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  1. Upregulation of Notch pathway molecules in oral squamous cell carcinoma

    OpenAIRE

    2010-01-01

    The constitutive activation of the Notch pathway has been demonstrated in various types of malignancies. However, it remains unclear how the Notch pathway is involved in the pathogenesis of oral squamous cell carcinoma (OSCC). We investigated the expression of Notch pathway molecules in OSCC cell lines and biopsy specimens and examined the effect of Notch pathway inhibition. Reverse transcription-polymerase chain reaction revealed upregulation of Notch1, Notch2, Jagged1, HES1 and HEY1 in both...

  2. Progesterone Upregulates Gene Expression in Normal Human Thyroid Follicular Cells

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    Ana Paula Santin Bertoni

    2015-01-01

    Full Text Available Thyroid cancer and thyroid nodules are more prevalent in women than men, so female sex hormones may have an etiological role in these conditions. There are no data about direct effects of progesterone on thyroid cells, so the aim of the present study was to evaluate progesterone effects in the sodium-iodide symporter NIS, thyroglobulin TG, thyroperoxidase TPO, and KI-67 genes expression, in normal thyroid follicular cells, derived from human tissue. NIS, TG, TPO, and KI-67 mRNA expression increased significantly after TSH 20 μUI/mL, respectively: 2.08 times, P<0.0001; 2.39 times, P=0.01; 1.58 times, P=0.0003; and 1.87 times, P<0.0001. In thyroid cells treated with 20 μUI/mL TSH plus 10 nM progesterone, RNA expression of NIS, TG, and KI-67 genes increased, respectively: 1.78 times, P<0.0001; 1.75 times, P=0.037; and 1.95 times, P<0.0001, and TPO mRNA expression also increased, though not significantly (1.77 times, P=0.069. These effects were abolished by mifepristone, an antagonist of progesterone receptor, suggesting that genes involved in thyroid cell function and proliferation are upregulated by progesterone. This work provides evidence that progesterone has a direct effect on thyroid cells, upregulating genes involved in thyroid function and growth.

  3. Upregulation of Notch pathway molecules in oral squamous cell carcinoma.

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    Hijioka, Hiroshi; Setoguchi, Takao; Miyawaki, Akihiko; Gao, Hui; Ishida, Takayuki; Komiya, Setsuro; Nakamura, Norifumi

    2010-04-01

    The constitutive activation of the Notch pathway has been demonstrated in various types of malignancies. However, it remains unclear how the Notch pathway is involved in the pathogenesis of oral squamous cell carcinoma (OSCC). We investigated the expression of Notch pathway molecules in OSCC cell lines and biopsy specimens and examined the effect of Notch pathway inhibition. Reverse transcription-polymerase chain reaction revealed upregulation of Notch1, Notch2, Jagged1, HES1 and HEY1 in both OSCC cell lines and biopsy specimens. Immunohistochemical examination showed that the Notch intracellular domain accumulates in the nucleus of cells in OSCC cell lines and biopsy specimens. In addition, Jagged1 is expressed in the cytoplasm of cells in OSCC cell lines and biopsy specimens. Furthermore, Notch pathway inhibition using a gamma-secretase inhibitor prevented the growth of OSCC in vitro. These findings suggest that inhibition of the Notch pathway suppresses OSCC growth and may be a useful approach for the treatment of patients with OSCC.

  4. Cell surface estrogen receptor alpha is upregulated during subchronic metabolic stress and inhibits neuronal cell degeneration.

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    Cristiana Barbati

    Full Text Available In addition to the classical nuclear estrogen receptor, the expression of non-nuclear estrogen receptors localized to the cell surface membrane (mER has recently been demonstrated. Estrogen and its receptors have been implicated in the development or progression of numerous neurodegenerative disorders. Furthermore, the pathogenesis of these diseases has been associated with disturbances of two key cellular programs: apoptosis and autophagy. An excess of apoptosis or a defect in autophagy has been implicated in neurodegeneration. The aim of this study was to clarify the role of ER in determining neuronal cell fate and the possible implication of these receptors in regulating either apoptosis or autophagy. The human neuronal cell line SH-SY5Y and mouse neuronal cells in primary culture were thus exposed to chronic minimal peroxide treatment (CMP, a form of subcytotoxic minimal chronic stress previously that mimics multiple aspects of long-term cell stress and represents a limited molecular proxy for neurodegenerative processes. We actually found that either E2 or E2-bovine serum albumin construct (E2BSA, i.e. a non-permeant form of E2 was capable of modulating intracellular cell signals and regulating cell survival and death. In particular, under CMP, the up-regulation of mERα, but not mERβ, was associated with functional signals (ERK phosphorylation and p38 dephosphorylation compatible with autophagic cytoprotection triggering and leading to cell survival. The mERα trafficking appeared to be independent of the microfilament system cytoskeletal network but was seemingly associated with microtubular apparatus network, i.e., to MAP2 molecular chaperone. Importantly, antioxidant treatments, administration of siRNA to ERα, or the presence of antagonist of ERα hindered these events. These results support that the surface expression of mERα plays a pivotal role in determining cell fate, and that ligand-induced activation of mER signalling exerts a

  5. Benfotiamine upregulates antioxidative system in activated BV-2 microglia cells.

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    Bozic, Iva; Savic, Danijela; Stevanovic, Ivana; Pekovic, Sanja; Nedeljkovic, Nadezda; Lavrnja, Irena

    2015-01-01

    Chronic microglial activation and resulting sustained neuroinflammatory reaction are generally associated with neurodegeneration. Activated microglia acquires proinflammatory cellular profile that generates oxidative burst. Their persistent activation exacerbates inflammation, which damages healthy neurons via cytotoxic mediators, such as superoxide radical anion and nitric oxide. In our recent study, we have shown that benfotiamine (S-benzoylthiamine O-monophosphate) possesses anti-inflammatory effects. Here, the effects of benfotiamine on the pro-oxidative component of activity of LPS-stimulated BV-2 cells were investigated. The activation of microglia was accompanied by upregulation of intracellular antioxidative defense, which was further promoted in the presence of benfotiamine. Namely, activated microglia exposed to non-cytotoxic doses of benfotiamine showed increased levels and activities of hydrogen peroxide- and superoxide-removing enzymes-catalase and glutathione system, and superoxide dismutase. In addition, benfotiamine showed the capacity to directly scavenge superoxide radical anion. As a consequence, benfotiamine suppressed the activation of microglia and provoked a decrease in NO and (·)O(-) 2 production and lipid peroxidation. In conclusion, benfotiamine might silence pro-oxidative activity of microglia to alleviate/prevent oxidative damage of neighboring CNS cells.

  6. Cytosolic DNA Sensor Upregulation Accompanies DNA Electrotransfer in B16.F10 Melanoma Cells.

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    Znidar, Katarina; Bosnjak, Masa; Cemazar, Maja; Heller, Loree C

    2016-06-07

    In several preclinical tumor models, antitumor effects occur after intratumoral electroporation, also known as electrotransfer, of plasmid DNA devoid of a therapeutic gene. In mouse melanomas, these effects are preceded by significant elevation of several proinflammatory cytokines. These observations implicate the binding and activation of intracellular DNA-specific pattern recognition receptors or DNA sensors in response to DNA electrotransfer. In tumors, IFNβ mRNA and protein levels significantly increased. The mRNAs of several DNA sensors were detected, and DAI, DDX60, and p204 tended to be upregulated. These effects were accompanied with reduced tumor growth and increased tumor necrosis. In B16.F10 cells in culture, IFNβ mRNA and protein levels were significantly upregulated. The mRNAs for several DNA sensors were present in these cells; DNA-dependent activator of interferon regulatory factor (DAI), DEAD (Asp-Glu-Ala-Asp) box polypeptide 60 (DDX60), and p204 were significantly upregulated while DDX60 protein levels were coordinately upregulated. Upregulation of DNA sensors in tumors could be masked by the lower transfection efficiency compared to in vitro or to dilution by other tumor cell types. Mirroring the observation of tumor necrosis, cells underwent a significant DNA concentration-dependent decrease in proliferation and survival. Taken together, these results indicate that DNA electrotransfer may cause the upregulation of several intracellular DNA sensors in B16.F10 cells, inducing effects in vitro and potentially in vivo.

  7. Upregulation of Phagocytic Clearance of Apoptotic Cells by Autoimmune Regulator

    Institute of Scientific and Technical Information of China (English)

    石亮; 胡丽华; 李一荣

    2010-01-01

    To investigate the effect of autoimmune regulator(AIRE) on phagocytic clearance of apoptotic cells,a recombinant expression vector containing full-length human AIRE cDNA was transfected into 16HBE cells.After incubation with transfected 16HBE cells,engulfment of apoptotic HL-60 cells induced by camptothecin was detected by myeloperoxidase(MPO) staining.The change in the expression of Rac 1 in transfected 16HBE cells was determined by RT-PCR and Western blotting.The results showed that the phagocytosis perce...

  8. T Cell Functional Disturbances in Patients with Pulmonary Tuberculosis.

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    Ostanin, Alexander A.; Khonina, Nataliya A.; Norkin, Maxim N.; Leplina, Olga Yu.; Nikonov, Sergey D.; Ogirenko, Anatoly P.; Chernykh, Helen R.

    2000-04-01

    The investigations of 38 patients with pulmonary tuberculosis (PT) revealed combined T cell and monocyte functional disturbances. Indeed, the percentages of CD4(+) and CD8(+) T lymphocytes, proliferative response and IL-2 production, as well as the percentages of HLA DR(+) monocytes and IL-1beta production were significantly decreased in PT patients as compared with normal individuals. Herewith the absolute T lymphocyte number did not undergo the pronounced changes. The decrease of T cell proliferative response was not mediated through immunosuppressive action of monocytes or T lymphocytes since removing of "adherent" cells from patient's peripheral blood mononuclear cells (PBMC) or pretreatment of PBMC with indomethacin and cyclophosphan failed to recover mitogenic reactivity in vitro. The patient's sera also did not significantly influence on PBMC proliferation. The decrease of IL-2 production and the stimulation of T cell proliferative response via TcR-CD3 complex, i.e. through the classic pathway of activation, indicated the anergy of T lymphocyte in tuberculosis patients. Furthermore, T lymphocytes were characterized by enhanced apoptosis. It should be noted, that patient's sera (especially in the patients with an initially high apoptosis) promoted significant anti-apoptotic activity. It is likely that this mechanism may be an explanation, why absolute T lymphopenia is absent during tuberculosis infection. Our findings suggest, that T lymphocyte dysfunctions in patients with PT are caused by impairments of T cell activation process, which lead to predominance of "negative" response (induction anergy, apoptosis) and to a lesser degree connected with direct suppressive mechanisms mediated by monocytes, T lymphocytes or serum factors.

  9. Neuropilin 1 Receptor Is Up-Regulated in Dysplastic Epithelium and Oral Squamous Cell Carcinoma

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    Shahrabi-Farahani, Shokoufeh; Gallottini, Marina; Martins, Fabiana; Li, Erik; Mudge, Dayna R.; Nakayama, Hironao; Hida, Kyoko; Panigrahy, Dipak; D'Amore, Patricia A.; Bielenberg, Diane R.

    2017-01-01

    Neuropilins are receptors for disparate ligands, including proangiogenic factors such as vascular endothelial growth factor and inhibitory class 3 semaphorin (SEMA3) family members. Differentiated cells in skin epithelium and cutaneous squamous cell carcinoma highly express the neuropilin-1 (NRP1) receptor. We examined the expression of NRP1 in human and mouse oral mucosa. NRP1 was significantly up-regulated in oral epithelial dysplasia and oral squamous cell carcinoma (OSCC). NRP1 receptor localized to the outer suprabasal epithelial layers in normal tongue, an expression pattern similar to the normal skin epidermis. However, dysplastic tongue epithelium and OSCC up-regulated NRP1 in basal and proliferating epithelial layers, a profile unseen in cutaneous squamous cell carcinoma. NRP1 up-regulation is observed in a mouse carcinogen-induced OSCC model and in human tongue OSCC biopsies. Human OSCC cell lines express NRP1 protein in vitro and in mouse tongue xenografts. Sites of capillary infiltration into orthotopic OSCC tumors correlate with high NRP1 expression. HSC3 xenografts, which express the highest NRP1 levels of the cell lines examined, showed massive intratumoral lymphangiogenesis. SEMA3A inhibited OSCC cell migration, suggesting that the NRP1 receptor was bioactive in OSCC. In conclusion, NRP1 is regulated in the oral epithelium and is selectively up-regulated during epithelial dysplasia. NRP1 may function as a reservoir to sequester proangiogenic ligands within the neoplastic compartment, thereby recruiting neovessels toward tumor cells. PMID:26877262

  10. Neuropilin 1 Receptor Is Up-Regulated in Dysplastic Epithelium and Oral Squamous Cell Carcinoma.

    Science.gov (United States)

    Shahrabi-Farahani, Shokoufeh; Gallottini, Marina; Martins, Fabiana; Li, Erik; Mudge, Dayna R; Nakayama, Hironao; Hida, Kyoko; Panigrahy, Dipak; D'Amore, Patricia A; Bielenberg, Diane R

    2016-04-01

    Neuropilins are receptors for disparate ligands, including proangiogenic factors such as vascular endothelial growth factor and inhibitory class 3 semaphorin (SEMA3) family members. Differentiated cells in skin epithelium and cutaneous squamous cell carcinoma highly express the neuropilin-1 (NRP1) receptor. We examined the expression of NRP1 in human and mouse oral mucosa. NRP1 was significantly up-regulated in oral epithelial dysplasia and oral squamous cell carcinoma (OSCC). NRP1 receptor localized to the outer suprabasal epithelial layers in normal tongue, an expression pattern similar to the normal skin epidermis. However, dysplastic tongue epithelium and OSCC up-regulated NRP1 in basal and proliferating epithelial layers, a profile unseen in cutaneous squamous cell carcinoma. NRP1 up-regulation is observed in a mouse carcinogen-induced OSCC model and in human tongue OSCC biopsies. Human OSCC cell lines express NRP1 protein in vitro and in mouse tongue xenografts. Sites of capillary infiltration into orthotopic OSCC tumors correlate with high NRP1 expression. HSC3 xenografts, which express the highest NRP1 levels of the cell lines examined, showed massive intratumoral lymphangiogenesis. SEMA3A inhibited OSCC cell migration, suggesting that the NRP1 receptor was bioactive in OSCC. In conclusion, NRP1 is regulated in the oral epithelium and is selectively up-regulated during epithelial dysplasia. NRP1 may function as a reservoir to sequester proangiogenic ligands within the neoplastic compartment, thereby recruiting neovessels toward tumor cells.

  11. Passive diffusion of naltrexone into human and animal cells and upregulation of cell proliferation.

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    Cheng, Fan; McLaughlin, Patricia J; Banks, William A; Zagon, Ian S

    2009-09-01

    Naltrexone (NTX) is a potent opioid antagonist that promotes cell proliferation by upregulating DNA synthesis through displacement of the tonically active inhibitory peptide, opioid growth factor (OGF) from its receptor (OGFr). To investigate how NTX enters cells, NTX was fluorescently labeled [1-(N)-fluoresceinyl NTX thiosemicarbazone; FNTX] to study its uptake by living cultured cells. When human head and neck squamous cell carcinoma cell line (SCC-1) was incubated with FNTX for as little as 1 min, cells displayed nuclear and cytoplasmic staining of FNTX as determined by fluorescent deconvolution microscopy, with enrichment of fluorescent signal in the nucleus and nucleolus. The same temporal-spatial distribution of FNTX was detected in a human pancreatic cancer cell line (MIA PaCa-2), African green monkey kidney cell line (COS-7), and human mesenchymal stem cells (hMSCs). FNTX remained in cells for as long as 48 h. FNTX was internalized in SCC-1 cells when incubation occurred at 4 degrees C, with the signal being comparable to that recorded at 37 degrees C. A 100-fold excess of NTX or a variety of other opioid ligands did not alter the temporal-spatial distribution of FNTX. Neither fluorescein-labeled dextran nor fluorescein alone entered the cells. To study the effect of FNTX on DNA synthesis, cells incubated with FNTX at concentrations ranging from 10(-5) to 10(-8) M had a 5-bromo-2'-deoxyuridine index that was 39-82% greater than for vehicle-treated cells and was comparable to that of unlabeled NTX (37-70%). Taken together, these results suggested that NTX enters cells by passive diffusion in a nonsaturable manner.

  12. To investigate the necessity of STRA6 upregulation in T cells during T cell immune responses.

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    Rafik Terra

    Full Text Available Our earlier study revealed that STRA6 (stimulated by retinoic acid gene 6 was up-regulated within 3 h of TCR stimulation. STRA6 is the high-affinity receptor for plasma retinol-binding protein (RBP and mediates cellular vitamin A uptake. We generated STRA6 knockout (KO mice to assess whether such up-regulation was critical for T-cell activation, differentiation and function. STRA6 KO mice under vitamin A sufficient conditions were fertile without apparent anomalies upon visual inspection. The size, cellularity and lymphocyte subpopulations of STRA6 KO thymus and spleen were comparable to those of their wild type (WT controls. KO and WT T cells were similar in terms of TCR-stimulated proliferation in vitro and homeostatic expansion in vivo. Naive KO CD4 cells differentiated in vitro into Th1, Th2, Th17 as well as regulatory T cells in an analogous manner as their WT counterparts. In vivo experiments revealed that anti-viral immune responses to lymphocytic choriomeningitis virus in KO mice were comparable to those of WT controls. We also demonstrated that STRA6 KO and WT mice had similar glucose tolerance. Total vitamin A levels are dramatically lower in the eyes of KO mice as compared to those of WT mice, but the levels in other organs were not significantly affected after STRA6 deletion under vitamin A sufficient conditions, indicating that the eye is the mouse organ most sensitive to the loss of STRA6. Our results demonstrate that 1 in vitamin A sufficiency, the deletion of STRA6 in T cells does no affect the T-cell immune responses so-far tested, including those depend on STAT5 signaling; 2 STRA6-independent vitamin A uptake compensated the lack of STRA6 in lymphoid organs under vitamin A sufficient conditions in mice; 3 STRA6 is critical for vitamin A uptake in the eyes even in vitamin A sufficiency.

  13. LncRNA TUG1 is upregulated and promotes cell proliferation in osteosarcoma

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    Yun-Bo Feng

    2016-01-01

    Full Text Available Objective: To examine the expression and function of long non-coding RNA taurine up-regulated 1 (TUG1 in human osteosarcoma cells. Methods: Real-time quantitive PCR was used to detect the transcription level of TUG1 in a series of osteosarcoma cell lines. Knockdown of TUG1 in U2OS cells was carried out by transient transfection of siRNAs. MTT assay was performed to access the cell growth rates. Afterwards, RNA and protein of these cells were extracted to analyze the transfection efficient as well as the expression of other molecules. Results: Compared to the normal cell line, TUG1 exhibited a significant upregulation in osteosarcoma cells. Phenotyping analysis showed the growth-promotion activity of TUG1, since knockdown of TUG1 resulted in declined proliferation. We also found that AKT phosphorylation was impaired after TUG1 was inhibited, suggesting that the AKT pathway was involved in the regulation of TUG1 in U2OS cells. Conclusion: Our data provided evidence that TUG1 was upregulated and acted as a possible oncogene via positively regulating cell proliferation in osteosarcoma cells.

  14. Quantum dot-induced cell death involves Fas upregulation and lipid peroxidation in human neuroblastoma cells

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    Lovrić Jasmina

    2007-02-01

    Full Text Available Abstract Background Neuroblastoma, a frequently occurring solid tumour in children, remains a therapeutic challenge as existing imaging tools are inadequate for proper and accurate diagnosis, resulting in treatment failures. Nanoparticles have recently been introduced to the field of cancer research and promise remarkable improvements in diagnostics, targeting and drug delivery. Among these nanoparticles, quantum dots (QDs are highly appealing due to their manipulatable surfaces, yielding multifunctional QDs applicable in different biological models. The biocompatibility of these QDs, however, remains questionable. Results We show here that QD surface modifications with N-acetylcysteine (NAC alter QD physical and biological properties. In human neuroblastoma (SH-SY5Y cells, NAC modified QDs were internalized to a lesser extent and were less cytotoxic than unmodified QDs. Cytotoxicity was correlated with Fas upregulation on the surface of treated cells. Alongside the increased expression of Fas, QD treated cells had increased membrane lipid peroxidation, as measured by the fluorescent BODIPY-C11 dye. Moreover, peroxidized lipids were detected at the mitochondrial level, contributing to the impairment of mitochondrial functions as shown by the MTT reduction assay and imaged with confocal microscopy using the fluorescent JC-1 dye. Conclusion QD core and surface compositions, as well as QD stability, all influence nanoparticle internalization and the consequent cytotoxicity. Cadmium telluride QD-induced toxicity involves the upregulation of the Fas receptor and lipid peroxidation, leading to impaired neuroblastoma cell functions. Further improvements of nanoparticles and our understanding of the underlying mechanisms of QD-toxicity are critical for the development of new nanotherapeutics or diagnostics in nano-oncology.

  15. Astrocytes Upregulate Survival Genes in Tumor Cells and Induce Protection from Chemotherapy

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    Sun-Jin Kim

    2011-03-01

    Full Text Available In the United States, more than 40% of cancer patients develop brain metastasis. The median survival for untreated patients is 1 to 2 months, which may be extended to 6 months with conventional radiotherapy and chemotherapy. The growth and survival of metastasis depend on the interaction of tumor cells with host factors in the organ microenvironment. Brain metastases are surrounded and infiltrated by activated astrocytes and are highly resistant to chemotherapy. We report here that coculture of human breast cancer cells or lung cancer cells with murine astrocytes (but not murine fibroblasts led to the up-regulation of survival genes, including GSTA5, BCL2L1, and TWIST1, in the tumor cells. The degree of up-regulation directly correlated with increased resistance to all tested chemotherapeutic agents. We further show that the up-regulation of the survival genes and consequent resistance are dependent on the direct contact between the astrocytes and tumor cells through gap junctions and are therefore transient. Knocking down these genes with specific small interfering RNA rendered the tumor cells sensitive to chemotherapeutic agents. These data clearly demonstrate that host cells in the microenvironment influence the biologic behavior of tumor cells and reinforce the contention that the organ microenvironment must be taken into consideration during the design of therapy.

  16. Fenofibrate suppressed proliferation and migration of human neuroblastoma cells via oxidative stress dependent of TXNIP upregulation

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    Su, Cunjin; Shi, Aiming; Cao, Guowen [Department of Pharmacy, The Second Affiliated Hospital of Soochow University, Suzhou, 215004 (China); Tao, Tao [Department of Urology, Zhongda Hospital, Medical School of Southeast University, Nanjing, 210009 (China); Chen, Ruidong [Department of Gastroenterology, The Second Affiliated Hospital of Soochow University, Suzhou, 215004 (China); Hu, Zhanhong; Shen, Zhu; Tao, Hong; Cao, Bin [Department of Pharmacy, The Second Affiliated Hospital of Soochow University, Suzhou, 215004 (China); Hu, Duanmin, E-mail: hudmsdfey@sina.com [Department of Gastroenterology, The Second Affiliated Hospital of Soochow University, Suzhou, 215004 (China); Bao, Junjie, E-mail: baojjsdfey@sina.com [Department of Pharmacy, The Second Affiliated Hospital of Soochow University, Suzhou, 215004 (China)

    2015-05-15

    There are no appropriate drugs for metastatic neuroblastoma (NB), which is the most common extra-cranial solid tumor for childhood. Thioredoxin binding protein (TXNIP), the endogenous inhibitor of ROS elimination, has been identified as a tumor suppressor in various solid tumors. It reported that fenofibrate exerts anti-tumor effects in several human cancer cell lines. However, its detail mechanisms remain unclear. The present study assessed the effects of fenofibrate on NB cells and investigated TXNIP role in its anti-tumor mechanisms. We used MTT assay to detect cells proliferation, starch wound test to investigate cells migration, H{sub 2}DCF-DA to detect intracellular ROS, siRNA to interfere TXNIP and peroxisome proliferator-androgen receptor-alpha (PPAR-α) expression, western blot to determine protein levels, flow cytometry to analyze apoptosis. Fenofibrate suppressed proliferation and migration of NB cells, remarkably increased intracellular ROS, upregulated TXNIP expression, promoted cell apoptosis. Furthermore, inhibition of TXNIP expression attenuated anti-tumor effects of fenofibrate, while inhibition of PPAR-α had no influences. Our results indicated the anti-tumor role of fenofibrate on NB cells by exacerbating oxidative stress and inducing apoptosis was dependent on the upregulation of TXNIP. - Highlights: • We found that fenofibrate suppressed proliferation and migration of NB cells. • We found that fenofibrate remarkably increased intracellular ROS, upregulated TXNIP expression, and promoted cell apoptosis. • Inhibition of TXNIP expression attenuated anti-tumor effects of fenofibrate, while inhibition of PPAR-α had no influences. • Our results indicated the anti-tumor role of fenofibrate on NB cells was dependent on the upregulation of TXNIP.

  17. Reciprocal upregulation of Notch signaling molecules in hematopoietic progenitor and mesenchymal stromal cells

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    Kikuchi Y

    2011-01-01

    Full Text Available Although mesenchymal stem cells (MSCs play pivotal supportive roles in hematopoiesis, how they interact with hematopoietic stem cells (HSCs is not well understood. We investigated the interaction between HSCs and surrogate MSCs (C3H10T1/2 stromal cells, focusing on the molecular events induced by cell contact of these bipartite populations. C3H10T1/2 is a mesenchymal stromal cell line that can be induced to differentiate into preadipocytes (A54 and myoblasts (M1601. The stromal cell derivatives were cocultured with murine HSCs (Lineage-Sca1+, and gene expression profiles in stromal cells and HSCs were compared before and after the coculture. HSCs gave rise to cobblestone areas only on A54 cells, with ninefold more progenitors than on M1601 or undifferentiated C3H10T1/2 cells. Microarray-based screening and a quantitative reverse transcriptase directed-polymerase chain reaction showed that the levels of Notch ligands (Jagged1 and Delta-like 3 were increased in A54 cells upon interaction with HSCs. On the other hand, the expression of Notch1 and Hes1 was upregulated in the HSCs cocultured with A54 cells. A transwell assay revealed that the reciprocal upregulation was dependent on cell-to-cell contact. The result suggested that in the hematopoietic niche, HSCs help MSCs to produce Notch ligands, and in turn, MSCs help HSCs to express Notch receptor. Such a reciprocal upregulation would reinforce the downstream signaling to determine the fate of hematopoietic cell lineage. Clarification of the initiating events on cell contact should lead to the identification of specific molecular targets to facilitate HSC engraftment in transplantation therapy.

  18. Up-regulation of cholesterol associated genes as novel resistance mechanism in glioblastoma cells in response to archazolid B

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    Hamm, Rebecca; Zeino, Maen [Institute of Pharmacy and Biochemistry, Department of Pharmaceutical Biology, Johannes Gutenberg University, Staudinger Weg 5, 55128 Mainz (Germany); Frewert, Simon [Helmholtz Institute for Pharmaceutical Research Saarland, Helmholtz Centre for Infection Research and Department of Pharmaceutical Biotechnology, Saarland University, Saarbrücken (Germany); Efferth, Thomas, E-mail: efferth@uni-mainz.de [Institute of Pharmacy and Biochemistry, Department of Pharmaceutical Biology, Johannes Gutenberg University, Staudinger Weg 5, 55128 Mainz (Germany)

    2014-11-15

    Treatment of glioblastoma multiforme (GBM), the most common and aggressive lethal brain tumor, represents a great challenge. Despite decades of research, the survival prognosis of GBM patients is unfavorable and more effective therapeutics are sorely required. Archazolid B, a potent vacuolar H{sup +}-ATPase inhibitor influencing cellular pH values, is a promising new compound exerting cytotoxicity in the nanomolar range on wild-type U87MG glioblastoma cells and U87MG.∆EGFR cells transfected with a mutant epidermal growth factor receptor (EGFR) gene. Gene expression profiling using microarray technology showed that archazolid B caused drastic disturbances in cholesterol homeostasis. Cholesterol, a main component of cellular membranes, is known to be essential for GBM growth and cells bearing EGFRvIII mutation are highly dependent on exogenous cholesterol. Archazolid B caused excessive accumulation of free cholesterol within intracellular compartments thus depleting cellular cholesterol and leading to up-regulation of SREBP targeted genes, including LDLR and HMGCR, the key enzyme of cholesterol biosynthesis. This cholesterol response is considered to be a novel resistance mechanism induced by archazolid B. We surmise that re-elevation of cholesterol levels in archazolid B treated cells may be mediated by newly synthesized cholesterol, since the drug leads to endosomal/lysosomal malfunction and cholesterol accumulation.

  19. ROS-dependent HMGA2 upregulation mediates Cd-induced proliferation in MRC-5 cells.

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    Xie, Huaying; Wang, Jiayue; Jiang, Liping; Geng, Chengyan; Li, Qiujuan; Mei, Dan; Zhao, Lian; Cao, Jun

    2016-08-01

    Cadmium (Cd) is a heavy metal widely found in a number of environmental matrices, and the exposure to Cd is increasing nowadays. In this study, the role of high mobility group A2 (HMGA2) in Cd-induced proliferation was investigated in MRC-5 cells. Exposure to Cd (2μM) for 48h significantly enhanced the growth of MRC-5 cells, increased reactive oxygen species (ROS) production, and induced both mRNA and protein expression of HMGA2. Evidence for Cd-induced reduction of the number of G0/G1 phase cells and an increase in the number of cells in S phase and G2/M phase was sought by flow cytometric analysis. Western blot analysis showed that cyclin D1, cyclin B1, and cyclin E were upregulated in Cd-treated cells. Further study revealed that N-acetyl cysteine (NAC) markedly prevented Cd-induced proliferation of MRC-5 cells, ROS generation, and the increasing protein level of HMGA2. Silencing of HMGA2 gene by siRNA blocked Cd-induced cyclin D1, cyclin B1, and cyclin E expression and reduction of the number of G0/G1 phase cells. Combining, our data showed that Cd-induced ROS formation provoked HMGA2 upregulation, caused cell cycle changes, and led to cell proliferation. This suggests that HMGA2 might be an important biomarker in Cd-induced cell proliferation.

  20. PI3K-delta mediates double-stranded RNA-induced upregulation of B7-H1 in BEAS-2B airway epithelial cells

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    Kan-o, Keiko [Research Institute for Diseases of the Chest, Graduate School of Medical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582 (Japan); Matsumoto, Koichiro, E-mail: koichi@kokyu.med.kyushu-u.ac.jp [Research Institute for Diseases of the Chest, Graduate School of Medical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582 (Japan); Asai-Tajiri, Yukari; Fukuyama, Satoru; Hamano, Saaka; Seki, Nanae; Nakanishi, Yoichi [Research Institute for Diseases of the Chest, Graduate School of Medical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582 (Japan); Inoue, Hiromasa [Research Institute for Diseases of the Chest, Graduate School of Medical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582 (Japan); Department of Pulmonary Medicine, Graduate School of Medical and Dental Sciences, Kagoshima University, 8-35-1 Sakuragaoka, Kagoshima 890-8520 (Japan)

    2013-05-31

    Highlights: •Double-stranded RNA upregulates B7-H1 on BEAS-2B airway epithelial cells. •The upregulation of B7-H1 is attenuated by inhibition of PI3Kδ isoform. •PI3Kδ-mediated upregulation of B7-H1 is independent of NF-κB activation. •Inhibition of PI3Kδ may prevent persistent viral infection induced by B7-H1. -- Abstract: Airway viral infection disturbs the health-related quality of life. B7-H1 (also known as PD-L1) is a coinhibitory molecule associated with the escape of viruses from the mucosal immunity, leading to persistent infection. Most respiratory viruses generate double-stranded (ds) RNA during replication. The stimulation of cultured airway epithelial cells with an analog of viral dsRNA, polyinosinic-polycytidylic acid (poly IC) upregulates the expression of B7-H1 via activation of the nuclear factor κB(NF-κB). The mechanism of upregulation was investigated in association with phosphatidylinositol 3-kinases (PI3Ks). Poly IC-induced upregulation of B7-H1 was profoundly suppressed by a pan-PI3K inhibitor and partially by an inhibitor or a small interfering (si)RNA for PI3Kδ in BEAS-2B cells. Similar results were observed in the respiratory syncytial virus-infected cells. The expression of p110δ was detected by Western blot and suppressed by pretreatment with PI3Kδ siRNA. The activation of PI3Kδ is typically induced by oxidative stress. The generation of reactive oxygen species was increased by poly IC. Poly IC-induced upregulation of B7-H1 was attenuated by N-acetyl-L-cysteine, an antioxidant, or by oxypurinol, an inhibitor of xanthine oxidase. Poly IC-induced activation of NF-κB was suppressed by a pan-PI3K inhibitor but not by a PI3Kδ inhibitor. These results suggest that PI3Kδ mediates dsRNA-induced upregulation of B7-H1 without affecting the activation of NF-κB.

  1. Overexpression of Ref-1 Inhibits Lead-induced Endothelial Cell Death via the Upregulation of Catalase.

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    Lee, Kwon Ho; Lee, Sang Ki; Kim, Hyo Shin; Cho, Eun Jung; Joo, Hee Kyoung; Lee, Eun Ji; Lee, Ji Young; Park, Myoung Soo; Chang, Seok Jong; Cho, Chung-Hyun; Park, Jin Bong; Jeon, Byeong Hwa

    2009-12-01

    The role of apurinic/apyrimidinic endonuclease1/redox factor-1 (Ref-1) on the lead (Pb)-induced cellular response was investigated in the cultured endothelial cells. Pb caused progressive cellular death in endothelial cells, which occurred in a concentration- and time-dependent manner. However, Ref-1 overexpression with AdRef-1 significantly inhibited Pb-induced cell death in the endothelial cells. Also the overexpression of Ref-1 significantly suppressed Pb-induced superoxide and hydrogen peroxide elevation in the endothelial cells. Pb exposure induced the downregulation of catalase, it was inhibited by the Ref-1 overexpression in the endothelial cells. Taken together, our data suggests that the overexpression of Ref-1 inhibited Pb-induced cell death via the upregulation of catalase in the cultured endothelial cells.

  2. Causes of upregulation of glycolysis in lymphocytes upon stimulation. A comparison with other cell types.

    Science.gov (United States)

    Stark, Heiko; Fichtner, Maximilian; König, Rainer; Lorkowski, Stefan; Schuster, Stefan

    2015-11-01

    In this review, we revisit the metabolic shift from respiration to glycolysis in lymphocytes upon activation, which is known as the Warburg effect in tumour cells. We compare the situation in lymphocytes with those in several other cell types, such as muscle cells, Kupffer cells, microglia cells, astrocytes, stem cells, tumour cells and various unicellular organisms (e.g. yeasts). We critically discuss and compare several explanations put forward in the literature for the observation that proliferating cells adopt this apparently less efficient pathway: hypoxia, poisoning of competitors by end products, higher ATP production rate, higher precursor supply, regulatory effects, and avoiding harmful effects (e.g. by reactive oxygen species). We conclude that in the case of lymphocytes, increased ATP production rate and precursor supply are the main advantages of upregulating glycolysis.

  3. Leptin upregulates beta3-integrin expression and interleukin-1beta, upregulates leptin and leptin receptor expression in human endometrial epithelial cell cultures.

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    Gonzalez, R R; Leavis, P

    2001-10-01

    Human endometrium and endometrial epithelial cells (EECs) either cultured alone or cocultured with human embryos express leptin and leptin receptor. This study compares the effect of leptin with that of interleukin-1beta (IL-1beta) on the expression of beta3-EEC integrin, a marker of endometrial receptivity. Both cytokines increased the expression of beta3-EEC at concentrations in the range of 0.06-3 nM; however, leptin exhibited a significantly greater effect than IL-1beta. We also determined the regulatory effects of IL-1beta on leptin secretion and on the expression of leptin and leptin receptor at the protein level in both EEC and endometrial stromal cell (ESC) cultures. In EEC cultures, IL-1beta upregulated secretion of leptin and expression of both leptin and leptin receptors. No effect of IL-1beta was found in the ESC cultures. However, leptin exhibited marginal upregulation of leptin receptor. The upregulation of beta3-integrin and leptin/leptin receptor expression by IL-1beta in EEC cultures indicates that both cytokines may be implicated in embryonic-maternal cross-talk during the early phase of human implantation. Our present data also raise the possibility that leptin is an endometrial molecular effector of IL-1beta action on beta3-integrin upregulation. Thus, a new role for leptin in human reproduction as an autocrine/paracrine regulator of endometrial receptivity is proposed.

  4. Microtubule stability and MAPI B upregulation control neuritogenesis in CAD cells

    Institute of Scientific and Technical Information of China (English)

    Wen LI; Jin-tang XIA; Yue FENG

    2006-01-01

    Aim: To study the role of microtubule dynamics and microtubule associated protein 1B (MAP1B) in regulation of the neurite extension in CAD catecholaminergic neuronal cell line. Methods: The neuritogenesis of the CAD cells was abolished by inhibiting microtubule polymerization with nocodazole and by blocking microtubule depolymerization with taxol. MAP1B and tubulin protein expression was detected by Western blot. Immunofluorescent staining of tubulins was observed by fluorescent and confocal microscopy. Results: Microtubule dynamics was essential for CAD neurite extension. Dosage analysis revealed that neurite extension was much more sensitive to nocodazole than to taxol, suggesting a functional requirement for highly active microtubule assembly. A remarkable upregulation of MAP1B protein was detected during neurite extension accompanied with increased microtubule stability. Conclusion: Upregulation of MAP1B leads to the stabilization of newly formed microtubules in the developing neurites, which in turn promotes neurite extension.

  5. MDP Up-Regulates the Gene Expression of Type I Interferons in Human Aortic Endothelial Cells

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    Xiumei Xie

    2012-03-01

    Full Text Available Muramyldipeptide (MDP, the minimum essential structure responsible for the immuno-adjuvant activity of peptidoglycan, is recognized by intracellular nuclear-binding oligomerization domain 2 (NOD2. Here, we obtained evidence that the treatment of human aortic endothelial cells (HAECs with MDP up-regulated the gene expression of type I interferons in a dose- and time-dependent manner. MDP also up-regulated the expression of the receptor NOD2, suggesting that MDP may induce a positive feedback response. The up-regulation of interferons was not dependent on the TNFa signaling, as HAECs did not express TNFa with the stimulation of MDP, and TNFa neutralizing antibody did not decrease the induction of IFNs induced by MDP. RT-PCR results showed that HAECs expressed the gene transcripts of interferon regulatory factor (IRF 1, 2, 3, 9. The western blot results showed that MDP induced the phosphorylation of IRF3. These results suggested that MDP induced the up-regulation of gene transcript of interferons through the activation of IRF3 signaling pathway. Meanwhile, MDP induced the gene expression of pro-inflammatory cytokines, including IL-1ß, IL-8, and MCP-1. Taken together, these results suggested that HAECs may play roles in the anti-infection immune response and in the induction of innate immunity.

  6. MDP up-regulates the gene expression of type I interferons in human aortic endothelial cells.

    Science.gov (United States)

    Lv, Qingshan; Yang, Mei; Liu, Xueting; Zhou, Lina; Xiao, Zhilin; Chen, Xiaobin; Chen, Meifang; Xie, Xiumei; Hu, Jinyue

    2012-03-23

    Muramyldipeptide (MDP), the minimum essential structure responsible for the immuno-adjuvant activity of peptidoglycan, is recognized by intracellular nuclear-binding oligomerization domain 2 (NOD2). Here, we obtained evidence that the treatment of human aortic endothelial cells (HAECs) with MDP up-regulated the gene expression of type I interferons in a dose- and time-dependent manner. MDP also up-regulated the expression of the receptor NOD2, suggesting that MDP may induce a positive feedback response. The up-regulation of interferons was not dependent on the TNFa signaling, as HAECs did not express TNFa with the stimulation of MDP, and TNFa neutralizing antibody did not decrease the induction of IFNs induced by MDP. RT-PCR results showed that HAECs expressed the gene transcripts of interferon regulatory factor (IRF) 1, 2, 3, 9. The western blot results showed that MDP induced the phosphorylation of IRF3. These results suggested that MDP induced the up-regulation of gene transcript of interferons through the activation of IRF3 signaling pathway. Meanwhile, MDP induced the gene expression of pro-inflammatory cytokines, including IL-1ß, IL-8, and MCP-1. Taken together, these results suggested that HAECs may play roles in the anti-infection immune response and in the induction of innate immunity.

  7. BLT2 up-regulates interleukin-8 production and promotes the invasiveness of breast cancer cells.

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    Hyunju Kim

    Full Text Available BACKGROUND: The elevated production of interleukin (IL-8 is critically associated with invasiveness and metastatic potential in breast cancer cells. However, the intracellular signaling pathway responsible for up-regulation of IL-8 production in breast cancer cells has remained unclear. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we report that the expression of BLT2 is markedly up-regulated in the highly aggressive human breast cancer cell lines MDA-MB-231 and MDA-MB-435 compared with MCF-10A immortalized human mammary epithelial cells, as determined by RT-PCR, real-time PCR and FACS analysis. Blockade of BLT2 with BLT2 siRNA knockdown or BLT2 inhibitor treatment downregulated IL-8 production and thereby diminished the invasiveness of aggressive breast cancer cells, analyzed by Matrigel invasion chamber assays. We further characterized the downstream signaling mechanism by which BLT2 stimulates IL-8 production and identified critical mediatory roles for the generation of reactive oxygen species (ROS and the consequent activation of the transcription factor NF-κB. Moreover, blockade of BLT2 suppressed the formation of metastatic lung nodules by MDA-MB-231 cells in both experimental and orthotopic metastasis models. CONCLUSIONS/SIGNIFICANCE: Taken together, our study demonstrates that a BLT2-ROS-NF-κB pathway up-regulates IL-8 production in MDA-MB-231 and MDA-MB-435 cells, thereby contributing to the invasiveness of these aggressive breast cancer cells. Our findings provide insight into the molecular mechanism of invasiveness in breast cancer.

  8. Neural cell 3D microtissue formation is marked by cytokines' up-regulation.

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    Yinzhi Lai

    Full Text Available Cells cultured in three dimensional (3D scaffolds as opposed to traditional two-dimensional (2D substrates have been considered more physiologically relevant based on their superior ability to emulate the in vivo environment. Combined with stem cell technology, 3D cell cultures can provide a promising alternative for use in cell-based assays or biosensors in non-clinical drug discovery studies. To advance 3D culture technology, a case has been made for identifying and validating three-dimensionality biomarkers. With this goal in mind, we conducted a transcriptomic expression comparison among neural progenitor cells cultured on 2D substrates, 3D porous polystyrene scaffolds, and as 3D neurospheres (in vivo surrogate. Up-regulation of cytokines as a group in 3D and neurospheres was observed. A group of 13 cytokines were commonly up-regulated in cells cultured in polystyrene scaffolds and neurospheres, suggesting potential for any or a combination from this list to serve as three-dimensionality biomarkers. These results are supportive of further cytokine identification and validation studies with cells from non-neural tissue.

  9. Upregulated expression of Ezrin and invasive phenotype in malignantly transformed esophageal epithelial cells

    Institute of Scientific and Technical Information of China (English)

    Zhong-Ying Shen; Li-Yan Xu; Ming-Hua Chen; En-Min Li; Jin-Tao Li; Xian-Ying Wu; Yi Zeng

    2003-01-01

    AIM: To investigate the correlation between ezrin expression and invasive phenotype formation in malignantly transformed esophageal epithelial cells. METHODS: The experimental cell line employed in the present study was originated form the progressive induction of a human embryonic esophageal epithelial cell line (SHEE)by the E6E7 genes of human papillomavirus (HPV) type 18.The cells at the 35th passage after induction called SHEEIMM were in a state of immortalized phase and used as the control,while that of the 85th passage denominated as SHEEMT represented the status of cells that were malignantly transformed. The expression changes of ezrin and its mRNA in both cell passages were respectively analyzed by RT-PCR and Western blot. Invasive phenotype was assessed in vivo by inoculating these cells into the severe combined immunodeficient (SCID) mice via subcutaneous and intraperitoneal injection, and in vitro by inoculating them on the surface of the amnion membranes, which then was determined by light microscopy and scanning electron microscopy. RESULTS: Upregulated expression of ezrin protein and its mRNA was observed in SHEEMT compared with that in SHEEIMM cells. The SHEEMT cells inoculated in SCID mice were observed forming tumor masses in both visceral organs and soft tissues in a period of 40 days with a special propensity to invading mesentery and pancreas, but did not exhibit hepatic metastases. Pathologically, these tumor cells harboring larger nucleus, nucleolus and less cytoplasm could infiltrate and destroy adjacent tissues. In the in vitro study,the inoculated SHEEMT cells could grow in cluster on the amniotic epithelial surface and intrude into the amniotic stroma. In contrast, unrestricted growth and invasiveness were not found in SHEEIMM cells in both in vivo and in vitroexperiment. CONCLUSION: The upregulated ezrin expression is one of the important factors that are possibly associated with the invasive phenotype formation in malignantly

  10. Hematopoietic transcription factor GATA-2 promotes upregulation of alpha globin and cell death in FL5.12 cells.

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    Brecht, K; Simonen, M; Kamke, M; Heim, J

    2005-10-01

    Recently we showed that alpha globin is a novel pro-apoptotic factor in programmed cell death in the pro-B cell line, FL5.12. Alpha globin was also upregulated in various other cell lines after different apoptotic stimuli. Under withdrawal of IL-3, overexpression of alpha globin accelerated apoptosis in FL5.12. Here, we have studied how transcription of alpha globin is placed in the broader context of apoptosis. We used Affymetrix chip technology and RT QPCR to compare expression patterns of FL5.12 cells growing with or without IL-3 to search for transcription factors which were concomitantly upregulated with alpha globin. The erythroid-specific transcription factor GATA-2 was the earliest and most prominently upregulated candidate. GATA-1 was expressed at low levels and was weakly induced while GATA-3 was completely absent. To evaluate the influence of GATA-2 on alpha globin expression and cell viability we overexpressed GATA-2 in FL5.12 cells. Interestingly, high expression of GATA-2 resulted in cell death and elevated alpha globin levels in FL5.12 cells. Transduction of antisense GATA-2 prevented both increase of GATA-2 and alpha globin under apoptotic conditions and delayed cell death. We suggest a role of GATA-2 in apoptosis besides its function in maintenance and proliferation of immature hematopoietic progenitors.

  11. WISP-1 overexpression upregulates cell proliferation in human salivary gland carcinomas via regulating MMP-2 expression

    Science.gov (United States)

    Li, Fu-Jun; Wang, Xin-Juan; Zhou, Xiao-Li

    2016-01-01

    Background WISP-1 is a member of the CCN family of growth factors and has been reported to play an important role in tumorigenesis by triggering downstream events via integrin signaling. However, little is known about the role of WISP-1 in proliferation of salivary gland carcinoma (SGC) cells. Methods In this study, we investigated the WISP-1 expression in SGC tissues via immunohistochemical staining, Western blotting assay, and real-time quantitative polymerase chain reaction method, and then evaluated the regulatory role of WISP-1 in the growth of SGC A-253 cells. In addition, the role of MMP-2 in the WISP-1-mediated growth regulation was also investigated. Results It was demonstrated that the WISP-1 expression was upregulated at both mRNA and protein levels in 15 of 21 SGC tumor tissues, compared to the non-tumor tissues (five of 21), associated with the lymph node dissection and bone invasion. The in vitro CCK-8 assay and colony-forming assay demonstrated that the exogenous WISP-1 treatment or the WISP-1 overexpression promoted the growth of A-253 cells. In addition, we confirmed that the WISP-1 overexpression upregulated the MMP-2 expression in A-253 cells with the gain-of-function and loss-of-function strategies, and that the MMP-2 knockdown attenuated the WISP-1-mediated growth promotion of A-253 cells. Conclusion We found that WISP-1 was overexpressed in the human SGCs, and the WISP-1 overexpression promoted the salivary gland cell proliferation via upregulating MMP-2 expression. Our study recognized the oncogenic role of WISP-1 in human SGCs, which could serve as a potential target for anticancer therapy. PMID:27799801

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

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    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.

