delannoy, Clément Philippe; Dewald, Justine Hélène; Gosselet, Fabien; Cecchelli, Romeo; Julien, Sylvain; Dehouck, Marie-Pierre; Delannoy, Philippe; Mysiorek, Caroline

    2016-01-01

    The ST6GALNAC5 gene that encodes an α2,6-sialyltransferase involved in the biosynthesis of α-series gangliosides, was previously identified as one of the genes that mediate breast cancer metastasis to the brain. We have shown that the expression of ST6GALNAC5 in MDA-MB-231 breast cancer cells resulted in the expression of GD1α ganglioside at the cell surface. By using a human blood-brain barrier in vitro model recently developed, consisting in CD34+ derived endothelial cells co-cultivated with pericytes, we show that ST6GALNAC5 expression decreased the interactions between the breast cancer cells and the human blood-brain barrier. PMID:27529215

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

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    Huang Wen-bin

    2009-09-01

    Full Text Available Abstract Background 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. Methods 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. Results 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. Conclusion 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.

  19. Telomerase inhibition decreases alpha-fetoprotein expression and secretion by hepatocellular carcinoma cell lines: in vitro and in vivo study.

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    Roula Tahtouh

    Full Text Available Alpha-fetoprotein (AFP is a diagnostic marker for hepatocellular carcinoma (HCC. A direct relationship between poor prognosis and the concentration of serum AFP has been observed. Telomerase, an enzyme that stabilizes the telomere length, is expressed by 90% of HCC. The aim of this study was to investigate the effect of telomerase inhibition on AFP secretion and the involvement of the PI3K/Akt/mTOR signaling pathway. Proliferation and viability tests were performed using tetrazolium salt. Apoptosis was determined through the Annexin V assay using flow cytometry. The concentrations of AFP were measured using ELISA kits. The AFP mRNA expression was evaluated using RT-PCR, and cell migration was evaluated using a Boyden chamber assay. The in vivo effect of costunolide on AFP production was tested in NSG mice. Telomerase inhibition by costunolide and BIBR 1532 at 5 and 10 μM decreased AFP mRNA expression and protein secretion by HepG2/C3A cells. The same pattern was obtained with cells treated with hTERT siRNA. This treatment exhibited no apoptotic effect. The AFP mRNA expression and protein secretion by PLC/PRF/5 was decreased after treatment with BIBR1532 at 10 μM. In contrast, no effect was obtained for PLC/PRF/5 cells treated with costunolide at 5 or 10 μM. Inhibition of the PI3K/Akt/mTOR signaling pathway decreased the AFP concentration. In contrast, the MAPK/ERK pathway appeared to not be involved in HepG2/C3A cells, whereas ERK inhibition decreased the AFP concentration in PLC/PRF/5 cells. Modulation of the AFP concentration was also obtained after the inhibition or activation of PKC. Costunolide (30 mg/kg significantly decreased the AFP serum concentration of NSG mice bearing HepG2/C3A cells. Both the inhibition of telomerase and the inhibition of the PI3K/Akt/mTOR signaling pathway decreased the AFP production of HepG2/C3A and PLC/PRF/5 cells, suggesting a relationship between telomerase and AFP expression through the PI3K

  20. Hydrogen sulfide induces hyperpolarization and decreases the exocytosis of secretory granules of rat GH3 pituitary tumor cells.

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    Mustafina, Alsu N; Yakovlev, Aleksey V; Gaifullina, Aisylu Sh; Weiger, Thomas M; Hermann, Anton; Sitdikova, Guzel F

    2015-10-02

    The aim of the present study was to evaluate the effects of hydrogen sulfide (H2S) on the membrane potential, action potential discharge and exocytosis of secretory granules in neurosecretory pituitary tumor cells (GH3). The H2S donor - sodium hydrosulfide (NaHS) induced membrane hyperpolarization, followed by truncation of spontaneous electrical activity and decrease of the membrane resistance. The NaHS effect was dose-dependent with an EC50 of 152 μM (equals effective H2S of 16-19 μM). NaHS effects were not altered after inhibition of maxi conductance calcium-activated potassium (BK) channels by tetraethylammonium or paxilline, but were significantly reduced after inhibition or activation of ATP-dependent potassium channels (KATP) by glibenclamide or by diazoxide, respectively. In whole-cell recordings NaHS increased the amplitude of KATP currents, induced by hyperpolarizing pulses and subsequent application of glibenclamide decreased currents to control levels. Using the fluorescent dye FM 1-43 exocytosis of secretory granules was analyzed in basal and stimulated conditions (high K(+) external solution). Prior application of NaHS decreased the fluorescence of the cell membrane in both conditions which links with activation of KATP currents (basal secretion) and activation of KATP currents and BK-currents (stimulated exocytosis). We suggest that H2S induces hyperpolarization of GH3 cells by activation of KATP channels which results in a truncation of spontaneous action potentials and a decrease of hormone release.

  1. Autophagy Induced by Areca Nut Extract Contributes to Decreasing Cisplatin Toxicity in Oral Squamous Cell Carcinoma Cells: Roles of Reactive Oxygen Species/AMPK Signaling

    Science.gov (United States)

    Xu, Zhi; Huang, Chun-Ming; Shao, Zhe; Zhao, Xiao-Ping; Wang, Meng; Yan, Ting-Lin; Zhou, Xiao-Cheng; Jiang, Er-Hui; Liu, Ke; Shang, Zheng-Jun

    2017-01-01

    Chewing areca nut is closely associated with oral squamous cell carcinoma (OSCC). The current study aimed to investigate potential associations between areca nut extract (ANE) and cisplatin toxicity in OSCC cells. OSCC cells (Cal-27 and Scc-9) viability and apoptosis were analyzed after treatment with ANE and/or cisplatin. The expressions of proteins associated with autophagy and the AMP-activated protein kinase (AMPK) signaling network were evaluated. We revealed that advanced OSCC patients with areca nut chewing habits presented higher LC3 expression and poorer prognosis. Reactive oxygen species (ROS)-mediated autophagy was induced after pro-longed treatment of ANE (six days, 3 μg). Cisplatin toxicity (IC50, 48 h) was decreased in OSCC cells after ANE treatment (six days, 3 μg). Cisplatin toxicity could be enhanced by reversed autophagy by pretreatment of 3-methyladenine (3-MA), N-acetyl-l-cysteine (NAC), or Compound C. Cleaved-Poly-(ADP-ribose) polymerase (cl-PARP) and cleaved-caspase 3 (cl-caspase 3) were downregulated in ANE-treated OSCC cells in the presence of cisplatin, which was also reversed by NAC and Compound C. Collectively, ANE could decrease cisplatin toxicity of OSCC by inducing autophagy, which involves the ROS and AMPK/mTOR signaling pathway. PMID:28257034

  2. Palm tocotrienols decrease levels of pro-angiogenic markers in human umbilical vein endothelial cells (HUVEC) and murine mammary cancer cells.

    Science.gov (United States)

    Selvaduray, Kanga Rani; Radhakrishnan, Ammu K; Kutty, Methil Kannan; Nesaretnam, Kalanithi

    2012-01-01

    Anti-angiogenic therapy is widely being used to halt tumour angiogenesis. In this study, the anti-angiogenic activity of palm tocotrienol-rich fraction (TRF) and its individual components (γ- and δ-tocotrienol) were first investigated in vitro in human umbilical vein endothelial cells (HUVEC) and 4T1 mouse mammary cancer cells. Results showed reduced levels of Interkeukin (IL)-8 and IL-6, two pro-angiogenic cytokines in HUVEC treated with palm tocotrienols compared with α-tocopherol (α-T) and control cells (P < 0.05). The production of IL-8 and IL-6 was lowest in δ-tocotrienol (δ-T3)-treated cells followed by γ-tocotrienol (γ-T3) and TRF. There was significant (P < 0.05) reduction in IL-8 and vascular endothelial growth factor (VEGF) production in 4T1 cells treated with TRF or δ-T3. There was decreased expression of VEGF and its receptors; VEGF-R1 (fms-like tyrosine kinase, Flt-1) and VEGF-R2 (Kinase-insert-domain-containing receptor, KDR/Flk-2) in tumour tissues excised from mice supplemented with TRF were observed. There was also decreased expression of VEGF-R2 in lung tissues of mice supplemented with TRF. These observations correlate with the smaller tumour size recorded in the tocotrienol-treated mice. This study confirms previous observations that palm tocotrienols exhibit anti-angiogenic properties that may inhibit tumour progression.

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

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    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.

  4. Moderate Hypothermia Significantly Decreases Hippocampal Cell Death Involving Autophagy Pathway after Moderate Traumatic Brain Injury.

    Science.gov (United States)

    Jin, Yichao; Lin, Yingying; Feng, Jun-feng; Jia, Feng; Gao, Guo-yi; Jiang, Ji-yao

    2015-07-15

    Here, we evaluated changes in autophagy after post-traumatic brain injury (TBI) followed by moderate hypothermia in rats. Adult male Sprague-Dawley rats were randomly divided into four groups: sham injury with normothermia group (37 °C); sham injury with hypothermia group (32 °C); TBI with normothermia group (TNG; 37 °C); and TBI with hypothermia group (THG; 32 °C). Injury was induced by a fluid percussion TBI device. Moderate hypothermia (32 °C) was achieved by partial immersion in a water bath (0 °C) under general anesthesia for 4 h. All rats were killed at 24 h after fluid percussion TBI. The ipsilateral hippocampus in all rats was analyzed with hematoxylin and eosin staining; terminal deoxynucleoitidyl transferase-mediated nick end labeling staining was used to determine cell death in ipsilateral hippocampus. Immunohistochemistry and western blotting of microtubule-associated protein light chain 3 (LC3), Beclin-1, as well as transmission electron microscopy performed to assess changes in autophagy. At 24 h after TBI, the cell death index was 27.90 ± 2.36% in TNG and 14.90 ± 1.52% in THG. Expression level of LC3 and Beclin-1 were significantly increased after TBI and were further up-regulated after post-TBI hypothermia. Further, ultrastructural observations showed that there was a marked increase of autophagosomes and autolysosomes in ipsilateral hippocampus after post-TBI hypothermia. Our data demonstrated that moderate hypothermia significantly attenuated cell death and increased autophagy in ipsilateral hippocampus after fluid percussion TBI. In conclusion, autophagy pathway may participate in the neuroprotective effect of post-TBI hypothermia.

  5. Fermented soybeans, Chungkookjang, prevent hippocampal cell death and β-cell apoptosis by decreasing pro-inflammatory cytokines in gerbils with transient artery occlusion.

    Science.gov (United States)

    Park, Sunmin; Kim, Da Sol; Kang, Sunna; Moon, Bo Reum

    2016-02-01

    Since Chungkookjang, a short-term fermented soybean, is known to improve glucose metabolism and antioxidant activity, it may prevent the neurological symptoms and glucose disturbance induced by artery occlusion. We investigated the protective effects and mechanisms of traditional (TFC) and standardized Chungkookjang fermented with Bacillus licheniformis (BLFC) against ischemia/reperfusion damage in the hippocampal CA1 region and against hyperglycemia after transient cerebral ischemia in gerbils. Gerbils were subjected to either an occlusion of the bilateral common carotid arteries for 8 min to render them ischemic or a sham operation. Ischemic gerbils were fed either a 40% fat diet containing 10% of either cooked soybean (CSB), TFC, or BLFC for 28 days. Neuronal cell death and cytokine expression in the hippocampus, neurological deficit, serum cytokine levels, and glucose metabolism were measured. TFC and BLFC contained more isoflavonoid aglycones than CSB. Artery occlusion increased the expressions of IL-1β and TNF-α as well as cell death in the hippocampal CA1 region and induced severe neurological symptoms. CSB, TFC, and BLFC prevented the neuronal cell death and the symptoms such as dropped eyelid, bristling hair, reduced muscle tone and flexor reflex, and abnormal posture and walking patterns, and suppressed cytokine expressions. CSB was less effective than TFC and BLFC. Artery occlusion induced glucose intolerance due to decreased insulin secretion and β-cell mass. TFC and BLFC prevented the impairment of glucose metabolism by artery occlusion. Especially TFC and BLFC increased β-cell proliferation and suppressed the β-cell apoptosis by suppressing TNF-α and IL-1β which in turn decreased cleaved caspase-3 that caused apoptosis. In conclusion, TFC and BLFC may prevent and alleviate neuronal cell death in the hippocampal CA1 region and neurological symptoms and poststroke hyperglycemia in gerbils with artery occlusion. This might be associated with

  6. Chlamydia trachomatis infection results in a modest pro-inflammatory cytokine response and a decrease in T cell chemokine secretion in human polarized endocervical epithelial cells.

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    Buckner, Lyndsey R; Lewis, Maria E; Greene, Sheila J; Foster, Timothy P; Quayle, Alison J

    2013-08-01

    The endocervical epithelium is a major reservoir for Chlamydia trachomatis in women, and genital infections are extended in their duration. Epithelial cells act as mucosal sentinels by secreting cytokines and chemokines in response to pathogen challenge and infection. We therefore determined the signature cytokine and chemokine response of primary-like endocervix-derived epithelial cells in response to a common genital serovar (D) of C. trachomatis. For these studies, we used a recently-established polarized, immortalized, endocervical epithelial cell model (polA2EN) that maintains, in vitro, the architectural and functional characteristics of endocervical epithelial cells in vivo including the production of pro-inflammatory cytokines. PolA2EN cells were susceptible to C. trachomatis infection, and chlamydiae in these cells underwent a normal developmental cycle as determined by a one-step growth curve. IL1α protein levels were increased in both apical and basolateral secretions of C. trachomatis infected polA2EN cells, but this response did not occur until 72h after infection. Furthermore, protein levels of the pro-inflammatory cytokines and chemokines IL6, TNFα and CXCL8 were not significantly different between C. trachomatis infected polA2EN cells and mock infected cells at any time during the chlamydial developmental cycle up to 120h post-infection. Intriguingly, C. trachomatis infection resulted in a significant decrease in the constitutive secretion of T cell chemokines IP10 and RANTES, and this required a productive C. trachomatis infection. Examination of anti-inflammatory cytokines revealed a high constitutive apical secretion of IL1ra from polA2EN cells that was not significantly modulated by C. trachomatis infection. IL-11 was induced by C. trachomatis, although only from the basolateral membrane. These results suggest that C. trachomatis can use evasion strategies to circumvent a robust pro-inflammatory cytokine and chemokine response. These evasion

  7. Neutrophil depletion-but not prevention of Kupffer cell activation-decreases the severity of cerulein-induced acute pancreatitis

    Institute of Scientific and Technical Information of China (English)

    Catherine M Pastor; Alain Vonlaufen; Fabianna Georgi; Antoine Hadengue; Philippe Morel; Jean-Louis Frossard

    2006-01-01

    AIM: To determine whether neutrophil depletion and Kupffer cell inhibition might combine their protective effects to decrease the severity of acute pancreatitis.METHODS: Mice had cerulein administration to induce acute pancreatitis and were pretreated with either anti-mouse neutrophil serum or gadolinium chloride (GdCl3) to prevent Kupffer cell activation, or both treatments. Injury was assessed in pancreas and lungs.Myeloperoxidases (MPO) assessed neutrophil infiltration.Interleukin-6 (IL-6) and IL-10 were measured in serum,pancreas, lungs and liver.RESULTS: In mice with acute pancreatitis, neutrophil depletion reduced the severity of pancreatitis and pancreatitis-associated lung injury. Kupffer cell inactivation by GdCl3 had less protective effect, although IL-6 and IL-10 concentrations were significantly decreased. The protective treatment brought by neutrophil depletion was not enhanced by Kupffer cell inactivation and both treatments did not combine their protective effects.CONCLUSION: Our results confirm the role of activated neutrophils in aggravating organ injury in acute pancreatitis while the role of Kupffer cell activation is less obvious.

  8. Chronic psychological stress suppresses contact hypersensitivity: potential roles of dysregulated cell trafficking and decreased IFN-γ production.

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    Hall, Jessica M F; Witter, Alexandra R; Racine, Ronny R; Berg, Rance E; Podawiltz, Alan; Jones, Harlan; Mummert, Mark E

    2014-02-01

    Increasing evidence shows that psychological stress can have dramatic impacts on the immune system, particularly the cutaneous immune response in dermatological disorders. While there have been many studies examining the impact of acute psychological stress on contact hypersensitivity there are relatively few studies concerning the impact of chronic psychological stress. Furthermore, the local immunological mechanisms by which chronic psychological stress impacts contact hypersensitivity still remain to be explored. Here we show that restraint-induced chronic psychological stress stimulates activation of the hypothalamus-pituitary-adrenal axis and delays weight gain in female BALB/c mice. We observed that chronic psychological stress reduces the cutaneous immune response as evidence by reduced ear swelling. This correlated with a significant decrease in the inflammatory cell infiltrate. On the other hand, chronic psychological stress does not influence T cell proliferation, activation, or sensitivity to corticosterone but does increase CD4(+) and CD8(+) T cell percentages in draining lymph nodes during a contact hypersensitivity reaction. Chronic psychological stress induces a decrease in overall circulating white blood cells, lymphocytes, and monocytes during a contact hypersensitivity reaction suggesting extravasation from the circulation. Finally, we found markedly reduced local IFN-γ production in chronically stressed animals. Based on these findings we propose that chronic psychological stress reduces contact hypersensitivity due to dysregulated cell trafficking and reduced production of IFN-γ.

  9. HDAC inhibitor AR-42 decreases CD44 expression and sensitizes myeloma cells to lenalidomide.

    Science.gov (United States)

    Canella, Alessandro; Cordero Nieves, Hector; Sborov, Douglas W; Cascione, Luciano; Radomska, Hanna S; Smith, Emily; Stiff, Andrew; Consiglio, Jessica; Caserta, Enrico; Rizzotto, Lara; Zanesi, Nicola; Stefano, Volinia; Kaur, Balveen; Mo, Xiaokui; Byrd, John C; Efebera, Yvonne A; Hofmeister, Craig C; Pichiorri, Flavia

    2015-10-13

    Multiple myeloma (MM) is a hematological malignancy of plasma cells in the bone marrow. Despite multiple treatment options, MM is inevitably associated with drug resistance and poor outcomes. Histone deacetylase inhibitors (HDACi's) are promising novel chemotherapeutics undergoing evaluation in clinical trials for the potential treatment of patients with MM. Although in preclinical studies HDACi's have proven anti-myeloma activity, but in the clinic single-agent HDACi treatments have been limited due to low tolerability. Improved clinical outcomes were reported only when HDACi's were combined with other drugs. Here, we show that a novel pan-HDACi AR-42 downregulates CD44, a glycoprotein that has been associated with lenalidomide and dexamethasone resistance in myeloma both in vitro and in vivo. We also show that this CD44 downregulation is in part mediated by miR-9-5p, targeting insulin-like growth factor 2 mRNA binding protein 3 (IGF2BP3), which directly binds to CD44 mRNA and increases its stability. Importantly, we also demonstrate that AR-42 enhances anti-myeloma activity of lenalidomide in primary MM cells isolated from lenalidomide resistant patients and in in vivo MM mouse model. Thus, our findings shed light on potential novel combinatorial therapeutic approaches modulating CD44 expression, which may help overcome lenalidomide resistance in myeloma patients.

  10. Baicalein Decreases Hydrogen Peroxide-Induced Damage to NG108-15 Cells via Upregulation of Nrf2.

    Science.gov (United States)

    Yeh, Chao-Hung; Ma, Kuo-Hsing; Liu, Pei-Shan; Kuo, Jung-Kuei; Chueh, Sheau-Huei

    2015-08-01

    Baicalein is a flavonoid inhibitor of 12-lipoxygenase. Here, we investigated its effect on hydrogen peroxide-induced damage to NG108-15 cells. Hydrogen peroxide activated the mitochondrial apoptotic pathway, decreased Nrf2 expression, increased reactive oxygen species (ROS) levels, reduced viability, and increased cell death after 2-24 h treatment of NG108-15 cells. Co-treatment with hydrogen peroxide and baicalein completely suppressed the activation of mitochondrial apoptotic pathway by upregulating Nrf2 expression and reducing ROS stress and partially inhibited the effects on cell viability and cell death. Silencing of 12-lipoxygenase had a similar protective effect to baicalein on hydrogen peroxide-induced damage by blocking the hydrogen peroxide-induced decrease in Nrf2 expression and increase in ROS levels. Neither protective effect was altered by addition of 12-hydroxyeicosatetraenoic acid, the product of 12-lipoxygenase, suggesting that hydrogen peroxide induced damage via 12-lipoxygenase by another, as yet unknown, mechanism, rather than activating it. Co-treatment of cells with hydrogen peroxide and N-acetylcysteine or the Nrf2 inducer sulforaphane reduced hydrogen peroxide-induced damage in a similar fashion to baicalein, while the Nrf2 inhibitor retinoic acid blocked the protective effect of baicalein. Silencing Nrf2 also inhibited the protective effects of baicalein, sulforaphane, and N-acetylcysteine and resulted in high ROS levels, suggesting ROS elimination was mediated by Nrf2. Taken together our results suggest that baicalein protects cells from hydrogen peroxide-induced activation of the mitochondrial apoptotic pathway by upregulating Nrf2 and inhibiting 12-lipoxygenase to block the increase in ROS levels. Hydrogen peroxide also activates a second mitochondrial dysfunction independent death pathway which is resistant to baicalein.

  11. Decreased NK Cell FcRgamma in HIV-1 infected individuals receiving combination antiretroviral therapy: a cross sectional study.

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    Edwin Leeansyah

    Full Text Available BACKGROUND: FcRgamma is an immunoreceptor tyrosine-based activation motif (ITAM-signalling protein essential for immunoreceptor signaling and monocyte, macrophage and NK cell function. Previous study from our laboratory showed that FcRgamma is down-regulated in HIV-infected macrophages in vitro. FcRgamma expression in immune cells present in HIV-infected individuals is unknown. METHODOLOGY/PRINCIPAL FINDINGS: We compared FcRgamma expression in peripheral blood mononuclear cells isolated from HIV-1-infected individuals receiving combination antiretroviral therapy and healthy, HIV-1-uninfected individuals. FcRgamma mRNA and protein levels were measured using quantitative real-time PCR and immunoblotting, respectively. CD56(+ CD94(+ lymphocytes isolated from blood of HIV-1 infected individuals had reduced FcRgamma protein expression compared to HIV-uninfected individuals (decrease = 76.8%, n = 18 and n = 12 respectively, p = 0.0036. In a second group of patients, highly purified NK cells had reduced FcRgamma protein expression compared to uninfected controls (decrease = 50.2%, n = 9 and n = 8 respectively, p = 0.021. Decreased FcRgamma expression in CD56+CD94+ lymphocytes was associated with reduced mRNA (51.7%, p = 0.021 but this was not observed for the smaller group of patients analysed for NK cell expression (p = 0.36. CONCLUSION/SIGNIFICANCE: These data suggest biochemical defects in ITAM-dependent signalling within NK cells in HIV-infected individuals which is present in the context of treatment with combination antiretroviral therapy.

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

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    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.

  13. mTOR inhibition decreases SOX2-SOX9 mediated glioma stem cell activity and temozolomide resistance

    Science.gov (United States)

    Garros-Regulez, Laura; Aldaz, Paula; Arrizabalaga, Olatz; Moncho-Amor, Veronica; Carrasco-Garcia, Estefania; Manterola, Lorea; Moreno-Cugnon, Leire; Barrena, Cristina; Villanua, Jorge; Ruiz, Irune; Pollard, Steven; Lovell-Badge, Robin; Sampron, Nicolas; Garcia, Idoia; Matheu, Ander

    2016-01-01

    ABSTRACT Background: SOX2 and SOX9 are commonly overexpressed in glioblastoma, and regulate the activity of glioma stem cells (GSCs). Their specific and overlapping roles in GSCs and glioma treatment remain unclear. Methods: SOX2 and SOX9 levels were examined in human biopsies. Gain and loss of function determined the impact of altering SOX2 and SOX9 on cell proliferation, senescence, stem cell activity, tumorigenesis and chemoresistance. Results: SOX2 and SOX9 expression correlates positively in glioma cells and glioblastoma biopsies. High levels of SOX2 bypass cellular senescence and promote resistance to temozolomide. Mechanistic investigations revealed that SOX2 acts upstream of SOX9. mTOR genetic and pharmacologic (rapamycin) inhibition decreased SOX2 and SOX9 expression, and reversed chemoresistance. Conclusions: Our findings reveal SOX2-SOX9 as an oncogenic axis that regulates stem cell properties and chemoresistance. We identify that rapamycin abrogate SOX protein expression and provide evidence that a combination of rapamycin and temozolomide inhibits tumor growth in cells with high SOX2/SOX9. PMID:26878385

  14. Deregulation of apoptotic volume decrease and ionic movements in multidrug-resistant tumor cells: role of chloride channels

    DEFF Research Database (Denmark)

    Poulsen, Kristian Arild; Andersen, E C; Hansen, C F;

    2010-01-01

    Changes in cell volume and ion gradients across the plasma membrane play a pivotal role in the initiation of apoptosis. Here we explore the kinetics of apoptotic volume decrease (AVD) and ion content dynamics in wild-type (WT) and multidrug-resistant (MDR) Ehrlich ascites tumor cells (EATC). In WT......3728 inhibited AVD and completely abolished the differences in AVD, ionic movements, and caspase 3 activation between WT and MDR EATC. Finally, the maximal capacity of volume-regulated anion channel was found to be strongly repressed in MDR EATC. Together, these data suggest that impairment of AVD...

  15. 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.

  16. Tolerogenic Dendritic Cells from Poorly Compensated Type 1 Diabetes Patients Have Decreased Ability To Induce Stable Antigen-Specific T Cell Hyporesponsiveness and Generation of Suppressive Regulatory T Cells

    DEFF Research Database (Denmark)

    Dáňová, Klára; Grohová, Anna; Strnadová, Pavla

    2017-01-01

    -loaded tolDCs from well-controlled patients decreased significantly primary Th1/Th17 responses, induced stable GAD65-specific T cell hyporesponsiveness, and suppressed markedly control DC-induced GAD65-specific T cell activation compared with poorly controlled patients. The ability of tolDCs from poorly...

  17. Monosodium Glutamate Dietary Consumption Decreases Pancreatic β-Cell Mass in Adult Wistar Rats.

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    Piyanard Boonnate

    Full Text Available The amount of dietary monosodium glutamate (MSG is increasing worldwide, in parallel with the epidemics of metabolic syndrome. Parenteral administration of MSG to rodents induces obesity, hyperglycemia, hyperlipidemia, insulin resistance, and type 2 diabetes. However, the impact of dietary MSG is still being debated. We investigated the morphological and functional effects of prolonged MSG consumption on rat glucose metabolism and on pancreatic islet histology.Eighty adult male Wistar rats were randomly subdivided into 4 groups, and test rats in each group were supplemented with MSG for a different duration (1, 3, 6, or 9 months, n=20 for each group. All rats were fed ad libitum with a standard rat chow and water. Ten test rats in each group were provided MSG 2 mg/g body weight/day in drinking water and the 10 remaining rats in each group served as non-MSG treated controls. Oral glucose tolerance tests (OGTT were performed and serum insulin measured at 9 months. Animals were sacrificed at 1, 3, 6, or 9 months to examine the histopathology of pancreatic islets.MSG-treated rats had significantly lower pancreatic β-cell mass at 1, 6 and 9 months of study. Islet hemorrhages increased with age in all groups and fibrosis was significantly more frequent in MSG-treated rats at 1 and 3 months. Serum insulin levels and glucose tolerance in MSG-treated and untreated rats were similar at all time points we investigated.Daily MSG dietary consumption was associated with reduced pancreatic β-cell mass and enhanced hemorrhages and fibrosis, but did not affect glucose homeostasis. We speculate that high dietary MSG intake may exert a negative effect on the pancreas and such effect might become functionally significant in the presence or susceptibility to diabetes or NaCl; future experiments will take these crucial cofactors into account.

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

    Institute of Scientific and Technical Information of China (English)

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

    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.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

  19. Luteolin decreases the attachment, invasion and cytotoxicity of UPEC in bladder epithelial cells and inhibits UPEC biofilm formation.

    Science.gov (United States)

    Shen, Xiao-fei; Ren, Lai-bin; Teng, Yan; Zheng, Shuang; Yang, Xiao-long; Guo, Xiao-juan; Wang, Xin-yuan; Sha, Kai-hui; Li, Na; Xu, Guang-ya; Tian, Han-wen; Wang, Xiao-ying; Liu, Xiao-kang; Li, Jingyu; Huang, Ning

    2014-10-01

    Urinary tract infection (UTI), primarily caused by uropathogenic Escherichia coli (UPEC), is one of the most common infectious diseases worldwide. Emerging antibiotic resistance requires novel treatment strategies. Luteolin, a dietary polyphenolic flavonoid, has been confirmed as a potential antimicrobial agent. Here, we evaluated the sub-MICs of luteolin for potential properties to modulate the UPEC infection. We found that luteolin significantly decreased the attachment and invasion of UPEC J96 or CFT073 in human bladder epithelial cell lines T24. Meanwhile, obvious decreased expression of type 1 fimbriae adhesin fimH gene, lower bacterial surface hydrophobicity and swimming motility, were observed in luteolin-pretreated UPEC. Furthermore, luteolin could attenuate UPEC-induced cytotoxicity in T24 cells, which manifested as decreased activity of lactate dehydrogenase (LDH). Simultaneously, the inhibition of luteolin on UPEC-induced cytotoxicity was confirmed by ethidium bromide/acridine orange staining. Finally, the luteolin-pretreated UPEC showed a lower ability of biofilm formation. Collectively, these results indicated that luteolin decreased the attachment and invasion of UPEC in bladder epithelial cells, attenuated UPEC-induced cytotoxicity and biofilm formation via down-regulating the expression of adhesin fimH gene, reducing the bacterial surface hydrophobicity and motility.

  20. The telomerase inhibitor imetelstat alone, and in combination with trastuzumab, decreases the cancer stem cell population and self-renewal of HER2+ breast cancer cells.

    Science.gov (United States)

    Koziel, Jillian E; Herbert, Brittney-Shea

    2015-02-01

    Cancer stem cells (CSCs) are thought to be responsible for tumor progression, metastasis, and recurrence. HER2 overexpression is associated with increased CSCs, which may explain the aggressive phenotype and increased likelihood of recurrence for HER2(+) breast cancers. Telomerase is reactivated in tumor cells, including CSCs, but has limited activity in normal tissues, providing potential for telomerase inhibition in anti-cancer therapy. The purpose of this study was to investigate the effects of a telomerase antagonistic oligonucleotide, imetelstat (GRN163L), on CSC and non-CSC populations of HER2(+) breast cancer cell lines. The effects of imetelstat on CSC populations of HER2(+) breast cancer cells were measured by ALDH activity and CD44/24 expression by flow cytometry as well as mammosphere assays for functionality. Combination studies in vitro and in vivo were utilized to test for synergism between imetelstat and trastuzumab. Imetelstat inhibited telomerase activity in both subpopulations. Moreover, imetelstat alone and in combination with trastuzumab reduced the CSC fraction and inhibited CSC functional ability, as shown by decreased mammosphere counts and invasive potential. Tumor growth rate was slower in combination-treated mice compared to either drug alone. Additionally, there was a trend toward decreased CSC marker expression in imetelstat-treated xenograft cells compared to vehicle control. Furthermore, the observed decrease in CSC marker expression occurred prior to and after telomere shortening, suggesting that imetelstat acts on the CSC subpopulation in telomere length-dependent and -independent mechanisms. Our study suggests addition of imetelstat to trastuzumab may enhance the effects of HER2 inhibition therapy, especially in the CSC population.

  1. Fasting serum levels of ferritin are associated with impaired pancreatic beta cell function and decreased insulin sensitivity

    DEFF Research Database (Denmark)

    Bonfils, Linéa; Ellervik, Christina; Friedrich, Nele

    2015-01-01

    Aims/hypothesis: Elevated serum ferritin levels are associated with an increased risk of type 2 diabetes, but the nature of this association remains elusive. The aim of this study was to test the hypothesis that an elevated fasting serum ferritin level is associated with an increased risk of type 2...... diabetes due to its association with impaired beta cell function and decreased insulin sensitivity. Methods: We investigated 6,392 individuals from the Danish general population. Surrogate measures of beta cell function and insulin sensitivity were calculated for approximately 6,100 individuals based...... glucose levels at 0, 30 and 120 min (p beta cell function estimated as the insulinogenic index and corrected insulin response (p 

  2. Sensitivity to PRIMA-1MET is associated with decreased MGMT in human glioblastoma cells and glioblastoma stem cells irrespective of p53 status.

    Science.gov (United States)

    Patyka, Mariia; Sharifi, Zeinab; Petrecca, Kevin; Mansure, Jose; Jean-Claude, Bertrand; Sabri, Siham

    2016-09-13

    Alterations of the TP53 tumor suppressor gene occur in ~30% of primary glioblastoma (GBM) with a high frequency of missense mutations associated with the acquisition of oncogenic "gain-of-function" (GOF) mutant (mut)p53 activities. PRIMA-1MET/APR-246, emerged as a promising compound to rescue wild-type (wt)p53 function in different cancer types. Previous studies suggested the role of wtp53 in the negative regulation of the DNA repair protein O6-methylguanine-DNA methyltransferase (MGMT), a major determinant in resistance to therapy in GBM treatment. The potential role of MGMT in expression of p53 and the efficacy of PRIMA-1MET with respect to TP53 status and expression of MGMT in GBM remain unknown. We investigated response to PRIMA-1MET of wtp53/MGMT-negative (U87MG, A172), mutp53/MGMT-positive U138, LN-18, T98/Empty vector (T98/EV) and its isogenic MGMT/shRNA gene knockdown counterpart (T98/shRNA). We show that MGMT silencing decreased expression of mutp53/GOF in T98/shRNA. PRIMA-1MET further cleared T98/shRNA cells of mutp53, decreased proliferation and clonogenic potential, abrogated the G2 checkpoint control, increased susceptibility to apoptotic cell death, expression of GADD45A and sustained expression of phosphorylated Erk1/2. PRIMA-1MET increased expression of p21 protein in U87MG and A172 and promoted senescence in U87MG cell line. Importantly, PRIMA-1MET decreased relative cell numbers, disrupted the structure of neurospheres of patient-derived GBM stem cells (GSCs) and enabled activation of wtp53 with decreased expression of MGMT in MGMT-positive GSCs or decreased expression of mutp53. Our findings highlight the cell-context dependent effects of PRIMA-1MET irrespective of p53 status and suggest the role of MGMT as a potential molecular target of PRIMA-1MET in MGMT-positive GSCs.

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

    Science.gov (United States)

    Herculano-Houzel, Suzana; Messeder, Débora J; Fonseca-Azevedo, Karina; Pantoja, Nilma A

    2015-01-01

    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.

  4. Cystine accumulation attenuates insulin release from the pancreatic β-cell due to elevated oxidative stress and decreased ATP levels.

    Science.gov (United States)

    McEvoy, Bernadette; Sumayao, Rodolfo; Slattery, Craig; McMorrow, Tara; Newsholme, Philip

    2015-12-01

    The pancreatic β-cell has reduced antioxidant defences making it more susceptible to oxidative stress. In cystinosis, a lysosomal storage disorder, an altered redox state may contribute to cellular dysfunction. This rare disease is caused by an abnormal lysosomal cystine transporter, cystinosin, which causes excessive accumulation of cystine in the lysosome. Cystinosis associated kidney damage and dysfunction leads to the Fanconi syndrome and ultimately end-stage renal disease. Following kidney transplant, cystine accumulation in other organs including the pancreas leads to multi-organ dysfunction. In this study, a Ctns gene knockdown model of cystinosis was developed in the BRIN-BD11 rat clonal pancreatic β-cell line using Ctns-targeting siRNA. Additionally there was reduced cystinosin expression, while cell cystine levels were similarly elevated to the cystinotic state. Decreased levels of chronic (24 h) and acute (20 min) nutrient-stimulated insulin secretion were observed. This decrease may be due to depressed ATP generation particularly from glycolysis. Increased ATP production and the ATP/ADP ratio are essential for insulin secretion. Oxidised glutathione levels were augmented, resulting in a lower [glutathione/oxidised glutathione] redox potential. Additionally, the mitochondrial membrane potential was reduced, apoptosis levels were elevated, as were markers of oxidative stress, including reactive oxygen species, superoxide and hydrogen peroxide. Furthermore, the basal and activated phosphorylated forms of the redox-sensitive transcription factor NF-κB were increased in cells with silenced Ctns. From this study, the cystinotic-like pancreatic β-cell model demonstrated that the altered oxidative status of the cell, resulted in depressed mitochondrial function and pathways of ATP production, causing reduced nutrient-stimulated insulin secretion.

  5. 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.

  6. High glucose decreases the expression of ATP-binding cassette transporter G1 in human vascular smooth muscle cells

    Institute of Scientific and Technical Information of China (English)

    Jiahong Xue; Zuyi Yuan; Yue Wu; Yan Zhao; Zhaofei Wan

    2008-01-01

    Objective:ATP-binding cassette transporters(ABC) A1 and G1 play an important role in mediating cholesterol efflux and preventing macrophage foam cell formation. In this study, we examined the regulation of ABC transporters by high glucose in human vascular smooth muscle cells(VSMCs), the other precursor of foam cells. Methods:Incubation of human VSMCs with D-ghicose(5 to 30 mM) for 1 to 7 days in the presence or absence of antioxidant and nuclear factor(NF)-kB inhibitors, the expressions of ABCA1 and ABCG1 were analyzed by real time PCR and Western blotting. Results:High glucose decreased ABCG1 mRNA and protein expression in cultured VSMCs, whereas the expression of ABCA1 was not significantly decreased. Down-regulation of ABCG1 mRNA expression by high glucose was abolished by antioxidant N-acetyl-L-cysteine(NAC) and NF-kB inhibitors, BAY 11-7085 and tosyl-phenylalanine chloromethyl-ketone(TPCK). Conclusion:High glucose suppresses the expression of ABCG1 in VSMCs, which is the possible mechanism of VSMC derived foam cell transformation.

  7. cDNA cloning of human myeloperoxidase: decrease in myeloperoxidase mRNA upon induction of HL-60 cells

    Energy Technology Data Exchange (ETDEWEB)

    Weil, S.C.; Rosner, G.L.; Reid, M.S.; Chisholm, R.L.; Farber, N.M.; Spitznagel, J.K.; Swanson, M.S.

    1987-04-01

    Myeloperoxidase (MPO), the most abundant neutrophil protein, is a bacteriocidal component of the primary granules and a critical marker in distinguishing acute myelogenous leukemia from acute lymphoid leukemia. A cDNA clone for human MPO was isolated by immunologic screening of human hematopoietic lambdagt11 expression vector libraries with specific anti-MPO antibody. The identity of the cDNA clone was confirmed by finding that (i) epitope-selected antibody against this clone recognizes purified MPO and MPO in human promyelocytic (HL-60) cell lysates by immunoblot analysis, and that (ii) hybrid section of HL-60 mRNA with this cDNA clone and translation in vitro results in the synthesis of an 80-kDa protein recognized by the anti-MPO antiserum. RNA blot analysis with this MPO cDNA clone detects hybridization to two polyadenylylated transcripts of approx. = 3.6 and approx. = 2.9 kilobases in HL-60 cells. No hybridization is detected to human placenta mRNA. Upon induction of HL-60 cells to differentiate by incubation for 4 days with dimethyl sulfoxide, a drastic decrease in the hybridization intensity of these two bands is seen. This is consistent with previous data suggesting a decrease in MPO synthesis upon such induction of these cells. The MPO cDNA should be useful for further molecular and genetic characterization of MPO and its expression and biosynthesis in normal and leukemic granulocytic differentiation.

  8. Human intestinal dendritic cells decrease cytokine release against Salmonella infection in the presence of Lactobacillus paracasei upon TLR activation.

    Directory of Open Access Journals (Sweden)

    Miriam Bermudez-Brito

    Full Text Available Probiotic bacteria have been shown to modulate immune responses and could have therapeutic effects in allergic and inflammatory disorders. However, little is known about the signalling pathways that are engaged by probiotics. Dendritic cells (DCs are antigen-presenting cells that are involved in immunity and tolerance. Monocyte-derived dendritic cells (MoDCs and murine DCs are different from human gut DCs; therefore, in this study, we used human DCs generated from CD34+ progenitor cells (hematopoietic stem cells harvested from umbilical cord blood; those DCs exhibited surface antigens of dendritic Langerhans cells, similar to the lamina propria DCs in the gut. We report that both a novel probiotic strain isolated from faeces of exclusively breast-fed newborn infants, Lactobacillus paracasei CNCM I-4034, and its cell-free culture supernatant (CFS decreased pro-inflammatory cytokines and chemokines in human intestinal DCs challenged with Salmonella. Interestingly, the supernatant was as effective as the bacteria in reducing pro-inflammatory cytokine expression. In contrast, the bacterium was a potent inducer of TGF-β2 secretion, whereas the supernatant increased the secretion of TGF-β1 in response to Salmonella. We also showed that both the bacteria and its supernatant enhanced innate immunity through the activation of Toll-like receptor (TLR signalling. These treatments strongly induced the transcription of the TLR9 gene. In addition, upregulation of the CASP8 and TOLLIP genes was observed. This work demonstrates that L. paracasei CNCM I-4034 enhanced innate immune responses, as evidenced by the activation of TLR signalling and the downregulation of a broad array of pro-inflammatory cytokines. The use of supernatants like the one described in this paper could be an effective and safe alternative to using live bacteria in functional foods.