  13. Moclobemide up-regulates proliferation of hippocampal progenitor cells in chronically stressed mice

    Institute of Scientific and Technical Information of China (English)

    Yun-fengLI; You-zhiZHANG; Yan-qinLIU; Heng-linWANG; LiYUAN; Zhi-puLUO

    2004-01-01

    AIM: To explore the action mechanism of antidepressants. METHODS: The PC 12 cell proliferation was detected by flow cytometry,. The proliferation of hippocampal progenitor cells and level of brain-derived neurotrophic factor (BDNF) were measured by immunohistochemistry. RESULTS: Treatment with N-methylaspartate (NMDA)600 μmol/L for 3 d significantly decreased the percentage of S-phase in PC12 cells, while in the presence of classical antidepressant, moclobemide (MOC) 2 and 10 μnol/L, the percentage in S-phase increased. Furthermore,the proliferation of progenitor cells in hippocampal dentate gyrus (subgranular zone), as well as the level of BDNF in hippocampus significantly decreased in chronically stressed mice, while chronic administration with MOC 40 mg/kg (ip) up-regulated the progenitor cell proliferation and BDNF level in the same time course. CONLUSION:Up-regulation of the proliferation of hippocampal progenitor cells is one of the action mechanisms for MOC, which may be closely related to the elevation of BDNF level at the same time. These results also extend evidence for our hypothesis that up-regulation of the hippocampal neurogenesis is one of the common mechanisms for antidepressants.

  14. Moclobemide up-regulates proliferation of hippocampal progenitor cells in chronically stressed mice

    Institute of Scientific and Technical Information of China (English)

    Yun-feng LI; You-zhi ZHANG; Yan-qin LIU; Heng-lin WANG; Li YUAN; Zhi-pu LUO

    2004-01-01

    AIM: To explore the action mechanism of antidepressants. METHODS: The PC12 cell proliferation was detected by flow cytometry,. The proliferation of hippocampal progenitor cells and level of brain-derived neurotrophic factor (BDNF) were measured by immunohistochemistry. RESULTS: Treatment with N-methylaspartate (NMDA)600 μmol/L for 3 d significantly decreased the percentage of S-phase in PC12 cells, while in the presence of classical antidepressant, moclobemide (MOC) 2 and 10 μmol/L, the percentage in S-phase increased. Furthermore,the proliferation of progenitor cells in hippocampal dentate gyrus (subgranular zone), as well as the level of BDNF in hippocampus significantly decreased in chronically stressed mice, while chronic administration with MOC 40mg/kg (ip) up-regulated the progenitor cell proliferation and BDNF level in the same time course. CONLUSION:Up-regulation of the proliferation of hippocampal progenitor cells is one of the action mechanisms for MOC, which may be closely related to the elevation of BDNF level at the same time. These results also extend evidence for our hypothesis that up-regulation of the hippocampal neurogenesis is one of the common mechanisms for antidepressants.

  15. Dux4 induces cell cycle arrest at G1 phase through upregulation of p21 expression

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    Xu, Hongliang; Wang, Zhaoxia; Jin, Suqin; Hao, Hongjun [Department of Neurology, Peking University First Hospital, Beijing 100034 (China); Zheng, Lemin [The Institute of Cardiovascular Sciences, Peking University Health Science Center, Key Laboratory of Molecular Cardiovascular Sciences of Education Ministry, Key Laboratory of Cardiovascular Molecular Biology and Regulatory Peptides of Health Ministry, Beijing 100191 (China); Zhou, Boda [The Department of Cardiology, Peking University Third Hospital, Beijing 100191 (China); Zhang, Wei; Lv, He [Department of Neurology, Peking University First Hospital, Beijing 100034 (China); Yuan, Yun, E-mail: yuanyun2002@sohu.com [Department of Neurology, Peking University First Hospital, Beijing 100034 (China)

    2014-03-28

    Highlights: • Dux4 induced TE671 cell proliferation defect and G1 phase arrest. • Dux4 upregulated p21 expression without activating p53. • Silencing p21 rescued Dux4 mediated proliferation defect and cell cycle arrest. • Sp1 binding site was required for Dux4-induced p21 promoter activation. - Abstract: It has been implicated that Dux4 plays crucial roles in development of facioscapulohumeral dystrophy. But the underlying myopathic mechanisms and related down-stream events of this retrogene were far from clear. Here, we reported that overexpression of Dux4 in a cell model TE671 reduced cell proliferation rate, and increased G1 phase accumulation. We also determined the impact of Dux4 on p53/p21 signal pathway, which controls the checkpoint in cell cycle progression. Overexpression of Dux4 increased p21 mRNA and protein level, while expression of p53, phospho-p53 remained unchanged. Silencing p21 rescued Dux4 mediated proliferation defect and cell cycle arrest. Furthermore, we demonstrated that enhanced Dux4 expression increased p21 promoter activity and elevated expression of Sp1 transcription factor. Mutation of Sp1 binding site decreased dux4 induced p21 promoter activation. Chromatin immunoprecipitation (ChIP) assays confirmed the Dux4-induced binding of Sp1 to p21 promoter in vivo. These results suggest that Dux4 might induce proliferation inhibition and G1 phase arrest through upregulation of p21.

  16. Oligomerization of Vibrio cholerae Hemolysin Induces CXCR3 Upregulation and Activation of B-1a Cell

    Institute of Scientific and Technical Information of China (English)

    Gayatri Mukherjee; Kalyan K Banerjee; Tapas Biswas

    2008-01-01

    The hemolysin oligomer promotes the proliferation of B-1a cells and the expression of CD25, which is indicative of cell activation, on B-1a cells. The upregulation of CD86 induced by the oligomer showed its selective bias for the B7-2 member of B7 family while the monomer failed to induce these effects. The oligomer induced the expression of CXCR3, associated with B cell activation, while the monomer induced the expression of CXCL4, a powerful angiostatic chemokine. In conclusion, we found that B-1a cells responded to the apoptogenic monomer by expressing CXCL4, whereas oligomerization of the immunogen induced CXCR3 to shift the response towards activation. Cellular & Molecular Immunology. 2008;5(3):231-234.

  17. Hypoxia increases the metastatic ability of breast cancer cells via upregulation of CXCR4

    LENUS (Irish Health Repository)

    Cronin, Patricia A

    2010-05-21

    Abstract Background Chemokine SDF1α and its unique receptor CXCR4 have been implicated in organ-specific metastases of many cancers including breast cancer. Hypoxia is a common feature of solid tumors and is associated with their malignant phenotype. We hypothesized that hypoxia would upregulate CXCR4 expression and lead to increased chemotactic responsiveness to its specific ligand SDF1α. Methods Three breast cancer cell lines MDA-MB-231, MCF7 and 4T1 were subjected to 48 hrs of hypoxia or normoxia. Cell surface receptor expression was evaluated using flow cytometry. An extracellular matrix invasion assay and microporous migration assay was used to assess chemotactic response and metastatic ability. Results CXCR4 surface expression was significantly increased in the two human breast cancer cell lines, MDA-MB-231 and MCF7, following exposure to hypoxia. This upregulation of CXCR4 cell surface expression corresponded to a significant increase in migration and invasion in response to SDF1-α in vitro. The increase in metastatic potential of both the normoxic and the hypoxic treated breast cancer cell lines was attenuated by neutralization of CXCR4 with a CXCR4 neutralizing mAb, MAB172 or a CXCR4 antagonist, AMD3100, showing the relationship between CXCR4 overexpression and increased chemotactic responsiveness. Conclusions CXCR4 expression can be modulated by the tissue microenvironment such as hypoxia. Upregulation of CXCR4 is associated with increased migratory and invasive potential and this effect can be abrogated by CXCR4 inhibition. Chemokine receptor CXCR4 is a potential therapeutic target in the adjuvant treatment of breast cancer.

  18. PPARγ inhibits ovarian cancer cells proliferation through upregulation of miR-125b

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    Luo, Shuang, E-mail: luoshuangsch@163.com [Department of Obstetrics and Gynecology, Suining Central Hospital, Suining (China); Wang, Jidong [Department of Gynecology and Obsterics, Jinan Central Hospital, Jinan (China); Ma, Ying [Department of Otorhinolaryngolgy, Suining Central Hospital, Suining (China); Yao, Zhenwei [Department of Gynecology and Obstetrics, The First Affiliated Hospital of Chongqing Medical University, Chongqing (China); Pan, Hongjuan [Department of Gynecology and Obsterics, Zhongshan Hospital, Wuhan (China)

    2015-06-26

    miR-125b has essential roles in coordinating tumor proliferation, angiogenesis, invasiveness, metastasis and chemotherapy recurrence. In ovarian cancer miR-125b has been shown to be downregulated and acts as a tumor suppressor by targeting proto-oncogene BCL3. PPARγ, a multiple functional transcription factor, has been reported to have anti-tumor effects through inhibition of proliferation and induction of differentiation and apoptosis by targeting the tumor related genes. However, it is unclear whether miR-125b is regulated by PPARγ in ovarian cancer. In this study, we demonstrated that the miR-125b downregulated in ovarian cancer tissues and cell lines. Ligands-activated PPARγ suppressed proliferation of ovarian cancer cells and this PPARγ-induced growth inhibition is mediated by the upregulation of miR-125b. PPARγ promoted the expression of miR-125b by directly binding to the responsive element in miR-125b gene promoter region. Thus, our results suggest that PPARγ can induce growth suppression of ovarian cancer by upregulating miR-125b which inhibition of proto-oncogene BCL3. These findings will extend our understanding of the function of PPARγ in tumorigenesis and miR-125b may be a therapeutic intervention of ovarian cancer. - Highlights: • miR-125b is down-regulated in ovarian cancer tissues and cells. • PPARγ upregulates miR-125b and downregulates its target gene BCL3 expression. • Silence of miR-125b attenuates PPARγ-mediated growth suppression of ovarian cancer cells. • PPARγ promotes the transcription of miR-125b via binding to PPARE in miR-125b gene promoter region.

  19. Estrogen-mediated upregulation of Noxa is associated with cell cycle progression in estrogen receptor-positive breast cancer cells.

    Science.gov (United States)

    Liu, Wensheng; Swetzig, Wendy M; Medisetty, Rajesh; Das, Gokul M

    2011-01-01

    Noxa is a Bcl-2-homology domain (BH3)-only protein reported to be a proapoptotic member of the Bcl-2 family. Estrogen has been well documented to stimulate cell growth and inhibit apoptosis in estrogen receptor (ER)-positive breast cancer cells. Intriguingly, recent reports have shown that 17β-estradiol (E2) induces Noxa expression, although the mechanisms underlying E2-mediated induction of Noxa and its functional significance are unknown. Using MCF7 human breast cancer cells as an experimental model, we show that Noxa is upregulated by E2 via p53-independent processes that involve c-Myc and ERα. Experiments using small interfering ribonucleic acids (siRNA) to specifically knock down p53, c-Myc, and ERα demonstrated that c-Myc and ERα, but not p53, are involved in the transcriptional upregulation of Noxa following E2 treatment. Furthermore, while E2 promoted the recruitment of c-Myc and ERα to the NOXA promoter in chromatin immunoprecipitation (ChIP) assays, E2 did not induce p53 recruitment. Interestingly, E2-mediated upregulation of Noxa was not associated with apoptosis. However, siRNA-mediated knockdown of Noxa resulted in cell cycle arrest in G(0)/G(1)-phase and significantly delayed the G(1)-to-S-phase transition following E2 treatment, indicating that Noxa expression is required for cell cycle progression in ER-positive breast cancer cells.

  20. Estrogen-mediated upregulation of Noxa is associated with cell cycle progression in estrogen receptor-positive breast cancer cells.

    Directory of Open Access Journals (Sweden)

    Wensheng Liu

    Full Text Available Noxa is a Bcl-2-homology domain (BH3-only protein reported to be a proapoptotic member of the Bcl-2 family. Estrogen has been well documented to stimulate cell growth and inhibit apoptosis in estrogen receptor (ER-positive breast cancer cells. Intriguingly, recent reports have shown that 17β-estradiol (E2 induces Noxa expression, although the mechanisms underlying E2-mediated induction of Noxa and its functional significance are unknown. Using MCF7 human breast cancer cells as an experimental model, we show that Noxa is upregulated by E2 via p53-independent processes that involve c-Myc and ERα. Experiments using small interfering ribonucleic acids (siRNA to specifically knock down p53, c-Myc, and ERα demonstrated that c-Myc and ERα, but not p53, are involved in the transcriptional upregulation of Noxa following E2 treatment. Furthermore, while E2 promoted the recruitment of c-Myc and ERα to the NOXA promoter in chromatin immunoprecipitation (ChIP assays, E2 did not induce p53 recruitment. Interestingly, E2-mediated upregulation of Noxa was not associated with apoptosis. However, siRNA-mediated knockdown of Noxa resulted in cell cycle arrest in G(0/G(1-phase and significantly delayed the G(1-to-S-phase transition following E2 treatment, indicating that Noxa expression is required for cell cycle progression in ER-positive breast cancer cells.

  1. Upregulation of metastasis-associated gene 2 promotes cell proliferation and invasion in nasopharyngeal carcinoma

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    Wu MH

    2016-03-01

    Full Text Available Minhua Wu,1,2,* Xiaoxia Ye,2,* Xubin Deng,3,* Yanxia Wu,4 Xiaofang Li,4 Lin Zhang11Department of Histology and Embryology, Southern Medical University, Guangzhou, 2Department of Histology and Embryology, Guangdong Medical University, Zhanjiang, 3Affiliated Cancer Hospital of Guangzhou Medical University, Cancer Center of Guangzhou Medical University, Guangzhou, 4Pathological Diagnosis and Research Center, Affiliated Hospital of Guangdong Medical University, Zhanjiang, People’s Republic of China*These authors contributed equally to this workAims: Metastasis-associated gene 2 (MTA2 is reported to play an important role in tumor progression, but little is known about the role of MTA2 in nasopharyngeal carcinoma (NPC. The aim of the study was to explore the expression and function of MTA2 in NPC.Methods: Expression of MTA2 in NPC tissues and cell lines was detected by immunohistochemistry and Western blotting. Relationship between MTA2 expression and clinicopathological features was analyzed. Stable MTA2-overexpressing and MTA2-siliencing NPC cells were established by transfection with plasmids encoding MTA2 cDNA and lentivirus-mediated short hairpin RNA, respectively. Cell viability was determined by Cell Counting Kit-8 and colony formation assay. Cell migration ability was evaluated by wound healing and transwell invasion assay. The impact of MTA2 knockdown on growth and metastasis of CNE2 cells in vivo was determined by nude mouse xenograft models. Expression of several Akt pathway proteins was detected by Western blotting.Results: MTA2 was upregulated in NPC tissues and three NPC cell lines detected (CNE1, CNE2, and HNE1. MTA2 expression was related to clinical stage and lymph node metastasis of patients with NPC. MTA2 upregulation promoted proliferation and invasion of CNE1 cells, while MTA2 depletion had opposite effects on CNE2 cells. Moreover, MTA2 depletion suppressed growth and metastasis of CNE2 cells in vivo. MTA2 overexpression

  2. Neural Stem Cell Transplantation Induces Stroke Recovery by Upregulating Glutamate Transporter GLT-1 in Astrocytes

    Science.gov (United States)

    Russo, Gianluca Luigi; Peruzzotti-Jametti, Luca; Rossi, Silvia; Sandrone, Stefano; Butti, Erica; De Ceglia, Roberta; Bergamaschi, Andrea; Motta, Caterina; Gallizioli, Mattia; Studer, Valeria; Colombo, Emanuela; Farina, Cinthia; Comi, Giancarlo; Politi, Letterio Salvatore; Muzio, Luca; Villani, Claudia; Invernizzi, Roberto William; Hermann, Dirk Matthias; Centonze, Diego

    2016-01-01

    Ischemic stroke is the leading cause of disability, but effective therapies are currently widely lacking. Recovery from stroke is very much dependent on the possibility to develop treatments able to both halt the neurodegenerative process as well as to foster adaptive tissue plasticity. Here we show that ischemic mice treated with neural precursor cell (NPC) transplantation had on neurophysiological analysis, early after treatment, reduced presynaptic release of glutamate within the ipsilesional corticospinal tract (CST), and an enhanced NMDA-mediated excitatory transmission in the contralesional CST. Concurrently, NPC-treated mice displayed a reduced CST degeneration, increased axonal rewiring, and augmented dendritic arborization, resulting in long-term functional amelioration persisting up to 60 d after ischemia. The enhanced functional and structural plasticity relied on the capacity of transplanted NPCs to localize in the peri-ischemic and ischemic area, to promote the upregulation of the glial glutamate transporter 1 (GLT-1) on astrocytes and to reduce peri-ischemic extracellular glutamate. The upregulation of GLT-1 induced by transplanted NPCs was found to rely on the secretion of VEGF by NPCs. Blocking VEGF during the first week after stroke reduced GLT-1 upregulation as well as long-term behavioral recovery in NPC-treated mice. Our results show that NPC transplantation, by modulating the excitatory–inhibitory balance and stroke microenvironment, is a promising therapy to ameliorate disability, to promote tissue recovery and plasticity processes after stroke. SIGNIFICANCE STATEMENT Tissue damage and loss of function occurring after stroke can be constrained by fostering plasticity processes of the brain. Over the past years, stem cell transplantation for repair of the CNS has received increasing interest, although underlying mechanism remain elusive. We here show that neural stem/precursor cell transplantation after ischemic stroke is able to foster

  3. Up-regulated expression of extracellular matrix remodeling genes in phagocytically challenged trabecular meshwork cells.

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    Kristine M Porter

    Full Text Available BACKGROUND: Cells in the trabecular meshwork (TM, the tissue responsible for draining aqueous humor out of the eye, are known to be highly phagocytic. Phagocytic function in TM cells is thought to play an important role in the normal functioning of the outflow pathway. Dysfunction of phagocytosis could lead to abnormalities of outflow resistance and increased intraocular pressure (IOP. However, the molecular mechanisms triggered by phagocytosis in TM cells are completely unknown. METHODOLOGY/PRINCIPAL FINDINGS: Gene expression profile analysis of human TM cells phagocytically challenged to E. coli or pigment under physiological and oxidative stress environment were performed using Affymetrix U133 plus 2.0 array and analyzed with Genespring GX. Despite the differential biological response elicited by E. coli and pigment particles, a number of genes, including MMP1, MMP3, TNFSF11, DIO2, KYNU, and KCCN2 showed differential expression with both phagocytic ligands in all conditions. Data was confirmed by qPCR in both human and porcine TM cells. Metacore pathway analysis and the usage of recombinant adenovirus encoding the dominant negative mutant of IkB identified NF-κB as a transcription factor mediating the up-regulation of at least MMP1 and MMP3 in TM cells with phagocytosis. In-gel zymography demonstrated increased collagenolytic and caseinolytic activities in the culture media of TM cells challenge to E. coli. In addition, collagenolytic I activity was further confirmed using the self-quenched fluorescent substrate DQ-Collagen I. CONCLUSIONS/SIGNIFICANCE: Here we report for the first time the differential gene expression profile of TM cells phagocytically challenged with either E. coli or pigment. Our data indicate a potential role of phagocytosis in outflow pathway tissue homeostasis through the up-regulation and/or proteolytic activation of extracellular matrix remodeling genes.

  4. Silencing GFAP isoforms in astrocytoma cells disturbs laminin-dependent motility and cell adhesion.

    Science.gov (United States)

    Moeton, Martina; Kanski, Regina; Stassen, Oscar M J A; Sluijs, Jacqueline A; Geerts, Dirk; van Tijn, Paula; Wiche, Gerhard; van Strien, Miriam E; Hol, Elly M

    2014-07-01

    Glial fibrillary acidic protein (GFAP) is an intermediate filament protein expressed in astrocytes and neural stem cells. The GFAP gene is alternatively spliced, and expression of GFAP is highly regulated during development, on brain damage, and in neurodegenerative diseases. GFAPα is the canonical splice variant and is expressed in all GFAP-positive cells. In the human brain, the alternatively spliced transcript GFAPδ marks specialized astrocyte populations, such as subpial astrocytes and the neurogenic astrocytes in the human subventricular zone. We here show that shifting the GFAP isoform ratio in favor of GFAPδ in astrocytoma cells, by selectively silencing the canonical isoform GFAPα with short hairpin RNAs, induced a change in integrins, a decrease in plectin, and an increase in expression of the extracellular matrix component laminin. Together, this did not affect cell proliferation but resulted in a significantly decreased motility of astrocytoma cells. In contrast, a down-regulation of all GFAP isoforms led to less cell spreading, increased integrin expression, and a >100-fold difference in the adhesion of astrocytoma cells to laminin. In summary, isoform-specific silencing of GFAP revealed distinct roles of a specialized GFAP network in regulating the interaction of astrocytoma cells with the extracellular matrix through laminin.-Moeton, M., Kanski, R., Stassen, O. M. J. A., Sluijs, J. A., Geerts, D., van Tijn, P., Wiche, G., van Strien, M. E., Hol, E. M. Silencing GFAP isoforms in astrocytoma cells disturbs laminin dependent motility and cell adhesion.

  5. Nicotine-induced upregulation of antioxidant protein Prx 1 in oral squamous cell carcinoma

    Institute of Scientific and Technical Information of China (English)

    ZHAO YanHua; ZHANG Min; YAN Fei; CASTO Bruce C; TANG XiaoFei

    2013-01-01

    Nicotine is a source of exogenous oxidative stress,which is associated with the pathogenesis of numerous diseases including oral squamous cell carcinoma (OSCC),whereas an antioxidant protein,peroxiredoxin 1 (Prx 1),plays an important role in the modulation of this condition.This study was to investigate the association between Prx 1 and tobacco-induced oxidative stress.The expression of Prx 1 and GST π in OSCC Tca8113 cells,which were pre-treated with nicotine,was determined.In the present study,MTT assay,reactive oxygen species (ROS) assay,RT-PCR and Western blot analyses,respectively,were conducted to assess cell viability,ROS level,and expression level of Prx 1 and GST π in nicotine-treated Tca8113 cells.Nuclear factor kappa B (NF-κB) expression was detected by immuno-fluorescence.Our results showed the growth of Tca8113 cells was increased in a dose-dependent manner when cells were treated with nicotine at concentrations from 0.1 to 10 μmol/L,but the proliferation of the ceils decreased at 100 μmol/L.ROS levels increased in all groups treated with nicotine at concentrations of 0.1,1,10,or 100/μmol/L for 24 h.Prx 1 and GST π mRNA and protein expression were up-regulated in cells treated with nicotine for the same time at different concentrations or at the same concentration for different times (P<0.05).NF-κB was translocated from cytoplasm to nucleus,the expression of NF-κB was increased in nucleus.These results suggest that up-regulation of Prx1 expression appears to be associated with tobacco-induced oxidative stress,which may play an important role in the pathogenesis of OSCC.

  6. DC electric stimulation upregulates angiogenic factors in endothelial cells through activation of VEGF receptors.

    Science.gov (United States)

    Bai, Huai; Forrester, John V; Zhao, Min

    2011-07-01

    Small direct current (DC) electric fields direct some important angiogenic responses of vascular endothelial cells. Those responses indicate promising use of electric fields to modulate angiogenesis. We sought to determine the regulation of electric fields on transcription and expression of a serial of import angiogenic factors by endothelial cells themselves. Using semi-quantitative PCR and ELISA we found that electric stimulation upregulates the levels of mRNAs and proteins of a number of angiogenic proteins, most importantly VEGF165, VEGF121 and IL-8 in human endothelial cells. The up-regulation of mRNA levels might be specific, as the mRNA encoding bFGF, TGF-beta and eNOS are not affected by DC electric stimulation at 24h time-point. Inhibition of VEGF receptor (VEGFR1 or VEGFR2) signaling significantly decreased VEGF production and completely abolished IL-8 production. DC electric stimulation selectively regulates production of some growth factors and cytokines important for angiogenesis through a feed-back loop mediated by VEGF receptors.

  7. Cannabidiol inhibits cancer cell invasion via upregulation of tissue inhibitor of matrix metalloproteinases-1.

    Science.gov (United States)

    Ramer, Robert; Merkord, Jutta; Rohde, Helga; Hinz, Burkhard

    2010-04-01

    Although cannabinoids exhibit a broad variety of anticarcinogenic effects, their potential use in cancer therapy is limited by their psychoactive effects. Here we evaluated the impact of cannabidiol, a plant-derived non-psychoactive cannabinoid, on cancer cell invasion. Using Matrigel invasion assays we found a cannabidiol-driven impaired invasion of human cervical cancer (HeLa, C33A) and human lung cancer cells (A549) that was reversed by antagonists to both CB(1) and CB(2) receptors as well as to transient receptor potential vanilloid 1 (TRPV1). The decrease of invasion by cannabidiol appeared concomitantly with upregulation of tissue inhibitor of matrix metalloproteinases-1 (TIMP-1). Knockdown of cannabidiol-induced TIMP-1 expression by siRNA led to a reversal of the cannabidiol-elicited decrease in tumor cell invasiveness, implying a causal link between the TIMP-1-upregulating and anti-invasive action of cannabidiol. P38 and p42/44 mitogen-activated protein kinases were identified as upstream targets conferring TIMP-1 induction and subsequent decreased invasiveness. Additionally, in vivo studies in thymic-aplastic nude mice revealed a significant inhibition of A549 lung metastasis in cannabidiol-treated animals as compared to vehicle-treated controls. Altogether, these findings provide a novel mechanism underlying the anti-invasive action of cannabidiol and imply its use as a therapeutic option for the treatment of highly invasive cancers.

  8. Signal peptide of eosinophil cationic protein upregulates transforming growth factor-alpha expression in human cells.

    Science.gov (United States)

    Chang, Hao-Teng; Kao, Yu-Lin; Wu, Chia-Mao; Fan, Tan-Chi; Lai, Yiu-Kay; Huang, Kai-Ling; Chang, Yuo-Sheng; Tsai, Jaw-Ji; Chang, Margaret Dah-Tsyr

    2007-04-01

    Eosinophil cationic protein (ECP) is a major component of eosinophil granule protein that is used as a clinical bio-marker for asthma and allergic inflammatory diseases. Previously, it has been reported that the signal peptide of human ECP (ECPsp) inhibits the cell growth of Escherichia coli (E. coli) and Pichia pastoris (P. pastoris), but not mammalian A431 cells. The inhibitory effect is due to the lack of human signal peptide peptidase (hSPP), a protease located on the endoplasmic reticulum (ER) membrane, in the lower organisms. In this study, we show that the epidermal growth factor receptor (EGFR) is upregulated by the exogenous ECPsp-eGFP as a result of the increased expression of the transforming growth factor-alpha (TGF-alpha) at both transcriptional and translational levels in A431 and HL-60 clone 15 cell lines. Furthermore, the N-terminus of ECPsp fragment generated by the cleavage of hSPP (ECPspM1-G17) gives rise to over threefold increase of TGF-alpha protein expression, whereas another ECPsp fragment (ECPspL18-A27) and the hSPP-resistant ECPsp (ECPspG17L) do not show similar effect. Our results indicate that the ECPspM1-G17 plays a crucial role in the upregulation of TGF-alpha, suggesting that the ECPsp not only directs the secretion of mature ECP, but also involves in the autocrine system.

  9. Curcumin Enhances the Radiosensitivity of U87 Cells by Inducing DUSP-2 Up-Regulation

    Directory of Open Access Journals (Sweden)

    Yu Qian

    2015-03-01

    Full Text Available Objective: Glioblastoma multiforme (GBM, an aggressive primary brain tumor, is radioresistant and recurs despite aggressive surgery, chemotherapy, and radiotherapy. Curcumin as a potential radiosensitizer has received extensive attention in cancer treatment. To explore an effectiveness of this radiosensitizer for GBM treatment, we evaluated the radiosensitizing effect of curcumin and investigated its potential molecular mechanisms in the human glioma cell line U87. Methods: The cytotoxic effects of curcumin on U87 cells were evaluated using the Cell Counting Kit-8 assay, and the radiosensitivity of U87 cells treated with curcumin was accessed by colony information assay. The effects of curcumin on cell proliferation and cell cycle regulation were determined using the 5-ethynyl-2-deoxyuridine incorporation assay and flow cytometry, respectively. Western blotting was applied to determine the effects of curcumin on protein expression of dual-specificity phosphatase-2 (DUSP-2, extracellular signal-regulated kinase (ERK, and c-Jun N-terminal kinase (JNK as well as phosphorylated ERK and JNK. Results: Curcumin significantly inhibited the proliferation of U87 cells in a dose-and time-dependent manner. Curcumin treatment at the concentrations of 5 µM and 10 M could significantly reduce the clonogenic activity and enhance the radiosensitivity of U87 cells with sensitive enhancement ratios (SERs of 1.71 and 4.65, respectively. Curcumin resulted in G2/M cell cycle arrest in U87 cells, which were radiosensitive. Pre-treatment of U87-MG cells with 5 µM curcumin enhanced radiation-induced cell proliferation inhibition and apoptosis. Furthermore, we observed that curcumin increased DUSP-2 protein expression and decreased the phosphorylation of ERK and JNK. Conclusion: Our results suggest that low-dose curcumin may enhance the radiosensitivity of human glioma U87 cells in vitro by inducing G2/M cell cycle arrest through up-regulation of DUSP-2 expression and

  10. Para-toluenesulfonamide induces tongue squamous cell carcinoma cell death through disturbing lysosomal stability.

    Science.gov (United States)

    Liu, Zhe; Liang, Chenyuan; Zhang, Zhuoyuan; Pan, Jian; Xia, Hui; Zhong, Nanshan; Li, Longjiang

    2015-11-01

    Para-toluenesulfonamide (PTS) has been implicated with anticancer effects against a variety of tumors. In the present study, we investigated the inhibitory effects of PTS on tongue squamous cell carcinoma (Tca-8113) and explored the lysosomal and mitochondrial changes after PTS treatment in vitro. High-performance liquid chromatography showed that PTS selectively accumulated in Tca-8113 cells with a relatively low concentration in normal fibroblasts. Next, the effects of PTS on cell viability, invasion, and cell death were determined. PTS significantly inhibited Tca-8113 cells' viability and invasive ability with increased cancer cell death. Flow cytometric analysis and the lactate dehydrogenase release assay showed that PTS induced cancer cell death by activating apoptosis and necrosis simultaneously. Morphological changes, such as cellular shrinkage, nuclear condensation as well as formation of apoptotic body and secondary lysosomes, were observed, indicating that PTS might induce cell death through disturbing lysosomal stability. Lysosomal integrity assay and western blot showed that PTS increased lysosomal membrane permeabilization associated with activation of lysosomal cathepsin B. Finally, PTS was shown to inhibit ATP biosynthesis and induce the release of mitochondrial cytochrome c. Therefore, our findings provide a novel insight into the use of PTS in cancer therapy.

  11. Aflatoxin B1 up-regulates insulin receptor substrate 2 and stimulates hepatoma cell migration.

    Directory of Open Access Journals (Sweden)

    Yanli Ma

    Full Text Available Aflatoxin B1 (AFB1 is a potent carcinogen that can induce hepatocellular carcinoma. AFB1-8,9-exo-epoxide, one of AFB1 metabolites, acts as a mutagen to react with DNA and induce gene mutations, including the tumor suppressor p53. In addition, AFB1 reportedly stimulates IGF receptor activation. Aberrant activation of IGF-I receptor (IGF-IR signaling is tightly associated with various types of human tumors. In the current study, we investigated the effects of AFB1 on key elements in IGF-IR signaling pathway, and the effects of AFB1 on hepatoma cell migration. The results demonstrated that AFB1 induced IGF-IR, Akt, and Erk1/2 phosphorylation in hepatoma cell lines HepG2 and SMMC-7721, and an immortalized human liver cell line Chang liver. AFB1 also down-regulated insulin receptor substrate (IRS 1 but paradoxically up-regulated IRS2 through preventing proteasomal degradation. Treatment of hepatoma cells and Chang liver cells with IGF-IR inhibitor abrogated AFB1-induced Akt and Erk1/2 phosphorylation. In addition, IRS2 knockdown suppressed AFB1-induced Akt and Erk1/2 phosphorylation. Finally, AFB1 stimulated hepatoma cell migration. IGF-IR inhibitor or IRS2 knockdown suppressed AFB1-induced hepatoma cell migration. These data demonstrate that AFB1 stimulates hepatoma cell migration through IGF-IR/IRS2 axis.

  12. Mangiferin Facilitates Islet Regeneration and β-Cell Proliferation through Upregulation of Cell Cycle and β-Cell Regeneration Regulators

    Science.gov (United States)

    Wang, Hai-Lian; Li, Chun-Yang; Zhang, Bin; Liu, Yuan-De; Lu, Bang-Min; Shi, Zheng; An, Na; Zhao, Liang-Kai; Zhang, Jing-Jing; Bao, Jin-Ku; Wang, Yi

    2014-01-01

    Mangiferin, a xanthonoid found in plants including mangoes and iris unguicularis, was suggested in previous studies to have anti-hyperglycemic function, though the underlying mechanisms are largely unknown. This study was designed to determine the therapeutic effect of mangiferin by the regeneration of β-cells in mice following 70% partial pancreatectomy (PPx), and to explore the mechanisms of mangiferin-induced β-cell proliferation. For this purpose, adult C57BL/6J mice after 7–14 days post-PPx, or a sham operation were subjected to mangiferin (30 and 90 mg/kg body weight) or control solvent injection. Mangiferin-treated mice exhibited an improved glycemia and glucose tolerance, increased serum insulin levels, enhanced β-cell hyperplasia, elevated β-cell proliferation and reduced β-cell apoptosis. Further dissection at the molecular level showed several key regulators of cell cycle, such as cyclin D1, D2 and cyclin-dependent kinase 4 (Cdk4) were significantly up-regulated in mangiferin-treated mice. In addition, critical genes related to β-cell regeneration, such as pancreatic and duodenal homeobox 1 (PDX-1), neurogenin 3 (Ngn3), glucose transporter 2 (GLUT-2), Forkhead box protein O1 (Foxo-1), and glucokinase (GCK), were found to be promoted by mangiferin at both the mRNA and protein expression level. Thus, mangiferin administration markedly facilitates β-cell proliferation and islet regeneration, likely by regulating essential genes in the cell cycle and the process of islet regeneration. These effects therefore suggest that mangiferin bears a therapeutic potential in preventing and/or treating the diabetes. PMID:24853132

  13. Mangiferin Facilitates Islet Regeneration and β-Cell Proliferation through Upregulation of Cell Cycle and β-Cell Regeneration Regulators

    Directory of Open Access Journals (Sweden)

    Hai-Lian Wang

    2014-05-01

    Full Text Available Mangiferin, a xanthonoid found in plants including mangoes and iris unguicularis, was suggested in previous studies to have anti-hyperglycemic function, though the underlying mechanisms are largely unknown. This study was designed to determine the therapeutic effect of mangiferin by the regeneration of β-cells in mice following 70% partial pancreatectomy (PPx, and to explore the mechanisms of mangiferin-induced β-cell proliferation. For this purpose, adult C57BL/6J mice after 7–14 days post-PPx, or a sham operation were subjected to mangiferin (30 and 90 mg/kg body weight or control solvent injection. Mangiferin-treated mice exhibited an improved glycemia and glucose tolerance, increased serum insulin levels, enhanced β-cell hyperplasia, elevated β-cell proliferation and reduced β-cell apoptosis. Further dissection at the molecular level showed several key regulators of cell cycle, such as cyclin D1, D2 and cyclin-dependent kinase 4 (Cdk4 were significantly up-regulated in mangiferin-treated mice. In addition, critical genes related to β-cell regeneration, such as pancreatic and duodenal homeobox 1 (PDX-1, neurogenin 3 (Ngn3, glucose transporter 2 (GLUT-2, Forkhead box protein O1 (Foxo-1, and glucokinase (GCK, were found to be promoted by mangiferin at both the mRNA and protein expression level. Thus, mangiferin administration markedly facilitates β-cell proliferation and islet regeneration, likely by regulating essential genes in the cell cycle and the process of islet regeneration. These effects therefore suggest that mangiferin bears a therapeutic potential in preventing and/or treating the diabetes.

  14. Homeobox A7 stimulates breast cancer cell proliferation by up-regulating estrogen receptor-alpha

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Yu [Department of Reproductive Endocrinology, Women’s Hospital, School of Medicine, Zhejiang University, Hangzhou 310006 (China); Department of Obstetrics and Gynaecology, Child and Family Research Institute, University of British Columbia, Vancouver, British Columbia V5Z 4H4 (Canada); Cheng, Jung-Chien [Department of Obstetrics and Gynaecology, Child and Family Research Institute, University of British Columbia, Vancouver, British Columbia V5Z 4H4 (Canada); Huang, He-Feng, E-mail: huanghefg@hotmail.com [Department of Reproductive Endocrinology, Women’s Hospital, School of Medicine, Zhejiang University, Hangzhou 310006 (China); Leung, Peter C.K., E-mail: peter.leung@ubc.ca [Department of Reproductive Endocrinology, Women’s Hospital, School of Medicine, Zhejiang University, Hangzhou 310006 (China); Department of Obstetrics and Gynaecology, Child and Family Research Institute, University of British Columbia, Vancouver, British Columbia V5Z 4H4 (Canada)

    2013-11-01

    Highlights: •HOXA7 regulates MCF7 cell proliferation. •HOXA7 up-regulates ERα expression. •HOXA7 mediates estrogen-induced MCF7 cell proliferation. -- Abstract: Breast cancer is the most common hormone-dependent malignancy in women. Homeobox (HOX) transcription factors regulate many cellular functions, including cell migration, proliferation and differentiation. The aberrant expression of HOX genes has been reported to be associated with human reproductive cancers. Estradiol (E2) and its nuclear receptors, estrogen receptor (ER)-alpha and ER-beta, are known to play critical roles in the regulation of breast cancer cell growth. However, an understanding of the potential relationship between HOXA7 and ER in breast cancer cells is limited. In this study, our results demonstrate that knockdown of HOXA7 in MCF7 cells significantly decreased cell proliferation and ERα expression. In addition, HOXA7 knockdown attenuated E2-induced cell proliferation as well as progesterone receptor (PR) expression. The stimulatory effects of E2 on cell proliferation and PR expression were abolished by co-treatment with ICI 182780, a selective ERα antagonist. In contrast, overexpression of HOXA7 significantly stimulated cell proliferation and ERα expression. Moreover, E2-induced cell proliferation, as well as PR expression, was enhanced by the overexpression of HOXA7. Neither knockdown nor overexpression of HOXA7 affected the ER-beta levels. Our results demonstrate a novel mechanistic role for HOXA7 in modulating breast cancer cell proliferation via regulation of ERα expression. This finding contributes to our understanding of the role HOXA7 plays in regulating the proliferation of ER-positive cancer cells.

  15. Down-regulation of the A3 adenosine receptor in human mast cells upregulates mediators of angiogenesis and remodeling.

    Science.gov (United States)

    Rudich, Noam; Dekel, Ornit; Sagi-Eisenberg, Ronit

    2015-05-01

    Adenosine activated mast cells have been long implicated in allergic asthma and studies in rodent mast cells have assigned the A3 adenosine receptor (A3R) a primary role in mediating adenosine responses. Here we analyzed the functional impact of A3R activation on genes that are implicated in tissue remodeling in severe asthma in the human mast cell line HMC-1 that shares similarities with lung derived human mast cells. Quantitative real time PCR demonstrated upregulation of IL6, IL8, VEGF, amphiregulin and osteopontin. Moreover, further upregulation of these genes was noted upon the addition of dexamethasone. Unexpectedly, activated A3R down regulated its own expression and knockdown of the receptor replicated the pattern of agonist induced gene upregulation. This study therefore identifies the human mast cell A3R as regulator of tissue remodeling gene expression in human mast cells and demonstrates a heretofore-unrecognized mode of feedback regulation that is exerted by this receptor.

  16. Metformin Induces Growth Inhibition and Cell Cycle Arrest by Upregulating MicroRNA34a in Renal Cancer Cells

    Science.gov (United States)

    Xie, Wei; Wang, Lei; Sheng, Halei; Qiu, Jing; Zhang, Di; Zhang, Le; Yang, Fan; Tang, Dahai; Zhang, Kebin

    2017-01-01

    Background Metformin is a widely used biguanide drug for the treatment of type 2 diabetes. It has been revaluated as a potential anti-cancer drug with promising activity in various tumors. However, the precise mechanisms underlying the suppression of cancer cells by metformin remain not well understood. Material/Methods In this study, human renal cell carcinoma cell line ACHN was used to investigate the anti-proliferation effect of metformin. A cell counting kit-8 assay was used to detect the cell viability. The cell cycle distribution and apoptosis were analyzed by flow cytometry. The expression of cyclin D1 and p27KIP1 was detected by Western blot. The underlying mechanism involving miRNA34a was further investigated by quantitative RT-PCR and transfection with miRNA inhibitor specific for miRNA34a in ACHN, 769-P, and A498 cells. Results Metformin could significantly inhibit the proliferation of ACHN cells in a dose- and time-dependent manner. In addition, the results showed that metformin induced G0/G1 phase arrest and delayed entry into S phase in ACHN cells. It was shown that metformin downregulates the expression of cyclin D1 and increases the p27KIP1 level. Furthermore, metformin increased ACHN cell death. Lastly, miRNA34a was found to be upregulated by metformin in ACHN, 769-P, and A498 cells. Subsequently, it was demonstrated that inhibition of miRNA34a could partially attenuate the suppressive effect of metformin on renal cancer cell proliferation. Conclusions The study data revealed that metformin induced cell growth inhibition and cell cycle arrest partially by upregulating miRNA34a in renal cancer cells. PMID:28045889

  17. PAX3-FOXO1 Induces Up-Regulation of Noxa Sensitizing Alveolar Rhabdomyosarcoma Cells to Apoptosis

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    Amy D. Marshall

    2013-07-01

    Full Text Available Alveolar rhabdomyosarcoma (ARMS has a much poorer prognosis than the more common embryonal subtype. Most ARMS tumors characteristically possess a specific genomic translocation between the genes of PAX3/7 and FOXO1 (FKHR, which forms fusion proteins possessing the DNA binding domains of PAX3/7 and the more transcriptionally potent transactivation domain of FOXO1. We have shown that the proapoptotic BH3-only family member Noxa is upregulated by the PAX3-FOXO1 fusion transcription factor in a p53-independent manner. The increased expression of Noxa renders PAX3-FOXO1-expressing cells more susceptible to apoptosis induced by a ă-secretase inhibitor (GSI1, Z-LLNle-CHO, the proteasome inhibitor bortezomib, and BH3 mimetic ABT-737. Apoptosis in response to bortezomib can be overcome by shRNA knockdown of Noxa. In vivo treatment with bortezomib reduced the growth of tumors derived from a PAX3-FOXO1-expressing primary myoblast tumor model and RH41 xenografts. We therefore demonstrate that PAX3-FOXO1 up-regulation of Noxa represents an unanticipated aspect of ARMS tumor biology that creates a therapeutic window to allow induction of apoptosis in ARMS cells.

  18. PAX3-FOXO1 induces up-regulation of Noxa sensitizing alveolar rhabdomyosarcoma cells to apoptosis.

    Science.gov (United States)

    Marshall, Amy D; Picchione, Fabrizio; Geltink, Ramon I Klein; Grosveld, Gerard C

    2013-07-01

    Alveolar rhabdomyosarcoma (ARMS) has a much poorer prognosis than the more common embryonal subtype. Most ARMS tumors characteristically possess a specific genomic translocation between the genes of PAX3/7 and FOXO1 (FKHR), which forms fusion proteins possessing the DNA binding domains of PAX3/7 and the more transcriptionally potent transactivation domain of FOXO1. We have shown that the proapoptotic BH3-only family member Noxa is upregulated by the PAX3-FOXO1 fusion transcription factor in a p53-independent manner. The increased expression of Noxa renders PAX3-FOXO1-expressing cells more susceptible to apoptosis induced by a γ-secretase inhibitor (GSI1, Z-LLNle-CHO), the proteasome inhibitor bortezomib, and BH3 mimetic ABT-737. Apoptosis in response to bortezomib can be overcome by shRNA knockdown of Noxa. In vivo treatment with bortezomib reduced the growth of tumors derived from a PAX3-FOXO1-expressing primary myoblast tumor model and RH41 xenografts. We therefore demonstrate that PAX3-FOXO1 up-regulation of Noxa represents an unanticipated aspect of ARMS tumor biology that creates a therapeutic window to allow induction of apoptosis in ARMS cells.

  19. Acute leptin exposure reduces megalin expression and upregulates TGFβ1 in cultured renal proximal tubule cells.

    Science.gov (United States)

    Briffa, Jessica F; Grinfeld, Esther; Mathai, Michael L; Poronnik, Phillip; McAinch, Andrew J; Hryciw, Deanne H

    2015-02-05

    Increased leptin concentrations observed in obesity can lead to proteinuria, suggesting that leptin may play a role in obesity-related kidney disease. Obesity reduces activation of AMP-activated protein kinase (AMPK) and increases transforming growth factor-β1 (TGF-β1) expression in the kidney, leading to albuminuria. Thus we investigated if elevated leptin altered AMPK and TGF-β1 signaling in proximal tubule cells (PTCs). In opossum kidney (OK) PTCs Western blot analysis demonstrated that leptin upregulates TGF-β1 secretion (0.50 µg/ml) and phosphorylated AMPKα (at 0.25, and 0.50 µg/ml), and downregulates megalin expression at all concentrations (0.05-0.50 µg/ml). Using the AMPK inhibitor, Compound C, leptin exposure regulated TGF-β1 expression and secretion in PTCs via an AMPK mediated pathway. In addition, elevated leptin exposure (0.50 µg/ml) reduced albumin handling in OK cells independently of megalin expression. This study demonstrates that leptin upregulates TGF-β1, reduces megalin, and reduces albumin handling in PTCs by an AMPK mediated pathway.

  20. PTEN overexpression improves cisplatin-resistance of human ovarian cancer cells through upregulating KRT10 expression

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    Wu, Huijuan; Wang, Ke; Liu, Wenxin; Hao, Quan, E-mail: quan_haotj@126.com

    2014-02-07

    Highlights: • Overexpression of PTEN enhanced the sensitivity of C13K cells to cisplatin. • KRT10 is a downstream molecule of PTEN involved in the resistance-reversing effect. • Overexpression of KRT10 enhanced the chemosensitivity of C13K cells to cisplatin. - Abstract: Multi-drug resistance (MDR) is a common cause of the failure of chemotherapy in ovarian cancer. PTEN, a tumor suppressor gene, has been demonstrated to be able to reverse cisplatin-resistance in ovarian cancer cell line C13K. However, the downstream molecules of PTEN involved in the resistance-reversing effect have not been completely clarified. Therefore, we screened the downstream molecules of PTEN and studied their interactions in C13K ovarian cancer cells using a 3D culture model. Firstly, we constructed an ovarian cancer cell line stably expressing PTEN, C13K/PTEN. MTT assay showed that overexpression of PTEN enhanced the sensitivity of C13K cells to cisplatin, but not to paclitaxel. Then we examined the differently expressed proteins that interacted with PTEN in C13K/PTEN cells with or without cisplatin treatment by co-immunoprecipitation. KRT10 was identified as a differently expressed protein in cisplatin-treated C13K/PTEN cells. Further study confirmed that cisplatin could induce upregulation of KRT10 mRNA and protein in C13K/PTEN cells and there was a directly interaction between KRT10 and PTEN. Forced expression of KRT10 in C13K cells also enhanced cisplatin-induced proliferation inhibition and apoptosis of C13K cells. In addition, KRT10 siRNA blocked cisplatin-induced proliferation inhibition of C13K/PTEN cells. In conclusion, our data demonstrate that KRT10 is a downstream molecule of PTEN which improves cisplatin-resistance of ovarian cancer and forced KRT10 overexpression may also act as a therapeutic method for overcoming MDR in ovarian cancer.

  1. Maggot debridement therapy promotes diabetic foot wound healing by up-regulating endothelial cell activity.

    Science.gov (United States)

    Sun, Xinjuan; Chen, Jin'an; Zhang, Jie; Wang, Wei; Sun, Jinshan; Wang, Aiping

    2016-03-01

    To determine the role of maggot debridement therapy (MDT) on diabetic foot wound healing, we compared growth related factors in wounds before and after treatment. Furthermore, we utilized human umbilical vein endothelial cells (HUVECs) to explore responses to maggot excretions/secretions on markers of angiogenesis and proliferation. The results showed that there was neo-granulation and angiogenesis in diabetic foot wounds after MDT. Moreover, significant elevation in CD34 and CD68 levels was also observed in treated wounds. In vitro, ES increased HUVEC proliferation, improved tube formation, and increased expression of vascular endothelial growth factor receptor 2 in a dose dependent manner. These results demonstrate that MDT and maggot ES can promote diabetic foot wound healing by up-regulating endothelial cell activity.

  2. Lysophosphatidic Acid Up-Regulates Hexokinase II and Glycolysis to Promote Proliferation of Ovarian Cancer Cells

    Directory of Open Access Journals (Sweden)

    Abir Mukherjee

    2015-09-01

    Full Text Available Lysophosphatidic acid (LPA, a blood-borne lipid mediator, is present in elevated concentrations in ascites of ovarian cancer patients and other malignant effusions. LPA is a potent mitogen in cancer cells. The mechanism linking LPA signal to cancer cell proliferation is not well understood. Little is known about whether LPA affects glucose metabolism to accommodate rapid proliferation of cancer cells. Here we describe that in ovarian cancer cells, LPA enhances glycolytic rate and lactate efflux. A real time PCR-based miniarray showed that hexokinase II (HK2 was the most dramatically induced glycolytic gene to promote glycolysis in LPA-treated cells. Analysis of the human HK2 gene promoter identified the sterol regulatory element-binding protein as the primary mediator of LPA-induced HK2 transcription. The effects of LPA on HK2 and glycolysis rely on LPA2, an LPA receptor subtype overexpressed in ovarian cancer and many other malignancies. We further examined the general role of growth factor-induced glycolysis in cell proliferation. Like LPA, epidermal growth factor (EGF elicited robust glycolytic and proliferative responses in ovarian cancer cells. Insulin-like growth factor 1 (IGF-1 and insulin, however, potently stimulated cell proliferation but only modestly induced glycolysis. Consistent with their differential effects on glycolysis, LPA and EGF-dependent cell proliferation was highly sensitive to glycolytic inhibition while the growth-promoting effect of IGF-1 or insulin was more resistant. These results indicate that LPA- and EGF-induced cell proliferation selectively involves up-regulation of HK2 and glycolytic metabolism. The work is the first to implicate LPA signaling in promotion of glucose metabolism in cancer cells.

  3. FOXO3-mediated up-regulation of Bim contributes to rhein-induced cancer cell apoptosis.

    Science.gov (United States)

    Wang, Jiao; Liu, Shu; Yin, Yancun; Li, Mingjin; Wang, Bo; Yang, Li; Jiang, Yangfu

    2015-03-01

    The anthraquinone compound rhein is a natural agent in the traditional Chinese medicine rhubarb. Preclinical studies demonstrate that rhein has anticancer activity. Treatment of a variety of cancer cells with rhein may induce apoptosis. Here, we report that rhein induces atypical unfolded protein response in breast cancer MCF-7 cells and hepatoma HepG2 cells. Rhein induces CHOP expression, eIF2α phosphorylation and caspase cleavage, while it does not induce glucose-regulated protein 78 (GRP78) expression in both MCF-7 and HepG2 cells. Meanwhile, rhein inhibits thapsigargin-induced GRP78 expression and X box-binding protein 1 splicing. In addition, rhein inhibits Akt phosphorylation and stimulates FOXO transactivation activity. Rhein induces Bim expression in MCF-7 and HepG2 cells, which can be abrogated by FOXO3a knockdown. Knockdown of FOXO3a or Bim abrogates rhein-induced caspase cleavage and apoptosis. The chemical chaperone 4-phenylbutyrate acid antagonizes the induction of FOXO activation, Bim expression and caspase cleavage by rhein, indicating that protein misfolding may be involved in triggering these deleterious effects. We conclude that FOXO3a-mediated up-regulation of Bim is a key mechanism underlying rhein-induced cancer cells apoptosis.

  4. TAZ promotes temozolomide resistance by upregulating MCL-1 in human glioma cells.

    Science.gov (United States)

    Tian, Tian; Li, Aimin; Lu, Hong; Luo, Ran; Zhang, Mingzhi; Li, Zhaoming

    2015-08-07

    Temozolomide is a novel cytotoxic agent currently used as first-line chemotherapy for glioblastoma multiforme (GBM). However, intrinsic or acquired chemoresistance to temozolomide remains the greatest obstacle to the successful treatment of human GBM. The principal mechanism responsible for this resistance is largely unknown. In the present study, we showed that expression of transcriptional co-activator with PDZ-binding motif (TAZ) in glioma cells correlated with temozolomide chemoresistance in human glioma cells. Overexpression of TAZ promoted temozolomide resistance in U-87MG cells, whereas knockdown of TAZ expression sensitized temozolomide-resistant U-251MG cells to temozolomide. Further, TAZ inhibits temozolomide induced apoptosis via upregulation of MCL-1 (myeloid cell leukemia 1) and high expression of TAZ predicts a poor prognosis for GBM patients. In conclusion, our results suggest that TAZ had a critical role in the resistance to temozolomide in glioma cells, and it may provide a promising target for improving the therapeutic outcome of temozolomide-resistant gliomas.

  5. Cell proliferation and cell death are disturbed during prenatal and postnatal brain development after uranium exposure.

    Science.gov (United States)

    Legrand, M; Elie, C; Stefani, J; N Florès; Culeux, C; Delissen, O; Ibanez, C; Lestaevel, P; Eriksson, P; Dinocourt, C

    2016-01-01

    The developing brain is more susceptible to neurotoxic compounds than adult brain. It is also well known that disturbances during brain development cause neurological disorders in adulthood. The brain is known to be a target organ of uranium (U) exposure and previous studies have noted that internal U contamination of adult rats induces behavioral disorders as well as affects neurochemistry and neurophysiological properties. In this study, we investigated whether depleted uranium (DU) exposure affects neurogenesis during prenatal and postnatal brain development. We examined the structural morphology of the brain, cell death and finally cell proliferation in animals exposed to DU during gestation and lactation compared to control animals. Our results showed that DU decreases cell death in the cortical neuroepithelium of gestational day (GD) 13 embryos exposed at 40mg/L and 120mg/L and of GD18 fetuses exposed at 120mg/L without modification of the number of apoptotic cells. Cell proliferation analysis showed an increase of BrdU labeling in the dentate neuroepithelium of fetuses from GD18 at 120mg/L. Postnatally, cell death is increased in the dentate gyrus of postnatal day (PND) 0 and PND5 exposed pups at 120mg/L and is associated with an increase of apoptotic cell number only at PND5. Finally, a decrease in dividing cells is observed in the dentate gyrus of PND21 rats developmentally exposed to 120mg/L DU, but not at PND0 and PND5. These results show that DU exposure during brain development causes opposite effects on cell proliferation and cell death processes between prenatal and postnatal development mainly at the highest dose. Although these modifications do not have a major impact in brain morphology, they could affect the next steps of neurogenesis and thus might disrupt the fine organization of the neuronal network.

  6. Exposure to cell phone radiation up-regulates apoptosis genes in primary cultures of neurons and astrocytes.

    Science.gov (United States)

    Zhao, Tian-Yong; Zou, Shi-Ping; Knapp, Pamela E

    2007-01-22

    The health effects of cell phone radiation exposure are a growing public concern. This study investigated whether expression of genes related to cell death pathways are dysregulated in primary cultured neurons and astrocytes by exposure to a working Global System for Mobile Communication (GSM) cell phone rated at a frequency of 1900MHz. Primary cultures were exposed to cell phone emissions for 2h. We used array analysis and real-time RT-PCR to show up-regulation of caspase-2, caspase-6 and Asc (apoptosis associated speck-like protein containing a card) gene expression in neurons and astrocytes. Up-regulation occurred in both "on" and "stand-by" modes in neurons, but only in "on" mode in astrocytes. Additionally, astrocytes showed up-regulation of the Bax gene. The effects are specific since up-regulation was not seen for other genes associated with apoptosis, such as caspase-9 in either neurons or astrocytes, or Bax in neurons. The results show that even relatively short-term exposure to cell phone radiofrequency emissions can up-regulate elements of apoptotic pathways in cells derived from the brain, and that neurons appear to be more sensitive to this effect than astrocytes.

  7. Could Upregulated Hsp70 Protein Compensate for the Hsp90-Silence-Induced Cell Death in Glioma Cells?

    Directory of Open Access Journals (Sweden)

    Chinmay Munje

    2014-01-01

    Full Text Available The molecular chaperone heat shock protein 90 alpha (Hsp90α has been recognized in various tumours including glioma. This pilot study using a proteomic approach analyses the downstream effects of Hsp90 inhibition using 17-allylamino-17-demethoxygeldanamycin (17AAG and a short hairpin RNA (shRNA oligonucleotide targeting hsp90α (shhsp90α in the U87-MG glioma cell line. Preliminary data coupled with bioinformatic analysis identified several known and unknown Hsp90 client proteins that demonstrated a change in their protein expression after Hsp90 inhibition, signifying an alteration in the canonical pathways of cell cycle progression, apoptosis, cell invasion, angiogenesis, and metastasis. Members of the glycolysis pathway were upregulated, demonstrating increased dependency on glycolysis for energy source by the treated glioma cells. Upregulated proteins also include Hsp70 and members of its family such as Hsp27 and gp96, thereby suggesting the role of Hsp90 co-chaperones in compensating for Hsp90 function after Hsp90 inhibition. Considering Hsp70’s role in antiapoptosis, it was postulated that a combination therapy involving a multitarget approach could be carried out. Consequently inhibition of both Hsp90 and Hsp70 in U87-MG glioma cells resulted in 60% cell death indicating the importance of combination therapy for glioma therapeutics.

  8. Amphiregulin activates human hepatic stellate cells and is upregulated in non alcoholic steatohepatitis

    Science.gov (United States)

    McKee, Chad; Sigala, Barbara; Soeda, Junpei; Mouralidarane, Angelina; Morgan, Maelle; Mazzoccoli, Gianluigi; Rappa, Francesca; Cappello, Francesco; Cabibi, Daniela; Pazienza, Valerio; Selden, Claire; Roskams, Tania; Vinciguerra, Manlio; Oben, Jude A.

    2015-01-01

    Amphiregulin (AR) involvement in liver fibrogenesis and hepatic stellate cells (HSC) regulation is under study. Non-alcoholic fatty liver disease (NAFLD) and its more severe form non-alcoholic steatohepatitis (NASH) may progress to cirrhosis and hepatocellular cancer (HCC). Our aim was to investigate ex vivo the effect of AR on human primary HSC (hHSC) and verify in vivo the relevance of AR in NAFLD fibrogenesis. hHSC isolated from healthy liver segments were analyzed for expression of AR and its activator, TNF-α converting enzyme (TACE). AR induction of hHSC proliferation and matrix production was estimated in the presence of antagonists. AR involvement in fibrogenesis was also assessed in a mouse model of NASH and in humans with NASH. hHSC time dependently expressed AR and TACE. AR increased hHSC proliferation through several mitogenic signaling pathways such as EGFR, PI3K and p38. AR also induced marked upregulation of hHSC fibrogenic markers and reduced hHSC death. AR expression was enhanced in the HSC of a murine model of NASH and of severe human NASH. In conclusion, AR induces hHSC fibrogenic activity via multiple mitogenic signaling pathways, and is upregulated in murine and human NASH, suggesting that AR antagonists may be clinically useful anti-fibrotics in NAFLD. PMID:25744849

  9. Radiation-induced cyclooxygenase 2 up-regulation is dependent on redox status in prostate cancer cells.

    Science.gov (United States)

    Li, Lingyun; Steinauer, Kirsten K; Dirks, Amie J; Husbeck, Bryan; Gibbs, Iris; Knox, Susan J

    2003-12-01

    Cyclooxygenase 2 (COX2) is the inducible isozyme of COX, a key enzyme in arachidonate metabolism and the conversion of arachidonic acid (AA) to prostaglandins (PGs) and other eicosanoids. Previous studies have demonstrated that the COX2 protein is up-regulated in prostate cancer cells after irradiation and that this results in elevated levels of PGE(2). In the present study, we further investigated whether radiation-induced COX2 up-regulation is dependent on the redox status of cells from the prostate cancer cell line PC-3. l-Buthionine sulfoximine (BSO), which inhibits gamma glutamyl cysteine synthetase (gammaGCS), and the antioxidants alpha-lipoic acid and N-acetyl-l-cysteine (NAC) were used to modulate the cellular redox status. BSO decreased the cellular GSH level and increased cellular reactive oxygen species (ROS) in PC-3 cells, whereas alpha-lipoic acid and NAC increased the GSH level and decreased cellular ROS. Both radiation and the oxidant H(2)O(2) had similar effects on COX2 up-regulation and PGE(2) production in PC-3 cells, suggesting that radiation-induced COX2 up-regulation is secondary to the production of ROS. The relative increases in COX2 expression and PGE(2) production induced by radiation and H(2)O(2) were even greater when PC-3 cells were pretreated with BSO. When the cells were pretreated with alpha-lipoic acid or NAC for 24 h, both radiation- and H(2)O(2)-induced COX2 up-regulation and PGE(2) production were markedly inhibited. These results demonstrate that radiation-induced COX2 up-regulation in prostate cancer cells is modulated by the cellular redox status. Radiation-induced increases in ROS levels contribute to the adaptive response of PC-3 cells, resulting in elevated levels of COX2.

  10. Circular RNA has_circ_0067934 is upregulated in esophageal squamous cell carcinoma and promoted proliferation

    Science.gov (United States)

    Xia, Wenjia; Qiu, Mantang; Chen, Rui; Wang, Siwei; Leng, Xuechun; Wang, Jie; Xu, Youtao; Hu, Jingwen; Dong, Gaochao; Xu, Prof Lin; Yin, Rong

    2016-01-01

    Esophageal squamous cell carcinoma (ESCC) is one of the most prevalent and deadly types of cancer worldwide especially in Eastern Asia and the prognosis of ESCC remain poor. Recent evidence suggests that circular RNAs (circRNAs) play important roles in multiple diseases, including cancer. In this study, we characterized a novel circRNA termed hsa_circ_0067934 in ESCC tumor tissues and cell lines. We analyzed a cohort of 51 patients and found that hsa_circ_0067934 was significantly overexpressed in ESCC tissues compared with paired adjacent normal tissues. The high expression level of hsa_circ_0067934 was associated with poor differentiation (P = 0.025), I-II T stage (P = 0.04), and I-II TNM stage (P = 0.021). The in vitro silence of hsa_circ_0067934 by siRNA inhibited the proliferation and migration of ESCC cells and blocked cell cycle progression. Cell fraction analyses and fluorescence in situ hybridization detected that hsa_circ_0067934 was mostly located in the cytoplasm. Our findings suggest that hsa_circ_0067934 is upregulated in ESCC tumor tissue. Our data suggest that hsa_circ_0067934 represents a novel potential biomarker and therapeutic target of ESCC. PMID:27752108

  11. Src-like adaptor protein (SLAP) is upregulated in antigen-stimulated mast cells and acts as a negative regulator.

    Science.gov (United States)

    Park, Seung-Kiel; Qiao, Huihong; Beaven, Michael A

    2009-06-01

    Our studies in the RBL-2H3 mast cell line suggest that responses to antigen (Ag) are negatively modulated through upregulation of Src-like adaptor protein (SLAP). Ag stimulation of RBL-2H3 cells leads to increased levels of SLAP (but not SLAP2) transcripts and protein over a period of several hours. The effects of pharmacologic inhibitors indicate that the upregulation of SLAP is dependent on multiple signaling pathways. Knockdown of SLAP with anti-SLAP siRNA is associated with enhanced phosphorylation of Syk, the linker for activation of T cells (LAT), phospholipase C gamma, MAP kinases, and various transcription factors. Production of IL-3 and MCP-1, but not degranulation, is also enhanced. The upregulation of SLAP may thus serve to limit the duration of cytokine production in Ag-stimulated cells.

  12. Cyclic AMP enhances TGFβ responses of breast cancer cells by upregulating TGFβ receptor I expression.

    Directory of Open Access Journals (Sweden)

    Ilka Oerlecke

    Full Text Available Cellular functions are regulated by complex networks of many different signaling pathways. The TGFβ and cAMP pathways are of particular importance in tumor progression. We analyzed the cross-talk between these pathways in breast cancer cells in 2D and 3D cultures. We found that cAMP potentiated TGFβ-dependent gene expression by enhancing Smad3 phosphorylation. Higher levels of total Smad3, as observed in 3D-cultured cells, blocked this effect. Two Smad3 regulating proteins, YAP (Yes-associated protein and TβRI (TGFβ receptor 1, were responsive to cAMP. While YAP had little effect on TGFβ-dependent expression and Smad3 phosphorylation, a constitutively active form of TβRI mimicked the cAMP effect on TGFβ signaling. In 3D-cultured cells, which show much higher levels of TβRI and cAMP, TβRI was unresponsive to cAMP. Upregulation of TβRI expression by cAMP was dependent on transcription. A proximal TβRI promoter fragment was moderately, but significantly activated by cAMP suggesting that cAMP increases TβRI expression at least partially by activating TβRI transcription. Neither the cAMP-responsive element binding protein (CREB nor the TβRI-regulating transcription factor Six1 was required for the cAMP effect. An inhibitor of histone deacetylases alone or together with cAMP increased TβRI expression by a similar extent as cAMP alone suggesting that cAMP may exert its effect by interfering with histone acetylation. Along with an additive stimulatory effect of cAMP and TGFβ on p21 expression an additive inhibitory effect of these agents on proliferation was observed. Finally, we show that mesenchymal stem cells that interact with breast cancer cells can simultaneously activate the cAMP and TGFβ pathways. In summary, these data suggest that combined effects of cAMP and TGFβ, as e.g. induced by mesenchymal stem cells, involve the upregulation of TβRI expression on the transcriptional level, likely due to changes in histone acetylation

  13. The oncoprotein HBXIP upregulates PDGFB via activating transcription factor Sp1 to promote the proliferation of breast cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Yingyi; Zhao, Yu; Li, Leilei; Shen, Yu; Cai, Xiaoli [Department of Biochemistry, College of Life Sciences, Nankai University, Tianjin 300071 (China); Zhang, Xiaodong, E-mail: zhangxd@nankai.edu.cn [Department of Cancer Research, Institute for Molecular Biology, College of Life Sciences, Nankai University, Tianjin 300071 (China); Ye, Lihong, E-mail: yelihong@nankai.edu.cn [Department of Biochemistry, College of Life Sciences, Nankai University, Tianjin 300071 (China)

    2013-05-03

    Highlights: •HBXIP is able to upregulate the expression of PDGFB in breast cancer cells. •HBXIP serves as a coactivator of activating transcription factor Sp1. •HBXIP stimulates the PDGFB promoter via activating transcription factor Sp1. •HBXIP promotes the proliferation of breast cancer cell via upregulating PDGFB. -- Abstract: We have reported that the oncoprotein hepatitis B virus X-interacting protein (HBXIP) acts as a novel transcriptional coactivator to promote proliferation and migration of breast cancer cells. Previously, we showed that HBXIP was able to activate nuclear factor-κB (NF-κB) in breast cancer cells. As an oncogene, the platelet-derived growth factor beta polypeptide (PDGFB) plays crucial roles in carcinogenesis. In the present study, we found that both HBXIP and PDGFB were highly expressed in breast cancer cell lines. Interestingly, HBXIP was able to increase transcriptional activity of NF-κB through PDGFB, suggesting that HBXIP is associated with PDGFB in the cells. Moreover, HBXIP was able to upregulate PDGFB at the levels of mRNA, protein and promoter in the cells. Then, we identified that HBXIP stimulated the promoter of PDGFB through activating transcription factor Sp1. In function, HBXIP enhanced the proliferation of breast cancer cells through PDGFB in vitro. Thus, we conclude that HBXIP upregulates PDGFB via activating transcription factor Sp1 to promote proliferation of breast cancer cells.

  14. Genomic amplification upregulates estrogen-related receptor alpha and its depletion inhibits oral squamous cell carcinoma tumors in vivo.

    Science.gov (United States)

    Tiwari, Ankana; Swamy, Shivananda; Gopinath, Kodaganur S; Kumar, Arun

    2015-12-07

    The ESRRA gene encodes a transcription factor and regulates several genes, such as WNT11 and OPN, involved in tumorigenesis. It is upregulated in several cancers, including OSCC. We have previously shown that the tumor suppressor miR-125a targets ESRRA, and its downregulation causes upregulation of ESRRA in OSCC. Upregulation of ESRRA in the absence of downregulation of miR-125a in a subset of OSCC samples suggests the involvement of an alternative mechanism. Using TaqMan(®) copy number assay, here we report for the first time that the genomic amplification of ESRRA causes its upregulation in a subset of OSCC samples. Ectopic overexpression of ESRRA led to accelerated cell proliferation, anchorage-independent cell growth and invasion, and inhibited apoptosis. Whereas, knockdown of ESRRA expression by siRNA led to reduced cell proliferation, anchorage-independent cell growth and invasion, and accelerated apoptosis. Furthermore, the delivery of a synthetic biostable ESRRA siRNA to OSCC cells resulted in regression of xenografts in nude mice. Thus, the genomic amplification of ESRRA is another novel mechanism for its upregulation in OSCC. Based on our in vitro and in vivo experiments, we suggest that targeting ESRRA by siRNA could be a novel therapeutic strategy for OSCC and other cancers.

  15. Up-regulation of Raf kinase inhibitor protein enhances chemosensitivity of cervical cancer cell

    Institute of Scientific and Technical Information of China (English)

    Xiao Chu; Xinqiang Ji; Mingcui Wang; Wenqing Zhang; Hui Ou; Chong Li

    2014-01-01

    Objective:The purpose of the study is to investigate the ef ects of up-regulation of Raf kinase inhibitor protein (RKlP) on the chemosensitivity of cervical cancer Hela cells. Methods:Eukaryotic expression plasmid pcDNA3.1(+)-ssRKIP containing human overal length RKIPcDNA was transfected into cervical cancer Hela cellby lipofectin assay, establishing a stable cellline containing a target gene by G418. Expression of RKIP in Hela cells was measured by Western blot analysis. After treatment with cisplatin of dif erent concentrations and intervals of time, the ef ect of RKIP on the proliferation of Hela cells was evaluated by MTT method. The flow cytometry was used to investigate whether the RKIP could inhibit apoptosis in Hela cells induced by cisplatin. Results:The expression of RKIP in Hela cells transfected with pcDNA3.1-ssRKIP was increased obviously. After dif erent concentrations of cisplatin treatment cells for 24, 48 and 72 h, the growth inhibition rate in Hela cells transfected with pcDNA3.1-ssRKIP was significantly higher than in control cells (P<0.05). With 5μg/mL cisplatin treatment for 24 h, pcDNA3.1-ssRKIP-transfected Hela cells had an obviously higher percentage of apoptosis (23.2 ± 0.24)%than non-transfected cells (12.4 ± 0.31)%and empty vector-transfected cells (13.4 ± 0.47)%. Without treatment of cisplatin, the percentage of apoptosis for Hela cells transfected with pcDNA3.1-ssRKIP was (5.7 ± 0.12)%, which was stil higher than those of the non-transfected cells (2.9 ± 0.21)%and empty vector-transfected cells (3 ± 0.08)%. Conclusion:Higher expres-sion of RKIP gene can improve chemosensitivitv of cervical cancer Hela cells to cisplatin.

  16. IL-21 and CD40L synergistically promote plasma cell differentiation through upregulation of Blimp-1 in human B cells.

    Science.gov (United States)

    Ding, B Belinda; Bi, Enguang; Chen, Hongshan; Yu, J Jessica; Ye, B Hilda

    2013-02-15

    After undergoing Ig somatic hypermutation and Ag selection, germinal center (GC) B cells terminally differentiate into either memory or plasma cells (PCs). It is known that the CD40L and IL-21/STAT3 signaling pathways play critical roles in this process, yet it is unclear how the B cell transcription program interprets and integrates these two types of T cell-derived signals. In this study, we characterized the role of STAT3 in the GC-associated PC differentiation using purified human tonsillar GC B cells and a GC B cell-like cell line. When primary GC B cells were cultured under PC differentiation condition, STAT3 inhibition by AG490 prevented the transition from GC centrocytes to preplasmablast, suggesting that STAT3 is required for the initiation of PC development. In a GC B cell-like human B cell line, although IL-21 alone can induce low-level Blimp-1 expression, maximum Blimp-1 upregulation and optimal PC differentiation required both IL-21 and CD40L. CD40L, although having no effect on Blimp-1 as a single agent, greatly augmented the amplitude and duration of IL-21-triggered Jak-STAT3 signaling. In the human PRDM1 locus, CD40L treatment enhanced the ability of STAT3 to upregulate Blimp-1 by removing BCL6, a potent inhibitor of Blimp-1 expression, from a shared BCL6/STAT3 site in intron 3. Thus, IL-21 and CD40L collaborate through at least two distinct mechanisms to synergistically promote Blimp-1 activation and PC differentiation.

  17. Hepatitis B virus X protein promotes hepatoma cell proliferation via upregulation of MEKK2

    Institute of Scientific and Technical Information of China (English)

    Guang-yao KONG; Jun-ping ZHANG; Shuai ZHANG; Chang-liang SHAN; Li-hong YE; Xiao-dong ZHANG

    2011-01-01

    To investigate the mechanism underlying the increase of hepatoma cell proliferation by hepatitis B virus X protein (HBx).Methods:HepG2,H7402 and HepG2.2.15 cells,which constitutively replicated hepatitis B virus were used.The effects of HBx on hepatoma cell proliferation were examined using 5-ethynyl-2-deoxyuridine (EdU) incorporation assay and MTT assay.The expression level of MEKK2 was measured using RT-PCR,Western blot and luciferase reporter gene assay.The activity of activator protein 1 (AP-1) was detected using luciferase reporter gene assay.The phosphorylation levels of JNK and c-Jun were measured using Western blot.The expression levels of HBx and MEKK2 in 11 clinical hepatocellular carcinoma (HCC) tissues were measured using real time PCR and Western blot.In addition,the expression of MEKK2 in 95 clinical HCC tissues was examined using immunohistochemistry.Results:HBx significantly enhanced HepG2-X cell proliferation.In HepG2-X,H7402-X and HepG2.2.15 cells,the expression level of MEKK2 was remarkably increased.In HepG2.2.15 cells,HBx was found to activate JNK and AP-1,which were the downstream effectors of MEKK2 in HepG2-X and HepG2.2.15 cells.In 11 clinical HCC tissues,both HBx and MEKK2 expression levels were remarkably increased,as compared to those in the corresponding peritumor tissues.In 95 clinical HCC tissues,the rate of detection of MEKK2 was 85.3%.Conclusion:HBx promotes hepatoma cell proliferation via upregulating MEKK2,which may be involved in hepatocarcinogenesis.

  18. Ibrutinib inhibits CD20 upregulation on CLL B cells mediated by the CXCR4/SDF-1 axis.

    Science.gov (United States)

    Pavlasova, Gabriela; Borsky, Marek; Seda, Vaclav; Cerna, Katerina; Osickova, Jitka; Doubek, Michael; Mayer, Jiri; Calogero, Raffaele; Trbusek, Martin; Pospisilova, Sarka; Davids, Matthew S; Kipps, Thomas J; Brown, Jennifer R; Mraz, Marek

    2016-09-22

    Agents targeting B-cell receptor (BCR) signaling-associated kinases such as Bruton tyrosine kinase (BTK) or phosphatidylinositol 3-kinase can induce mobilization of neoplastic B cells from the lymphoid tissues into the blood, which makes them potentially ideal to combine with anti-CD20 monoclonal antibodies (such as rituximab, obinutuzumab, or ofatumumab) for treatment of B-cell lymphomas and chronic lymphocytic leukemia (CLL). Here we show that interactions between leukemia cells and stromal cells (HS-5) upregulate CD20 on CLL cells and that administering ibrutinib downmodulates CD20 (MS4A1) expression in vivo. We observed that CLL cells that have recently exited the lymph node microenvironment and moved into the peripheral blood (CXCR4(dim)CD5(bright) subpopulation) have higher cell surface levels of CD20 than the cells circulating in the bloodstream for a longer time (CXCR4(bright)CD5(dim) cells). We found that CD20 is directly upregulated by CXCR4 ligand stromal cell-derived factor 1 (SDF-1α, CXCL12) produced by stromal cells, and BTK-inhibitor ibrutinib and CXCR4-inhibitor plerixafor block SDF-1α-mediated CD20 upregulation. Ibrutinib also downmodulated Mcl1 levels in CLL cells in vivo and in coculture with stromal cells. Overall, our study provides a first detailed mechanistic explanation of CD20 expression regulation in the context of chemokine signaling and microenvironmental interactions, which may have important implications for microenvironment-targeting therapies.

  19. Na(+)-K(+)-ATPase expression in alveolar epithelial cells: upregulation of active ion transport by KGF.

    Science.gov (United States)

    Borok, Z; Danto, S I; Dimen, L L; Zhang, X L; Lubman, R L

    1998-01-01

    We evaluated the effects of keratinocyte growth factor (KGF) on alveolar epithelial cell (AEC) active ion transport and on rat epithelial Na channel (rENaC) subunit and Na(+)-K(+)-adenosinetriphosphatase (ATPase) subunit isoform expression using monolayers of AEC grown in primary culture. Rat alveolar type II cells were plated on polycarbonate filters in serum-free medium, and KGF (10 ng/ml) was added to confluent AEC monolayers on day 4 in culture. Exposure of AEC monolayers to KGF on day 4 resulted in dose-dependent increases in short-circuit current (Isc) compared with controls by day 5, with further increases occurring through day 8. Relative Na(+)-K(+)-ATPase alpha 1-subunit mRNA abundance was increased by 41% on days 6 and 8 after exposure to KGF, whereas alpha 2-subunit mRNA remained only marginally detectable in both the absence and presence of KGF. Levels of mRNA for the beta 1-subunit of Na(+)-K(+)-ATPase did not increase, whereas cellular alpha 1- and beta 1-subunit protein increased 70 and 31%, respectively, on day 6. mRNA for alpha-, beta-, and gamma-rENaC all decreased in abundance after treatment with KGF. These results indicate that KGF upregulates active ion transport across AEC monolayers via a KGF-induced increase in Na pumps, primarily due to increased Na(+)-K(+)-ATPase alpha 1-subunit mRNA expression. We conclude that KGF may enhance alveolar fluid clearance after acute lung injury by upregulating Na pump expression and transepithelial Na transport across the alveolar epithelium.

  20. Metformin ameliorates insulin resistance in L6 rat skeletal muscle cells through upregulation of SIRT3

    Institute of Scientific and Technical Information of China (English)

    Song Yuping; Shi Jingli; Wu Ying; Han Chong; Zou Junjie; Shi Yongquan; Liu Zhimin

    2014-01-01

    Background SIRT3 is an important regulator in cell metabolism,and recent studies have shown that it may be involved in the pharmacological effects of mefformin.However,the molecular mechanisms underlying this process are unclear.Methods The effects of SIRT3 on the regulation of oxidative stress and insulin resistance in skeletal muscle were evaluated in vitro.Differentiated L6 skeletal muscle cells were treated with 750 μmol/L palmitic acid to induce insulin resistance.SIRT3 was knocked down and overexpressed in L6 cells.SIRT3,nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) p65,c-Jun N-terminal kinase 1 (JNK1),and superoxide dismutase 2 (SOD2) were evaluated by Western blotting.Results Over expression of SIRT3 increased glucose uptake and decreased ROS production in L6-IR cells as well as in L6 cells.Knock-down of SIRT3 induced increased production of ROS while decreased glucose uptake in both L6 and L6-IR cells,and these effects were reversed by N-acetyl-L-cysteine (NAC).Metformin increased the expression of SIRT3 (1.5-fold) and SOD2 (2-fold) while down regulating NF-κB p65 (1.5-fold) and JNK1 (1.5-fold).Knockdown of SIRT3 (P<0.05)reversed the metformin-induced decreases in NF-κB p65 and JNK1 and the mefformin-induced increase in SOD2 (P<0.05).Conclusions Upregulated SIRT3 is involved in the pharmacological mechanism by which mefformin promotes glucose uptake.Additionally,SIRT3 may function as an important regulator of oxidative stress and a new alternative approach for targeting insulin resistance-related diseases.

  1. Stat3 promotes invasion of esophageal squamous cell carcinoma through up-regulation of MMP2.

    Science.gov (United States)

    Xuan, Xaioyan; Li, Shanshan; Lou, Xi; Zheng, Xianzhao; Li, Yunyun; Wang, Feng; Gao, Yuan; Zhang, Hongyan; He, Hongliu; Zeng, Qingru

    2015-05-01

    Stat3 alters the expression of its downstream genes and is associated with tumor invasion and metastasis in several human cancers. Its role in esophageal squamous cell carcinoma (ESCC) has not been well characterized. We examined the tumor sections of 100 cases of ESCC by immunohistochemistry and observed significant overexpression of Stat3 in the cytoplasm of 89% of ESCC cells and of phosphorylated Stat3 (p-Stat3) in the nuclei of 71% of ESCC when compare with normal esophageal mucosa (72%, p = 0.02; and 31%, p = 0.001). Overexpression of Stat3 and p-Stat3 positively correlated with that of matrix metalloproteinase-2 (MMP2), a known regulator for cell migration, in 65% of ESCC while only 26% shown in benign esophageal mucosa. To further investigate the association of Stat3 with tumor metastasis in vitro, invasion of EC-1 cells (a human ESCC cell line) were investigated with Boyden chambers. The results showed that transfection of Stat3 not only promoted invasion of EC-1 cells but also significantly induced MMP2 expression in a dose-dependent manner. In contrast, suppressing expression of endogenous Stat3 mRNA and protein by Stat3 siRNA significantly reduced EC-1 cell invasion and MMP2 expression. A high-affinity Stat3-binding element was localized to the positions of 648-641 bp (TTCTCGAA) in the MMP2 promoter with electrophoretic mobility shift assay. Our results suggest that Stat3, p-Stat3, and MMP2 were overexpressed in ESCC and associated with invasion of ESCC; and Stat3 up-regulated expression of MMP2 in ESCC through directly binding to the MMP2 promoter.

  2. p53-independent upregulation of p21WAF1 in NIH 3T3 cells malignantly transformed by mot-2

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    ot-2 protein is shown to interact with p53 and inhibit its transcriptional activation function.Mot-2 overexpressing stable clones of NIH 3T3 cells were malignantly transformed,however,they had a high level of expression of a p53 downstream gene,P21waf1.The present study was undertaken to elucidate possible molecular mechanism(s) of such upregulation.An increased level of P21waf1 expression was detected in stable transfectants although an exogenous reporter gene driven by P21waf1 promoter exhibited lower activity in these cells suggesting that some post-transcriptional mechanism contributes to upregulation.Western analyses of transient and stable clones revealed that upregulation of P21waf1 in stable NIH 3T3/mot-2 cells may be mediated by cyclin D1 and cdk-2.

  3. Effects of notoginosides on proliferation and upregulation of GR nuclear transcription factor in hematopoietic cells

    Institute of Scientific and Technical Information of China (English)

    Rui-lan GAO; Xiao-hong CHEN; Xiao-jie LIN; Xu-dai QIAN; Wei-hong XU; Beng Hock CHONC

    2007-01-01

    Aim: To investigate the effects of panax notoginosides (PNS) on the proliferation of human hematopoietic stem/progenitor cells, and to explore the signaling path-way of the nuclear transcription factor of the glucocorticoid receptor (GR-NTF) initiated by PNS related with the proliferation. Methods: The human CD34+ cells and bone marrow nuclear cells were exposed to PNS at a concentration of 0, 10, 25,50, and 100 mg/L, respectively, in semi-solid culture system to observe colony forming unite of all lineages, granulocyte, erythrocyte, and megakaryocyte (CFU-GEMM, CFU-GM, CFU-E, and CFU-MK). Three lineages of human hematopoietic cell lines, including granulocytic HL-60, erythrocytic K562, megakaryocytic CHRF-288, and Meg-01 cells were incubated with PNS at 20 mg/L for 14 d. Meanwhile,dexamethasone (Dex) was used as a positive control. The nuclear protein of the cells was analyzed by Western blotting with monoclonal antibodies against the amino or carboxyl terminus of GR-NTF. Electrophoretic mobility shift assay per-formed by using the 32p-radiolabeled GR-NTF consensus oligonucleotide. Results:PNS promoted the proliferation of CD34+ cells and significantly raised the colony numbers of CFU-GEMM by 34.7%~±16.0% over the non-PNS control (P<0.01).PNS also enhanced the proliferation of CFU-GM, CFU-E, and CFU-MK by 39.3%±5.7%, 33.3%±7.3%, and 26.2%±3.2%, respectively. GR-NTF protein levels of either the amino or carboxyl terminus in K562, CHRF-288, and Meg-01 treated by PNS increased by 2.4- 2.8 fold and 1.3- 3.9 fold over the untreated cells. GR-NTF binding activity, initiated by either PNS or Dex, was apparently elevated to form the complex of GR-NTF with DNA as higher density bands in K562 and CHRF-288 cells, and some activity appeared as a band in HL-60 cells induced by PNS.Conclusion: PNS displayed the action of hematopoietic growth factor-like or syn-ergistic efficacy to promote proliferation of human progenitor cells, may play a role in the upregulation of gene

  4. Tangeretin induces cell cycle arrest and apoptosis through upregulation of PTEN expression in glioma cells.

    Science.gov (United States)

    Ma, Li-Li; Wang, Da-Wei; Yu, Xu-Dong; Zhou, Yan-Ling

    2016-07-01

    Tangeretin (TANG), present in peel of citrus fruits, has been shown to various medicinal properties such as chemopreventive and neuroprotective. However, the chemopreventive effect of TANG on glioblastoma cells has not been examined. The present study was designed to explore the anticancer potential of TANG in glioblastoma cells and to investigate the related mechanism. Human glioblastoma U-87MG and LN-18 cells were treated with 45μM concentration of TANG and cell growth was measured by MTT assay. The cell cycle distribution and cell death were measured by flow cytometry. The expression of cell cycle and apoptosis related genes were analyzed by quantitative RT-PCR and western blot. The cells treated with TANG were significantly increased cell growth suppression and cell death effects than vehicle treated cells. Further, TANG treatment increases G2/M arrest and apoptosis by modulating PTEN and cell-cycle regulated genes such as cyclin-D and cdc-2 mRNA and protein expressions. Moreover, the ability of TANG to decrease cell growth and to induce cell death was compromised when PTEN was knockdown by siRNA. Taken together, the chemopreventive effect of TANG is associated with regulation of cell-cycle and apoptosis in glioblastoma, thereby attenuating glioblastoma cell growth. Hence, the present findings suggest that TANG may be a therapeutic agent for glioblastoma treatment.

  5. Rab25 upregulation correlates with the proliferation, migration, and invasion of renal cell carcinoma

    Energy Technology Data Exchange (ETDEWEB)

    Li, Yuanyuan; Jia, Qingzhu [Biomedical Analysis Center, Third Military Medical University, Chongqing (China); Chongqing Key Laboratory of Cytomics, Chongqing (China); Zhang, Qian [Department of Urology, Xinqiao Hospital, Third Military Medical University, Chongqing (China); Wan, Ying, E-mail: wanying_cn@163.com [Biomedical Analysis Center, Third Military Medical University, Chongqing (China); Chongqing Key Laboratory of Cytomics, Chongqing (China)

    2015-03-20

    Renal cell carcinoma (RCC) is a common urological cancer with a poor prognosis. A recent cohort study revealed that the median survival of RCC patients was only 1.5 years and that <10% of the patients in the study survived up to 5 years. In tumor development, Rab GTPase are known to play potential roles such as regulation of cell proliferation, migration, invasion, communication, and drug resistance in multiple tumors. However, the correlation between Rabs expression and the occurrence, development, and metastasis of RCC remains unclear. In this study, we analyzed the transcriptional levels of 52 Rab GTPases in RCC patients. Our results showed that high levels of Rab25 expression were significantly correlated with RCC invasion classification (P < 0.01), lymph-node metastasis (P < 0.001), and pathological stage (P < 0.01). Conversely, in 786-O and A-498 cells, knocking down Rab25 protein expression inhibited cell proliferation, migration, and invasion. Our results also demonstrated that Rab25 is a target gene of let-7d, and further suggested that Rab25 upregulation in RCC is due to diminished expression of let-7d. These findings indicate that Rab25 might be a novel candidate molecule involved in RCC development, thus identifying a potential biological therapeutic target for RCC. - Highlights: • The transcriptional levels of 52 Rab GTPases were analyzed in renal cell carcinoma (RCC). • High levels of Rab25 expression were significantly correlated with clinicopathological factors of RCC. • Knockdown of Rab25 protein expression reduced RCC cells proliferation, migration, and invasion. • Rab25 is a target gene of let-7d in RCC.

  6. IL-1β up-regulates expression of IL-8 in endometrial stromal cells in vitro

    Institute of Scientific and Technical Information of China (English)

    Zhang Guiyu; Ren Shuwen; Zhang Youzhong; Yang Xingsheng

    2005-01-01

    Objective:To investigate the effects of interleukin-1beta (IL-1β) on expression of IL-8 in endometrial stromal cells (ESC) and evaluate the relationship between IL1 β and IL-8 ,and the significance of IL-1β in the development of endometriosis. Methods:The endometrial stromal cells obtained from patient with and without endometriosis cultured within 3 ~5 passage were exposed to various concentrations of IL-1β. The amount of IL-8 protein was assessed by ELISA. The expression of IL-8 mRNA was determined by RT-PCR. Results: 1. IL-8 protein was detected in culture supernatant of which the cells were not treated with IL-1β. The amount of IL-8 protein secretion increased obviously after stimulation with IL-1β at 1.0ng/ml for 4h and the peak of secretion was at 12h. 2. Expression of IL-8 mRNA was positive in unstimulated endometrial stromal cells. However, after stromal cells were incubated with IL-1β, the intensity of expression of IL-8 mRNA was obviously increased and demonstrated a dose-and timedependent manner. Increase of IL-8 mRNA was observed following stimulation with IL-1β for 4h ,and the peak at 12h. Conclusions:IL-1β induces endometrial stromal cell of endometriosis to express IL-8 not only at transcription level but also at post-transcription level. This up-regulation is dose-and time-dependent. IL-1β may play an important role in the onset of endometriosis.

  7. Up-regulated manganese superoxide dismutase expression increases apoptosis resistance in human esophageal squamous cell carcinomas

    Institute of Scientific and Technical Information of China (English)

    HU Hai; WANG Ming-rong; LUO Man-li; DU Xiao-li; FENG Yan-bin; ZHANG Yu; SHEN Xiao-ming; XU Xin; CAI Yan; HAN Ya-ling

    2007-01-01

    Background Esophageal cancer is one of the most common malignancies in the world.In order to identify the proteins associated with esophageal squamous cell carcinomas(ESCC),we analyzed the protein profiles of ESCC cases with tumor and matched adjacent normal tissues.Methods Two-dimensional electrophoresis(2-DE)was carried out to analyze the protein profiles.Dysregulated protein spots were identified by Matrix-Assisted Laser Desorption Ionization Time-of-Flight(MALDI-TOF)and verified by liquid chromatography/electrospray ionization ion trap-mass spectrometry/mass spectrometry(LC-ESI-IT MS).RT-PCR and immunohistochemistry on tissue microarray were performed to confirm the gene dysregulation in esophageal cancerous tissues.RNA interference (RNAi)was used to knock down the gene expression in ESCC cell lines.Apoptosis assay with annexin V-FITC/PI staining was conducted and cells were analyzed by flow cytometry.Results 2-DE showed that two protein spots with approximate molecular weights and different pl were elevated in 12 out of 18 ESCCs as compared to the corresponding normal tissues.Both the two spots were identified as MnSOD by MALDI-TOF and were verified by LC-ESI-IT MS.MnSOD overexpression was detected in 14 tumors out of 24 cases by RT-PCR and 52 tumors out of 116 cases by immunohistochemistry comparing to normal epithelia.siRNA-mediated silencing of MnSOD in KYSE450 and KYSE150 cell lines revealed that MnSOD protected ESCC cells from apoptosis induced by ultraviolet(UV)and doxorubicin(DOX).Conclusions These findings suggest that there existed two isoforms of MnSOD protein in normal and tumor esophageal tissues.MnSOD was overexpressed in ESCC and its up-regulation in esophageal cancer cells was associated with apoptosis resistance.

  8. On active disturbance rejection in temperature regulation of the proton exchange membrane fuel cells

    Science.gov (United States)

    Li, Dazi; Li, Chong; Gao, Zhiqiang; Jin, Qibing

    2015-06-01

    Operating a Proton Exchange Membrane fuel cell (PEMFC) system to maintain the stack temperature stable is one of the key issues in PEMFC's normal electrochemical reaction process. Its temperature characteristic is easily affected by inlet gas humidity, external disturbances, and electrical load changes and so on. Because of the complexity and nonlinearity of the reaction process, it is hard to build a model totally consistent with the real characteristic of the process. If model uncertainty, external disturbances, parameters changes can be regarded as "total disturbance", which is then estimated and compensated, the accurate model is no longer required and the control design can be greatly simplified to meet the practical needs. Based on this idea, an active disturbance rejection control (ADRC) with a switching law is proposed for the problem of precise temperature regulation in PEMFC. Results of the work show that the proposed control system allows the PEMFC to operate successfully at the temperature of 343 K point in the presence of two different disturbances.

  9. Up-regulation of hexokinaseII in myeloma cells: targeting myeloma cells with 3-bromopyruvate.

    Science.gov (United States)

    Nakano, Ayako; Miki, Hirokazu; Nakamura, Shingen; Harada, Takeshi; Oda, Asuka; Amou, Hiroe; Fujii, Shiro; Kagawa, Kumiko; Takeuchi, Kyoko; Ozaki, Shuji; Matsumoto, Toshio; Abe, Masahiro

    2012-02-01

    Hexokinase II (HKII), a key enzyme of glycolysis, is widely over-expressed in cancer cells. However, HKII levels and its roles in ATP production and ATP-dependent cellular process have not been well studied in hematopoietic malignant cells including multiple myeloma (MM) cells.We demonstrate herein that HKII is constitutively over-expressed in MM cells. 3-bromopyruvate (3BrPA), an inhibitor of HKII, promptly and substantially suppresses ATP production and induces cell death in MM cells. Interestingly, cocultures with osteoclasts (OCs) but not bone marrow stromal cells (BMSCs) enhanced the phosphorylation of Akt along with an increase in HKII levels and lactate production in MM cells. The enhancement of HKII levels and lactate production in MM cells by OCs were mostly abrogated by the PI3K inhibitor LY294002, suggesting activation of glycolysis in MM cells by OCs via the PI3K-Akt-HKII pathway. Although BMSCs and OCs stimulate MM cell growth and survival, 3BrPA induces cell death in MM cells even in cocultures with OCs as well as BMSCs. Furthermore, 3BrPA was able to diminish ATP-dependent ABC transporter activity to restore drug retention in MM cells in the presence of OCs. These results may underpin possible clinical application of 3BrPA in patients with MM.