  9. Human placenta-derived stromal cells decrease inflammation, placental injury and blood pressure in hypertensive pregnant mice.

    Science.gov (United States)

    Chatterjee, Piyali; Chiasson, Valorie L; Pinzur, Lena; Raveh, Shani; Abraham, Eytan; Jones, Kathleen A; Bounds, Kelsey R; Ofir, Racheli; Flaishon, Liat; Chajut, Ayelet; Mitchell, Brett M

    2016-04-01

    Pre-eclampsia, the development of hypertension and proteinuria or end-organ damage during pregnancy, is a leading cause of both maternal and fetal morbidity and mortality, and there are no effective clinical treatments for pre-eclampsia aside from delivery. The development of pre-eclampsia is characterized by maladaptation of the maternal immune system, excessive inflammation and endothelial dysfunction. We have reported that detection of extracellular RNA by the Toll-like receptors (TLRs) 3 and 7 is a key initiating signal that contributes to the development of pre-eclampsia. PLacental eXpanded (PLX-PAD) cells are human placenta-derived, mesenchymal-like, adherent stromal cells that have anti-inflammatory, proangiogenic, cytoprotective and regenerative properties, secondary to paracrine secretion of various molecules in response to environmental stimulation. We hypothesized that PLX-PAD cells would reduce the associated inflammation and tissue damage and lower blood pressure in mice with pre-eclampsia induced by TLR3 or TLR7 activation. Injection of PLX-PAD cells on gestational day 14 significantly decreased systolic blood pressure by day 17 in TLR3-induced and TLR7-induced hypertensive mice (TLR3 144-111 mmHg; TLR7 145-106 mmHg; both Pinflammation and placental injury, increased markedly in both groups of TLR-induced hypertensive mice, were reduced by PLX-PAD cells. Importantly, PLX-PAD cell therapy had no effects on these measures in pregnant control mice or on the fetuses. These data demonstrate that PLX-PAD cell therapy can safely reverse pre-eclampsia-like features during pregnancy and have a potential therapeutic role in pre-eclampsia treatment.

  10. Paradoxical growth of Candida albicans in the presence of caspofungin is associated with multiple cell wall rearrangements and decreased virulence.

    Science.gov (United States)

    Rueda, Cristina; Cuenca-Estrella, Manuel; Zaragoza, Oscar

    2014-01-01

    In the last decade, echinocandins have emerged as an important family of antifungal drugs because of their fungicidal activity against Candida spp. Echinocandins inhibit the enzyme β-1,3-d-glucan synthase, encoded by the FKS genes, and resistance to echinocandins is associated with mutations in this gene. In addition, echinocandin exposure can produce paradoxical growth, defined as the ability to grow at high antifungal concentrations but not at intermediate concentrations. In this work, we have demonstrated that paradoxical growth of Candida albicans in the presence of caspofungin is not due to antifungal degradation or instability. Media with high caspofungin concentrations recovered from wells where C. albicans showed paradoxical growth inhibited the growth of a Candida krusei reference strain. Cells exhibiting paradoxical growth at high caspofungin concentrations showed morphological changes such as enlarged size, abnormal septa, and absence of filamentation. Chitin content increased from the MIC to high caspofungin concentrations. Despite the high chitin levels, around 23% of cells died after treatment with caspofungin, indicating that chitin is required but not sufficient to protect the cells from the fungicidal effect of caspofungin. Moreover, we found that after paradoxical growth, β-1,3-glucan was exposed at the cell wall surface. Cells grown at high caspofungin concentrations had decreased virulence in the invertebrate host Galleria mellonella. Cells grown at high caspofungin concentrations also induced a proinflammatory response in murine macrophages compared to control cells. Our work highlights important aspects about fungal adaptation to caspofungin, and although this adaptation is associated with reduced virulence, the clinical implications remain to be elucidated.

  11. Betaine treatment decreased oxidative stress, inflammation, and stellate cell activation in rats with alcoholic liver fibrosis.

    Science.gov (United States)

    Bingül, İlknur; Başaran-Küçükgergin, Canan; Aydın, A Fatih; Çoban, Jale; Doğan-Ekici, Işın; Doğru-Abbasoğlu, Semra; Uysal, Müjdat

    2016-07-01

    The aim of this study was to investigate the effect of betaine (BET) on alcoholic liver fibrosis in rats. Fibrosis was experimentally generated with ethanol plus carbon tetrachloride (ETH+CCl4) treatment. Rats were treated with ETH (5% v/v in drinking water) for 14 weeks. CCl4 was administered intraperitoneally (i.p.) 0.2mL/kg twice a week to rats in the last 6 weeks with/without commercial food containing BET (2% w/w). Serum hepatic damage markers, tumor necrosis factor-α, hepatic triglyceride (TG) and hydroxyproline (HYP) levels, and oxidative stress parameters were measured together with histopathologic observations. In addition, α-smooth muscle-actin (α-SMA), transforming growth factor-β1 (TGF-β1) and type I collagen (COL1A1) protein expressions were assayed immunohistochemically to evaluate stellate cell (HSC) activation. mRNA expressions of matrix metalloproteinase-2 (MMP-2) and its inhibitors (TIMP-1 and TIMP-2) were also determined. BET treatment diminished TG and HYP levels; prooxidant status and fibrotic changes; α-SMA, COL1A1 and TGF-β protein expressions; MMP-2, TIMP-1 and TIMP-2 mRNA expressions in the liver of fibrotic rats. In conclusion, these results indicate that the antifibrotic effect of BET may be related to its suppressive effects on oxidant and inflammatory processes together with HSC activation in alcoholic liver fibrosis.

  12. 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

    2012-03-25

    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.

  13. Men with Sickle Cell Anemia and Priapism Exhibit Increased Hemolytic Rate, Decreased Red Blood Cell Deformability and Increased Red Blood Cell Aggregate Strength.

    Directory of Open Access Journals (Sweden)

    Kizzy-Clara Cita

    Full Text Available To investigate the association between priapism in men with sickle cell anemia (SCA and hemorheological and hemolytical parameters.Fifty-eight men with SCA (median age: 38 years were included; 28 who had experienced priapism at least once during their life (priapism group and 30 who never experienced this complication (control group. Twenty-two patients were treated with hydroxycarbamide, 11 in each group. All patients were at steady state at the time of inclusion. Hematological and biochemical parameters were obtained through routine procedures. The Laser-assisted Optical Rotational Cell Analyzer was used to measure red blood cell (RBC deformability at 30 Pa (ektacytometry and RBC aggregation properties (laser backscatter versus time. Blood viscosity was measured at a shear rate of 225 s-1 using a cone/plate viscometer. A principal component analysis was performed on 4 hemolytic markers (i.e., lactate dehydrogenase (LDH, aspartate aminotransferase (ASAT, total bilirubin (BIL levels and reticulocyte (RET percentage to calculate a hemolytic index.Compared to the control group, patients with priapism exhibited higher ASAT (p = 0.01, LDH (p = 0.03, RET (p = 0.03 levels and hemolytic indices (p = 0.02. Higher RBC aggregates strength (p = 0.01 and lower RBC deformability (p = 0.005 were observed in patients with priapism compared to controls. After removing the hydroxycarbamide-treated patients, RBC deformability (p = 0.01 and RBC aggregate strength (p = 0.03 were still different between the two groups, and patients with priapism exhibited significantly higher hemolytic indices (p = 0.01 than controls.Our results confirm that priapism in SCA is associated with higher hemolytic rates and show for the first time that this complication is also associated with higher RBC aggregate strength and lower RBC deformability.

  14. Cyclosporine restores hematopoietic function by compensating for decreased Tregs in patients with pure red cell aplasia and acquired aplastic anemia.

    Science.gov (United States)

    Dao, An T T; Yamazaki, Hirohito; Takamatsu, Hiroyuki; Sugimori, Chiharu; Katagiri, Takamasa; Maruyama, Hiroyuki; Zaimoku, Yoshitaka; Maruyama, Kana; Ly, Trung Q; Espinoza, Luis; Nakao, Shinji

    2016-04-01

    Most patients with acquired pure red cell aplasia (PRCA) and some with acquired aplastic anemia (AA) respond well to cyclosporine (CsA), but thereafter often show CsA dependency. The mechanism underlying this dependency remains unknown. We established a reliable method for measuring the regulatory T cell (Treg) count using FoxP3 and Helios expression as markers and determined the balance between Tregs and other helper T cell subsets in 16 PRCA and 29 AA patients. The ratios of interferon-γ-producing CD4(+) (Th1) T cells to Tregs in untreated patients and CsA-dependent patients were significantly higher (PRCA 5.77 ± 1.47 and 7.38 ± 2.58; AA 6.18 ± 2.35 and 8.94 ± 4.06) than in healthy volunteers (HVs; 3.33 ± 0.90) due to the profound decrease in the percentage of Tregs. In contrast, the ratios were comparable to HVs in convalescent CsA-treated AA patients (4.74 ± 2.10) and AA patients in remission after the cessation of CsA treatment (4.24 ± 1.67). Low-dose CsA (100 ng/ml) inhibited the proliferation of conventional T cells (Tconv) to a similar degree to the inhibition by Tregs in a co-culture with a 1:1 Treg/Tconv ratio. The data suggest that CsA may reverse the hematopoietic suppression in PRCA and AA patients by compensating for the inadequate immune regulatory function that occurs due to a profound decrease in the Treg count.

  15. Enrichment of LDL with EPA and DHA decreased oxidized LDL-induced apoptosis in U937 cells.

    Science.gov (United States)

    Wu, Tianying; Geigerman, Cissy; Lee, Ye-Sun; Wander, Rosemary C

    2002-08-01

    Oxidized LDL (oxLDL) may contribute to the accumulation of apoptotic cells in atherosclerotic plaques. Although it is well established in monophasic chemical systems that the highly unsaturated EPA and DHA will oxidize more readily than FA that contain fewer double bonds, our previous studies showed that enrichment of LDL, which has discrete polar and nonpolar phases, with these FA did not increase oxidation. The objective of this study was to compare the extent of apoptosis induced by EPA/DHA-rich oxLDL to that induced by EPA/DHA-non-rich oxLDL in U937 cells. LDL was obtained from one healthy subject three times before and after supplementation for 5 wk with 15 g/d of fish oil (FO), an amount easily obtainable from a diet that contains fatty fish. After supplementation, an EPA/DHA-rich LDL was obtained. Oxidative susceptibility of LDL, as determined by measuring the formation of conjugated dienes and the accumulation of cholesteryl ester hydroperoxides, was not higher in EPA/DHA-rich LDL. The oxLDL-induced cell apoptosis was detected by the activation of caspase-3, the translocation of PS to the outer surface of the plasma membrane using the Annexin V-fluorescein isothiocyanate binding assay, and the presence of chromatin condensation and nuclear fragmentation using the 4,6-diamidino-2-phenylindole staining assay. All three measures showed that after FO supplementation, EPA/DHA-rich oxLDL-induced cell apoptosis decreased. The decrease was not related to the concentration of lipid hydroperoxides. This study suggests that a possible protective effect of EPA/DHA-rich diets on atherosclerosis may be through lessening cell apoptosis in the arterial wall.

  16. Adoptive transfer of bone marrow-derived dendritic cells decreases inhibitory and regulatory T-cell differentiation and improves survival in murine polymicrobial sepsis.

    Science.gov (United States)

    Wang, Hong-Wei; Yang, Wen; Gao, Lei; Kang, Jia-Rui; Qin, Jia-Jian; Liu, Yue-Ping; Lu, Jiang-Yang

    2015-05-01

    A decrease in the number of dendritic cells (DCs) is a major cause of post-sepsis immunosuppression and opportunistic infection and is closely associated with poor prognosis. Increasing the number of DCs to replenish their numbers post sepsis can improve the condition. This therapeutic approach could improve recovery after sepsis. Eighty C57BL/6 mice were subjected to sham or caecal ligation and puncture (CLP) surgery. Mice were divided into four groups: (i) Sham + vehicle, (ii) Sham + DC, (iii) CLP + vehicle, and (iv) CLP + DC. Bone-marrow-derived DCs (BMDCs) were administered at 6, 12 and 24 hr after surgery. After 3 days, we assessed serum indices of organ function (alanine aminotransferase, aspartate aminotransferase, creatinine, amylase and lipase), organ tissue histopathology (haematoxylin and eosin staining), cytokine [interferon-γ (IFN-γ), tumour necrosis factor-α, interleukin-12p70 (IL-12p70), IL-6 and IL-10] levels in the serum, programmed death-1 (PD-1) expression on T cells, regulatory T-cell differentiation in the spleen, and the survival rate (monitored for 7 days). BMDC transfer resulted in the following changes: a significant reduction in damage to the liver, kidney and pancreas in the CLP-septic mice as well as in the pathological changes seen in the liver, lung, small intestine and pancreas; significantly elevated levels of the T helper type 1 (Th1) cytokines IFN-γ and IL-12p70 in the serum; decreased levels of the Th2 cytokines IL-6 and IL-10 in the serum; reduced expression of PD-1 molecules on CD4(+) T cells; reduced the proliferation and differentiation of splenic suppressor T cells and CD4(+)  CD25(+)  Foxp3(+) regulatory T cells, and a significant increase in the survival rate of the septic animals. These results show that administration of BMDCs may have modulated the differentiation and immune function of T cells and contributed to alleviate immunosuppression, hence reducing organ damage and mortality post sepsis. Hence

  17. Nε-(carboxymethyl) lysine-induced mitochondrial fission and mitophagy cause decreased insulin secretion from β-cells.

    Science.gov (United States)

    Lo, Mei-Chen; Chen, Ming-Hong; Lee, Wen-Sen; Lu, Chin-I; Chang, Chuang-Rung; Kao, Shu-Huei; Lee, Horng-Mo

    2015-11-15

    Nε-(carboxymethyl) lysine-conjugated bovine serum albumin (CML-BSA) is a major component of advanced glycation end products (AGEs). We hypothesised that AGEs reduce insulin secretion from pancreatic β-cells by damaging mitochondrial functions and inducing mitophagy. Mitochondrial morphology and the occurrence of autophagy were examined in pancreatic islets of diabetic db/db mice and in the cultured CML-BSA-treated insulinoma cell line RIN-m5F. In addition, the effects of α-lipoic acid (ALA) on mitochondria in AGE-damaged tissues were evaluated. The diabetic db/db mouse exhibited an increase in the number of autophagosomes in damaged mitochondria and receptor for AGEs (RAGE). Treatment of db/db mice with ALA for 12 wk increased the number of mitochondria with well-organized cristae and fewer autophagosomes. Treatment of RIN-m5F cells with CML-BSA increased the level of RAGE protein and autophagosome formation, caused mitochondrial dysfunction, and decreased insulin secretion. CML-BSA also reduced mitochondrial membrane potential and ATP production, increased ROS and lipid peroxide production, and caused mitochondrial DNA deletions. Elevated fission protein dynamin-related protein 1 (Drp1) level and mitochondrial fragmentation demonstrated the unbalance of mitochondrial fusion and fission in CML-BSA-treated cells. Additionally, increased levels of Parkin and PTEN-induced putative kinase 1 protein suggest that fragmented mitochondria were associated with increased mitophagic activity, and ALA attenuated the CML-BSA-induced mitophage formation. Our study demonstrated that CML-BSA induced mitochondrial dysfunction and mitophagy in pancreatic β-cells. The findings from this study suggest that increased concentration of AGEs may damage β-cells and reduce insulin secretion.

  18. Mutations Decreasing Intrinsic β-Lactam Resistance Are Linked to Cell Division in the Nosocomial Pathogen Acinetobacter baumannii.

    Science.gov (United States)

    Knight, Daniel; Dimitrova, Daniela D; Rudin, Susan D; Bonomo, Robert A; Rather, Philip N

    2016-06-01

    Transposon mutagenesis was used to identify novel determinants of intrinsic β-lactam resistance in Acinetobacter baumannii An EZ-Tn5 transposon insertion in a gene corresponding to the A1S_0225 sequence resulted in a 4-fold decrease in resistance to ampicillin, cefotaxime, imipenem, and ceftriaxone but did not alter resistance to other classes of antibiotics. Based on this phenotype, the gene was designated blhA (β-lactam hypersusceptibility). The blhA::EZ-Tn5 mutation conferred a similar phenotype in A. baumannii strain ATCC 17978. The wild-type blhA gene complemented the blhA::EZTn5 insertion and restored β-lactam resistance levels back to wild-type levels. The blhA mutation also increased β-lactam susceptibility in an adeB adeJ double mutant, indicating that the blhA mutation acted independently of these efflux systems to mediate susceptibility. In addition, mRNA levels for the blaOXA and blaADC β-lactamase genes were not altered by the blhA mutation. The blhA mutation resulted in a prominent cell division and morphological defect, with cells exhibiting a highly elongated phenotype, combined with large bulges in some cells. The blhA gene is unique to Acinetobacter and likely represents a novel gene involved in cell division. Three additional mutations, in zipA, zapA, and ftsK, each of which encode predicted cell division proteins, also conferred increased β-lactam susceptibility, indicating a common link between cell division and intrinsic β-lactam resistance in A. baumannii.

  19. Nucleolin antagonist triggers autophagic cell death in human glioblastoma primary cells and decreased in vivo tumor growth in orthotopic brain tumor model.

    Science.gov (United States)

    Benedetti, Elisabetta; Antonosante, Andrea; d'Angelo, Michele; Cristiano, Loredana; Galzio, Renato; Destouches, Damien; Florio, Tiziana Marilena; Dhez, Anne Chloé; Astarita, Carlo; Cinque, Benedetta; Fidoamore, Alessia; Rosati, Floriana; Cifone, Maria Grazia; Ippoliti, Rodolfo; Giordano, Antonio; Courty, José; Cimini, Annamaria

    2015-12-01

    Nucleolin (NCL) is highly expressed in several types of cancer and represents an interesting therapeutic target. It is expressed at the plasma membrane of tumor cells, a property which is being used as a marker for several human cancer including glioblastoma. In this study we investigated targeting NCL as a new therapeutic strategy for the treatment of this pathology. To explore this possibility, we studied the effect of an antagonist of NCL, the multivalent pseudopeptide N6L using primary culture of human glioblastoma cells. In this system, N6L inhibits cell growth with different sensitivity depending to NCL localization. Cell cycle analysis indicated that N6L-induced growth reduction was due to a block of the G1/S transition with down-regulation of the expression of cyclin D1 and B2. By monitoring autophagy markers such as p62 and LC3II, we demonstrate that autophagy is enhanced after N6L treatment. In addition, N6L-treatment of mice bearing tumor decreased in vivo tumor growth in orthotopic brain tumor model and increase mice survival. The results obtained indicated an anti-proliferative and pro-autophagic effect of N6L and point towards its possible use as adjuvant agent to the standard therapeutic protocols presently utilized for glioblastoma.

  20. The endocannabinoid 2-arachidonylglycerol decreases the immunological activation of Guinea pig mast cells: involvement of nitric oxide and eicosanoids.

    Science.gov (United States)

    Vannacci, Alfredo; Giannini, Lucia; Passani, Maria Beatrice; Di Felice, Annamaria; Pierpaoli, Simone; Zagli, Giovanni; Fantappiè, Ornella; Mazzanti, Roberto; Masini, Emanuela; Mannaioni, Pier Francesco

    2004-10-01

    The antigen-induced release of histamine from sensitized guinea pig mast cells was dose-dependently reduced by endogenous (2-arachidonylglycerol; 2AG) and exogenous [(1R,3R,4R)-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-4-(3-hydroxypropyl)cyclohexan-1-ol (CP55,940)] cannabinoids. The inhibitory action afforded by 2AG and CP55,940 was reversed by N-[(1S)-endo-1,3,3-trimethylbicyclo[2.2.1]heptan-2-yl]5-(4-chloro-3-methylphenyl)-1-(4-methylbenzyl)pyrazole-3-carboxamide (SR144528), a selective cannabinoid 2 (CB(2)) receptor antagonist, and left unchanged by the selective CB(1) antagonist N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (AM251). The inhibitory action of 2AG and CP55,940 was reduced by the unselective nitric-oxide synthase (NOS) inhibitor N-monomethyl-L-arginine methylester (l-NAME) and reinstated by L-arginine, the physiological substrate. The inhibitory action of 2AG and CP55,940 was also reduced by the unselective cyclooxygenase (COX) inhibitor indomethacin and the selective COX-2 blocker rofecoxib. Both 2AG and CP55,940 significantly increased the production of nitrite from mast cells, which was abrogated by L-NAME and N-(3-(aminomethyl)benzyl)acetamidine (1400W), a selective inducible NOS (iNOS) inhibitor. Nitrite production consistently paralleled a CP55,940-induced increase in the expression of iNOS protein in mast cells. Both 2AG and CP55,940 increased the generation of prostaglandin E(2) from mast cells, which was abrogated by indomethacin and rofecoxib and parallel to the CP55,940-induced expression of COX-2 protein. Mast cell challenge with antigen was accompanied by a net increase in intracellular calcium levels. Both cannabinoid receptor ligands decreased the intracellular calcium levels, which were reversed by SR144528 and l-NAME. In unstimulated mast cells, both ligands increased cGMP levels. The increase was abrogated by SR144528, l-NAME, indomethacin, and rofecoxib. Our results suggest that 2

  1. Triomics Analysis of Imatinib-Treated Myeloma Cells Connects Kinase Inhibition to RNA Processing and Decreased Lipid Biosynthesis.

    Science.gov (United States)

    Breitkopf, Susanne B; Yuan, Min; Helenius, Katja P; Lyssiotis, Costas A; Asara, John M

    2015-11-03

    The combination of metabolomics, lipidomics, and phosphoproteomics that incorporates triple stable isotope labeling by amino acids in cell culture (SILAC) protein labeling, as well as (13)C in vivo metabolite labeling, was demonstrated on BCR-ABL-positive H929 multiple myeloma cells. From 11 880 phosphorylation sites, we confirm that H929 cells are primarily signaling through the BCR-ABL-ERK pathway, and we show that imatinib treatment not only downregulates phosphosites in this pathway but also upregulates phosphosites on proteins involved in RNA expression. Metabolomics analyses reveal that BCR-ABL-ERK signaling in H929 cells drives the pentose phosphate pathway (PPP) and RNA biosynthesis, where pathway inhibition via imatinib results in marked PPP impairment and an accumulation of RNA nucleotides and negative regulation of mRNA. Lipidomics data also show an overall reduction in lipid biosynthesis and fatty acid incorporation with a significant decrease in lysophospholipids. RNA immunoprecipitation studies confirm that RNA degradation is inhibited with short imatinib treatment and transcription is inhibited upon long imatinib treatment, validating the triomics results. These data show the utility of combining mass spectrometry-based "-omics" technologies and reveals that kinase inhibitors may not only downregulate phosphorylation of their targets but also induce metabolic events via increased phosphorylation of other cellular components.

  2. Decreased Laminin Expression by Human Lung Epithelial Cells and Fibroblasts Cultured in Acellular Lung Scaffolds from Aged Mice.

    Science.gov (United States)

    Godin, Lindsay M; Sandri, Brian J; Wagner, Darcy E; Meyer, Carolyn M; Price, Andrew P; Akinnola, Ifeolu; Weiss, Daniel J; Panoskaltsis-Mortari, Angela

    2016-01-01

    The lung changes functionally and structurally with aging. However, age-related effects on the extracellular matrix (ECM) and corresponding effects on lung cell behavior are not well understood. We hypothesized that ECM from aged animals would induce aging-related phenotypic changes in healthy inoculated cells. Decellularized whole organ scaffolds provide a powerful model for examining how ECM cues affect cell phenotype. The effects of age on ECM composition in both native and decellularized mouse lungs were assessed as was the effect of young vs old acellular ECM on human bronchial epithelial cells (hBECs) and lung fibroblasts (hLFs). Native aged (1 year) lungs demonstrated decreased expression of laminins α3 and α4, elastin and fibronectin, and elevated collagen, compared to young (3 week) lungs. Proteomic analyses of decellularized ECM demonstrated similar findings, and decellularized aged lung ECM contained less diversity in structural proteins compared to young ECM. When seeded in old ECM, hBECs and hLFs demonstrated lower gene expression of laminins α3 and α4, respectively, as compared to young ECM, paralleling the laminin deficiency of aged ECM. ECM changes appear to be important factors in potentiating aging-related phenotypes and may provide clues to mechanisms that allow for aging-related lung diseases.

  3. Decreased Laminin Expression by Human Lung Epithelial Cells and Fibroblasts Cultured in Acellular Lung Scaffolds from Aged Mice.

    Directory of Open Access Journals (Sweden)

    Lindsay M Godin

    Full Text Available The lung changes functionally and structurally with aging. However, age-related effects on the extracellular matrix (ECM and corresponding effects on lung cell behavior are not well understood. We hypothesized that ECM from aged animals would induce aging-related phenotypic changes in healthy inoculated cells. Decellularized whole organ scaffolds provide a powerful model for examining how ECM cues affect cell phenotype. The effects of age on ECM composition in both native and decellularized mouse lungs were assessed as was the effect of young vs old acellular ECM on human bronchial epithelial cells (hBECs and lung fibroblasts (hLFs. Native aged (1 year lungs demonstrated decreased expression of laminins α3 and α4, elastin and fibronectin, and elevated collagen, compared to young (3 week lungs. Proteomic analyses of decellularized ECM demonstrated similar findings, and decellularized aged lung ECM contained less diversity in structural proteins compared to young ECM. When seeded in old ECM, hBECs and hLFs demonstrated lower gene expression of laminins α3 and α4, respectively, as compared to young ECM, paralleling the laminin deficiency of aged ECM. ECM changes appear to be important factors in potentiating aging-related phenotypes and may provide clues to mechanisms that allow for aging-related lung diseases.

  4. Locked nucleic acid-inhibitor of miR-205 decreases endometrial cancer cells proliferation in vitro and in vivo.

    Science.gov (United States)

    Torres, Anna; Kozak, Joanna; Korolczuk, Agnieszka; Rycak, Dominika; Wdowiak, Paulina; Maciejewski, Ryszard; Torres, Kamil

    2016-11-08

    Pathogenesis of endometrial cancer has been connected with alterations of microRNA expression and in particular miR-205 up-regulation was consistently reported in this carcinoma. Presented study aimed to investigate if inhibition of miR-205 expression using LNA-modified-nucleotide would attenuate endometrial cancer cells proliferation in vitro and in vivo.In the course of the study we found that the proliferation of endometrial cancer cells (HEC-1-B, RL-95, KLE, Ishikawa) transfected with LNA-miR-205-inhibitor and evaluated using real time cell monitoring as well as standard cell proliferation assay, was significantly decreased. Next, LNA-miR-205-inhibitor was used to assess the in vivo effects of miR-205 inhibition of endometrial cancer growth. Cby.Cg-Foxn1/cmdb mice bearing endometrial cancer xenografts were intraperitoneally injected with nine dosages of 25mg/kg of miR-205-LNA-inhibitor or scramble control or phosphatase buffered saline and were observed for 32 days. We found that systemic administration of miR-205-LNA-inhibitor was technically possible, and exerted inhibitory effect on endometrial cancer xenograft growth in vivo with only mild toxic effects in treated animals.In conclusion our results suggest that systemic delivery of miR-205-LNA-inhibitor is feasible, devoid of significant toxicity, and could be a promising treatment strategy for endometrial cancer. Therefore it warrants further studies in other animal models.

  5. Palmitic acid but not palmitoleic acid induces insulin resistance in a human endothelial cell line by decreasing SERCA pump expression.

    Science.gov (United States)

    Gustavo Vazquez-Jimenez, J; Chavez-Reyes, Jesus; Romero-Garcia, Tatiana; Zarain-Herzberg, Angel; Valdes-Flores, Jesus; Manuel Galindo-Rosales, J; Rueda, Angelica; Guerrero-Hernandez, Agustin; Olivares-Reyes, J Alberto

    2016-01-01

    Palmitic acid is a negative regulator of insulin activity. At the molecular level, palmitic acid reduces insulin stimulated Akt Ser473 phosphorylation. Interestingly, we have found that incubation with palmitic acid of human umbilical vein endothelial cells induced a biphasic effect, an initial transient elevation followed by a sustained reduction of SERCA pump protein levels. However, palmitic acid produced a sustained inhibition of SERCA pump ATPase activity. Insulin resistance state appeared before there was a significant reduction of SERCA2 expression. The mechanism by which palmitic acid impairs insulin signaling may involve endoplasmic reticulum stress, because this fatty acid induced activation of both PERK, an ER stress marker, and JNK, a kinase associated with insulin resistance. None of these effects were observed by incubating HUVEC-CS cells with palmitoleic acid. Importantly, SERCA2 overexpression decreased the palmitic acid-induced insulin resistance state. All these results suggest that SERCA pump might be the target of palmitic acid to induce the insulin resistance state in a human vascular endothelial cell line. Importantly, these data suggest that HUVEC-CS cells respond to palmitic acid-exposure with a compensatory overexpression of SERCA pump within the first hour, which eventually fades out and insulin resistance prevails.

  6. Increased levels of p21((CIP1/WAF1)) correlate with decreased chondrogenic differentiation potential in synovial membrane progenitor cells.

    Science.gov (United States)

    Masson, Anand Oliveira; Hess, Ricarda; O'Brien, Kate; Bertram, Karri L; Tailor, Pankaj; Irvine, Edward; Ren, Guomin; Krawetz, Roman J

    2015-07-01

    Cartilage injuries are a major concern in the field of orthopedics. They occur following trauma, as well as from a variety of pathological conditions including Osteoarthritis (OA). Although cartilage does not exhibit robust endogenous repair, it has been demonstrated that modulating the activity of p21 can increase the regenerative abilities of cartilage in vitro and in vivo. Since the synovial membrane is abundant with mesenchymal progenitor cells (MPCs) capable of differentiating into cartilage both in vitro and in vivo, we examined if p21 expression levels varied between MPCs derived from normal vs. OA knee joints. Analysis of p21 at the mRNA and protein levels within normal and OA MPCs demonstrated differential levels of expression between these two groups, with OA MPCs having higher p21 expression levels. The higher levels of p21 in OA MPCs are also correlated with a decreased chondrogenic differentiation capacity and synovial inflammation, however, there was no evidence of senescence in the OA cells. The results of this study suggest that cell cycle regulation in MPCs may be altered in OA and that modulation of this pathway may have therapeutic potential once the mechanism by which this regulates stem/progenitor cells is better understood.

  7. Suppression of dynamin GTPase decreases α-synuclein uptake by neuronal and oligodendroglial cells: a potent therapeutic target for synucleinopathy

    Directory of Open Access Journals (Sweden)

    Konno Masatoshi

    2012-08-01

    Full Text Available Abstract Background The intracellular deposition of misfolded proteins is a common neuropathological hallmark of most neurodegenerative disorders. Increasing evidence suggests that these pathogenic proteins may spread to neighboring cells and induce the propagation of neurodegeneration. Results In this study, we have demonstrated that α-synuclein (αSYN, a major constituent of intracellular inclusions in synucleinopathies, was taken up by neuronal and oligodendroglial cells in both a time- and concentration-dependent manner. Once incorporated, the extracellular αSYN was immediately assembled into high-molecular-weight oligomers and subsequently formed cytoplasmic inclusion bodies. Furthermore, αSYN uptake by neurons and cells of the oligodendroglial lineage was markedly decreased by the genetic suppression and pharmacological inhibition of the dynamin GTPases, suggesting the involvement of the endocytic pathway in this process. Conclusions Our findings shed light on the mode of αSYN uptake by neuronal and oligodendroglial cells and identify therapeutic strategies aimed at reducing the propagation of protein misfolding.

  8. Persistent Morbillivirus Infection Leads to Altered Cortactin Distribution in Histiocytic Sarcoma Cells with Decreased Cellular Migration Capacity

    Science.gov (United States)

    Pfankuche, Vanessa Maria; Sayed-Ahmed, Mohamed; Contioso, Vanessa Bono; Spitzbarth, Ingo; Rohn, Karl; Ulrich, Reiner; Deschl, Ulrich; Kalkuhl, Arno; Baumgärtner, Wolfgang; Puff, Christina

    2016-01-01

    Histiocytic sarcomas represent rare but fatal neoplasms in humans. Based on the absence of a commercially available human histiocytic sarcoma cell line the frequently affected dog displays a suitable translational model. Canine distemper virus, closely related to measles virus, is a highly promising candidate for oncolytic virotherapy. Therapeutic failures in patients are mostly associated with tumour invasion and metastasis often induced by misdirected cytoskeletal protein activities. Thus, the impact of persistent canine distemper virus infection on the cytoskeletal protein cortactin, which is frequently overexpressed in human cancers with poor prognosis, was investigated in vitro in a canine histiocytic sarcoma cell line (DH82). Though phagocytic activity, proliferation and apoptotic rate were unaltered, a significantly reduced migration activity compared to controls (6 hours and 1 day after seeding) accompanied by a decreased number of cortactin mRNA transcripts (1 day) was detected. Furthermore, persistently canine distemper virus infected DH82 cells showed a predominant diffuse intracytoplasmic cortactin distribution at 6 hours and 1 day compared to controls with a prominent membranous expression pattern (p ≤ 0.05). Summarized, persistent canine distemper virus infection induces reduced tumour cell migration associated with an altered intracellular cortactin distribution, indicating cytoskeletal changes as one of the major pathways of virus-associated inhibition of tumour spread. PMID:27911942

  9. Production of cloned dogs by decreasing the interval between fusion and activation during somatic cell nuclear transfer.

    Science.gov (United States)

    Kim, Sue; Park, Sun Woo; Hossein, Mohammad Shamim; Jeong, Yeon Woo; Kim, Joung Joo; Lee, Eugine; Kim, Yeun Wook; Hyun, Sang Hwan; Shin, Taeyoung; Hwang, Woo Suk

    2009-05-01

    To improve the efficiency of somatic cell nuclear transfer (SCNT) in dogs, we evaluated whether or not the interval between fusion and activation affects the success rate of SCNT. Oocytes retrieved from outbred dogs were reconstructed with adult somatic cells from a male or female Golden Retriever. In total, 151 and 225 reconstructed oocytes were transferred to 9 and 14 naturally synchronized surrogates for male and female donor cells, respectively. Chromosomal morphology was evaluated in 12 oocytes held for an interval of 2 hr between fusion and activation and 14 oocytes held for an interval of 4 hr. Three hundred seventy-six and 288 embryos were transferred to 23 and 16 surrogates for the 2 and 4 hr interval groups, respectively. Both the male (two pregnant surrogates gave birth to three puppies) and female (one pregnant surrogate gave birth to one puppy) donor cells gave birth to live puppies (P > 0.05). In the 2 hr group, significantly more reconstructed oocytes showed condensed, metaphase-like chromosomes compared to the 4 hr group (P dogs carried pregnancies to term and four puppies were born. These results demonstrate that decreasing the interval between fusion and activation increases the success rate of clone production and pregnancy. These results may increase the overall efficiency of SCNT in the canine family.

  10. Methotrexate decreases hippocampal cell proliferation and induces memory deficits in rats

    NARCIS (Netherlands)

    Seigers, Riejanne; Schagen, Sanne B.; Coppens, Caroline M.; van der Most, Peter J.; van Dam, Frits S. A. M.; Koolhaas, Jaap M.; Buwalda, Bauke

    2009-01-01

    Methotrexate (MTX) is a cytostatic agent used in adjuvant chemotherapy for treatment of breast cancer and is associated with cognitive impairment in a subgroup of patients. The aim of this paper is to test whether MTX can rapidly affect various brain structures resulting in decreased hippocampal cel

  11. 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 

  12. 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.

  13. TOX3 (TNRC9) Over Expression in Bladder Cancer Cells Decreases Cellular Proliferation and Triggers an Interferon-Like Response

    DEFF Research Database (Denmark)

    Birkenkamp-Demtroder, Karin; Mansilla Castaño, Francisco; Dyrskjøt, Lars

    2013-01-01

    urothelium. Microarray expression profiling of human bladder cancer cells over expressing TOX3 followed by Pathway analysis showed that TOX3 Overexpression mainly affected the Interferon Signaling Pathway. TOX3 up regulation induced the expression of several genes with a gamma interferon activation site (GAS......), e.g. STAT1. In vitro functional studies showed that TOX3 was able to bind to the GAS-sequence located at the STAT1 promoter. siRNA mediated knockdown of TOX3 in RT4 bladder cancer cells decreased STAT1 expression suggesting a direct impact of TOX3 on STAT1. Immunoprecipitation of TOX3 over......Background: Human TOX3 (TOX high mobility group box family member 3) regulates Ca2+ dependent transcription in neurons and has been associated with breast cancer susceptibility. Aim of the study was to investigate the expression of TOX3 in bladder cancer tissue samples and to identify genes...

  14. TOX3 (TNRC9) overexpression in bladder cancer cells decreases cellular proliferation and triggers an interferon-like response

    DEFF Research Database (Denmark)

    Birkenkamp-Demtröder, Karin; Mansilla, Francisco; Andersen, Lars Dyrskjøt

    2013-01-01

    urothelium. Microarray expression profiling of human bladder cancer cells overexpressing TOX3 followed by Pathway analysis showed that TOX3 overexpression mainly affected the Interferon Signaling Pathway. TOX3 upregulation induced the expression of several genes with a gamma interferon activation site (GAS......), e.g. STAT1. In vitro functional studies showed that TOX3 was able to bind to the GAS-sequence located at the STAT1 promoter. siRNA mediated knockdown of TOX3 in RT4 bladder cancer cells decreased STAT1 expression suggesting a direct impact of TOX3 on STAT1. Immunoprecipitation of TOX3 overexpressing......Background Human TOX3 (TOX high mobility group box family member 3) regulates Ca2+-dependent transcription in neurons and has been associated with breast cancer susceptibility. Aim of the study was to investigate the expression of TOX3 in bladder cancer tissue samples and to identify genes...

  15. Heat stress decreases testicular germ cell proliferation and increases apoptosis in short term: an immunohistochemical and ultrastructural study.

    Science.gov (United States)

    Kanter, Mehmet; Aktas, Cevat; Erboga, Mustafa

    2013-03-01

    Scrotal hyperthermia has been known as a cause of male infertility but the exact mechanism leading to impaired spermatogenesis is unknown. This work was aimed to investigate the role of scrotal hyperthermia on cell proliferation and apoptosis in testes. The rats were randomly allotted into one of the four experimental groups: A (control), B (1 day after scrotal hyperthermia), C (14 days after scrotal hyperthermia), and D (35 days after scrotal hyperthermia); each group comprised 7 animals. Scrotal hyperthermia was carried out in a thermostatically controlled water bath at 43°C for 30 min once daily for 6 consecutive days. Control rats were treated in the same way, except the testes were immersed in a water bath maintained at 22°C. Hyperthermia-exposed rats were killed under 50 mg/kg ketamine anaesthesia and tissue samples were obtained for biochemical and histopathological investigations. Hyperthermia treatment significantly decreased the testicular antioxidant system, including decreases in the glutathione level, superoxide dismutase, and glutathione peroxidase activities. Moreover, exposure to hyperthermia resulted in lipid peroxidation increase in testes. Our data indicate a significant reduction in the expression of proliferating cell nuclear antigen and an enhancement in the activity of terminal deoxynucleotidyl transferase dUTP nick end labelling after scrotal hyperthermia. In scrotal hyperthermia, the mitochondrial degeneration, dilatation of smooth endoplasmic reticulum, and enlarged intercellular spaces were observed in both Sertoli and spermatid cells. Scrotal hyperthermia is one of the major factors that impair spermatogenesis in testis. This heat stress is shown to be closely associated with oxidative stress, followed by apoptosis of germ cells.

  16. 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

  17. Knockdown of Rab5a expression decreases cancer cell motility and invasion through integrin-mediated signaling pathway

    Directory of Open Access Journals (Sweden)

    Shi Shu-liang

    2011-08-01

    Full Text Available Abstract Background Rab GTPases function as modulators in intracellular transport. Rab5a, a member of the Rab subfamily of small GTPases, is an important regulator of vesicle traffic from the plasma membrane to early endosomes. Recent findings have reported that Rab5a gene was involved in the progression of cancer. In the present study, we investigated the effect of Rab5a on cervical cancer invasion and metastasis and the molecular mechanism underlying the involvement of Rab5a. Methods Rab5a expression was assessed by immunohistochemical analysis on a cervical cancer tissue microarray. RNA interference (RNAi was performed to knock down the endogenous expression of Rab5a gene in HeLa and SiHa cells. Cell motility was evaluated using invasion assay and wound migration assay in vitro. The expression levels of integrin-associated molecules were detected by Western blot and immunofluorescence. Results We found that Rab5a was expressed at a high level in cervical cancer tissues. Silencing of Rab5a expression significantly decreased cancer cell motility and invasiveness. The down-regulation of integrin-associated focal adhesion signaling molecules was further detected in Rab5a knockdown cells. Meanwhile, active GTP-bound Rac1, Cdc42, and RhoA were also down-regulated, accompanied with the reduction in the number and size of filopodia and lamellipodia. Conclusions Taken together, these data suggest that Rab5a functions in regulating the invasion phenotype, and we propose that this regulation may be via integrin-mediated signaling pathway in cervical cancer cells.