  10. Mutant p53 confers chemoresistance in non-small cell lung cancer by upregulating Nrf2.

    Science.gov (United States)

    Tung, Min-Che; Lin, Po-Lin; Wang, Yao-Chen; He, Tsung-Ying; Lee, Ming-Ching; Yeh, Sauh D; Chen, Chih-Yi; Lee, Huei

    2015-12-08

    Nrf2 is a key transcription factor for genes coding for antioxidants, detoxification enzymes, and multiple drug resistance and it also confers resistance to anticancer drugs. Here, we hypothesized that mutant p53 could upregulate Nrf2 expression at the transcriptional level, thereby conferring cisplatin resistance in non-small cell lung cancer (NSCLC). Luciferase reporter assays and real-time PCR analysis indicated that the Nrf2 promoter activity and its mRNA levels were markedly suppressed by wild-type p53, but not by mutant p53. Chromatin immunoprecipitation (ChIP) further confirmed that wild-type p53 binds at the p53 putative binding site to block Sp1 binding to the Nrf2 promoter and consequently to suppress the Nrf2 promoter activity. The MTT assay indicated that an increase in Nrf2 expression by mutant p53 is responsible for cisplatin resistance. Among the Nrf2 downstream genes, Bcl-2 and Bcl-xL contribute more strongly to Nrf2-mediated cisplatin resistance when compared with heme oxygenase 1 (HO-1). Cox regression analysis showed that patients with high-Nrf2, high-Bcl-2, high-Bcl-xL mRNA tumors were more commonly occurred unfavorable response to cisplatin-based chemotherapy than their counterparts. The prognostic significance of Nrf2 mRNA levels on OS and RFS was also observed in patients who have received cisplatin-based chemotherapy, particularly in p53-mutant patients. Collectively, mutant p53 may confer cisplatin resistance via upregulation of Nrf2 expression, and Nrf2 mRNA level may predict chemotherapeutic response and outcomes in NSCLC.

  11. Coordinating Etk/Bmx activation and VEGF upregulation to promote cell survival and proliferation.

    Science.gov (United States)

    Chau, Cindy H; Chen, Kai-Yun; Deng, Hong-Tao; Kim, Kwang-Jin; Hosoya, Ken-ichi; Terasaki, Tetsuya; Shih, Hsiu-Ming; Ann, David K

    2002-12-12

    Etk/Bmx, a member of the Tec family of non-receptor tyrosine kinase, is characterized by an N-terminal PH domain and has recently been shown to be involved in the regulation of various cellular processes, including proliferation, differentiation, motility and apoptosis. Since VEGF and the activation of its signaling pathway have been implicated in modulating a variety of biological responses, we characterized the role of Etk-dependent signaling pathways involved in the upregulation of VEGF expression, and explored the functional implications of this enhancement in sustaining cell proliferation and survival. Using Northern and Western analyses, transient transfections, and pharmacological agents, we demonstrate that Etk activation alone is sufficient to transcriptionally induce VEGF expression, independent of the previously identified hypoxia response element (HRE), in both Pa-4 epithelial and TR-BBB endothelial cells under normoxia. In addition, Etk utilizes both MEK/ERK and PI3-K/Pak1 signaling pathways in concert to activate VEGF transcription. Functionally, Etk activation elicits a profound stimulatory effect on TR-BBB cell proliferation and formation of capillary-like networks in Matrigel containing reduced levels of growth factors. Finally, antisense oligonucleotides against either endogenous VEGF or Etk abrogate the proliferation of Etk-activated TR-BBB cells, and exogenous VEGF treatment stimulates endogenous Etk tyrosine phosphorylation in HUVECs. Taken together, these results indicate that VEGF is both an Etk downstream target gene and an Etk upstream activator, constituting a reciprocal Etk-VEGF autoregulatory loop. These findings, to our knowledge, are the first delineation of a network of positive feedforward signaling pathways that converge on the Etk-VEGF axis, causally associating Etk-mediation of VEGF induction with enhanced cellular processes in both epithelial and endothelial cells.

  12. NRF2 activation is involved in ozonated human serum upregulation of HO-1 in endothelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Pecorelli, Alessandra [Department of Molecular and Developmental Medicine, University of Siena (Italy); Child Neuropsychiatry Unit, University Hospital, AOUS, Siena (Italy); Bocci, Velio [Department of Physiology, University of Siena (Italy); Acquaviva, Alessandra [Department of Molecular and Developmental Medicine, University of Siena (Italy); Belmonte, Giuseppe [Department of Biomedical Sciences, University of Siena (Italy); Gardi, Concetta [Department of Molecular and Developmental Medicine, University of Siena (Italy); Virgili, Fabio [INRAN, Rome (Italy); Ciccoli, Lucia [Department of Molecular and Developmental Medicine, University of Siena (Italy); Valacchi, Giuseppe, E-mail: giuseppe.valacchi@unife.it [Department of Life Sciences and Biotechnology, University of Ferrara (Italy); Department of Food and Nutrition, Kyung Hee University, Seoul (Korea, Republic of)

    2013-02-15

    During the last decade, it has been shown that the activation of NRF2 and the binding to electrophile-responsive element (EpREs), stimulates the expression of a great number of genes responsible for the synthesis of phase I and phase II proteins, including antioxidants enzymes and heme oxygenase-1 (HO-1). This critical cell response occurs in cardiovascular, degenerative and chronic infective diseases aggravated by a chronic oxidative stress. In our previous reports we have shown that ozonated plasma is able to up-regulate HO-1 expression in endothelial cells. In the present work we investigated a candidate mechanism involved in this process. After treatment with increasing doses of ozonated serum (20, 40 and 80 μg/mL O{sub 3} per mL of serum), a clear dose dependent activation of NRF2 and the subsequent induction of HO-1 and NAD(P)H quinone oxidoreductase 1(NQO1) was observed. This effect was also present when cells were treated with serum and hydrogen peroxide (H{sub 2}O{sub 2}) or serum and 4-hydroxynonenal (4HNE). Moreover, the treatment with ozonated serum was associated with a dose-dependent activation of extracellular-signal-regulated kinases (ERK1/2) and p38 MAP kinases (p38), not directly involved in NRF2 activation. These data, provide a new insight on the mechanism responsible for the induction of HO-1 expression by ozonated serum in the endothelium, and have a practical importance as an expedient approach to the treatment of patients with both effective orthodox drugs and ozonated autohemotherapy, targeted to the restoration of redox homeostasis. - Highlights: ► Endothelial HO1 is upregulated by ozonated plasma ► This activation is induced by NRF2 and it is ERK independent. ► 4HNE and H{sub 2}O{sub 2} are the main molecules involved in this process. ► Ozonated plasma induced a hormetic effect ► Combination of orthodox medicine and ozonated plasma can be a useful treatment.

  13. Upregulation of Renin-Angiotensin System in Bone Marrow Mesenchymal Stem Cells Under Hypoxia Conditions

    Institute of Scientific and Technical Information of China (English)

    XIAO Rong-rong; GAO Jing-hong; LI Qing-ping

    2014-01-01

    Objective:To investigate the expressions of AT1-R, AT2-R and angiotensin converting enzyme (ACE) in mesenchymal stem cells (MSCs) under hypoxia and serum deprivation condition. Methods:Bone MSCs were isolated, cultured and identiifed by anti-CD29 and anti-CD11b/c with flow cytometry. The ischemic injury model was established by exposing MSCs to hypoxia and serum deprivation (Hypoxia/SD). Cell viability and apoptotic rate were detected by trypan blue staining, CCK8 assays and Annexin V-FITC staining. The mRNA expressions of AT1-R, AT2-R and ACE were determined by Reverse Transcription-PCR and Real-time Quantitative PCR, The expression of AT1-R, AT2-R and ACE protein were measured by Western-blot. Results:MSCs expressed CD29, but not the CD11b/c. The purity of MSCs employed was up to 97%. The results of trypan blue staining along with CCK8 and Annexin V-FITC staining proved that the injury model induced by Hypoxia/SD was successfully established. MSCs under hypoxia and serum deprivation for 24 h induced a rapid increase in mRNA expression of AT1-R, AT2-R and ACE as well as their protein expressions. Conclusion:The local RAS in MSCs is activated by Hypoxia/SD stimulation and the mRNA and protein expressions of AT1-R, AT2-R and ACE are up-regulated.

  14. LeY oligosaccharide upregulates DAG/PKC signaling pathway in the human endometrial cells.

    Science.gov (United States)

    Li, Yali; Ma, Keli; Sun, Ping; Liu, Shuai; Qin, Huamin; Zhu, Zhengmei; Wang, Xiaoqi; Yan, Qiu

    2009-11-01

    LeY oligosaccharide is stage specifically expressed by the embryo and uterine endometrium, and it plays important roles in embryo implantation. In addition to participating in the recognition and adhesion on fetal-maternal interface, LeY potentially regulates the expression of some implantation-related factors. However, it remains elusive whether it can mediate the involved signaling pathway. In this study, agarose-LeY beads were used to mimic the embryos, and the effects of LeY oligosaccharide on DAG/PKC signaling pathway was studied in human endometrial epithelial cells. Results showed that LeY could significantly trigger the activation of cPKCalpha and cPKCbeta2, and their translocation from the cytosol to the plasma membrane. The cellular DAG content was also upregulated, and the activation of PLCgamma1 was promoted. On the contrary, DAG/PKC signaling pathway was significantly inhibited when anti-LeY antibody was used after confirmation of LeY expression in human endometrial epithelial cells by immunohistochemistry and flow cytometry. These results suggest that LeY oligosaccharide acts as a signal molecule to modulate DAG/PKC signaling pathway.

  15. Growth Arrest Specific 2 Is Up-Regulated in Chronic Myeloid Leukemia Cells and Required for Their Growth

    OpenAIRE

    Haixia Zhou; Yue Ge; Lili Sun; Wenjuan Ma; Jie Wu; Xiuyan Zhang; Xiaohui Hu; Eaves, Connie J; Depei Wu; Yun Zhao

    2014-01-01

    Although the generation of BCR-ABL is the molecular hallmark of chronic myeloid leukemia (CML), the comprehensive molecular mechanisms of the disease remain unclear yet. Growth arrest specific 2 (GAS2) regulates multiple cellular functions including cell cycle, apoptosis and calpain activities. In the present study, we found GAS2 was up-regulated in CML cells including CD34+ progenitor cells compared to their normal counterparts. We utilized RNAi and the expression of dominant negative form o...

  16. Hyperforin induces apoptosis of chronic lymphocytic leukemia cells through upregulation of the BH3-only protein Noxa.

    Science.gov (United States)

    Zaher, Murhaf; Tang, Ruoping; Bombarda, Isabelle; Merhi, Faten; Bauvois, Brigitte; Billard, Christian

    2012-01-01

    We previously reported that hyperforin, a phloroglucinol purified from Hypericum perforatum, induces the mitochondrial pathway of caspase-dependent apoptosis in chronic lymphocytic leukemia (CLL) cells ex vivo, and that this effect is associated with upregulation of Noxa, a BH3-only protein of the Bcl-2 family. Here, we investigated the role of this upregulation in the pro-apoptotic activity of hyperforin in the cells of CLL patients and MEC-1 cell line. We found that the increase in Noxa expression is a time- and concentration-dependent effect of hyperforin occurring without change in Noxa mRNA levels. A post-translational regulation is suggested by the capacity of hyperforin to inhibit proteasome activity in CLL cells. Noxa silencing by siRNA reduces partially hyperforin-elicited apoptosis. Furthermore, treatment with hyperforin, which has no effect on the expression of the prosurvival protein Mcl-1, induces the interaction of Noxa with Mcl-1 and the dissociation of Mcl-1/Bak complex, revealing that upregulated Noxa displaces the proapoptotic protein Bak from Mcl-1. This effect is accompanied with Bak activation, known to allow the release of apoptogenic factors from mitochondria. Our data indicate that Noxa upregulation is one of the mechanisms by which hyperforin triggers CLL cell apoptosis. They also favor that new agents capable of mimicking specifically the BH3-only protein Noxa should be developed for apoptosis-based therapeutic strategy in CLL.

  17. Upregulation of the cell-cycle regulator RGC-32 in Epstein-Barr virus-immortalized cells.

    Directory of Open Access Journals (Sweden)

    Sandra N Schlick

    Full Text Available Epstein-Barr virus (EBV is implicated in the pathogenesis of multiple human tumours of lymphoid and epithelial origin. The virus infects and immortalizes B cells establishing a persistent latent infection characterized by varying patterns of EBV latent gene expression (latency 0, I, II and III. The CDK1 activator, Response Gene to Complement-32 (RGC-32, C13ORF15, is overexpressed in colon, breast and ovarian cancer tissues and we have detected selective high-level RGC-32 protein expression in EBV-immortalized latency III cells. Significantly, we show that overexpression of RGC-32 in B cells is sufficient to disrupt G2 cell-cycle arrest consistent with activation of CDK1, implicating RGC-32 in the EBV transformation process. Surprisingly, RGC-32 mRNA is expressed at high levels in latency I Burkitt's lymphoma (BL cells and in some EBV-negative BL cell-lines, although RGC-32 protein expression is not detectable. We show that RGC-32 mRNA expression is elevated in latency I cells due to transcriptional activation by high levels of the differentially expressed RUNX1c transcription factor. We found that proteosomal degradation or blocked cytoplasmic export of the RGC-32 message were not responsible for the lack of RGC-32 protein expression in latency I cells. Significantly, analysis of the ribosomal association of the RGC-32 mRNA in latency I and latency III cells revealed that RGC-32 transcripts were associated with multiple ribosomes in both cell-types implicating post-initiation translational repression mechanisms in the block to RGC-32 protein production in latency I cells. In summary, our results are the first to demonstrate RGC-32 protein upregulation in cells transformed by a human tumour virus and to identify post-initiation translational mechanisms as an expression control point for this key cell-cycle regulator.

  18. Upregulation of MiR-122 via Trichostatin A Treatments in Hepatocyte-like Cells Derived from Mesenchymal Stem Cells.

    Science.gov (United States)

    Alizadeh, Effat; Eslaminejad, MohamadReza Baghaban; Akbarzadeh, Abolfazl; Sadeghi, Zohre; Abasi, Mozghan; Herizchi, Roya; Zarghami, Nosratollah

    2016-02-01

    The miR-122 is a tissue-specific miRNA; its expression is abundant in liver. MiR-122 upregulation is crucial for differentiation, functionality, and maintenance of differentiated phenotype in hepatocytes. The improving effects of trichostatin A (TSA) on hepatic differentiation have been reported previously. The aim of this study was to determine whether TSA can affect the expression of miR-122 in hepatocyte-like cells (HLCs) generated from human adipose tissue-derived mesenchymal stem cells (hAT-MSCs). The hepatic differentiation of hAT-MSCs induced by a mixture of growth factors and cytokines either with or without TSA treatments. The functionality of HLCs generated with or without TSA and the expression levels of miR-122 were studied. The expression levels of miR-122 in TSA-treated HLCs was significantly (p < 0.05) higher than those generated by growth factors and cytokines, only. The downregulation of a-fetoprotein (AFP) levels but enhanced albumin synthesis (p < 0.05) and upregulation of liver-enriched transcription factors (LETFs) HNF4α (hepatocyte nuclear factor 4α) and HNF6 (hepatocyte nuclear factor 6) were observed in TSA-treated HLCs (p < 0.05). In conclusion, administration of TSA in hepatogenic differentiation of hAT-MSCs resulted in higher expression levels of miR-122, facilitation of differentiation, and subsequently attenuation of AFP levels.

  19. Antitumor effects of a sirtuin inhibitor, tenovin-6, against gastric cancer cells via death receptor 5 up-regulation.

    Directory of Open Access Journals (Sweden)

    Sachiko Hirai

    Full Text Available Up-regulated sirtuin 1 (SIRT1, an NAD+-dependent class III histone deacetylase, deacetylates p53 and inhibits its transcriptional activity, leading to cell survival. SIRT1 overexpression has been reported to predict poor survival in some malignancies, including gastric cancer. However, the antitumor effect of SIRT1 inhibition remains elusive in gastric cancer. Here, we investigated the antitumor mechanisms of a sirtuin inhibitor, tenovin-6, in seven human gastric cancer cell lines (four cell lines with wild-type TP53, two with mutant-type TP53, and one with null TP53. Interestingly, tenovin-6 induced apoptosis in all cell lines, not only those with wild-type TP53, but also mutant-type and null versions, accompanied by up-regulation of death receptor 5 (DR5. In the KatoIII cell line (TP53-null, DR5 silencing markedly attenuated tenovin-6-induced apoptosis, suggesting that the pivotal mechanism behind its antitumor effects is based on activation of the death receptor signal pathway. Although endoplasmic reticulum stress caused by sirtuin inhibitors was reported to induce DR5 up-regulation in other cancer cell lines, we could not find marked activation of its related molecules, such as ATF6, PERK, and CHOP, in gastric cancer cells treated with tenovin-6. Tenovin-6 in combination with docetaxel or SN-38 exerted a slight to moderate synergistic cytotoxicity against gastric cancer cells. In conclusion, tenovin-6 has potent antitumor activity against human gastric cancer cells via DR5 up-regulation. Our results should be helpful for the future clinical development of sirtuin inhibitors.

  20. Epidrug-induced upregulation of functional somatostatin type 2 receptors in human pancreatic neuroendocrine tumor cells.

    Science.gov (United States)

    Veenstra, Marije J; van Koetsveld, Peter M; Dogan, Fadime; Farrell, William E; Feelders, Richard A; Lamberts, Steven W J; de Herder, Wouter W; Vitale, Giovanni; Hofland, Leo J

    2016-05-19

    Somatostatin receptors are a pivotal target for treatment of pancreatic neuroendocrine tumors (pNET), either with somatostatin analogues (SSA) or radiolabeled SSA. The highest affinity target for the most commonly used SSA is the somatostatin receptor type 2 (sst2). An important factor that may complicate treatment efficacy, is the variable number of receptors expressed on pNETs. Gene expression is subject to complex regulation, in which epigenetics has a central role. In this study we explored the possible role of epigenetic modifications in the variations in sst2 expression levels in two human pNET cell lines, BON-1 and QGP-1. We found upregulation of sst2 mRNA after treatment with the epidrugs 5-aza-2'-deoxycytidine (5-aza-dC) and valproic acid (VPA), an increased uptake of radiolabeled octreotide, as well as increased sensitivity to the SSA octreotide in functional cAMP inhibition. At epigenetic level we observed low methylation levels of the sst2 gene promoter region irrespective of expression. Activating histone mark H3K9Ac can be regulated with epidrug treatment, with an angle of effect corresponding to the effect on mRNA expression. Repressive histone mark H3K27me3 is not regulated by either 5-aza-dC or VPA. We conclude that epidrug treatment, in particular with combined 5-aza-dC and VPA treatment, might hold promise for improving and adding to current SSA treatment strategies of patients with pNETs.

  1. Respiratory virus infection up-regulates TRPV1, TRPA1 and ASICS3 receptors on airway cells

    Science.gov (United States)

    Omar, Shadia; Clarke, Rebecca; Abdullah, Haniah; Brady, Clare; Corry, John; Winter, Hanagh; Touzelet, Olivier; Power, Ultan F.; Lundy, Fionnuala; McGarvey, Lorcan P. A.

    2017-01-01

    Receptors implicated in cough hypersensitivity are transient receptor potential vanilloid 1 (TRPV1), transient receptor potential cation channel, Subfamily A, Member 1 (TRPA1) and acid sensing ion channel receptor 3 (ASIC3). Respiratory viruses, such as respiratory syncytial virus (RSV) and measles virus (MV) may interact directly and/or indirectly with these receptors on sensory nerves and epithelial cells in the airways. We used in vitro models of sensory neurones (SHSY5Y or differentiated IMR-32 cells) and human bronchial epithelium (BEAS-2B cells) as well as primary human bronchial epithelial cells (PBEC) to study the effect of MV and RSV infection on receptor expression. Receptor mRNA and protein levels were examined by qPCR and flow cytometry, respectively, following infection or treatment with UV inactivated virus, virus-induced soluble factors or pelleted virus. Concentrations of a range of cytokines in resultant BEAS-2B and PBEC supernatants were determined by ELISA. Up-regulation of TRPV1, TRPA1 and ASICS3 expression occurred by 12 hours post-infection in each cell type. This was independent of replicating virus, within the same cell, as virus-induced soluble factors alone were sufficient to increase channel expression. IL-8 and IL-6 increased in infected cell supernatants. Antibodies against these factors inhibited TRP receptor up-regulation. Capsazepine treatment inhibited virus induced up-regulation of TRPV1 indicating that these receptors are targets for treating virus-induced cough. PMID:28187208

  2. Disturbance of copper homeostasis is a mechanism for homocysteine-induced vascular endothelial cell injury.

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    Daoyin Dong

    Full Text Available Elevation of serum homocysteine (Hcy levels is a risk factor for cardiovascular diseases. Previous studies suggested that Hcy interferes with copper (Cu metabolism in vascular endothelial cells. The present study was undertaken to test the hypothesis that Hcy-induced disturbance of Cu homeostasis leads to endothelial cell injury. Exposure of human umbilical vein endothelial cells (HUVECs to concentrations of Hcy at 0.01, 0.1 or 1 mM resulted in a concentration-dependent decrease in cell viability and an increase in necrotic cell death. Pretreatment of the cells with a final concentration of 5 µM Cu in cultures prevented the effects of Hcy. Hcy decreased intracellular Cu concentrations. HPLC-ICP-MS analysis revealed that Hcy caused alterations in the distribution of intracellular Cu; more Cu was redistributed to low molecular weight fractions. ESI-Q-TOF detected the formation of Cu-Hcy complexes. Hcy also decreased the protein levels of Cu chaperone COX17, which was accompanied by a decrease in the activity of cytochrome c oxidase (CCO and a collapse of mitochondrial membrane potential. These effects of Hcy were all preventable by Cu pretreatment. The study thus demonstrated that Hcy disturbs Cu homeostasis and limits the availability of Cu to critical molecules such as COX17 and CCO, leading to mitochondrial dysfunction and endothelial cell injury.

  3. MAPK15 upregulation promotes cell proliferation and prevents DNA damage in male germ cell tumors

    Science.gov (United States)

    Ilardi, Gennaro; Acunzo, Mario; Nigita, Giovanni; Sasdelli, Federica; Celetti, Angela; Strambi, Angela; Staibano, Stefania; Croce, Carlo Maria; Chiariello, Mario

    2016-01-01

    Germ cell tumors (GCT) are the most common malignancies in males between 15 and 35 years of age. Despite the high cure rate, achieved through chemotherapy and/or surgery, the molecular basis of GCT etiology is still largely obscure. Here, we show a positive correlation between MAPK15 (ERK8; ERK7) expression and specific GCT subtypes, with the highest levels found in the aggressive embryonal carcinomas (EC). Indeed, in corresponding cellular models for EC, MAPK15 enhanced tumorigenicity in vivo and promoted cell proliferation in vitro, supporting a role for this kinase in human GCT. At molecular level, we demonstrated that endogenous MAPK15 is necessary to sustain cell cycle progression of EC cells, by limiting p53 activation and preventing the triggering of p53-dependent mechanisms resulting in cell cycle arrest. To understand MAPK15-dependent mechanisms impinging on p53 activation, we demonstrate that this kinase efficiently protects cells from DNA damage. Moreover, we show that the ability of MAPK15 to control the autophagic process is necessary for basal management of DNA damage and for tumor formation controlled by the kinase. In conclusion, our findings suggest that MAPK15 overexpression may contribute to the malignant transformation of germ cells by controlling a “stress support” autophagic pathway, able to prevent DNA damage and the consequent activation of the p53 tumor suppressor. Moreover, in light of these results, MAPK15-specific inhibitors might represent new tools to enhance the therapeutic index of cytotoxic therapy in GCT treatment, and to increase the sensitivity to DNA-damaging drugs in other chemotherapy-resistant human tumors. PMID:26988910

  4. PDGF-DD, a novel mediator of smooth muscle cell phenotypic modulation, is upregulated in endothelial cells exposed to atherosclerosis-prone flow patterns.

    Science.gov (United States)

    Thomas, James A; Deaton, Rebecca A; Hastings, Nicole E; Shang, Yueting; Moehle, Christopher W; Eriksson, Ulf; Topouzis, Stavros; Wamhoff, Brian R; Blackman, Brett R; Owens, Gary K

    2009-02-01

    Platelet-derived growth factor (PDGF)-BB is a well-known smooth muscle (SM) cell (SMC) phenotypic modulator that signals by binding to PDGF alphaalpha-, alphabeta-, and betabeta-membrane receptors. PDGF-DD is a recently identified PDGF family member, and its role in SMC phenotypic modulation is unknown. Here we demonstrate that PDGF-DD inhibited expression of multiple SMC genes, including SM alpha-actin and SM myosin heavy chain, and upregulated expression of the potent SMC differentiation repressor gene Kruppel-like factor-4 at the mRNA and protein levels. On the basis of the results of promoter-reporter assays, changes in SMC gene expression were mediated, at least in part, at the level of transcription. Attenuation of the SMC phenotypic modulatory activity of PDGF-DD by pharmacological inhibitors of ERK phosphorylation and by a small interfering RNA to Kruppel-like factor-4 highlight the role of these two pathways in this process. PDGF-DD failed to repress SM alpha-actin and SM myosin heavy chain in mouse SMCs lacking a functional PDGF beta-receptor. Importantly, PDGF-DD expression was increased in neointimal lesions in the aortic arch region of apolipoprotein C-deficient (ApoE(-/-)) mice. Furthermore, human endothelial cells exposed to an atherosclerosis-prone flow pattern, as in vascular regions susceptible to the development of atherosclerosis, exhibited a significant increase in PDGF-DD expression. These findings demonstrate a novel activity for PDGF-DD in SMC biology and highlight the potential contribution of this molecule to SMC phenotypic modulation in the setting of disturbed blood flow.

  5. Investigation and process optimization of SONOS cell's drain disturb in 2-transistor structure flash arrays

    Science.gov (United States)

    Xu, Zhaozhao; Qian, Wensheng; Chen, Hualun; Xiong, Wei; Hu, Jun; Liu, Donghua; Duan, Wenting; Kong, Weiran; Na, Wei; Zou, Shichang

    2017-03-01

    The mechanism and distribution of drain disturb (DD) are investigated in silicon-oxide-nitride-oxide-silicon (SONOS) flash cells. It is shown that DD is the only concern in this paper. First, the distribution of trapped charge in nitride layer is found to be non-localized (trapped in entire nitride layer along the channel) after programming. Likewise, the erase is also non-localized. Then, the main disturb mechanism: Fowler Nordheim tunneling (FNT) has been confirmed in this paper with negligible disturb effect from hot-hole injection (HHI). And then, distribution of DD is confirmed to be non-localized similarly, which denotes that DD exists in entire tunneling oxide (Oxide for short). Next, four process optimization ways are proposed for minimization of DD, and VTH shift is measured. It reveals that optimized lightly doped drain (LDD), halo, and channel implant are required for the fabrication of a robust SONOS cell. Finally, data retention and endurance of the optimized SONOS are demonstrated.

  6. Exposure to Cell Phone Radiation Up-Regulates Apoptosis Genes in Primary Cultures of Neurons and Astrocytes

    OpenAIRE

    Zhao, Tian-Yong; Zou, Shi-Ping; Pamela E Knapp

    2006-01-01

    The health effects of cell phone radiation exposure are a growing public concern. This study investigated whether expression of genes related to cell death pathways are dysregulated in primary cultured neurons and astrocytes by exposure to a working GSM (Global System for Mobile Communication) cell phone rated at a frequency of 1900 MHz. Primary cultures were exposed to cell phone emissions for 2 hrs. We used array analysis and real-time RT-PCR to show up-regulation of caspase-2, caspase-6 an...

  7. Noxa upregulation by oncogenic activation of MEK/ERK through CREB promotes autophagy in human melanoma cells.

    Science.gov (United States)

    Liu, Yi Lun; Lai, Fritz; Wilmott, James S; Yan, Xu Guang; Liu, Xiao Ying; Luan, Qi; Guo, Su Tang; Jiang, Chen Chen; Tseng, Hsin-Yi; Scolyer, Richard A; Jin, Lei; Zhang, Xu Dong

    2014-11-30

    Reduction in the expression of the anti-survival BH3-only proteins PUMA and Bim is associated with the pathogenesis of melanoma. However, we have found that the expression of the other BH3-only protein Noxa is commonly upregulated in melanoma cells, and that this is driven by oncogenic activation of MEK/ERK. Immunohistochemistry studies showed that Noxa was expressed at higher levels in melanomas than nevi. Moreover, the expression of Noxa was increased in metastatic compared to primary melanomas, and in thick primaries compared to thin primaries. Inhibition of oncogenic BRAFV600E or MEK downregulated Noxa, whereas activation of MEK/ERK caused its upregulation. In addition, introduction of BRAFV600E increased Noxa expression in melanocytes. Upregulation of Noxa was due to a transcriptional increase mediated by cAMP responsive element binding protein, activation of which was also increased by MEK/ERK signaling in melanoma cells. Significantly, Noxa appeared necessary for constitutive activation of autophagy, albeit at low levels, by MEK/ERK in melanoma cells. Furthermore, it was required for autophagy activation that delayed apoptosis in melanoma cells undergoing nutrient deprivation. These results reveal that oncogenic activation of MEK/ERK drives Noxa expression to promote autophagy, and suggest that Noxa has an indirect anti-apoptosis role in melanoma cells under nutrient starvation conditions.

  8. HDAC up-regulation in early colon field carcinogenesis is involved in cell tumorigenicity through regulation of chromatin structure.

    Directory of Open Access Journals (Sweden)

    Yolanda Stypula-Cyrus

    Full Text Available Normal cell function is dependent on the proper maintenance of chromatin structure. Regulation of chromatin structure is controlled by histone modifications that directly influence chromatin architecture and genome function. Specifically, the histone deacetylase (HDAC family of proteins modulate chromatin compaction and are commonly dysregulated in many tumors, including colorectal cancer (CRC. However, the role of HDAC proteins in early colorectal carcinogenesis has not been previously reported. We found HDAC1, HDAC2, HDAC3, HDAC5, and HDAC7 all to be up-regulated in the field of human CRC. Furthermore, we observed that HDAC2 up-regulation is one of the earliest events in CRC carcinogenesis and observed this in human field carcinogenesis, the azoxymethane-treated rat model, and in more aggressive colon cancer cell lines. The universality of HDAC2 up-regulation suggests that HDAC2 up-regulation is a novel and important early event in CRC, which may serve as a biomarker. HDAC inhibitors (HDACIs interfere with tumorigenic HDAC activity; however, the precise mechanisms involved in this process remain to be elucidated. We confirmed that HDAC inhibition by valproic acid (VPA targeted the more aggressive cell line. Using nuclease digestion assays and transmission electron microscopy imaging, we observed that VPA treatment induced greater changes in chromatin structure in the more aggressive cell line. Furthermore, we used the novel imaging technique partial wave spectroscopy (PWS to quantify nanoscale alterations in chromatin. We noted that the PWS results are consistent with the biological assays, indicating a greater effect of VPA treatment in the more aggressive cell type. Together, these results demonstrate the importance of HDAC activity in early carcinogenic events and the unique role of higher-order chromatin structure in determining cell tumorigenicity.

  9. HDAC up-regulation in early colon field carcinogenesis is involved in cell tumorigenicity through regulation of chromatin structure.

    Science.gov (United States)

    Stypula-Cyrus, Yolanda; Damania, Dhwanil; Kunte, Dhananjay P; Cruz, Mart Dela; Subramanian, Hariharan; Roy, Hemant K; Backman, Vadim

    2013-01-01

    Normal cell function is dependent on the proper maintenance of chromatin structure. Regulation of chromatin structure is controlled by histone modifications that directly influence chromatin architecture and genome function. Specifically, the histone deacetylase (HDAC) family of proteins modulate chromatin compaction and are commonly dysregulated in many tumors, including colorectal cancer (CRC). However, the role of HDAC proteins in early colorectal carcinogenesis has not been previously reported. We found HDAC1, HDAC2, HDAC3, HDAC5, and HDAC7 all to be up-regulated in the field of human CRC. Furthermore, we observed that HDAC2 up-regulation is one of the earliest events in CRC carcinogenesis and observed this in human field carcinogenesis, the azoxymethane-treated rat model, and in more aggressive colon cancer cell lines. The universality of HDAC2 up-regulation suggests that HDAC2 up-regulation is a novel and important early event in CRC, which may serve as a biomarker. HDAC inhibitors (HDACIs) interfere with tumorigenic HDAC activity; however, the precise mechanisms involved in this process remain to be elucidated. We confirmed that HDAC inhibition by valproic acid (VPA) targeted the more aggressive cell line. Using nuclease digestion assays and transmission electron microscopy imaging, we observed that VPA treatment induced greater changes in chromatin structure in the more aggressive cell line. Furthermore, we used the novel imaging technique partial wave spectroscopy (PWS) to quantify nanoscale alterations in chromatin. We noted that the PWS results are consistent with the biological assays, indicating a greater effect of VPA treatment in the more aggressive cell type. Together, these results demonstrate the importance of HDAC activity in early carcinogenic events and the unique role of higher-order chromatin structure in determining cell tumorigenicity.

  10. Green tea polyphenol stimulates cancer preventive effects of celecoxib in human lung cancer cells by upregulation of GADD153 gene.

    Science.gov (United States)

    Suganuma, Masami; Kurusu, Miki; Suzuki, Kaori; Tasaki, Emi; Fujiki, Hirota

    2006-07-01

    To more clearly understand the molecular mechanisms involved in synergistic enhancement of cancer preventive activity with the green tea polyphenol (-)-epigallocatechin gallate (EGCG), we examined the effects of cotreatment with EGCG plus celecoxib, a cyclooxygenase-2 selective inhibitor. We specifically looked for induction of apoptosis and expression of apoptosis related genes, with emphasis on growth arrest and DNA damage-inducible 153 (GADD153) gene, in human lung cancer cell line PC-9: Cotreatment with EGCG plus celecoxib strongly induced the expression of both GADD153 mRNA level and protein in PC-9 cells, while neither EGCG nor celecoxib alone did. However, cotreatment did not induce expression of other apoptosis related genes, p21(WAF1) and GADD45. Judging by upregulation of GADD153, only cotreatment with EGCG plus celecoxib synergistically induced apoptosis of PC-9 cells. Synergistic effects with the combination were also observed in 2 other lung cancer cell lines, A549 and ChaGo K-1. Furthermore, EGCG did not enhance GADD153 gene expression or apoptosis induction in PC-9 cells in combination with N-(4-hydroxyphenyl)retinamide or with aspirin. Thus, upregulation of GADD153 is closely correlated with synergistic enhancement of apoptosis with EGCG. Cotreatment also activated the mitogen-activated protein kinases (MAPKs), such as ERK1/2 and p38 MAPK: Preteatment with PD98059 (ERK1/2 inhibitor) and UO126 (selective MEK inhibitor) abrogated both upregulation of GADD153 and synergistic induction of apoptosis of PC-9 cells, while SB203580 (p38 MAPK inhibitor) did not do so, indicating that GADD153 expression was mediated through the ERK signaling pathway. These findings indicate that high upregulation of GADD153 is a key requirement for cancer prevention in combination with EGCG.

  11. Upregulation of long non-coding RNA PRNCR1 in colorectal cancer promotes cell proliferation and cell cycle progression.

    Science.gov (United States)

    Yang, Liu; Qiu, Mantang; Xu, Youtao; Wang, Jie; Zheng, Yanyan; Li, Ming; Xu, Lin; Yin, Rong

    2016-01-01

    Colorectal cancer (CRC) is one of the most common cancers worldwide. Long non-coding RNAs (lncRNAs) have been confirmed to play a critical regulatory role in various biological processes including carcinogenesis, which indicates that lncRNAs are valuable biomarkers and therapeutic targets. The novel lncRNA prostate cancer non-coding RNA 1 (PRNCR1) is located in the susceptible genomic area of CRC, however the functional role of PRNCR1 remains unknown. Thus, we aimed to investigate the clinical significance and biological function of PRNCR1 in CRC. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to assess the expression profile of PRNCR1 in CRC tissues and cell lines. An antisense oligonucleotide (ASO) was designed to knock down PRNCR1. In a cohort of 63 patients, PRNCR1 was significantly overexpressed in CRC tissues compared with the expression in adjacent tissues, with an average fold increase of 10.55 (P=0.006). Additionally, a high level of PRNCR1 was associated with large tumor volume (Pline (FHC), PRNCR1 was upregulated in most CRC cell lines (HCT116, SW480, LoVo and HT-29). After knockdown of PRNCR1 by ASO, CRC cell proliferation ability was significantly inhibited. We further found that PRNCR1 knockdown induced cell cycle arrest in the G0/G1 phase and a significant decrease in the proportion of cells in the S phases. In contrast, PRNCR1 knockdown did not affect cell apoptosis or invasive ability. Hence, these data indicate that PRNCR1 promotes the proliferation of CRC cells and is a potential oncogene of CRC.

  12. Inhibition of proliferation of human lung cancer cells by green tea catechins is mediated by upregulation of let-7.

    Science.gov (United States)

    Zhong, Zhiwei; Dong, Zhuo; Yang, Lihua; Chen, Xiaoqiang; Gong, Zhaohui

    2012-08-01

    Green tea catechins are known to function as anticancer agents via inhibition of carcinogenesis during the initiation, promotion and progression stages. Many potential mechanisms have been proposed, yet the precise mechanism of lung cancer prevention by green tea catechins remains unclear. microRNAs (miRs) are a class of 21-24 nucleotide small non-coding RNAs and play critical roles throughout cellular development and regulation. Emerging evidence demonstrates that tea catechins influence the expression of miRs in human cancer cells to inhibit tumorigenesis. Both let-7a-1 and let-7g were detected in the human lung cancer cells treated with tea catechins. The cell viability and cell cycle were analyzed after tea catechins treatment. In the present study, we found that tea catechins upregulated the tumor-suppressor miRs, let-7a-1 and let-7g, in lung cancer cell lines. The upregulation of let-7a/7g repressed the expression of their targets, C-MYC and the regulatory protein of LIN-28, at the mRNA and protein levels. Moreover, the cell growth assay indicated that tea catechins significantly inhibited cell proliferation, and the flow cytometric analysis revealed an increase in the number of cells in the G2/M phase and a decrease in the number of cells in the S phase after treatment with tea catechins. These observations suggest that green tea catechins mediate the inhibition of proliferation of lung cancer cells through the let-7 signaling pathway.

  13. Theobromine Upregulates Osteogenesis by Human Mesenchymal Stem Cells In Vitro and Accelerates Bone Development in Rats.

    Science.gov (United States)

    Clough, Bret H; Ylostalo, Joni; Browder, Elizabeth; McNeill, Eoin P; Bartosh, Thomas J; Rawls, H Ralph; Nakamoto, Tetsuo; Gregory, Carl A

    2017-03-01

    Theobromine (THB) is one of the major xanthine-like alkaloids found in cacao plant and a variety of other foodstuffs such as tea leaves, guarana and cola nuts. Historically, THB and its derivatives have been utilized to treat cardiac and circulatory disorders, drug-induced nephrotoxicity, proteinuria and as an immune-modulator. Our previous work demonstrated that THB has the capacity to improve the formation of hydroxyl-apatite during tooth development, suggesting that it may also enhance skeletal development. With its excellent safety profile and resistance to pharmacokinetic elimination, we reasoned that it might be an excellent natural osteoanabolic supplement during pregnancy, lactation and early postnatal growth. To determine whether THB had an effect on human osteoprogenitors, we subjected primary human bone marrow mesenchymal stem cells (hMSCs) to osteogenic assays after exposure to THB in vitro and observed that THB exposure increased the rate of osteogenesis and mineralization by hMSCs. Moreover, THB exposure resulted in a list of upregulated mRNA transcripts that best matched an osteogenic tissue expression signature as compared to other tissue expression signatures archived in several databases. To determine whether oral administration of THB resulted in improved skeletal growth, we provided pregnant rats with chow supplemented with THB during pregnancy and lactation. After weaning, offspring received THB continuously until postnatal day 50 (approximately 10 mg kg(-1) day(-1)). Administration of THB resulted in neonates with larger bones, and 50-day-old offspring accumulated greater body mass, longer and thicker femora and superior tibial trabecular parameters. The accelerated growth did not adversely affect the strength and resilience of the bones. These results indicate that THB increases the osteogenic potential of bone marrow osteoprogenitors, and dietary supplementation of a safe dose of THB to expectant mothers and during the postnatal period

  14. SOX2 expression is upregulated in adult spinal cord after contusion injury in both oligodendrocyte lineage and ependymal cells.

    Science.gov (United States)

    Lee, Hyun Joon; Wu, Junfang; Chung, Jumi; Wrathall, Jean R

    2013-02-01

    The upregulation of genes normally associated with development may occur in the adult after spinal cord injury (SCI). To test this, we performed real-time RT-PCR array analysis of mouse spinal cord mRNAs comparing embryonic day (E)14.5 spinal cord with intact adult and adult cord 1 week after a clinically relevant standardized contusion SCI. We found significantly increased expression of a large number of neural development- and stem cell-associated genes after SCI. These included Sox2 (sex determining region Y-box 2), a transcription factor that regulates self-renewal and potency of embryonic neural stem cells and is one of only a few key factors needed to induce pluripotency. In adult spinal cord of Sox2-EGFP mice, Sox2-EGFP was found mainly in the ependymal cells of the central canal. After SCI, both mRNA and protein levels of Sox2 were significantly increased at and near the injury site. By 1 day, Sox2 was upregulated in NG2(+) oligodendrocyte progenitor cells (OPC) in the spared white matter. By 3 days, Sox2-EGFP ependymal cells had increased proliferation and begun to form multiple layers and clusters of cells in the central lesion zone of the cord. Expression of Sox2 by NG2(+) cells had declined by 1 week, but increased numbers of other Sox2-expressing cells persisted for at least 4 weeks after SCI in both mouse and rat models. Thus, SCI upregulates many genes associated with development and neural stem cells, including the key transcription factor Sox2, which is expressed in a pool of cells that persists for weeks after SCI.

  15. Titanium dioxide induces apoptotic cell death through reactive oxygen species-mediated Fas upregulation and Bax activation

    Directory of Open Access Journals (Sweden)

    Yoon TH

    2012-03-01

    Full Text Available Ki-Chun Yoo1, Chang-Hwan Yoon1, Dongwook Kwon2, Kyung-Hwan Hyun1, Soo Jung Woo1, Rae-Kwon Kim1, Eun-Jung Lim1, Yongjoon Suh1, Min-Jung Kim1, Tae Hyun Yoon2, Su-Jae Lee11Laboratory of Molecular Biochemistry, 2Laboratory of Nanoscale Characterization and Environmental Chemistry, Department of Chemistry, Hanyang University, Seoul, Republic of KoreaBackground: Titanium dioxide (TiO2 has been widely used in many areas, including biomedicine, cosmetics, and environmental engineering. Recently, it has become evident that some TiO2 particles have a considerable cytotoxic effect in normal human cells. However, the molecular basis for the cytotoxicity of TiO2 has yet to be defined.Methods and results: In this study, we demonstrated that combined treatment with TiO2 nanoparticles sized less than 100 nm and ultraviolet A irradiation induces apoptotic cell death through reactive oxygen species-dependent upregulation of Fas and conformational activation of Bax in normal human cells. Treatment with P25 TiO2 nanoparticles with a hydrodynamic size distribution centered around 70 nm (TiO2P25–70 together with ultraviolet A irradiation-induced caspase-dependent apoptotic cell death, accompanied by transcriptional upregulation of the death receptor, Fas, and conformational activation of Bax. In line with these results, knockdown of either Fas or Bax with specific siRNA significantly inhibited TiO2-induced apoptotic cell death. Moreover, inhibition of reactive oxygen species with an antioxidant, N-acetyl-L-cysteine, clearly suppressed upregulation of Fas, conformational activation of Bax, and subsequent apoptotic cell death in response to combination treatment using TiO2P25–70 and ultraviolet A irradiation.Conclusion: These results indicate that sub-100 nm sized TiO2 treatment under ultraviolet A irradiation induces apoptotic cell death through reactive oxygen species-mediated upregulation of the death receptor, Fas, and activation of the preapoptotic protein

  16. Promotion of cell proliferation by HBXIP via upregulation of human telomerase reverse transcriptase in human mesenchymal stem cells1

    Institute of Scientific and Technical Information of China (English)

    Feng-ze WANG; Li SHA; Li-hong YE; Xiao-dong ZHANG

    2008-01-01

    Aim: We previously found that the hepatitis B X-interacting protein (HBXIP) was able to promote the proliferation of cells. Telomerase activity is known to be critical in cellular senescence and its level is modulated by the regulation of the telomerase catalytic subunit, telomerase reverse transcriptase (TERT), at both the transcriptional and post-transcriptional levels. To investigate the mechanism of promoting proliferation by HBXIP, the effect of HBXIP on human TERT (hTERT) was investigated in human mesenchymal stem cells (hMSC). Methods: BMMS-03 cells and hMSC from the bone marrow of a 4-month-old elicited fetus, were tran-siently transfected with the pcDNA3-hbxip plasmid encoding the HBXIP gene and pSilencer-hbxip plasmid encoding RNA interference (RNAi) targeting HBXIP mRNA, followed by the examination of the hTERT promoter reporter gene by luciferase assay, and the detection of telomerase activity by telomeric repeat amplication protocol, respectively, as well as the expression levels of hTERT, c-Myc, and Bcl-2 by Western blot analysis. Results: The overexpression of HBXIP led to a significant upregulation of hTERT promoter activity, telomerase activity, and the expression levels of hTERT, c-Myc, and Bcl-2 in BMMS-03 cells. RNAi target-ing HBXIP mRNA produced the opposite results completely. Conclusion: Our data demonstrated that HBXIP significantly stimulated the transcription and ex-pression of hTERT and increased the activity of telomerase in BMMS-03 cells, which provides a new insight into the mechanism of promoting cell proliferation by HBXIP.

  17. Histone deacetylase inhibitor upregulates peroxisomal fatty acid oxidation and inhibits apoptotic cell death in abcd1-deficient glial cells.

    Directory of Open Access Journals (Sweden)

    Jaspreet Singh

    Full Text Available In X-ALD, mutation/deletion of ALD gene (ABCD1 and the resultant very long chain fatty acid (VLCFA derangement has dramatically opposing effects in astrocytes and oligodendrocytes. While loss of Abcd1 in astrocytes produces a robust inflammatory response, the oligodendrocytes undergo cell death leading to demyelination in X-linked adrenoleukodystrophy (X-ALD. The mechanisms of these distinct pathways in the two cell types are not well understood. Here, we investigated the effects of Abcd1-knockdown and the subsequent alteration in VLCFA metabolism in human U87 astrocytes and rat B12 oligodendrocytes. Loss of Abcd1 inhibited peroxisomal β-oxidation activity and increased expression of VLCFA synthesizing enzymes, elongase of very long chain fatty acids (ELOVLs (1 and 3 in both cell types. However, higher induction of ELOVL's in Abcd1-deficient B12 oligodendrocytes than astrocytes suggests that ELOVL pathway may play a prominent role in oligodendrocytes in X-ALD. While astrocytes are able to maintain the cellular homeostasis of anti-apoptotic proteins, Abcd1-deletion in B12 oligodendrocytes downregulated the anti-apototic (Bcl-2 and Bcl-xL and cell survival (phospho-Erk1/2 proteins, and upregulated the pro-apoptotic proteins (Bad, Bim, Bax and Bid leading to cell loss. These observations provide insights into different cellular signaling mechanisms in response to Abcd1-deletion in two different cell types of CNS. The apoptotic responses were accompanied by activation of caspase-3 and caspase-9 suggesting the involvement of mitochondrial-caspase-9-dependent mechanism in Abcd1-deficient oligodendrocytes. Treatment with histone deacetylase (HDAC inhibitor suberoylanilide hydroxamic acid (SAHA corrected the VLCFA derangement both in vitro and in vivo, and inhibited the oligodendrocytes loss. These observations provide a proof-of principle that HDAC inhibitor SAHA may have a therapeutic potential for X-ALD.

  18. Upregulated HSP27 in human breast cancer cells reduces Herceptin susceptibility by increasing Her2 protein stability

    Directory of Open Access Journals (Sweden)

    Kong Sun-Young

    2008-10-01

    Full Text Available Abstract Background Elucidating the molecular mechanisms by which tumors become resistant to Herceptin is critical for the treatment of Her2-overexpressed metastatic breast cancer. Methods To further understand Herceptin resistance mechanisms at the molecular level, we used comparative proteome approaches to analyze two human breast cancer cell lines; Her2-positive SK-BR-3 cells and its Herceptin-resistant SK-BR-3 (SK-BR-3 HR cells. Results Heat-shock protein 27 (HSP27 expression was shown to be upregulated in SK-BR-3 HR cells. Suppression of HSP27 by specific siRNA transfection increased the susceptibility of SK-BR-3 HR cells to Herceptin. In the presence of Herceptin, Her2 was downregulated in both cell lines. However, Her2 expression was reduced by a greater amount in SK-BR-3 parent cells than in SK-BR-3 HR cells. Interestingly, co-immunoprecipitation analysis showed that HSP27 can bind to Her2. In the absence of Herceptin, HSP27 expression is suppressed and Her2 expression is reduced, indicating that downregulation of Her2 by Herceptin can be obstructed by the formation of a Her2-HSP27 complex. Conclusion Our present study demonstrates that upregulated HSP27 in human breast cancer cells can reduce Herceptin susceptibility by increasing Her2 protein stability.

  19. PRL-3 promotes the proliferation of LoVo cells via the upregulation of KCNN4 channels.

    Science.gov (United States)

    Lai, Wei; Chen, Shuang; Wu, Heng; Guan, Yufeng; Liu, Lu; Zeng, Yujie; Zhao, Haiyan; Jiang, Jianmin; Chu, Zhonghua

    2011-10-01

    Previous studies have shown that phosphatase of regenerating liver-3 (PRL-3) plays an important role in the metastasis and proliferation of tumor cells. However, the mechanism by which PRL-3 controls the cell cycle of tumor cells remains unknown. In the present study, considering that the K+ channels strictly control cell proliferation, we examined whether K+ channels participate in the proliferation of tumor cells induced by PRL-3. Interestingly, the expression of intermediate-conductance Ca2+-activated K+ channels (KCNN4) was upregulated in an NF-κB-dependent manner when PRL-3 was transfected into LoVo cells. Also, we identified two NF-κB binding sites in the promoter region of KCNN4. Use of the specific inhibitor 1-[(2-chlorophenyl) diphenylmethyl]-1H-pyrazole (TRAM-34) significantly inhibited the proliferation induced by PRL-3 and blocked the cell cycle at the G2/M phase. Meanwhile, the level of phosphorylation of Cdc2 was increased in a dose-dependent manner. Furthermore, TRAM-34 also inhibited tumor formation of PRL-3 cell xenografts implanted by injection in nude mice. In conclusion, PRL-3 promoted the proliferation of LoVo cells through upregulation of KCNN4 channels which facilitated the G2/M transition.

  20. Disturbance of DKK1 level is partly involved in survival of lung cancer cells via regulation of ROMO1 and γ-radiation sensitivity

    Energy Technology Data Exchange (ETDEWEB)

    Kim, In Gyu, E-mail: igkim@kaeri.re.kr [Department of Radiation Biology, Environmental Radiation Research Group, Korea Atomic Energy Research Institute, P.O. Box 105, Yuseong, Daejeon 305-600 (Korea, Republic of); Department of Radiation Biotechnology and Applied Radioisotope, University of Science and Technology (UST), 989-111 Daedeok-daero, Yuseong-gu, Daejeon 305-353 (Korea, Republic of); Kim, Seo Yoen [Department of Radiation Biology, Environmental Radiation Research Group, Korea Atomic Energy Research Institute, P.O. Box 105, Yuseong, Daejeon 305-600 (Korea, Republic of); Biomedical Translational Research Center, Korea Research Institute of Bioscience and Biotechnology, 125 Gwahak-ro, Yuseong-gu, Daejeon 305-806 (Korea, Republic of); Kim, Hyun A; Kim, Jeong Yul [Department of Radiation Biology, Environmental Radiation Research Group, Korea Atomic Energy Research Institute, P.O. Box 105, Yuseong, Daejeon 305-600 (Korea, Republic of); Lee, Jae Ha; Choi, Soo Im [Department of Radiation Biology, Environmental Radiation Research Group, Korea Atomic Energy Research Institute, P.O. Box 105, Yuseong, Daejeon 305-600 (Korea, Republic of); Department of Radiation Biotechnology and Applied Radioisotope, University of Science and Technology (UST), 989-111 Daedeok-daero, Yuseong-gu, Daejeon 305-353 (Korea, Republic of); Han, Jeong Ran; Kim, Kug Chan [Department of Radiation Biology, Environmental Radiation Research Group, Korea Atomic Energy Research Institute, P.O. Box 105, Yuseong, Daejeon 305-600 (Korea, Republic of); Cho, Eun Wie [Biomedical Translational Research Center, Korea Research Institute of Bioscience and Biotechnology, 125 Gwahak-ro, Yuseong-gu, Daejeon 305-806 (Korea, Republic of)

    2014-01-03

    Highlights: •DKK1 was expressed differently among non-small-cell lung cancer cell lines. •DKK1 negatively regulated ROMO1 gene expression. •Disturbance of DKK1 level induced the imbalance of cellular ROS. •DKK1/ROMO1-induced ROS imbalance is involved in cell survival in NSCLC. -- Abstract: Dickkopf1 (DKK1), a secreted protein involved in embryonic development, is a potent inhibitor of the Wnt signaling pathway and has been postulated to be a tumor suppressor or tumor promoter depending on the tumor type. In this study, we showed that DKK1 was expressed differently among non-small-cell lung cancer cell lines. The DKK1 expression level was much higher in A549 cells than in H460 cells. We revealed that blockage of DKK1 expression by silencing RNA in A549 cells caused up-regulation of intracellular reactive oxygen species (ROS) modulator (ROMO1) protein, followed by partial cell death, cell growth inhibition, and loss of epithelial–mesenchymal transition property caused by ROS, and it also increased γ-radiation sensitivity. DKK1 overexpression in H460 significantly inhibited cell survival with the decrease of ROMO1 level, which induced the decrease of cellular ROS. Thereafter, exogenous N-acetylcysteine, an antioxidant, or hydrogen peroxide, a pro-oxidant, partially rescued cells from death and growth inhibition. In each cell line, both overexpression and blockage of DKK1 not only elevated p-RB activation, which led to cell growth arrest, but also inactivated AKT/NF-kB, which increased radiation sensitivity and inhibited cell growth. This study is the first to demonstrate that strict modulation of DKK1 expression in different cell types partially maintains cell survival via tight regulation of the ROS-producing ROMO1 and radiation resistance.

  1. Mutant KRas-Induced Mitochondrial Oxidative Stress in Acinar Cells Upregulates EGFR Signaling to Drive Formation of Pancreatic Precancerous Lesions

    Directory of Open Access Journals (Sweden)

    Geou-Yarh Liou

    2016-03-01

    Full Text Available The development of pancreatic cancer requires the acquisition of oncogenic KRas mutations and upregulation of growth factor signaling, but the relationship between these is not well established. Here, we show that mutant KRas alters mitochondrial metabolism in pancreatic acinar cells, resulting in increased generation of mitochondrial reactive oxygen species (mROS. Mitochondrial ROS then drives the dedifferentiation of acinar cells to a duct-like progenitor phenotype and progression to PanIN. This is mediated via the ROS-receptive kinase protein kinase D1 and the transcription factors NF-κB1 and NF-κB2, which upregulate expression of the epidermal growth factor, its ligands, and their sheddase ADAM17. In vivo, interception of KRas-mediated generation of mROS reduced the formation of pre-neoplastic lesions. Hence, our data provide insight into how oncogenic KRas interacts with growth factor signaling to induce the formation of pancreatic cancer.

  2. Upregulation of T-Cell-Specific Transcription Factor Expression in Pediatric T-Cell Acute Lymphoblastic Leukemia (T-ALL

    Directory of Open Access Journals (Sweden)

    Müge Sayitoğlu

    2012-12-01

    Full Text Available OBJECTIVE: T-cell acute lymphoblastic leukemia (T-ALL is associated with recurrent chromosomal aberrations and abnormal ectopic gene expression during T-cell development. In order to gain insight into the pathogenesis of T-ALL this study aimed to measure the level of expression of 7 T-cell oncogenes (LMO2, LYL1, TAL1, TLX1, TLX3, BMI1, and CALM-AF10 in pediatric T-ALL patients. METHODS: LMO2, LYL1, TLX1, TLX3, BMI1, TAL1, and CALM-AF10 expression was measured using quantitative real-time PCR in 43 pediatric T-ALL patients. RESULTS: A high level of expression of LMO2, LYL1, TAL1, and BMI1 genes was observed in a large group of T-ALL. Several gene expression signatures indicative of leukemic arrest at specific stages of normal thymocyte development (LYL1 and LMO2 were highly expressed during the cortical and mature stages of T-cell development. Furthermore, upregulated TAL1 and BMI1 expression was observed in all phenotypic subgroups. In all, 6 of the patients had TLX1 and TLX3 proto-oncogene expression, which does not occur in normal cells, and none of the patients had CALM-AF10 fusion gene transcription. Expression of LYL1 alone and LMO2-LYL1 co-expression were associated with mediastinal involvement; however, high-level oncogene expression was not predictive of outcome in the present pediatric T-ALL patient group, but there was a trend towards a poor prognostic impact of TAL1 and/or LMO2 and/or LYL1 protooncogene expression. CONCLUSION: Poor prognostic impact of TAL1 and/or LMO2 and/or LYL1 proto-oncogene expression indicate the need for extensive study on oncogenic rearrangement and immunophenotypic markers in T-ALL, and their relationship to treatment outcome.

  3. Serum amyloid A stimulates macrophage foam cell formation via lectin-like oxidized low-density lipoprotein receptor 1 upregulation

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Ha Young, E-mail: hayoung@skku.edu [Department of Biological Science, Sungkyunkwan University, Suwon 440-746 (Korea, Republic of); Mitochondria Hub Regulation Center, Dong-A University, Busan 602-714 (Korea, Republic of); Kim, Sang Doo [Department of Biological Science, Sungkyunkwan University, Suwon 440-746 (Korea, Republic of); Baek, Suk-Hwan [Department of Biochemistry and Molecular Biology, College of Medicine, Yeungnam University, Daegu 705-717 (Korea, Republic of); Choi, Joon Hyuk [Department of Pathology, College of Medicine, Yeungnam University, Daegu 705-717 (Korea, Republic of); Cho, Kyung-Hyun [School of Biotechnology, Yeungnam University, Gyeongsan 712-749 (Korea, Republic of); Zabel, Brian A. [Palo Alto Institute for Research and Education, Veterans Affairs Hospital, Palo Alto, CA 94304 (United States); Bae, Yoe-Sik, E-mail: yoesik@skku.edu [Department of Biological Science, Sungkyunkwan University, Suwon 440-746 (Korea, Republic of); Mitochondria Hub Regulation Center, Dong-A University, Busan 602-714 (Korea, Republic of); Samsung Advanced Institute for Health Sciences and Technology, Sungkyunkwan University, Seoul 135-710 (Korea, Republic of)

    2013-03-29

    Highlights: ► SAA induced macrophage foam cell formation. ► SAA stimulated upregulation of lectin-like oxidized low-density lipoprotein receptor 1 (LOX1). ► SAA-induced LOX1 expression and foam cell formation is mediated by JNK/NF-κB signaling. ► HDL-conjugated SAA also stimulates foam cell formation via LOX1 upregulation. ► The finding reveals a novel mechanism of action of SAA in the pathogenesis of atherosclerosis. -- Abstract: Elevated levels of serum amyloid A (SAA) is a risk factor for cardiovascular diseases, however, the role of SAA in the pathophysiology of atherosclerosis remains unclear. Here we show that SAA induced macrophage foam cell formation. SAA-stimulated foam cell formation was mediated by c-jun N-terminal kinase (JNK) signaling. Moreover, both SAA and SAA-conjugated high density lipoprotein stimulated the expression of the important scavenger receptor lectin-like oxidized low-density lipoprotein receptor 1 (LOX1) via nuclear factor-κB (NF-κB). A LOX1 antagonist carrageenan significantly blocked SAA-induced foam cell formation, indicating that SAA promotes foam cell formation via LOX1 expression. Our findings therefore suggest that SAA stimulates foam cell formation via LOX1 induction, and thus likely contributes to atherogenesis.

  4. Growth arrest specific 2 is up-regulated in chronic myeloid leukemia cells and required for their growth.

    Directory of Open Access Journals (Sweden)

    Haixia Zhou

    Full Text Available Although the generation of BCR-ABL is the molecular hallmark of chronic myeloid leukemia (CML, the comprehensive molecular mechanisms of the disease remain unclear yet. Growth arrest specific 2 (GAS2 regulates multiple cellular functions including cell cycle, apoptosis and calpain activities. In the present study, we found GAS2 was up-regulated in CML cells including CD34+ progenitor cells compared to their normal counterparts. We utilized RNAi and the expression of dominant negative form of GAS2 (GAS2DN to target GAS2, which resulted in calpain activity enhancement and growth inhibition of both K562 and MEG-01 cells. Targeting GAS2 also sensitized K562 cells to Imatinib mesylate (IM. GAS2DN suppressed the tumorigenic ability of MEG-01 cells and impaired the tumour growth as well. Moreover, the CD34+ cells from CML patients and healthy donors were transduced with control and GAS2DN lentiviral vectors, and the CD34+ transduced (YFP+ progeny cells (CD34+YFP+ were plated for colony-forming cell (CFC assay. The results showed that GAS2DN inhibited the CFC production of CML cells by 57±3% (n = 3, while affected those of normal hematopoietic cells by 31±1% (n = 2. Next, we found the inhibition of CML cells by GAS2DN was dependent on calpain activity but not the degradation of beta-catenin. Lastly, we generated microarray data to identify the differentially expressed genes upon GAS2DN and validated that the expression of HNRPDL, PTK7 and UCHL5 was suppressed by GAS2DN. These 3 genes were up-regulated in CML cells compared to normal control cells and the growth of K562 cells was inhibited upon HNRPDL silence. Taken together, we have demonstrated that GAS2 is up-regulated in CML cells and the inhibition of GAS2 impairs the growth of CML cells, which indicates GAS2 is a novel regulator of CML cells and a potential therapeutic target of this disease.

  5. Myeloid zinc finger 1 mediates sulindac sulfide-induced upregulation of death receptor 5 of human colon cancer cells

    OpenAIRE

    Mano Horinaka; Tatsushi Yoshida; Mitsuhiro Tomosugi; Shusuke Yasuda; Yoshihiro Sowa; Toshiyuki Sakai

    2014-01-01

    A combined therapy of sulindac sulfide and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising strategy for the treatment of cancer. Sulindac sulfide had been shown to induce the expression of death receptor 5 (DR5), a receptor for TRAIL, and sensitize cancer cells to TRAIL-induced apoptosis; however, the molecular mechanism underlying the upregulation of DR5 has not yet been elucidated. We demonstrate here that myeloid zinc finger 1 (MZF1) mediates the induction of...

  6. Disturbed Th17/Treg Balance in Patients with Non-small Cell Lung Cancer.

    Science.gov (United States)

    Duan, Min-Chao; Han, Wei; Jin, Pei-Wen; Wei, Yu-Ping; Wei, Qiu; Zhang, Liang-Ming; Li, Jun-Chen

    2015-12-01

    The fine balance of T help-17 (Th17)/regulatory T(Treg) cells is crucial for maintenance of immune homeostasis. However, there is little information concerning the role played in non-small cell lung cancer (NSCLC) by Th17/Treg cells. The objective of this study was to investigate the variation of Th17 and Treg cells in the peripheral blood of patients with NSCLC. Blood samples were collected from 19 patients with NSCLC and 19 healthy donors. Samples were processed to detect CD4(+)IL-17(+) Th17 cells and CD4(+)CD25(+)Foxp3(+) Treg cells by flow cytometry, and related gene expressions were assessed by real-time quantitative polymerase chain reaction. The concentrations of interleukin (IL)-1β, IL-6, IL-10, IL-17, IL-23, and transforming growth factor-beta (TGF-β1) were also measured by enzyme-linked immunosorbent assay analysis (ELISA). The frequency of circulating Th17 cells and Treg cells was increased in samples derived from patients with NSCLC, accompanied by the upregulation of Foxp3 and RORγt. However, a negative correlation between Treg cells and Th17 cells was found in patients with NSCLC. Additionally, the Th17/Treg ratio and the related cytokines were also significantly higher in patients with NSCLC than in healthy controls. Furthermore, the frequency of Th17 cells was positively correlated with IL-1β, IL-6, and IL-23 in patients with NSCLC, and the frequency of Treg cells was positively correlated with TGF-β1 and IL-10. More importantly, the Th17/Treg ratio was positively correlated with the CEA concentrations in patients with NSCLC. Our data indicated that Th17 and Treg subset are involved in the immunopathology of NSCLC. Distinct cytokine environment might play a key role in the differentiation of the Th17 and Treg cells in NSCLC. Reconstituting an adequate balance between Th17 and Treg may be beneficial in the treatment of NSCLC.

  7. Hypoxia upregulates Bcl-2 expression and suppresses interferon-gamma induced antiangiogenic activity in human tumor derived endothelial cells.

    LENUS (Irish Health Repository)

    Wang, Jiang Huai

    2012-02-03

    BACKGROUND: Hypoxia in solid tumors potentially stimulates angiogenesis by promoting vascular endothelial growth factor (VEGF) production and upregulating VEGF receptor expression. However, it is unknown whether hypoxia can modulate the effect of anti-angiogenic treatment on tumor-derived endothelium. METHODS: Human tumor-derived endothelial cells (HTDEC) were freshly isolated from surgically removed human colorectal tumors by collagenase\\/DNase digestion and Percol gradient sedimentation. Cell proliferation was assessed by measuring BrdU incorporation, and capillary tube formation was measured using Matrigel. Cell apoptosis was assessed by flow cytometry and ELISA, and Bcl-2 expression was detected by Western blot analysis. RESULTS: Under aerobic culture conditions (5% CO2 plus 21% O2) HTDEC expressed less Bcl-2 and were more susceptible to IFN-gamma-induced apoptosis with significant reductions in both cell proliferation and capillary tube formation, when compared with normal human macrovascular and microvascular EC. Following exposure of HTDEC to hypoxia (5% CO2 plus 2% O2), IFN-gamma-induced cell apoptosis, and antiangiogenic activity (i.e. an inhibition in cell proliferation and capillary tube formation) in HTDEC were markedly attenuated. This finding correlated with hypoxia-induced upregulation of Bcl-2 expression in HTDEC. CONCLUSIONS: These results indicate that hypoxia can protect HTDEC against IFN-gamma-mediated cell death and antiangiogenic activity, and suggest that improvement of tumor oxygenation may potentiate the efficacy of anti-cancer therapies specifically targeting the inhibition of tumor angiogenesis.

  8. Gas6 induces cancer cell migration and epithelial–mesenchymal transition through upregulation of MAPK and Slug

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Yunhee [Department of Chemistry, Korea Advanced Institute of Science and Technology, Daejeon (Korea, Republic of); Immunotherapy Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon (Korea, Republic of); Lee, Mira [Immunotherapy Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon (Korea, Republic of); Kim, Semi, E-mail: semikim@kribb.re.kr [Department of Chemistry, Korea Advanced Institute of Science and Technology, Daejeon (Korea, Republic of); Immunotherapy Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon (Korea, Republic of)

    2013-04-26

    Highlights: •We investigated the molecular mechanisms underlying Gas6-mediated cancer cell migration. •Gas6 treatment and subsequent Axl activation induce cell migration and EMT via upregulation of Slug. •Slug expression mediated by Gas6 is mainly through c-Jun and ATF-2 in an ERK1/2 and JNK-dependent manner. •The Gas6/Axl-Slug axis may be exploited as a target for anti-cancer metastasis therapy. -- Abstract: Binding of Gas6 to Axl (Gas6/Axl axis) alters cellular functions, including migration, invasion, proliferation, and survival. However, the molecular mechanisms underlying Gas6-mediated cell migration remain poorly understood. In this study, we found that Gas6 induced the activation of JNK and ERK1/2 signaling in cancer cells expressing Axl, resulting in the phosphorylation of activator protein-1 (AP-1) transcription factors c-Jun and ATF-2, and induction of Slug. Depletion of c-Jun or ATF-2 by siRNA attenuated the Gas6-induced expression of Slug. Slug expression was required for cell migration and E-cadherin reduction/vimentin induction induced by Gas6. These results suggest that Gas6 induced cell migration via Slug upregulation in JNK- and ERK1/2-dependent mechanisms. These data provide an important insight into the molecular mechanisms mediating Gas6-induced cell migration.

  9. EFFECT OF UP-REGULATION OF S-ADOMET SYNTHETASE ON TAXOL-INDUCED APOPTOSIS IN HUMAN BREAST CANCER CELLS

    Institute of Scientific and Technical Information of China (English)

    Chen Lirong; Zheng Shu; Fan Weimin; Zhang Suzhan

    1998-01-01

    Objective:To investigate the gene regulation of taxolinduced apoptosis. Methods: Northern blot hybridization,enzyme activity assay of S-AdoMet synthetase and flow cytometry were performed in the investigation of expression in the Mrna level and biological action of SAdoMet synthetase in taxol-induced apoptosis in human breast cancer cell line (Bcap 37). Results: Up-regulation of S-AdoMet synthetase expression was resulted by taxol treatment and the expression peaked at 48hours. Moreover,the up-regulation of S-AdoMet synthetase was associated with cytotoxicity of antimicrotubule agents including taxol and colchicine.Inhibition rate of S-AdoMet synthetase activity by 1%DMSO was 34% in taxol-treated cells and 14% in taxoluntreated cells compared to control groups, respectively.Posttreatment with 1% DMSO following pretreatment with individual antitumor agent for 3 hrs promoted apoptotic cell death of taxol-,colchicine-,and adriamycintreated Bcap37 cells. Conclusion : The induction of apoptosis enhanced by post-treatment with DMSO in taxol-treated cells is probably linked to its inhibition on enzyme activity of S-AdoMet synthetase ,suggesting that the increased expression of S-AdoMet synthetase possibly plays an important role in protecting cells from DNA fragmentation in taxol-induced apoptosis.

  10. Parathyroid hormone inhibits TGF-β/Smad signaling and extracellular matrix proteins upregulation in rat mesangial cells.

    Science.gov (United States)

    Peng, Fang-Fang; Xiao, Ze-Ling; Chen, Hong-Min; Chen, Yan; Zhou, Jian; Yu, Hong; Zhang, Bai-Fang

    2016-09-23

    Accumulation of glomerular matrix is a hallmark of diabetic nephropathy. TGF-β1 is a major cytokine mediating the production of various extracellular matrix (ECM) proteins. The aim of this study is to elucidate the effect of parathyroid hormone (PTH) on TGF-β1 and high glucose-induced upregulation of ECM proteins in primary mesangial cells from Sprague-Dawley rat. The results showed that PTH pretreatment prevented TGF-β1 and high glucose-induced Smad2/3 phosphorylation and consequent upregulation of fibronectin and type IV collagen within 4 h. The inhibitory effect of PTH is due to PTH1R activation, because knocking down PTH 1 receptor (PTH1R) by RNA interference reversed the inhibitory effect of PTH on TGF-β1 and high glucose-induced Smad2/3 phosphorylation and ECM upregulation. Furthermore, it is found that PTH1R associated with TGF-β type II receptor (TβR II) and both receptors internalized into the cytoplasm when mesangial cells were stimulated with PTH alone. The internalization of TβR II might reduce the amount of membrane TβR II, attenuate the sensitivity of mesangial cells to TGF-β1, and therefore inhibit Smad activation and ECM upregulation induced by TGF-β1 and high glucose. Further studies are needed to know whether the endocytic receptors are to be degraded or recycled, and evaluate the role of PTH in TGF-β1 signaling more comprehensively.

  11. Long non-coding RNA small nucleolar RNA host gene 12 (SNHG12) promotes cell proliferation and migration by upregulating angiomotin gene expression in human osteosarcoma cells.

    Science.gov (United States)

    Ruan, Wendong; Wang, Pei; Feng, Shiqing; Xue, Yuan; Li, Yulin

    2016-03-01

    The long non-coding RNA (lncRNA) small nucleolar RNA host gene 12 (SNHG12) has a role in cell proliferation and migration. Angiomotin, encoded by the AMOT gene, is a protein that regulates the migration and organization of endothelial cells. SNHG12 and AMOT have been shown to play a role in a variety of human cancers but have yet to be studied in detail in human osteosarcoma. Tissue samples from primary osteosarcoma (n = 20) and adjacent normal tissues (n = 20), the osteosarcoma cell lines, SAOS-2, MG-63, U-2 OS, and the human osteoblast cell line hFOB (OB3) were studied using Western blot for angiomotin, and quantitative real-time polymerase chain reaction for the expression of SNHG12 and AMOT. The expression of SNHG12 was knocked down using RNA interference. Cell migration assays were performed. Cell apoptosis was studied using flow cytometry. SNHG12 and AMOT messenger RNA (mRNA) expression was upregulated in osteosarcoma tissues and cell lines when compared with normal tissues and cells. Upregulation of AMOT mRNA was associated with upregulation of SNHG12. Knockdown of SNHG12 reduced the expression of angiomotin in osteosarcoma cells and suppressed cell proliferation and migration but did not affect cell apoptosis. This preliminary study has shown that the lncRNA SNHG12 promotes cell proliferation and migration by upregulating AMOT gene expression in osteosarcoma cells in vivo and in vitro. Further studies are recommended to investigate the role of SNHG12 and AMOT expression in tumor cell proliferation and migration and angiogenesis in osteosarcoma and a range of malignant mesenchymal tumors.

  12. Isoreserpine promotes {beta}-catenin degradation via Siah-1 up-regulation in HCT116 colon cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Gwak, Jungsug; Song, Taeyun [PharmacoGenomics Research Center, Inje University, Busan 614-735 (Korea, Republic of); Song, Jie-Young; Yun, Yeon-Sook [Laboratory of Radiation Cancer Science, Korea Institute of Radiological and Medical Sciences, Seoul 139-706 (Korea, Republic of); Choi, Il-Whan [Department of Microbiology, Center for Viral Disease Research, Inje University College of Medicine, Busan 614-735 (Korea, Republic of); Jeong, Yongsu [Department of Genetic Engineering, and Graduate School of Biotechnology, Kyung Hee University, Yongin 446-701 (Korea, Republic of); Shin, Jae-Gook [PharmacoGenomics Research Center, Inje University, Busan 614-735 (Korea, Republic of); Department of Clinical Pharmacology, Inje University Busan Paik Hospital, Busan 614-735 (Korea, Republic of); Oh, Sangtaek, E-mail: ohsa@inje.ac.kr [PharmacoGenomics Research Center, Inje University, Busan 614-735 (Korea, Republic of)

    2009-09-25

    Aberrant accumulation of intracellular {beta}-catenin in intestinal epithelial cells is a frequent early event during the development of colon cancer. To identify small molecules that decrease the level of intracellular {beta}-catenin, we performed cell-based chemical screening using genetically engineered HEK293 reporter cells to detect compounds that inhibit TOPFlash reporter activity, which was stimulated by Wnt3a-conditioned medium. We found that isoreserpine promoted the degradation of intracellular {beta}-catenin by up-regulation of Siah-1 in HEK293 and HCT116 colon cancer cells. Moreover, isoreserpine repressed the expression of {beta}-catenin/T-cell factor (TCF)-dependent genes, such as cyclin D1 and c-myc, resulting in the suppression of HCT116 cell proliferation. Our findings suggest that isoreserpine can potentially be used as a chemotherapeutic agent against colon cancer.

  13. 5-Azacytidine suppresses EC9706 cell proliferation and metastasis by upregulating the expression of SOX17 and CDH1.

    Science.gov (United States)

    Li, Wenli; Wu, Dan; Niu, Ziyu; Jiang, Dalei; Ma, Huan; He, Heming; Zuo, Xiuli; Xie, Xiangjun; He, Yuanlong

    2016-10-01

    5-Azacytidine is a well-known anticancer drug that is clinically used in the treatment of breast cancer, melanoma and colon cancer. It has been reported that 5-azacytidine suppresses the biological behavior of esophageal cancer cells. However, corresponding mechanisms remain unclear. In this study, using Transwell invasion and cell proliferation assays, we demonstrated that 5-azacytidine significantly inhibited the metastasis and proliferation of EC9706 cells, and upregulated the expression of cadherin 1 (CDH1) and SRY-box containing gene 17 (SOX17). Moreover, the inhibition of the metastasis of the 5-azacytidine-treated EC9706 cells was impaired following transfection with siRNA targeting CDH1 (CDH1 siRNA), and the inhibition of cell proliferation was attenuated following the downregulation of SOX17 by siRNA targeting SOX17 (SOX17 siRNA). Furthermore, 5-azacytidine remarkably reduced the CDH1 and SOX17 promoter methylation levels, suggesting that 5-azacytidine upregulates the expression of SOX17 and CDH1 by inhibiting the methylation of the SOX17 and CDH1 promoter. The findings of our study confirm that 5-azacytidine suppresses the proliferation and metastasis of EC9706 esophageal cancer cells by upregulating the expression of CDH1 and SOX17. The expression levels of CDH1 and SOX17 negatively correlate with the promoter methylation levels. CDH1 and SOX17 are potential indicators of the clinical application of 5-azacytidine.

  14. Increasing matrix stiffness upregulates vascular endothelial growth factor expression in hepatocellular carcinoma cells mediated by integrin β1.

    Science.gov (United States)

    Dong, Yinying; Xie, Xiaoying; Wang, Zhiming; Hu, Chao; Zheng, Qiongdan; Wang, Yaohui; Chen, Rongxin; Xue, Tongchun; Chen, Jie; Gao, Dongmei; Wu, Weizhong; Ren, Zhenggang; Cui, Jiefeng

    2014-02-14

    Matrix stiffness as a novel regulation factor involves in modulating the pathogenesis of hepatocellular carcinoma (HCC) invasion or metastasis. However, the mechanism by which matrix stiffness modulates HCC angiogenesis remains unknown. Here, using buffalo rat HCC models with different liver matrix stiffness backgrounds and an in vitro cell culture system of mechanically tunable Collagen1 (COL1)-coated polyacrylamide gel, we investigated the effects of different matrix stiffness levels on vascular endothelial growth factor (VEGF) expression in HCC cells and explored its regulatory mechanism for controlling HCC angiogenesis. Tissue microarray analysis showed that the expression levels of VEGF and CD31 were gradually upregulated in tumor tissues with increasing COL1 and lysyl oxidase (LOX) expression, indicating a positive correlation between tumor angiogenesis and matrix rigidity. The expression of VEGF and the phosphorylation levels of PI3K and Akt were all upregulated in HCC cells on high-stiffness gel than on low-stiffness gel. Meanwhile, alteration of intergrin β1 expression was found to be the most distinctive, implying that it might mediate the response of HCC cells to matrix stiffness simulation. After integrin β1 was blocked in HCC cells using specific monoclonal antibody, the expression of VEGF and the phosphorylation levels of PI3K and Akt at different culture times were accordingly suppressed and downregulated in the treatment group as compared with those in the control group. All data suggested that the extracellular matrix stiffness stimulation signal was transduced into HCC cells via integrin β1, and this signal activated the PI3K/Akt pathway and upregulated VEGF expression. This study unveils a new paradigm in which matrix stiffness as initiators to modulate HCC angiogenesis.

  15. Fenofibrate inhibited pancreatic cancer cells proliferation via activation of p53 mediated by upregulation of LncRNA MEG3.

    Science.gov (United States)

    Hu, Duanmin; Su, Cunjin; Jiang, Min; Shen, Yating; Shi, Aiming; Zhao, Fenglun; Chen, Ruidong; Shen, Zhu; Bao, Junjie; Tang, Wen

    2016-03-04

    There is still no suitable drug for pancreatic cancer treatment, which is one of the most aggressive human tumors. Maternally expressed gene 3 (MEG3), a LncRNA, has been suggested as a tumor suppressor in a range of human tumors. Studies found fenofibrate exerted anti-tumor roles in various human cancer cell lines. However, its role in pancreatic cancer remains unknown. The present study aimed to explore the impacts of fenofibrate on pancreatic cancer cell lines, and to investigate MEG3 role in its anti-tumor mechanisms. We used MTT assay to determine cells proliferation, genome-wide LncRNA microarray analysis to identify differently expressed LncRNAs, siRNA or pCDNA-MEG3 transfection to interfere or upregulate MEG3 expression, western blot to detect protein levels, real-time PCR to determine MEG3 level. Fenofibrate significantly inhibited proliferation of pancreatic cancer cells, increased MEG3 expression and p53 levels. Moreover, knockdown of MEG3 attenuated cytotoxicity induced by fenofibrate. Furthermore, overexpression of MEG3 induced cells death and increased p53 expression. Our results indicated fenofibrate inhibited pancreatic cancer cells proliferation via activation of p53 mediated by upregulation of MEG3.

  16. Myeloid zinc finger 1 mediates sulindac sulfide-induced upregulation of death receptor 5 of human colon cancer cells.

    Science.gov (United States)

    Horinaka, Mano; Yoshida, Tatsushi; Tomosugi, Mitsuhiro; Yasuda, Shusuke; Sowa, Yoshihiro; Sakai, Toshiyuki

    2014-08-08

    A combined therapy of sulindac sulfide and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising strategy for the treatment of cancer. Sulindac sulfide had been shown to induce the expression of death receptor 5 (DR5), a receptor for TRAIL, and sensitize cancer cells to TRAIL-induced apoptosis; however, the molecular mechanism underlying the upregulation of DR5 has not yet been elucidated. We demonstrate here that myeloid zinc finger 1 (MZF1) mediates the induction of DR5 by sulindac sulfide. Sulindac sulfide induced the expression of DR5 at the protein and mRNA levels in colon cancer SW480 cells. Furthermore, sulindac sulfide increased DR5 promoter activity. We showed that sulindac sulfide stimulated DR5 promoter activity via the -301 to -253 region. This region contained a putative MZF1-binding site. Site-directed mutations in the site abrogated the enhancement in DR5 promoter activity by sulindac sulfide. MZF1 directly bound to the putative MZF1-binding site of the DR5 promoter and the binding was increased by sulindac sulfide. The expression of MZF1 was also increased by sulindac sulfide, and MZF1 siRNA attenuated the upregulation of DR5 by sulindac sulfide. These results indicate that sulindac sulfide induces the expression of DR5 by up-regulating MZF1.

  17. Hydrogen sulfide inhibits formaldehyde-induced endoplasmic reticulum stress in PC12 cells by upregulation of SIRT-1.

    Directory of Open Access Journals (Sweden)

    Xiang Li

    Full Text Available BACKGROUND: Formaldehyde (FA, a well-known environmental pollutant, has been classified as a neurotoxic molecule. Our recent data demonstrate that hydrogen sulfide (H2S, the third gaseous transmitter, has a protective effect on the neurotoxicity of FA. However, the exact mechanisms underlying this protection remain largely unknown. Endoplasmic reticulum (ER stress has been implicated in the neurotoxicity of FA. Silent mating type information regulator 2 homolog 1 (SIRT-1, a histone deacetylases, has various biological activities, including the extension of lifespan, the modulation of ER stress, and the neuroprotective action. OBJECTIVE: We hypothesize that the protection of H2S against FA-induced neurotoxicity involves in inhibiting ER stress by upregulation of SIRT-1. The present study attempted to investigate the protective effect of H2S on FA-induced ER stress in PC12 cells and the contribution of SIRT-1 to the protection of H2S against FA-induced injuries, including ER stress, cytotoxicity and apoptosis. PRINCIPAL FINDINGS: We found that exogenous application of sodium hydrosulfide (NaHS; an H2S donor significantly attenuated FA-induced ER stress responses, including the upregulated levels of glucose-regulated protein 78, C/EBP homologous protein, and cleaved caspase-12 expression. We showed that NaHS upregulates the expression of SIRT-1 in PC12 cells. Moreover, the protective effects of H2S on FA-elicited ER stress, cytotoxicity and apoptosis were reversed by Sirtinol, a specific inhibitor of SIRT-1. CONCLUSION/SIGNIFICANCE: These data indicate that H2S exerts its protection against the neurotoxicity of FA through overcoming ER stress via upregulation of SIRT-1. Our findings provide novel insights into the protective mechanisms of H2S against FA-induced neurotoxicity.

  18. KAI1/CD82 suppresses hepatocyte growth factor-induced migration of hepatoma cells via upregulation of Sprouty2

    Institute of Scientific and Technical Information of China (English)

    MU ZhenBin; WANG Hua; ZHANG Jing; LI QingFang; WANG LiSheng; GUO XiaoZhong

    2008-01-01

    We conducted a study concerning the suppressive mechanism of KAI1/CD82 on hepatoma cell metas-tasis. Hepatocyte growth factor (HGF) induces the migration of hepatoma cells through activation of cellular sphingosine kinase 1 (SphK1). Adenovirus-mediated gene transfer of KAI1 (Ad-KAI1) down-regulates the SphK1 expression and suppresses the HGF-induced migration of SMMC-7721 human hepatocellcular carcinoma cells. Overexpression of KAI1/CD82 significantly elevates Sprouty2 at the protein level. Ablation of Sprouty2 with RNA interference can block the KAI1/CD82-induced suppres-sion of hepatoma cell migration and downregulation of SphK1 expression. It is demonstrated that KAI1/CD82 suppresses HGF-induced migration of hepatoma cells via upregulation of Sprouty2.

  19. Expression of nerve growth factor is upregulated in the rat thymic epithelial cells during thymus regeneration following acute thymic involution.

    Science.gov (United States)

    Lee, Hee-Woo; Kim, Sung-Min; Shim, Na-Ri; Bae, Soo-Kyung; Jung, Il-Gun; Kwak, Jong-Young; Kim, Bong-Seon; Kim, Jae-Bong; Moon, Jeon-Ok; Chung, Joo-Seop; Yoon, Sik

    2007-06-07

    Neuroimmune networks in the thymic microenvironment are thought to be involved in the regulation of T cell development. Nerve growth factor (NGF) is increasingly recognized as a potent immunomodulator, promoting "cross-talk" between various types of immune system cells. The present study describes the expression of NGF during thymus regeneration following acute involution induced by cyclophosphamide in the rat. Immunohistochemical stain demonstrated not only the presence of NGF but also its upregulated expression mainly in the subcapsular, paraseptal, and perivascular epithelial cells, and medullary epithelial cells including Hassall's corpuscles in both the normal and regenerating thymus. Biochemical data obtained using Western blot and RT-PCR supported these results and showed that thymic extracts contain NGF protein and mRNA, at higher levels during thymus regeneration. Thus, our results suggest that NGF expressed in these thymic epithelial cells plays a role in the T lymphopoiesis associated with thymus regeneration during recovery from acute thymic involution.

  20. KAI1/CD82 suppresses hepatocyte growth factorinduced migration of hepatoma cells via upregulation of Sprouty2

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    We conducted a study concerning the suppressive mechanism of KAI1/CD82 on hepatoma cell metastasis.Hepatocyte growth factor(HGF)induces the migration of hepatoma cells through activation of cellular sphingosine kinase 1(SphK1).Adenovirus-mediated gene transfer of KAI1(Ad-KAI1)downregulates the SphK1 expression and suppresses the HGF-induced migration of SMMC-7721 human hepatocellcular carcinoma cells.Overexpression of KAI1/CD82 significantly elevates Sprouty2 at the protein level.Ablation of Sprouty2 with RNA interference can block the KAI1/CD82-induced suppression of hepatoma cell migration and downregulation of SphK1 expression.It is demonstrated that KAI1/CD82 suppresses HGF-induced migration of hepatoma cells via upregulation of Sprouty2.

  1. JNK-dependent Atg4 upregulation mediates asperphenamate derivative BBP-induced autophagy in MCF-7 cells

    Energy Technology Data Exchange (ETDEWEB)

    Li, Yanchun; Luo, Qiyu [Department of Pharmacology, Shenyang Pharmaceutical University, Shenyang 110016 (China); Yuan, Lei [School of Pharmaceutical Engineering, Shenyang Pharmaceutical University, Shenyang 110016 (China); Miao, Caixia; Mu, Xiaoshuo [Department of Pharmacology, Shenyang Pharmaceutical University, Shenyang 110016 (China); Xiao, Wei [Jiangsu Kanion Pharmaceutical Co., Ltd., Nanjing 222001 (China); Li, Jianchun [Department of Pharmacology, Shenyang Pharmaceutical University, Shenyang 110016 (China); Sun, Tiemin, E-mail: suntiemin@126.com [School of Pharmaceutical Engineering, Shenyang Pharmaceutical University, Shenyang 110016 (China); Ma, Enlong, E-mail: maenlong@hotmail.com [Department of Pharmacology, Shenyang Pharmaceutical University, Shenyang 110016 (China); Jiangsu Kanion Pharmaceutical Co., Ltd., Nanjing 222001 (China)

    2012-08-15

    N-Benzoyl-O-(N′-(1-benzyloxycarbonyl-4-piperidiylcarbonyl) -D-phenylalanyl)-D-phenylalaninol (BBP), a novel synthesized asperphenamate derivative with the increased solubility, showed growth inhibitory effect on human breast carcinoma MCF-7 cells in a time- and concentration-dependent manner. The growth inhibitory effect of BBP was associated with induction of autophagy, which was demonstrated by the development of acidic vesicular organelles, cleavage of LC3 and upregulation of Atg4 in BBP-treated MCF-7 cells. Since the application of Atg4 siRNA totally blocked the cleavage of LC3, we demonstrated a central role of Atg4 in BBP-induced autophagy. The further studies showed that BBP increased the levels of reactive oxygen species (ROS), and pretreatment with NAC effectively blocked the accumulation of ROS, autophagy and growth inhibition triggered by BBP. Moreover, BBP induced the activation of JNK, and JNK inhibitor SP600125 reversed autophagy, the increase of Atg4 levels, conversion of LC3 and growth inhibition induced by BBP. Knockdown of JNK by siRNA efficiently inhibited ROS production and autophagy, but antioxidant NAC failed to block JNK activation induced by BBP, indicating that JNK activation may be a upstream signaling of ROS and should be a core component in BBP-induced autophagic signaling pathway. These results suggest that BBP produces its growth inhibitory effect through induction of the autophagic cell death in MCF-7 cells, which is modulated by a JNK-dependent Atg4 upregulation involving ROS production. -- Highlights: ► Asperphenamate derivative BBP with increased solubility was synthesized. ► BBP selectively inhibited the growth of human breast tumor cells. ► The growth inhibitory effect of BBP was associated with induction of autophagy. ► JNK-dependent Atg4 upregulation mediated BBP-induced autophagy.

  2. Critical role of p53 upregulated modulator of apoptosis in benzyl isothiocyanate-induced apoptotic cell death.

    Directory of Open Access Journals (Sweden)

    Marie Lue Antony

    Full Text Available Benzyl isothiocyanate (BITC, a constituent of edible cruciferous vegetables, decreases viability of cancer cells by causing apoptosis but the mechanism of cell death is not fully understood. The present study was undertaken to determine the role of Bcl-2 family proteins in BITC-induced apoptosis using MDA-MB-231 (breast, MCF-7 (breast, and HCT-116 (colon human cancer cells. The B-cell lymphoma 2 interacting mediator of cell death (Bim protein was dispensable for proapoptotic response to BITC in MCF-7 and MDA-MB-231 cells as judged by RNA interference studies. Instead, the BITC-treated MCF-7 and MDA-MB-231 cells exhibited upregulation of p53 upregulated modulator of apoptosis (PUMA protein. The BITC-mediated induction of PUMA was relatively more pronounced in MCF-7 cells due to the presence of wild-type p53 compared with MDA-MB-231 with mutant p53. The BITC-induced apoptosis was partially but significantly attenuated by RNA interference of PUMA in MCF-7 cells. The PUMA knockout variant of HCT-116 cells exhibited significant resistance towards BITC-induced apoptosis compared with wild-type HCT-116 cells. Attenuation of BITC-induced apoptosis in PUMA knockout HCT-116 cells was accompanied by enhanced G2/M phase cell cycle arrest due to induction of p21 and down regulation of cyclin-dependent kinase 1 protein. The BITC treatment caused a decrease in protein levels of Bcl-xL (MCF-7 and MDA-MB-231 cells and Bcl-2 (MCF-7 cells. Ectopic expression of Bcl-xL in MCF-7 and MDA-MB-231 cells and that of Bcl-2 in MCF-7 cells conferred protection against proapoptotic response to BITC. Interestingly, the BITC-treated MDA-MB-231 cells exhibited induction of Bcl-2 protein expression, and RNA interference of Bcl-2 in this cell line resulted in augmentation of BITC-induced apoptosis. The BITC-mediated inhibition of MDA-MB-231 xenograft growth in vivo was associated with the induction of PUMA protein in the tumor. In conclusion, the results of the present study

  3. Up-Regulated Expression of LAMP2 and Autophagy Activity during Neuroendocrine Differentiation of Prostate Cancer LNCaP Cells

    Science.gov (United States)

    Vara-Ciruelos, Diana; Ramos-Torres, Ágata; Altamirano-Dimas, Manuel; Díaz-Laviada, Inés; Rodríguez-Henche, Nieves

    2016-01-01

    Neuroendocrine (NE) prostate cancer (PCa) is a highly aggressive subtype of prostate cancer associated with resistance to androgen ablation therapy. In this study, we used LNCaP prostate cancer cells cultured in a serum-free medium for 6 days as a NE model of prostate cancer. Serum deprivation increased the expression of NE markers such as neuron-specific enolase (NSE) and βIII tubulin (βIII tub) and decreased the expression of the androgen receptor protein in LNCaP cells. Using cDNA microarrays, we compared gene expression profiles of NE cells and non-differentiated LNCaP cells. We identified up-regulation of 155 genes, among them LAMP2, a lysosomal membrane protein involved in lysosomal stability and autophagy. We then confirmed up-regulation of LAMP2 in NE cells by qRT-PCR, Western blot and confocal microscopy assays, showing that mRNA up-regulation correlated with increased levels of LAMP2 protein. Subsequently, we determined autophagy activity in NE cells by assessing the protein levels of SQSTM/p62 and LC3 by Western blot and LC3 and Atg5 mRNAs content by qRT-PCR. The decreased levels of SQSTM/p62 was accompanied by an enhanced expression of LC3 and ATG5, suggesting activation of autophagy in NE cells. Blockage of autophagy with 1μM AKT inhibitor IV, or by silencing Beclin 1 and Atg5, prevented NE cell differentiation, as revealed by decreased levels of the NE markers. In addition, AKT inhibitor IV as well as Beclin1 and Atg5 kwockdown attenuated LAMP2 expression in NE cells. On the other hand, LAMP2 knockdown by siRNA led to a marked blockage of autophagy, prevention of NE differentiation and decrease of cell survival. Taken together, these results suggest that LAMP2 overexpression assists NE differentiation of LNCaP cells induced by serum deprivation and facilitates autophagy activity in order to attain the NE phenotype and cell survival. LAMP2 could thus be a potential biomarker and potential target for NE prostate cancer. PMID:27627761

  4. Up-regulation of CHAF1A, a poor prognostic factor, facilitates cell proliferation of colon cancer

    Energy Technology Data Exchange (ETDEWEB)

    Wu, Zehua; Cui, Feifei; Yu, Fudong; Peng, Xiao; Jiang, Tao; Chen, Dawei [Department of General Surgery, Shanghai Jiaotong University Affiliated First People’s Hospital, 85 Wujin Road, Shanghai 200080 (China); Lu, Su [Department of Pathology, Shanghai Jiaotong University Affiliated First People’s Hospital, 85 Wujin Road, Shanghai 200080 (China); Tang, Huamei, E-mail: tanghuamei@gmail.com [Department of Pathology, Shanghai Jiaotong University Affiliated First People’s Hospital, 85 Wujin Road, Shanghai 200080 (China); Peng, Zhihai, E-mail: zhihai.peng@hotmail.com [Department of General Surgery, Shanghai Jiaotong University Affiliated First People’s Hospital, 85 Wujin Road, Shanghai 200080 (China)

    2014-06-27

    Highlights: • We identified that CHAF1A was up-regulated in colon tumor mucosa in TMA. • The expression pattern of CHAF1A was validated with qPCR and western-blot. • CHAF1A overexpression is an independent indicator for poor colon cancer survival. • CHAF1A facilitates cell proliferation of colon cancer both in vitro and in vivo. - Abstract: Deregulation of chromatin assembly factor 1, p150 subunit A (CHAF1A) has recently been reported to be involved in the development of some cancer types. In this study, we identified that the frequency of positive CHAF1A staining in primary tumor mucosa (45.8%, 93 of 203 samples) was significantly elevated compared to that in paired normal mucosa (18.7%, 38 of 203 samples). The increased expression was strongly associated with cancer stage, tumor invasion, and histological grade. The five-year survival rate of patients with CHAF1A-positive tumors was remarkably lower than that of patients with CHAF1A-negative tumors. Colon cancer cells with CHAF1A knockdown exhibited decreased cell growth index, reduction in colony formation ability, elevated cell apoptosis rate as well as impaired colon tumorigenicity in nude mice. Hence, CHAF1A upregulation functions as a poor prognostic indicator of colon cancer, potentially contributing to its progression by mediating cancer cell proliferation.