  18. C3a Increases VEGF and Decreases PEDF mRNA Levels in Human Retinal Pigment Epithelial Cells

    Science.gov (United States)

    Long, Qin; Cao, Xiaoguang; Bian, Ailing

    2016-01-01

    Complement activation, specifically complement 3 (C3) activation and C3a generation, contributes to an imbalance between angiogenic stimulation by vascular endothelial growth factor (VEGF) and angiogenic inhibition by pigment epithelial derived factor (PEDF), leading to pathological angiogenesis. This study aimed to investigate the effects of C3a and small interfering RNA (siRNA) targeting C3 on the levels of VEGF and PEDF mRNAs in human retinal pigment epithelial (RPE) cells. ARPE-19 cells were cultured in the presence of exogenous C3a at 0.1 μM and 0.3 μM C3a for 24, 48, and 72 hours. 0.1 pmol/μL duplexes of siRNA targeting C3 were applied for C3a inhibition by transfecting ARPE-19 cells for 48 hours. RT-PCR was performed to examine the level of VEGF and PEDF mRNA. A random siRNA duplex was set for control siRNA. Results demonstrated that exogenous C3a significantly upregulated VEGF and downregulated PEDF mRNA levels in cultured ARPE-19 cells, and siRNA targeting C3 transfection reversed the above changes, significantly reducing VEGF and enhancing PEDF mRNAs level in ARPE-19 cells compared to the control. The present data provided evidence that reducing C3 activation can decreases VEGF and increase PEDF mRNA level in RPE and may serve as a potential therapy in pathological angiogenesis.

  19. The flavanone homoeriodictyol increases SGLT-1-mediated glucose uptake but decreases serotonin release in differentiated Caco-2 cells

    Science.gov (United States)

    Hoi, Julia Katharina; Holik, Ann-Katrin; Geissler, Katrin; Hans, Joachim; Friedl, Barbara; Liszt, Kathrin; Krammer, Gerhard E.; Ley, Jakob P.; Somoza, Veronika

    2017-01-01

    Flavanoids and related polyphenols, among them hesperitin, have been shown to modulate cellular glucose transport by targeting SGLT-1 and GLUT-2 transport proteins. We aimed to investigate whether homoeriodictyol, which is structurally related to hesperitin, affects glucose uptake in differentiated Caco-2 cells as a model for the intestinal barrier. The results revealed that, in contrast to other polyphenols, the flavanon homoeriodictyol promotes glucose uptake by 29.0 ± 3.83% at a concentration of 100 μM. The glucose uptake stimulating effect was sensitive to phloridzin, but not to phloretin, indicating an involvement of the sodium-coupled glucose transporter SGLT-1, but not of sodium-independent glucose transporters (GLUT). In addition, in contrast to the increased extracellular serotonin levels by stimulation with 500 mM D-(+)-glucose, treatment with 100 μM homoeriodictyol decreased serotonin release by –48.8 ± 7.57% in Caco-2 cells via a phloridzin-sensitive signaling pathway. Extracellular serotonin levels were also reduced by –57.1 ± 5.43% after application of 0.01 μM homoeriodictyol to human neural SH-SY5Y cells. In conclusion, we demonstrate that homoeriodictyol affects both the glucose metabolism and the serotonin system in Caco-2 cells via a SGLT-1-meditated pathway. Furthermore, the results presented here support the usage of Caco-2 cells as a model for peripheral serotonin release. Further investigations may address the value of homoeriodictyol in the treatment of anorexia and malnutrition through the targeting of SGLT-1. PMID:28192456

  20. Prolonged renal allograft survival by donor interleukin-6 deficiency: association with decreased alloantibodies and increased intragraft T regulatory cells.

    Science.gov (United States)

    Wang, Hao; Guan, Qiunong; Lan, Zhu; Li, Shuyuan; Ge, Wei; Chen, Huifang; Nguan, Christopher Y C; Du, Caigan

    2012-01-15

    Both humoral and cellular immune responses are involved in renal allograft rejection. Interleukin (IL)-6 is a regulatory cytokine for both B and Foxp3 (forkhead box P3)-expressing regulatory T (Treg) cells. This study was designed to investigate the impact of donor IL-6 production on renal allograft survival. Donor kidneys from IL-6 knockout (KO) vs. wild-type (WT) C57BL/6 mice (H-2(b)) were orthotopically transplanted to nephrotomized BALB/c mice (H-2(d)). Alloantibodies and Treg cells were examined by fluorescence-activated cell sorting analysis. Graft survival was determined by the time to graft failure. Here, we showed that a deficiency in IL-6 expression in donor kidneys significantly prolonged renal allograft survival compared with WT controls. IL-6 protein was upregulated in renal tubules and endothelium of renal allografts following rejection, which correlated with an increase in serum IL-6 compared with that in those receiving KO grafts or naive controls. The absence of graft-producing IL-6 or lower levels of serum IL-6 in the recipients receiving IL-6 KO allografts was associated with decreased circulating anti-graft alloantibodies and increased the percentage of intragraft CD4(+)CD25(+)Foxp3(+) Treg cells compared with those with WT allografts. In conclusion, the lack of graft-producing IL-6 significantly prolongs renal allograft survival, which is associated with reduced alloantibody production and/or increased intragraft Treg cell population, implying that targeting donor IL-6 may effectively prevent both humoral and cellular rejection of kidney transplants.

  1. 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.

  2. Saturated lipids decrease mitofusin 2 leading to endoplasmic reticulum stress activation and insulin resistance in hypothalamic cells.

    Science.gov (United States)

    Diaz, Brenda; Fuentes-Mera, Lizeth; Tovar, Armando; Montiel, Teresa; Massieu, Lourdes; Martínez-Rodríguez, Herminia Guadalupe; Camacho, Alberto

    2015-11-19

    Endoplasmic reticulum (ER) and mitochondria dysfunction contribute to insulin resistance generation during obesity and diabetes. ER and mitochondria interact through Mitofusin 2 (MTF2), which anchors in the outer mitochondrial and ER membranes regulating energy metabolism. Ablation of MTF2 leads to ER stress activation and insulin resistance. Here we determine whether lipotoxic insult induced by saturated lipids decreases MTF2 expression leading to ER stress response in hypothalamus and its effects on insulin sensitivity using in vitro and in vivo models. We found that lipotoxic stimulation induced by palmitic acid, but not the monounsaturated palmitoleic acid, decreases MTF2 protein levels in hypothalamic mHypoA-CLU192 cells. Also, palmitic acid incubation activates ER stress response evidenced by increase in the protein levels of GRP78/BIP marker at later stage than MTF2 downregulation. Additionally, we found that MTF2 alterations induced by palmitic, but not palmitoleic, stimulation exacerbate insulin resistance in hypothalamic cells. Insulin resistance induced by palmitic acid is prevented by pre-incubation of the anti-inflammatory and the ER stress release reagents, sodium salicylate and 4 phenylbutirate, respectively. Finally, we demonstrated that lipotoxic insult induced by high fat feeding to mice decreases MTF2 proteins levels in arcuate nucleus of hypothalamus. Our data indicate that saturated lipids modulate MTF2 expression in hypothalamus coordinating the ER stress response and the susceptibility to insulin resistance.

  3. Polyclonal neural cell adhesion molecule antibody prolongs the effective duration time of botulinum toxin in decreasing muscle strength.

    Science.gov (United States)

    Guo, Yan; Pan, Lizhen; Liu, Wuchao; Pan, Yougui; Nie, Zhiyu; Jin, Lingjing

    2015-11-01

    This study aimed to investigate if the effective duration time of botulinum toxin A (Btx-A) could be prolonged by polyclonal neural cell adhesion molecule antibody (P-NCAM-Ab). 175 male SD rats were randomly divided into three major groups: control group (n = 25), Btx-A group (n = 25), and P-NCAM-Ab groups. P-NCAM-Ab groups were composed of five sub-groups, with 25 rats each in the dose-response study. Muscle strength of rat lower limbs was determined using a survey system. The expressions of muscle-specific receptor tyrosine kinase (MuSK) and neural cell adhesion molecule (NCAM) were determined by real-time polymerase chain reactions (RT-PCR) and western blotting (WB). The muscle strength was significantly decreased by Btx-A in Btx-A/P-NCAM-Ab groups compared with normal control group. Besides, the muscle strength of P-NCAM-Ab group was significantly decreased compared with the Btx-A group. The recovery time of muscle strength in P-NCAM-Ab group was significantly longer compared with Btx-A group. RT-PCR and WB assay showed that PNCAM-Ab delayed the increase of MuSK and NCAM after Btx-A injection. P-NCAM-Ab prolongs the effective duration time of Btx-A in decreasing muscle strength, which could provide a novel enhancement in clinical application.

  4. 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.

  5. Ataxia-telangiectasia mutated (ATM) deficiency decreases reprogramming efficiency and leads to genomic instability in iPS cells.

    Science.gov (United States)

    Kinoshita, Taisuke; Nagamatsu, Go; Kosaka, Takeo; Takubo, Keiyo; Hotta, Akitsu; Ellis, James; Suda, Toshio

    2011-04-08

    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. Decreased ADP-Ribosyl Cyclase Activity in Peripheral Blood Mononuclear Cells from Diabetic Patients with Nephropathy

    Directory of Open Access Journals (Sweden)

    Michio Ohtsuji

    2008-01-01

    Results. ADPRCA negatively correlated with the level of HbA1c (=.040, 2=.073, although ADPRCA showed no significant correlation with gender, age, BMI, blood pressure, level of fasting plasma glucose and lipid levels, as well as type, duration, or medication of diabetes. Interestingly, patients with nephropathy, but not other complications, presented significantly lower ADPRCA than those without nephropathy (=.0198 and diabetes (=.0332. ANCOVA analysis adjusted for HbA1c showed no significant correlation between ADPRCA and nephropathy. However, logistic regression analyses revealed that determinants for nephropathy were systolic blood pressure and ADPRCA, not HbA1c. Conclusion/interpretation. Decreased ADPRCA significantly correlated with diabetic nephropathy. ADPRCA in PBMCs would be an important marker associated with diabetic nephropathy.

  7. TPA decreases 1,25(OH)2D3 binding and calbindin D-28K in renal (MDBK) cells.

    Science.gov (United States)

    Simboli-Campbell, M; Gagnon, A M; Franks, D J; Welsh, J

    1992-02-01

    The effect of the phorbol ester TPA (12-O-tetradecanoylphorbol 13-acetate) on vitamin D receptors (VDRs) was studied in MDBK cells, a normal bovine renal epithelial cell line. 24 h treatment of MDBK cells with TPA resulted in down-regulation of VDR number, with no change in the binding affinity for 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) or approximate molecular weight determined by fast protein liquid chromatography (FPLC). TPA treatment also reduced the level of calbindin D-28K, a vitamin D-dependent renal protein. 4 alpha-Phorbol 12,13-didecanoate (4 alpha-PDD), an inactive phorbol ester, did not affect either 1,25(OH)2D3 binding or calbindin D-28K levels. TPA elicited a significant decrease in membrane-associated protein kinase C (PKC) activity which coincided with the reduction in VDR number and calbindin D-28K. These data support a link between TPA, PKC activity and vitamin D actions in kidney.

  8. HU-446 and HU-465, Derivatives of the Non-psychoactive Cannabinoid Cannabidiol, Decrease the Activation of Encephalitogenic T Cells.

    Science.gov (United States)

    Kozela, Ewa; Haj, Christeene; Hanuš, Lumir; Chourasia, Mukesh; Shurki, Avital; Juknat, Ana; Kaushansky, Nathali; Mechoulam, Raphael; Vogel, Zvi

    2016-01-01

    Cannabidiol (CBD), the non-psychoactive cannabinoid, has been previously shown by us to decrease peripheral inflammation and neuroinflammation in mouse experimental autoimmune encephalomyelitis (EAE) model of multiple sclerosis (MS). Here we have studied the anti-inflammatory effects of newly synthesized derivatives of natural (-)-CBD ((-)-8,9-dihydro-7-hydroxy-CBD; HU-446) and of synthetic (+)-CBD ((+)-8,9-dihydro-7-hydroxy-CBD; HU-465) on activated myelin oligodendrocyte glycoprotein (MOG)35-55-specific mouse encephalitogenic T cells (T(MOG) ) driving EAE/MS-like pathologies. Binding assays followed by molecular modeling revealed that HU-446 has negligible affinity toward the cannabinoid CB1 and CB2 receptors while HU-465 binds to both CB1 and CB2 receptors at the high nanomolar concentrations (Ki = 76.7 ± 5.8 nm and 12.1 ± 2.3 nm, respectively). Both, HU-446 and HU-465, at 5 and 10 μm (but not at 0.1 and 1 μm), inhibited the MOG35-55-induced proliferation of autoreactive T(MOG) cells via CB1/CB2 receptor independent mechanisms. Moreover, both HU-446 and HU-465, at 5 and 10 μm, inhibited the release of IL-17, a key autoimmune cytokine, from MOG35-55-stimulated T(MOG) cells. These results suggest that HU-446 and HU-465 have anti-inflammatory potential in inflammatory and autoimmune diseases.

  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. Saccharomyces cerevisiae decreases inflammatory responses induced by F4+ enterotoxigenic Escherichia coli in porcine intestinal epithelial cells.

    Science.gov (United States)

    Zanello, Galliano; Meurens, François; Berri, Mustapha; Chevaleyre, Claire; Melo, Sandrine; Auclair, Eric; Salmon, Henri

    2011-05-15

    Probiotic yeasts may provide protection against intestinal inflammation induced by enteric pathogens. In piglets, infection with F4+ enterotoxigenic Escherichia coli (ETEC) leads to inflammation, diarrhea and intestinal damage. In this study, we investigated whether the yeast strains Saccharomyces cerevisiae (Sc, strain CNCM I-3856) and S. cerevisiae variety boulardii (Sb, strain CNCM I-3799) decreased the expression of pro-inflammatory cytokines and chemokines in intestinal epithelial IPI-2I cells cultured with F4+ ETEC. Results showed that viable Sc inhibited the ETEC-induced TNF-α gene expression whereas Sb did not. In contrast, killed Sc failed to inhibit the expression of pro-inflammatory genes. This inhibition was dependent on secreted soluble factors. Sc culture supernatant decreased the TNF-α, IL-1α, IL-6, IL-8, CXCL2 and CCL20 ETEC-induced mRNA. Furthermore, Sc culture supernatant filtrated fraction yeast strains onto inflammation.

  11. Lenalidomide enhances antigen-specific activity and decreases CD45RA expression of T cells from patients with multiple myeloma.

    Science.gov (United States)

    Neuber, Brigitte; Herth, Isabelle; Tolliver, Claudia; Schoenland, Stefan; Hegenbart, Ute; Hose, Dirk; Witzens-Harig, Mathias; Ho, Anthony D; Goldschmidt, Hartmut; Klein, Bernard; Hundemer, Michael

    2011-07-15

    The aim of this study was to investigate whether the specific T cell response against the multiple myeloma Ag HM1.24 is enhanced by the immunomodulatory drug lenalidomide (Revlimid). Ag-specific CD3(+)CD8(+) T cells against the HM1.24 Ag were expanded in vitro by dendritic cells in 29 healthy donors and 26 patients with plasma cell dyscrasias. Ag-specific activation was analyzed by IFN-γ, granzyme B, and perforin secretion using ELISA, ELISPOT assay, and intracellular staining, and generation of Ag-specific T cells was analyzed by tetramer staining. Expression of T cell maturation markers (CD45RA, CD45R0, CCR7, and CD28) was investigated by flow cytometry. We found that activation of HM1.24-specific T cells from healthy donors and patients with plasma cell dyscrasias was enhanced significantly by lenalidomide and furthermore that the impact of lenalidomide on T cells depends on the duration of the exposure. Notably, lenalidomide supports the downregulation of CD45RA on T cells upon activation, observed in healthy donors and in patients in vitro and also in patients during lenalidomide therapy in vivo. We showed for the first time, to our knowledge, that lenalidomide enhances the Ag-specific activation of T cells and the subsequent downregulation of CD45RA expression of T cells in vitro and in vivo.

  12. Phenotypic Modulation of Mesenteric Vascular Smooth Muscle Cells from Type 2 Diabetic Rats is Associated with Decreased Caveolin-1 Expression

    Directory of Open Access Journals (Sweden)

    Maria Alicia Carrillo-Sepulveda

    2014-10-01

    Full Text Available Aims: Diabetes-induced vascular complications are associated with vascular smooth muscle cell (VSMC phenotypic modulation, switching from a contractile to a synthetic-proliferative phenotype. Loss of caveolin-1 is involved with proliferation of VSMCs. We tested the hypothesis that mesenteric VSMCs from type 2 diabetic Goto-Kakizaki (GK rat undergo phenotypic modulation and it is linked to decreased caveolin-1 expression. Methods: VSMCs were isolated from mesenteric arteries from GK rats and age-matched control Wistar rats. Western blotting was used to determine expression of target proteins such as caveolin-1, calponin (marker of differentiation, and proliferating cell nuclear antigen (PCNA, marker of proliferation. In addition, we measured intracellular reactive oxygen species (ROS production using H2DCF-DA and activation of extracellular signal-regulated kinase (ERK1/2 by western blotting in VSMCs from GK stimulated with lipopolysaccharide (LPS, an endotoxin upregulated in diabetes. Results: Mesenteric VSMCs from diabetic GK rats exhibited decreased caveolin-1 and calponin expression and increased PCNA expression compared to control. Increased levels of ROS and phospho-ERK1/2 expression were also found in GK VSMCs. LPS augmented ROS and phosphorylated ERK1/2 levels to a greater extent in GK VSMCs than in control. Likewise, high glucose decreased caveolin-1 and calponin expression, increased PCNA expression and augmented ROS production in control mesenteric VSMCs. Conclusion: These results suggest that mesenteric VSMCs from diabetic GK rats undergo phenotypic modulation and it is associated with decreased caveolin-1 expression. These alterations may be due to enhanced inflammatory stimuli and glucose levels present in diabetic milieu.

  13. Prognostic Significance of Decreased Expression of Six Large Common Fragile Site Genes in Oropharyngeal Squamous Cell Carcinomas

    Directory of Open Access Journals (Sweden)

    Ge Gao

    2014-12-01

    Full Text Available Common fragile sites (CFSs are large regions with profound genomic instability that often span extremely large genes a number of which have been found to be important tumor suppressors. RNA sequencing previously revealed that there was a group of six large CFS genes which frequently had decreased expression in oropharyngeal squamous cell carcinomas (OPSCCs and real-time reverse transcriptase polymerase chain reaction experiments validated that these six large CFS genes (PARK2, DLG2, NBEA, CTNNA3, DMD, and FHIT had decreased expression in most of the tumor samples. In this study, we investigated whether the decreased expression of these genes has any clinical significance in OPSCCs. We analyzed the six CFS large genes in 45 OPSCC patients and found that 27 (60% of the OPSCC tumors had decreased expression of these six genes. When we correlated the expression of these six genes to each patient’s clinical records, for 11 patients who had tumor recurrence, 10 of them had decreased expression of almost all 6 genes. When we divided the patients into two groups, one group with decreased expression of the six genes and the other group with either slight changes or increased expression of the six genes, we found that there is significant difference in the incidence of tumor recurrence between these two groups by Kaplan-Meier plot analysis (P < .05. Our results demonstrated that those OPSCC tumors with decreased expression of this select group of six large CFS genes were much more likely to be associated with tumor recurrence and these genes are potential prognostic markers for predicting tumor recurrence in OPSCC.

  14. 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.

  15. Rapid copper acquisition by developing murine mesothelioma: decreasing bioavailable copper slows tumor growth, normalizes vessels and promotes T cell infiltration.

    Directory of Open Access Journals (Sweden)

    Andrew Crowe

    Full Text Available Copper, an essential trace element acquired through nutrition, is an important co-factor for pro-angiogenic factors including vascular endothelial growth factor (VEGF. Decreasing bioavailable copper has been used as an anti-angiogenic and anti-cancer strategy with promising results. However, the role of copper and its potential as a therapy in mesothelioma is not yet well understood. Therefore, we monitored copper levels in progressing murine mesothelioma tumors and analyzed the effects of lowering bioavailable copper. Copper levels in tumors and organs were assayed using atomic absorption spectrophotometry. Mesothelioma tumors rapidly sequestered copper at early stages of development, the copper was then dispersed throughout growing tumor tissues. These data imply that copper uptake may play an important role in early tumor development. Lowering bioavailable copper using the copper chelators, penicillamine, trientine or tetrathiomolybdate, slowed in vivo mesothelioma growth but did not provide any cures similar to using cisplatin chemotherapy or anti-VEGF receptor antibody therapy. The impact of copper lowering on tumor blood vessels and tumor infiltrating T cells was measured using flow cytometry and confocal microscopy. Copper lowering was associated with reduced tumor vessel diameter, reduced endothelial cell proliferation (reduced Ki67 expression and lower surface ICAM/CD54 expression implying reduced endothelial cell activation, in a process similar to endothelial normalization. Copper lowering was also associated with a CD4(+ T cell infiltrate. In conclusion, these data suggest copper lowering is a potentially useful anti-mesothelioma treatment strategy that slows tumor growth to provide a window of opportunity for inclusion of other treatment modalities to improve patient outcomes.

  16. Specific siRNA Targeting Receptor for Advanced Glycation End Products (RAGE Decreases Proliferation in Human Breast Cancer Cell Lines

    Directory of Open Access Journals (Sweden)

    Sheng Liu

    2013-04-01

    Full Text Available Receptor for Advanced Glycation End Products (RAGE is an oncogenic trans-membranous receptor overexpressed in various human cancers. However, the role of RAGE in breast cancer development and proliferation is still unclear. In this study, we demonstrated that RAGE expression levels are correlated to the degree of severity of breast cancer. Furthermore, there is a decrease in the proliferation of all sub-types of breast cancer, MCF-7, SK-Br-3 and MDA-MB-231, as a result of the effect of RAGE siRNA. RAGE siRNA arrested cells in the G1 phase and inhibited DNA synthesis (p < 0.05. Moreover, qRT-PCR and Western Blot results demonstrated that RAGE siRNA decreases the expression of transcriptional factor NF-κB p65 as well as the expression of cell proliferation markers PCNA and cyclinD1. RAGE and RAGE ligands can thus be considered as possible targets for breast cancer management and therapy.

  17. Cyclosporine A decreases the fluconazole minimum inhibitory concentration of Candida albicans clinical isolates but not biofilm formation and cell growth.

    Science.gov (United States)

    Wibawa, T; Nurrokhman; Baly, I; Daeli, P R; Kartasasmita, G; Wijayanti, N

    2015-03-01

    Among the genus Candida, Candida albicans is the most abundant species in humans. One of the virulent factors of C. albicans is its ability to develop biofilm. Biofilm forming microbes are characterized by decreasing of its susceptibility to antibiotics and antifungal. The fungicidal effect of fluconazole may be enhanced by cyclosporine A in laboratory engineered C. albicans strains. The aim of this work is to analyze the synergistic effect of cyclosporine A with fluconazole in C. albicans clinical isolates and the effect of cycolsporine A alone in the biofilm formation. Six fluconazole resistant and six sensitive C. albicans clinical isolates were analyzed for its minimum inhibitory concentration (MICs), biofilm formation, and cell growths. A semi-quantitative XTT [2,3-bis(2-methoxy-4-nitro-5- sulfo-phenyl)-2H-tetrazolium-5-carboxanilide] reduction assay was conducted to measure the biofilm formation. Cyclosporine A has synergistic effect with fluconazole that was shown by decreasing MICs of both fluconazole resistant and sensitive C. albicans clinical isolates. However, cyclosporine A alone did not influence the biofilm formation and cell growth of both fluconazole resistant and sensitive C. albicans clinical isolates. These results indicated that cyclosporine A might be a promising candidate of adjuvant therapy for fluconazole against both fluconazole resistant and sensitive C. albicans clinical isolates.

  18. Isoorientin induces apoptosis, decreases invasiveness, and downregulates VEGF secretion by activating AMPK signaling in pancreatic cancer cells

    Directory of Open Access Journals (Sweden)

    Ye T

    2016-12-01

    Full Text Available Tingting Ye,1 Jiadong Su,1 Chaohao Huang,1 Dinglai Yu,1 Shengjie Dai,1 Xince Huang,1 Bicheng Chen,1,2 Mengtao Zhou1 1Department of Surgery, The First Affiliated Hospital, Wenzhou Medical University, 2Zhejiang Provincial Top Key Discipline in Surgery, Wenzhou Key Laboratory of Surgery, Wenzhou, Zhejiang Province, People’s Republic of China Abstract: Isoorientin (or homoorientin is a flavone, which is a chemical flavonoid-like compound, and a 6-C-glucoside of luteolin. Isoorientin has been demonstrated to have anti-cancer activities against various tumors, but its effects on pancreatic cancer (PC have not been studied in detail. In this study, we aim to investigate whether isoorientin has potential anti-PC effects and its underlying mechanism. In PC, isoorientin strongly inhibited the survival of the cells, induced cell apoptosis, and decreased its malignancy by reversing the expression of epithelial–mesenchymal transition and matrix metalloproteinase and decreased vascular endothelial growth factor expression. Meanwhile, we investigated the activity of the AMP-activated protein kinase (AMPK signaling pathway after isoorientin treatment, which was forcefully activated by isoorientin, as expected. In addition, in the PC cells that were transfected with lentivirus to interfere with the expression of the gene PRKAA1, there were no differences in the apoptosis rate and the expression of malignancy biomarkers in the tumors of the isoorientin-treated and untreated groups. Thus, we demonstrated that isoorientin has potential antitumor effects via the AMPK signaling pathway, and isoorientin merits further investigation. Keywords: pancreatic cancer, AMPK, isoorientin, apoptosis, invasiveness, VEGF

  19. In vivo Cigarette Smoke Exposure Decreases CCL20, SLPI, and BD-1 Secretion by Human Primary Nasal Epithelial Cells

    Science.gov (United States)

    Jukosky, James; Gosselin, Benoit J.; Foley, Leah; Dechen, Tenzin; Fiering, Steven; Crane-Godreau, Mardi A.

    2016-01-01

    Smokers and individuals exposed to second-hand cigarette smoke have a higher risk of developing chronic sinus and bronchial infections. This suggests that cigarette smoke (CS) has adverse effects on immune defenses against pathogens. Epithelial cells are important in airway innate immunity and are the first line of defense against infection. Airway epithelial cells not only form a physical barrier but also respond to the presence of microbes by secreting antimicrobials, cytokines, and chemokines. These molecules can lyse infectious microorganisms and/or provide signals critical to the initiation of adaptive immune responses. We examined the effects of CS on antimicrobial secretions of primary human nasal epithelial cells (PHNECs). Compared to non-CS-exposed individuals, PHNEC from in vivo CS-exposed individuals secreted less chemokine ligand (C-C motif) 20 (CCL20), Beta-defensin 1 (BD-1), and SLPI apically, less BD-1 and SLPI basolaterally, and more CCL20 basolaterally. Cigarette smoke extract (CSE) exposure in vitro decreased the apical secretion of CCL20 and beta-defensin 1 by PHNEC from non-CS-exposed individuals. Exposing PHNEC from non-CS exposed to CSE also significantly decreased the levels of many mRNA transcripts that are involved in immune signaling. Our results show that in vivo or in vitro exposure to CS alters the secretion of key antimicrobial peptides from PHNEC, but that in vivo CS exposure is a much more important modifier of antimicrobial peptide secretion. Based on the gene expression data, it appears that CSE disrupts multiple immune signaling pathways in PHNEC. Our results provide mechanistic insight into how CS exposure alters the innate immune response and increases an individual’s susceptibility to pathogen infection. PMID:26793127

  20. Overexpression of CD97 confers an invasive phenotype in glioblastoma cells and is associated with decreased survival of glioblastoma patients.

    Directory of Open Access Journals (Sweden)

    Michael Safaee

    Full Text Available Mechanisms of invasion in glioblastoma (GBM relate to differential expression of proteins conferring increased motility and penetration of the extracellular matrix. CD97 is a member of the epidermal growth factor seven-span transmembrane family of adhesion G-protein coupled receptors. These proteins facilitate mobility of leukocytes into tissue. In this study we show that CD97 is expressed in glioma, has functional effects on invasion, and is associated with poor overall survival. Glioma cell lines and low passage primary cultures were analyzed. Functional significance was assessed by transient knockdown using siRNA targeting CD97 or a non-target control sequence. Invasion was assessed 48 hours after siRNA-mediated knockdown using a Matrigel-coated invasion chamber. Migration was quantified using a scratch assay over 12 hours. Proliferation was measured 24 and 48 hours after confirmed protein knockdown. GBM cell lines and primary cultures were found to express CD97. Knockdown of CD97 decreased invasion and migration in GBM cell lines, with no difference in proliferation. Gene-expression based Kaplan-Meier analysis was performed using The Cancer Genome Atlas, demonstrating an inverse relationship between CD97 expression and survival. GBMs expressing high levels of CD97 were associated with decreased survival compared to those with low CD97 (p = 0.007. CD97 promotes invasion and migration in GBM, but has no effect on tumor proliferation. This phenotype may explain the discrepancy in survival between high and low CD97-expressing tumors. This data provides impetus for further studies to determine its viability as a therapeutic target in the treatment of GBM.

  1. TLR9-induced miR-155 and Ets-1 decrease expression of CD1d on B cells in SLE.

    Science.gov (United States)

    Liu, Fei; Fan, Hongye; Ren, Deshan; Dong, Guanjun; Hu, Erling; Ji, Jianjian; Hou, Yayi

    2015-07-01

    B cells present lipid antigens to CD1d-restricted invariant natural killer T (iNKT) cells to maintain autoimmune tolerance, and this process is disrupted in systemic lupus erythematosus (SLE). Inflammation may inhibit CD1d expression to exacerbate the pathology of lupus. However, how inflammation regulates CD1d expression on B cells is unclear in SLE. In the present study, we showed that the surface expression of CD1d on B cells from SLE mice was decreased and that stimulation of inflammatory responses through TLR9 decreased the membrane and total CD1d levels of CD1d on B cells. Moreover, inflammation-related microRNA-155 (miR-155) negatively correlated with the expression of CD1d in B cells. miR-155 directly targeted the 3'-untranslated region (3'-UTR) of CD1d upon TLR9 activation in both humans and mice. The inhibitory effects of miR-155 on CD1d expression in B cells impaired their antigen-presenting capacity to iNKT cells. In addition, Ets-1, a susceptibility gene of SLE, also directly regulated the expression of the CD1d gene at the transcriptional level. These findings provide new insight into the mechanism underlying decreased CD1d expression on B cells in SLE, suggesting that inhibition of inflammation may increase CD1d expression in B cells to ameliorate SLE via modulating iNKT cells.

  2. miR-143 decreases COX-2 mRNA stability and expression in pancreatic cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Pham, Hung [Department of Surgery, UCLA Center of Excellence in Pancreatic Diseases, UCLA David Geffen School of Medicine, University of California – Los Angeles, Los Angeles, CA 90095 (United States); Department of Medicine, Veterans Affair Greater Los Angeles Healthcare System, Los Angeles, CA 90073 (United States); Ekaterina Rodriguez, C.; Donald, Graham W.; Hertzer, Kathleen M.; Jung, Xiaoman S.; Chang, Hui-Hua; Moro, Aune; Reber, Howard A.; Hines, O. Joe [Department of Surgery, UCLA Center of Excellence in Pancreatic Diseases, UCLA David Geffen School of Medicine, University of California – Los Angeles, Los Angeles, CA 90095 (United States); Eibl, Guido, E-mail: geibl@mednet.ucla.edu [Department of Surgery, UCLA Center of Excellence in Pancreatic Diseases, UCLA David Geffen School of Medicine, University of California – Los Angeles, Los Angeles, CA 90095 (United States)

    2013-09-13

    Highlights: •Pancreatic cancer cells express low miR-143 levels and elevated p-MEK, p-MAPK and RREB1. •MEK inhibitors U0126 and PD98059 increase miR-143 expression. •miR-143 decreases COX-2 mRNA stability and expression and PGE{sub 2}. •miR-143 decreases p-p38MAPK, p-MEK, p-MAPK and RREB1 expression. -- Abstract: Small non-coding RNAs, microRNAs (miRNA), inhibit the translation or accelerate the degradation of message RNA (mRNA) by targeting the 3′-untranslated region (3′-UTR) in regulating growth and survival through gene suppression. Deregulated miRNA expression contributes to disease progression in several cancers types, including pancreatic cancers (PaCa). PaCa tissues and cells exhibit decreased miRNA, elevated cyclooxygenase (COX)-2 and increased prostaglandin E{sub 2} (PGE{sub 2}) resulting in increased cancer growth and metastases. Human PaCa cell lines were used to demonstrate that restoration of miRNA-143 (miR-143) regulates COX-2 and inhibits cell proliferation. miR-143 were detected at fold levels of 0.41 ± 0.06 in AsPC-1, 0.20 ± 0.05 in Capan-2 and 0.10 ± 0.02 in MIA PaCa-2. miR-143 was not detected in BxPC-3, HPAF-II and Panc-1 which correlated with elevated mitogen-activated kinase (MAPK) and MAPK kinase (MEK) activation. Treatment with 10 μM of MEK inhibitor U0126 or PD98059 increased miR-143, respectively, by 187 ± 18 and 152 ± 26-fold in BxPC-3 and 182 ± 7 and 136 ± 9-fold in HPAF-II. miR-143 transfection diminished COX-2 mRNA stability at 60 min by 2.6 ± 0.3-fold in BxPC-3 and 2.5 ± 0.2-fold in HPAF-II. COX-2 expression and cellular proliferation in BxPC-3 and HPAF-II inversely correlated with increasing miR-143. PGE{sub 2} levels decreased by 39.3 ± 5.0% in BxPC-3 and 48.0 ± 3.0% in HPAF-II transfected with miR-143. Restoration of miR-143 in PaCa cells suppressed of COX-2, PGE{sub 2}, cellular proliferation and MEK/MAPK activation, implicating this pathway in regulating miR-143 expression.

  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 cells with TRECs was 3.6% +/- 0.7% compared with 14.3% +/- 2.2% in controls, P ... cytometric determination of lymphocyte subsets, including the naive CD4 count. Furthermore, to determine thymic output, cord blood mononuclear cells were used for determination of T-cell receptor excision circles (TRECs). Evaluation of progenitor cell function was done by means of colony-forming cell assay......). 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...

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

    Science.gov (United States)

    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 the DHA-induced acidification, we conducted experiments in Ca2+-free (0% Ca2+) and Ca2+-containing (100% Ca2+) buffer. We observed that there was no difference in the degree of DHA-induced transient acidification in both the experimental conditions, though pHi recovery was faster in 0% Ca2+ medium than that in 100% Ca2+ medium. In the presence of BAPTA, a calcium chelator, a rapid recovery of DHA-induced acidosis was observed. Furthermore, addition of CaCl2 into 0% Ca2+ medium curtailed DHA-evoked rapid pHi recovery. In 0% Ca2+ medium, containing BAPTA, DHA did not evoke increases in [Ca2+]i, though this fatty acid still induced a rapid acidification in these cells. These observations suggest that calcium is implicated in the long-lasting DHA-induced acidosis. DHA-induced rapid acidification may be due to its deprotonation in the plasma membrane (flip-flop model), as suggested by the following observations: (1) DHA with a –COOH group induced intracellular acidification, but this fatty acid with a –COOCH3 group failed to do so, and (2) DHA, but not propionic acid, -induced acidification was completely reversed by addition of fatty acid-free bovine serum albumin in these cells. These results suggest that DHA induces acidosis via deprotonation and Ca2+ mobilization in human T-cells. PMID:14645139

  5. 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.

  6. The effect of decrease in salinity on the dynamics of abundance and the cell size of Corethron Hystrix (Bacillariophyta) in laboratory culture

    Science.gov (United States)

    Aizdaicher, Nina A.; Markina, Zhanna V.

    2010-03-01

    Effect of salinity on abundance dynamics and cell size of microalga Corethron hystrix Hensen (Bacillariophyta) were studied. C. hystrix can normally grow within a rather narrow salinity range between 32 and 28‰. The viable cells of this microalga change their morphological characters at a salinity of 24‰. This salinity level probably marks the beginning of cell division restriction, because the general number of cells by the end of the experiment was lower than in the control. The decrease of salinity to 16‰ caused pronounced irreversible morphological changes: cell height increased, chloroplasts compressed, protoplasm became granular, cytoplasm retracted, and spines shortened.

  7. Decrease of reactive oxygen species-related biomarkers in the tissue-mimic 3D spheroid culture of human lung cells exposed to zinc oxide nanoparticles.

    Science.gov (United States)

    Kim, Eunjoo; Jeon, Won Bae; Kim, Soonhyun; Lee, Soo-Keun

    2014-05-01

    Common 2-dimensional (2D) cell cultures do not adequately represent cell-cell and cell-matrix signaling and substantially different diffusion/transport pathways. To obtain tissue-mimic information on nanoparticle toxicity from in vitro cell tests, we used a 3-dimensional (3D) culture of human lung cells (A549) prepared with elastin-like peptides modified with an arginine-glycine-aspartate motif. The 3D cells showed different cellular phenotypes, gene expression profiles, and functionalities compared to the 2D cultured cells. In gene array analysis, 3D cells displayed the induced extracellular matrix (ECM)-related biological functions such as cell-to-cell signaling and interaction, cellular function and maintenance, connective tissue development and function, molecular transport, and tissue morphology. Additionally, the expression of ECM-related molecules, such as laminin, fibronectin, and insulin-like growth factor binding protein 3 (IGFBP3), was simultaneously induced at both mRNA and protein levels. When 0.08-50 microg/ml zinc oxide nanoparticles (ZnO-NPs) were administered to 2D and 3D cells, the cell proliferation was not significantly changed. The level of molecular markers for oxidative stress, such as superoxide dismutase (SOD), Bcl-2, ATP synthase, and Complex IV (cytochrome C oxidase), was significantly reduced in 2D culture when exposed to 10 microg/ml ZnO-NPs, but no significant decrease was detected in 3D culture when exposed to the same concentration of ZnO-NPs. In conclusion, the tissue-mimic phenotype and functionality of 3D cells could be achieved through the elevated expression of ECM components. The 3D cells were expected to help to better predict the nanotoxicity of ZnO-NPs at tissue-level by increased cell-cell and cell-ECM adhesion and signaling. The tissue-mimic morphology would also be useful to simulate the diffusion/transport of the nanoparticles in vitro.

  8. Decrease of back recombination rate in CdS quantum dots sensitized solar cells using reduced graphene oxide

    Institute of Scientific and Technical Information of China (English)

    Ali Badawi

    2015-01-01

    The photovoltaic performance of CdS quantum dots sensitized solar cells (QDSSCs) using the 0.2 wt%of reduced graphene oxide and TiO2 nanoparticles (RGO+TiO2 nanocomposite) photoanode is investigated. CdS QDs are adsorbed onto RGO+TiO2 nanocomposite films by the successive ionic layer adsorption and reaction (SILAR) technique for several cycles. The current density–voltage (J–V ) characteristic curves of the assembled QDSSCs are measured at AM1.5 sim-ulated sunlight. The optimal photovoltaic performance for CdS QDSSC was achieved for six SILAR cycles. Solar cells based on the RGO+TiO2 nanocomposite photoanode achieve a 33%increase in conversion efficiency (η) compared with those based on plain TiO2 nanoparticle (NP) photoanodes. The electron back recombination rates decrease significantly for CdS QDSSCs based on RGO+TiO2 nanocomposite photoanodes. The lifetime constant (τ) for CdS QDSSC based on the RGO+TiO2 nanocomposite photoanode is at least one order of magnitude larger than that based on the bare TiO2NPs photoanode.

  9. Decrease of back recombination rate in CdS quantum dots sensitized solar cells using reduced graphene oxide

    Science.gov (United States)

    Badawi, Ali

    2015-04-01

    The photovoltaic performance of CdS quantum dots sensitized solar cells (QDSSCs) using the 0.2 wt% of reduced graphene oxide and TiO2 nanoparticles (RGO+TiO2 nanocomposite) photoanode is investigated. CdS QDs are adsorbed onto RGO+TiO2 nanocomposite films by the successive ionic layer adsorption and reaction (SILAR) technique for several cycles. The current density-voltage (J-V) characteristic curves of the assembled QDSSCs are measured at AM1.5 simulated sunlight. The optimal photovoltaic performance for CdS QDSSC was achieved for six SILAR cycles. Solar cells based on the RGO+TiO2 nanocomposite photoanode achieve a 33% increase in conversion efficiency (η) compared with those based on plain TiO2 nanoparticle (NP) photoanodes. The electron back recombination rates decrease significantly for CdS QDSSCs based on RGO+TiO2 nanocomposite photoanodes. The lifetime constant (τ) for CdS QDSSC based on the RGO+TiO2 nanocomposite photoanode is at least one order of magnitude larger than that based on the bare TiO2 NPs photoanode. Project supported by the Fund from Taif University, Saudi Arabia (Grant No. 1/435/3524).

  10. Hyperglycemia alters the schwann cell mitochondrial proteome and decreases coupled respiration in the absence of superoxide production.

    Science.gov (United States)

    Zhang, Liang; Yu, Cuijuan; Vasquez, Francisco E; Galeva, Nadya; Onyango, Isaac; Swerdlow, Russell H; Dobrowsky, Rick T

    2010-01-01

    Hyperglycemia-induced mitochondrial dysfunction contributes to sensory neuron pathology in diabetic neuropathy. Although Schwann cells (SCs) also undergo substantial degeneration in diabetic neuropathy, the effect of hyperglycemia on the SC mitochondrial proteome and mitochondrial function has not been examined. Stable isotope labeling with amino acids in cell culture (SILAC) was used to quantify the temporal effect of hyperglycemia on the mitochondrial proteome of primary SCs isolated from neonatal rats. Of 317 mitochondrial proteins identified, about 78% were quantified and detected at multiple time points. Pathway analysis indicated that proteins associated with mitochondrial dysfunction, oxidative phosphorylation, the TCA cycle, and detoxification were significantly increased in expression and over-represented. Assessing mitochondrial respiration in intact SCs indicated that hyperglycemia increased the overall rate of oxygen consumption but decreased the efficiency of coupled respiration. Although a glucose-dependent increase in superoxide production occurs in embryonic sensory neurons, hyperglycemia did not induce a substantial change in superoxide levels in SCs. This correlated with a 1.9-fold increase in Mn superoxide dismutase expression, which was confirmed by immunoblot and enzymatic activity assays. These data support that hyperglycemia alters mitochondrial respiration and can cause remodeling of the SC mitochondrial proteome independent of significant contributions from glucose-induced superoxide production.

  11. Histone deacetylase inhibition decreases cholesterol levels in neuronal cells by modulating key genes in cholesterol synthesis, uptake and efflux.

    Directory of Open Access Journals (Sweden)

    Maria João Nunes

    Full Text Available Cholesterol is an essential component of the central nervous system and increasing evidence suggests an association between brain cholesterol metabolism dysfunction and the onset of neurodegenerative disorders. Interestingly, histone deacetylase inhibitors (HDACi such as trichostatin A (TSA are emerging as promising therapeutic approaches in neurodegenerative diseases, but their effect on brain cholesterol metabolism is poorly understood. We have previously demonstrated that HDACi up-regulate CYP46A1 gene transcription, a key enzyme in neuronal cholesterol homeostasis. In this study, TSA was shown to modulate the transcription of other genes involved in cholesterol metabolism in human neuroblastoma cells, namely by up-regulating genes that control cholesterol efflux and down-regulating genes involved in cholesterol synthesis and uptake, thus leading to an overall decrease in total cholesterol content. Furthermore, co-treatment with the amphipathic drug U18666A that can mimic the intracellular cholesterol accumulation observed in cells of Niemman-Pick type C patients, revealed that TSA can ameliorate the phenotype induced by pathological cholesterol accumulation, by restoring the expression of key genes involved in cholesterol synthesis, uptake and efflux and promoting lysosomal cholesterol redistribution. These results clarify the role of TSA in the modulation of neuronal cholesterol metabolism at the transcriptional level, and emphasize the idea of HDAC inhibition as a promising therapeutic tool in neurodegenerative disorders with impaired cholesterol metabolism.

  12. Maraviroc decreases CCL8-mediated migration of CCR5(+) regulatory T cells and reduces metastatic tumor growth in the lungs.

    Science.gov (United States)

    Halvorsen, E C; Hamilton, M J; Young, A; Wadsworth, B J; LePard, N E; Lee, H N; Firmino, N; Collier, J L; Bennewith, K L

    2016-06-01

    Regulatory T cells (Tregs) play a crucial physiological role in the regulation of immune homeostasis, although recent data suggest Tregs can contribute to primary tumor growth by suppressing antitumor immune responses. Tregs may also influence the development of tumor metastases, although there is a paucity of information regarding the phenotype and function of Tregs in metastatic target organs. Herein, we demonstrate that orthotopically implanted metastatic mammary tumors induce significant Treg accumulation in the lungs, which is a site of mammary tumor metastasis. Tregs in the primary tumor and metastatic lungs express high levels of C-C chemokine receptor type 5 (CCR5) relative to Tregs in the mammary fat pad and lungs of tumor-free mice, and Tregs in the metastatic lungs are enriched for CCR5 expression in comparison to other immune cell populations. We also identify that C-C chemokine ligand 8 (CCL8), an endogenous ligand of CCR5, is produced by F4/80(+) macrophages in the lungs of mice with metastatic primary tumors. Migration of Tregs toward CCL8 ex vivo is reduced in the presence of the CCR5 inhibitor Maraviroc. Importantly, treatment of mice with Maraviroc (MVC) reduces the level of CCR5(+) Tregs and metastatic tumor burden in the lungs. This work provides evidence of a CCL8/CCR5 signaling axis driving Treg recruitment to the lungs of mice bearing metastatic primary tumors, representing a potential therapeutic target to decrease Treg accumulation and metastatic tumor growth.

  13. Inhibition of CXCR4 activity with AMD3100 decreases invasion of human colorectal cancer cells in vitro

    Institute of Scientific and Technical Information of China (English)

    Ji-Kun Li; Liang Yu; Yun Shen; Li-Sheng Zhou; Yi-Cheng Wang; Jian-Hai Zhang

    2008-01-01

    AIM: To investigate the effect and mechanism of blockade of the CXC chemokine receptor-4 (CXCR4) signaling pathway by AMD3100, a small non-peptide CXCR4 inhibitor, on invasion and metastasis of colorectal cancer cells in vitro.METHODS: Human colorectal cancer cell line SW480 was treated with AMD3100 at different final concentrations.3-(4,5-dimethylthiazole-2-yl)-2.5-dipheny-ltetrazolium bromide (MTT) assay was used to detect the effect of AMD3100 on cell proliferation. The invasion ability of SW480 cells was determined by cell invasion assay kit.In the presence of AMD3100, the CXCL12-mediated migratory response of SW480 cells was tested by classical chemotaxis assays. RT-PCR analysis and Western blotting were used to detect the expression of vascular endothelial growth factor (VEGF), matrix metalloproteinase-2 (MMP-2)and -9 (MMP-9) in SW480 cells.RESULTS: Cell viability was significantly suppressed by AMD3100 in a dose-dependent manner. AMD3100 (100and 1000 ng/mL) significantly inhibited the invasion ability of SW480 cells. Treatment with AMD3100markedly reduced the expression of VEGF and MMP-9but not MMP-2 in SW480 cells.CONCLUSION: The CXCL12/CXCR4 system is an important mediator of proliferation and invasion of CXCR4-expressing colorectal cancer cells, AMD3100inhibited invasion and metastasis activity of the colorectal cancer cell line SW480 through down-regulation of VEGF and MMP-9 expression.

  14. AGE-modified basement membrane cooperates with Endo180 to promote epithelial cell invasiveness and decrease prostate cancer survival

    DEFF Research Database (Denmark)

    Rodriguez-Teja, Mercedes; Gronau, Julian H; Breit, Claudia

    2015-01-01

    Biomechanical strain imposed by age-related thickening of the basal lamina and augmented tissue stiffness in the prostate gland coincides with increased cancer risk. Here we hypothesized that the structural alterations in the basal lamina associated with age can induce mechanotransduction pathways...... in prostate epithelial cells (PECs) to promote invasiveness and cancer progression. To demonstrate this, we developed a 3D model of PEC acini in which thickening and stiffening of basal lamina matrix was induced by advanced glycation end-product (AGE)-dependent non-enzymatic crosslinking of its major......(Δ) (Ex2-6/) (Δ) (Ex2-6) mice, with constitutively exposed CTLD2 and decreased survival of men with early (non-invasive) prostate cancer with high epithelial Endo180 expression and levels of AGE. These findings indicate that AGE-dependent modification of the basal lamina induces invasive behaviour...

  15. XRCC3 C18067T polymorphism contributes a decreased risk to both basal cell carcinoma and squamous cell carcinoma: evidence from a meta-analysis.

    Directory of Open Access Journals (Sweden)

    Xu Chen

    Full Text Available BACKGROUND: The X-ray repair cross-complementing group 3 (XRCC3 in homologous recombination repair (HRR pathway plays a very important role in DNA double-strand break repair (DSBR. Variations in the XRCC3 gene might lead to altered protein structure or function which may change DSBR efficiency and result in cancer. The XRCC3 C18067T polymorphism has been reported to be associated with skin cancer susceptibility, yet the results of these previous results have been inconsistent or controversial. To derive a more precise estimation of the association, we conducted a meta-analysis. METHODS: The quality of the studies was assessed according to a predefined scale. The association between the XRCC3 C18067T polymorphism and skin cancer risk was assessed by odds ratios (ORs together with their 95% confidence intervals (CIs. RESULTS: Overall, no significant association was observed between XRCC3 C18067T polymorphism and skin cancer risk in any genetic model. Stratified analyses according to tumor type, significant association was found in the relationship between XRCC3 C18067T polymorphism and nonmelanoma skin cancer risk (homozygote comparison TT versus CC: OR = 0.74, 95%CI = 0.61-0.90, P = 0.003; recessive model TT versus TC/CC: OR = 0.81, 95%CI = 0.68-0.95, P = 0.01. Furthermore, significant association was also observed in XRCC3 C18067T polymorphism with both basal cell carcinoma risk (homozygote comparison TT versus CC: OR = 0.70, 95%CI = 0.53-0.92, P = 0.011; recessive model TT versus. TC/CC: OR = 0.74, 95%CI = 0.60-0.92, P = 0.007 and squamous cell carcinoma risk (heterozygote comparison TT versus .CC: OR = 0.81, 95%CI = 0.67-0.99, P = 0.04; dominant model TT/TC versus .CC: OR = 0.81, 95%CI = 0.68-0.98, P = 0.029. CONCLUSION: The present meta-analysis demonstrates that XRCC3 C18067T polymorphism was not associated with risk of cutaneous melanoma but contributed a decreased risk to both basal cell carcinoma and squamous cell carcinoma.

  16. Ascorbic acid inhibits TPA-induced HL-60 cell differentiation by decreasing cellular H₂O₂ and ERK phosphorylation.

    Science.gov (United States)

    Yiang, Giou-Teng; Chen, Jen-Ni; Wu, Tsai-Kun; Wang, Hsueh-Fang; Hung, Yu-Ting; Chang, Wei-Jung; Chen, Chinshuh; Wei, Chyou-Wei; Yu, Yung-Luen

    2015-10-01

    Retinoic acid (RA), vitamin D and 12-O‑tetradecanoyl phorbol-13-acetate (TPA) can induce HL-60 cells to differentiate into granulocytes, monocytes and macrophages, respectively. Similar to RA and vitamin D, ascorbic acid also belongs to the vitamin family. High‑dose ascorbic acid (>100 µM) induces HL‑60 cell apoptosis and induces a small fraction of HL‑60 cells to express the granulocyte marker, CD66b. In addition, ascorbic acid exerts an anti‑oxidative stress function. Oxidative stress is required for HL‑60 cell differentiation following treatment with TPA, however, the effect of ascorbic acid on HL‑60 cell differentiation in combination with TPA treatment remains to be fully elucidated. The aim of the present study was to investigate the cellular effects of ascorbic acid treatment on TPA-differentiated HL-60 cells. TPA-differentiated HL-60 cells were used for this investigation, this study and the levels of cellular hydrogen peroxide (H2O2), caspase activity and ERK phosphorylation were determined following combined treatment with TPA and ascorbic acid. The results demonstrated that low‑dose ascorbic acid (5 µM) reduced the cellular levels of H2O2 and inhibited the differentiation of HL‑60 cells into macrophages following treatment with TPA. In addition, the results of the present study further demonstrated that low‑dose ascorbic acid inactivates the ERK phosphorylation pathway, which inhibited HL‑60 cell differentiation following treatment with TPA.

  17. Increased sesquiterpenoid biosynthesis and an apparent decrease in sterol biosynthesis in elicitor-treated tobacco cell suspension cultures

    Energy Technology Data Exchange (ETDEWEB)

    Voegeli, U.; Bhatt, P.N.; Chappell, J.

    1987-04-01

    Addition of fungel elicitor prepared from Phytophthora parasitica to tobacco cell suspension cultures leads to an increased production of the phytoalexin capsidiol. Capsidiol is a sesquiterpenoid which is most likely synthesized from farnesylpyrophosphat (FPP) by a bicyclic cyclase reaction. Because FPP is also a substrate for squalene synthetase and therefore a precursor of sterol biosynthesis, the question arises whether or not the accumulation of capsidiol in elicitor-treated cells occurs at the expense of sterol biosynthesis. (/sup 14/C)-acetate was given to elicitor-treated and control (no treatment) cell cultures and incorporation into sterols and capsidiol determined. No labeled capsidiol was detected in control cells. In elicitor-treated cells about 12-15% of the radioactivity taken up by the cells was incorporated into capsidiol. In contrast, control cells incorporated 4 times more radioactivity into sterols than elicitor-treated cells. Similar results were obtained using (/sup 3/H)-mevalonate as a precursor of capsidiol and sterol biosynthesis. Likely explanations for the apparently decline in sterol biosynthesis in elicitor-treated cells include: (1) inhibition of squalene synthetase; (2) induction of capsidiol synthesizing enzymes; and (3) metabolic channeling of FPP into capsidiol versus sterols. These possibilities will be discussed further together with other results.

  18. 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

    transport capacity and expression in the rat proximal tubule cell line SKPT0193 cl.2 (SKPT), which expresses rat PEPT2 (rPEPT2) in the apical membrane. Treatment of SKPT cells with EGF during cell culture growth caused a dose-dependent decrease in rPEPT2 transport capacity and expression, as determined...... suggests that this might be disadvantageous when studying PEPT2-mediated transport phenomena. These findings demonstrate for the first time EGF-mediated regulation of PEPT2 expression in a kidney cell line. The relevance for kidney regulation of peptide transport activity in physiological and...... 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...

  19. Iron deficiency decreases hemolysis in sickle cell anemia Anemia ferropriva diminui hemólise em anemia falciforme

    Directory of Open Access Journals (Sweden)

    Oswaldo Castro

    2009-02-01

    Full Text Available A woman with homozygous sickle cell disease developed severe iron deficiency due to long-standing uterine bleeding. At this point, the serum lactic dehydrogenase level was normal and the reticulocyte count was only minimally elevated. This suggested that the low red cell hemoglobin concentration that resulted from iron deficiency also decreased Hb S polymerization and lowered the hemolytic rate. Iron replacement led first to a substantially improved hemoglobin concentration with only a minimal increase in the hemolytic rate and secondarily to a modest further improvement in the hemoglobin concentration and a marked increase in the hemolytic rate. The hematologic changes observed in this patient, and those in other iron deficient sickle cell patients reported in the literature, suggest that it may be appropriate to consider the induction of an intermediate iron deficient stage as experimental treatment in adult sickle cell patients.Uma mulher com anemia falciforme homozigose para a Hb S evoluiu com anemia ferropriva grave devido a sangramento uterino prolongado. A dosagem de dehidrogenase lática era normal e a contagem de reticulócitos estava levemente aumentada. Isto sugere que concentrações baixas de hemoglobina, que resulta de anemia ferropriva, também diminuem a polimeração de Hb S e reduz a taxa de hemólise. O complemento de ferro levou, primeiramente, a uma concentração substancialmente maior de hemoglobina com apenas um aumento mínimo na taxa hemolítica e subsequentemente a um aumento leve adicional na concentração da hemoglobina e um aumento notável na taxa hemolítica. As mudanças hematológicas observadas nesta paciente e aquelas em outras pacientes com anemia falciforme e também deficientes de ferro relatadas na literatura sugerem que pode ser interessante considerar a indução de deficiência de ferro como tratamento experimental em pacientes adultos com anemia falciforme.

  20. Loss of CD28 on Peripheral T Cells Decreases the Risk for Early Acute Rejection after Kidney Transplantation.

    Directory of Open Access Journals (Sweden)

    Burç Dedeoglu

    Full Text Available End-stage renal disease patients have a dysfunctional, prematurely aged peripheral T-cell system. Here we hypothesized that the degree of premature T-cell ageing before kidney transplantation predicts the risk for early acute allograft rejection (EAR.222 living donor kidney transplant recipients were prospectively analyzed. EAR was defined as biopsy proven acute allograft rejection within 3 months after kidney transplantation. The differentiation status of circulating T cells, the relative telomere length and the number of CD31+ naive T cells were determined as T-cell ageing parameters.Of the 222 patients analyzed, 30 (14% developed an EAR. The donor age and the historical panel reactive antibody score were significantly higher (p = 0.024 and p = 0.039 respectively and the number of related donor kidney transplantation was significantly lower (p = 0.018 in the EAR group. EAR-patients showed lower CD4+CD28null T-cell numbers (p<0.01 and the same trend was observed for CD8+CD28null T-cell numbers (p = 0.08. No differences regarding the other ageing parameters were found. A multivariate Cox regression analysis showed that higher CD4+CD28null T-cell numbers was associated with a lower risk for EAR (HR: 0.65, p = 0.028. In vitro, a significant lower percentage of alloreactive T cells was observed within CD28null T cells (p<0.001.Immunological ageing-related expansion of highly differentiated CD28null T cells is associated with a lower risk for EAR.

  1. 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....... Leydig cell clusters of more than 15 cells in a cross-section, for which we proposed the name 'micronodules', were frequently seen in testicles exhibiting Sertoli-cell-only syndrome (SCO), a mixed pattern of impaired spermatogenesis, or complete spermatogenesis in combination with elevated FSH. Median......), 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...

  2. MicroRNA-29a is up-regulated in beta-cells by glucose and decreases glucose-stimulated insulin secretion

    DEFF Research Database (Denmark)

    Bagge, Annika; Clausen, Trine R; Larsen, Sylvester;

    2012-01-01

    Chronically elevated levels of glucose impair pancreatic beta-cell function while inducing beta-cell proliferation. MicroRNA-29a (miR-29a) levels are increased in several tissues in diabetic animals and mediate decreased insulin-stimulated glucose-transport of adipocytes. The aim was to investiga...

  3. Nampt Expression Decreases Age-Related Senescence in Rat Bone Marrow Mesenchymal Stem Cells by Targeting Sirt1

    Science.gov (United States)

    Ma, Cao; Pi, Chenchen; Yang, Yue; Lin, Lin; Shi, Yingai; Li, Yan; Li, Yulin; He, Xu

    2017-01-01

    Senescence restricts the development of applications involving mesenchymal stem cells (MSCs) in research fields, such as tissue engineering, and stem cell therapeutic strategies. Understanding the mechanisms underlying natural aging processes may contribute to the development of novel approaches to preventing age-related diseases or slowing individual aging processes. Nampt is a rate-limiting NAD biosynthetic enzyme that plays critical roles in energy metabolism, cell senescence and maintaining life spans. However, it remains unknown whether Nampt influences stem cell senescence. In this study, the function of Nampt was investigated using a rat model of natural aging. Our data show that Nampt expression was significantly lower in MSCs obtained from aged rats than in those obtained from young rats during physiological aging. Reducing the level of Nampt in aged MSCs resulted in lower intracellular concentrations of NAD+ and downregulated Sirt1 expression and activity. After the Nampt inhibitor FK866 was added, young MSCs were induced to become aged cells. The enhanced senescence was correlated with NAD+ depletion and Sirt1 activity attenuation. In addition, Nampt overexpression attenuated cell senescence in aged MSCs. Our findings provide a new explanation for the mechanisms underlying stem cell senescence and a novel target for delaying stem cell senescence and preventing and treating age-related diseases. PMID:28125705

  4. The selective adhesion molecule inhibitor Natalizumab decreases multiple myeloma cell growth in the bone marrow microenvironment: therapeutic implications.

    Science.gov (United States)

    Podar, Klaus; Zimmerhackl, Alexander; Fulciniti, Mariateresa; Tonon, Giovanni; Hainz, Ursula; Tai, Yu-Tzu; Vallet, Sonia; Halama, Niels; Jäger, Dirk; Olson, Dian L; Sattler, Martin; Chauhan, Dharminder; Anderson, Kenneth C

    2011-11-01

    Recent advances regarding the introduction of anti-adhesion strategies as a novel therapeutic concept in oncology hold great promise. Here we evaluated the therapeutic potential of the new-in-class-molecule selective-adhesion-molecule (SAM) inhibitor Natalizumab, a recombinant humanized IgG4 monoclonal antibody, which binds integrin-α4, in multiple myeloma (MM). Natalizumab, but not a control antibody, inhibited adhesion of MM cells to non-cellular and cellular components of the microenvironment as well as disrupted the binding of already adherent MM cells. Consequently, Natalizumab blocked both the proliferative effect of MM-bone marrow (BM) stromal cell interaction on tumour cells, and vascular endothelial growth factor (VEGF)-induced angiogenesis in the BM milieu. Moreover, Natalizumab also blocked VEGF- and insulin-like growth factor 1 (IGF-1)-induced signalling sequelae triggering MM cell migration. In agreement with our in vitro results, Natalizumab inhibited tumour growth, VEGF secretion, and angiogenesis in a human severe combined immunodeficiency murine model of human MM in the human BM microenvironment. Importantly, Natalizumab not only blocked tumour cell adhesion, but also chemosensitized MM cells to bortezomib, in an in vitro therapeutically representative human MM-stroma cell co-culture system model. Our data therefore provide the rationale for the clinical evaluation of Natalizumab, preferably in combination with novel agents (e.g. bortezomib) to enhance MM cytotoxicity and improve patient outcome.

  5. The mast cell stabiliser ketotifen decreases visceral hypersensitivity and improves intestinal symptoms in patients with irritable bowel syndrome

    NARCIS (Netherlands)

    Klooker, Tamira K.; Braak, Breg; Koopman, Karin E.; Welting, Olaf; Wouters, Mira M.; van der Heide, Sicco; Schemann, Michael; Bischoff, Stephan C.; van den Wijngaard, Rene M.; Boeckxstaens, Guy E.

    2010-01-01

    Background Mast cell activation is thought to be involved in visceral hypersensitivity, one of the main characteristics of the irritable bowel syndrome (IBS). A study was therefore undertaken to investigate the effect of the mast cell stabiliser ketotifen on rectal sensitivity and symptoms in patien

  6. Early transcutaneous electrical nerve stimulation reduces hyperalgesia and decreases activation of spinal glial cells in mice with neuropathic pain.

    Science.gov (United States)

    Matsuo, Hideaki; Uchida, Kenzo; Nakajima, Hideaki; Guerrero, Alexander Rodriguez; Watanabe, Shuji; Takeura, Naoto; Sugita, Daisuke; Shimada, Seiichiro; Nakatsuka, Terumasa; Baba, Hisatoshi

    2014-09-01

    Although transcutaneous electrical nerve stimulation (TENS) is widely used for the treatment of neuropathic pain, its effectiveness and mechanism of action in reducing neuropathic pain remain uncertain. We investigated the effects of early TENS (starting from the day after surgery) in mice with neuropathic pain, on hyperalgesia, glial cell activation, pain transmission neuron sensitization, expression of proinflammatory cytokines, and opioid receptors in the spinal dorsal horn. Following nerve injury, TENS and behavioral tests were performed every day. Immunohistochemical, immunoblot, and flow cytometric analysis of the lumbar spinal cord were performed after 8 days. Early TENS reduced mechanical and thermal hyperalgesia and decreased the activation of microglia and astrocytes (PEarly TENS decreased p-p38 within microglia (Pearly TENS relieved hyperalgesia in our mouse model of neuropathic pain by inhibiting glial activation, MAP kinase activation, PKC-γ, and p-CREB expression, and proinflammatory cytokines expression, as well as maintenance of spinal opioid receptors. The findings indicate that TENS treatment is more effective when applied as early after nerve injury as possible.

  7. Decreased glial cell line-derived neurotrophic factor levels in patients with depression: a meta-analytic study.

    Science.gov (United States)

    Lin, Pao-Yen; Tseng, Ping-Tao

    2015-04-01

    Glial cell-line derived neurotrophic factor (GDNF) has been shown to promote development, differentiation, and protection of CNS neurons and was thought to play an important role in various neuropsychiatric disorders. Several studies have examined the GDNF levels in patients with depression but shown inconsistent results. In this study, we compared blood GDNF levels between depressive patients and control subjects through meta-analytic method. The effect sizes (ESs) from all eligible studies were synthesized by using a random effect model. In this meta-analysis, we included 526 patients and 502 control subjects from 12 original articles. Compared to control subjects, blood GDNF levels are significantly decreased in patients with depression (ES = -0.62, p = 0.0011). However, significant heterogeneity was found among included studies. Through subgroup analysis, we found that GDNF was still decreased in studies with major depressive disorder (ES = -0.73, p = 0.0001); in studies with non-old-age depression (ES = -1.25, p = 0.0001), but not with old-age depression; and in studies using serum samples (ES = -0.86, p GDNF levels as a biomarker of depression as a whole, but the results were modulated by psychiatric diagnosis, age of included subjects, and sampling sources. With these results, future studies are required to examine whether effective antidepressant treatment is associated with an increase in serum GDNF levels.

  8. Transplantation of ATP7B-transduced bone marrow mesenchymal stem cells decreases copper overload in rats.

    Directory of Open Access Journals (Sweden)

    Shenglin Chen

    Full Text Available BACKGROUND: Recent studies have demonstrated that transplantation of ATP7B-transduced hepatocytes ameliorates disease progression in LEC (Long-Evans Cinnamon rats, a model of Wilson's disease (WD. However, the inability of transplanted cells to proliferate in a normal liver hampers long-term treatment. In the current study, we investigated whether transplantation of ATP7B-transduced bone marrow mesenchymal stem cells (BM-MSCs could decrease copper overload in LEC rats. MATERIALS AND METHODS: The livers of LEC rats were preconditioned with radiation (RT and/or ischemia-reperfusion (IRP before portal vein infusion of ATP7B-transduced MSCs (MSCsATP7B. The volumes of MSCsATP7B or saline injected as controls were identical. The expression of ATP7B was analyzed by real-time quantitative polymerase chain reaction (RT-PCR at 4, 12 and 24 weeks post-transplantation. MSCATP7B repopulation, liver copper concentrations, serum ceruloplasmin levels, and alanine transaminase (ALT and aspartate transaminase (AST levels were also analyzed at each time-point post-transplantation. RESULTS: IRP-plus-RT preconditioning was the most effective strategy for enhancing the engraftment and repopulation of transplanted MSCsATP7B. This strategy resulted in higher ATP7B expression and serum ceruloplasmin, and lower copper concentration in this doubly preconditioned group compared with the saline control group, the IRP group, and the RT group at all three time-points post-transplantation (p<0.05 for all. Moreover, 24 weeks post-transplantation, the levels of ALT and AST in the IRP group, the RT group, and the IRP-plus-RT group were all significantly decreased compared to those of the saline group (p<0.05 compared with the IRP group and RT group, p<0.01 compared with IRP-plus-RT group; ALT and AST levels were significantly lower in the IRP-plus-RT group compared to either the IRP group or the RT group (p<0.01 and p<0.05. respectively. CONCLUSIONS: These results demonstrate

  9. SET overexpression decreases cell detoxification efficiency: ALDH2 and GSTP1 are downregulated, DDR is impaired and DNA damage accumulates.

    Science.gov (United States)

    Almeida, Luciana O; Goto, Renata N; Pestana, Cezar R; Uyemura, Sérgio A; Gutkind, Silvio; Curti, Carlos; Leopoldino, Andréia M

    2012-12-01

    Alcohol and tobacco consumption are risk factors for head and neck squamous cell carcinoma (HNSCC). Aldehyde dehydrogenase 2 (ALDH2) and glutathione S-transferase pi 1 (GSTP1) are important enzymes for cellular detoxification and low efficiencies are implicated in cancer. We assessed the potential role of SET protein overexpression, a histone acetylation modulator accumulated in HNSCC, in gene regulation and protein activity of ALDH2 and GSTP1. SET was knocked down in HN13, HN12 and Cal27, and overexpressed in HEK293 cells; ethanol and cisplatin were the chemical agents. Cells with SET overexpression (HEK293/SET, HN13 and HN12) showed lower ALDH2 and GSTP1 mRNA levels and trichostatin A increased them (real-time PCR). Ethanol upregulated GSTP1 and ALDH2 mRNAs, whereas cisplatin upregulated GSTP1 in HEK293 cells. SET-chromatin binding revealed SET interaction with ALDH2 and GSTP1 promoters, specifically via SET NAP domain; ethanol and cisplatin abolished SET binding. ALDH2 and GSTP1 efficiency was assessed by enzymatic and comet assay. A lower ALDH2 activity was associated with greater DNA damage (tail intensity) in HEK293/SET compared with HEK293 cells, whereas HN13/siSET showed ALDH2 activity higher than HN13 cells. HN13/siSET cells showed increased tail intensity. Cisplatin-induced DNA damage response showed negative relationship between SET overexpression and BRCA2 recruitment. SET downregulated repair genes ATM, BRCA1 and CHEK2 and upregulated TP53. Cisplatin-induced cell-cycle arrest occurred in G(0) /G(1) and S in HEK293 cells, whereas HEK293/SET showed G(2) /M stalling. Overall, cisplatin was more cytotoxic for HN13 than HN13/siSET cells. Our data suggest a role for SET in cellular detoxification, DNA damage response and genome integrity.

  10. 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.

    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 μg/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, 18 mg/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 PGE2 levels, they may prove beneficial in the chemoprevention and/or therapy of NSCLC, PMID:19628293

  11. Functional decreases in P2X7 receptors are associated with retinoic acid-induced neuronal differentiation of Neuro-2a neuroblastoma cells.

    Science.gov (United States)

    Wu, Pei-Yu; Lin, Yu-Chia; Chang, Chia-Ling; Lu, Hsing-Tsen; Chin, Chia-Hsuan; Hsu, Tsan-Ting; Chu, Dachen; Sun, Synthia H

    2009-06-01

    Neuro-2a (N2a) cells are derived from spontaneous neuroblastoma of mouse and capable to differentiate into neuronal-like cells. Recently, P2X7 receptor has been shown to sustain growth of human neuroblastoma cells but its role during neuronal differentiation remains unexamined.We characterized the role of P2X7 receptors in the retinoic acid (RA)-differentiated N2a cells. RA induced N2a cells differentiation into neurite bearing and neuronal specific proteins, microtubule-associated protein 2 (MAP2) and neuronal specific nuclear protein (NeuN), expressing neuronal-like cells. Interestingly, the RA-induced neuronal differentiation was associated with decreases in the expression and function of P2X7 receptors. Functional inhibition of P2X7 receptors by P2X7 receptor selective antagonists, 5'-triphosphate, periodate-oxidized 2',3'-dialdehyde ATP (oATP), brilliant blue G (BBG) or A438079 induced neurite outgrowth. In addition, RA and oATP treatment stimulated the expression of neuron-specific class III beta-tubulin (TuJ1), and knockdown of P2X7 receptor expression by siRNA induced neurite outgrowth. To elucidate the possible mechanism, we found the levels of basal intracellular Ca2+ concentrations ([Ca2+]i) were decreased in either RA- or oATP-differentiated or P2X7receptor knockdown N2a cells. Simply cultured N2a cells in low Ca2+ medium induced a 2-fold increase in neurite length. Treatment of N2a cells with ATP hydrolase apyrase and the P2X7 receptors selective antagonist oATP or BBG decreased cell viability and cell number. Nevertheless, oATP but not BBG decreased cell proliferation and cell cycle progression. These results suggest for the first time that decreases in expression/function of P2X7 receptors are involved in neuronal differentiation.We provide additional evidence shown that the ATP release-activated P2X7 receptor is important in maintaining cell survival of N2a neuroblastoma cells.

  12. Decreased expression of IL-27 and its correlation with Th1 and Th17 cells in progressive multiple sclerosis.

    Science.gov (United States)

    Tang, Shao-can; Fan, Xiao-hua; Pan, Qing-min; Sun, Qiang-san; Liu, Yu

    2015-01-15

    Progressive multiple sclerosis (MS) is an immune-mediated demyelinating disease in which both imbalanced T helper (Th) subsets and aberrant cytokine profiles have been found. Interleukin-27 (IL-27), a cytokine with pro-inflammatory and anti-inflammatory effects, plays pleiotropic roles in immunomodulation. In the present study, plasma levels of IL-27, interferon-gamma (IFN-γ), IL-17 and frequencies of peripheral Th1, Th17 cells were determined by enzyme-linked immunosorbent assay (ELISA) and flow cytometry in 45 progressive MS and 25 healthy controls. mRNA expression levels of IL-27, IFN-γ, T-bet, IL-17 and RAR-related orphan receptor gamma t (RORγt) in peripheral blood mononuclear cells (PBMCs) were also quantified by real-time polymerase chain reaction. Plasma and mRNA levels of IL-27 in progressive MS patients were significantly lower than those in healthy controls, while plasma concentrations of IL-17, frequencies of circulating Th17, and mRNA expression levels of IL-17 as well as RORγt were all increased remarkably compared with healthy controls. No statistical significance was observed in IFN-γ and T-bet mRNA expression or plasma IFN-γ levels between progressive MS patients and healthy controls. Moreover, plasma levels of IL-27 were found to be negatively correlated to the percentages of circulating Th17 or plasma IL-17 concentrations in patients with progressive MS. Our data showed that progressive MS patients had decreased plasma and mRNA expression levels of IL-27, suggesting that it might be involved in the pathophysiological process of MS.

  13. Astaxanthin Normalizes Epigenetic Modifications of Bovine Somatic Cell Cloned Embryos and Decreases the Generation of Lipid Peroxidation.

    Science.gov (United States)

    Li, R; Wu, H; Zhuo, W W; Mao, Q F; Lan, H; Zhang, Y; Hua, S

    2015-10-01

    Astaxanthin is an extremely common antioxidant scavenging reactive oxygen species (ROS) and blocking lipid peroxidation. This study was conducted to investigate the effects of astaxanthin supplementation on oocyte maturation, and development of bovine somatic cell nuclear transfer (SCNT) embryos. Cumulus-oocyte complexes were cultured in maturation medium with astaxanthin (0, 0.5, 1.0, or 1.5 mg/l), respectively. We found that 0.5 mg/l astaxanthin supplementation significantly increased the proportion of oocyte maturation. Oocytes cultured in 0.5 mg/l astaxanthin supplementation were used to construct SCNT embryos and further cultured with 0, 0.5, 1.0 or 1.5 mg/l astaxanthin. The results showed that the supplementation of 0.5 mg/l astaxanthin significantly improved the proportions of cleavage and blastulation, as well as the total cell number in blastocysts compared with the control group, yet this influence was not concentration dependent. Chromosomal analyses revealed that more blastomeres showed a normal chromosomal complement in 0.5 mg/l astaxanthin treatment group, which was similar to that in IVF embryos. The methylation levels located on the exon 1 of the imprinted gene H19 and IGF2, pluripotent gene OCT4 were normalized, and global DNA methylation, H3K9 and H4K12 acetylation were also improved significantly, which was comparable to that in vitro fertilization (IVF) embryos. Moreover, we also found that astaxanthin supplementation significantly decreased the level of lipid peroxidation. Our findings showed that the supplementation of 0.5 mg/l astaxanthin to oocyte maturation medium and embryo culture medium improved oocyte maturation, SCNT embryo development, increased chromosomal stability and normalized the epigenetic modifications, as well as inhibited overproduction of lipid peroxidation.

  14. Glucocorticoid receptor knockdown decreases the antioxidant protection of B16 melanoma cells: an endocrine system-related mechanism that compromises metastatic cell resistance to vascular endothelium-induced tumor cytotoxicity.

    Science.gov (United States)

    Obrador, Elena; Valles, Soraya L; Benlloch, María; Sirerol, J Antoni; Pellicer, José A; Alcácer, Javier; Coronado, Javier Alcácer-F; Estrela, José M

    2014-01-01

    We previously reported an interorgan system in which stress-related hormones (corticosterone and noradrenaline), interleukin-6, and glutathione (GSH) coordinately regulate metastatic growth of highly aggressive B16-F10 melanoma cells. Corticosterone, at levels measured in tumor-bearing mice, also induces apoptotic cell death in metastatic cells with low GSH content. In the present study we explored the potential role of glucocorticoids in the regulation of metastatic cell death/survival during the early stages of organ invasion. Glucocorticoid receptor (GCR) knockdown decreased the expression and activity of γ-glutamylcysteine synthetase (γ-GCS), the rate-limiting step in GSH synthesis, in metastatic cells in vivo independent of the tumor location (liver, lung, or subcutaneous). The decrease in γ-GCS activity was associated with lower intracellular GSH levels. Nrf2- and p53-dependent down-regulation of γ-GCS was associated with a decrease in the activities of superoxide dismutase 1 and 2, catalase, glutathione peroxidase, and glutathione reductase, but not of the O2--generating NADPH oxidase. The GCR knockdown-induced decrease in antioxidant protection caused a drastic decrease in the survival of metastatic cells during their interaction with endothelial cells, both in vitro and in vivo; only 10% of cancer cells attached to the endothelium survived compared to 90% survival observed in the controls. This very low rate of metastatic cell survival was partially increased (up to 52%) in vivo by inoculating B16-F10 cells preloaded with GSH ester, which enters the cell and delivers free GSH. Taken together, our results indicate that glucocorticoid signaling influences the survival of metastatic cells during their interaction with the vascular endothelium.

  15. Addition of maraviroc to antiretroviral therapy decreased interferon-γ mRNA in the CD4+ T cells of patients with suboptimal CD4+ T-cell recovery.

    Science.gov (United States)

    Minami, Rumi; Takahama, Soichiro; Kaku, Yu; Yamamoto, Masahiro

    2017-01-01

    The CCR5 antagonist, maraviroc (MVC), is associated with an enhanced CD4+ T-cell response independent of virological suppression; however, its mechanism of action has not been elucidated. In this study, we confirmed the effect of MVC on CD4+ T-cell count recovery in immunological non-responders, and compared the conventional combination antiretroviral therapy (cART) with MVC-intensified cART. We also investigated the effect of MVC on interferon-γ (IFN-γ) production in CD4+ T cells in vitro and in vivo, and evaluated the relationship between the mRNA level of IFN-γ and the degree of CD4+ T-cell count recovery. In vitro analysis indicated that MVC significantly decreased mRNA levels of IFN-γ in HIV-Tat stimulated CD4+ T cells from healthy donor peripheral blood mononuclear cells. Of the 18 HIV-infected patients treated with MVC-intensified cART, 12 had a significantly increased CD4+ T-cell count after 24 weeks of additional treatment with MVC. In patients exhibiting a response in CD4+ T-cell counts, mRNA levels of IFN-γ in CD4+ T cells were lower than those in patients showing a non-response at baseline and at week 24, while mRNA levels of IFN-γ decreased in both groups at 24 weeks. In conclusion, MVC decreased the mRNA level of IFN-γ in CD4+ T cells in vitro and in vivo, especially in patients whose CD4+ T-cell count increased significantly. We also found that the lower baseline IFN-γ mRNA level and the larger decreased rate of IFN-γ mRNA in CD4+ T cells were associated with a good response to MVC regarding CD4+ T-cell recovery.

  16. Notch signaling mediates the age-associated decrease in adhesion of germline stem cells to the niche.

    Science.gov (United States)

    Tseng, Chen-Yuan; Kao, Shih-Han; Wan, Chih-Ling; Cho, Yueh; Tung, Shu-Yun; Hsu, Hwei-Jan

    2014-12-01

    Stem cells have an innate ability to occupy their stem cell niche, which in turn, is optimized to house stem cells. Organ aging is associated with reduced stem cell occupancy in the niche, but the mechanisms involved are poorly understood. Here, we report that Notch signaling is increased with age in Drosophila female germline stem cells (GSCs), and this results in their removal from the niche. Clonal analysis revealed that GSCs with low levels of Notch signaling exhibit increased adhesiveness to the niche, thereby out-competing their neighbors with higher levels of Notch; adhesiveness is altered through regulation of E-cadherin expression. Experimental enhancement of Notch signaling in GSCs hastens their age-dependent loss from the niche, and such loss is at least partially mediated by Sex lethal. However, disruption of Notch signaling in GSCs does not delay GSC loss during aging, and nor does it affect BMP signaling, which promotes self-renewal of GSCs. Finally, we show that in contrast to GSCs, Notch activation in the niche (which maintains niche integrity, and thus mediates GSC retention) is reduced with age, indicating that Notch signaling regulates GSC niche occupancy both intrinsically and extrinsically. Our findings expose a novel role of Notch signaling in controlling GSC-niche adhesion in response to aging, and are also of relevance to metastatic cancer cells, in which Notch signaling suppresses cell adhesion.

  17. Notch signaling mediates the age-associated decrease in adhesion of germline stem cells to the niche.

    Directory of Open Access Journals (Sweden)

    Chen-Yuan Tseng

    2014-12-01

    Full Text Available Stem cells have an innate ability to occupy their stem cell niche, which in turn, is optimized to house stem cells. Organ aging is associated with reduced stem cell occupancy in the niche, but the mechanisms involved are poorly understood. Here, we report that Notch signaling is increased with age in Drosophila female germline stem cells (GSCs, and this results in their removal from the niche. Clonal analysis revealed that GSCs with low levels of Notch signaling exhibit increased adhesiveness to the niche, thereby out-competing their neighbors with higher levels of Notch; adhesiveness is altered through regulation of E-cadherin expression. Experimental enhancement of Notch signaling in GSCs hastens their age-dependent loss from the niche, and such loss is at least partially mediated by Sex lethal. However, disruption of Notch signaling in GSCs does not delay GSC loss during aging, and nor does it affect BMP signaling, which promotes self-renewal of GSCs. Finally, we show that in contrast to GSCs, Notch activation in the niche (which maintains niche integrity, and thus mediates GSC retention is reduced with age, indicating that Notch signaling regulates GSC niche occupancy both intrinsically and extrinsically. Our findings expose a novel role of Notch signaling in controlling GSC-niche adhesion in response to aging, and are also of relevance to metastatic cancer cells, in which Notch signaling suppresses cell adhesion.

  18. Electrical Stimulation Decreases Coupling Efficiency Between Beta-Adrenergic Receptors and Cyclic AMP Production in Cultured Muscle Cells

    Science.gov (United States)

    Young, R. B.; Bridge, K. Y.