  5. Sesamin Inhibits PDGF-Mediated Proliferation of Vascular Smooth Muscle Cells by Upregulating p21 and p27.

    Science.gov (United States)

    Han, Joo-Hui; Lee, Sang-Gil; Jung, Sang-Hyuk; Lee, Jung-Jin; Park, Hyun-Soo; Kim, Young Ho; Myung, Chang-Seon

    2015-08-26

    Sesamin, an active ingredient of Asiasarum heterotropoides, is known to exhibit many bioactive functions, but the effect thereof on vascular smooth muscle cell (VSMC) proliferation remains poorly understood. Hence, we explored the antiproliferative action of sesamin on VSMCs and the underlying mechanism thereof, focusing on possible effects of sesamin on cell cycle progression. Sesamin significantly inhibited platelet-derived growth factor (PDGF)-induced VSMC proliferation (inhibition percentage at 1, 5, and 10 μM sesamin was 49.8 ± 22.0%, 74.6 ± 19.9%, and 87.8 ± 13.0%, respectively) in the absence of cytotoxicity and apoptosis, and PDGF-induced DNA synthesis; and arrested cell cycle progression in the G0/G1-to-S phase. Sesamin potently inhibited cyclin D1 and CDK4 expression, pRb phosphorylation, and expression of the proliferating cell nuclear antigen (PCNA); and upregulated p27(KIP1), p21(CIP1), and p53. The results thus indicate that the antiproliferative effect of sesamin on PDGF-stimulated VSMCs is attributable to arrest of the cell cycle in G0/G1 caused, in turn, by upregulation of p27(KIP1), p21(CIP1), and p53, and inhibition of cyclin E-CDK2 and cyclin D1-CDK4 expression.

  6. Thymoquinone up-regulates PTEN expression and induces apoptosis in doxorubicin-resistant human breast cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Arafa, El-Shaimaa A.; Zhu Qianzheng [Department of Radiology, Ohio State University, Columbus, OH 43210 (United States); Shah, Zubair I. [James Cancer Hospital and Solove Research Institute, Ohio State University, Columbus, OH 43210 (United States); Wani, Gulzar; Barakat, Bassant M.; Racoma, Ira [Department of Radiology, Ohio State University, Columbus, OH 43210 (United States); El-Mahdy, Mohamed A., E-mail: Mohamed.el-mahdy@osumc.edu [Department of Radiology, Ohio State University, Columbus, OH 43210 (United States); Wani, Altaf A., E-mail: wani.2@osu.edu [Department of Radiology, Ohio State University, Columbus, OH 43210 (United States); Department of Molecular and Cellular Biochemistry, Ohio State University, Columbus, OH 43210 (United States); James Cancer Hospital and Solove Research Institute, Ohio State University, Columbus, OH 43210 (United States); DNA Research Chair, King Saud University, Riyadh (Saudi Arabia)

    2011-01-10

    The use of innocuous naturally occurring compounds to overcome drug resistance and cancer recalcitrance is now in the forefront of cancer research. Thymoquinone (TQ) is a bioactive constituent of the volatile oil derived from seeds of Nigella sativa Linn. TQ has shown promising anti-carcinogenic and anti-tumor activities through different mechanisms. However, the effect of TQ on cell signaling and survival pathways in resistant cancer cells has not been fully delineated. Here, we report that TQ greatly inhibits doxorubicin-resistant human breast cancer MCF-7/DOX cell proliferation. TQ treatment increased cellular levels of PTEN proteins, resulting in a substantial decrease of phosphorylated Akt, a known regulator of cell survival. The PTEN expression was accompanied with elevation of PTEN mRNA. TQ arrested MCF-7/DOX cells at G2/M phase and increased cellular levels of p53 and p21 proteins. Flow cytometric analysis and agarose gel electrophoresis revealed a significant increase in Sub-G1 cell population and appearance of DNA ladders following TQ treatment, indicating cellular apoptosis. TQ-induced apoptosis was associated with disrupted mitochondrial membrane potential and activation of caspases and PARP cleavage in MCF-7/DOX cells. Moreover, TQ treatment increased Bax/Bcl2 ratio via up-regulating Bax and down-regulating Bcl2 proteins. More importantly, PTEN silencing by target specific siRNA enabled the suppression of TQ-induced apoptosis resulting in increased cell survival. Our results reveal that up-regulation of the key upstream signaling factor, PTEN, in MCF-7/DOX cells inhibited Akt phosphorylation, which ultimately causes increase in their regulatory p53 levels affecting the induction of G2/M cell cycle arrest and apoptosis. Overall results provide mechanistic insights for understanding the molecular basis and utility of the anti-tumor activity of TQ.

  7. Upregulation of 25-hydroxyvitamin D3-1α-hydroxylase by butyrate in Caco-2 cells

    Institute of Scientific and Technical Information of China (English)

    Oliver Schr(o)der; Sinan Turak; Carolin Daniel; Tanja Gaschott; Jürgen Stein

    2005-01-01

    AIM: To investigate the possible involvement of 25-hydroxyvitamin D3-1α-hydroxylase [1α-25(OH)2D3] in butyrate-induced differentiation in human intestinal cell line Caco-2 cells.METHODS: Caco-2 cells were incubated either with 3 mmol/L butyrate and 1 μmol/L 25(OH)2D3 or with 1μmol/L 1α-25(OH)2D3 for various time intervals ranging from 0 to 72 h. Additionally, cells were co-incubated with butyrate and either 25(OH)2D3 or 1α-25(OH)2D3.1α-25(OH)2D3 mRNA was determined semi-quantitatively using the fluorescent dye PicoGreen. Immunoblotting was used for the detection of 1α-25(OH)2D3 protein.Finally, enzymatic activity was measured by ELISA.RESULTS: Both butyrate and 1α-25(OH)2D3 stimulated differentiation of Caco-2 cells after a 48 h incubation period, while 25(OH)2D3 had no impact on cell differentiation. Synergistic effects on differentiation were observed when cells were co-incubated with butyrate and vitamin D metabolite. Butyrate transiently upregulated 1α-25(OH)2D3 mRNA followed by a timely delayed protein upregulation. Coincidently, enzymatic activity was enhanced significantly. The induction of the enzyme allowed for comparable differentiating effects of both vitamin D metabolites.CONCLUSION: Our experimental data pr ovide a further mechanism for the involvement of the vitamin D signaling pathway in colonic epithelial cell differentiation by butyrate. The enhancement of 1α-25(OH)2D3 followed by antiproliferative effects of the vitamin D prohormone in the Caco-2 cell line suggest that 25(OH)2D3 in combination with butyrate may offer a new therapeutic approach for the treatment of colon cancer.

  8. Upregulation of the SERCA-type Ca2+ pump activity in response to endoplasmic reticulum stress in PC12 cells

    Directory of Open Access Journals (Sweden)

    Frandsen Aase

    2001-04-01

    Full Text Available Abstract Background Ca2+-ATPases of endoplasmic reticulum (SERCAs are responsible for maintenance of the micro- to millimolar Ca2+ ion concentrations within the endoplasmic reticulum (ER of eukaryotic cells. This intralumenal Ca2+ storage is important for the generation of Ca2+ signals as well as for the correct folding and posttranslational processing of proteins entering ER after synthesis. ER perturbations such as depletion of Ca2+ or abolishing the oxidative potential, inhibition of glycosylation, or block of secretory pathway, activate the Unfolded Protein Response, consisting of an upregulation of a number of ER-resident chaperones/stress proteins in an effort to boost the impaired folding capacity. Results We show here that in PC12 cells, depletion of ER Ca2+ by EGTA, as well as inhibition of disulphide bridge formation within the ER by dithiotreitol or inhibition of N-glycosylation by tunicamycin, led to a 2- to 3-fold increase of the SERCA-mediated 45Ca2+ transport to microsomes isolated from cells exposed to these stress agents. The time course of this response corresponded to that for transcriptional upregulation of ER stress proteins, as well as to the increase in the SERCA2b mRNA, as we recently observed in an independent study. Conclusions These findings provide the first functional evidence for the increase of SERCA pumping capacity in cells subjected to the ER stress. Since at least three different and unrelated mechanisms of eliciting the ER stress response were found to cause this functional upregulation of Ca2+ transport into the ER, these results support the existence of a coupling between the induction of the UPR pathway in general, and the regulation of expression of at least one of the SERCA pump isoforms.

  9. Restoration of p53 Expression in Human Cancer Cell Lines Upregulates the Expression of Notch1: Implications for Cancer Cell Fate Determination after Genotoxic Stress

    Directory of Open Access Journals (Sweden)

    Fatouma Alimirah

    2007-05-01

    Full Text Available Following genotoxic stress, transcriptional activation of target genes by p53 tumor suppressor is critical in cell fate determination. Here we report that the restoration of p53 function in human cancer cell lines that are deficient in p53 function upregulated the expression of Notch1. Interestingly, the expression of wild-type p53 in human prostate and breast cancer cell lines correlated well with increased expression of Notch1. Furthermore, knockdown of p53 expression in cancer cells that express wild-type p53 resulted in reduced expression of Notch1. Importantly, genotoxic stress to cancer cells that resulted in activation of p53 also upregulated the expression of Notch1. Moreover, p53mediated induction of Notch1 expression was associated with stimulation of the activity of Notch-responsive reporters. Notably, p53 differentially regulated the expression of Notch family members: expression of Notch2 and Notch4 was not induced by p53. Significantly, treatment of cells with gamma secretase inhibitor, an inhibitor of Notch signaling, increased susceptibility to apoptosis in response to genotoxic stress. Together, our observations suggest that p53mediated upregulation of Notch1 expression in human cancer cell lines contributes to cell fate determination after genotoxic stress.

  10. Restoration of p53 Expression in Human Cancer Cell Lines Upregulates the Expression of Notch1: Implications for Cancer Cell Fate Determination after Genotoxic Stress1

    Science.gov (United States)

    Alimirah, Fatouma; Panchanathan, Ravichandran; Davis, Francesca J; Chen, Jianming; Choubey, Divaker

    2007-01-01

    Following genotoxic stress, transcriptional activation of target genes by p53 tumor suppressor is critical in cell fate determination. Here we report that the restoration of p53 function in human cancer cell lines that are deficient in p53 function upregulated the expression of Notch1. Interestingly, the expression of wild-type p53 in human prostate and breast cancer cell lines correlated well with increased expression of Notch1. Furthermore, knockdown of p53 expression in cancer cells that express wild-type p53 resulted in reduced expression of Notch1. Importantly, genotoxic stress to cancer cells that resulted in activation of p53 also upregulated the expression of Notch1. Moreover, p53-mediated induction of Notch1 expression was associated with stimulation of the activity of Notch-responsive reporters. Notably, p53 differentially regulated the expression of Notch family members: expression of Notch2 and Notch4 was not induced by p53. Significantly, treatment of cells with gamma secretase inhibitor, an inhibitor of Notch signaling, increased susceptibility to apoptosis in response to genotoxic stress. Together, our observations suggest that p53-mediated upregulation of Notch1 expression in human cancer cell lines contributes to cell fate determination after genotoxic stress. PMID:17534448

  11. Cathepsin B is up-regulated and mediates extracellular matrix degradation in trabecular meshwork cells following phagocytic challenge.

    Directory of Open Access Journals (Sweden)

    Kristine Porter

    Full Text Available Cells in the trabecular meshwork (TM, a tissue responsible for draining aqueous humor out of the eye, are known to be highly phagocytic. Phagocytic activity in TM cells is thought to play an important role in outflow pathway physiology. However, the molecular mechanisms triggered by phagocytosis in TM cells are unknown. Here we investigated the effects of chronic phagocytic stress on lysosomal function using different phagocytic ligands (E. coli, carboxylated beads, collagen I-coated beads, and pigment. Lysotracker red co-localization and electron micrographs showed the maturation of E. coli- and collagen I-coated beads-containing phagosomes into phagolysosomes. Maturation of phagosomes into phagolysosomes was not observed with carboxylated beads or pigment particles. In addition, phagocytosis of E. coli and collagen I-coated beads led to increased lysosomal mass, and the specific up-regulation and activity of cathepsin B (CTSB. Higher levels of membrane-bound and secreted CTSB were also detected. Moreover, in vivo zymography showed the intralysosomal degradation of ECM components associated with active CTSB, as well as an overall increased gelatinolytic activity in phagocytically challenged TM cells. This increased gelatinolytic activity with phagocytosis was partially blocked with an intracellular CTSB inhibitor. Altogether, these results suggest a potential role of phagocytosis in outflow pathway tissue homeostasis through the up-regulation and/or proteolytic activation of extracellular matrix remodeling genes.

  12. C-Myc negatively controls the tumor suppressor PTEN by upregulating miR-26a in glioblastoma multiforme cells

    Energy Technology Data Exchange (ETDEWEB)

    Guo, Pin; Nie, Quanmin; Lan, Jin; Ge, Jianwei [Department of Neurosurgery, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 200127 (China); Qiu, Yongming, E-mail: qiuzhoub@hotmail.com [Department of Neurosurgery, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 200127 (China); Shanghai Institute of Head Trauma, Shanghai 200127 (China); Mao, Qing, E-mail: maoq@netease.com [Department of Neurosurgery, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 200127 (China); Shanghai Institute of Head Trauma, Shanghai 200127 (China)

    2013-11-08

    Highlights: •The c-Myc oncogene directly upregulates miR-26a expression in GBM cells. •ChIP assays demonstrate that c-Myc interacts with the miR-26a promoter. •Luciferase reporter assays show that PTEN is a specific target of miR-26a. •C-Myc–miR-26a suppression of PTEN may regulate the PTEN/AKT pathway. •Overexpression of c-Myc enhances the proliferative capacity of GBM cells. -- Abstract: The c-Myc oncogene is amplified in many tumor types. It is an important regulator of cell proliferation and has been linked to altered miRNA expression, suggesting that c-Myc-regulated miRNAs might contribute to tumor progression. Although miR-26a has been reported to be upregulated in glioblastoma multiforme (GBM), the mechanism has not been established. We have shown that ectopic expression of miR-26a influenced cell proliferation by targeting PTEN, a tumor suppressor gene that is inactivated in many common malignancies, including GBM. Our findings suggest that c-Myc modulates genes associated with oncogenesis in GBM through deregulation of miRNAs via the c-Myc–miR-26a–PTEN signaling pathway. This may be of clinical relevance.

  13. Short-term uvb-irradiation leads to putative limbal stem cell damage and niche cell-mediated upregulation of macrophage recruiting cytokines

    Directory of Open Access Journals (Sweden)

    Maria Notara

    2015-11-01

    Full Text Available Ultraviolet light B (UVB-irradiation is linked to various ocular pathologies such as limbal stem cell defects in pterygium. Despite the large circumstantial evidence linking UVB irradiation and limbal epithelial stem cell damage, the precise molecular responses of limbal stem cells to UVB irradiation are unclear. Here the effect of UVB irradiation on the putative stem cell phenotype, limbal niche cells and the subsequent effects on corneal (lymphangiogenic privilege were investigated. Primary human limbal epithelial stem cells and fibroblasts were irradiated with 0.02 J/cm2 of UVB, a low dose corresponding to 3 min of solar irradiation. UVB irradiation caused significant reduction of limbal epithelial and limbal fibroblast proliferation for 24 h, but apoptosis of limbal epithelial stem cells only. Moreover, UVB induced stem-like character loss of limbal epithelial cells, as their colony forming efficiency and putative stem cell marker expression significantly decreased. Interestingly, limbal epithelial cells co-cultured with UVB-irradiated limbal fibroblasts also exhibited loss of stem cell character and decrease of colony forming efficiency. Conditioned media from limbal epithelial cells inhibited lymphatic endothelial cell proliferation and tube network complexity; however this effect diminished following UVB irradiation. In contrast, pro-inflammatory and macrophage-recruiting cytokines such as TNFα, IFNγ and MCP1 were significantly upregulated following cell irradiation of limbal fibroblasts. These data demonstrate the key role of the limbal stem cell niche in response to UVB and subsequent (lymphangiogenic and inflammatory events. These data suggest that the known pro(lymphangiogenic effect of UVB irradiation in pterygium is not linked to a direct up-regulation of pro-angiogenic cytokines, but rather to indirect macrophage-recruiting cytokines being upregulated after UVB irradiation.

  14. Perifosine induces protective autophagy and upregulation of ATG5 in human chronic myelogenous leukemia cells in vitro

    Institute of Scientific and Technical Information of China (English)

    Yin TONG; Yan-yan LIU; Liang-shun YOU; Wen-bin QIAN

    2012-01-01

    Aim:The efficacy of the Akt inhibitor perifosine against chronic myeloid leukemia (CML)cells and its mechanisms of action are unknown.In this study,the cytotoxic effects of perifosine on CML and acute myeloid leukemia (AML)cell lines were compared to elucidate the mechanisms underlying the differences.Methods:Human AML cell lines Kasumi-1 and HL-60,and the CML cell line K562 were used.Cell viability was quantitated using MTT assay.Apoptosis was determined using Annexin V-FITC/propidium iodide and Hoechst staining,which were followed by flow cytometry and fluorescence microscopy analysis,respectively.Caspase pathway activation and the expression of autophagy-related genes were examined using Western blot.Autophagy was studied using electron microscopy,the acridine orange staining method,and GFP-LC3 was examined with fluorescence microscopy.Results:In contrast to AML cell lines,the CML cell lines K562 and K562/G (an imatinib-insensitive CML cell line)were resistant to perifosine (2.5-20 μmol/L)in respect to inhibiting cell growth and inducing apoptosis.Perifosine (2.5,5,and 10 μmol/L)inhibited Akt and its phosphorylation in AML cells,but not in CML cells.Treatment with perifosine (20 μmol/L)resulted in autophagy in CML cells as shown by the increased formation of acidic vesicular organelles and the accumulation of LC3-II.Treatment of CML cells with perifosine (5,10,and 20 μmol/L)dose-dependently upregulated AGT5,but not Beclin 1 at the protein level.Furthermore,inhibition of autophagyby chloroquine (40 nmol/L)significantly suppressed the cell growth and induced apoptosis in CML cells treated with perifosine (20 μmol/L).Conclusion:Our results show that CML cell lines were resistant to the Akt inhibitor perifosine in vitro,which is due to perifosine-induced protective autophagy and upregulation of ATG5.

  15. NADPH OXIDASE AND LIPID RAFT-ASSOCIATED REDOX SIGNALING ARE REQUIRED FOR PCB153-INDUCED UPREGULATION OF CELL ADHESION MOLECULES IN HUMAN BRAIN ENDOTHELIAL CELLS

    Science.gov (United States)

    Eum, Sung Yong; Andras, Ibolya; Hennig, Bernhard; Toborek, Michal

    2009-01-01

    Exposure to persistent organic pollutants, such as polychlorinated biphenyls (PCBs), can lead to chronic inflammation and the development of vascular diseases. Because cell adhesion molecules (CAMs) of the cerebrovascular endothelium regulate infiltration of inflammatory cells into the brain, we have explored the molecular mechanisms by which ortho-substituted polychlorinated biphenyls (PCBs), such as PCB153, can upregulate CAMs in brain endothelial cells. Exposure to PCB153 increased expression of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1), as well as elevated adhesion of leukocytes to brain endothelial cells. These effects were impeded by inhibitors of EGFR, JAKs, or Src activity. In addition, pharmacological inhibition of NADPH oxidase or disruption of lipid rafts by cholesterol depleting agents blocked PCB153-induced phosphorylation of JAK and Src kinases and upregulation of CAMs. In contrast, silencing of caveolin-1 by siRNA interference did not affect upregulation of ICAM-1 and VCAM-1 in brain endothelial cells stimulated by PCB153. Results of the present study indicate that lipid raft-dependent NADPH oxidase/JAK/EGFR signaling mechanisms regulate the expression of CAMs in brain endothelial cells and adhesion of leukocytes to endothelial monolayers. Due to its role in leukocyte infiltration, induction of CAMs may contribute to PCB-induced cerebrovascular disorders and neurotoxic effects in the CNS. PMID:19632255

  16. Interaction of apoptotic cells with macrophages upregulates COX-2/PGE2 and HGF expression via a positive feedback loop.

    Science.gov (United States)

    Byun, Ji Yeon; Youn, Young-So; Lee, Ye-Ji; Choi, Youn-Hee; Woo, So-Yeon; Kang, Jihee Lee

    2014-01-01

    Recognition of apoptotic cells by macrophages is crucial for resolution of inflammation, immune tolerance, and tissue repair. Cyclooxygenase-2 (COX-2)/prostaglandin E2 (PGE2) and hepatocyte growth factor (HGF) play important roles in the tissue repair process. We investigated the characteristics of macrophage COX-2 and PGE2 expression mediated by apoptotic cells and then determined how macrophages exposed to apoptotic cells in vitro and in vivo orchestrate the interaction between COX-2/PGE2 and HGF signaling pathways. Exposure of RAW 264.7 cells and primary peritoneal macrophages to apoptotic cells resulted in induction of COX-2 and PGE2. The COX-2 inhibitor NS-398 suppressed apoptotic cell-induced PGE2 production. Both NS-398 and COX-2-siRNA, as well as the PGE2 receptor EP2 antagonist, blocked HGF expression in response to apoptotic cells. In addition, the HGF receptor antagonist suppressed increases in COX-2 and PGE2 induction. The in vivo relevance of the interaction between the COX-2/PGE2 and HGF pathways through a positive feedback loop was shown in cultured alveolar macrophages following in vivo exposure of bleomycin-stimulated lungs to apoptotic cells. Our results demonstrate that upregulation of the COX-2/PGE2 and HGF in macrophages following exposure to apoptotic cells represents a mechanism for mediating the anti-inflammatory and antifibrotic consequences of apoptotic cell recognition.

  17. Interaction of Apoptotic Cells with Macrophages Upregulates COX-2/PGE2 and HGF Expression via a Positive Feedback Loop

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    Ji Yeon Byun

    2014-01-01

    Full Text Available Recognition of apoptotic cells by macrophages is crucial for resolution of inflammation, immune tolerance, and tissue repair. Cyclooxygenase-2 (COX-2/prostaglandin E2 (PGE2 and hepatocyte growth factor (HGF play important roles in the tissue repair process. We investigated the characteristics of macrophage COX-2 and PGE2 expression mediated by apoptotic cells and then determined how macrophages exposed to apoptotic cells in vitro and in vivo orchestrate the interaction between COX-2/PGE2 and HGF signaling pathways. Exposure of RAW 264.7 cells and primary peritoneal macrophages to apoptotic cells resulted in induction of COX-2 and PGE2. The COX-2 inhibitor NS-398 suppressed apoptotic cell-induced PGE2 production. Both NS-398 and COX-2-siRNA, as well as the PGE2 receptor EP2 antagonist, blocked HGF expression in response to apoptotic cells. In addition, the HGF receptor antagonist suppressed increases in COX-2 and PGE2 induction. The in vivo relevance of the interaction between the COX-2/PGE2 and HGF pathways through a positive feedback loop was shown in cultured alveolar macrophages following in vivo exposure of bleomycin-stimulated lungs to apoptotic cells. Our results demonstrate that upregulation of the COX-2/PGE2 and HGF in macrophages following exposure to apoptotic cells represents a mechanism for mediating the anti-inflammatory and antifibrotic consequences of apoptotic cell recognition.

  18. Uvangoletin induces mitochondria-mediated apoptosis in HL-60 cells in vitro and in vivo without adverse reactions of myelosuppression, leucopenia and gastrointestinal tract disturbances.

    Science.gov (United States)

    Zheng, Zhuanzhen; Qiao, Zhenhua; Gong, Rong; Wang, Yalin; Zhang, Yiqun; Ma, Yanping; Zhang, Li; Lu, Yujin; Jiang, Bo; Li, Guoxia; Dong, Chunxia; Chen, Wenliang

    2016-02-01

    This study investigated the cytotoxic effect of uvangoletin on HL-60 cells, and the effects of uvangoletin on myelosuppression, leucopenia, gastrointestinal tract disturbances and the possible cytotoxic mechanisms by using CCK-8, flow cytometry, western blot, xenograft, cyclophosphamide-induced leucopenia, copper sulfate-induced emesis and ethanol-induced gastric mucosal lesions assays. The results of CCK-8, flow cytometry and western blot assays indicated that uvangoletin showed the cytotoxic effect on HL-60 cells and induced the apoptosis of HL-60 cells by downregulating the expression levels of anti-apoptotic proteins (Survivin, Bcl-xl and Bcl-2), upregulating the expression levels of pro-apoptotic proteins (Smac, Bax, Bad, c-caspase-3 and c-caspase-9), and promoting the release of cytochrome c from mitochondria to cytoplasm. Further, the results of xenograft assay suggested that uvangoletin inhibited the HL-60-induced tumor growth without adverse effect on body weight of nude mice in vivo by regulating the expression levels of above apoptotic proteins. The results indicated that the reductions of WBCs count and thighbone marrow granulocytes percentage in cyclophosphamide-induced leucopenia assay, the incubation period and number of emesis in copper sulfate-induced emesis assay and the gastric mucosal lesions in ethanol-induced gastric mucosal lesions assay were not exacerbated or reversed by uvangoletin. In conclusion, the research preliminarily indicated that uvangoletin induced apoptosis of HL-60 cells in vitro and in vivo without adverse reactions of myelosuppression, leucopenia and gastrointestinal tract disturbances, and the pro-apoptotic mechanisms may be related to mitochondria-mediated apoptotic pathway.

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

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    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.

  20. AGEs-Induced IL-6 Synthesis Precedes RAGE Up-Regulation in HEK 293 Cells: An Alternative Inflammatory Mechanism?

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    Andreea Iren Serban

    2015-08-01

    Full Text Available Advanced glycation end products (AGEs can activate the inflammatory pathways involved in diabetic nephropathy. Understanding these molecular pathways could contribute to therapeutic strategies for diabetes complications. We evaluated the modulation of inflammatory and oxidative markers, as well as the protective mechanisms employed by human embryonic kidney cells (HEK 293 upon exposure to 200 μg/mL bovine serum albumine (BSA or AGEs–BSA for 12, 24 and 48 h. The mRNA and protein expression levels of AGEs receptor (RAGE and heat shock proteins (HSPs 27, 60 and 70, the activity of antioxidant enzymes and the expression levels of eight cytokines were analysed. Cell damage via oxidative mechanisms was evaluated by glutathione and malondialdehyde levels. The data revealed two different time scale responses. First, the up-regulation of interleukin-6 (IL-6, HSP 27 and high catalase activity were detected as early as 12 h after exposure to AGEs–BSA, while the second response, after 24 h, consisted of NF-κB p65, RAGE, HSP 70 and inflammatory cytokine up-regulation, glutathione depletion, malondialdehyde increase and the activation of antioxidant enzymes. IL-6 might be important in the early ignition of inflammatory responses, while the cellular redox imbalance, RAGE activation and NF-κB p65 increased expression further enhance inflammatory signals in HEK 293 cells.

  1. Rosemary Extracts Upregulate Nrf2, Sestrin2, and MRP2 Protein Level in Human Hepatoma HepG2 Cells

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    Xiao-pei Tong

    2017-01-01

    Full Text Available In the past few decades, the incidence of liver cancer has been rapidly rising across the world. Rosemary is known to possess antioxidant activity and is used as natural antioxidant food preservative. It is proposed to have anticancer activity in treating different tumor models. In this study, we try to explore the impact of rosemary extracts on upregulating the level of Nrf2 and Nrf2-regulatory proteins, Sestrin2 and MRP2 in HepG2 cells, and to speculate its potential mechanism. The anticancer activity of rosemary extract, including its polyphenolic diterpenes carnosic acid and carnosol, was evaluated to understand the potential effect on HepG2 cells. Rosemary extract, carnosic acid, and carnosol induced the expression of Sestrin2 and MRP2 associate with enhancement of Nrf2 protein level in HepG2 cells, in which carnosic acid showed most obvious effect. Although the activation pathway of Nrf2/ARE was not exactly assessed, it can be assumed that the enhancement of expression of Sestrin2 and MRP2 may result from upregulation of Nrf2.

  2. Stimulation of T cells up-regulates expression of Ifi202, an interferon-inducible lupus susceptibility gene, through activation of JNK/c-Jun pathway

    Science.gov (United States)

    Chen, Jianming; Panchanathan, Ravichandran; Choubey, Divaker

    2008-01-01

    Studies have revealed that increased expression of interferon (IFN)-inducible Ifi202 gene (encoding p202 protein) in splenic B and T cells from B6.Nba2 congenic (congenic for Nb2 locus derived from NZB mice) female mice is associated with lupus susceptibility. However, signaling pathways that regulate Ifi202 expression in immune cells remain to be elucidated. Here we report that stimulation of T cells up-regulates the Ifi202 expression. We found that steady-state levels of Ifi202 mRNA and protein were detectable in splenic T cells from NZB mice and stimulation of T cells with anti-CD3 and anti-CD28 up-regulated expression of the Ifi202 gene. Similarly, stimulation of cells of a mouse T-cell hybridoma cell line (2B4.11) also activated transcription of the Ifi202 gene. Significantly, up-regulation of Ifi202 expression in stimulated T cells was inhibited by treatment of cells with SP600125, a specific inhibitor of c-Jun N-terminal kinase (JNK). Conversely, treatment of cells with anisomycin, a potent activator of the JNK and c-Jun, up-regulated Ifi202 expression. Consistent with the activation of JNK/c-Jun pathway by T cell stimulation, forced expression of c-Jun in 2B4 T-cells and in mouse embryonic fibroblasts (MEFs) also up-regulated the Ifi202 expression. Furthermore, we found that stimulation of T cells increased association of the activated c-Jun to the 5′-regulatory region of the Ifi202 gene in chromatin immunoprecipitation assays (ChIPs). Together, our observations demonstrate that stimulation of T cells up-regulates the Ifi202 expression in part through the JNK/c-Jun pathway. PMID:18374989

  3. HBV X Gene Transfection Upregulates IL-1β and IL-6 Gene Expression and Induces Rat Glomerular Mesangial Cell Proliferation

    Institute of Scientific and Technical Information of China (English)

    Hongzhu LU; Jianhua ZHOU

    2008-01-01

    The X gene of HBV encodes a 17-KD protein, termed HBx, which has been shown to function as a transcriptional trans-activator of a variety of viral and cellular promoter/enhancer elements. The aim of this study was to investigate the effect of HBx on gene expression of interleukin (IL)-1β and IL-6, and proliferation of rat mesangial cells in vitro. The X gene of HBV was amplified by PCR assay, and inserted into the eukaryotic expression vector pCI-neo. The structure of recombinant pCI-neo-X plasmid was proved by restrict endonuclease digestion and sequencing analysis. pCI-neo-X was transfected into cultured rat mesangial cell line in vitro via liposome. HBx expression in transfected mesangial cells was detected by Western blot. The IL-1β and IL-6 mRNA expression in those cells was assayed by semiquantitative RT-PCR. Mesangial cell proliferation was tested by MTT. The results showed that HBx was obviously expressed in cultured mesangial cell line at 36th and 48th h after transfection. The expression of IL-1β and IL-6 mRNA was simultaneously increased. The cell proliferation was also obvious at the same time. It was concluded that HBx gene transfection could induce IL-1β and IL-6 gene expression and mesangial cell proliferation. HBx may play a critical role in mesangial cell proliferation through upregulation of the IL-1β and IL-6 gene expression.

  4. Up-regulation of P2X7 receptors mediating proliferation of Schwann cells after sciatic nerve injury.

    Science.gov (United States)

    Song, Xian-min; Xu, Xiao-hui; Zhu, Jiao; Guo, Zhili; Li, Jian; He, Cheng; Burnstock, Geoffrey; Yuan, Hongbin; Xiang, Zhenghua

    2015-06-01

    Peripheral nerve injury (PNI) is a common disease, which results in a partial or total loss of motor, sensory and autonomic functions, leading to a decrease in quality of life. Schwann cells play a vital role in maintaining the peripheral nervous system and in injury and repair. Using immunohistochemistry, Western blot, calcium assay and bromodeoxyuridine (BrdU) proliferation assay, the present study clearly demonstrated that P2X7 receptors (R) were expressed in myelinating and non-myelinating Schwann cells in longitudinal sections of sciatic nerves. After sciatic nerve injury (SNI), P2X7R expression in Schwann cells of injured sciatic nerves was significantly up-regulated during the early days of SNI. Double immunofluorescence of proliferating cell nuclear antigen (PCNA) and P2X7R implied that P2X7R may be involved in proliferation of Schwann cells. Further experiments on primary cultures of Schwann cells showed that P2X7R are functionally expressed in Schwann cells of rat sciatic nerves; ATP via P2X7R can promote Schwann cell proliferation, possibly via the MAPK/ERK intracellular signalling pathway. Other possible roles of P2X7R on Schwann cells are discussed.

  5. Up-regulation of K{sub ir}2.1 by ER stress facilitates cell death of brain capillary endothelial cells

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    Kito, Hiroaki [Department of Molecular and Cellular Pharmacology, Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya (Japan); Yamazaki, Daiju [Department of Molecular and Cellular Pharmacology, Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya (Japan); Department of Biological Chemistry, Kyoto University, Graduate School of Pharmaceutical Sciences, Kyoto (Japan); Department of Molecular Neurobiology, Graduate School of Medical Sciences, Nagoya City University, Nagoya (Japan); Ohya, Susumu; Yamamura, Hisao [Department of Molecular and Cellular Pharmacology, Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya (Japan); Asai, Kiyofumi [Department of Molecular Neurobiology, Graduate School of Medical Sciences, Nagoya City University, Nagoya (Japan); Imaizumi, Yuji, E-mail: yimaizum@phar.nagoya-cu.ac.jp [Department of Molecular and Cellular Pharmacology, Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya (Japan)

    2011-07-29

    Highlights: {yields} We found that application of endoplasmic reticulum (ER) stress with tunicamycin to brain capillary endothelial cells (BCECs) induced cell death. {yields} The ER stress facilitated the expression of inward rectifier K{sup +} channel (K{sub ir}2.1) and induced sustained membrane hyperpolarization. {yields} The membrane hyperpolarization induced sustained Ca{sup 2+} entry through voltage-independent nonspecific cation channels and consequently facilitated cell death. {yields} The K{sub ir}2.1 up-regulation by ER stress is, at least in part, responsible for cell death of BCECs under pathological conditions. -- Abstract: Brain capillary endothelial cells (BCECs) form blood brain barrier (BBB) to maintain brain homeostasis. Cell turnover of BCECs by the balance of cell proliferation and cell death is critical for maintaining the integrity of BBB. Here we found that stimuli with tunicamycin, endoplasmic reticulum (ER) stress inducer, up-regulated inward rectifier K{sup +} channel (K{sub ir}2.1) and facilitated cell death in t-BBEC117, a cell line derived from bovine BCECs. The activation of K{sub ir} channels contributed to the establishment of deeply negative resting membrane potential in t-BBEC117. The deep resting membrane potential increased the resting intracellular Ca{sup 2+} concentration due to Ca{sup 2+} influx through non-selective cation channels and thereby partly but significantly regulated cell death in t-BBEC117. The present results suggest that the up-regulation of K{sub ir}2.1 is, at least in part, responsible for cell death/cell turnover of BCECs induced by a variety of cellular stresses, particularly ER stress, under pathological conditions.

  6. Up-regulation of lymphocyte antigen 6 complex expression in side-population cells derived from a human trophoblast cell line HTR-8/SVneo.

    Science.gov (United States)

    Inagaki, Tetsunori; Kusunoki, Soshi; Tabu, Kouichi; Okabe, Hitomi; Yamada, Izumi; Taga, Tetsuya; Matsumoto, Akemi; Makino, Shintaro; Takeda, Satoru; Kato, Kiyoko

    2016-01-01

    The continual proliferation and differentiation of trophoblasts are critical for the maintenance of pregnancy. It is well known that the tissue stem cells are associated with the development of tissues and pathologies. It has been demonstrated that side-population (SP) cells identified by fluorescence-activated cell sorting (FACS) are enriched with stem cells. The SP cells in HTR-8/SVneo cells derived from human primary trophoblast cells were isolated by FACS. HTR-8/SVneo-SP cell cultures generated both SP and non-SP (NSP) subpopulations. In contrast, NSP cell cultures produced NSP cells and failed to produce SP cells. These SP cells showed self-renewal capability by serial colony-forming assay. Microarray expression analysis using a set of HTR-8/SVneo-SP and -NSP cells revealed that SP cells overexpressed several stemness genes including caudal type homeobox2 (CDX2) and bone morphogenic proteins (BMPs), and lymphocyte antigen 6 complex locus D (LY6D) gene was the most highly up-regulated in HTR-8/SVneo-SP cells. LY6D gene reduced its expression in the course of a 7-day cultivation in differentiation medium. SP cells tended to reduce its fraction by treatment of LY6D siRNA indicating that LY6D had potential to maintain cell proliferation of HTR-8/SVneo-SP cells. On ontology analysis, epithelial-mesenchymal transition (EMT) pathway was involved in the up-regulated genes on microarray analysis. HTR-SVneo-SP cells showed enhanced migration. This is the first report that LY6D was important for the maintenance of HTR-8/SVneo-SP cells. EMT was associated with the phenotype of these SP cells.

  7. Immune tolerance induced by intravenous transfer of immature dendritic cells via up-regulating numbers of suppressive IL-10(+) IFN-γ(+)-producing CD4(+) T cells.

    Science.gov (United States)

    Zhou, Fang; Ciric, Bogoljub; Zhang, Guang-Xian; Rostami, Abdolmohamad

    2013-05-01

    Dendritic cells (DCs) regulate immunity and immune tolerance in vivo. However, the mechanisms of DC-mediated tolerance have not been fully elucidated. Here, we demonstrate that intravenous (i.v.) transfer of bone marrow-derived DCs pulsed with myelin oligodendrocyte glycoprotein (MOG) peptide blocks the development of experimental autoimmune encephalomyelitis in C57BL/6J mice. i.v. transfer of MOG-pulsed DCs leads to the down-regulation of the production of IL-17A and IFN-γ and up-regulation of IL-10 secretion. The development of regulatory T cells (Tregs) is facilitated via up-regulation of FoxP3 expression and production of IL-10. The number of suppressive CD4(+)IL-10(+)IFN-γ(+) T cells is also improved. The expression of OX40, CD154, and CD28 is down-regulated, but the expression of CD152, CD80, PD-1, ICOS, and BTLA is up-regulated on CD4(+) T cells after i.v. transfer of immature DCs. The expression of CCR4, CCR5, and CCR7 on CD4(+) T cells is also improved. Our results suggest that immature DCs may induce tolerance via facilitating the development of CD4(+)FoxP3(+) Tregs and suppressive CD4(+)IL-10(+)IFN-γ(+) T cells in vivo.

  8. LINE-1 family member GCRG123 gene is up-regulated in human gastric signet-ring cell carcinoma

    Institute of Scientific and Technical Information of China (English)

    Gang-Shi Wang; Meng-Wei Wang; Ben-Yan Wu; Xin-Yan Yang; Wei-Hua Wang; Wei-Di You

    2008-01-01

    AIM:To analyze the expression profiles of a human gastric-cancer-related gene,GCRG123,in human gastric signet-ring cell carcinoma tissues,and to perform bioinformatics analysis on GCRG123.METHODS:In situ hybridization was used to explore the GCRG123 expression pattern in paraffin-embedded gastric tissues,including 15 cases of signet-ring cell carcinoma,15 of intestinal-type adenocarcinoma,and 15 of normal gastric mucosa.Northnem blotting was used to analyze the differences in GCRG123 expression between stomach signet-ring cell carcinoma and intestinal-type adenocarcinoma tissues.Online software,including BLAST,Multalin and BLAT,were applied for bioinformatics analysis.National Center for Biotechnology Information (NCBI) and the University of California Santa Cruz (UCSC) databases were used for the analyses.RESULTS:The in situ hybridization signal appeared as blue precipitates restricted to the cytoplasm.Ten out of 15 cases of gastric signet ring cell carcinoma,normal gastric mucosal epithelium and pyloric glands showed high GCRG123 expression.Low GCRG123 expressionv was observed in gastric intestinal-type adenocarcinoma and normal gastric glands.Northern blotting revealed that GCRG123 was up-regulated in signet-ring cell carcinoma tissue but down-regulated in intestinal-type adenocarcinoma tissue.BLAST and Multalin analyses revealed that the GCRG123 sequence had 92% similarity with the ORF2 sequence of human long interspersed nuclear element retrotransposons (LINE-1,L1).BLAT analysis indicated that GCRG123 mapped to all chromosomes.GCRG123 was found to integrate in the intron-17 and -23 of Rb,5' flanking region of IL-2 and clotting factor IX genes.CONCLUSION:GCRG123,an active member of the L1family,was up-regulated in human gastric signet-ring cell carcinoma.

  9. Immunomodulatory drugs act as inhibitors of DNA methyltransferases and induce PU.1 up-regulation in myeloma cells.

    Science.gov (United States)

    Endo, Shinya; Amano, Masayuki; Nishimura, Nao; Ueno, Niina; Ueno, Shikiko; Yuki, Hiromichi; Fujiwara, Shiho; Wada, Naoko; Hirata, Shinya; Hata, Hiroyuki; Mitsuya, Hiroaki; Okuno, Yutaka

    2016-01-08

    Immunomodulatory drugs (IMiDs) such as thalidomide, lenalidomide, and pomalidomide are efficacious in the treatment of multiple myeloma and significantly prolong their survival. However, the mechanisms of such effects of IMiDs have not been fully elucidated. Recently, cereblon has been identified as a target binding protein of thalidomide. Lenalidomide-resistant myeloma cell lines often lose the expression of cereblon, suggesting that IMiDs act as an anti-myeloma agent through interacting with cereblon. Cereblon binds to damaged DNA-binding protein and functions as a ubiquitin ligase, inducing degradation of IKZF1 and IKZF3 that are essential transcription factors for B and T cell development. Degradation of both IKZF1 and IKZF3 reportedly suppresses myeloma cell growth. Here, we found that IMiDs act as inhibitors of DNA methyltransferases (DMNTs). We previously reported that PU.1, which is an ETS family transcription factor and essential for myeloid and lymphoid development, functions as a tumor suppressor in myeloma cells. PU.1 induces growth arrest and apoptosis of myeloma cell lines. In this study, we found that low-dose lenalidomide and pomalidomide up-regulate PU.1 expression through inducing demethylation of the PU.1 promoter. In addition, IMiDs inhibited DNMT1, DNMT3a, and DNMT3b activities in vitro. Furthermore, lenalidomide and pomalidomide decreased the methylation status of the whole genome in myeloma cells. Collectively, IMiDs exert demethylation activity through inhibiting DNMT1, 3a, and 3b, and up-regulating PU.1 expression, which may be one of the mechanisms of the anti-myeloma activity of IMiDs.

  10. Adenosine A(2A receptor up-regulates retinal wave frequency via starburst amacrine cells in the developing rat retina.

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    Pin-Chien Huang

    Full Text Available BACKGROUND: Developing retinas display retinal waves, the patterned spontaneous activity essential for circuit refinement. During the first postnatal week in rodents, retinal waves are mediated by synaptic transmission between starburst amacrine cells (SACs and retinal ganglion cells (RGCs. The neuromodulator adenosine is essential for the generation of retinal waves. However, the cellular basis underlying adenosine's regulation of retinal waves remains elusive. Here, we investigated whether and how the adenosine A(2A receptor (A(2AR regulates retinal waves and whether A(2AR regulation of retinal waves acts via presynaptic SACs. METHODOLOGY/PRINCIPAL FINDINGS: We showed that A(2AR was expressed in the inner plexiform layer and ganglion cell layer of the developing rat retina. Knockdown of A(2AR decreased the frequency of spontaneous Ca²⁺ transients, suggesting that endogenous A(2AR may up-regulate wave frequency. To investigate whether A(2AR acts via presynaptic SACs, we targeted gene expression to SACs by the metabotropic glutamate receptor type II promoter. Ca²⁺ transient frequency was increased by expressing wild-type A(2AR (A2AR-WT in SACs, suggesting that A(2AR may up-regulate retinal waves via presynaptic SACs. Subsequent patch-clamp recordings on RGCs revealed that presynaptic A(2AR-WT increased the frequency of wave-associated postsynaptic currents (PSCs or depolarizations compared to the control, without changing the RGC's excitability, membrane potentials, or PSC charge. These findings suggest that presynaptic A(2AR may not affect the membrane properties of postsynaptic RGCs. In contrast, by expressing the C-terminal truncated A(2AR mutant (A(2AR-ΔC in SACs, the wave frequency was reduced compared to the A(2AR-WT, but was similar to the control, suggesting that the full-length A(2AR in SACs is required for A(2AR up-regulation of retinal waves. CONCLUSIONS/SIGNIFICANCE: A(2AR up-regulates the frequency of retinal waves via

  11. Palladium Nanoparticles Induce Disturbances in Cell Cycle Entry and Progression of Peripheral Blood Mononuclear Cells: Paramount Role of Ions

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    Claudia Petrarca

    2014-01-01

    Full Text Available There is concern about the possible toxicity of palladium nanoparticles (Pd-NP, as they are released in the environment through many applications. We previously studied the toxicity of Pd-NP at high concentrations; here we address the possible toxicity of Pd-NP at low, subtoxic doses. In particular, we have exposed normal human PBMC entering into the first in vitro mitotic division to Pd-NP and to Pd(IV ions to evaluate ROS generation and cell cycle progression. We have measured a statistically significant increase of intracellular ROS in Pd(IV exposed cells, but not in Pd-NP exposed cells. TEM revealed accumulation of lipid droplets and autophagic and mitophagic vacuoles, which appeared more conspicuous in cells exposed to Pd(IV ions than to Pd-NP. Pd-NP were visible in the cytoplasm of Pd-NP exposed cells. Pd-NP addition was associated with a significant increase of cells within the G0/G1-phase and a significant reduction in GS- and G2/M-phases. Cells exposed to Pd(IV ions showed a significant amplification of these cell cycle alterations. These results suggest that ions, per se or released by NPs, are the true inducers of Pd toxicity. It will be essential to verify whether the observed disturbance represents a temporary response or might result in permanent alterations.