    1999-01-01

    Electrical stimulation of skeletal muscle cells in culture is an effective way to simulate the effects of muscle contraction and its effects on gene expression in muscle cells. Expression of the beta-adrenergic receptor and its coupling to cyclic AMP synthesis are important components of the signaling system that controls muscle atrophy and hypertrophy, and the goal of this project was to determine if electrical stimulation altered the beta-adrenergic response in muscle cells. Chicken skeletal muscle cells that had been grown for seven days in culture were subjected to electrical stimulation for an additional two days at a pulse frequency of 0.5 pulses/sec and a pulse duration of 200 msec. At the end of this two-day stimulation period, beta-adrenergic receptor population was measured by the binding of tritium-labeled CGP-12177 to muscle cells, and coupling to cAMP synthesis was measured by Radioimmunoassay (RIA) after treating the cells for 10 min with the potent (beta)AR agonist, isoproterenol. The number of beta adrenergic receptors and the basal levels of intracellular cyclic AMP were not affected by electrical stimulation. However, the ability of these cells to synthesize cyclic AMP was reduced by approximately 50%. Thus, an enhanced level of contraction reduces the coupling efficiency of beta-adrenergic receptors for cyclic AMP production.

  19. Scorpion venom heat-resistant protein decreases immunoreactivity of OX-42-positive microglia cells in MPTP-treated mice

    Institute of Scientific and Technical Information of China (English)

    Shengming Yin; Deqin Yu; Xi Gao; Yan Peng; Yanhui Feng; Jie Zhao; Yiyuan Tang; Wanqin Zhang

    2008-01-01

    BACKGROUND: Microglia function as the immune surveyors of the brain under normal physiological conditions. However, microglia become activated in response to brain injuries and immunological stimulation. OBJECTIVE: To explore the influence of scorpion venom (SV) heat-resistant protein on frontal cortex and hippocampal microglia cells in a mice model of Parkinson's disease. DESIGN, TIME AND SETTING: Randomized, controlled, cellular immunity study. The experiment was performed at the Physiology Department Laboratory in Dalian Medical University between June 2005 and July 2008. MATERIALS: Ninety-six healthy, C57BI/6 mice; 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) from Sigma, USA; SV heat-resistant protein (Experimental Base Institute in Dalian Medical University). The mice were randomly divided into tour groups (n = 24): normal control, negative control, model, and SV heat-resistant protein. METHODS: Mice in the model and SV heat-resistant protein groups were subcutaneously injected with MPTP (20 mg/kg) to model Parkinson's disease, while the normal control and negative control groups were injected with physiological saline in the neck for 8 successive days. In addition, mice in the model and normal control groups were intraperitoneally injected with physiological saline 2 hours following administration, while SV heat-resistant protein and negative control groups were injected SV heat-resistant protein (0.01 mg/kg). MAIN OUTCOME MEASURES: Immunoreactivity of microglia cells in MPTP-treated mice. RESULTS: Compared with normal control mice, MPTP-treated mice displayed increased OX-42 expression in the brain. However, in the SV heat-resistant protein-treated mice, OX-42 expression was decreased, compared to the model group. In the model mouse group, the number of OX-42-positive microglia was increased in the frontal cortex, caudatum, and hippocampal hilus, compared to the normal control mice (P < 0.01). However, in the SV heat-resistant protein-treated mice

  20. Age-related decrease in the proportion of germinal center B cells from mouse Peyer's patches is accompanied by an accumulation of somatic mutations in their immunoglobulin genes.

    Science.gov (United States)

    González-Fernández, A; Gilmore, D; Milstein, C

    1994-11-01

    Somatic hypermutation of immunoglobulin genes and the generation of memory B cells seems to take place in germinal centers, which are chronically present in Peyer's patches. Age-associated changes in the germinal center B cell compartment of Peyer's patches and in the mutations of a kappa light chain transgene were analyzed in unimmunized mice. Somatic mutations accumulate in germinal center B cells slowly and continuously to reach an apparent plateau when the animals are around 5 months old. In contrast, the proportion of germinal center B cells reaches a maximum in very young mice (about 2 months old) and decreases progressively thereafter. These results suggest that the highly mutated B cells in older mice arise by the successive accumulation of mutations in memory cells. The data also show that the optimum time for the analysis of hypermutation of transgenes in Peyer's patches is when the mice are about 5 months old.

  1. Inhibition of connective tissue growth factor overexpression decreases growth of hepatocellular carcinoma cells in vitro and in vivo

    Institute of Scientific and Technical Information of China (English)

    JIA Xiao-qin; CHENG Hai-qing; LI Hong; ZHU Yan; LI Yu-hua; FENG Zhen-qing; ZHANG Jian-ping

    2011-01-01

    Background We have previously found that connective tissue growth factor (CTGF) is highly expressed in a rat model of liver cancer.Here,we examined expression of CTGFin human hepatocellular carcinoma (HCC) cells and its effect on cell growth.Methods Real-time PCR was used to observe expression of CTGF in human HCC cell lines HepG2,SMMC-7721,MHCC-97H and LO2.siRNA for the CTGFgene was designed,synthesized and cloned into a Plk0.1-GFP-SP6 vector to construct a lentivirus-mediated shRNA/CTGF.CTGF mRNA and protein expression in HepG2 cells treated by CTGF-specific shRNA was evaluated by real-time PCR and Western blotting.3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was utilized to evaluate the growth effect,and a colony formation assay was used for observing clonogenic growth.In vivo,tumor cell proliferation was evaluated in a nude mouse model of xenotransplantation.Statistical significance was determined by t test for comparison between two groups,or analysis of variance (ANOVA) for multiple groups.Results Immunohistochemical staining of CTGF was seen in 35 of 40 HCC samples (87.5%).CTGF was overexpressed 5-fold in 20 HCC tissues,compared with surrounding non-tumor liver tissue.CTGF mRNA level was 5-8-fold higher in HepG2,SMMC-7721 and MHCC-97H than in LO2 cells.This indicated that the inhibition rate of cell growth was 43% after knockdown of CTGF expression (P <0.05).Soft agar colony formation assay showed that siRNA mediated knockdown of CTGF inhibited colony formation in soft agar of HepG2 cells (P <0.05).The volume of tumors from CTGF-shRNA-expressing cells only accounted for 35% of the tumors from the scrambled control-infected HepG2 cells (P <0.05).Conclusions CTGF was overexpressed in human HCC cells and downregulation of CTGF inhibited HCC growth in vitro and in vivo.Knockdown of CTGF may be a potential therapeutic strategy for treatment of HCC.

  2. Tumor cells have decreased ability to metabolize H2O2: Implications for pharmacological ascorbate in cancer therapy

    Directory of Open Access Journals (Sweden)

    Claire M. Doskey

    2016-12-01

    Full Text Available Ascorbate (AscH− functions as a versatile reducing agent. At pharmacological doses (P-AscH−; [plasma AscH−] ≥≈20 mM, achievable through intravenous delivery, oxidation of P-AscH− can produce a high flux of H2O2 in tumors. Catalase is the major enzyme for detoxifying high concentrations of H2O2. We hypothesize that sensitivity of tumor cells to P-AscH− compared to normal cells is due to their lower capacity to metabolize H2O2. Rate constants for removal of H2O2 (kcell and catalase activities were determined for 15 tumor and 10 normal cell lines of various tissue types. A differential in the capacity of cells to remove H2O2 was revealed, with the average kcell for normal cells being twice that of tumor cells. The ED50 (50% clonogenic survival of P-AscH− correlated directly with kcell and catalase activity. Catalase activity could present a promising indicator of which tumors may respond to P-AscH−.

  3. Loss of Selenium-Binding Protein 1 Decreases Sensitivity to Clastogens and Intracellular Selenium Content in HeLa Cells

    Science.gov (United States)

    Zhao, Changhui; Zeng, Huawei; Wu, Ryan T. Y.; Cheng, Wen-Hsing

    2016-01-01

    Selenium-binding protein 1 (SBP1) is not a selenoprotein but structurally binds selenium. Loss of SBP1 during carcinogenesis usually predicts poor prognosis. Because genome instability is a hallmark of cancer, we hypothesize that SBP1 sequesters cellular selenium and sensitizes cancer cells to DNA-damaging agents. To test this hypothesis, we knocked down SBP1 expression in HeLa cervical cancer cells by employing a short hairpin RNA (shRNA) approach. Reduced sensitivity to hydrogen peroxide, paraquat and camptothecin, reactive oxygen species content, and intracellular retention of selenium after selenomethionine treatment were observed in SBP1 shRNA HeLa cells. Results from Western analyses showed that treatment of HeLa cells with selenomethionine resulted in increased SBP1 protein expression in a dose-dependent manner. Knockdown of SBP1 rendered HeLa cells increased expression of glutathione peroxidase-1 but not glutathione peroxidase-4 protein levels and accelerated migration from a wound. Altogether, SBP1 retains supplemental selenium and sensitizes HeLa cancer cells to clastogens, suggesting a new cancer treatment strategy by sequestering selenium through SBP1. PMID:27404728

  4. Antagonism of serotonin receptor 1B decreases viability and promotes apoptosis in the COS canine osteosarcoma cell line.

    Science.gov (United States)

    Viall, A K; Goodall, C P; Stang, B; Marley, K; Chappell, P E; Bracha, S

    2016-06-01

    Serotonin receptor 1B (5HTR1B) traditionally exhibits anti-proliferative activity in osteoblasts. We examined the expression and function of 5HTR1B in the COS canine osteosarcoma cell line and normal canine osteoblasts. Equal levels of 5HTR1B gene and protein expression were found between normal and malignant osteoblasts. Treatment with serotonin enhanced viability of osteosarcoma cells but not normal osteoblasts. Challenge with the 5HTR1B agonist anpirtoline caused no change in cell viability. Rather incubation with the specific receptor antagonist SB224289 caused reduction in osteoblast viability, with this effect more substantial in osteosarcoma cells. Investigation of this inhibitory activity showed 5HTR1B antagonism induces apoptosis in malignant cells. Evaluation of phosphorylated levels of CREB and ERK, transcriptional regulators associated with serotonin receptor signalling in osteoblasts, revealed aberrant 5HTR1B signalling in COS. Our results confirm the presence of 5HTR1B in a canine osteosarcoma cell line and highlight this receptor as a possible novel therapeutic target.

  5. GroEL1, a heat shock protein 60 of Chlamydia pneumoniae, impairs neovascularization by decreasing endothelial progenitor cell function.

    Directory of Open Access Journals (Sweden)

    Yi-Wen Lin

    Full Text Available The number and function of endothelial progenitor cells (EPCs are sensitive to hyperglycemia, hypertension, and smoking in humans, which are also associated with the development of atherosclerosis. GroEL1 from Chlamydia pneumoniae has been found in atherosclerotic lesions and is related to atherosclerotic pathogenesis. However, the actual effects of GroEL1 on EPC function are unclear. In this study, we investigate the EPC function in GroEL1-administered hind limb-ischemic C57BL/B6 and C57BL/10ScNJ (a toll-like receptor 4 (TLR4 mutation mice and human EPCs. In mice, laser Doppler imaging, flow cytometry, and immunohistochemistry were used to evaluate the degree of neo-vasculogenesis, circulating level of EPCs, and expression of CD34, vWF, and endothelial nitric oxide synthase (eNOS in vessels. Blood flow in the ischemic limb was significantly impaired in C57BL/B6 but not C57BL/10ScNJ mice treated with GroEL1. Circulating EPCs were also decreased after GroEL1 administration in C57BL/B6 mice. Additionally, GroEL1 inhibited the expression of CD34 and eNOS in C57BL/B6 ischemic muscle. In vitro, GroEL1 impaired the capacity of differentiation, mobilization, tube formation, and migration of EPCs. GroEL1 increased senescence, which was mediated by caspases, p38 MAPK, and ERK1/2 signaling in EPCs. Furthermore, GroEL1 decreased integrin and E-selectin expression and induced inflammatory responses in EPCs. In conclusion, these findings suggest that TLR4 and impaired NO-related mechanisms could contribute to the reduced number and functional activity of EPCs in the presence of GroEL1 from C. pneumoniae.

  6. Microglial numbers attain adult levels after undergoing a rapid decrease in cell number in the third postnatal week.

    Science.gov (United States)

    Nikodemova, Maria; Kimyon, Rebecca S; De, Ishani; Small, Alissa L; Collier, Lara S; Watters, Jyoti J

    2015-01-15

    During postnatal development, microglia, CNS resident innate immune cells, are essential for synaptic pruning, neuronal apoptosis and remodeling. During this period microglia undergo morphological and phenotypic transformations; however, little is known about how microglial number and density is regulated during postnatal CNS development. We found that after an initial increase during the first 14 postnatal days, microglial numbers in mouse brain began declining in the third postnatal week and were reduced by 50% by 6weeks of age; these "adult" levels were maintained until at least 9months of age. Microglial CD11b levels increased, whereas CD45 and ER-MP58 declined between P10 and adulthood, consistent with a maturing microglial phenotype. Our data indicate that both increased microglial apoptosis and a decreased proliferative capacity contribute to the developmental reduction in microglial numbers. We found no correlation between developmental reductions in microglial numbers and brain mRNA levels of Cd200, Cx3Cl1, M-Csf or Il-34. We tested the ability of M-Csf-overexpression, a key growth factor promoting microglial proliferation and survival, to prevent microglial loss in the third postnatal week. Mice overexpressing M-Csf in astrocytes had higher numbers of microglia at all ages tested. However, the developmental decline in microglial numbers still occurred, suggesting that chronically elevated M-CSF is unable to overcome the developmental decrease in microglial numbers. Whereas the identity of the factor(s) regulating microglial number and density during development remains to be determined, it is likely that microglia respond to a "maturation" signal since the reduction in microglial numbers coincides with CNS maturation.

  7. 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.

  8. Morphological abnormalities, impaired fetal development and decrease in myostatin expression following somatic cell nuclear transfer in dogs.

    Science.gov (United States)

    Hong, Il-Hwa; Jeong, Yeon-Woo; Shin, Taeyoung; Hyun, Sang-Hwan; Park, Jin-Kyu; Ki, Mi-Ran; Han, Seon-Young; Park, Se-Il; Lee, Ji-Hyun; Lee, Eun-Mi; Kim, Ah-Young; You, Sang-Young; Hwang, Woo-Suk; Jeong, Kyu-Shik

    2011-05-01

    Several mammals, including dogs, have been successfully cloned using somatic cell nuclear transfer (SCNT), but the efficiency of generating normal, live offspring is relatively low. Although the high failure rate has been attributed to incomplete reprogramming of the somatic nuclei during the cloning process, the exact cause is not fully known. To elucidate the cause of death in cloned offspring, 12 deceased offspring cloned by SCNT were necropsied. The clones were either stillborn just prior to delivery or died with dyspnea shortly after birth. On gross examination, defects in the anterior abdominal wall and increased heart and liver sizes were found. Notably, a significant increase in muscle mass and macroglossia lesions were observed in deceased SCNT-cloned dogs. Interestingly, the expression of myostatin, a negative regulator of muscle growth during embryogenesis, was down-regulated at the mRNA level in tongues and skeletal muscles of SCNT-cloned dogs compared with a normal dog. Results of the present study suggest that decreased expression of myostatin in SCNT-cloned dogs may be involved in morphological abnormalities such as increased muscle mass and macroglossia, which may contribute to impaired fetal development and poor survival rates.

  9. 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.

  10. Lipoxin A4 decreases human memory B cell antibody production via an ALX/FPR2-dependent mechanism: A link between resolution signals and adaptive immunity

    Science.gov (United States)

    Ramon, Sesquile; Bancos, Simona; Serhan, Charles N.; Phipps, Richard P.

    2013-01-01

    Summary Specialized proresolving mediators (SPMs) are endogenous bioactive lipid molecules that play a fundamental role in the regulation of inflammation and its resolution. SPMs are classified into lipoxins, resolvins, protectins and maresins. Lipoxins and other SPMs have been identified in important immunological tissues including bone marrow, spleen and blood. Lipoxins regulate functions of the innate immune system including the promotion of monocyte recruitment and increase macrophage phagocytosis of apoptotic neutrophils. A major knowledge gap is whether lipoxins influence adaptive immune cells. Here, we analyzed the actions of lipoxin A4 (LXA4) and its receptor ALX/FPR2 on human B cells. LXA4 decreased IgM and IgG production on activated B cells through ALX/FPR2-dependent signaling, which downregulated NF-κB p65 nuclear translocation. LXA4 also inhibited human memory B cell antibody production and proliferation, but not naïve B cell function. Lastly, LXA4 decreased antigen-specific antibody production in vivo. To our knowledge, this is the first description of the actions of lipoxins on human B cells, which shows a link between resolution signals and adaptive immunity. Regulating antibody production is crucial to prevent unwanted inflammation. Harnessing the ability of lipoxins to decrease memory B cell antibody production can be beneficial to threat inflammatory and autoimmune disorders. PMID:24166736

  11. Scrapie infection in experimental rodents and SMB-S15 cells decreased the brain endogenous levels and activities of Sirt1.

    Science.gov (United States)

    Wang, Jing; Zhang, Jin; Shi, Qi; Zhang, Bao-Yun; Chen, Cao; Chen, Li-Na; Sun, Jing; Wang, Hui; Xiao, Kang; Dong, Xiao-Ping

    2015-04-01

    Prion diseases are composed of a group of fatal neurodegenerative disorders resulting from misfolding of cellular prion (PrP(C)) into scrapie prion (PrP(Sc)). Sirt1, a class III histone deacetylase, has been reported to protect neuronal cells against PrP (106-126)-induced cell death. To address the potential role of Sirt1 during prion infection, the levels and enzyme activities of Sirt1 in the brains of scrapie-infected rodents, including hamsters infected with strain 263K, mice infected with strains 139A and ME7, and in prion infected SMB-S15 cells, were analyzed. Western blots revealed that endogenous Sirt1 levels were significantly decreased in all tested scrapie-infected models. Dynamic assays of brain Sirt1 levels in 263K-infected hamsters during incubation period showed a time-dependent decrease. The acetylating forms of Sirt1 target proteins, P53, PGC-1, and STAT3, markedly increased both in the brains of scrapie-infected rodents and in SMB-S15 cells, representing decreased Sirt1 activity. Immunofluorescent assays illustrated that Sirt1 predominately localized in cytosol of SMB-S15 cells but clearly distributed in nucleus of its normal partner cell line, SMB-PS. Moreover, accompanying with increase of Sirt1 level and decrease of acetyl-P53 level, treatments with Sirt1 activators SRT1720 and resveratrol in SMB-S15 cells significantly reduced PrP(Sc); at the same time, the cellular distribution of PrP proteins became normal, and the cell proliferating state was slightly improved. These data indicate that prion infection notably attenuates the Sirt1 activity in host cells. Sensitivity of the PrP(Sc) to Sirt1 activators highlights a potential role of Sirt1 in prion therapeutics.

  12. Differentiation of glioblastoma multiforme stem-like cells leads to downregulation of EGFR and EGFRvIII and decreased tumorigenic and stem-like cell potential

    DEFF Research Database (Denmark)

    Stockhausen, Marie-Thérése; Kristoffersen, Karina; Stobbe Olsen, Marie-Louise;

    2014-01-01

    Glioblastoma multiforme (GBM) is the most common and devastating primary brain tumor among adults. Despite recent treatment progress, most patients succumb to their disease within 2 years of diagnosis. Current research has highlighted the importance of a subpopulation of cells, assigned brain...... cancer stem-like cells (bCSC), to play a pivotal role in GBM malignancy. bCSC are identified by their resemblance to normal neural stem cells (NSC), and it is speculated that the bCSC have to be targeted in order to improve treatment outcome for GBM patients. One hallmark of GBM is aberrant expression...... and activation of the epidermal growth factor receptor (EGFR) and expression of a deletion variant EGFRvIII. In the normal brain, EGFR is expressed in neurogenic areas where also NSC are located and it has been shown that EGFR is involved in regulation of NSC proliferation, migration, and differentiation...

  13. T helper 17 and T helper 1 cells are increased but regulatory T cells are decreased in subchondral bone marrow microenvironment of patients with rheumatoid arthritis

    OpenAIRE

    Wang, Ting; Li, Shufeng; YANG, YUN; Zhang, Kaining; Dong, Shixiao; Wang, Xiuhua; Liu, Xinguang; Ren, Yanjun; Ming ZHANG; Yan, Xinfeng; Li, Jianmin; Zhang, Lei

    2016-01-01

    Objectives: The present study is to investigate the profiles of Th17, Th1 and Treg cells in bone marrow of patients with rheumatoid arthritis (RA). Methods: Flow cytometry was used to analyze the frequencies of Th17, Th1 and Treg cells in paired peripheral blood and bone marrow of 26 RA patients and 11 osteoarthritis (OA) patients, as well as 10 healthy controls. In addition, the disease activity was analyzed by the 28-joint disease activity score (DAS28). Results: The frequencies of Th17 and...

  14. 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.

  15. Anti-CD25 treatment depletes Treg cells and decreases disease severity in susceptible and resistant mice infected with Paracoccidioides brasiliensis.

    Directory of Open Access Journals (Sweden)

    Maíra Felonato

    Full Text Available Regulatory T (Treg cells are fundamental in the control of immunity and excessive tissue pathology. In paracoccidioidomycosis, an endemic mycosis of Latin America, the immunoregulatory mechanisms that control the progressive and regressive forms of this infection are poorly known. Due to its modulatory activity on Treg cells, we investigated the effects of anti-CD25 treatment over the course of pulmonary infection in resistant (A/J and susceptible (B10.A mice infected with Paracoccidioides brasiliensis. We verified that the resistant A/J mice developed higher numbers and more potent Treg cells than susceptible B10.A mice. Compared to B10.A cells, the CD4(+CD25(+Foxp3(+ Treg cells of A/J mice expressed higher levels of CD25, CTLA4, GITR, Foxp3, LAP and intracellular IL-10 and TGF-β. In both resistant and susceptible mice, anti-CD25 treatment decreased the CD4(+CD25(+Foxp3(+ Treg cell number, impaired indoleamine 2,3-dioxygenase expression and resulted in decreased fungal loads in the lungs, liver and spleen. In A/J mice, anti-CD25 treatment led to an early increase in T cell immunity, demonstrated by the augmented influx of activated CD4(+ and CD8(+ T cells, macrophages and dendritic cells to the lungs. At a later phase, the mild infection was associated with decreased inflammatory reactions and increased Th1/Th2/Th17 cytokine production. In B10.A mice, anti-CD25 treatment did not alter the inflammatory reactions but increased the fungicidal mechanisms and late secretion of Th1/Th2/Th17 cytokines. Importantly, in both mouse strains, the early depletion of CD25(+ cells resulted in less severe tissue pathology and abolished the enhanced mortality observed in susceptible mice. In conclusion, this study is the first to demonstrate that anti-CD25 treatment is beneficial to the progressive and regressive forms of paracoccidioidomycosis, potentially due to the anti-CD25-mediated reduction of Treg cells, as these cells have suppressive effects on the

  16. Targeting cancer stem cells expressing an embryonic signature with anti-proteases to decrease their tumor potential.

    Science.gov (United States)

    Darini, C Y; Martin, P; Azoulay, S; Drici, M-D; Hofman, P; Obba, S; Dani, C; Ladoux, A

    2013-07-04

    Cancer stem cells (CSCs) are a specific subset of cancer cells that sustain tumor growth and dissemination. They might represent a significant treatment target to reduce malignant progression and prevent tumor recurrence. In solid tumors, several hierarchically organized CSC clones coexist, even within a single tumor. Among them, CSCs displaying an embryonic stem cell 'stemness' signature, based on the expression of Oct-4, Nanog and Sox2, are present in distinct high-grade tumor types associated with poor prognosis. We previously designed a model to isolate pure populations of these CSCs from distinct solid tumors and used it to screen for molecules showing selective toxicity for this type of CSC. Here we show that human immunodeficiency virus (HIV)-protease inhibitors (HIV-PIs) specifically target CSCs expressing an embryonic signature derived from tumors with distinct origins. They reduced proliferation in a dose-dependent manner with a higher specificity as compared with the total population of cancer cells and/or healthy stem cells, and they were efficient in inducing cell death. Lopinavir was the most effective HIV-PI among those tested. It reduced self-renewal and induced apoptosis of CSCs, subsequently impairing in vivo CSC-induced allograft formation. Two key pharmacophores in the LPV structure were also identified. They are responsible for the specificity of CSC targeting and also for the overall antitumoral activity. These results contribute to the identification of molecules presenting selective toxicity for CSCs expressing an embryonic stemness signature. This paves the way to promising therapeutic opportunities for patients suffering from solid cancer tumors of poor prognosis.

  17. Downregulation of β-catenin decreases the tumorigenicity, but promotes epithelial-mesenchymal transition in breast cancer cells

    OpenAIRE

    Kai Cai; Longwei Jiang; Jing Wang; Hongyi Zhang; Xiaoying Wang; Dengyu Cheng; Jun Dou

    2014-01-01

    Background: Wnt/β-catenin signaling pathway plays a key role in human breast cancer progression. In this study, we down regulated β-catenin expression in human breast cancer MDA-MB-231 cells and investigated the effect of β-catenin knockdown on the cell biological characteristics. Materials and Methods: The recombinant plasmids of pSUPER-enhancement green fluorescent protein 1 (EGFP1)-scrabble-β-catenin-short hairpin ribonucleic acid (shRNA) and pSUPER-EGFP1-β-catenin-shRNA-1 were transfe...

  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. Erythropoietin inhibits gamma-irradiation-induced apoptosis by upregulation of Bcl-2 and decreasing the activation of caspase 3 in human UT-7/erythropoietin cell line.

    Science.gov (United States)

    Liu, Yuan-Yuan; She, Zhen-Jue; Yao, Ming-Hui

    2010-05-01

    1. Erythropoietin (EPO) can reverse radiotherapy-induced anaemia by stimulating bone marrow cells to produce erythrocytes. However, there are limited studies that address the mechanisms by which EPO exerts its beneficial effects in radiotherapy-induced anaemia. In the present study, we used a human bone marrow-derived EPO-dependent leukaemia cell line UT-7/EPO that progressed further in erythroid development to evaluate the anti-apoptotic effects of EPO on irradiated human erythroid progenitor. 2. The UT-7/EPO cells exposed to gamma-irradiation were cultured in the presence or absence of EPO at a concentration of 7 U/mL. The cell viability, cell apoptosis and the expression of apoptosis-related proteins Bcl-2, Bax and caspase 3 were examined. 3. The results showed that EPO protected the viability of human UT-7/EPO cells exposed to gamma-irradiation. EPO significantly inhibited gamma-irradiation-induced apoptosis in human UT-7/EPO cells: a significant decrease in the percentage of apoptotic cells was observed (62, 69 and 62% at 24, 48 and 72 h, respectively). Furthermore, EPO significantly increased the expression of Bcl-2 protein and the relative Bcl-2/Bax ratio, and decreased the activation of caspase 3 and formation of the p17 and p12 cleavage in similar conditions. 4. In conclusion, EPO exerts anti-apoptotic effects on irradiated human UT-7/EPO cells through upregulation of Bcl-2 protein and the relative Bcl-2/Bax ratio, and by decreasing the activation of caspase 3. These findings may contribute to our understanding of the beneficial function of EPO in radiotherapy-induced anaemia.

  20. P-glycoprotein (ABCB1) activity decreases raltegravir disposition in primary CD4+P-gphigh cells and correlates with HIV-1 viral load

    Science.gov (United States)

    Minuesa, Gerard; Arimany-Nardi, Cristina; Erkizia, Itziar; Cedeño, Samandhy; Moltó, José; Clotet, Bonaventura; Pastor-Anglada, Marçal; Martinez-Picado, Javier

    2016-01-01

    Objectives To evaluate the role of P-glycoprotein (P-gp) and multidrug-resistant-protein 1 (MRP1) on raltegravir intracellular drug disposition in CD4+ T cells, investigate the effect of HIV-1 infection on P-gp expression and correlate HIV-1 viraemia with P-gp activity in primary CD4+ T cell subsets. Methods The cellular accumulation ratio of [3H]raltegravir was quantified in CD4+ T cell lines overexpressing either P-gp (CEM-P-gp) or MRP1 (CEM-MRP1) and in primary CD3+CD4+ T cells with high (P-gphigh) and low P-gp activity (P-gplow); inhibition of efflux transporters was confirmed by the intracellular retention of calcein-AM. The correlation of P-gp activity with HIV-1 viraemia was assessed in naive and memory T cell subsets from 21 HIV-1-infected treatment-naive subjects. Results [3H]Raltegravir cellular accumulation ratio decreased in CEM-P-gp cells (P < 0.0001). XR9051 (a P-gp inhibitor) and HIV-1 PIs reversed this phenomenon. Primary CD4+P-gphigh cells accumulated less raltegravir (38.4% ± 9.6%) than P-gplow cells, whereas XR9051 also reversed this effect. In vitro HIV-1 infection of PBMCs and stimulation of CD4+ T cells increased P-gp mRNA and P-gp activity, respectively, while primary CD4+P-gphigh T cells sustained a higher HIV-1 replication than P-gplow cells. A significant correlation between HIV-1 viraemia and P-gp activity was found in different CD4+ T cell subsets, particularly memory CD4+ T cells (r = 0.792, P < 0.0001). Conclusions Raltegravir is a substrate of P-gp in CD4+ T cells. Primary CD4+P-gphigh T cells eliminate intracellular raltegravir more readily than P-gplow cells and HIV-1 viraemia correlates with P-gp overall activity. Specific CD4+P-gphigh T cell subsets could facilitate the persistence of viral replication in vivo and ultimately promote the appearance of drug resistance. PMID:27334660

  1. Activation of Estrogen Receptor Transfected into a Receptor-Negative Brest Cancer Cell Line Decreases the Metastatic and Invasive Potential of the Cells

    Science.gov (United States)

    Garcia, Marcel; Derocq, Danielle; Freiss, Gilles; Rochefort, Henri

    1992-12-01

    Breast cancers containing estrogen receptors are responsive to antiestrogen treatment and have a better prognosis than estrogen receptor-negative tumors. The loss of estrogen and progesterone receptors appears to be associated with a progression to less-differentiated tumors. We transfected the human estrogen receptor into the estrogen receptor-negative metastatic breast cancer cell line MDA-MB-231 in an attempt to restore their sensitivity to antiestrogens. Two stable sublines of MDA-MB-231 cells (HC1 and HE5) expressing functional estrogen receptors were studied for their ability to grow and invade in vitro and to metastasize in athymic nude mice. The number and size of lung metastases developed by these two sublines in ovariectomized nude mice was not markedly altered by tamoxifen but was inhibited 3-fold by estradiol. Estradiol also significantly inhibited in vitro cell proliferation of these sublines and their invasiveness in Matrigel, a reconstituted basement membrane, whereas the antiestrogens 4-hydroxytamoxifen and ICI 164,384 reversed these effects. These results show that estradiol inhibits the metastatic ability of estrogen receptornegative breast cancer cells following transfection with the estrogen receptor, whereas estrogen receptor-positive breast cancers are stimulated by estrogen, indicating that factors other than the estrogen receptor are involved in progression toward hormone independence. Reactivation or transfer of the estrogen receptor gene can therefore be considered as therapeutic approaches to hormone-independent cancers

  2. Aging correlates with decreased beta-cell proliferative capacity and enhanced sensitivity to apoptosis: a potential role for Fas and pancreatic duodenal homeobox-1

    OpenAIRE

    Maedler, Kathrin; Schumann, Desiree; Schulthess, Fabienne; Oberholzer, José; Bosco, Domenico; Berney, Thierry; Donath, Marc Y

    2006-01-01

    Type 2 diabetes is characterized by a deficit in beta-cell mass, and its incidence increases with age. Here, we analyzed beta-cell turnover in islets from 2- to 3- compared with 7- to 8-month-old rats and in human islets from 53 organ donors with ages ranging from 17 to 74 years. In cultured islets from 2- to 3-month-old rats, the age at which rats are usually investigated, increasing glucose from 5.5 to 11.1 mmol/l decreased beta-cell apoptosis, which was augmented when glucose was further i...

  3. Decreased helenalin-induced cytotoxicity by flavonoids from Arnica as studied in a human lung carcinoma cell line

    NARCIS (Netherlands)

    Woerdenbag, HJ; Merfort, [No Value; Schmidt, TJ; Passreiter, CM; Willuhn, G; vanUden, W; Pras, N; Konings, AWT

    1995-01-01

    The effect of the flavones apigenin, luteolin, hispidulin and eupafolin, and of the flavonols kaempferol, quercetin, 6-methoxykaempferol and patuletin from Amica spp, on the cytotoxicity of the sesquiterpene lactone helenalin was studied in the human lung carcinoma cell line GLC(4) using the microcu

  4. Argininosuccinate Synthase 1-Deficiency Enhances the Cell Sensitivity to Arginine through Decreased DEPTOR Expression in Endometrial Cancer

    Science.gov (United States)

    Ohshima, Kenji; Nojima, Satoshi; Tahara, Shinichiro; Kurashige, Masako; Hori, Yumiko; Hagiwara, Kohei; Okuzaki, Daisuke; Oki, Shinya; Wada, Naoki; Ikeda, Jun-ichiro; Kanai, Yoshikatsu; Morii, Eiichi

    2017-01-01

    Argininosuccinate synthetase 1 (ASS1) is a rate-limiting enzyme in arginine biosynthesis. Although ASS1 expression levels are often reduced in several tumors and low ASS1 expression can be a poor prognostic factor, the underlying mechanism has not been elucidated. In this study, we reveal a novel association between ASS1 and migration/invasion of endometrial tumors via regulation of mechanistic target of rapamycin complex (mTORC) 1 signaling. ASS1-knockout cells showed enhanced migration and invasion in response to arginine following arginine starvation. In ASS1-knockout cells, DEPTOR, an inhibitor of mTORC1 signal, was downregulated and mTORC1 signaling was more activated in response to arginine. ASS1 epigenetically enhanced DEPTOR expression by altering the histone methylation. Consistent with these findings, tumor cells at the invasive front of endometrioid carcinoma cases showed lower ASS1 and DEPTOR expression. Our findings suggest that ASS1 levels in each tumor cell are associated with invasion capability in response to arginine within the tumor microenvironment through mTORC1 signal regulation. PMID:28358054

  5. Decreased gene expression of human beta-defensin-1 in the development of squamous cell carcinoma of the oral cavity.

    NARCIS (Netherlands)

    Wenghoefer, M.H.; Pantelis, A.; Dommisch, H.; Reich, R.; Martini, M.; Allam, J.P.; Novak, N.; Berge, S.; Jepsen, S.; Winter, J.

    2008-01-01

    The aim of this study was to investigate the gene expression of human beta-defensin-1, -2, -3 (hBD-1, -2, -3), interleukin-1beta, tumour necrosis factor-alpha and cyclooxygenase-2 in oral squamous cell carcinoma (OSCC) compared to benign and premalignant lesions as well as healthy controls. Biopsies

  6. Extracellular ATP decreases trophoblast invasion, spiral artery remodeling and immune cells in the mesometrial triangle in pregnant rats

    NARCIS (Netherlands)

    Spaans, F.; Melgert, B. N.; Chiang, C.; Borghuis, T.; Klok, P. A.; de Vos, P.; van Goor, H.; Bakker, W.W.; Faas, M. M.

    2014-01-01

    Introduction: Preeclampsia is characterized by deficient trophoblast invasion and spiral artery remodeling, a process governed by inflammatory cells. High levels of the danger signal extracellular adenosine triphosphate (ATP) have been found in women with preeclampsia and infusion of ATP in pregnant

  7. Diet-induced glucose intolerance in mice with decreased beta-cell ATP-sensitive K+ channels.

    Science.gov (United States)

    Remedi, Maria S; Koster, Joseph C; Markova, Kamelia; Seino, Susumu; Miki, Takashi; Patton, Brian L; McDaniel, Michael L; Nichols, Colin G

    2004-12-01

    ATP-sensitive K+ channels (K(ATP) channels) control electrical activity in beta-cells and therefore are key players in excitation-secretion coupling. Partial suppression of beta-cell K(ATP) channels in transgenic (AAA) mice causes hypersecretion of insulin and enhanced glucose tolerance, whereas complete suppression of these channels in Kir6.2 knockout (KO) mice leads to hyperexcitability, but mild glucose intolerance. To test the interplay of hyperexcitability and dietary stress, we subjected AAA and KO mice to a high-fat diet. After 3 months on the diet, both AAA and KO mice converted to an undersecreting and markedly glucose-intolerant phenotype. Although Kir6.2 is expressed in multiple tissues, its primary functional consequence in both AAA and KO mice is enhanced beta-cell electrical activity. The results of our study provide evidence that, when combined with dietary stress, this hyperexcitability is a causal diabetic factor. We propose an "inverse U" model for the response to enhanced beta-cell excitability: the expected initial hypersecretion can progress to undersecretion and glucose-intolerance, either spontaneously or in response to dietary stress.

  8. Optimization of temperature, sugar concentration, and inoculum size to maximize ethanol production without significant decrease in yeast cell viability.

    Science.gov (United States)

    Laluce, Cecilia; Tognolli, João Olimpio; de Oliveira, Karen Fernanda; Souza, Crisla Serra; Morais, Meline Rezende

    2009-06-01

    Aiming to obtain rapid fermentations with high ethanol yields and a retention of high final viabilities (responses), a 2(3) full-factorial central composite design combined with response surface methodology was employed using inoculum size, sucrose concentration, and temperature as independent variables. From this statistical treatment, two well-fitted regression equations having coefficients significant at the 5% level were obtained to predict the viability and ethanol production responses. Three-dimensional response surfaces showed that increasing temperatures had greater negative effects on viability than on ethanol production. Increasing sucrose concentrations improved both ethanol production and viability. The interactions between the inoculum size and the sucrose concentrations had no significant effect on viability. Thus, the lowering of the process temperature is recommended in order to minimize cell mortality and maintain high levels of ethanol production when the temperature is on the increase in the industrial reactor. Optimized conditions (200 g/l initial sucrose, 40 g/l of dry cell mass, 30 degrees C) were experimentally confirmed and the optimal responses are 80.8 +/- 2.0 g/l of maximal ethanol plus a viability retention of 99.0 +/- 3.0% for a 4-h fermentation period. During consecutive fermentations with cell reuse, the yeast cell viability has to be kept at a high level in order to prevent the collapse of the process.

  9. Decrease of FOXP3 mRNA in CD4~+ T cells in latent autoimmune diabetes in adult

    Institute of Scientific and Technical Information of China (English)

    杨治芳

    2006-01-01

    Objective To study the percentage of peripheral blood CD4+ CD25+ T cells and the expression of F0XP3 mRNA in patients with latent autoimmune diabetes in adult (LADA). Methods Fresh peripheral blood samples were obtained from 60 patients with LADA,30 patients with type 2 diabetes and 30 age- and sex-matched

  10. Decreasing Pin1 suppresses telomerase activity by NF-κB in HCT116 cells colorectal carcinoma

    Institute of Scientific and Technical Information of China (English)

    Jianwen Sun; Lijun Fan; Meining Li; Yuehong Zhang; Niuliang Cheng

    2013-01-01

    Objective: The aim of our study was to investigate the effect of Pin1 on the telomerase activity in human colorectal carcinoma HCT116 cells. Methods: Firstly, we transfected plasmid pGenesil-1-Pin1 (p-shRNA) using liposome (Lipofectamine 2000) into colorectal cancer HCT-116 cells to down-regulate the expression of Pin1. To detect the apoptotic rate of HCT116 cells was by cytometry (FCM). The expression of Pin1 and hTERT at RNA levels in human colorectal cancer HCT116 cells were determined by RT-PCR. To evaluate the activity of telomerase was by TRAP-silver staining. The subcellular localization and accumulative level of p-NF-κB/p65 protein at the nuclear was detected by Immunofluorescence and Western blotting. The DNA-binding activity of NF-κB/p65 was detected by electrophoretic mobility shift assay (EMSA). Results: Using liposome into colorectal cancer HCT-116 cells, and down-regulate the expression of Pin1 (0.392 ± 0.072-fold; P = 0.001), and the apoptotic rate was increased (11.40% ± 1.54%; P < 0.05). Compared with transfected p-CON cell group, in transfected p-shRNA cell group, the transcription of hTERT was lower (0.171 ± 0.060-fold; P = 0.001) by quantitative real-time RT-PCR, and the results of TRAP-silver staining analysis suggested that the telomerase activity was significantly declined (0.384 ± 0.015-fold; P < 0.05). Furthermore, it was demonstrated by Immunofluorescence that p-NF-κB/p65 had a nuclear localization, and the level of p-NF-κB/p65 protein at the nuclear was reduced with silencing the expression of Pin1 by Western blotting. Using EMSA, it was suggested that NF-κB/p65 was able to bind to hTERT promoter, and the direct interaction was declined with silencing the expression of Pin1. Conclusion: Taken together, silencing Pin1 may suppress activity of telomerase and the expression of hTERT by inhibiting NF-κB/p65 activity and reducing the combination of NF-κB/p65 and hTERT gene promoter.

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

    Science.gov (United States)

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

    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 600mg/kg body weight/day for 28days. In the subgranular zone (SGZ), 600mg/kg CPZ increased the number of cleaved caspase-3(+) apoptotic cells. At ≥120mg/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 ≥120mg/kg decreased the number of postmitotic granule cells immunoreactive for NEUN, CHRNA7, ARC or FOS. In the dentate hilus, CPZ at ≥120mg/kg decreased phosphorylated TRKB(+) interneurons, although the number of reelin(+) interneurons was unchanged. At 600mg/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.

  12. Methamphetamine decreases CD4 T cell frequency and alters pro-inflammatory cytokine production in a model of drug abuse.

    Science.gov (United States)

    Mata, Mariana M; Napier, T Celeste; Graves, Steven M; Mahmood, Fareeha; Raeisi, Shohreh; Baum, Linda L

    2015-04-01

    The reason co-morbid methamphetamine use and HIV infection lead to more rapid progression to AIDS is unclear. We used a model of methamphetamine self-administration to measure the effect of methamphetamine on the systemic immune system to better understand the co-morbidity of methamphetamine and HIV. Catheters were implanted into the jugular veins of male, Sprague Dawley rats so they could self-administer methamphetamine (n=18) or be given saline (control; n=16) for 14 days. One day after the last operant session, blood and spleens were collected. We measured serum levels of pro-inflammatory cytokines, intracellular IFN-γ and TNF-α, and frequencies of CD4(+), CD8(+), CD200(+) and CD11b/c(+) lymphocytes in the spleen. Rats that self-administered methamphetamine had a lower frequency of CD4(+) T cells, but more of these cells produced IFN-γ. Methamphetamine did not alter the frequency of TNF-α-producing CD4(+) T cells. Methamphetamine using rats had a higher frequency of CD8(+) T cells, but fewer of them produced TNF-α. CD11b/c and CD200 expression were unchanged. Serum cytokine levels of IFN-γ, TNF-α and IL-6 in methamphetamine rats were unchanged. Methamphetamine lifetime dose inversely correlated with serum TNF-α levels. Our data suggest that methamphetamine abuse may exacerbate HIV disease progression by activating CD4 T cells, making them more susceptible to HIV infection, and contributing to their premature demise. Methamphetamine may also increase susceptibility to HIV infection, explaining why men who have sex with men (MSM) and frequently use methamphetamine are at the highest risk of HIV infection.

  13. 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.

  14. 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.

  15. Decrease of breast cancer cell invasiveness by sodium phenylacetate (NaPa) is associated with an increased expression of adhesive molecules.

    Science.gov (United States)

    Vasse, M; Thibout, D; Paysant, J; Legrand, E; Soria, C; Crépin, M

    2001-03-23

    Sodium phenylacetate (NaPa), a non-toxic phenylalanine metabolite, has been shown to induce in vivo and in vitro cytostatic and antiproliferative effects on various cell types. In this work, we analysed the effect of NaPa on the invasiveness of breast cancer cell (MDA-MB-231, MCF-7 and MCF-7 ras). Using the highly invasive breast cancer cell line MDA-MB-231, we demonstrated that an 18-hour incubation with NaPa strongly inhibits the cell invasiveness through Matrigel (86% inhibition at 20 mM of NaPa). As cell invasiveness is greatly influenced by the expression of urokinase (u-PA) and its cell surface receptor (u-PAR) as well as the secretion of matrix metalloproteinases (MMP), we tested the effect of NaPa on these parameters. An 18-hour incubation with NaPa did not modify u-PA expression, either on MDA-MB-231 or on MCF-7 and MCF-7 ras cell lines, and induced a small u-PA decrease after 3 days of treatment of MDA-MB-321 with NaPa. In contrast, an 18 h incubation of MDA-MB-231 increased the expression of u-PAR and the secretion of MMP-9. As u-PAR is a ligand for vitronectin, a composant of the extracellular matrix, these data could explain the increased adhesion of MDA-MB-231 to vitronectin, while cell adhesivity of MCF-7 and MCF-7 ras was unmodified by NaPa treatment. NaPa induced also an increased expression of both Lymphocyte Function-Associated-1 (LFA-1) and Intercellular Adhesion Molecule-1 (ICAM-1), which was obvious from 18 hour incubation with NaPa for the MDA-MB-231 cells, but was delayed (3 days) for MCF-7 and MCF-7 ras. Only neutralizing antibodies against LFA-1 reversed the decreased invasiveness of NaPa-treated cells. Therefore we can conclude that the strong inhibition of MDA-MB-231 invasiveness is not due to a decrease in proteases involved in cell migration (u-PA and MMP) but could be related both to the modification of cell structure and an increased expression of adhesion molecules such as u-PAR and LFA-1.

  16. 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.

  17. 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.

  18. Severe feline sporotrichosis associated with an increased population of CD8low cells and a decrease in CD4⁺ cells.

    Science.gov (United States)

    Miranda, Luisa H M; Santiago, Marta de A; Schubach, Tânia M P; Morgado, Fernanda N; Pereira, Sandro A; de Oliveira, Raquel de V C; Conceição-Silva, Fátima

    2016-01-01

    Sporotrichosis is a subcutaneous mycosis with worldwide distribution, especially in tropical and subtropical areas. Zoonotic transmission is described with cats being the main animal species involved. The occurrence of severe feline sporotrichosis with high fungal levels demonstrates the susceptibility of cats to this disease and the importance of studying its pathogenesis. This study describes the leukocytes profile in blood of cats with sporotrichosis by flow cytometry and its correlation with histopathology and fungal load. The cats with sporotrichosis were separated into groups L1, L2, and L3 (lesions at one, two, and three or more noncontiguous skin locations, respectively) and were classified as good, fair, or poor general conditions. The highest percentage of CD4+ cells was associated to L1 (P = .04) and to good general condition (P = .03). The percentage of CD8+ cells was greater in L2 and L3 (P = .01). CD8(low) expression occurred in 20 animals with sporotrichosis, mainly in L3 (P = .01) and was not observed in healthy controls. This expression was related to macrophage granulomas (P = .01) and predominated in cases with high fungal load. Altogether, the results indicated that control over feline sporotrichosis, with maintenance of a good general condition, fixed lesions, well-organized response and lower fungal load, is associated with increased CD4+ cells percentages. In contrast, a poor general condition, disseminated lesions and high fungal load were related to increased CD8+ cell percentages and increased expression of CD8(low). As conclusion these results point to an important role of the CD4:CD8 balance in determining the clinical outcome in feline sporotrichosis.

  19. Global Decrease of Histone H3K27 Acetylation in ZEB1-Induced Epithelial to Mesenchymal Transition in Lung Cancer Cells

    Energy Technology Data Exchange (ETDEWEB)

    Roche, Joëlle, E-mail: joelle.roche@univ-poitiers.fr [Department of Medicine, Hematology Oncology Division, MUSC, 96 Jonathan Lucas St., Charleston, SC 29425 (United States); CNRS FRE 3511, University of Poitiers, 1 rue Georges Bonnet, F-86022 Poitiers Cédex (France); Nasarre, Patrick; Gemmill, Robert [Department of Medicine, Hematology Oncology Division, MUSC, 96 Jonathan Lucas St., Charleston, SC 29425 (United States); Baldys, Aleksander [Department of Medicine, Nephrology Division, MUSC, Ralph H. Johnson Veterans Affairs Medical Center, Charleston, SC 29425 (United States); Pontis, Julien [Epigénétique & Destin Cellulaire, CNRS UMR 7216, University of Paris Diderot, Sorbonne Paris Cité, F-75013 Paris (France); Korch, Christopher [CU DNA Sequencing and Analysis Core, University of Colorado, School of Medicine, Anschutz Medical Campus, 12801 E. 17th Ave., Aurora, CO 80045 (United States); Guilhot, Joëlle [INSERM, CIC 0802, CHU de Poitiers, F-86021 (France); Ait-Si-Ali, Slimane [Epigénétique & Destin Cellulaire, CNRS UMR 7216, University of Paris Diderot, Sorbonne Paris Cité, F-75013 Paris (France); Drabkin, Harry [Department of Medicine, Hematology Oncology Division, MUSC, 96 Jonathan Lucas St., Charleston, SC 29425 (United States)

    2013-04-03

    The epithelial to mesenchymal transition (EMT) enables epithelial cells with a migratory mesenchymal phenotype. It is activated in cancer cells and is involved in invasion, metastasis and stem-like properties. ZEB1, an E-box binding transcription factor, is a major suppressor of epithelial genes in lung cancer. In the present study, we show that in H358 non-small cell lung cancer cells, ZEB1 downregulates EpCAM (coding for an epithelial cell adhesion molecule), ESRP1 (epithelial splicing regulatory protein), ST14 (a membrane associated serine protease involved in HGF processing) and RAB25 (a small G-protein) by direct binding to these genes. Following ZEB1 induction, acetylation of histone H4 and histone H3 on lysine 9 (H3K9) and 27 (H3K27) was decreased on ZEB1 binding sites on these genes as demonstrated by chromatin immunoprecipitation. Of note, decreased H3K27 acetylation could be also detected by western blot and immunocytochemistry in ZEB1 induced cells. In lung cancers, H3K27 acetylation level was higher in the tumor compartment than in the corresponding stroma where ZEB1 was more often expressed. Since HDAC and DNA methylation inhibitors increased expression of ZEB1 target genes, targeting these epigenetic modifications would be expected to reduce metastasis.

  20. Salinomycin enhances doxorubicin-induced cytotoxicity in multidrug resistant MCF-7/MDR human breast cancer cells via decreased efflux of doxorubicin.

    Science.gov (United States)

    Kim, Kwang-Youn; Kim, Sang-Hun; Yu, Sun-Nyoung; Park, Suel-Ki; Choi, Hyeun-Deok; Yu, Hak-Sun; Ji, Jae-Hoon; Seo, Young-Kyo; Ahn, Soon-Cheol

    2015-08-01

    Salinomycin is a monocarboxylic polyether antibiotic, which is widely used as an anticoccidial agent. The anticancer property of salinomycin has been recognized and is based on its ability to induce apoptosis in human multidrug resistance (MDR). The present study investigated whether salinomycin reverses MDR towards chemotherapeutic agents in doxorubicin-resistant MCF-7/MDR human breast cancer cells. The results demonstrated that doxorubicin-mediated cytotoxicity was significantly enhanced by salinomycin in the MCF-7/MDR cells, and this occurred in a dose-dependent manner. This finding was consistent with subsequent observations made under a confocal microscope, in which the doxorubicin fluorescence signals of the salinomycin-treated cells were higher compared with the cells treated with doxorubicin alone. In addition, flow cytometric analysis revealed that salinomycin significantly increased the net cellular uptake and decreased the efflux of doxorubicin. The expression levels of MDR-1 and MRP-1 were not altered at either the mRNA or protein levels in the cells treated with salinomycin. These results indicated that salinomycin was mediated by its ability to increase the uptake and decrease the efflux of doxorubicin in MCF-7/MDR cells. Salinomycin reversed the resistance of doxorubicin, suggesting that chemotherapy in combination with salinomycin may benefit MDR cancer therapy.

  1. High Population Density of Juvenile Chum Salmon Decreased the Number and Sizes of Growth Hormone Cells in the Pituitary

    OpenAIRE

    Salam, Md. Abdus; Ota, Yuki; Ando, Hironori; Fukuwaka, Masa-aki; Kaeriyama, Masahide; Urano, Akihisa

    1999-01-01

    Juveniles of chum salmon (Oncorhynchus keta) held at high population density were apparently smaller than those held at medium and low population densities. The effects of high population density on pituitary growth hormone (GH) cells in juvenile chum salmon were examined using immunocytochemical and in situ hybridization techniques. The ratio of GH-immunoreactive (ir) area to the whole pituitary was almost constant in all of the high, medium and low population density groups, although the nu...

  2. Cytotoxic isolates of Helicobacter pylori from Peptic Ulcer Diseases decrease K+-dependent ATPase Activity in HeLa cells

    Directory of Open Access Journals (Sweden)

    Archana Ayyagari

    2003-11-01

    Full Text Available Abstract Background Helicobacter pylori is a Gram negative bacterium that plays a central role in the etiology of chronic gastritis and peptic ulcer diseases. However, not all H. pylori positive cases develop advanced disease. This discriminatory behavior has been attributed to the difference in virulence of the bacteria. Among all virulence factors, cytotoxin released by H. pylori is the most important factor. In this work, we studied variation in H. pylori isolates from Indian dyspeptic patients on the basis of cytotoxin production and associated changes in K+-dependent ATPase (one of its targets enzyme activity in HeLa cells. Methods The patients were retrospectively grouped on the basis of endoscopic and histopathological observation as having gastritis or peptic ulcer. The HeLa cells were incubated with the broth culture filtrates (BCFs of H. pylori isolates from patients of both groups and observed for the cytopathic effects: morphological changes and viability. In addition, the K+-dependent ATPase activity was measured in HeLa cells extracts. Results The cytotoxin production was observed in 3/7 (gastritis and 4/4 (peptic ulcer H. pylori isolates. The BCFs of cytotoxin producing H. pylori strains reduced the ATPase activity of HeLa cells to 40% of that measured with non-cytotoxin producing H. pylori strains (1.33 μmole Pi/mg protein and 3.36 μmole Pi/mg protein, respectively, p Conclusions Our results suggest that the isolation of cytotoxic H. pylori is more common in severe form of acid peptic diseases (peptic ulcer than in gastritis patients from India. Also the cytotoxin released by H. pylori impairs the ion-transporting ATPase and is a measure of cytotoxicity.

  3. Disodium pentaborate decahydrate (DPD) induced apoptosis by decreasing hTERT enzyme activity and disrupting F-actin organization of prostate cancer cells.

    Science.gov (United States)

    Korkmaz, Mehmet; Avcı, Cigir Biray; Gunduz, Cumhur; Aygunes, Duygu; Erbaykent-Tepedelen, Burcu

    2014-02-01

    Animal and cell culture studies have showed that boron and its derivatives may be promising anticancer agents in prostate cancer treatment. Thus, DU145 cells were treated with disodium pentaborate decahydrate (DPD) for 24, 48, and 72 h in order to investigate the inhibitor effect and mechanisms of DPD. Then, cell proliferation, telomerase enzyme activity, actin polymerization, and apoptosis were detected by WST-1 assay, qRT-PCR, immunofluorescence labeling, and flow cytometry, respectively. We found that DPD inhibited the growth of human prostate cancer cell line DU145 at the concentration of 3.5 mM for 24 h. Our results demonstrated that 7 mM of DPD treatment prevented the telomerase enzyme activity at the rate of 38 %. Furthermore, DPD has an apoptotic effect on DU145 cells which were examined by labeling DNA breaks. With 7 mM of DPD treatment, 8, 14, and 41 % of apoptotic cells were detected for 24, 48, and 72 h, respectively. Additionally, immunofluorescence labeling showed that the normal organization of actin filaments was disrupted in DPD-exposed cells, which is accompanied by the alteration of cell shape and by apoptosis in targeted cells. Taken together, the results indicate that DPD may exert its cytotoxicity at least partly by interfering with the dynamic properties of actin polymerization and decreasing the telomerase activity. Eventually, for the first time, the results of this study showed that DPD suppressed the activity of telomerase in DU145 cells, and therefore, we suggested that DPD could be an important agent for its therapeutic potential in the treatment of prostate cancer.

  4. A nonconjugated bridge in dimer-sensitized solar cells retards charge recombination without decreasing charge injection efficiency.

    Science.gov (United States)

    Sunahara, Kenji; Griffith, Matthew J; Uchiyama, Takayuki; Wagner, Pawel; Officer, David L; Wallace, Gordon G; Mozer, Attila J; Mori, Shogo

    2013-11-13

    Dye sensitized solar cells (DSSCs) employing a dimer porphyrin, which was synthesised with two porphyrin units connected without conjugation, have shown that both porphyrin components can contribute to photocurrent generation, that is, more than 50 % internal quantum efficiency. In addition, the open-circuit voltage (Voc) of the DSSCs was higher than that of DSSCs using monomer porphyrins. In this paper, we first optimized cell structure and fabrication conditions. We obtained more than 80% incident photon to current conversion efficiency from the dimer porphyrin sensitized DSSCs and higher Voc and energy conversion efficiency than monomer porphyrin sensitized solar cells. To examine the origin of the higher Voc, we measured electron lifetime in the DSSCs with various conditions, and found that the dimer system increased the electron lifetime by improving the steric blocking effect of the dye layer, whilst the lack of a conjugated linker prevents an increase in the attractive force between conjugated sensitizers and the acceptor species in the electrolyte. The results support a hypothesis; dispersion force is one of the factors influencing the electron lifetime in DSSCs.

  5. Purple carrot (Daucus carota L.) polyacetylenes decrease lipopolysaccharide-induced expression of inflammatory proteins in macrophage and endothelial cells.

    Science.gov (United States)

    Metzger, Brandon T; Barnes, David M; Reed, Jess D

    2008-05-28

    Carrots ( Daucus carota L.) contain phytochemicals including carotenoids, phenolics, polyacetylenes, isocoumarins, and sesquiterpenes. Purple carrots also contain anthocyanins. The anti-inflammatory activity of extracts and phytochemicals from purple carrots was investigated by determining attenuation of the response to lipopolysaccharide (LPS). A bioactive chromatographic fraction (Sephadex LH-20) reduced LPS inflammatory response. There was a dose-dependent reduction in nitric oxide production and mRNA of pro-inflammatory cytokines (IL-6, IL-1beta, TNF-alpha) and iNOS in macrophage cells. Protein secretions of IL-6 and TNF-alpha were reduced 77 and 66% in porcine aortic endothelial cells treated with 6.6 and 13.3 microg/mL of the LH-20 fraction, respectively. Preparative liquid chromatography resulted in a bioactive subfraction enriched in the polyacetylene compounds falcarindiol, falcarindiol 3-acetate, and falcarinol. The polyacetylenes were isolated and reduced nitric oxide production in macrophage cells by as much as 65% without cytotoxicity. These results suggest that polyacetylenes, not anthocyanins, in purple carrots are responsible for anti-inflammatory bioactivity.

  6. Inhibition of host extracellular signal-regulated kinase (ERK) activation decreases new world alphavirus multiplication in infected cells

    Energy Technology Data Exchange (ETDEWEB)

    Voss, Kelsey; Amaya, Moushimi [National Center for Biodefense and Infectious Diseases, School of Systems Biology, George Mason University, 10650 Pyramid Place, Manassas, VA (United States); Mueller, Claudius [Center for Applied Proteomics and Personalized Medicine, George Mason University, 10900 University Boulevard, Manassas, VA (United States); Roberts, Brian [Leidos Health Life Sciences, 5202 Presidents Court, Suite 110, Frederick, MD (United States); Kehn-Hall, Kylene; Bailey, Charles [National Center for Biodefense and Infectious Diseases, School of Systems Biology, George Mason University, 10650 Pyramid Place, Manassas, VA (United States); Petricoin, Emanuel [Center for Applied Proteomics and Personalized Medicine, George Mason University, 10900 University Boulevard, Manassas, VA (United States); Narayanan, Aarthi, E-mail: anaraya1@gmu.edu [National Center for Biodefense and Infectious Diseases, School of Systems Biology, George Mason University, 10650 Pyramid Place, Manassas, VA (United States)

    2014-11-15

    New World alphaviruses belonging to the family Togaviridae are classified as emerging infectious agents and Category B select agents. Our study is focused on the role of the host extracellular signal-regulated kinase (ERK) in the infectious process of New World alphaviruses. Infection of human cells by Venezuelan equine encephalitis virus (VEEV) results in the activation of the ERK-signaling cascade. Inhibition of ERK1/2 by the small molecule inhibitor Ag-126 results in inhibition of viral multiplication. Ag-126-mediated inhibition of VEEV was due to potential effects on early and late stages of the infectious process. While expression of viral proteins was down-regulated in Ag-126 treated cells, we did not observe any influence of Ag-126 on the nuclear distribution of capsid. Finally, Ag-126 exerted a broad-spectrum inhibitory effect on New World alphavirus multiplication, thus indicating that the host kinase, ERK, is a broad-spectrum candidate for development of novel therapeutics against New World alphaviruses. - Highlights: • VEEV infection activated multiple components of the ERK signaling cascade. • Inhibition of ERK activation using Ag-126 inhibited VEEV multiplication. • Activation of ERK by Ceramide C6 increased infectious titers of TC-83. • Ag-126 inhibited virulent strains of all New World alphaviruses. • Ag-126 treatment increased percent survival of infected cells.

  7. Environmental enrichment and working memory tasks decrease hippocampal cell proliferation after wheel running--A role for the prefrontal cortex in hippocampal plasticity?

    Science.gov (United States)

    Schaefers, Andrea T U

    2015-10-22

    Despite an increasing amount of evidence about the regulation of adult hippocampal neurogenesis on the local level, less attention has been paid to its systemic embedding in wider brain circuits. The aim of the present study was to obtain evidence for a potential role of the prefrontal cortex in the regulation of adult hippocampal neurogenesis. We hypothesised that activation of the prefrontal cortex by environmental enrichment or a working-memory task would decrease previously enhanced cell proliferation rates. Wheel running was applied as a common stimulator of cell proliferation in CD1 mice reared under deprivation of natural environmental stimulation. Next, the animals were assigned to four groups for different treatments in the following three days: housing under continued deprivation, environmental enrichment, a spatial-delayed alternation task in an automated T-maze that activates the prefrontal cortex by working-memory requirements or a control task in the automated T-maze differing only in the single parameter working-memory-associated delay. Both the environmental enrichment and spatial-delayed alternation tasks decreased cell proliferation rates in the dentate gyrus compared to deprived housing and the control task in the T-maze. As the control animals underwent the same procedures and stressors and differed only in the single parameter working-memory-associated delay, the working-memory requirement seems to be the crucial factor for decreasing cell proliferation rates. Taken together, these results suggest that the prefrontal cortex may play a role in the regulation of hippocampal cell proliferation.

  8. Decreasing methylation of pectin caused by nitric oxide leads to higher aluminium binding in cell walls and greater aluminium sensitivity of wheat roots.

    Science.gov (United States)

    Sun, Chengliang; Lu, Lingli; Yu, Yan; Liu, Lijuan; Hu, Yan; Ye, Yiquan; Jin, Chongwei; Lin, Xianyong

    2016-02-01

    Nitric oxide (NO) is an important bioactive molecule involved in cell wall metabolism, which has been recognized as a major target of aluminium (Al) toxicity. We have investigated the effects of Al-induced NO production on cell wall composition and the subsequent Al-binding capacity in roots of an Al-sensitive cultivar of wheat (Triticum aestivum L. cv. Yang-5). We found that Al exposure induced NO accumulation in the root tips. Eliminating NO production with an NO scavenger (cPTIO) significantly alleviated the Al-induced inhibition of root growth and thus reduced Al accumulation. Elimination of NO, however, did not significantly affect malate efflux or rhizosphere pH changes under Al exposure. Levels of cell wall polysaccharides (pectin, hemicelluloses 1, and hemicelluloses 2) and pectin methylesterase activity, as well as pectin demethylation in the root apex, significantly increased under Al treatment. Exogenous cPTIO application significantly decreased pectin methylesterase activity and increased the degree of methylation of pectin in the root cell wall, thus decreasing the Al-binding capacity of pectin. These results suggest that the Al-induced enhanced production of NO decreases cell wall pectin methylation, thus increasing the Al-binding capacity of pectin and negatively regulating Al tolerance in wheat.

  9. 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

  10. Cultured senescent myoblasts derived from human vastus lateralis exhibit normal mitochondrial ATP synthesis capacities with correlating concomitant ROS production while whole cell ATP production is decreased

    DEFF Research Database (Denmark)

    Minet, Ariane D; Gaster, Michael

    2012-01-01

    The free radical theory of aging says that increased oxidative stress and mitochondrial dysfunction are associated with old age. In the present study we have investigated the effects of cellular senescence on muscle energetic by comparing mitochondrial content and function in cultured muscle...... satellite cells at early and late passage numbers. We show that cultured muscle satellite cells undergoing senescence express a reduced mitochondrial mass, decreased whole cell ATP level, normal to increased mitochondrial ATP production under ATP utilization, increased mitochondrial membrane potential......, but the existent mitochondria express normal to increased functional capabilities. The present data suggest that the origin of aging lies outside the mitochondria and that a malfunction in the cell might be preceding and initiating the increase of mitochondrial ATP synthesis and concomitant ROS production...

  11. 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

    . Leydig cell clusters of more than 15 cells in a cross-section, for which we proposed the name 'micronodules', were frequently seen in testicles exhibiting Sertoli-cell-only syndrome (SCO), a mixed pattern of impaired spermatogenesis, or complete spermatogenesis in combination with elevated FSH. Median...... numbers of micronodules per 1.77 mm(2) (four fields of vision) in these three histological patterns were 6, 4, and 3.5, respectively. In contrast, micronodules were only occasionally observed in testicular biopsies from patients with complete spermatogenesis and normal gonadotrophin levels (median 1...... 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...

  12. Apoptosis is increased and cell proliferation is decreased in out-of-phase endometria from infertile and recurrent abortion patients

    Directory of Open Access Journals (Sweden)

    Irigoyen Marcela

    2010-10-01

    Full Text Available Abstract Background Various endometrial abnormalities have been associated with luteal phase deficiency: a significant dyssynchrony in the maturation of the glandular epithelium and the stroma and a prevalence of out-of-phase endometrial biopsy specimens. Out-of phase endometrium is a controversial disorder related to failed implantation, infertility and early pregnancy loss. Given that the regulation of the apoptotic process in endometrium of luteal phase deficiency is still unknown, the aim of this study was to evaluate cell proliferation, apoptosis and the levels of the main effector caspase, caspase-3 in the luteal in-phase and out-of-phase endometrium. Methods Thirty-seven endometrial samples from sterile or recurrent abortion patients were included in this study: 21 in-phase samples (controls and 16 samples with out-of-phase endometrium. Biopsy specimens of eutopic endometrium were obtained from all subjects during days 21-25 of the menstrual cycle. The endometrium with endometrial maturity of cycle day 25 or less at the time of menstruation was considered out-of phase. Endometrial tissues were fixed in 10% buffered formaldehyde. For apoptosis quantification, sections were processed for in situ immunohistochemical localization of nuclei exhibiting DNA fragmentation, by the terminal deoxynucleotidyl transferase (TdT-mediated dUTP digoxygenin nick-end labeling (TUNEL technique. Expressions of Proliferating Cell Nuclear Antigen (PCNA as a marker of cell proliferation, and of cleaved caspase-3 as a marker of apoptosis, were assessed by immunohistochemistry in the luteal in-phase and out-of-phase endometrium from infertile and recurrent abortion patients. Results Luteal out-of-phase endometrium had increased apoptosis levels compared to in-phase endometrium (p Conclusions this study represents the first report describing variations at the cell proliferation and cell death levels in the out-of-phase endometrium in comparison with in

  13. Loss of Sparc in p53-null Astrocytes Promotes Macrophage Activation and Phagocytosis Resulting in Decreased Tumor Size and Tumor Cell Survival.

    Science.gov (United States)

    Thomas, Stacey L; Schultz, Chad R; Mouzon, Ezekiell; Golembieski, William A; El Naili, Reima; Radakrishnan, Archanna; Lemke, Nancy; Poisson, Laila M; Gutiérrez, Jorge A; Cottingham, Sandra; Rempel, Sandra A

    2015-07-01

    Both the induction of SPARC expression and the loss of the p53 tumor suppressor gene are changes that occur early in glioma development. Both SPARC and p53 regulate glioma cell survival by inverse effects on apoptotic signaling. Therefore, during glioma formation, the upregulation of SPARC may cooperate with the loss of p53 to enhance cell survival. This study determined whether the loss of Sparc in astrocytes that are null for p53 would result in reduced cell survival and tumor formation and increased tumor immunogenicity in an in vivo xenograft brain tumor model. In vitro, the loss of Sparc in p53-null astrocytes resulted in an increase in cell proliferation, but a loss of tumorigenicity. At 7 days after intracranial implantation, Sparc-null tumors had decreased tumor cell survival, proliferation and reduced tumor size. The loss of Sparc promoted microglia/macrophage activation and phagocytosis of tumor cells. Our results indicate that the loss of p53 by deletion/mutation in the early stages of glioma formation may cooperate with the induction of SPARC to potentiate cancer cell survival and escape from immune surveillance.

  14. Adenovirus-mediated hAQP1 expression in irradiated mouse salivary glands causes recovery of saliva secretion by enhancing acinar cell volume decrease.

    Science.gov (United States)

    Teos, L Y; Zheng, C-Y; Liu, X; Swaim, W D; Goldsmith, C M; Cotrim, A P; Baum, B J; Ambudkar, I S

    2016-07-01

    Head and neck irradiation (IR) during cancer treatment causes by-stander effects on the salivary glands leading to irreversible loss of saliva secretion. The mechanism underlying loss of fluid secretion is not understood and no adequate therapy is currently available. Delivery of an adenoviral vector encoding human aquaporin-1 (hAQP1) into the salivary glands of human subjects and animal models with radiation-induced salivary hypofunction leads to significant recovery of saliva secretion and symptomatic relief in subjects. To elucidate the mechanism underlying loss of salivary secretion and the basis for AdhAQP1-dependent recovery of salivary gland function we assessed submandibular gland function in control mice and mice 2 and 8 months after treatment with a single 15-Gy dose of IR (delivered to the salivary gland region). Salivary secretion and neurotransmitter-stimulated changes in acinar cell volume, an in vitro read-out for fluid secretion, were monitored. Consistent with the sustained 60% loss of fluid secretion following IR, a carbachol (CCh)-induced decrease in acinar cell volume from the glands of mice post IR was transient and attenuated as compared with that in cells from non-IR age-matched mice. The hAQP1 expression in non-IR mice induced no significant effect on salivary fluid secretion or CCh-stimulated cell volume changes, except in acinar cells from 8-month group where the initial rate of cell shrinkage was increased. Importantly, the expression of hAQP1 in the glands of mice post IR induced recovery of salivary fluid secretion and a volume decrease in acinar cells to levels similar to those in cells from non-IR mice. The initial rates of CCh-stimulated cell volume reduction in acinar cells from hAQP1-expressing glands post IR were similar to those from control cells. Altogether, the data suggest that expression of hAQP1 increases the water permeability of acinar cells, which underlies the recovery of fluid secretion in the salivary glands

  15. Moraxella catarrhalis decreases antiviral innate immune responses by down-regulation of TLR3 via inhibition of p53 in human bronchial epithelial cells.

    Science.gov (United States)

    Heinrich, Annina; Haarmann, Helge; Zahradnik, Sabrina; Frenzel, Katrin; Schreiber, Frauke; Klassert, Tilman E; Heyl, Kerstin A; Endres, Anne-Sophie; Schmidtke, Michaela; Hofmann, Jörg; Slevogt, Hortense

    2016-06-01

    Chronic obstructive pulmonary disease (COPD) is complicated by infectious exacerbations with acute worsening of respiratory symptoms. Coinfections of bacterial and viral pathogens are associated with more severe exacerbations. Moraxella catarrhalis is one of the most frequent lower respiratory tract pathogens detected in COPD. We therefore studied the impact of M. catarrhalis on the antiviral innate immune response that is mediated via TLR3 and p53. Molecular interactions between M. catarrhalis and normal human bronchial epithelial (NHBE) cells as well as Beas-2B cells were studied using flow cytometry, quantitative PCR analysis, chromatin immunoprecipitation, RNA interference, and ELISA. M. catarrhalis induces a significant down-regulation of TLR3 in human bronchial epithelial cells. In M. catarrhalis-infected cells, expression of p53 was decreased. We detected a reduced binding of p53 to the tlr3 promoter, resulting in reduced TLR3 gene transcription. M. catarrhalis diminished the TLR3-dependent secretion of IFN-β, IFN-λ, and chemokine (C-X-C motif) ligand 8. In addition in M. catarrhalis infected cells, expression of rhinovirus type 1A RNA was increased compared with uninfected cells. M. catarrhalis reduces antiviral defense functions of bronchial epithelial cells, which may increase susceptibility to viral infections.-Heinrich, A., Haarmann, H., Zahradnik, S., Frenzel, K., Schreiber, F., Klassert, T. E., Heyl, K. A., Endres, A.-S., Schmidtke, M., Hofmann, J., Slevogt, H. Moraxella catarrhalis decreases antiviral innate immune responses by down-regulation of TLR3 via inhibition of p53 in human bronchial epithelial cells.

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

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    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.

  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. 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.

  19. Decreased TFR/TFH ratio in SIV-infected rhesus macaques may contribute to accumulation of TFH cells in chronic infection

    Science.gov (United States)

    Chowdhury, Ankita; Estrada Del Rio, Perla Maria; Tharp, Greg K; Trible, Ronald P; Amara, Rama R; Chahroudi, Ann; Reyes-Teran, Gustavo; Bosinger, Steven E.; Silvestri, Guido

    2015-01-01

    T follicular helper cells (TFH) are critical for the development and maintenance of germinal centers (GC) and humoral immune responses. During chronic HIV/SIV infection TFH accumulate, possibly as a result of antigen persistence. The HIV/SIV-associated TFH expansion may also reflect lack of regulation by suppressive follicular regulatory CD4+ T-cells (TFR). TFR are natural regulatory T-cells (TREG) that migrate into the follicle and, similarly to TFH, up-regulate CXCR5, Bcl-6, and PD1. Here we identified TFR as CD4+CD25+FoxP3+CXCR5+PD1hiBcl-6+ within lymph nodes of rhesus macaques (RM) and confirmed their localization within the GC by immunohistochemistry. RNA sequencing showed that TFR exhibit a distinct transcriptional profile with shared features of both TFH and TREG, including intermediate expression of FoxP3, Bcl-6, PRDM1, IL-10, and IL-21. In healthy, SIV-uninfected RM, we observed a negative correlation between frequencies of TFR and both TFH and GC B-cells as well as levels of CD4+ T-cell proliferation. Following SIV infection, the TFR/TFH ratio was reduced with no change in the frequency of TREG or TFR within the total CD4+ T-cell pool. Finally, we examined whether higher levels of direct virus infection of TFR were responsible for their relative depletion post-SIV infection. We found that TFH, TFR and TREG sorted from SIV- infected RM harbor comparable levels of cell-associated viral DNA. Our data suggests that TFR may contribute to the regulation and proliferation of TFH and GC B-cells in vivo and that a decreased TFR/TFH ratio in chronic SIV infection may lead to unchecked expansion of both TFH and GC B-cells. PMID:26297764

  20. Dual silencing of insulin-like growth factor-I receptor and epidermal growth factor receptor in colorectal cancer cells is associated with decreased proliferation and enhanced apoptosis.

    Science.gov (United States)

    Kaulfuss, Silke; Burfeind, Peter; Gaedcke, Jochen; Scharf, Jens-Gerd

    2009-04-01

    Overexpression and activation of tyrosine kinase receptors are common features of colorectal cancer. Using the human colorectal cancer cell lines DLD-1 and Caco-2, we evaluated the role of the insulin-like growth factor-I (IGF-I) receptor (IGF-IR) and epidermal growth factor receptor (EGFR) in cellular functions of these cells. We used the small interfering RNA (siRNA) technology to specifically down-regulate IGF-IR and EGFR expression. Knockdown of IGF-IR and EGFR resulted in inhibition of cell proliferation of DLD-1 and Caco-2 cells. An increased rate of apoptosis was associated with siRNA-mediated silencing of IGF-IR and EGFR as assessed by activation of caspase-3/caspase-7. The combined knockdown of both EGFR and IGF-IR decreased cell proliferation and induced cell apoptosis more effectively than did silencing of either receptor alone. Comparable effects on cell proliferation and apoptosis were observed after single and combinational treatment of cells by the IGF-IR tyrosine kinase inhibitor NVP-AEW541 and/or the EGFR tyrosine kinase inhibitor erlotinib. Combined IGF-IR and EGFR silencing by either siRNAs or tyrosine kinase inhibitors diminished the phosphorylation of downstream signaling pathways AKT and extracellular signal-regulated kinase (ERK)-1/2 more effectively than did the single receptor knockdown. Single IGF-IR knockdown inhibited IGF-I-dependent phosphorylation of AKT but had no effect on IGF-I- or EGF-dependent phosphorylation of ERK1/2, indicating a role of EGFR in ligand-dependent ERK1/2 phosphorylation. The present data show that inhibition of the IGF-IR transduction cascade augments the antipoliferative and proapoptotic effects of EGFR inhibition in colorectal cancer cells. A clinical application of combination therapy targeting both EGFR and IGF-IR could be a promising therapeutic strategy.

  1. Synergistic decrease of DNA single-strand break repair rates in mouse neural cells lacking both Tdp1 and aprataxin

    Science.gov (United States)

    El-Khamisy, Sherif F.; Katyal, Sachin; Patel, Poorvi; Ju, Limei; McKinnon, Peter J.; Caldecott, Keith W.

    2009-01-01

    Ataxia oculomotor apraxia-1 (AOA1) is an autosomal recessive neurodegenerative disease that results from mutations of aprataxin (APTX). APTX associates with the DNA single- and double-strand break repair machinery and is able to remove AMP from 5′-termini at DNA strand breaks in vitro. However, attempts to establish a DNA strand break repair defect in APTX-defective cells have proved conflicting and unclear. We reasoned that this may reflect that DNA strand breaks with 5′-AMP represent only a minor subset of breaks induced in cells, and/or the availability of alternative mechanisms for removing AMP from 5′-termini. Here, we have attempted to increase the dependency of chromosomal single- and double-strand break repair on aprataxin activity by slowing the rate of repair of 3′-termini in aprataxin-defective neural cells, thereby increasing the likelihood that the 5′-termini at such breaks become adenylated and/or block alternative repair mechanisms. To do this, we generated a mouse model in which APTX is deleted together with tyrosyl DNA phosphodiesterase (TDP1), an enzyme that repairs 3′-termini at a subset of single-strand breaks (SSBs), including those with 3′-topoisomerase-1 (Top1) peptide. Notably, the global rate of repair of oxidative and alkylation-induced SSBs was significantly slower in Tdp1−/−/Aptx−/− double knockout quiescent mouse astrocytes compared with Tdp1−/− or Aptx−/− single knockouts. In contrast, camptothecin-induced Top1-SSBs accumulated to similar levels in Tdp1−/− and Tdp1−/−/Aptx−/− double knockout astrocytes. Finally, we failed to identify a measurable defect in double-strand break repair in Tdp1−/−, Aptx−/− or Tdp1−/−/Aptx−/− astrocytes. These data provide direct evidence for a requirement for aprataxin during chromosomal single-strand break repair in primary neural cells lacking Tdp1. PMID:19303373

  2. 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.

  3. Decreased H3K27 and H3K4 trimethylation on mortal chromosomes in distributed stem cells.

    Science.gov (United States)

    Huh, Y H; Sherley, J L

    2014-12-04

    The role of immortal DNA strands that co-segregate during mitosis of asymmetrically self-renewing distributed stem cells (DSCs) is unknown. Previously, investigation of immortal DNA strand function and molecular mechanisms responsible for their nonrandom co-segregation was precluded by difficulty in identifying DSCs and immortal DNA strands. Here, we report the use of two technological innovations, selective DSC expansion and establishment of H2A.Z chromosomal asymmetry as a specific marker of 'immortal chromosomes,' to investigate molecular properties of immortal chromosomes and opposing 'mortal chromosomes' in cultured mouse hair follicle DSCs. Although detection of the respective suppressive and activating H3K27me3 and H3K4me3 epigenetic marks on immortal chromosomes was similar to randomly segregated chromosomes, detection of both was lower on mortal chromosomes destined for lineage-committed sister cells. This global epigenomic feature of nonrandom co-segregation may reveal a mechanism that maintains an epigenome-wide 'poised' transcription state, which preserves DSC identity, while simultaneously activating sister chromosomes for differentiation.

  4. Increase of microRNA-210, decrease of raptor gene expression and alteration of mammalian target of rapamycin regulated proteins following mithramycin treatment of human erythroid cells.

    Directory of Open Access Journals (Sweden)

    Nicoletta Bianchi

    Full Text Available Expression and regulation of microRNAs is an emerging issue in erythroid differentiation and globin gene expression in hemoglobin disorders. In the first part of this study microarray analysis was performed both in mithramycin-induced K562 cells and erythroid precursors from healthy subjects or β-thalassemia patients producing low or high levels of fetal hemoglobin. We demonstrated that: (a microRNA-210 expression is higher in erythroid precursors from β-thalassemia patients with high production of fetal hemoglobin; (b microRNA-210 increases as a consequence of mithramycin treatment of K562 cells and human erythroid progenitors both from healthy and β-thalassemia subjects; (c this increase is associated with erythroid induction and elevated expression of γ-globin genes; (d an anti-microRNA against microRNA-210 interferes with the mithramycin-induced changes of gene expression. In the second part of the study we have obtained convergent evidences suggesting raptor mRNA as a putative target of microRNA-210. Indeed, microRNA-210 binding sites of its 3'-UTR region were involved in expression and are targets of microRNA-210-mediated modulation in a luciferase reporter assays. Furthermore, (i raptor mRNA and protein are down-regulated upon mithramycin-induction both in K562 cells and erythroid progenitors from healthy and β-thalassemia subjects. In addition, (ii administration of anti-microRNA-210 to K562 cells decreased endogenous microRNA-210 and increased raptor mRNA and protein expression. Finally, (iii treatment of K562 cells with premicroRNA-210 led to a decrease of raptor mRNA and protein. In conclusion, microRNA-210 and raptor are involved in mithramycin-mediated erythroid differentiation of K562 cells and participate to the fine-tuning and control of γ-globin gene expression in erythroid precursor cells.

  5. 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.

  6. The Over-expression of the β2 Catalytic Subunit of the Proteasome Decreases Homologous Recombination and Impairs DNA Double-Strand Break Repair in Human Cells

    Directory of Open Access Journals (Sweden)

    Anita Collavoli

    2011-01-01

    Full Text Available By a human cDNA library screening, we have previously identified two sequences coding two different catalytic subunits of the proteasome which increase homologous recombination (HR when overexpressed in the yeast Saccharomyces cerevisiae. Here, we investigated the effect of proteasome on spontaneous HR and DNA repair in human cells. To determine if the proteasome has a role in the occurrence of spontaneous HR in human cells, we overexpressed the β2 subunit of the proteasome in HeLa cells and determined the effect on intrachromosomal HR. Results showed that the overexpression of β2 subunit decreased HR in human cells without altering the cell proteasome activity and the Rad51p level. Moreover, exposure to MG132 that inhibits the proteasome activity reduced HR in human cells. We also found that the expression of the β2 subunit increases the sensitivity to the camptothecin that induces DNA double-strand break (DSB. This suggests that the β2 subunit has an active role in HR and DSB repair but does not alter the intracellular level of the Rad51p.