  12. Small Molecule Cardiogenol C Upregulates Cardiac Markers and Induces Cardiac Functional Properties in Lineage-Committed Progenitor Cells

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    Agnes K. Mike

    2014-01-01

    Full Text Available Background/Aims: Cell transplantation into the heart is a new therapy after myocardial infarction. Its success, however, is impeded by poor donor cell survival and by limited transdifferentiation of the transplanted cells into functional cardiomyocytes. A promising strategy to overcome these problems is the induction of cardiomyogenic properties in donor cells by small molecules. Methods: Here we studied cardiomyogenic effects of the small molecule compound cardiogenol C (CgC, and structural derivatives thereof, on lineage-committed progenitor cells by various molecular biological, biochemical, and functional assays. Results: Treatment with CgC up-regulated cardiac marker expression in skeletal myoblasts. Importantly, the compound also induced cardiac functional properties: first, cardiac-like sodium currents in skeletal myoblasts, and secondly, spontaneous contractions in cardiovascular progenitor cell-derived cardiac bodies. Conclusion: CgC induces cardiomyogenic function in lineage-committed progenitor cells, and can thus be considered a promising tool to improve cardiac repair by cell therapy.

  13. Pyridoxine enhances cell proliferation and neuroblast differentiation by upregulating the GABAergic system in the mouse dentate gyrus.

    Science.gov (United States)

    Yoo, Dae Young; Kim, Woosuk; Kim, Dae Won; Yoo, Ki-Yeon; Chung, Jin Young; Youn, Hwa Young; Yoon, Yeo Sung; Choi, Soo Young; Won, Moo-Ho; Hwang, In Koo

    2011-05-01

    We investigated the effects of pyridoxine (vitamin B(6)) on cell death, cell proliferation, neuroblast differentiation, and the GABAergic system in the mouse dentate gyrus. We administered pyridoxine (350 mg/kg intraperitoneally) to 8 week old mice twice a day for 14 days and sacrificed them at 10 weeks of age. Pyridoxine treatment did not induce neuronal death or activate microglia in the dentate gyrus, while glial fibrillary acidic protein (GFAP)-positive cells were significantly increased in the subgranular zone of the dentate gyrus. The increase in GFAP-positive cells was confirmed to be due to proliferating cells based on double immunofluorescence staining. GFAP-positive cells, which were also labeled with Ki67, a marker for cell proliferation, and doublecortin, a marker for neuroblast differentiation, were significantly increased in the pyridoxine-treated group compared to those in the vehicle-treated group. Pyridoxine treatment also increased the protein levels of glutamic acid decarboxylase (GAD) 67, an enzyme for GABA synthesis, and pyridoxal 5'-phosphate (PNP) oxidase, an enzyme for pyridoxal phosphate synthesis, in the dentate gyrus. These results suggest that pyridoxine treatment distinctly increases cell proliferation, neuroblast differentiation, and upregulated the GABAergic system, as revealed by the increases of GAD67 and PNP oxidase in the mouse dentate gyrus.

  14. α-Hispanolol sensitizes hepatocellular carcinoma cells to TRAIL-induced apoptosis via death receptor up-regulation

    Energy Technology Data Exchange (ETDEWEB)

    Mota, Alba, E-mail: amota@iib.uam.es [Unidad de Terapias Farmacológicas, Área de Genética Humana, Instituto de Investigación de Enfermedades Raras (IIER), Instituto de Salud Carlos III, Madrid (Spain); Jiménez-Garcia, Lidia, E-mail: ljimenez@isciii.es [Unidad de Terapias Farmacológicas, Área de Genética Humana, Instituto de Investigación de Enfermedades Raras (IIER), Instituto de Salud Carlos III, Madrid (Spain); Herránz, Sandra, E-mail: sherranz@isciii.es [Unidad de Terapias Farmacológicas, Área de Genética Humana, Instituto de Investigación de Enfermedades Raras (IIER), Instituto de Salud Carlos III, Madrid (Spain); Heras, Beatriz de las, E-mail: lasheras@ucm.es [Departamento de Farmacología, Facultad de Farmacia, Universidad Complutense de Madrid (UCM), Madrid (Spain); Hortelano, Sonsoles, E-mail: shortelano@isciii.es [Unidad de Terapias Farmacológicas, Área de Genética Humana, Instituto de Investigación de Enfermedades Raras (IIER), Instituto de Salud Carlos III, Madrid (Spain)

    2015-08-01

    Hispanolone derivatives have been previously described as anti-inflammatory and antitumoral agents. However, their effects on overcoming Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) resistance remain to be elucidated. In this study, we analyzed the cytotoxic effects of the synthetic hispanolone derivative α-hispanolol (α-H) in several tumor cell lines, and we evaluated the induction of apoptosis, as well as the TRAIL-sensitizing potential of α-H in the hepatocellular carcinoma cell line HepG2. Our data show that α-H decreased cell viability in a dose-dependent manner in HeLa, MDA-MB231, U87 and HepG2 cell lines, with a more prominent effect in HepG2 cells. Interestingly, α-H had no effect on non-tumoral cells. α-H induced activation of caspase-8 and caspase-9 and also increased levels of the proapoptotic protein Bax, decreasing antiapoptotic proteins (Bcl-2, X-IAP and IAP-1) in HepG2 cells. Specific inhibition of caspase-8 abrogated the cascade of caspase activation, suggesting that the extrinsic pathway has a critical role in the apoptotic events induced by α-H. Furthermore, combined treatment of α-H with TRAIL enhanced apoptosis in HepG2 cells, activating caspase-8 and caspase-9. This correlated with up-regulation of both the TRAIL death receptor DR4 and DR5. DR4 or DR5 neutralizing antibodies abolished the effect of α-H on TRAIL-induced apoptosis, suggesting that sensitization was mediated through the death receptor pathway. Our results demonstrate that α-H induced apoptosis in the human hepatocellular carcinoma cell line HepG2 through activation of caspases and induction of the death receptor pathway. In addition, we describe a novel function of α-H as a sensitizer on TRAIL-induced apoptotic cell death in HepG2 cells. - Highlights: • α-Hispanolol induced apoptosis in the human hepatocellular carcinoma cell line HepG2. • α-Hispanolol induced activation of caspases and the death receptor pathway. • α-Hispanolol enhanced

  15. TNF-α regulates the proteolytic degradation of ST6Gal-1 and endothelial cell-cell junctions through upregulating expression of BACE1

    Science.gov (United States)

    Deng, Xiao; Zhang, Jun; Liu, Yan; Chen, Linmu; Yu, Chao

    2017-01-01

    Endothelial dysfunction and monocyte adhesion to vascular endothelial cells are two critical steps in atherosclerosis development, and emerging evidence suggests that protein sialylation is involved in these processes. However, the mechanism underlying this phenomenon remains incompletely elucidated. In this study, we demonstrated that treatment with the proinflammatory cytokine TNF-α disrupted vascular endothelial cell-cell tight junctions and promoted monocyte endothelial cell adhesion. Western blotting and Sambucus nigra lectin (SNA) blotting analyses revealed that TNF-α treatment decreased α-2, 6-sialic acid transferase 1 (ST6Gal-I) levels and downregulated VE-Cadherin α-2, 6 sialylation. Further analysis demonstrated that TNF-α treatment upregulated β-site amyloid precursor protein enzyme 1 (BACE1) expression, thus resulting in sequential ST6Gal-I proteolytic degradation. Furthermore, our results revealed that PKC signaling cascades were involved in TNF-α-induced BACE1 upregulation. Together, these results indicated that the proinflammatory cytokine TNF-α impairs endothelial tight junctions and promotes monocyte-endothelial cell adhesion by upregulating BACE1 expression through activating PKC signaling and sequentially cleaving ST6Gal-I. Thus, inhibition of BACE1 expression may be a new approach for treating atherosclerosis. PMID:28091531

  16. Disturbed B cell subpopulations and increased plasma cells in myasthenia gravis patients.

    Science.gov (United States)

    Kohler, Siegfried; Keil, Thomas Oskar Philipp; Swierzy, Marc; Hoffmann, Sarah; Schaffert, Hanne; Ismail, Mahmoud; Rückert, Jens Carsten; Alexander, Tobias; Hiepe, Falk; Gross, Christian; Thiel, Andreas; Meisel, Andreas

    2013-11-15

    Whether there is a general perturbation of B and plasma cell subsets in myasthenia gravis (MG) has not been investigated so far. Here we performed a detailed flow cytometric analysis of blood and if available thymic tissue in order to detect MG-specific and therapy-induced changes. We observed significant differences in the distribution of B cell subsets in MG patients, yet these were mainly attributable to medical treatment. Furthermore MG is associated with significantly increased frequencies of plasma cells that were especially activated in purely ocular disease manifestation. In contrast to thymoma, B cell subset distribution in hyperplastic thymus could be distinguished from peripheral blood, however both tissues were not significantly enriched with plasma cells. Thus B cell differentiation in general is not defective in MG, but modified by therapy and enhanced frequencies of plasma cells can be detected in MG patients.

  17. Cell Density-Dependent Upregulation of PDCD4 in Keratinocytes and Its Implications for Epidermal Homeostasis and Repair

    Directory of Open Access Journals (Sweden)

    Tao Wang

    2015-12-01

    Full Text Available Programmed cell death 4 (PDCD4 is one multi-functional tumor suppressor inhibiting neoplastic transformation and tumor invasion. The role of PDCD4 in tumorigenesis has attracted more attention and has been systematically elucidated in cutaneous tumors. However, the normal biological function of PDCD4 in skin is still unclear. In this study, for the first time, we find that tumor suppressor PDCD4 is uniquely induced in a cell density-dependent manner in keratinocytes. To determine the potential role of PDCD4 in keratinocyte cell biology, we show that knockdown of PDCD4 by siRNAs can promote cell proliferation in lower cell density and partially impair contact inhibition in confluent HaCaT cells, indicating that PDCD4 serves as an important regulator of keratinocytes proliferation and contact inhibition in vitro. Further, knockdown of PDCD4 can induce upregulation of cyclin D1, one key regulator of the cell cycle. Furthermore, the expression patterns of PDCD4 in normal skin, different hair cycles and the process of wound healing are described in detail in vivo, which suggest a steady-state regulatory role of PDCD4 in epidermal homeostasis and wound healing. These findings provide a novel molecular mechanism for keratinocytes’ biology and indicate that PDCD4 plays a role in epidermal homeostasis.

  18. Up-regulation of interleukin-22 mediates liver fibrosis via activating hepatic stellate cells in patients with hepatitis C.

    Science.gov (United States)

    Wu, Li-Yuan; Liu, Shuhong; Liu, Yuan; Guo, Chaonan; Li, Hanwei; Li, Wenshu; Jin, Xueyuan; Zhang, Keming; Zhao, Ping; Wei, Lai; Zhao, Jingmin

    2015-05-01

    Interleukin-22 (IL-22) is known to play a critical role in liver immunity. However, the role of IL-22 in HCV-associated liver fibrosis is poorly understood. In this study, patients with HCV infection disclosed significant increases in peripheral numbers of IL-22-producing cells as well as in IL-22 plasma levels. In the liver, the increased intrahepatic IL-22(+) cells were positively correlated with fibrotic staging scores and clinical progression from CHC to cirrhosis. Moreover, the majority of IL-22(+) cells were located in fibrotic areas in the liver of patients with cirrhosis and co-localized with α-smooth muscle actin (α-SMA) positive hepatic stellate cells (HSCs). In vitro, administration of IL-22 was accompanied with inhibited LX-2 cell apoptosis, promoted LX-2 cell proliferation, increased expression of α-SMA, and up-regulated collagen production by LX-2 cells. Collectively, our data provide evidence that IL-22 may contribute to the fibrogenesis of HCV-associated liver fibrosis by activating HSCs.

  19. Upregulation of CD11A on Hematopoietic Stem Cells Denotes the Loss of Long-Term Reconstitution Potential

    Directory of Open Access Journals (Sweden)

    John W. Fathman

    2014-11-01

    Full Text Available Small numbers of hematopoietic stem cells (HSCs generate large numbers of mature effector cells through the successive amplification of transiently proliferating progenitor cells. HSCs and their downstream progenitors have been extensively characterized based on their cell-surface phenotype and functional activities during transplantation assays. These cells dynamically lose and acquire specific sets of surface markers during differentiation, leading to the identification of markers that allow for more refined separation of HSCs from early hematopoietic progenitors. Here, we describe a marker, CD11A, which allows for the enhanced purification of mouse HSCs. We show through in vivo transplantations that upregulation of CD11A on HSCs denotes the loss of their long-term reconstitution potential. Surprisingly, nearly half of phenotypic HSCs (defined as Lin−KIT+SCA-1+CD150+CD34− are CD11A+ and lack long-term self-renewal potential. We propose that CD11A+Lin−KIT+SCA-1+CD150+CD34− cells are multipotent progenitors and CD11A−Lin−KIT+SCA-1+CD150+CD34− cells are true HSCs.

  20. Disturbing forest disturbances

    Energy Technology Data Exchange (ETDEWEB)

    Volney, W.J.A.; Hirsch, K.G. [Natural Resources Canada, Canadian Forest Service, Northern Forestry Centre, Edmonton, AB (Canada)

    2005-10-01

    This paper described the role that disturbances play in maintaining the ecological integrity of Canadian boreal forests. Potential adaptation options to address the challenges that these disturbances present were also examined. Many forest ecosystems need fire for regeneration, while other forests rely on a cool, wet disintegration process driven by insects and commensal fungi feeding on trees to effect renewal. While there are characteristic natural, temporal and spatial patterns to these disturbances, recent work has demonstrated that the disturbances are being perturbed by climatic change that has been compounded by anthropogenic disturbances in forests. Fire influences species composition and age structure, regulates forest insects and diseases, affects nutrient cycling and energy fluxes, and maintains the productivity of different habitats. Longer fire seasons as a result of climatic change will lead to higher intensity fires that may more easily evade initial attacks and become problematic. Fire regimes elevated beyond the range of natural variation will have a dramatic effect on the regional distribution and functioning of forest ecosystems and pose a threat to the safety and prosperity of people. While it was acknowledged that if insect outbreaks were to be controlled on the entire forest estate, the productivity represented by dead wood would be lost, it was suggested that insects such as the forest tent caterpillar and the spruce bud worm may also pose a greater threat as the climate gets warmer and drier. Together with fungal associates, saproxylic arthropods are active in nutrient cycling and ultimately determine the fertility of forest sites. It was suggested that the production of an age class structure and forest mosaic would render the forest landscape less vulnerable to the more negative aspects of climate change on vegetation response. It was concluded that novel management design paradigms are needed to successfully reduce the risk from threats

  1. Temperature shift and host cell contact up-regulate sporozoite expression of Plasmodium falciparum genes involved in hepatocyte infection.

    Directory of Open Access Journals (Sweden)

    Anthony Siau

    Full Text Available Plasmodium sporozoites are deposited in the skin by Anopheles mosquitoes. They then find their way to the liver, where they specifically invade hepatocytes in which they develop to yield merozoites infective to red blood cells. Relatively little is known of the molecular interactions during these initial obligatory phases of the infection. Recent data suggested that many of the inoculated sporozoites invade hepatocytes an hour or more after the infective bite. We hypothesised that this pre-invasive period in the mammalian host prepares sporozoites for successful hepatocyte infection. Therefore, the genes whose expression becomes modified prior to hepatocyte invasion would be those likely to code for proteins implicated in the subsequent events of invasion and development. We have used P. falciparum sporozoites and their natural host cells, primary human hepatocytes, in in vitro co-culture system as a model for the pre-invasive period. We first established that under co-culture conditions, sporozoites maintain infectivity for an hour or more, in contrast to a drastic loss in infectivity when hepatocytes were not included. Thus, a differential transcriptome of salivary gland sporozoites versus sporozoites co-cultured with hepatocytes was established using a pan-genomic P. falciparum microarray. The expression of 532 genes was found to have been up-regulated following co-culture. A fifth of these genes had no orthologues in the genomes of Plasmodium species used in rodent models of malaria. Quantitative RT-PCR analysis of a selection of 21 genes confirmed the reliability of the microarray data. Time-course analysis further indicated two patterns of up-regulation following sporozoite co-culture, one transient and the other sustained, suggesting roles in hepatocyte invasion and liver stage development, respectively. This was supported by functional studies of four hitherto uncharacterized proteins of which two were shown to be sporozoite surface

  2. Acquisition of docetaxel resistance in breast cancer cells reveals upregulation of ABCB1 expression as a key mediator of resistance accompanied by discrete upregulation of other specific genes and pathways

    DEFF Research Database (Denmark)

    Ninel Hansen, Stine; Westergaard, David; Borg Houlberg Thomsen, Mathilde

    2015-01-01

    to be prominent at higher docetaxel concentrations (second-phase response). Additional resistance mechanisms were indicated by gene expression profiling, including genes in the interferon-inducible protein family in MCF7RES and cancer testis antigen family in MDARES. Also, upregulated expression of various ABC...... resistance and thereby identify key molecular mechanisms and predictive molecular characteristics to docetaxel resistance. Two docetaxel-resistant cell lines, MCF7RES and MDARES, were generated from their respective parental cell lines MCF-7 and MDA-MB-231 by stepwise selection in docetaxel dose increments...... analysis singled out ABCB1, which encodes permeability glycoprotein (Pgp), as the top upregulated gene in both MCF7RES and MDARES. Functional validation revealed Pgp as a key resistance mediator at low docetaxel concentrations (first-phase response), whereas additional resistance mechanisms appeared...

  3. Para-toluenesulfonamide induces tongue squamous cell carcinoma cell death through disturbing lysosomal stability

    OpenAIRE

    Liu, Zhe; Liang, Chenyuan; Zhang, Zhuoyuan; Pan, Jian; Xia, Hui; Zhong, Nanshan; Li, Longjiang

    2015-01-01

    Para-toluenesulfonamide (PTS) has been implicated with anticancer effects against a variety of tumors. In the present study, we investigated the inhibitory effects of PTS on tongue squamous cell carcinoma (Tca-8113) and explored the lysosomal and mitochondrial changes after PTS treatment in vitro. High-performance liquid chromatography showed that PTS selectively accumulated in Tca-8113 cells with a relatively low concentration in normal fibroblasts. Next, the effects of PTS on cell viability...

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

    Science.gov (United States)

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

    2013-01-01

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

  5. Leptin upregulates telomerase activity and transcription of human telomerase reverse transcriptase in MCF-7 breast cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Ren, He, E-mail: herenrh@yahoo.com.cn [Key Laboratory of Breast Cancer Prevention and Therapy, Tianjin Medical University, Ministry of Education, Tianjin Medical University Cancer Hospital, Tianjin (China); Zhao, Tiansuo; Wang, Xiuchao; Gao, Chuntao; Wang, Jian; Yu, Ming [Key Laboratory of Breast Cancer Prevention and Therapy, Tianjin Medical University, Ministry of Education, Tianjin Medical University Cancer Hospital, Tianjin (China); Hao, Jihui, E-mail: jihuihao@yahoo.com [Key Laboratory of Breast Cancer Prevention and Therapy, Tianjin Medical University, Ministry of Education, Tianjin Medical University Cancer Hospital, Tianjin (China)

    2010-03-26

    The aim was to analyze the mechanism of leptin-induced activity of telomerase in MCF-7 breast cancer cells. We found that leptin activated telomerase in a dose-dependent manner; leptin upregulated the expression of Human Telomerase Reverse Transcriptase (hTERT) at mRNA and protein levels; blockade of signal transducer and activator of transcription 3 (STAT3) phosphorylation significantly counteracted leptin-induced hTERT transcription and protein expression; chromatin immunoprecipitation analysis showed that leptin enhanced the binding of STAT3 to the hTERT promoter. This study uncovers a new mechanism of the proliferative effect of leptin on breast cancer cells and provides a new explanation of obesity-related breast cancer.

  6. Vesnarinone downregulates CXCR4 expression via upregulation of Krüppel-like factor 2 in oral cancer cells

    Directory of Open Access Journals (Sweden)

    Uchida Daisuke

    2009-08-01

    Full Text Available Abstract Background We have demonstrated that the stromal cell-derived factor-1 (SDF-1; CXCL12/CXCR4 system is involved in the establishment of lymph node metastasis in oral squamous cell carcinoma (SCC. Chemotherapy is a powerful tool for the treatment of oral cancer, including oral SCC; however, the effects of chemotherapeutic agents on the expression of CXCR4 are unknown. In this study, we examined the expression of CXCR4 associated with the chemotherapeutic agents in oral cancer cells. Results The expression of CXCR4 was examined using 3 different chemotherapeutic agents; 5-fluorouracil, cisplatin, and vesnarinone (3,4-dihydro-6-[4-(3,4-dimethoxybenzoyl-1-piperazinyl]-2-(1H-quinolinone in B88, a line of oral cancer cells that exhibits high levels of CXCR4 and lymph node metastatic potential. Of the 3 chemotherapeutic agents that we examined, only vesnarinone downregulated the expression of CXCR4 at the mRNA as well as the protein level. Vesnarinone significantly inhibited lymph node metastasis in tumor-bearing nude mice. Moreover, vesnarinone markedly inhibited 2.7-kb human CXCR4 promoter activity, and we identified the transcription factor, Krüppel-like factor 2 (KLF2, as a novel vesnarinone-responsive molecule, which was bound to the CXCR4 promoter at positions -300 to -167 relative to the transcription start site. The forced-expression of KLF2 led to the downregulation of CXCR4 mRNA and impaired CXCR4 promoter activity. The use of siRNA against KLF2 led to an upregulation of CXCR4 mRNA. Conclusion These Results indicate that vesnarinone downregulates CXCR4 via the upregulation of KLF2 in oral cancer.

  7. Cadmium induces Wnt signaling to upregulate proliferation and survival genes in sub-confluent kidney proximal tubule cells

    Directory of Open Access Journals (Sweden)

    Wolff Natascha A

    2010-05-01

    Full Text Available Abstract Background The class 1 carcinogen cadmium (Cd2+ disrupts the E-cadherin/β-catenin complex of epithelial adherens junctions (AJs and causes renal cancer. Deregulation of E-cadherin adhesion and changes in Wnt/β-catenin signaling are known to contribute to carcinogenesis. Results We investigated Wnt signaling after Cd2+-induced E-cadherin disruption in sub-confluent cultured kidney proximal tubule cells (PTC. Cd2+ (25 μM, 3-9 h caused nuclear translocation of β-catenin and triggered a Wnt response measured by TOPflash reporter assays. Cd2+ reduced the interaction of β-catenin with AJ components (E-cadherin, α-catenin and increased binding to the transcription factor TCF4 of the Wnt pathway, which was upregulated and translocated to the nucleus. While Wnt target genes (c-Myc, cyclin D1 and ABCB1 were up-regulated by Cd2+, electromobility shift assays showed increased TCF4 binding to cyclin D1 and ABCB1 promoter sequences with Cd2+. Overexpression of wild-type and mutant TCF4 confirmed Cd2+-induced Wnt signaling. Wnt signaling elicited by Cd2+ was not observed in confluent non-proliferating cells, which showed increased E-cadherin expression. Overexpression of E-cadherin reduced Wnt signaling, PTC proliferation and Cd2+ toxicity. Cd2+ also induced reactive oxygen species dependent expression of the pro-apoptotic ER stress marker and Wnt suppressor CHOP/GADD153 which, however, did not abolish Wnt response and cell viability. Conclusions Cd2+ induces Wnt signaling in PTC. Hence, Cd2+ may facilitate carcinogenesis of PTC by promoting Wnt pathway-mediated proliferation and survival of pre-neoplastic cells.

  8. Magnetic ferroferric oxide nanoparticles induce vascular endothelial cell dysfunction and inflammation by disturbing autophagy.

    Science.gov (United States)

    Zhang, Lu; Wang, XueQin; Miao, YiMing; Chen, ZhiQiang; Qiang, PengFei; Cui, LiuQing; Jing, Hongjuan; Guo, YuQi

    2016-03-01

    Despite the considerable use of magnetic ferroferric oxide nanoparticles (Fe3O4NPs) worldwide, their safety is still an important topic of debate. In the present study, we detected the toxicity and biological behavior of bare-Fe3O4NPs (B-Fe3O4NPs) on human umbilical vascular endothelial cells (HUVECs). Our results showed that B-Fe3O4NPs did not induce cell death within 24h even at concentrations up to 400 μg/ml. The level of nitric oxide (NO) and the activity of endothelial NO synthase (eNOS) were decreased after exposure to B-Fe3O4NPs, whereas the levels of proinflammatory cytokines were elevated. Importantly, B-Fe3O4NPs increased the accumulation of autophagosomes and LC3-II in HUVECs through both autophagy induction and the blockade of autophagy flux. The levels of Beclin 1 and VPS34, but not phosphorylated mTOR, were increased in the B-Fe3O4NP-treated HUVECs. Suppression of autophagy induction or stimulation of autophagy flux, at least partially, attenuated the B-Fe3O4NP-induced HUVEC dysfunction. Additionally, enhanced autophagic activity might be linked to the B-Fe3O4NP-induced production of proinflammatory cytokines. Taken together, these results demonstrated that B-Fe3O4NPs disturb the process of autophagy in HUVECs, and eventually lead to endothelial dysfunction and inflammation.

  9. Upregulated Expression of microRNA-16 Correlates with Th17/Treg Cell Imbalance in Patients with Rheumatoid Arthritis.

    Science.gov (United States)

    Wu, Yuan-Hao; Liu, Wei; Xue, Bin; Zhang, Lei; Liu, Xiao-Ya; Liu, Bin; Wang, Yi; Cai, Yue; Duan, Ran

    2016-12-01

    To explore the correlation between miR-16 expression in T cells of peripheral blood mononuclear cells (PBMCs) and Th17/Treg imbalance in rheumatoid arthritis (RA) patients. Forty RA patients were recruited as the case group and further grouped as active RA and inactive RA groups; 21 healthy individuals were selected as the control group. Th17 and Treg were measured by flow cytometry, and their related cytokines were measured by FlowCytomix. RORγt, FoxP3 mRNA, and miR-16 expression in T cells was determined by real-time quantitative polymerase chain reaction. Western blotting was performed to measure RORγt and FoxP3 protein expression. RA patients showed upregulated Th17 and RORγt mRNA and protein expression compared with the controls (all p Treg and FoxP3 mRNA and protein expression compared with inactive RA patients and controls (all p Treg-related cytokines were lower in active RA patients than in controls (all p Treg cells of PBMCs (both p Treg cells was positively related with FoxP3 mRNA expression (both p Treg cells of PBMCs in RA patients was closely associated with the expression of RORγt and FoxP3. MiR-16 may be involved in Th17/Treg imbalance of RA patients by affecting the expression of RORγt and FoxP3.

  10. Acceleration of lung metastasis by up-regulation of CD44 expression in osteosarcoma-derived cell transplanted mice.

    Science.gov (United States)

    Shiratori, H; Koshino, T; Uesugi, M; Nitto, H; Saito, T

    2001-09-20

    The effect of CD44-phenotypic expression on metastasis to the lung was studied using a spontaneous murine osteosarcoma-derived cell line, POS-1, stimulated with lipopolysaccharide (LPS). POS-1 cells were inoculated into the hind paws of 20 C3H/HeJ mice and produced a visible mass in all mice in 5 weeks, and these transplanted tumors resulted in lung metastasis in all mice. The number of metastatic foci in the lungs was 12.0+/-2.1 (mean+/-SD) with LPS-stimulated cells, which was significantly higher than that of unstimulated cells (5.8+/-1.4; N=10 for each; P<0.05). Hyaluronate (HA), a ligand of CD44, inhibited a number of lung metastases in a dose-dependent manner (0.5% HA, 3.0+/-1.1; 0.005% HA, 5.1+/-1.5; without HA, 8.6+/-1.7; N=10 for each; P<0.05, each group with HA versus the group without HA). Adhesion assay by coculturing POS-1 cells and lung microvascular endothelial cells on culture plate showed that the adhesion was significantly lower in HA treated POS-1 than those without HA (1.18+/-0.12 and 2.74+/-0.17, respectively, P<0.05). These results suggest that lung metastasis was accelerated by up-regulation of CD44.

  11. Poncirin Induces Apoptosis in AGS Human Gastric Cancer Cells through Extrinsic Apoptotic Pathway by up-Regulation of Fas Ligand.

    Science.gov (United States)

    Saralamma, Venu Venkatarame Gowda; Nagappan, Arulkumar; Hong, Gyeong Eun; Lee, Ho Jeong; Yumnam, Silvia; Raha, Suchismita; Heo, Jeong Doo; Lee, Sang Joon; Lee, Won Sup; Kim, Eun Hee; Kim, Gon Sup

    2015-09-18

    Poncirin, a natural bitter flavanone glycoside abundantly present in many species of citrus fruits, has various biological benefits such as anti-oxidant, anti-microbial, anti-inflammatory and anti-cancer activities. The anti-cancer mechanism of Poncirin remains elusive to date. In this study, we investigated the anti-cancer effects of Poncirin in AGS human gastric cancer cells (gastric adenocarcinoma). The results revealed that Poncirin could inhibit the proliferation of AGS cells in a dose-dependent manner. It was observed Poncirin induced accumulation of sub-G1 DNA content, apoptotic cell population, apoptotic bodies, chromatin condensation, and DNA fragmentation in a dose-dependent manner in AGS cells. The expression of Fas Ligand (FasL) protein was up-regulated dose dependently in Poncirin-treated AGS cells Moreover, Poncirin in AGS cells induced activation of Caspase-8 and -3, and subsequent cleavage of poly(ADP-ribose) polymerase (PARP). Inhibitor studies' results confirm that the induction of caspase-dependent apoptotic cell death in Poncirin-treated AGS cells was led by the Fas death receptor. Interestingly, Poncirin did not show any effect on mitochondrial membrane potential (ΔΨm), pro-apoptotic proteins (Bax and Bak) and anti-apoptotic protein (Bcl-xL) in AGS-treated cells followed by no activation in the mitochondrial apoptotic protein caspase-9. This result suggests that the mitochondrial-mediated pathway is not involved in Poncirin-induced cell death in gastric cancer. These findings suggest that Poncirin has a potential anti-cancer effect via extrinsic pathway-mediated apoptosis, possibly making it a strong therapeutic agent for human gastric cancer.

  12. Poncirin Induces Apoptosis in AGS Human Gastric Cancer Cells through Extrinsic Apoptotic Pathway by up-Regulation of Fas Ligand

    Directory of Open Access Journals (Sweden)

    Venu Venkatarame Gowda Saralamma

    2015-09-01

    Full Text Available Poncirin, a natural bitter flavanone glycoside abundantly present in many species of citrus fruits, has various biological benefits such as anti-oxidant, anti-microbial, anti-inflammatory and anti-cancer activities. The anti-cancer mechanism of Poncirin remains elusive to date. In this study, we investigated the anti-cancer effects of Poncirin in AGS human gastric cancer cells (gastric adenocarcinoma. The results revealed that Poncirin could inhibit the proliferation of AGS cells in a dose-dependent manner. It was observed Poncirin induced accumulation of sub-G1 DNA content, apoptotic cell population, apoptotic bodies, chromatin condensation, and DNA fragmentation in a dose-dependent manner in AGS cells. The expression of Fas Ligand (FasL protein was up-regulated dose dependently in Poncirin-treated AGS cells Moreover, Poncirin in AGS cells induced activation of Caspase-8 and -3, and subsequent cleavage of poly(ADP-ribose polymerase (PARP. Inhibitor studies’ results confirm that the induction of caspase-dependent apoptotic cell death in Poncirin-treated AGS cells was led by the Fas death receptor. Interestingly, Poncirin did not show any effect on mitochondrial membrane potential (ΔΨm, pro-apoptotic proteins (Bax and Bak and anti-apoptotic protein (Bcl-xL in AGS-treated cells followed by no activation in the mitochondrial apoptotic protein caspase-9. This result suggests that the mitochondrial-mediated pathway is not involved in Poncirin-induced cell death in gastric cancer. These findings suggest that Poncirin has a potential anti-cancer effect via extrinsic pathway-mediated apoptosis, possibly making it a strong therapeutic agent for human gastric cancer.

  13. Up-regulation of Store-operated Ca2+ Entry and Nuclear Factor of Activated T Cells Promote the Acinar Phenotype of the Primary Human Salivary Gland Cells.

    Science.gov (United States)

    Jang, Shyh-Ing; Ong, Hwei Ling; Liu, Xibao; Alevizos, Ilias; Ambudkar, Indu S

    2016-04-15

    The signaling pathways involved in the generation and maintenance of exocrine gland acinar cells have not yet been established. Primary human salivary gland epithelial cells, derived from salivary gland biopsies, acquired an acinar-like phenotype when the [Ca(2+)] in the serum-free medium (keratinocyte growth medium, KGM) was increased from 0.05 mm (KGM-L) to 1.2 mm (KGM-H). Here we examined the mechanism underlying this Ca(2+)-dependent generation of the acinar cell phenotype. Compared with cells in KGM-L, those in KGM-H display enhancement of Orai1, STIM1, STIM2, and nuclear factor of activated T cells 1 (NFAT1) expression together with an increase in store-operated Ca(2+) entry (SOCE), SOCE-dependent nuclear translocation of pGFP-NFAT1, and NFAT-dependent but not NFκB-dependent gene expression. Importantly, AQP5, an acinar-specific protein critical for function, is up-regulated in KGM-H via SOCE/NFAT-dependent gene expression. We identified critical NFAT binding motifs in the AQP5 promoter that are involved in Ca(2+)-dependent up-regulation of AQP5. These important findings reveal that the Ca(2+)-induced switch of salivary epithelial cells to an acinar-like phenotype involves remodeling of SOCE and NFAT signaling, which together control the expression of proteins critically relevant for acinar cell function. Our data provide a novel strategy for generating and maintaining acinar cells in culture.

  14. Acquisition of anoikis resistance up-regulates syndecan-4 expression in endothelial cells.

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    Bruna Ribeiro Carneiro

    Full Text Available Anoikis is a programmed cell death induced upon cell detachment from extracellular matrix, behaving as a critical mechanism in preventing adherent-independent cell growth and attachment to an inappropriate matrix, thus avoiding colonization of distant organs. Cell adhesion plays an important role in neoplastic transformation. Tumors produce several molecules that facilitate their proliferation, invasion and maintenance, especially proteoglycans. The syndecan-4, a heparan sulfate proteoglycan, can act as a co-receptor of growth factors and proteins of the extracellular matrix by increasing the affinity of adhesion molecules to their specific receptors. It participates together with integrins in cell adhesion at focal contacts connecting the extracellular matrix to the cytoskeleton. Changes in the expression of syndecan-4 have been observed in tumor cells, indicating its involvement in cancer. This study investigates the role of syndecan-4 in the process of anoikis and cell transformation. Endothelial cells were submitted to sequential cycles of forced anchorage impediment and distinct lineages were obtained. Anoikis-resistant endothelial cells display morphological alterations, high rate of proliferation, poor adhesion to fibronectin, laminin and collagen IV and deregulation of the cell cycle, becoming less serum-dependent. Furthermore, anoikis-resistant cell lines display a high invasive potential and a low rate of apoptosis. This is accompanied by an increase in the levels of heparan sulfate and chondroitin sulfate as well as by changes in the expression of syndecan-4 and heparanase. These results indicate that syndecan-4 plays a important role in acquisition of anoikis resistance and that the conferral of anoikis resistance may suffice to transform endothelial cells.

  15. Gefitinib upregulates death receptor 5 expression to mediate rmhTRAIL-induced apoptosis in Gefitinib-sensitive NSCLC cell line

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    Yan D

    2015-07-01

    TRAIL synergistically interact to inhibit cell proliferation, and apoptosis assessment demonstrated that associations of drug increased the apoptotic index. rmhTRAIL when used alone downregulated DR5 and upregulated BAX, FLIP, and cleaved-caspase3 proteins expressions. However, results obtained in Western blot analyses demonstrated that the combined treatment-induced cell apoptosis was achieved involving upregulated DR5, cleaved-caspase3, and BAX proteins expression and downregulated FLIP protein expression. Moreover, quantitative polymerase chain reaction showed that gefitinib modulated the expression of targets related to rmhTRAIL activity.Conclusion: These results indicate that epidermal growth factor receptor inhibitors enhance rmhTRAIL antitumor activity in the gefitinib-responsive PC9 cell line, and upregulated DR5 expression plays a critical role in activating caspase-signaling apoptotic pathway.Keywords: gefitinib, rmhTRAIL, apoptosis, DR5

  16. Toll-like receptor 3 signalling up-regulates expression of the HIV co-receptor G-protein coupled receptor 15 on human CD4+ T cells.

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    Miriam Kiene

    Full Text Available BACKGROUND: Many HIV-2 and SIV isolates, as well as some HIV-1 strains, can use the orphan 7-transmembrane receptor GPR15 as co-receptor for efficient entry into host cells. GPR15 is expressed on central memory and effector memory CD4(+ T cells in healthy individuals and a subset of these cells is susceptible to HIV-1 and SIV infection. However, it has not been determined whether GPR15 expression is altered in the context of HIV-1 infection. RESULTS: Here, we show that GPR15 expression in CD4(+ T cells is markedly up-regulated in some HIV-1 infected individuals compared to the rest of the infected patients and to healthy controls. Infection of the PM1 T cell line with primary HIV-1 isolates was found to up-regulate GPR15 expression on the infected cells, indicating that viral components can induce GPR15 expression. Up-regulation of GPR15 expression on CD4(+ T cells was induced by activation of Toll-like receptor 3 signalling via TIR-domain-containing adapter-inducing interferon-β (TRIF and was more prominent on gut-homing compared to lymph node-homing CD4(+ T cells. CONCLUSION: These results suggest that infection-induced up-regulation of GPR15 expression could increase susceptibility of CD4(+ T cells to HIV infection and target cell availability in the gut in some infected individuals.

  17. Upregulation of immunoproteasome subunits in myositis indicates active inflammation with involvement of antigen presenting cells, CD8 T-cells and IFNΓ.

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    Khetam Ghannam

    Full Text Available OBJECTIVE: In idiopathic inflammatory myopathies (IIM infiltration of immune cells into muscle and upregulation of MHC-I expression implies increased antigen presentation and involvement of the proteasome system. To decipher the role of immunoproteasomes in myositis, we investigated individual cell types and muscle tissues and focused on possible immune triggers. METHODS: Expression of constitutive (PSMB5, -6, -7 and corresponding immunoproteasomal subunits (PSMB8, -9, -10 was analyzed by real-time RT-PCR in muscle biopsies and sorted peripheral blood cells of patients with IIM, non-inflammatory myopathies (NIM and healthy donors (HD. Protein analysis in muscle biopsies was performed by western blot. Affymetrix HG-U133 platform derived transcriptome data from biopsies of different muscle diseases and from immune cell types as well as monocyte stimulation experiments were used for validation, coregulation and coexpression analyses. RESULTS: Real-time RT-PCR revealed significantly increased expression of immunoproteasomal subunits (PSMB8/-9/-10 in DC, monocytes and CD8+ T-cells in IIM. In muscle biopsies, the immunosubunits were elevated in IIM compared to NIM and exceeded levels of matched blood samples. Proteins of PSMB8 and -9 were found only in IIM but not NIM muscle biopsies. Reanalysis of 78 myositis and 20 healthy muscle transcriptomes confirmed these results and revealed involvement of the antigen processing and presentation pathway. Comparison with reference profiles of sorted immune cells and healthy muscle confirmed upregulation of PSMB8 and -9 in myositis biopsies beyond infiltration related changes. This upregulation correlated highest with STAT1, IRF1 and IFNγ expression. Elevation of T-cell specific transcripts in active IIM muscles was accompanied by increased expression of DC and monocyte marker genes and thus reflects the cell type specific involvement observed in peripheral blood. CONCLUSIONS: Immunoproteasomes seem to indicate

  18. Hepatitis B Virus X Upregulates HuR Protein Level to Stabilize HER2 Expression in Hepatocellular Carcinoma Cells

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    Chao-Ming Hung

    2014-01-01

    Full Text Available Hepatitis B virus- (HBV- associated hepatocellular carcinoma (HCC is the most common type of liver cancer. However, the underlying mechanism of HCC tumorigenesis is very complicated and HBV-encoded X protein (HBx has been reported to play the most important role in this process. Activation of downstream signal pathways of epidermal growth factor receptor (EGFR family is known to mediate HBx-dependent HCC tumor progression. Interestingly, HER2 (also known as ErbB2/Neu/EGFR2 is frequently overexpressed in HBx-expressing HCC patients and is associated with their poor prognosis. However, it remains unclear whether and how HBx regulates HER2 expression. In this study, our data showed that HBx expression increased HER2 protein level via enhancing its mRNA stability. The induction of RNA-binding protein HuR expression by HBx mediated the HER2 mRNA stabilization. Finally, the upregulated HER2 expression promoted the migration ability of HBx-expressing HCC cells. These findings deciphered the molecular mechanism of HBx-mediated HER2 upregulation in HBV-associated HCC.

  19. Hypoxia induces upregulation of the deoxyribonuclease I gene in the human pancreatic cancer cell line QGP-1.

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    Kominato, Yoshihiko; Iida, Reiko; Nakajima, Tamiko; Tajima, Yutaka; Takagi, Rie; Makita, Chikako; Kishi, Koichiro; Ueki, Misuzu; Kawai, Yasuyuki; Yasuda, Toshihiro

    2007-11-01

    We have previously demonstrated that ischemia caused by acute myocardial infarction induces an abrupt increase of serum deoxyribonuclease I (DNase I) activity. In this study, we examined whether hypoxia can affect the levels of DNase I activity and/or its transcripts in vitro. We first exposed the human pancreatic cancer cell line QGP-1, which is the first documented DNase-I-producing cell line, to hypoxia (2% O2), and found that this induced a significant increase in both the activity and transcripts of DNase I. This response was mediated by increased transcription only from exon 1a of the two alternative transcription-initiating exons utilized simultaneously in the human DNase I gene (DNASE1); exposure of QGP-1 cells to hypoxia for 24 h resulted in a 15-fold increase of DNASE1 transcripts starting from exon 1a compared with the expression level under normoxic conditions. Promoter, electrophoretic mobility shift, and chromatin immunoprecipitation assays with QGP-1 cells exposed to hypoxia or normoxia showed that the region just upstream from exon 1a was involved in this response in a hypoxia-induced factor-1-independent, but at least in a Sp1 transcription factor-dependent manner possibly through enhanced binding of Sp1 protein to the promoter. These results indicate that DNASE1 expression is upregulated by hypoxia in the cells.

  20. Cinnamon and its Components Suppress Vascular Smooth Muscle Cell Proliferation by Up-Regulating Cyclin-Dependent Kinase Inhibitors.

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    Kwon, Hyeeun; Lee, Jung-Jin; Lee, Ji-Hye; Cho, Won-Kyung; Gu, Min Jung; Lee, Kwang Jin; Ma, Jin Yeul

    2015-01-01

    Cinnamomum cassia bark has been used in traditional herbal medicine to treat a variety of cardiovascular diseases. However, the antiproliferative effect of cinnamon extract on vascular smooth muscle cells (VSMCs) and the corresponding restenosis has not been explored. Hence, after examining the effect of cinnamon extract on VSMC proliferation, we investigated the possible involvement of signal transduction pathways associated with early signal and cell cycle analysis, including regulatory proteins. Besides, to identify the active components, we investigated the components of cinnamon extract on VSMC proliferation. Cinnamon extract inhibited platelet-derived growth factor (PDGF)-BB-induced VSMC proliferation and suppressed the PDGF-stimulated early signal transduction. In addition, cinnamon extract arrested the cell cycle and inhibited positive regulatory proteins. Correspondingly, the protein levels of p21 and p27 not only were increased in the presence of cinnamon extract, also the expression of proliferating cell nuclear antigen (PCNA) was inhibited by cinnamon extract. Besides, among the components of cinnamon extract, cinnamic acid (CA), eugenol (EG) and cinnamyl alcohol significantly inhibited the VSMC proliferation. Overall, the present study demonstrates that cinnamon extract inhibited the PDGF-BB-induced proliferation of VSMCs through a G0/G1 arrest, which down-regulated the expression of cell cycle positive regulatory proteins by up-regulating p21 and p27 expression.

  1. CIZ1 is upregulated in hepatocellular carcinoma and promotes the growth and migration of the cancer cells.

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    Wu, Jinsheng; Lei, Liu; Gu, Dianhua; Liu, Hui; Wang, Shaochuang

    2016-04-01

    Hepatocellular carcinoma (HCC) is one of the most common malignancies in the world, and the prognosis for the HCC remains very poor. Although dys-regulation of CIZ1 (Cip1 interacting zinc finger protein 1) has been observed in various cancer types, its expression and functions in HCC remain unknown. In this study, the mRNA level of CIZ1 in the HCC tissues were examined using real-time polymerase chain reaction, and the effects of CIZ1 on the growth, migration, and metastasis of HCC cells were examined by crystal violet assay, Boyden chamber assay, and in vivo image system, respectively. In addition, the molecular mechanisms were investigated by luciferase assay. Upregulation of CIZ1 in the clinical HCC samples was observed. Forced expression of CIZ1 promoted the growth and migration of HCC cells, while knocking down the expression of CIZ1 inhibited the growth, migration, and metastasis of HCC cells. Molecular mechanism studies revealed that CIZ1 activated YAP/TAZ signaling in HCC cells. Taken together, our study demonstrated the oncogenic roles of CIZ1 in HCC cells and CIZ1 might be a promising therapeutic target for HCC.

  2. Amphotericin B Increases Transglutaminase 2 Expression Associated with Upregulation of Endocytotic Activity in Mouse Microglial Cell Line BV-2.

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    Kawabe, Kenji; Takano, Katsura; Moriyama, Mitsuaki; Nakamura, Yoichi

    2017-02-21

    Amphotericin B (AmB), a polyene antibiotic, is reported to cause the microglial activation to induce nitric oxide (NO) production and proinflammatory cytokines expression, and change neurotrophic factors expression in cultured microglia (Motoyoshi et al. in Neurochem Int 52:1290-1296, 2008). On the other hand, tissue-type transglutaminase (TG2) is involved in connection to phagocytes with apoptotic cells. Engulfment of neurons by activated microglia is thought to cause neurodegenerative diseases but detail is unclear, and involvement of TG2 in phagocytosis has been reported in our previous study using lipopolysaccharide-stimulated BV-2 cells (Kawabe et al. in Neuroimmunomodulation 22(4):243-249, 2015). In the present study, we examined the changes of TG2 expression, phagocytosis and pinocytosis in BV-2 cells stimulated by AmB. AmB stimulation increased TG2 expression and TG activity. Phagocytosis of dead cells and pinocytosis of fluorescent microbeads were also up-regulated by AmB stimulation in BV-2 cells. Blockade of TG activity by cystamine, an inhibitor of TGs, suppressed AmB-enhanced TG2 expression, TG activity, NO production, phagocytosis and pinocytosis. Excessive NO production from microglia and/or facilitation of phagocytosis might be involved in neuronal death. To control TG activity might make possible to protect neurons and care for CNS diseases.

  3. PTX3 is up-regulated in epithelial mammary cells during S. aureus intramammary infection in goat

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    Joel Fernando Soares Filipe

    2015-07-01

    Full Text Available Pentraxins are a superfamily of conserved molecules with immune functions such as complement activation and opsonization. PTX3 is the prototypic long pentraxin and is produced by different cell populations after pro-inflammatory stimuli. Different studies have demonstrated the up-regulation of PTX3 during ruminant mastitis, but its role is still unknown.The aim of this study was to elucidate the role of PTX3 in the immune response to S. aureus intra-mammary infection (IMI. Given that no data are available on PTX3 expression in goat tissues, we first studied its pattern of expression  in goat normal tissues. Then we investigated the role of PTX3 during mammary infection, comparing its expression in healthy and infected blood, milk and tissues.Six healthy goats were infused with PBS in the right udder and with S. aureus in the left udder. Mammary biopsies from udders were collected 30h post infection, formalin fixed and routinely processed for microscopic evaluation or immediately stored in RNAlater.Tissue samples were collected at the slaughterhouse from healthy goats and were immediately stored in RNAlater.Blood and milk were collected from healthy and infected goats; cells from blood and milk were isolated and processed for RNA extraction or for cytospins; milk fat globules were obtained through milk centrifugation and immediately processed for RNA extraction.Total RNA from different organs, blood or milk cells, milk fat globules and mammary tissues was extracted and used as template in qPCR for PTX3.PTX3 expression was investigated by immunohistochemistry on formalin fixed paraffin embedded mammary tissue samples and on cytospins of isolated goat blood and milk cells.PTX3 mRNA was expressed with very high levels in bone marrow, mammary gland, aorta, pancreas, skin and lung. Given the high expression in the mammary gland, we investigated which cell population expressed PTX3. PTX3 was mainly expressed in the apical cytoplasmic portion of

  4. The antidiabetic drug ciglitazone induces high grade bladder cancer cells apoptosis through the up-regulation of TRAIL.