  7. Cells lacking Rieske iron-sulfur protein have a reactive oxygen species-associated decrease in respiratory complexes I and IV.

    Science.gov (United States)

    Diaz, Francisca; Enríquez, José Antonio; Moraes, Carlos T

    2012-01-01

    Mitochondrial respiratory complexes of the electron transport chain (CI, CIII, and CIV) can be assembled into larger structures forming supercomplexes. We analyzed the assembly/stability of respiratory complexes in mouse lung fibroblasts lacking the Rieske iron-sulfur protein (RISP knockout [KO]cells), one of the catalytic subunits of CIII. In the absence of RISP, most of the remaining CIII subunits were able to assemble into a large precomplex that lacked enzymatic activity. CI, CIV, and supercomplexes were decreased in the RISP-deficient cells. Reintroduction of RISP into KO cells restored CIII activity and increased the levels of active CI, CIV, and supercomplexes. We found that hypoxia (1% O(2)) resulted in increased levels of CI, CIV, and supercomplex assembly in RISP KO cells. In addition, treatment of control cells with different oxidative phosphorylation (OXPHOS) inhibitors showed that compounds known to generate reactive oxygen species (ROS) (e.g., antimycin A and oligomycin) had a negative impact on CI and supercomplex levels. Accordingly, a superoxide dismutase (SOD) mimetic compound and SOD2 overexpression provided a partial increase in supercomplex levels in the RISP KO cells. Our data suggest that the stability of CI, CIV, and supercomplexes is regulated by ROS in the context of defective oxidative phosphorylation.

  8. Processing of Kansui Roots Stir-Baked with Vinegar Reduces Kansui-Induced Hepatocyte Cytotoxicity by Decreasing the Contents of Toxic Terpenoids and Regulating the Cell Apoptosis Pathway

    Directory of Open Access Journals (Sweden)

    Xiaojing Yan

    2014-06-01

    Full Text Available Euphorbia kansui is a Traditional Chinese Medicine widely used for the treatment of oedema, ascites and asthma. However, its serious hepatotoxicity hinders its safe clinical application. The process of stir-baking with vinegar is regularly used to reduce the toxicity of kansui. Up till now, the exact mechanism of the reduction in hepatotoxicity of kansui stir-baked with vinegar has been poorly defined. In this study, decreased  contents of five diterpene and one triterpene in kansui (GS-1 after stir-baking with vinegar (GS-2 was investigated by UPLC-QTOF/MS. Flow cytometry and Hoechst staining were used to show that the stir-baking with vinegar process reduces kansui-induced cell apoptosis. Furthermore, the result also indicated that kansui stir-baked with vinegar protects LO2 cells from apoptosis by increasing the cell mitochondrial membrane potential (ΔΨm, decreasing the release of cytochrome c and inhibiting the activities of caspase-9 and caspase-3 as evidenced by means of high content screening (HCS, ELISA and western blotting. These results suggested that the stir-baking vinegar could reduce the hepatotoxicity of kansui by effectively decreasing the contents of toxic terpenoids and inhibiting the intrinsic pathway of hepatocyte cell apoptosis. In conclusion, the study provided significant data for promoting safer and better clinical use of this herb.

  9. Piperine, a component of black pepper, decreases eugenol-induced cAMP and calcium levels in non-chemosensory 3T3-L1 cells.

    Science.gov (United States)

    Yoon, Yeo Cho; Kim, Sung-Hee; Kim, Min Jung; Yang, Hye Jeong; Rhyu, Mee-Ra; Park, Jae-Ho

    2015-01-01

    This study investigated the effects of an ethanol extract of black pepper and its constituent, piperine, on odorant-induced signal transduction in non-chemosensory cells. An ethanol extract of black pepper decreased eugenol-induced cAMP and calcium levels in preadipocyte 3T3-L1 cells with no toxicity. Phosphorylation of CREB (cAMP response element-binding protein) was down-regulated by the black pepper extract. The concentration (133.8 mg/g) and retention time (5.5 min) of piperine in the ethanol extract were quantified using UPLC-MS/MS. Pretreatment with piperine decreased eugenol-induced cAMP and calcium levels in 3T3-L1 cells. Piperine also decreased the phosphorylation of CREB, which is up-regulated by eugenol. These results suggest that piperine inhibits the eugenol-induced signal transduction pathway through modulation of cAMP and calcium levels and phosphorylation of CREB in non-chemosensory cells.

  10. Transplantation of bone marrow mesenchymal stem cells decreases oxidative stress, apoptosis, and hippocampal damage in brain of a spontaneous stroke model.

    Science.gov (United States)

    Calió, Michele Longoni; Marinho, Darci Sousa; Ko, Gui Mi; Ribeiro, Renata Rodrigues; Carbonel, Adriana Ferraz; Oyama, Lila Missae; Ormanji, Milene; Guirao, Tatiana Pinoti; Calió, Pedro Luiz; Reis, Luciana Aparecida; Simões, Manuel de Jesus; Lisbôa-Nascimento, Telma; Ferreira, Alice Teixeira; Bertoncini, Clélia Rejane Antônio

    2014-05-01

    Stroke is the most common cause of motor disabilities and is a major cause of mortality worldwide. Adult stem cells have been shown to be effective against neuronal degeneration through mechanisms that include both the recovery of neurotransmitter activity and a decrease in apoptosis and oxidative stress. We chose the lineage stroke-prone spontaneously hypertensive rat (SHRSP) as a model for stem cell therapy. SHRSP rats can develop such severe hypertension that they generally suffer a stroke at approximately 1 year of age. The aim of this study was to evaluate whether mesenchymal stem cells (MSCs) decrease apoptotic death and oxidative stress in existing SHRSP brain tissue. The results of qRT-PCR assays showed higher levels of the antiapoptotic Bcl-2 gene in the MSC-treated animals, compared with untreated. Our study also showed that superoxide, apoptotic cells, and by-products of lipid peroxidation decreased in MSC-treated SHRSP to levels similar those found in the animal controls, Wistar Kyoto rats. In addition, we saw a repair of morphological damage at the hippocampal region after MSC transplantation. These data suggest that MSCs have neuroprotective and antioxidant potential in stroke-prone spontaneously hypertensive rats.

  11. Ornithine and Homocitrulline Impair Mitochondrial Function, Decrease Antioxidant Defenses and Induce Cell Death in Menadione-Stressed Rat Cortical Astrocytes: Potential Mechanisms of Neurological Dysfunction in HHH Syndrome.

    Science.gov (United States)

    Zanatta, Ângela; Rodrigues, Marília Danyelle Nunes; Amaral, Alexandre Umpierrez; Souza, Débora Guerini; Quincozes-Santos, André; Wajner, Moacir

    2016-09-01

    Hyperornithinemia-hyperammonemia-homocitrullinuria (HHH) syndrome is caused by deficiency of ornithine translocase leading to predominant tissue accumulation and high urinary excretion of ornithine (Orn), homocitrulline (Hcit) and ammonia. Although affected patients commonly present neurological dysfunction manifested by cognitive deficit, spastic paraplegia, pyramidal and extrapyramidal signs, stroke-like episodes, hypotonia and ataxia, its pathogenesis is still poorly known. Although astrocytes are necessary for neuronal protection. Therefore, in the present study we investigated the effects of Orn and Hcit on cell viability (propidium iodide incorporation), mitochondrial function (thiazolyl blue tetrazolium bromide-MTT-reduction and mitochondrial membrane potential-ΔΨm), antioxidant defenses (GSH) and pro-inflammatory response (NFkB, IL-1β, IL-6 and TNF-α) in unstimulated and menadione-stressed cortical astrocytes that were previously shown to be susceptible to damage by neurotoxins. We first observed that Orn decreased MTT reduction, whereas both amino acids decreased GSH levels, without altering cell viability and the pro-inflammatory factors in unstimulated astrocytes. Furthermore, Orn and Hcit decreased cell viability and ΔΨm in menadione-treated astrocytes. The present data indicate that the major compounds accumulating in HHH syndrome impair mitochondrial function and reduce cell viability and the antioxidant defenses in cultured astrocytes especially when stressed by menadione. It is presumed that these mechanisms may be involved in the neuropathology of this disease.

  12. 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

  13. The red-vine-leaf extract AS195 increases nitric oxide synthase-dependent nitric oxide generation and decreases oxidative stress in endothelial and red blood cells.

    Science.gov (United States)

    Grau, Marijke; Bölck, Birgit; Bizjak, Daniel Alexander; Stabenow, Christina Julia Annika; Bloch, Wilhelm

    2016-02-01

    The red-vine-leaf extract AS195 improves cutaneous oxygen supply and the microcirculation in patients suffering from chronic venous insufficiency. Regulation of blood flow was associated to nitric oxide synthase (NOS)-dependent NO (nitric oxide) production, and endothelial and red blood cells (RBC) have been shown to possess respective NOS isoforms. It was hypothesized that AS195 positively affects NOS activation in human umbilical vein endothelial cells (HUVECs) and RBC. Because patients with microvascular disorders show increased oxidative stress which limits NO bioavailability, it was further hypothesized that AS195 increases NO bioavailability by decreasing the content of reactive oxygen species (ROS) and increasing antioxidant capacity. Cultured HUVECs and RBCs from healthy volunteers were incubated with AS195 (100 μmol/L), tert-butylhydroperoxide (TBHP, 1 mmol/L) to induce oxidative stress and with both AS195 and TBHP. Endothelial and red blood cell-nitric oxide synthase (RBC-NOS) activation significantly increased after AS195 incubation. Nitrite concentration, a marker for NO production, increased in HUVEC but decreased in RBC after AS195 application possibly due to nitrite scavenging potential of flavonoids. S-nitrosylation of RBC cytoskeletal spectrins and RBC deformability were increased after AS195 incubation. TBHP-induced ROS were decreased by AS195, and antioxidative capacity was significantly increased in AS195-treated cells. TBHP also reduced RBC deformability, but reduction was attenuated by parallel incubation with AS195. Adhesion of HUVEC was also reduced after AS195 treatment. Red-vine-leaf extract AS195 increases NOS activation and decreases oxidative stress. Both mechanisms increase NO bioavailability, improve cell function, and may thus account for enhanced microcirculation in both health and disease.

  14. Stem Cells

    Science.gov (United States)

    Stem cells are cells with the potential to develop into many different types of cells in the body. ... the body. There are two main types of stem cells: embryonic stem cells and adult stem cells. Stem ...

  15. Decreased expression of intercellular adhesion molecule-1 (ICAM-1) and urokinase-type plasminogen activator receptor (uPAR) is associated with tumor cell spreading in vivo.

    Science.gov (United States)

    Donadio, Ana C; Remedi, María M; Frede, Silvia; Bonacci, Gustavo R; Chiabrando, Gustavo A; Pistoresi-Palencia, María C

    2002-01-01

    The development of an effective antitumor immune response to control tumor growth is influenced by the tumor cell itself and/or by the tumor microenvironment. Tumor invasion and tumor cell spreading require a finely tuned regulation of the formation and loosening of adhesive contacts of tumor cells with the extracellular matrix (ECM). In our laboratory, a rat tumor cell line derived from a spontaneous rat sarcoma revealed, by flow cytometry, a high frequency of intercellular adhesion molecule-1 (ICAM-1, 70.1 +/- 8.7%) and urokinase-type plaminogen activator receptor (uPAR, 51.2 +/- 5.2%) positive cells, while a weak expression of MHC class II (IA, 2.2 +/- 0.2% and IE, 17.4 +/- 3.7%) and B7 (12.1 +/- 2.2%) antigens was detected. In our tumor experimental model, after implantation of tumor cells, visible tumor masses were present at days 5-7 with a relatively fast tumor growth until day 15 (progressive phase) followed by a suppression of the tumor growth (regressive phase). Here we present data that correlates a significant decrease in the frequency of ICAM-1 and uPAR expressing tumor cells with the appearance of tumor cells in sites distant from that of the primary tumor. In addition we describe the development of a cellular immune response which controls the tumor progression and is associated with an increase in the expression of major histocompatibility complex (MHC) class II IA antigen during tumor development. The histological examination at tumor progressive and regressive time points revealed the relevant presence of polymorphonuclear neutrophils (PMNs) evidencing colliquative necrosis in tumor growth areas. Taken together, these results support the idea that the balance between adhesive interactions, proteolytic activity and tumorigenicity may lead to a tumor invasive phenotype.

  16. Cell volume control in phospholemman (PLM) knockout mice: do cardiac myocytes demonstrate a regulatory volume decrease and is this influenced by deletion of PLM?

    Science.gov (United States)

    Bell, James R; Lloyd, David; Curl, Claire L; Delbridge, Lea M D; Shattock, Michael J

    2009-03-01

    In addition to modulatory actions on Na+-K+-ATPase, phospholemman (PLM) has been proposed to play a role in cell volume regulation. Overexpression of PLM induces ionic conductances, with 'PLM channels' exhibiting selectivity for taurine. Osmotic challenge of host cells overexpressing PLM increases taurine efflux and augments the cellular regulatory volume decrease (RVD) response, though a link between PLM and cell volume regulation has not been studied in the heart. We recently reported a depressed cardiac contractile function in PLM knockout mice in vivo, which was exacerbated in crystalloid-perfused isolated hearts, indicating that these hearts were osmotically challenged. To address this, the present study investigated the role of PLM in osmoregulation in the heart. Isolated PLM wild-type and knockout hearts were perfused with a crystalloid buffer supplemented with mannitol in a bid to prevent perfusate-induced cell swelling and maintain function. Accordingly, and in contrast to wild-type control hearts, contractile function was improved in PLM knockout hearts with 30 mM mannitol. To investigate further, isolated PLM wild-type and knockout cardiomyocytes were subjected to increasing hyposmotic challenges. Initial validation studies showed the IonOptix video edge-detection system to be a simple and accurate 'real-time' method for tracking cell width as a marker of cell size. Myocytes swelled equally in both genotypes, indicating that PLM, when expressed at physiological levels in cardiomyocytes, is not essential to limit water accumulation in response to a hyposmotic challenge. Interestingly, freshly isolated adult cardiomyocytes consistently failed to mount RVDs in response to cell swelling, adding to conflicting reports in the literature. A proposed perturbation of the RVD response as a result of the cell isolation process was not restored, however, with short-term culture in either adult or neonatal cardiomyocytes.

  17. The acquired radioresistance in HeLa cells under conditions mimicking hypoxia was attenuated by a decreased expression of HIF subunit genes induced by RNA interference

    Energy Technology Data Exchange (ETDEWEB)

    Doi, Nobutaka [Department of Radiological Sciences, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Toyama 930-0194 (Japan); New Products Research & Development, Gene Engineering Division, NIPPON GENE Co., Ltd. (Japan); Ogawa, Ryohei, E-mail: ogawa@med.u-toyama.ac.jp [Department of Radiological Sciences, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Toyama 930-0194 (Japan); Cui, Zheng-Guo [Department of Public Health, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama (Japan); Morii, Akihiro; Watanabe, Akihiko [Department of Urology, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama (Japan); Kanayama, Shinji; Yoneda, Yuko [New Products Research & Development, Gene Engineering Division, NIPPON GENE Co., Ltd. (Japan); Kondo, Takashi [Department of Radiological Sciences, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Toyama 930-0194 (Japan)

    2015-05-01

    The cancer cells residing in the hypoxic layer are resistant to radiation and these are ones responsible for cancer recurrence after radiation therapy. One of the reasons why hypoxic cancer cells acquire radioresistance may be attributable to changes in the gene expression profile by the activation of hypoxia inducible factors (HIFs). However, the details underlying this process remain unknown. In this study, we investigated the effects of knockdown of HIF subunit genes to elucidate how HIF subunit genes may be involved in the radioresistance acquired by HeLa cells following exposure to a hypoxia mimic. Interestingly, HIF-1α and HIF-2α seemed mutually complementary for each other when either of them was suppressed. We thus suppressed the expression of both genes simultaneously. To do this, we developed a short hairpin RNA (shRNA) targeting a high homology region between HIF-1α and HIF-2α. It was shown that the expression of the shRNA effectively suppressed the acquisition of radioresistance following the hypoxia mimic. Moreover, it was confirmed that suppression of both subunits resulted in the downregulation of stem cell markers and the suppression of spheroid formation during the hypoxia mimicking-conditions. This shRNA-mediated knockdown method targeting a common region shared by a family of genes may offer a new candidate cancer treatment. - Highlights: • Incubation with CoCl{sub 2} confers radioresistance to HeLa cells. • Both HIF-1α and HIF-2α are involved in the acquisition of radioresistance. • An shRNA to a homology region of HIF-1α and HIF-2α suppressed the radioresistance. • The shRNA decreased cells with stem cell markers and a stem cell phenotype.

  18. Increased level of both CD4+FOXP3+ regulatory T cells and CD14+HLA-DR⁻/low myeloid-derived suppressor cells and decreased level of dendritic cells in patients with multiple myeloma

    DEFF Research Database (Denmark)

    Brimnes, M K; Vangsted, Annette Juul; Knudsen, L M

    2010-01-01

    Patients with multiple myeloma (MM) suffer from a general impaired immunity comprising deficiencies in humoral responses, T-cell responses as well as dendritic cell (DC) function. Thus, to achieve control of tumour growth through immune therapy constitutes a challenge. Careful evaluation...

  19. Selective decrease in cell surface expression and mRNA level of the 55-kDa tumor necrosis factor receptor during differentiation of HL-60 cells into macrophage-like but not granulocyte-like cells

    DEFF Research Database (Denmark)

    Winzen, R; Wallach, D; Engelmann, H;

    1992-01-01

    Expression of the two known receptors for TNF was studied in the promyelocytic leukemia cell line HL-60 before and after differentiation of the cells along the granulocyte lineage (induced by incubation with retinoic acid), or along the macrophage lineage (induced by incubation with the phorbol d...

  20. Over-expression of Arabidopsis CAP causes decreased cell expansion leading to organ size reduction in transgenic tobacco plants.

    Science.gov (United States)

    Barrero, Roberto A; Umeda, Masaaki; Yamamura, Saburo; Uchimiya, Hirofumi

    2003-04-01

    Cyclase-associated proteins (CAP) are multifunctional proteins involved in Ras-cAMP signalling and regulation of the actin cytoskeleton. It has recently been demonstrated that over-expression of AtCAP1 in transgenic arabidopsis plants causes severe morphological defects owing to loss of actin filaments. To test the generality of the function of AtCAP1 in plants, transgenic tobacco plants over-expressing an arabidopsis CAP (AtCAP1) under the regulation of a glucocorticoid-inducible promoter were produced. Over-expression of AtCAP1 in transgenic tobacco plants led to growth abnormalities, in particular a reduction in the size of leaves. Morphological alterations in leaves were the result of reduced elongation of epidermal and mesophyll cells.

  1. Decreased [{sup 18}F]fluoro-2-deoxy-D-glucose incorporation and increased glucose transport are associated with resistance to 5FU in MCF7 cells in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Smith, Tim A.D. [PET Unit, Department of Biomedical Physics, University of Aberdeen, Foresterhill, Aberdeen AB25 2ZD (United Kingdom)], E-mail: t.smith@biomed.abdn.ac.uk; Sharma, Rituka I. [PET Unit, Department of Biomedical Physics, University of Aberdeen, Foresterhill, Aberdeen AB25 2ZD (United Kingdom); Wang, Weiguang G. [Department of Medicine and Therapeutics, University of Aberdeen, Foresterhill, Aberdeen AB25 2ZD (United Kingdom); School of Applied Sciences, University of Wolverhampton, City Campus-South, Wolverhampton WV1 1SB (United Kingdom); Welch, Andy E.; Schweiger, Lutz F. [PET Unit, Department of Biomedical Physics, University of Aberdeen, Foresterhill, Aberdeen AB25 2ZD (United Kingdom); Collie-Duguid, Elaina S.R. [Department of Medicine and Therapeutics, University of Aberdeen, Foresterhill, Aberdeen AB25 2ZD (United Kingdom)

    2007-11-15

    Introduction: Tumor refractoriness to chemotherapy is frequently due to the acquisition of resistance. Resistant cells selected by exposure to chemotherapy agents may exhibit differences in [{sup 18}F]fluoro-2-deoxy-D-glucose (FDG) incorporation, as compared with sensitive cells. Methods: FDG incorporation, hexokinase (HK) activity, glucose transport and ATP content were determined in clones of 5-fluorouracil (5FU)-resistant MCF7 cells, established by long-term exposure to increasing 5FU concentrations, and in parental MCF7 cells. Results: FDG incorporation was decreased in MCF7 cells resistant to 5FU; HK activity was similar in the resistant and sensitive cells, while glucose transport was increased, as compared with sensitive cells. Treatment of cells with the glucose efflux inhibitor phloretin increased FDG incorporation to similar levels in the resistant and sensitive cells. Analysis of microarray data demonstrated the expression of GLUT1, 8 and 10 transporters in MCF7 cells. GLUT8 and 10 expression was decreased in the resistant cells, while GLUT1 was only increased in cells resistant to the lowest 5FU concentration. Conclusion: FDG incorporation in 5FU-resistant MCF7 cells is decreased, as compared with sensitive cells. Our findings also suggest that this may be due to high rates of membrane glucose transport in the resistant cells resulting in enhanced efflux of FDG. We believe that this is the first demonstration that facilitative glucose transporters can actually decrease the incorporation of FDG.

  2. Comparison of Monoamine Oxidase Inhibitors in Decreasing Production of the Autotoxic Dopamine Metabolite 3,4-Dihydroxyphenylacetaldehyde in PC12 Cells.

    Science.gov (United States)

    Goldstein, David S; Jinsmaa, Yunden; Sullivan, Patti; Holmes, Courtney; Kopin, Irwin J; Sharabi, Yehonatan

    2016-02-01

    According to the catecholaldehyde hypothesis, the toxic dopamine metabolite 3,4-dihydroxyphenylacetaldehyde (DOPAL) contributes to the loss of nigrostriatal dopaminergic neurons in Parkinson's disease. Monoamine oxidase-A (MAO-A) catalyzes the conversion of intraneuronal dopamine to DOPAL and may serve as a therapeutic target. The "cheese effect"-paroxysmal hypertension evoked by tyramine-containing foodstuffs-limits clinical use of irreversible MAO-A inhibitors. Combined MAO-A/B inhibition decreases DOPAL production in rat pheochromocytoma PC12 cells, but whether reversible MAO-A inhibitors or MAO-B inhibitors decrease endogenous DOPAL production is unknown. We compared the potencies of MAO inhibitors in attenuating DOPAL production and examined possible secondary effects on dopamine storage, constitutive release, synthesis, and auto-oxidation. Catechol concentrations were measured in cells and medium after incubation with the irreversible MAO-A inhibitor clorgyline, three reversible MAO-A inhibitors, or the MAO-B inhibitors selegiline or rasagiline for 180 minutes. Reversible MAO-A inhibitors were generally ineffective, whereas clorgyline (1 nM), rasagiline (500 nM), and selegiline (500 nM) decreased DOPAL levels in the cells and medium. All three drugs also increased dopamine and norepinephrine, decreased 3,4-dihydroxyphenylalanine, and increased cysteinyl-dopamine concentrations in the medium, suggesting increased vesicular uptake and constitutive release, decreased dopamine synthesis, and increased dopamine spontaneous oxidation. In conclusion, clorgyline, rasagiline, and selegiline decrease production of endogenous DOPAL. At relatively high concentrations, the latter drugs probably lose their selectivity for MAO-B. Possibly offsetting increased formation of potentially toxic oxidation products and decreased formation of DOPAL might account for the failure of large clinical trials of MAO-B inhibitors to demonstrate slowing of neurodegeneration in Parkinson

  3. L-FABP T94A decreased fatty acid uptake and altered hepatic triglyceride and cholesterol accumulation in Chang liver cells stably transfected with L-FABP.

    Science.gov (United States)

    Gao, Na; Qu, Xia; Yan, Jin; Huang, Qi; Yuan, Hao-Yong; Ouyang, Dong-Sheng

    2010-12-01

    Liver fatty acid-binding protein (L-FABP, FABP1) is a highly conserved key factor in lipid metabolism. This study was undertaken to verify whether the T94A mutation in the L-FABP gene affects fatty acid uptake and intracellular esterification into specific lipid pools. Candidate SNPs were recreated using site-directed mutagenesis and tested for physical function in stably transfected Chang liver cell lines. We found that the T94A mutant of L-FABP lowered FFA uptake but had no effect on FFA efflux. L-FABP T94A-expressing cells showed decreased triglyceride content and increased cholesterol accumulation compared to the wild-type control for cells incubated with an FFA mixture (oleate: palmitate, 2:1 ratio). In conclusion, our study provided additional indications of the functional relevance of the L-FABP T94A SNP in hepatic fatty acid and lipid metabolism in humans.

  4. Aberrant decrease of microRNA19b regulates TSLP expression and contributes to Th17 cells development in myasthenia gravis related thymomas.

    Science.gov (United States)

    Wang, Zhongkui; Chen, Yuping; Xu, Shengjie; Yang, Yanhua; Wei, Dongning; Wang, Wei; Huang, Xusheng

    2015-11-15

    Myasthenia gravis (MG) is an organ-specific autoimmune disease. The imbalance of T helper type 17 cells (Th17) plays a key role in the pathogenesis of thymomatous MG. But the regulatory mechanism for Th17 cell development in MG-related thymoma remains undefined. Here we demonstrated that thymic stromal lymphopoietin (TSLP) is significantly decreased in thymomas. We also proved that TSLP was post-trancriptionally regulated by microRNA-19b. The expression of microRNA-19b was negatively correlated with the expression of TSLP mRNA and protein in thymomas. This study indicated that the elevation of microRNA-19b suppressed TSLP expression and then influenced T cell development in thymomatous MG.

  5. Inhibition of hydrogen peroxide signaling by 4-hydroxynonenal due to differential regulation of Akt1 and Akt2 contributes to decreases in cell survival and proliferation in hepatocellular carcinoma cells.

    Science.gov (United States)

    Shearn, Colin T; Reigan, Philip; Petersen, Dennis R

    2012-07-01

    Dysregulation of cell signaling by electrophiles such as 4-hydroxynonenal (4-HNE) is a key component in the pathogenesis of chronic inflammatory liver disease. Another consequence of inflammation is the perpetuation of oxidative damage by the production of reactive oxidative species such as hydrogen peroxide. Previously, we have demonstrated Akt2 as a direct target of 4-HNE in hepatocellular carcinoma cells. In the present study, we used the hepatocellular carcinoma cell line HepG2 as model to understand the combinatorial effects of 4-HNE and hydrogen peroxide. We demonstrate that 4-HNE inhibits hydrogen peroxide-mediated phosphorylation of Akt1 but not Akt2. Pretreatment of HepG2 cells with 4-HNE prevented hydrogen peroxide stimulation of Akt-dependent phosphorylation of downstream targets and intracellular Akt activity compared with untreated control cells. Using biotin hydrazide capture, it was confirmed that 4-HNE treatment resulted in carbonylation of Akt1, which was not observed in untreated control cells. Using a synthetic GSK3α/β peptide as a substrate, treatment of recombinant human myristoylated Akt1 (rAkt1) with 20 or 40 μΜ 4-HNE inhibited rAkt1 activity by 29 and 60%, respectively. We further demonstrate that 4-HNE activates Erk via a PI3 kinase and PP2A-dependent mechanism leading to increased Jnk phosphorylation. At higher concentrations, 4-HNE decreased both cell survival and proliferation as evidenced by MTT assays and EdU incorporation as well as decreased expression of cyclin D1 and β-catenin, an effect only moderately increased by the addition of hydrogen peroxide. The ability of 4-HNE to exert combinatorial effects on Erk, Jnk, and Akt-dependent cell survival pathways provides additional insight into the mechanisms of cellular damage associated with chronic inflammation.

  6. Deficiency of G1 regulators P53, P21Cip1 and/or pRb decreases hepatocyte sensitivity to TGFβ cell cycle arrest

    Directory of Open Access Journals (Sweden)

    Harrison David J

    2007-11-01

    Full Text Available Abstract Background TGFβ is critical to control hepatocyte proliferation by inducing G1-growth arrest through multiple pathways leading to inhibition of E2F transcription activity. The retinoblastoma protein pRb is a key controller of E2F activity and G1/S transition which can be inhibited in viral hepatitis. It is not known whether the impairment of pRb would alter the growth inhibitory potential of TGFβ in disease. We asked how Rb-deficiency would affect responses to TGFβ-induced cell cycle arrest. Results Primary hepatocytes isolated from Rb-floxed mice were infected with an adenovirus expressing CRE-recombinase to delete the Rb gene. In control cells treatment with TGFβ prevented cells to enter S phase via decreased cMYC activity, activation of P16INK4A and P21Cip and reduction of E2F activity. In Rb-null hepatocytes, cMYC activity decreased slightly but P16INK4A was not activated and the great majority of cells continued cycling. Rb is therefore central to TGFβ-induced cell cycle arrest in hepatocytes. However some Rb-null hepatocytes remained sensitive to TGFβ-induced cell cycle arrest. As these hepatocytes expressed very high levels of P21Cip1 and P53 we investigated whether these proteins regulate pRb-independent signaling to cell cycle arrest by evaluating the consequences of disruption of p53 and p21Cip1. Hepatocytes deficient in p53 or p21Cip1 showed diminished growth inhibition by TGFβ. Double deficiency had a similar impact showing that in cells containing functional pRb; P21Cip and P53 work through the same pathway to regulate G1/S in response to TGFβ. In Rb-deficient cells however, p53 but not p21Cip deficiency had an additive effect highlighting a pRb-independent-P53-dependent effector pathway of inhibition of E2F activity. Conclusion The present results show that otherwise genetically normal hepatocytes with disabled p53, p21Cip1 or Rb genes respond less well to the antiproliferative effects of TGFβ. As the function of

  7. Elevation of arginine decarboxylase-dependent putrescine production enhances aluminum tolerance by decreasing aluminum retention in root cell walls of wheat.

    Science.gov (United States)

    Yu, Yan; Jin, Chongwei; Sun, Chengliang; Wang, Jinghong; Ye, Yiquan; Lu, Lingli; Lin, Xianyong

    2015-12-15

    Aluminum (Al) stress induces putrescine (Put) accumulation in several plants and this response is proposed to alleviate Al toxicity. However, the mechanisms underlying this alleviation remain largely unknown. Here, we show that exposure to Al clearly increases Put accumulation in the roots of wheat plants (Triticum aestivum L. 'Xi Aimai-1') and that this was accompanied by significant increase in the activity of arginine decarboxylase (ADC), a Put producing enzyme. Application of an ADC inhibitor (d-arginine) terminated the Al-induced Put accumulation, indicating that increased ADC activity may be responsible for the increase in Put accumulation in response to Al. The d-arginine treatment also increased the Al-induced accumulation of cell wall polysaccharides and the degree of pectin demethylation in wheat roots. Thus, it elevated Al retention in cell walls and exacerbated Al accumulation in roots, both of which aggravate Al toxicity in wheat plants. The opposite effects were true for exogenous Put application. These results suggest that ADC-dependent Put accumulation plays important roles in providing protection against Al toxicity in wheat plants through decreasing cell wall polysaccharides and increasing the degree of pectin methylation, thus decreasing Al retention in the cell walls.

  8. Cultured senescent myoblasts derived from human vastus lateralis exhibit normal mitochondrial ATP synthesis capacities with correlating concomitant ROS production while whole cell ATP production is decreased.

    Science.gov (United States)

    Minet, Ariane D; Gaster, Michael

    2012-06-01

    The free radical theory of aging says that increased oxidative stress and mitochondrial dysfunction are associated with old age. In the present study we have investigated the effects of cellular senescence on muscle energetic by comparing mitochondrial content and function in cultured muscle satellite cells at early and late passage numbers. We show that cultured muscle satellite cells undergoing senescence express a reduced mitochondrial mass, decreased whole cell ATP level, normal to increased mitochondrial ATP production under ATP utilization, increased mitochondrial membrane potential and increased superoxide/mitochondrial mass and hydrogen peroxide/mitochondrial mass ratios. Moreover, the increased ROS production correlates with the corresponding mitochondrial ATP production. Thus, myotubes differentiated from human myoblasts undergoing senescence have a reduced mitochondrial content, but the existent mitochondria express normal to increased functional capabilities. The present data suggest that the origin of aging lies outside the mitochondria and that a malfunction in the cell might be preceding and initiating the increase of mitochondrial ATP synthesis and concomitant ROS production in the single mitochondrion in response to decreased mitochondrial mass and reduced extra-mitochondrial energy supply. This then can lead to the increased damage of DNA, lipids and proteins of the mitochondria as postulated by the free radical theory of aging.

  9. Juxtaglomerular cell CaSR stimulation decreases renin release via activation of the PLC/IP(3) pathway and the ryanodine receptor.

    Science.gov (United States)

    Ortiz-Capisano, M Cecilia; Reddy, Mahendranath; Mendez, Mariela; Garvin, Jeffrey L; Beierwaltes, William H

    2013-02-01

    The calcium-sensing receptor (CaSR) is a G-coupled protein expressed in renal juxtaglomerular (JG) cells. Its activation stimulates calcium-mediated decreases in cAMP content and inhibits renin release. The postreceptor pathway for the CaSR in JG cells is unknown. In parathyroids, CaSR acts through G(q) and/or G(i). Activation of G(q) stimulates phospholipase C (PLC), and inositol 1,4,5-trisphosphate (IP(3)), releasing calcium from intracellular stores. G(i) stimulation inhibits cAMP formation. In afferent arterioles, the ryanodine receptor (RyR) enhances release of stored calcium. We hypothesized JG cell CaSR activation inhibits renin via the PLC/IP(3) and also RyR activation, increasing intracellular calcium, suppressing cAMP formation, and inhibiting renin release. Renin release from primary cultures of isolated mouse JG cells (n = 10) was measured. The CaSR agonist cinacalcet decreased renin release 56 ± 7% of control (P PLC inhibitor U73122 reversed cinacalcet inhibition of renin (104 ± 11% of control). The IP(3) inhibitor 2-APB also reversed inhibition of renin from 56 ± 6 to 104 ± 11% of control (P PLC/IP(3) pathway, activating RyR, increasing intracellular calcium, and resulting in calcium-mediated renin inhibition.

  10. Relationship between the induction of heat shock proteins and the decrease in glucocorticoid receptor during heat shock response in human osteosarcoma cells

    Institute of Scientific and Technical Information of China (English)

    宋亮年

    1995-01-01

    Previously,it has been found that glucocorticoid receptor(GR)binding activity decreasedrapidly during heat shock response in HOS-8603,a human osteosarcorna cell line.In this study,Therelationship between the induction of heat shock proteins(HSPs)and the decrease in GR was furtherstudied in the same cell line.It was found that even though quercetin could specifically inhibit the ex-pression of hsp90α and hsp70 mRNA,it could not prevent GR from the decrease in response to the heatshock treatment.This represents the first reported evidence that the induction of HSPs and the decrease inGR during heat shock response were 2 independent biological events.The results of the present study furthershowed that although the heat shock treatment alone had no effects on alkaline phosphatase(AKP)activity,itcould completely block the induction of AKP activity in HOS-8603 cells by dexamethasone(Dex),a syntheticglucocorticoid.These results demonstrate that the heat shock-induced alteration in GR was accompanied by adecrease in GR functional activity.Furthermore,when the induction of HSPs was inhibited by the treatmentof cells with quercetin,the stimulatory effects of Dex on AKP activity could still be inhibited completely bythe heat shock treatment.The results of this part,on the basis of GR functional activity,further demonstratethat quercetin could not inhibit the heat shock-induced decrease in GR,even though it could inhibit the induc-tion of HSPs.To clarify further the effects of quercetin alone on GR binding activity in HOS-8603 cells,theregulation of GR by quercetin was also studied.It was found for the first time that quercetin coulddown-regulate GR in a time-dependent manner significantly,and that the down-regulation of GR by quercetinin HOS-8603 cells paralelled with a decrease in glucocorticoid-mediated functional responses,suggesting thatthe down-regulation of GR by quercetin is of biological significance.

  11. An altered gp100 peptide ligand with decreased binding by TCR and CD8alpha dissects T cell cytotoxicity from production of cytokines and activation of NFAT

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    Niels eSchaft

    2013-09-01

    Full Text Available Altered peptide ligands (APLs provide useful tools to study T cell activation and potentially direct immune responses to improve treatment of cancer patients. To better understand and exploit APLs, we studied the relationship between APLs and T cell function in more detail. Here, we tested a broad panel of gp100(280-288 APLs with respect to T cell cytotoxicity, production of cytokines and activation of Nuclear Factor of Activated T cells (NFAT by human T cells gene-engineered with a gp100-HLA-A2-specific TCRalpha/beta. We demonstrated that gp100-specific cytotoxicity, production of cytokines, and activation of NFAT were not affected by APLs with single amino acid substitutions, except for an APL with an amino acid substitution at position 3 (APL A3, which did not elicit any T cell response. A gp100 peptide with a double amino acid mutation (APL S4S6 elicited T cell cytotoxicity and production of IFNgamma, and to a lesser extent TNFalpha, IL-4, and IL-5, but not production of IL-2 and IL-10, or activation of NFAT. Notably, TCR-mediated functions showed decreases in sensitivities for S4S6 versus gp100 wt peptide, which were minor for cytotoxicity but at least a 1000-fold more prominent for the production of cytokines. TCR-engineered T cells did not bind A3-HLA-A2, but did bind S4S6-HLA-A2 although to a lowered extent compared to wt peptide-HLA-A2. Moreover, S4S6-induced T cell function demonstrated an enhanced dependency on CD8alpha. Taken together, most gp100 APLs functioned as agonists, but A3 and S4S6 peptides acted as a null ligand and partial agonist, respectively. Our results further suggest that TCR-mediated cytotoxicity can be dissected from production of cytokines and activation of NFAT, and that the agonist potential of peptide mutants relates to the extent of binding by TCR and CD8alpha. These findings may facilitate the design of APLs to advance the study of T cell activation and their use for therapeutic applications.

  12. High Fat Diet-Induced Skeletal Muscle Wasting Is Decreased by Mesenchymal Stem Cells Administration: Implications on Oxidative Stress, Ubiquitin Proteasome Pathway Activation, and Myonuclear Apoptosis

    Directory of Open Access Journals (Sweden)

    Johanna Abrigo

    2016-01-01

    Full Text Available Obesity can lead to skeletal muscle atrophy, a pathological condition characterized by the loss of strength and muscle mass. A feature of muscle atrophy is a decrease of myofibrillar proteins as a result of ubiquitin proteasome pathway overactivation, as evidenced by increased expression of the muscle-specific ubiquitin ligases atrogin-1 and MuRF-1. Additionally, other mechanisms are related to muscle wasting, including oxidative stress, myonuclear apoptosis, and autophagy. Stem cells are an emerging therapy in the treatment of chronic diseases such as high fat diet-induced obesity. Mesenchymal stem cells (MSCs are a population of self-renewable and undifferentiated cells present in the bone marrow and other mesenchymal tissues of adult individuals. The present study is the first to analyze the effects of systemic MSC administration on high fat diet-induced skeletal muscle atrophy in the tibialis anterior of mice. Treatment with MSCs reduced losses of muscle strength and mass, decreases of fiber diameter and myosin heavy chain protein levels, and fiber type transitions. Underlying these antiatrophic effects, MSC administration also decreased ubiquitin proteasome pathway activation, oxidative stress, and myonuclear apoptosis. These results are the first to indicate that systemically administered MSCs could prevent muscle wasting associated with high fat diet-induced obesity and diabetes.

  13. High Fat Diet-Induced Skeletal Muscle Wasting Is Decreased by Mesenchymal Stem Cells Administration: Implications on Oxidative Stress, Ubiquitin Proteasome Pathway Activation, and Myonuclear Apoptosis

    Science.gov (United States)

    Aravena, Javier; Cabrera, Daniel; Simon, Felipe; Ezquer, Fernando

    2016-01-01

    Obesity can lead to skeletal muscle atrophy, a pathological condition characterized by the loss of strength and muscle mass. A feature of muscle atrophy is a decrease of myofibrillar proteins as a result of ubiquitin proteasome pathway overactivation, as evidenced by increased expression of the muscle-specific ubiquitin ligases atrogin-1 and MuRF-1. Additionally, other mechanisms are related to muscle wasting, including oxidative stress, myonuclear apoptosis, and autophagy. Stem cells are an emerging therapy in the treatment of chronic diseases such as high fat diet-induced obesity. Mesenchymal stem cells (MSCs) are a population of self-renewable and undifferentiated cells present in the bone marrow and other mesenchymal tissues of adult individuals. The present study is the first to analyze the effects of systemic MSC administration on high fat diet-induced skeletal muscle atrophy in the tibialis anterior of mice. Treatment with MSCs reduced losses of muscle strength and mass, decreases of fiber diameter and myosin heavy chain protein levels, and fiber type transitions. Underlying these antiatrophic effects, MSC administration also decreased ubiquitin proteasome pathway activation, oxidative stress, and myonuclear apoptosis. These results are the first to indicate that systemically administered MSCs could prevent muscle wasting associated with high fat diet-induced obesity and diabetes. PMID:27579157

  14. Decrease in immune activation in HIV-infected patients treated with highly active antiretroviral therapy correlates with the function of hematopoietic progenitor cells and the number of naive CD4+ cells

    DEFF Research Database (Denmark)

    Nielsen, S D; Sørensen, T U; Ersbøll, A K;

    2000-01-01

    determined. During the study period, the naive CD4+ count and the cloning efficiency increased significantly. Immune activation was found in HIV-infected patients and decreased during HAART. The level of immune activation correlated negatively with both the naive CD4+ count and the function of progenitor...... cells. A negative correlation was found between apoptosis and the naive CD4+ count. Alterations in cytokine production during HAART or correlation between cytokine production and the naive CD4+ count or the cloning efficiency of progenitor cells were not detected. In conclusion, immune activation in HIV...

  15. 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.

  16. The pore size of non-graminaceous plant cell walls is rapidly decreased by borate ester cross-linking of the pectic polysaccharide rhamnogalacturonan II

    Energy Technology Data Exchange (ETDEWEB)

    Fleischer, A.; O' Neill, M.A.; Ehwald, R.

    1999-11-01

    The walls of suspension-cultured Chenopodium album L. cells grown continually for more than 1 year on B-deficient medium contained monomeric rhamnogalacturonan (mRG-II) but not the borate ester cross-linked RG II dimer (dRG-II-B). The walls of these cells had an increased size limit for dextran permeation, which is a measure of wall pore size. Adding boric acid to growing B-deficient cells resulted in B binding to the wall, the formation of dRG-II-B from mRG-II, and a reduction in wall pore size within 10 min. The wall pore size of denatured B-grown cells was increased by treatment at pH {le} 2.0 or by treatment with Ca{sup 2+}-chelating agents. The acid-mediated increase in wall pore size was prevented by boric acid alone at pH 2.0 and by boric acid together with Ca{sup 2+}, but not by Na{sup +} or Mg{sup 2+} ions at pH 1.5. The Ca{sup 2+}-chelator-mediated increase in pore size was partially reduced by boric acid. Their results suggest that B-mediated cross-linking of RG-II in the walls of living plant cells generates a pectin network with a decreased size exclusion limit for polymers. The formation, stability, and possible functions of a borate ester cross-linked pectic network in the primary walls of nongraminaceous plant cells are discussed.

  17. Trans-1O,12,not cis-9,trans-11,conjugated linoleic acid decreases ErbB3 expression in HT-29 human colon cancer cells

    Institute of Scientific and Technical Information of China (English)

    Han Jin Cho; Woo Kyoung Kim; Jae In Jung; Eun Ji Kim; Soon Sung Lim; Dae Young Kwon; Jung Han Yoon Park

    2005-01-01

    AIM: To examine whether trans-10, cis-12 CLA (t10c12)or cis-9, trans-11 CLA (c9 t11) inhibits heregulin (H RG)-β-stimulated cell growth and HRG-β-ErbB3 signaling in HT-29 cells.METHODS: We cultured HT-29 cells in the absence or presence of the CLA isomers and/or the ErbB3 ligand HRG-β. MTT assay, [3H]thymidine incorporation, Annexin V staining, RT-PCR, Western blotting, immunoprecipitation,and in vitro kinase assay were performed.RESULTS: HRG-β increased cell growth, but did not prevent t10c12-induced growth inhibition. T10c12 inhibited DNA synthesis and induced apoptosis of HT-29 cells, whereas c9t11 had no effect. T10c12 decreased the levels of ErbB1,ErbB2, and ErbB3 proteins and transcripts in a dose-dependent manner, whereas cgt11 had no effect. Immunoprecipitation/Western blot studies revealed that t10c12 inhibited HRG-β-stimulated phosphorylation of ErbB3, recruitment of the p85 subunit of phosphoinositide 3-kinase (PI3K) to ErbB3, ErbB3-associated PI3K activities, and phosphorylation ofAkt. However, c9t11 had no effect on phospho Akt levels.Neither t10c12 nor c9t11 had any effect on HRG-β-induced phosphorylation of ERK-1/2.CONCLUSION: These results indicate that the inhibition of HT-29 cell growth by t10c12 may be induced via its modulation of ErbB3 signaling leading to inhibition of Akt activation.

  18. Major Components of Energy Drinks (Caffeine, Taurine, and Guarana Exert Cytotoxic Effects on Human Neuronal SH-SY5Y Cells by Decreasing Reactive Oxygen Species Production

    Directory of Open Access Journals (Sweden)

    Fares Zeidán-Chuliá

    2013-01-01

    Full Text Available Scope. To elucidate the morphological and biochemical in vitro effects exerted by caffeine, taurine, and guarana, alone or in combination, since they are major components in energy drinks (EDs. Methods and Results. On human neuronal SH-SY5Y cells, caffeine (0.125–2 mg/mL, taurine (1–16 mg/mL, and guarana (3.125–50 mg/mL showed concentration-dependent nonenzymatic antioxidant potential, decreased the basal levels of free radical generation, and reduced both superoxide dismutase (SOD and catalase (CAT activities, especially when combined together. However, guarana-treated cells developed signs of neurite degeneration in the form of swellings at various segments in a beaded or pearl chain-like appearance and fragmentation of such neurites at concentrations ranging from 12.5 to 50 mg/mL. Swellings, but not neuritic fragmentation, were detected when cells were treated with 0.5 mg/mL (or higher doses of caffeine, concentrations that are present in EDs. Cells treated with guarana also showed qualitative signs of apoptosis, including membrane blebbing, cell shrinkage, and cleaved caspase-3 positivity. Flow cytometric analysis confirmed that cells treated with 12.5–50 mg/mL of guarana and its combinations with caffeine and/or taurine underwent apoptosis. Conclusion. Excessive removal of intracellular reactive oxygen species, to nonphysiological levels (or “antioxidative stress”, could be a cause of in vitro toxicity induced by these drugs.

  19. Hypoxia preconditioning increases survival and decreases expression of Toll-like receptor 4 in pulmonary artery endothelial cells exposed to lipopolysaccharide.

    Science.gov (United States)

    Ali, Irshad; Nanchal, Rahul; Husnain, Fouad; Audi, Said; Konduri, G Ganesh; Densmore, John C; Medhora, Meetha; Jacobs, Elizabeth R

    2013-09-01

    Abstract Pulmonary or systemic infections and hypoxemic respiratory failure are among the leading causes of admission to intensive care units, and these conditions frequently exist in sequence or in tandem. Inflammatory responses to infections are reproduced by lipopolysaccharide (LPS) engaging Toll-like receptor 4 (TLR4). Apoptosis is a hallmark of lung injury in sepsis. This study was conducted to determine whether preexposure to LPS or hypoxia modulated the survival of pulmonary artery endothelial cells (PAECs). We also investigated the role TLR4 receptor expression plays in apoptosis due to these conditions. Bovine PAECs were cultured in hypoxic or normoxic environments and treated with LPS. TLR4 antagonist TAK-242 was used to probe the role played by TLR4 receptors in cell survival. Cell apoptosis and survival were measured by caspase 3 activity and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) incorporation. TLR4 expression and tumor necrosis factor α (TNF-α) production were also determined. LPS increased caspase 3 activity in a TAK-242-sensitive manner and decreased MTT incorporation. Apoptosis was decreased in PAECs preconditioned with hypoxia prior to LPS exposure. LPS increased TNF-α production, and hypoxic preconditioning blunted it. Hypoxic preconditioning reduced LPS-induced TLR4 messenger RNA and TLR4 protein. TAK-242 decreased to baseline the LPS-stimulated expression of TLR4 messenger RNA regardless of environmental conditions. In contrast, LPS followed by hypoxia substantially increased apoptosis and cell death. In conclusion, protection from LPS-stimulated PAEC apoptosis by hypoxic preconditioning is attributable in part to reduction in TLR4 expression. If these signaling pathways apply to septic patients, they may account for differing sensitivities of individuals to acute lung injury depending on oxygen tensions in PAECs in vivo.

  20. Clinico-pathological analysis of renal cell carcinoma demonstrates decreasing tumour grade over a 17-year period

    Science.gov (United States)

    Nason, Gregory J.; McGuire, Barry B.; Kelly, Michael E.; Murphy, Theodore M.; Looney, Aisling T.; Byrne, Damien P.; Mulvin, David W.; Galvin, David J.; Quinlan, David M.; Lennon, Gerald M.

    2014-01-01

    Introduction: Renal cell carcinoma (RCC) represents about 3% of adult malignancies in Ireland. Worldwide there is a reported increasing incidence and recent studies report a stage migration towards smaller tumours. We assess the clinico-pathological features and survival of patients with RCC in a surgically treated cohort. Methods: A retrospective analysis of all nephrectomies carried out between 1995 and 2012 was carried out in an Irish tertiary referral university hospital. Data recorded included patient demographics, size of tumour, tumour-node-metastasis (TNM) classification, operative details and final pathology. The data were divided into 3 equal consecutive time periods for comparison purposes: Group 1 (1995–2000), Group 2 (2001–2006) and Group 3 (2007–2012). Survival data were verified with the National Cancer Registry of Ireland. Results: In total, 507 patients underwent nephrectomies in the study period. The median tumour size was 5.8 cm (range: 1.2–20 cm) and there was no statistical reduction in size observed over time (p = 0.477). A total of 142 (28%) RCCs were classified as pT1a, 111 (21.9%) were pT1b, 67 (13.2%) were pT2, 103 (20.3%) were pT3a, 75 (14.8%) were pT3b and 9 (1.8%) were pT4. There was no statistical T-stage migration observed (p = 0.213). There was a significant grade reduction over time (p = 0.017). There was significant differences noted in overall survival between the T-stages (p < 0.001), nuclear grades (p < 0.001) and histological subtypes (p = 0.022). Conclusion: There was a rising incidence in the number of nephrectomies over the study period. Despite previous reports, a stage migration was not evident; however, a grade reduction was apparent in this Irish surgical series. We can demonstrate that tumour stage, nuclear grade and histological subtype are significant prognosticators of relative survival in RCC. PMID:24839483

  1. Conditional Knockout of the MicroRNA 17-92 Cluster in Type-I Collagen-Expressing Cells Decreases Alveolar Bone Size and Incisor Tooth Mechanical Properties.

    Science.gov (United States)

    Ibrahim, M; Mohan, S; Xing, M J; Kesavan, C

    2016-01-01

    To test the role of the miR17-92 (miR) cluster in dental bones, we evaluated the incisor tooth phenotype by micro-CT in 5- and 12-week-old conditional knockout (CKO) mice deficient in the miR17-92 cluster in type-I collagen-expressing cells and bone strength by finite element analysis. The incisor teeth of CKO mice showed a 23-30 % reduction in tissue volume and bone volume. Accordingly, the stiffness and failure load of incisor teeth assessed by finite element analysis showed an 18-40 % decrease in CKO compared to wild-type mice. A positive correlation between bone parameters and strength data suggests that the decreased mechanical properties of incisor teeth are due to decreased tissue volume and bone volume. Subsequently, we found that the width of alveolar bone was reduced by 25 % with a 16 % increase in periodontal ligament space, suggesting that the CKO mice are more susceptible to tooth movement. Since alveolar bone is populated primarily by osteoblast lineage cells, it is likely that the reduction in periosteal expansion of alveolar bone in the lower jaw of CKO mice results from decreased periosteal bone formation. Overall, our phenotype analysis demonstrates that the miR17-92 cluster is essential for development and maintenance of tooth strength by regulating its tooth size.

  2. Ion-neutral gas reactions in a collision/reaction cell in inductively coupled plasma mass spectrometry: Correlation of ion signal decrease to kinetic rate constants

    Energy Technology Data Exchange (ETDEWEB)

    Gray, Patrick J. [Trace Element Research Laboratory, School of Earth Sciences, The Ohio State University, 125 S. Oval Mall, Columbus, OH 43210 (United States); Department of Chemistry, The Ohio State University, 120 18th Avenue, Columbus, OH 43210 (United States); Olesik, John W., E-mail: olesik.2@osu.edu [Trace Element Research Laboratory, School of Earth Sciences, The Ohio State University, 125 S. Oval Mall, Columbus, OH 43210 (United States)

    2015-03-01

    Reaction gas flow rate dependent Ar{sub 2}{sup +} and Ar{sup +} signals are correlated to fundamental kinetic rate coefficients. A simple calculation, assuming that gas exits the reaction cell due only to effusion, is described to estimate the gas pressure in the reaction cell. The value of the product of the kinetic rate constant and the ion residence time in the reaction cell can be determined from experimental measurement of the decrease in an ion signal as a function of reaction gas flow rate. New kinetic rate constants are determined for the reaction of CH{sub 3}F with Ar{sup +} and Ar{sub 2}{sup +}. - Highlights: • How to determine pressure and the product of the kinetic rate constant times the ion residence time in reaction cell • Relate measured ICP-DRC-MS signals versus gas flow rate to kinetic rate constants measured previously using SIFT-MS • Describe how to determine previously unmeasured kinetic rate constants using ICP-DRC-MS.

  3. Iron-induced oxidative stress activates AKT and ERK1/2 and decreases Dyrk1B and PRMT1 in neuroblastoma SH-SY5Y cells.

    Science.gov (United States)

    Bautista, Elizabeth; Vergara, Paula; Segovia, José

    2016-03-01

    Iron is essential for proper neuronal functioning; however, excessive accumulation of brain iron is reported in Parkinson's, Alzheimer's, Huntington's diseases and amyotrophic lateral sclerosis. This indicates that dysregulated iron homeostasis is involved in the pathogenesis of these diseases. To determinate the effect of iron on oxidative stress and on cell survival pathways, such as AKT, ERK1/2 and DyrK1B, neuroblastoma SH-SY5Y cells were exposed to different concentration of FeCl2 (iron). We found that iron induced cell death in SH-SY5Y cells in a concentration-dependent manner. Detection of iNOS and 3-nitrotyrosine confirms the presence of increased nitrogen species. Furthermore, we found a decrease of catalase and protein arginine methyl-transferase 1 (PRMT1). Interestingly, iron increased the activity of ERK and AKT and reduced DyrK1B. Moreover, after FeCl2 treatment, the transcription factors c-Jun and pSmad1/5 were activated. These results indicate that the presence of high levels of iron increase the vulnerability of neurons to oxidative stress.

  4. Non-thermal plasma inhibits human cervical cancer HeLa cells invasiveness by suppressing the MAPK pathway and decreasing matrix metalloproteinase-9 expression

    Science.gov (United States)

    Li, Wei; Yu, K. N.; Bao, Lingzhi; Shen, Jie; Cheng, Cheng; Han, Wei

    2016-01-01

    Non-thermal plasma (NTP) has been proposed as a novel therapeutic method for anticancer treatment. However, the mechanism underlying its biological effects remains unclear. In this study, we investigated the inhibitory effect of NTP on the invasion of HeLa cells, and explored the possible mechanism. Our results showed that NTP exposure for 20 or 40 s significantly suppressed the migration and invasion of HeLa cells on the basis of matrigel invasion assay and wound healing assay, respectively. Moreover, NTP reduced the activity and protein expression of the matrix metalloproteinase (MMP)-9 enzyme. Western blot analysis indicated that NTP exposure effectively decreased phosphorylation level of both ERK1/2 and JNK, but not p38 MAPK. Furthermore, treatment with MAPK signal pathway inhibitors or NTP all exhibited significant depression of HeLa cells migration and MMP-9 expression. The result showed that NTP synergistically suppressed migration and MMP-9 expression in the presence of ERK1/2 inhibitor and JNK inhibitor, but not p38 MAPK inhibitor. Taken together, these findings suggested that NTP exposure inhibited the migration and invasion of HeLa cells via down-regulating MMP-9 expression in ERK1/2 and JNK signaling pathways dependent manner. These findings provide hints to the potential clinical research and therapy of NTP on cervical cancer metastasis.

  5. Ginsenoside Rg3-induced EGFR/MAPK pathway deactivation inhibits melanoma cell proliferation by decreasing FUT4/LeY expression.

    Science.gov (United States)

    Shan, Xiu; Aziz, Faisal; Tian, Li Li; Wang, Xiao Qi; Yan, Qiu; Liu, Ji Wei

    2015-04-01

    Malignant melanoma is a destructive and lethal form of skin cancer with poor prognosis. An effective treatment for melanoma is greatly needed. Ginsenoside Rg3 is a herbal medicine with high antitumor activity. It is reported that abnormal glycosylation is correlated with the tumor cell growth. However, the antitumor effect of Rg3 on melanoma and its mechanism on regulating glycosylation are unknown. We found that Rg3 did not only inhibit A375 melanoma cell proliferation in a dose-dependent manner, but also decreased the expression of fucosyltransferase IV (FUT4) and its synthetic product Lewis Y (LeY), a tumor-associated carbohydrate antigen (TACA). Knocking down FUT4 expression by siRNA dramatically reduced FUT4/LeY level and inhibited cell proliferation through preventing the activation of EGFR/MAPK pathway. Consistently, the inhibitory effect of the Rg3 and FUT4 knockdown on melanoma growth was also seen in a xenograft melanoma mouse model. In conclusion, Rg3 effectively inhibited melanoma cell growth by downregulating FUT4 both in vitro and in vivo. Targeting FUT4/LeY mediated fucosylation by Rg3 inhibited the activation of EGFR/MAPK pathway and prevented melanoma growth. Results from this study suggest Rg3 is a potential novel therapy agent for melanoma treatment.

  6. Decreased infiltration of macrophage scavenger receptor-positive cells in initial negative biopsy specimens is correlated with positive repeat biopsies of the prostate.

    Science.gov (United States)

    Nonomura, Norio; Takayama, Hitoshi; Kawashima, Atsunari; Mukai, Masatoshi; Nagahara, Akira; Nakai, Yasutomo; Nakayama, Masashi; Tsujimura, Akira; Nishimura, Kazuo; Aozasa, Katsuyuki; Okuyama, Akihiko

    2010-06-01

    Macrophage scavenger receptor (MSR)-positive inflammatory cells and tumor-associated macrophages (TAMs) have been reported to regulate the growth of various cancers. In this study, the infiltration of MSR-positive cells and TAMs was analyzed to predict the outcome of repeat biopsy in men diagnosed as having no malignancy at the first prostate biopsy. Repeat biopsy of the prostate was carried out in 92 patients who were diagnosed as having no malignancy at the first biopsy. Of these, 30 patients (32.6%) were positive for prostate cancer at the repeat biopsy. Tumor-associated macrophages and MSR-positive cells were immunohistochemically stained with mAbs CD68 and CD204, respectively. Six ocular measuring fields were chosen randomly under a microscope at x400 power in the initial negative biopsy specimens, and the mean TAM and MSR counts for each case were determined. No difference in TAM count was found between the cases with or without prostate cancer. By contrast, the MSR count in patients with cancer was significantly lower than that in patients without cancer at the repeat biopsy (P biopsies, or TAM count. Decreased infiltration of MSR-positive cells in negative first biopsy specimens was correlated with positive findings in the repeat biopsy. The MSR count might be a good indicator for avoiding unnecessary repeat biopsies.

  7. Lysosomal-associated Transmembrane Protein 4B (LAPTM4B) Decreases Transforming Growth Factor β1 (TGF-β1) Production in Human Regulatory T Cells.

    Science.gov (United States)

    Huygens, Caroline; Liénart, Stéphanie; Dedobbeleer, Olivier; Stockis, Julie; Gauthy, Emilie; Coulie, Pierre G; Lucas, Sophie

    2015-08-14

    Production of active TGF-β1 is one mechanism by which human regulatory T cells (Tregs) suppress immune responses. This production is regulated by glycoprotein A repetitions predominant (GARP), a transmembrane protein present on stimulated Tregs but not on other T lymphocytes (Th and CTLs). GARP forms disulfide bonds with proTGF-β1, favors its cleavage into latent inactive TGF-β1, induces the secretion and surface presentation of GARP·latent TGF-β1 complexes, and is required for activation of the cytokine in Tregs. We explored whether additional Treg-specific protein(s) associated with GARP·TGF-β1 complexes regulate TGF-β1 production in Tregs. We searched for such proteins by yeast two-hybrid assay, using GARP as a bait to screen a human Treg cDNA library. We identified lysosomal-associated transmembrane protein 4B (LAPTM4B), which interacts with GARP in mammalian cells and is expressed at higher levels in Tregs than in Th cells. LAPTM4B decreases cleavage of proTGF-β1, secretion of soluble latent TGF-β1, and surface presentation of GARP·TGF-β1 complexes by Tregs but does not contribute to TGF-β1 activation. Therefore, LAPTM4B binds to GARP and is a negative regulator of TGF-β1 production in human Tregs. It may play a role in the control of immune responses by decreasing Treg immunosuppression.

  8. Tunable Biodegradable Nanocomposite Hydrogel for Improved Cisplatin Efficacy on HCT-116 Colorectal Cancer Cells and Decreased Toxicity in Rats.

    Science.gov (United States)

    Abdel-Bar, Hend Mohamed; Osman, Rihab; Abdel-Reheem, Amal Youssef; Mortada, Nahed; Awad, Gehanne A S

    2016-02-08

    This work describes the development of a modified nanocomposite thermosensitive hydrogel for controlled cisplatin release and improved cytotoxicity with decreased side effects. The system was characterized in terms of physical properties, morphological architecture and in vitro cisplatin release. Cytotoxicity was tested against human colorectal carcinoma HCT-116. In vivo studies were conducted to evaluate the acute toxicity in terms of rats' survival rate and body weight loss. Nephro and hepatotoxicities were evaluated followed by histopathological alterations of various tissue organs. Nanocomposite thermosensitive hydrogel containing nanosized carrier conferred density and stiffness allowing a zero order drug release for 14 days. Enhanced cytotoxicity with 2-fold decrease in cisplatin IC50 was accomplished. A linear in vivo-in vitro correlation was proved for the system degradation. Higher animal survival rate and lower tissue toxicities proved the decreased toxicity of cisplatin nanocomposite compared to its solution.

  9. Symptomatic dermographism: wealing, mast cells and histamine are decreased in the skin following long-term application of a potent topical corticosteroid.

    Science.gov (United States)

    Lawlor, F; Black, A K; Murdoch, R D; Greaves, M W

    1989-11-01

    Clobetasol propionate 0.05% ointment and an otherwise identical steroid-free base were applied topically to a 10 cm2 area on the anterior thighs of six patients with symptomatic dermographism for 6 weeks. Four patients showed a significantly decreased wealing response to stroking of steroid pretreated skin compared to that of control sites. There was a parallel decrease in mast cell numbers and histamine levels in skin biopsies taken from the steroid treated areas. At 6 weeks two patients demonstrated a decrease in flare areas following the intradermal injection of compound 48/80 in steroid pretreated skin compared to base treated sites. Flare areas following intradermal injection of histamine in these two patients were equivalent in base and steroid treated skin.

  10. Alpha-thalassemia is associated with a decreased occurrence and a delayed age-at-onset of albuminuria in sickle cell anemia patients.

    Science.gov (United States)

    Nebor, Danitza; Broquere, Cédric; Brudey, Karine; Mougenel, Danielle; Tarer, Vanessa; Connes, Philippe; Elion, Jacques; Romana, Marc

    2010-08-15

    The aim of this study was to identify possible risk factors for albuminuria, an early marker of sickle cell anemia (SCA) glomerulopathy, in a cohort of 189 SCA adult patients followed at the Sickle Cell Center of Guadeloupe, a French Caribbean island. Biological parameters obtained at baseline, alpha-globin gene status, and beta(S) haplotypes were compared in patients stratified accordingly to graded albuminuria. Abnormal albumin excretion rate was detected in half of the studied adult patients and macroalbuminuria occurred in 21.6%. Graded albuminuria was associated with advanced age (p=0.006), systolic blood pressure (p=0.031), and worsened anemia, i.e. low hemoglobin rate (pcell count (phaplotype. Our results strongly suggest a protective effect of alpha-thalassemia against glomerulopathy in SCA adult patients which could be related to a decreased hemolytic rate.

  11. Bradykinin activates ADP-ribosyl cyclase in neuroblastoma cells: intracellular concentration decrease in NAD and increase in cyclic ADP-ribose.

    Science.gov (United States)

    Higashida, Haruhiro; Salmina, Alla; Hashii, Minako; Yokoyama, Shigeru; Zhang, Jia-Sheng; Noda, Mami; Zhong, Zen-Guo; Jin, Duo

    2006-09-04

    ADP-ribosyl cyclase activity in the crude membrane fraction of neuroblastomaxglioma NGPM1-27 hybrid cells was measured by monitoring [(3)H] cyclic ADP-ribose (cADPR) formation from [(3)H] NAD(+). Bradykinin (BK) at 100nM increased ADP-ribosyl cyclase activity by about 2.5-fold. Application of 300nM BK to living NGPM1-27 cells decreased NAD(+) to 78% of the prestimulation level at 30s. In contrast, intracellular cADPR concentrations were increased by 2-3-fold during the period from 30 to 120s after the same treatment. Our results suggest that cADPR is one of the second messengers downstream of B(2) BK receptors.

  12. R59949, a diacylglycerol kinase inhibitor, inhibits inducible nitric oxide production through decreasing transplasmalemmal L-arginine uptake in vascular smooth muscle cells.

    Science.gov (United States)

    Shimomura, Tomoko; Nakano, Tomoyuki; Goto, Kaoru; Wakabayashi, Ichiro

    2017-02-01

    Although diacylglycerol kinase (DGK) is known to be expressed in vascular smooth muscle cell, its functional significance remains to be clarified. We hypothesized that DGK is involved in the pathway of cytokine-induced nitric oxide (NO) production in vascular smooth muscle cells. The purpose of this study was to investigate the effects of R59949, a diacylglycerol kinase inhibitor, on inducible nitric oxide production in vascular smooth muscle cell. Cultured rat aortic smooth muscle cells (RASMCs) were used to elucidate the effects of R59949 on basal and interleukin-1β (IL-1β)-induced NO production. The effects of R59949 on protein and mRNA expression of induced nitric oxide synthase (iNOS) and on transplasmalemmal L-arginine uptake were also evaluated using RASMCs. Treatment of RASMCs with R59949 (10 μM) inhibited IL-1β (10 ng/ml)-induced NO production but not basal NO production. Neither protein nor mRNA expression level of iNOS after stimulation with IL-1β was significantly affected by R59949. Estimated enzymatic activities of iNOS in RASMCs were comparable in the absence and presence of R59949. Stimulation of RASMCs with IL-1β caused a marked increase in transplasmalemmal L-arginine uptake into RASMCs. L-Arginine uptake in the presence of IL-1β was markedly inhibited by R59949, while basal L-arginine uptake was not significantly affected by R59949. Both IL-1β-induced NO production and L-arginine uptake were abolished in the presence of cycloheximide (1 μM). The results indicate that R59949 inhibits inducible NO production through decreasing transplasmalemmal L-arginine uptake. DGK is suggested to be involved in cytokine-stimulated L-arginine transport and regulate its intracellular concentration in vascular smooth muscle cell.

  13. Polyimide-coated carbon electrodes combined with redox mediators for superior Li-O2 cells with excellent cycling performance and decreased overpotential

    Science.gov (United States)

    Yoon, Seon Hye; Park, Yong Joon

    2017-02-01

    We report an air electrode employing polyimide-coated carbon nanotubes (CNTs) combined with a redox mediator for Li-O2 cells with enhanced electrochemical performance. The polyimide coating on the carbon surface suppresses unwanted side reactions, which decreases the amount of accumulated reaction products on the surface of the air electrode during cycling. The redox mediators lower the overpotential of the Li-O2 cells because they can easily transfer electrons from the electrode to the reaction products. The low overpotential can also decrease the side reactions that activate at a high potential range. Specifically, the CsI redox mediator effectively interrupted dendrite growth on the Li anode during cycling due to the shielding effect of its Cs+ ions and acted as a redox mediator due to its I‑ ions. LiNO3 also facilitates the decrease in side reactions and the stabilization of the Li anode. The synergic effect of the polyimide coating and the electrolyte containing the LiNO3/CsI redox mediator leads to a low overpotential and excellent cycling performance (over 250 cycles with a capacity of 1,500 mAh·gelectrode‑1).

  14. Nonivamide enhances miRNA let-7d expression and decreases adipogenesis PPARγ expression in 3T3-L1 cells.

    Science.gov (United States)

    Rohm, Barbara; Holik, Ann-Katrin; Kretschy, Nicole; Somoza, Mark M; Ley, Jakob P; Widder, Sabine; Krammer, Gerhard E; Marko, Doris; Somoza, Veronika

    2015-06-01

    Red pepper and its major pungent principle, capsaicin (CAP), have been shown to be effective anti-obesity agents by reducing energy intake, enhancing energy metabolism, decreasing serum triacylglycerol content, and inhibiting adipogenesis via activation of the transient receptor potential cation channel subfamily V member 1 (TRPV1). However, the binding of CAP to the TRPV1 receptor is also responsible for its pungent sensation, strongly limiting its dietary intake. Here, the effects of a less pungent structural CAP-analog, nonivamide, on adipogenesis and underlying mechanisms in 3T3-L1 cells were studied. Nonivamide was found to reduce mean lipid accumulation, a marker of adipogenesis, to a similar extent as CAP, up to 10.4% (P < 0.001). Blockage of the TRPV1 receptor with the specific inhibitor trans-tert-butylcyclohexanol revealed that the anti-adipogenic activity of nonivamide depends, as with CAP, on TRPV1 receptor activation. In addition, in cells treated with nonivamide during adipogenesis, protein levels of the pro-adipogenic transcription factor peroxisome-proliferator activated receptor γ (PPARγ) decreased. Results from miRNA microarrays and digital droplet PCR analysis demonstrated an increase in the expression of the miRNA mmu-let-7d-5p, which has been associated with decreased PPARγ levels.

  15. Polyimide-coated carbon electrodes combined with redox mediators for superior Li-O2 cells with excellent cycling performance and decreased overpotential

    Science.gov (United States)

    Yoon, Seon Hye; Park, Yong Joon

    2017-01-01

    We report an air electrode employing polyimide-coated carbon nanotubes (CNTs) combined with a redox mediator for Li-O2 cells with enhanced electrochemical performance. The polyimide coating on the carbon surface suppresses unwanted side reactions, which decreases the amount of accumulated reaction products on the surface of the air electrode during cycling. The redox mediators lower the overpotential of the Li-O2 cells because they can easily transfer electrons from the electrode to the reaction products. The low overpotential can also decrease the side reactions that activate at a high potential range. Specifically, the CsI redox mediator effectively interrupted dendrite growth on the Li anode during cycling due to the shielding effect of its Cs+ ions and acted as a redox mediator due to its I− ions. LiNO3 also facilitates the decrease in side reactions and the stabilization of the Li anode. The synergic effect of the polyimide coating and the electrolyte containing the LiNO3/CsI redox mediator leads to a low overpotential and excellent cycling performance (over 250 cycles with a capacity of 1,500 mAh·gelectrode−1). PMID:28198419

  16. Cobalt chloride decreases fibroblast growth factor-21 expression dependent on oxidative stress but not hypoxia-inducible factor in Caco-2 cells

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Yanlong [School of Pharmacy, Wenzhou Medical College, Wenzhou (China); Department of Medicine, University of Louisville, Louisville, KY (United States); Wang, Chunhong [Second Hospital, Jilin University, Changchun (China); Department of Medicine, University of Louisville, Louisville, KY (United States); Wang, Yuhua [College of Food Science and Engineering, Jilin Agricultural University, Changchun (China); Department of Medicine, University of Louisville, Louisville, KY (United States); Ma, Zhenhua [First Hospital, Xi' an Jiaotong University, Xi' an (China); Department of Medicine, University of Louisville, Louisville, KY (United States); Xiao, Jian [School of Pharmacy, Wenzhou Medical College, Wenzhou (China); McClain, Craig [Department of Medicine, University of Louisville, Louisville, KY (United States); Department of Pharmacology and Toxicology, University of Louisville, Louisville, KY (United States); Alcohol Research Center, University of Louisville, Louisville, KY (United States); Robley Rex Veterans Affairs Medical Center, Louisville, KY (United States); Li, Xiaokun [School of Pharmacy, Wenzhou Medical College, Wenzhou (China); Feng, Wenke, E-mail: wenke.feng@louisville.edu [School of Pharmacy, Wenzhou Medical College, Wenzhou (China); Department of Medicine, University of Louisville, Louisville, KY (United States); Alcohol Research Center, University of Louisville, Louisville, KY (United States)

    2012-10-15

    Fibroblast growth factor-21 (FGF21) is a potential metabolic regulator with multiple beneficial effects on metabolic diseases. FGF21 is mainly expressed in the liver, but is also found in other tissues including the intestine, which expresses β-klotho abundantly. The intestine is a unique organ that operates in a physiologically hypoxic environment, and is responsible for the fat absorption processes including triglyceride breakdown, re-synthesis and absorption into the portal circulation. In the present study, we investigated the effects of hypoxia and the chemical hypoxia inducer, cobalt chloride (CoCl{sub 2}), on FGF21 expression in Caco-2 cells and the consequence of fat accumulation. Physical hypoxia (1% oxygen) and CoCl{sub 2} treatment decreased both FGF21 mRNA and secreted protein levels. Gene silence and inhibition of hypoxia-inducible factor-α (HIFα) did not affect the reduction of FGF21 mRNA and protein levels by hypoxia. However, CoCl{sub 2} administration caused a significant increase in oxidative stress. The addition of n-acetylcysteine (NAC) suppressed CoCl{sub 2}-induced reactive oxygen species (ROS) formation and completely negated CoCl{sub 2}-induced FGF21 loss. mRNA stability analysis demonstrated that the CoCl{sub 2} administration caused a remarkable reduction in FGF21 mRNA stability. Furthermore, CoCl{sub 2} increased intracellular triglyceride (TG) accumulation, along with a reduction in mRNA levels of lipid lipase, hormone sensitive lipase (HSL) and adipose triglyceride lipase (ATGL), and an increase of sterol regulatory element-binding protein-1c (SREBP1c) and stearoyl-coenzyme A (SCD1). Addition of both NAC and recombinant FGF21 significantly attenuated the CoCl{sub 2}-induced TG accumulation. In conclusion, the decrease of FGF21 in Caco-2 cells by chemical hypoxia is independent of HIFα, but dependent on an oxidative stress-mediated mechanism. The regulation of FGF21 by hypoxia may contribute to intestinal lipid metabolism and

  17. Autologous hematopoietic stem cell transplantation in lymphoma patients is associated with a decrease in the double strand break repair capacity of peripheral blood lymphocytes

    Science.gov (United States)

    Lacoste, Sandrine; Bhatia, Smita; Chen, Yanjun; Bhatia, Ravi; O’Connor, Timothy R.

    2017-01-01

    Patients who undergo autologous hematopoietic stem cell transplantation (aHCT) for treatment of a relapsed or refractory lymphoma are at risk of developing therapy related- myelodysplasia/acute myeloid leukemia (t-MDS/AML). Part of the risk likely resides in inherent interindividual differences in their DNA repair capacity (DRC), which is thought to influence the effect chemotherapeutic treatments have on the patient’s stem cells prior to aHCT. Measuring DRC involves identifying small differences in repair proficiency among individuals. Initially, we investigated the cell model in healthy individuals (primary lymphocytes and/or lymphoblastoid cell lines) that would be appropriate to measure genetically determined DRC using host-cell reactivation assays. We present evidence that interindividual differences in DRC double-strand break repair (by non-homologous end-joining [NHEJ] or single-strand annealing [SSA]) are better preserved in non-induced primary lymphocytes. In contrast, lymphocytes induced to proliferate are required to assay base excision (BER) or nucleotide excision repair (NER). We established that both NHEJ and SSA DRCs in lymphocytes of healthy individuals were inversely correlated with the age of the donor, indicating that DSB repair in lymphocytes is likely not a constant feature but rather something that decreases with age (~0.37% NHEJ DRC/year). To investigate the predictive value of pre-aHCT DRC on outcome in patients, we then applied the optimized assays to the analysis of primary lymphocytes from lymphoma patients and found that individuals who later developed t-MDS/AML (cases) were indistinguishable in their DRC from controls who never developed t-MDS/AML. However, when DRC was investigated shortly after aHCT in the same individuals (21.6 months later on average), aHCT patients (both cases and controls) showed a significant decrease in DSB repair measurements. The average decrease of 6.9% in NHEJ DRC observed among aHCT patients was much

  18. Autologous hematopoietic stem cell transplantation in lymphoma patients is associated with a decrease in the double strand break repair capacity of peripheral blood lymphocytes.

    Science.gov (United States)

    Lacoste, Sandrine; Bhatia, Smita; Chen, Yanjun; Bhatia, Ravi; O'Connor, Timothy R

    2017-01-01

    Patients who undergo autologous hematopoietic stem cell transplantation (aHCT) for treatment of a relapsed or refractory lymphoma are at risk of developing therapy related- myelodysplasia/acute myeloid leukemia (t-MDS/AML). Part of the risk likely resides in inherent interindividual differences in their DNA repair capacity (DRC), which is thought to influence the effect chemotherapeutic treatments have on the patient's stem cells prior to aHCT. Measuring DRC involves identifying small differences in repair proficiency among individuals. Initially, we investigated the cell model in healthy individuals (primary lymphocytes and/or lymphoblastoid cell lines) that would be appropriate to measure genetically determined DRC using host-cell reactivation assays. We present evidence that interindividual differences in DRC double-strand break repair (by non-homologous end-joining [NHEJ] or single-strand annealing [SSA]) are better preserved in non-induced primary lymphocytes. In contrast, lymphocytes induced to proliferate are required to assay base excision (BER) or nucleotide excision repair (NER). We established that both NHEJ and SSA DRCs in lymphocytes of healthy individuals were inversely correlated with the age of the donor, indicating that DSB repair in lymphocytes is likely not a constant feature but rather something that decreases with age (~0.37% NHEJ DRC/year). To investigate the predictive value of pre-aHCT DRC on outcome in patients, we then applied the optimized assays to the analysis of primary lymphocytes from lymphoma patients and found that individuals who later developed t-MDS/AML (cases) were indistinguishable in their DRC from controls who never developed t-MDS/AML. However, when DRC was investigated shortly after aHCT in the same individuals (21.6 months later on average), aHCT patients (both cases and controls) showed a significant decrease in DSB repair measurements. The average decrease of 6.9% in NHEJ DRC observed among aHCT patients was much higher

  19. Decreased expression of BRCA1 in SK-BR-3 cells is the result of aberrant activation of the GABP Beta promoter by an NRF-1-containing complex

    Directory of Open Access Journals (Sweden)

    MacDonald Gwen

    2011-05-01

    Full Text Available Abstract Background BRCA1 has recently been identified as a potential regulator of mammary stem/progenitor cell differentiation, and this function may explain the high prevalence of breast cancer in BRCA1 mutation carriers, as well as the downregulation of BRCA1 in a large proportion of sporadic breast cancers. That is, loss of BRCA1 function results in blocked differentiation with expansion of the mammary stem/progenitor cells. Because BRCA1 also maintains genomic integrity, its loss could produce a pool of genetically unstable stem/progenitor cells that are prime targets for further transforming events. Thus, elucidating the regulatory mechanisms of BRCA1 expression is important to our understanding of normal and malignant breast differentiation. Results Loss of BRCA1 expression in the ErbB2-amplified SK-BR-3 cell line was found to be the result of loss of activity of the ets transcription factor GABP, a previously characterized regulator of BRCA1 transcription. The expression of the non-DNA binding GABPβ subunit was shown to be deficient, while the DNA binding subunit, GABPα was rendered unstable by the absence of GABPβ. Deletion analysis of the GABPβ proximal promoter identified a potential NRF-1 binding site as being critical for expression. Supershift analysis, the binding of recombinant protein and chromatin immunoprecipitation confirmed the role of NRF-1 in regulating the expression of GABPβ. The siRNA knockdown of NRF-1 resulted in decreased GABPβ and BRCA1 expression in MCF-7 cells indicating that they form a transcriptional network. NRF-1 levels and activity did not differ between SK-BR-3 and MCF-7 cells, however the NRF-1 containing complex on the GABPβ promoter differed between the two lines and appears to be the result of altered coactivator binding. Conclusions Both NRF-1 and GABP have been linked to the regulation of nuclear-encoded mitochondrial proteins, and the results of this study suggest their expression is

  20. Plasma miR-185 is decreased in patients with esophageal squamous cell carcinoma and might suppress tumor migration and invasion by targeting RAGE.

    Science.gov (United States)

    Jing, Rongrong; Chen, Wen; Wang, Huimin; Ju, Shaoqing; Cong, Hui; Sun, Baolan; Jin, Qin; Chu, Shaopeng; Xu, Lili; Cui, Ming

    2015-11-01

    The receptor for advanced-glycation end products (RAGE) is upregulated in various cancers and has been associated with tumor progression, but little is known about its expression and regulation by microRNAs (miRNAs) in esophageal squamous cell carcinoma (ESCC). Here, we describe miR-185, which represses RAGE expression, and investigate the biological role of miR-185 in ESCC. In this study, we found that the high level of RAGE expression in 29 pairs of paraffin-embedded ESCC tissues was correlated positively with the depth of invasion by immunohistochemistry, suggesting that RAGE was involved in ESCC. We used bioinformatics searches and luciferase reporter assays to investigate the prediction that RAGE was regulated directly by miR-185. Besides, overexpression of miR-185 in ESCC cells was accompanied by 27% (TE-11) and 49% (Eca-109) reduced RAGE expression. The effect was further confirmed in RAGE protein by immunofluorescence in both cell lines. The effects were reversed following cotransfection with miR-185 and high-level expression of the RAGE vector. Furthermore, the biological role of miR-185 in ESCC cell lines was investigated using assays of cell viability, Ki-67 staining, and c