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    Marie-Laure Plissonnier

    Full Text Available BACKGROUND: Ciglitazone belongs to the thiazolidinediones class of antidiabetic drug family and is a high-affinity ligand for the Peroxisome Proliferator-Activated Receptor γ (PPARγ. Apart from its antidiabetic activity, this molecule shows antineoplastic effectiveness in numerous cancer cell lines. METHODOLOGY/PRINCIPAL FINDINGS: Using RT4 (derived from a well differentiated grade I papillary tumor and T24 (derived from an undifferentiated grade III carcinoma bladder cancer cells, we investigated the potential of ciglitazone to induce apoptotic cell death and characterized the molecular mechanisms involved. In RT4 cells, the drug induced G2/M cell cycle arrest characterized by an overexpression of p53, p21(waf1/CIP1 and p27(Kip1 in concomitance with a decrease of cyclin B1. On the contrary, in T24 cells, it triggered apoptosis via extrinsic and intrinsic pathways. Cell cycle arrest and induction of apoptosis occurred at high concentrations through PPARγ activation-independent pathways. We show that in vivo treatment of nude mice by ciglitazone inhibits high grade bladder cancer xenograft development. We identified a novel mechanism by which ciglitazone kills cancer cells. Ciglitazone up-regulated soluble and membrane-bound TRAIL and let TRAIL-resistant T24 cells to respond to TRAIL through caspase activation, death receptor signalling pathway and Bid cleavage. We provided evidence that TRAIL-induced apoptosis is partially driven by ciglitazone-mediated down-regulation of c-FLIP and survivin protein levels through a proteasome-dependent degradation mechanism. CONCLUSIONS/SIGNIFICANCE: Therefore, ciglitazone could be clinically relevant as chemopreventive or therapeutic agent for the treatment of TRAIL-refractory high grade urothelial cancers.

  5. MUC5AC EXPRESSION UP-REGULATION GOBLET CELL HYPERPLASIA IN THE AIRWAY OF PATIENTS WITH CHRONIC OBSTRUCTIVE PULMONARY DISEASE

    Institute of Scientific and Technical Information of China (English)

    Rui Ma; Ying Wang; Gang Cheng; Hui-zhen Zhang; Huan-ying Wan; Shao-guang Huang

    2005-01-01

    Objective To determine the number of goblet cells, the change of MUC5AC expression in chronic obstructive pul monary disease (COPD) patients and the relationship of smoking with goblet cell, MUC5AC, and lung function. Methods Eighteen patients undergoing lung resections for a solitary peripheral carcinoma were classified by lung function as having COPD. Twenty patients with normal lung function served as the control group. Normal lobe bronchioles far away from the lesion site were taken for paraffin section. Goblet cells were identified by AB/PAS staining and the ex pressionof MUC5AC in the paraffin's section was tested by immunohistochemistry. Results Goblet cell hyperplasia was observed in the COPD group. The positive rate of goblet cell in COPD group (0.20% ± 0.10%) was significantly higher than that in the normal lung function group (0.13% ± 0.06%, P < 0.05). The positive rate of MUC5AC expression in the COPD group (0.27% ± 0.09%) was higher than that in the normal lung function group (0.20% ± 0.10%, P <0.05).The positive rate of goblet cell in smokers (27.93% ± 9.00%) of the COPD group and normal lung function group was higher than that in non-smokers (17.70% ± 9.37%, P < 0.05), while MUC5AC expression had no significant difference between smokers and non-smokers (17.88% ± 6.44% and 10.88% ± 7.10%, respectively). Conclusion For COPD patients with declined lung function, there were goblet cell hyperplasia and increased expres sion of MUC5AC. MUC5AC expression up-regulation may due to goblet cell hyperplasia. Smoking may be an important factor for goblet cell hyperplasia.

  6. Cytokeratin 15 marks basal epithelia in developing ureters and is upregulated in a subset of urothelial cell carcinomas.

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    Guangping Tai

    Full Text Available The mammalian ureter contains a water-tight epithelium surrounded by smooth muscle. Key molecules have been defined which regulate ureteric bud initiation and drive the differentiation of ureteric mesenchyme into peristaltic smooth muscle. Less is known about mechanisms underlying the developmental patterning of the multilayered epithelium characterising the mature ureter. In skin, which also contains a multilayered epithelium, cytokeratin 15 (CK15, an acidic intermediate filament protein, marks cells whose progeny contribute to epidermal regeneration following wounding. Moreover, CK15+ precursor cells in skin can give rise to basal cell carcinomas. In the current study, using transcriptome microarrays of embryonic wild type mouse ureters, Krt15, coding for CK15, was detected. Quantitative polymerase chain reaction analyses confirmed the initial finding and demonstrated that Krt15 levels increased during the fetal period when the ureteric epithelium becomes multilayered. CK15 protein was undetectable in the ureteric bud, the rudiment from which the ureter grows. Nevertheless, later in fetal development, CK15 was immunodetected in a subset of basal urothelial cells in the ureteric stalk. Superficial epithelial cells, including those positive for the differentiation marker uroplakin III, were CK15-. Transformation-related protein 63 (P63 has been implicated in epithelial differentiation in murine fetal urinary bladders. In wild type fetal ureters, CK15+ cells were positive for P63, and p63 homozygous null mutant ureters lacked CK15+ cells. In these mutant ureters, sections of the urothelium were monolayered versus the uniform multilayering found in wild type littermates. Human urothelial cell carcinomas account for considerable morbidity and mortality. CK15 was upregulated in a subset of invasive ureteric and urinary bladder cancers. Thus, in ureter development, the absence of CK15 is associated with a structurally simplified urothelium whereas

  7. Osteoblast-secreted collagen upregulates paracrine Sonic hedgehog signaling by prostate cancer cells and enhances osteoblast differentiation

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    Zunich Samantha M

    2012-07-01

    Full Text Available Abstract Background Induction of osteoblast differentiation by paracrine Sonic hedgehog (Shh signaling may be a mechanism through which Shh-expressing prostate cancer cells initiate changes in the bone microenvironment and promote metastases. A hallmark of osteoblast differentiation is the formation of matrix whose predominant protein is type 1 collagen. We investigated the formation of a collagen matrix by osteoblasts cultured with prostate cancer cells, and its effects on interactions between prostate cancer cells and osteoblasts. Results In the presence of exogenous ascorbic acid (AA, a co-factor in collagen synthesis, mouse MC3T3 pre-osteoblasts in mixed cultures with human LNCaP prostate cancer cells or LNCaP cells modified to overexpress Shh (LNShh cells formed collagen matrix with distinct fibril ultrastructural characteristics. AA increased the activity of alkaline phosphatase and the expression of the alkaline phosphatase gene Akp2, markers of osteoblast differentiation, in MC3T3 pre-osteoblasts cultured with LNCaP or LNShh cells. However, the AA-stimulated increase in Akp2 expression in MC3T3 pre-osteoblasts cultured with LNShh cells far exceeded the levels observed in MC3T3 cells cultured with either LNCaP cells with AA or LNShh cells without AA. Therefore, AA and Shh exert a synergistic effect on osteoblast differentiation. We determined whether the effect of AA on LNShh cell-induced osteoblast differentiation was mediated by Shh signaling. AA increased the expression of Gli1 and Ptc1, target genes of the Shh pathway, in MC3T3 pre-osteoblasts cultured with LNShh cells to at least twice their levels without AA. The ability of AA to upregulate Shh signaling and enhance alkaline phosphatase activity was blocked in MC3T3 cells that expressed a dominant negative form of the transcription factor GLI1. The AA-stimulated increase in Shh signaling and Shh-induced osteoblast differentiation was also inhibited by the specific collagen synthesis

  8. Upregulated and prolonged differentiation potential of the ependymal cells lining the ventriculus terminalis in human fetuses.

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    Song, Dae Yong; Cho, Byung Pil; Choi, Byoung Young; Yang, Young Chul; Lee, Bong Hee; Lim, Chang Kyo; Kang, Ho Suck

    2005-09-23

    The ventriculus terminalis (VT) is a dilated cavity within the conus medullaris of the spinal cord. Although the VT was discovered in the mid-nineteenth century, little is known about its characteristics during development in human fetuses. Ependymal cells lining the cavities within the CNS retain high differentiation potential, and are believed to be responsible for the postnatal neurogenesis. To evaluate the differentiation capacity of the ependymal cells lining the VT during development, we examined glial fibrillary acidic protein (GFAP) and proliferating cell nuclear antigen (PCNA) expression in the spinal cord of 18-24-week-old human fetuses. GFAP is a marker for the degree of ependymal cell differentiation in the human fetus, and PCNA is a well-known marker for cell division. Morphological characteristics of the VT were also examined. At the lower portion of the conus medullaris, the central canal abruptly expands dorsally to become the VT. Then the VT widens bilaterally while its anteroposterior diameter reduces gradually in a caudal direction. Finally, the VT becomes a narrow, transverse slit at the level of the lowermost conus medullaris. Compared with those lining the central canal, more numerous ependymal cells lining the VT showed more intensive GFAP and PCNA expression throughout all gestational ages examined. This suggests that, in the developing human spinal cord, ependymal cells lining the VT retain their differentiation potential, including a higher proliferative capacity, until a later stage of development than those lining the central canal.

  9. Copper deficiency alters cell bioenergetics and induces mitochondrial fusion through up-regulation of MFN2 and OPA1 in erythropoietic cells

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    Bustos, Rodrigo I.; Jensen, Erik L.; Ruiz, Lina M.; Rivera, Salvador; Ruiz, Sebastián [Center for Biomedical Research, Faculty of Biological Sciences and Faculty of Medicine, Universidad Andres Bello, Santiago (Chile); Simon, Felipe; Riedel, Claudia [Center for Biomedical Research, Faculty of Biological Sciences and Faculty of Medicine, Universidad Andres Bello, Santiago (Chile); Millennium Institute of Immunology and Immunotherapy, Santiago (Chile); Ferrick, David [Seahorse Bioscience, Billerica, MA (United States); Elorza, Alvaro A., E-mail: aelorza@unab.cl [Center for Biomedical Research, Faculty of Biological Sciences and Faculty of Medicine, Universidad Andres Bello, Santiago (Chile); Millennium Institute of Immunology and Immunotherapy, Santiago (Chile)

    2013-08-02

    Highlights: •In copper deficiency, cell proliferation is not affected. In turn, cell differentiation is impaired. •Enlarged mitochondria are due to up-regulation of MNF2 and OPA1. •Mitochondria turn off respiratory chain and ROS production. •Energy metabolism switch from mitochondria to glycolysis. -- Abstract: Copper is essential in cell physiology, participating in numerous enzyme reactions. In mitochondria, copper is a cofactor for respiratory complex IV, the cytochrome c oxidase. Low copper content is associated with anemia and the appearance of enlarged mitochondria in erythropoietic cells. These findings suggest a connection between copper metabolism and bioenergetics, mitochondrial dynamics and erythropoiesis, which has not been explored so far. Here, we describe that bathocuproine disulfonate-induced copper deficiency does not alter erythropoietic cell proliferation nor induce apoptosis. However it does impair erythroid differentiation, which is associated with a metabolic switch between the two main energy-generating pathways. That is, from mitochondrial function to glycolysis. Switching off mitochondria implies a reduction in oxygen consumption and ROS generation along with an increase in mitochondrial membrane potential. Mitochondrial fusion proteins MFN2 and OPA1 were up-regulated along with the ability of mitochondria to fuse. Morphometric analysis of mitochondria did not show changes in total mitochondrial biomass but rather bigger mitochondria because of increased fusion. Similar results were also obtained with human CD34+, which were induced to differentiate into red blood cells. In all, we have shown that adequate copper levels are important for maintaining proper mitochondrial function and for erythroid differentiation where the energy metabolic switch plus the up-regulation of fusion proteins define an adaptive response to copper deprivation to keep cells alive.

  10. IL7Rα expression and upregulation by IFNβ in dendritic cell subsets is haplotype-dependent.

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    Fiona C McKay

    Full Text Available The IL7Rα gene is unequivocally associated with susceptibility to multiple sclerosis (MS. Haplotype 2 (Hap 2 confers protection from MS, and T cells and dendritic cells (DCs of Hap 2 exhibit reduced splicing of exon 6, resulting in production of relatively less soluble receptor, and potentially more response to ligand. We have previously shown in CD4 T cells that IL7Rα haplotypes 1 and 2, but not 4, respond to interferon beta (IFNβ, the most commonly used immunomodulatory drug in MS, and that haplotype 4 (Hap 4 homozygotes have the highest risk of developing MS. We now show that IL7R expression increases in myeloid cells in response to IFNβ, but that the response is haplotype-dependent, with cells from homozygotes for Hap 4 again showing no response. This was shown using freshly derived monocytes, in vitro cultured immature and mature monocyte-derived dendritic cells, and by comparing homozygotes for the common haplotypes, and relative expression of alleles in heterozygotes (Hap 4 vs not Hap 4. As for T cells, in all myeloid cell subsets examined, Hap 2 homozygotes showed a trend for reduced splicing of exon 6 compared to the other haplotypes, significantly so in most conditions. These data are consistent with increased signaling being protective from MS, constitutively and in response to IFNβ. We also demonstrate significant regulation of immune response, chemokine activity and cytokine biosynthesis pathways by IL7Rα signaling in IFNβ -treated myeloid subsets. IFNβ-responsive genes are over-represented amongst genes associated with MS susceptibility. IL7Rα haplotype may contribute to MS susceptibility through reduced capacity for IL7Rα signalling in myeloid cells, especially in the presence of IFNβ, and is currently under investigation as a predictor of therapeutic response.

  11. Bortezomib reduces the tumorigenicity of multiple myeloma via downregulation of upregulated targets in clonogenic side population cells.

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    Miho Nara

    Full Text Available Side population (SP cells in cancers, including multiple myeloma, exhibit tumor-initiating characteristics. In the present study, we isolated SP cells from human myeloma cell lines and primary tumors to detect potential therapeutic targets specifically expressed in SP cells. We found that SP cells from myeloma cell lines (RPMI 8226, AMO1, KMS-12-BM, KMS-11 express CD138 and that non-SP cells include a CD138-negative population. Serial transplantation of SP and non-SP cells into NOD/Shi-scid IL-2γnul mice revealed that clonogenic myeloma SP cells are highly tumorigenic and possess a capacity for self-renewal. Gene expression analysis showed that SP cells from five MM cell lines (RPMI 8226, AMO1, KMS-12-BM, KMS-11, JJN3 express genes involved in the cell cycle and mitosis (e.g., CCNB1, CDC25C, CDC2, BIRC5, CENPE, SKA1, AURKB, KIFs, TOP2A, ASPM, polycomb (e.g., EZH2, EPC1 and ubiquitin-proteasome (e.g., UBE2D3, UBE3C, PSMA5 more strongly than do non-SP cells. Moreover, CCNB1, AURKB, EZH2 and PSMA5 were also upregulated in the SPs from eight primary myeloma samples. On that basis, we used an aurora kinase inhibitor (VX-680 and a proteasome inhibitor (bortezomib with RPMI 8226 and AMO1 cells to determine whether these agents could be used to selectively target the myeloma SP. We found that both these drugs reduced the SP fraction, though bortezomib did so more effectively than VX-680 due to its ability to reduce levels of both phospho-histone H3 (p-hist. H3 and EZH2; VX-680 reduced only p-hist. H3. This is the first report to show that certain oncogenes are specifically expressed in the myeloma SP, and that bortezomib effectively downregulates expression of their products. Our approach may be useful for screening new agents with which to target a cell population possessing strong tumor initiating potential in multiple myeloma.

  12. Unsaturated compounds induce up-regulation of CD86 on dendritic cells in the in vitro sensitization assay LCSA.

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    Frohwein, Thomas Armin; Sonnenburg, Anna; Zuberbier, Torsten; Stahlmann, Ralf; Schreiner, Maximilian

    2016-04-01

    Unsaturated compounds are known to cause false-positive reactions in the local lymph node assay (LLNA) but not in the guinea pig maximization test. We have tested a panel of substances (succinic acid, undecylenic acid, 1-octyn-3-ol, fumaric acid, maleic acid, linoleic acid, oleic acid, alpha-linolenic acid, squalene, and arachidonic acid) in the loose-fit coculture-based sensitization assay (LCSA) to evaluate whether unspecific activation of dendritic cells is a confounder for sensitization testing in vitro. Eight out of 10 tested substances caused significant up-regulation of CD86 on dendritic cells cocultured with keratinocytes and would have been classified as sensitizers; only succinic acid was tested negative, and squalene had to be excluded from data analysis due to poor solubility in cell culture medium. Based on human data, only undecylenic acid can be considered a true sensitizer. The true sensitizing potential of 1-octyn-3-ol is uncertain. Fumaric acid and its isomer maleic acid are not known as sensitizers, but their esters are contact allergens. A group of 18- to 20-carbon chain unsaturated fatty acids (linoleic acid, oleic acid, alpha-linolenic acid, and arachidonic acid) elicited the strongest reaction in vitro. This is possibly due to the formation of pro-inflammatory lipid mediators in the cell culture causing nonspecific activation of dendritic cells. In conclusion, both the LLNA and the LCSA seem to provide false-positive results for unsaturated fatty acids. The inclusion of T cells in dendritic cell-based in vitro sensitization assays may help to eliminate false-positive results due to nonspecific dendritic cell activation. This would lead to more accurate prediction of sensitizers, which is paramount for consumer health protection and occupational safety.

  13. Upregulation of microRNA-98 increases radiosensitivity in esophageal squamous cell carcinoma

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    Jin, Ying-Ying; Chen, Qing-Juan; Wei, Yang; Wang, Ya-Li; Wang, Zhong-Wei; Xu, Kun; He, Yun; Ma, Hong-Bing

    2016-01-01

    Although radiation resistance is a common challenge in the clinical treatment of esophageal squamous cell carcinoma (ESCC), an effective treatment strategy has yet to be developed. Aberrant expression of microRNAs (miRNAs) is responsible for cancer sensitivity to radiation. In this study, we aimed to identify the miRNAs that are associated with radioresistance in ESCC. We used a miRNA microarray to perform a comparison of miRNA expression in both ESCC parental and acquired radioresistance cell lines. qRT-PCR was used to confirm the alterations. Cell radiosensitivity was determined with a survival fraction assay. Functional analyses of the identified miRNA in ESCC cells with regard to metastasis and apoptosis were performed by transwell assays and flow cytometry. The miRNA targets were identified with pathway analysis and confirmed with a luciferase assay. miR-98 was recognized as the most downregulated miRNA in established radioresistant cell line. AmiR-98 mimic enforced the expression of miRNA-98 and made ESCC cells sensitive to radiotherapy, while anti-miR-98 reversed this process. Optimal results were achieved by decreasing cellular proliferation, decreasing cell migration and inducing apoptosis. The luciferase target gene analysis results showed that the overexpression of miRNA-98 inhibited tumor growth and resistance tolerance by directly binding to the BCL-2 gene. Our study indicated that increasing miRNA-98 expression can be used as a potential radiosensitive therapeutic strategy for treating esophageal cancer cells. PMID:27422937

  14. Silencing GFAP isoforms in astrocytoma cells disturbs laminin-dependent motility and cell adhesion

    NARCIS (Netherlands)

    Moeton, Martina; Kanski, Regina; Stassen, Oscar M J A; Sluijs, Jacqueline A; Geerts, Dirk; van Tijn, P.; Wiche, Gerhard; van Strien, Miriam E; Hol, Elly M

    2014-01-01

    Glial fibrillary acidic protein (GFAP) is an intermediate filament protein expressed in astrocytes and neural stem cells. The GFAP gene is alternatively spliced, and expression of GFAP is highly regulated during development, on brain damage, and in neurodegenerative diseases. GFAPα is the canonical

  15. Porcine EPCs downregulate stem cell markers and upregulate endothelial maturation markers during in vitro cultivation.

    Science.gov (United States)

    Avci-Adali, Meltem; Nolte, Andrea; Simon, Perikles; Ziemer, Gerhard; Wendel, Hans P

    2009-10-01

    In recent years, interest in endothelial progenitor cells (EPCs) in the field of tissue engineering and regenerative medicine has increased tremendously. However, each clinical stem cell application requires prior validation through animal experiments. This study investigates the isolation and characterization of porcine EPCs from peripheral blood and the change of their cell surface marker expression during in vitro cultivation. RT-PCR demonstrated that the EPCs express stem cell markers CD34 and CD133, which decrease with in vitro cultivation time. Throughout the cultivation process EPCs did not express monocytic (CD14) or haematopoietic marker (CD45). Surprisingly, the CD31 and VE-cadherin expression in EPCs was significantly higher than in endothelial cells (ECs). In contrast, the VEGFR2 and E-selectin expression was significantly lower than in ECs, but increased during the expansion process. This study clarifies the characteristic properties of porcine EPCs during cell culture and may help to improve the impact of EPC-based therapies in porcine animal studies.

  16. MAPKK-dependent growth factor-induced upregulation of P2Y2 receptors in vascular smooth muscle cells

    DEFF Research Database (Denmark)

    Hou, M; Möller, S; Edvinsson, L;

    1999-01-01

    The ATP- and UTP-sensitive P2Y2 receptor which mediates both contractile and mitogenic effects has recently been shown to be upregulated in the synthetic phenotype of the vascular smooth muscle cell (VSMC). Using a competitive RT-PCR we demonstrate that the P2Y2 receptor mRNA is increased by fetal...... calf serum and other growth factors in a MAPKK-dependent way. This was confirmed at the functional level by examining UTP-stimulated release of intracellular Ca2+. Furthermore, the P2Y2 receptor mRNA is positively autoregulated by ATP and the mRNA is rapidly degraded with only 26% remaining after 1 h...... in the presence of actinomycin D. Our results indicate growth factor regulation and rapid turnover of the P2Y2 receptor mRNA, which may be of importance in atherosclerosis and neointima formation after balloon angioplasty....

  17. Reconstitution of TGFBR2-Mediated Signaling Causes Upregulation of GDF-15 in HCT116 Colorectal Cancer Cells.

    Directory of Open Access Journals (Sweden)

    Jennifer Lee

    Full Text Available Although inactivating frameshift mutations in the Transforming growth factor beta receptor type 2 (TGFBR2 gene are considered as drivers of microsatellite unstable (MSI colorectal tumorigenesis, consequential alterations of the downstream target proteome are not resolved completely. Applying a click-it chemistry protein labeling approach combined with mass spectrometry in a MSI colorectal cancer model cell line, we identified 21 de novo synthesized proteins differentially expressed upon reconstituted TGFBR2 expression. One candidate gene, the TGF-ß family member Growth differentiation factor-15 (GDF-15, exhibited TGFBR2-dependent transcriptional upregulation causing increased intracellular and extracellular protein levels. As a new TGFBR2 target gene it may provide a link between the TGF-ß branch and the BMP/GDF branch of SMAD-mediated signaling.

  18. Upregulation of glycolytic enzymes, mitochondrial dysfunction and increased cytotoxicity in glial cells treated with Alzheimer's disease plasma.

    Directory of Open Access Journals (Sweden)

    Tharusha Jayasena

    Full Text Available Alzheimer's disease (AD is a neurodegenerative disorder associated with increased oxidative stress and neuroinflammation. Markers of increased protein, lipid and nucleic acid oxidation and reduced activities of antioxidant enzymes have been reported in AD plasma. Amyloid plaques in the AD brain elicit a range of reactive inflammatory responses including complement activation and acute phase reactions, which may also be reflected in plasma. Previous studies have shown that human AD plasma may be cytotoxic to cultured cells. We investigated the effect of pooled plasma (n = 20 each from healthy controls, individuals with amnestic mild cognitive impairment (aMCI and Alzheimer's disease (AD on cultured microglial cells. AD plasma and was found to significantly decrease cell viability and increase glycolytic flux in microglia compared to plasma from healthy controls. This effect was prevented by the heat inactivation of complement. Proteomic methods and isobaric tags (iTRAQ found the expression level of complement and other acute phase proteins to be altered in MCI and AD plasma and an upregulation of key enzymes involved in the glycolysis pathway in cells exposed to AD plasma. Altered expression levels of acute phase reactants in AD plasma may alter the energy metabolism of glia.

  19. Distinctive adaptive response to repeated exposure to hydrogen peroxide associated with upregulation of DNA repair genes and cell cycle arrest

    Directory of Open Access Journals (Sweden)

    Gloria A. Santa-Gonzalez

    2016-10-01

    Full Text Available Many environmental and physiological stresses are chronic. Thus, cells are constantly exposed to diverse types of genotoxic insults that challenge genome stability, including those that induce oxidative DNA damage. However, most in vitro studies that model cellular response to oxidative stressors employ short exposures and/or acute stress models. In this study, we tested the hypothesis that chronic and repeated exposure to a micromolar concentration of hydrogen peroxide (H2O2 could activate DNA damage responses, resulting in cellular adaptations. For this purpose, we developed an in vitro model in which we incubated mouse myoblast cells with a steady concentration of ~50 μM H2O2 for one hour daily for seven days, followed by a final challenge of a 10 or 20X higher dose of H2O2 (0.5 or 1 mM. We report that intermittent long-term exposure to this oxidative stimulus nearly eliminated cell toxicity and significantly decreased genotoxicity (in particular, a >5-fold decreased in double-strand breaks resulting from subsequent acute exposure to oxidative stress. This protection was associated with cell cycle arrest in G2/M and induction of expression of nine DNA repair genes. Together, this evidence supports an adaptive response to chronic, low-level oxidative stress that results in genomic protection and up-regulated maintenance of cellular homeostasis.

  20. Upregulation of ULK1 expression in PC-3 cells following tumor protein P53 transfection by sonoporation

    Science.gov (United States)

    WANG, YU; CHEN, YI-NI; ZHANG, WEI; YANG, YU; BAI, WEN-KUN; SHEN, E; HU, BING

    2016-01-01

    The aim of the present study was to investigate whether ultrasound combined with microbubbles was able to enhance liposome-mediated transfection of genes into human prostate cancer cells, and to examine the association between autophagy and tumor protein P53 (P53). An MTT assay was used to evaluate cell viability, while flow cytometry and fluorescence microscopy were used to measure gene transfection efficiency. Autophagy was observed using transmission electron microscopy. Reverse transcription-polymerase chain reaction (RT-PCR) and western blot analysis were used to assess the expression of autophagy-associated genes. The results of the present study revealed that cell viability was significantly reduced following successfully enhanced transfection of P53 by ultrasound combined with microbubbles. In addition, serine/threonine-protein kinase ULK1 levels were simultaneously upregulated. Castration-resistant prostate cancer is difficult to treat and is investigated in the present study. P53 has a significant role in a number of key biological functions, including DNA repair, apoptosis, cell cycle, autophagy, senescence and angiogenesis. Prior to the present study, to the best of our knowledge, increased transfection efficiency and reduced side effects have been difficult to achieve. Ultrasound is considered to be a ‘gentle’ technique that may be able to achieve increased transfection efficiency and reduced side effects. The results of the present study highlight a potential novel therapeutic strategy for the treatment of prostate cancer. PMID:26870270

  1. Laminar shear stress up-regulates peroxiredoxins (PRX) in endothelial cells: PRX 1 as a mechanosensitive antioxidant.

    Science.gov (United States)

    Mowbray, Amy L; Kang, Dong-Hoon; Rhee, Sue Goo; Kang, Sang Won; Jo, Hanjoong

    2008-01-18

    Shear stress plays a significant role in endothelial cell biology and atherosclerosis development. Previous work by our group has shown that fluid flow stimulates important functional changes in cells through protein expression regulation. Peroxiredoxins (PRX) are a family of antioxidant enzymes but have yet to be investigated in response to shear stress. Studies have shown that oscillatory shear stress (OS) increases reactive oxygen species (ROS) levels in endothelial cells, whereas laminar shear stress (LS) blocks this response. We hypothesized that PRX are responsible for the anti-oxidative effect of LS. To test this hypothesis, bovine aortic endothelial cells (BAEC) were subjected to LS (15 dyn/cm(2)), OS (+/-5 dyn/cm(2), 1 Hz), or static conditions for 24 h. Using Western blot and immunofluorescence staining, all six isoforms of PRX were identified in BAEC. When compared with OS and static, exposure to chronic LS up-regulated PRX 1 levels intracellularly. LS also increased expression of PRX 5 relative to static controls, but not OS. PRX exhibited broad subcellular localization, with distribution in the cytoplasm, Golgi, mitochondria, and intermediate filaments. In addition, PRX 1 knock down, using specific small interference RNA, attenuated LS-dependent reactive oxygen species reduction in BAEC. However, PRX 5 depletion did not. Together, these results suggest that PRX 1 is a novel mechanosensitive antioxidant, playing an important role in shear-dependent regulation of endothelial biology and atherosclerosis.

  2. Salmonella infection upregulates the leaky protein claudin-2 in intestinal epithelial cells.

    Directory of Open Access Journals (Sweden)

    Yong-guo Zhang

    Full Text Available BACKGROUND: Tight junctions seal the space between adjacent epithelial cells. Mounting evidence suggests that tight junction proteins play a key role in the pathogenesis of human disease. Claudin is a member of the tight junction protein family, which has 24 members in humans. To regulate cellular function, claudins interact structurally and functionally with membrane and scaffolding proteins via their cytoplasmic domain. In particular, claudin-2 is known to be a leaky protein that contributes to inflammatory bowel disease and colon cancer. However, the involvement of claudin-2 in bacterial infection in the intestine remains unknown. METHODS/PRINCIPAL FINDINGS: We hypothesized that Salmonella elevates the leaky protein claudin-2 for its own benefit to facilitate bacterial invasion in the colon. Using a Salmonella-colitis mouse model and cultured colonic epithelial cells, we found that pathogenic Salmonella colonization significantly increases the levels of claudin-2 protein and mRNA in the intestine, but not that of claudin-3 or claudin-7 in the colon, in a time-dependent manner. Immunostaining studies showed that the claudin-2 expression along the crypt-villous axis postinfection. In vitro, Salmonella stimulated claudin-2 expression in the human intestinal epithelial cell lines SKCO15 and HT29C19A. Further analysis by siRNA knockdown revealed that claudin-2 is associated with the Salmonella-induced elevation of cell permeability. Epithelial cells with claudin-2 knockdown had significantly less internalized Salmonella than control cells with normal claudin-2 expression. Inhibitor assays demonstrated that this regulation is mediated through activation of the EGFR pathway and the downstream protein JNK. CONCLUSION/SIGNIFICANCE: We have shown that Salmonella targets the tight junction protein claudin-2 to facilitate bacterial invasion. We speculate that this disruption of barrier function contributes to a new mechanism by which bacteria interact

  3. Upregulated microRNA-224 promotes ovarian cancer cell proliferation by targeting KLLN.

    Science.gov (United States)

    Hu, Ke; Liang, Meng

    2017-02-01

    Human epithelial ovarian cancer is a complex disease, with low 5-yr survival rate largely due to the terminal stage at diagnosis in most patients. MicroRNAs play critical roles during epithelial ovarian cancer progression in vivo and have also been shown to regulate characteristic of ovarian cancer cell line in vitro. Alterative microRNA-224 (microRNA-224) expression affects human epithelial ovarian cancer cell survival, apoptosis, and metastasis. However, people know little about the effects of microRNA-224 on epithelial ovarian cancer cell proliferation. In the current study, we found that the microRNA-224 expression level of human syngeneic epithelial ovarian cancer cells HO8910 (low metastatic ability) was lower than that of HO8910PM (high metastatic ability). Furthermore, microRNA-224 was confirmed to target KLLN in HO8910 and HO8910PM. The known KLLN downstream target cyclin A was regulated by microRNA-224 in HO8910 and HO8910PM. In addition, overexpression of microRNA-224 enhanced the proliferation abilities of HO8910 and knockdown of microRNA-224 suppressed the proliferation abilities of HO8910PM by KLLN-cyclin A pathway. Our results provide new data about microRNAs and their targets involved in proliferation of epithelial ovarian cancer cells by modulating the downstream signaling.

  4. Rad6 upregulation promotes stem cell-like characteristics and platinum resistance in ovarian cancer

    Science.gov (United States)

    Somasagara, Ranganatha R.; Tripathi, Kaushlendra; Spencer, Sebastian M.; Clark, David W.; Barnett, Reagan; Bachaboina, Lavanya; Scalici, Jennifer; Rocconi, Rodney P.; Piazza, Gary A.; Palle, Komaraiah

    2015-01-01

    Ovarian cancer is the fifth most deadly cancer in women in the United States and despite advances in surgical and chemotherapeutic treatments survival rates have not significantly improved in decades. The poor prognosis for ovarian cancer patients is largely due to the extremely high (80%) recurrence rate of ovarian cancer and because the recurrent tumors are often resistant to the widely utilized platinum-based chemotherapeutic drugs. In this study, expression of Rad6, an E2 ubiquitin-conjugating enzyme, was found to strongly correlate with ovarian cancer progression. Furthermore, in ovarian cancer cells Rad6 was found to stabilize β-catenin promoting stem cell-related characteristics, including expression of stem cell markers and anchorage-independent growth. Cancer stem cells can promote chemoresistance, tumor recurrence and metastasis, all of which are limiting factors in treating ovarian cancer. Thus it is significant that Rad6 overexpression led to increased resistance to the chemotherapeutic drug carboplatin and correlated with tumor cell invasion. These findings show the importance of Rad6 in ovarian cancer and emphasize the need for further studies of Rad6 as a potential target for the treatment of ovarian cancer. PMID:26679603

  5. Flavonoids from Triticum aestivum inhibit adipogenesis in 3T3-L1 cells by upregulating the insig pathway.

    Science.gov (United States)

    Poudel, Barun; Nepali, Sarmila; Xin, Mingjie; Ki, Hyeon-Hui; Kim, Young-Ho; Kim, Dae-Ki; Lee, Young-Mi

    2015-08-01

    The present study aimed to compare the potential anti-adipogenic effects and underlying mechanisms of the luteolin, isoscoparin and isoorientin flavonoids, purified from Triticum aestivum sprout (TA) in 3T3-L1 cells. The cells were treated with different concentrations of flavonoids for 8 days and the lipid accumulation was assessed using Oil-Red-O staining. The expression levels of the transcription factors and the genes involved in adipogenesis in the cells were assessed by reverse transcription-quantitative polymerase chain reaction and western blotting. The results demonstrated that 10 μM luteolin, isoscoparin or isoorientin inhibited lipid deposition in the cells by 74, 63 and 65%, respectively. The flavonoids also significantly inhibited the transcriptional regulators of adipogenesis, including peroxisome proliferator-activated receptor-γ, CAAT/enhancer binding protein-α and sterol regulatory element binding protein (SREBP)-1c, compared with the control cells. Similarly, there was a significant downregulation of the adipocyte specific markers associated with lipid metabolism, including activating protein-2, fatty acid synthase, hormone-sensitive lipase and lipoprotein lipase, in the flavonoid treated cells. Notably, the cells treated with the flavonoids demonstrated increased expression levels of the insulin-induced genes, insig-1 and insig-2, which may have inhibited the activation of the adipogenic transcription factor, SREBP, eventually leading to the inhibition of adipogenesis. Taken together, these results revealed that the flavonoids from TA possessed an inhibitory effect on adipogenesis through downregulation of adipogenic transcription factors and genes associated with lipid metabolism, and the upregulation of insig 1 and 2, suggesting that the flavonoids from TA may be potential therapeutic agents for the prevention and treatment of obesity.

  6. miR-92a is upregulated in cervical cancer and promotes cell proliferation and invasion by targeting FBXW7

    Energy Technology Data Exchange (ETDEWEB)

    Zhou, Chuanyi [Department of Oncology, Xiangya Hospital, Central South University, Changsha 410008 (China); Shen, Liangfang, E-mail: lfshen2008@163.com [Department of Oncology, Xiangya Hospital, Central South University, Changsha 410008 (China); Mao, Lei; Wang, Bing; Li, Yang; Yu, Huizhi [Department of Radiation Oncology, Yueyang Second People' s Hospital, Yueyang 414000 (China)

    2015-02-27

    MicroRNAs (miRNAs) are involved in the cervical carcinogenesis and progression. In this study, we investigated the role of miR-92a in progression and invasion of cervical cancer. MiR-92a was significantly upregulated in cervical cancer tissues and cell lines. Overexpression of miR-92a led to remarkably enhanced proliferation by promoting cell cycle transition from G1 to S phase and significantly enhanced invasion of cervical cancer cells, while its knockdown significantly reversed these cellular events. Bioinformatics analysis suggested F-box and WD repeat domain-containing 7 (FBXW7) as a novel target of miR-92a, and miR-92a suppressed the expression level of FBXW7 mRNA by direct binding to its 3′-untranslated region (3′UTR). Expression of miR-92a was negatively correlated with FBXW7 in cervical cancer tissues. Furthermore, Silencing of FBXW7 counteracted the effects of miR-92a suppression, while its overexpression reversed oncogenic effects of miR-92a. Together, these findings indicate that miR-92a acts as an onco-miRNA and may contribute to the progression and invasion of cervical cancer, suggesting miR-92a as a potential novel diagnostic and therapeutic target of cervical cancer. - Highlights: • miR-92a is elevated in cervical cancer tissues and cell lines. • miR-92a promotes cervical cancer cell proliferation, cell cycle transition from G1 to S phase and invasion. • FBXW7 is a direct target of miR-92a. • FBXW7 counteracts the oncogenic effects of miR-92a on cervical cancer cells.

  7. Ulmus davidiana var. japonica Nakai upregulates eosinophils and suppresses Th1 and Th17 cells in the small intestine.

    Directory of Open Access Journals (Sweden)

    Han-Sung Lee

    Full Text Available The bark of Ulmus davidiana var. japonica Nakai (Ulmaceae has been used in traditional Korean medicine for chronic inflammation in the gastrointestinal tract. Here we investigated the frequency and cytokine profile of the major immune cells in the small intestinal lamina propria (SI LP, spleen, and mesenteric lymph nodes (MLNs of mice treated orally with Ulmus davidiana var. japonica Nakai bark water extract (UDE to address the immunomodulatory role of this herb in intestinal homeostasis. B6 mice were given 5g/kg UDE once daily for 14 days. They were then sacrificed, and cells were isolated from the spleen, MLNs, and SI LP. The proportion of B versus T lymphocytes, CD4(+ versus CD8(+ T lymphocytes, Th1 and Th17 cells, and Foxp3(+ regulatory T cells in the spleen, MLNs, and SI LP were analyzed. The frequency of antigen-presenting cells (APCs, including dendritic cells, macrophages, and eosinophils in the SI LP and the expression of costimulatory molecules on APCs were also evaluated. The numbers and frequencies of Th1 and Th17 cells in the SI LP were significantly reduced in the UDE-treated mice compared with PBS controls. In addition, the proportion of IL-4-producing eosinophils in the SI LP was significantly elevated in the UDE-treated mice compared with controls. Taken together, these data indicate that UDE up-regulates the number and frequency of SI LP eosinophils, which can down-regulate the Th1 and Th17 responses via IL-4 secretion and contribute to intestinal homeostasis.

  8. Pro-Inflammatory Cytokine IL-1β Up-Regulates CXC Chemokine Receptor 4 via Notch and ERK Signaling Pathways in Tongue Squamous Cell Carcinoma.

    Directory of Open Access Journals (Sweden)

    Yi Sun

    Full Text Available Chronic inflammation contributes to tumor development through the induction of oncogenic mutations, genomic instability, early tumor promotion, and enhanced angiogenesis. Here, we report that IL-1 receptor 1 (IL-1R1 was expressed in 40 of 41 human tongue squamous cell carcinomas (TSCC. IL-1β up-regulated the expression of CXCR4, a CXC chemokine receptor that mediates cancer growth and metastasis, at both mRNA and protein levels in Tca8113 TSCC cells. IL-1β treatment of Tca8113 cells promoted migration in response to CXCR4 ligand stromal-derived factor α (SDF-1α. The inhibition of IL-1R1 by its antagonist IL-1Ra or RNA interference significantly reversed the up-regulation of CXCR4 induced by IL-1β. IL-1R1 activation also up-regulated the expression of IL-1β itself, suggesting a positive feedback regulation of CXCR4 expression. Furthermore, IL-1β induced the activation of Notch, which was originally considered a stem cell regulator. Pharmacological inhibition of Notch signaling reversed the up-regulation of CXCR4 induced by IL-1β, suggesting that Notch signaling may be involved in the growth and metastasis of cancers via up-regulation of CXCR4. In addition, IL-1β induced the activation of extracellular signal regulated kinase (ERK and ERK inhibition decreased the up-regulation of CXCR4 induced by IL-1β, suggesting the involvement of ERK signaling in cancer metastasis. Taken together these data suggest that IL-1β and IL-1R1 promote cancer growth and metastasis by up-regulating CXCR4 expression and that CXCR4 may be a link between inflammation and cancer.

  9. Monocyte chemoattractant protein-1 secreted by decidual stromal cells inhibits NK cells cytotoxicity by up-regulating expression of SOCS3.

    Directory of Open Access Journals (Sweden)

    Xiaofei Xu

    Full Text Available BACKGROUND: Decidual stromal cells (DSCs are of particular importance due to their pleiotropic functions during pregnancy. Although previous research has demonstrated that DSCs participated in the regulation of immune cells during pregnancy, the crosstalk between DSCs and NK cells has not been fully elucidated. To address this issue, we investigated the effect of DSCs on perforin expression in CD56(+ NK cells and explored the underlying mechanism. METHODOLOGY/PRINCIPAL FINDINGS: Flow cytometry analysis showed perforin production in NK cells was attenuated by DSC media, and it was further suppressed by media from DSCs pretreated with lipopolysaccharide (LPS. However, the expression of granzyme A and apoptosis of NK cells were not influenced by DSC media. ELISA assays to detect cytokine production indicated that monocyte chemoattractant protein-1 (MCP-1 in the supernatant of DSCs conditioned culture significantly increased after LPS stimulation. The inhibitory effect of DSC media on perforin was abolished by the administration of anti-MCP-1 neutralizing antibody. Notably, reduced perforin expression attenuated the cytotoxic potential of CD56(+ NK cells to K562 cells. Moreover, Suppressor of cytokine signaling 3 (SOCS3 expression in NK cells was enhanced by treatment with MCP-1, as measured by RT-PCR and western blot. Interestingly, MCP-1-induced perforin expression was partly abolished by the siRNA induced SOCS3 knockdown. Western blot analysis suggested that both NF-κB and ERK/MAPKs pathway were involved in the LPS-induced upregulation of MCP-1 in DSCs. CONCLUSIONS/SIGNIFICANCE: Our results demonstrate that LPS induces upregulation of MCP-1 in DSCs, which may play a critical role in inhibiting the cytotoxicity of NK cells partly by promoting SOCS3 expression. These findings suggest that the crosstalk between DSCs and NK cells may be crucial to maintain pregnancy homeostasis.

  10. CD147 is increased in HCC cells under starvation and reduces cell death through upregulating p-mTOR in vitro.

    Science.gov (United States)

    Gou, Xingchun; Tang, Xu; Kong, Derek Kai; He, Xinying; Gao, Xingchun; Guo, Na; Hu, Zhifang; Zhao, Zhaohua; Chen, Yanke

    2016-01-01

    Transarterial chemoembolization (TACE) is the standard of care for treatment of intermediate hepatocellular carcinoma (HCC), however, key molecules involved in HCC cell survival and tumor metastasis post-TACE remain unclear. CD147 is a member of the immunoglobulin superfamily that is overexpressed on the surface of HCC cells and is associated with malignant potential and poor prognosis in HCC patients. In this study, using an Earle's Balanced Salt Solution medium culture model that mimics nutrient deprivation induced by TACE, we investigated the regulation of CD147 expression on HCC cells under starvation conditions and its functional effects on HCC cell death. During early stages of starvation, the expression of CD147 was considerably upregulated in SMMC7721, HepG2 and HCC9204 hepatoma cell lines at the protein levels. Downregulation of CD147 by specific small interfering RNA (siRNA) significantly promoted starvation-induced cell death. In addition, CD147 siRNA-transfected SMMC7721 cells demonstrated significantly increased levels of both apoptosis and autophagy as compared to cells transfected with control siRNA under starvation conditions, whereas no difference was observed between the two treatment groups under normal culture conditions. Furthermore, silencing of CD147 resulted in a remarkable downregulation of phosphorylated mammalian target of rapamycin (p-mTOR) in starved SMMC7721 cells. Finally, the combined treatment of starvation and anti-CD147 monoclonal antibody exhibited a synergistic HCC cell killing effect. Our study suggests that upregulation of CD147 under starvation may reduce hepatoma cell death by modulating both apoptosis and autophagy through mTOR signaling, and that CD147 may be a novel potential molecular target to improve the efficacy of TACE.

  11. Regulation of store-operated Ca{sup 2+} entry activity by cell cycle dependent up-regulation of Orai2 in brain capillary endothelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Kito, Hiroaki [Department of Molecular & Cellular Pharmacology, Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya (Japan); Department of Pharmacology, Division of Pathological Sciences, Kyoto Pharmaceutical University, Kyoto (Japan); Yamamura, Hisao; Suzuki, Yoshiaki; Yamamura, Hideto [Department of Molecular & Cellular Pharmacology, Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya (Japan); Ohya, Susumu [Department of Molecular & Cellular Pharmacology, Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya (Japan); Department of Pharmacology, Division of Pathological Sciences, Kyoto Pharmaceutical University, Kyoto (Japan); Asai, Kiyofumi [Department of Molecular Neurobiology, Graduate School of Medical Sciences, Nagoya City University, Nagoya (Japan); Imaizumi, Yuji, E-mail: yimaizum@phar.nagoya-cu.ac.jp [Department of Molecular & Cellular Pharmacology, Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya (Japan)

    2015-04-10

    Store-operated Ca{sup 2+} entry (SOCE) via Orai1 and STIM1 complex is supposed to have obligatory roles in the regulation of cellular functions of vascular endothelial cells, while little is known about the contribution of Orai2. Quantitative PCR and Western blot analyses indicated the expression of Orai2 and STIM2, in addition to Orai1 and STIM1 in bovine brain capillary endothelial cell line, t-BBEC117. During the exponential growth of t-BBEC117, the knockdown of Orai1 and STIM1 significantly reduced the SOCE activity, whereas Orai2 and STIM2 siRNAs had no effect. To examine whether endogenous SOCE activity contributes to the regulation of cell cycle progression, t-BBEC117 were synchronized using double thymidine blockage. At the G2/M phase, Ca{sup 2+} influx via SOCE was decreased and Orai2 expression was increased compared to the G0/G1 phase. When Orai2 was knocked down at the G2/M phase, the decrease in SOCE was removed, and cell proliferation was partly attenuated. Taken together, Orai1 significantly contributes to cell proliferation via the functional expression, which is presumably independent of the cell cycle phases. In construct, Orai2 is specifically up-regulated during the G2/M phase, negatively modulates the SOCE activity, and may contribute to the regulation of cell cycle progression in brain capillary endothelial cells. - Highlights: • Orai1 is essential for SOCE activity in brain capillary endothelial cells (BCECs). • Cell cycle independent expression of Orai1 regulated SOCE and cell proliferation. • Orai2 was up-regulated only at G2/M phase and this consequently reduced SOCE. • Orai2 as well as Orai1 is a key player controlling SOCE and proliferation in BCECs.

  12. Upregulation of macrophage migration inhibitory factor and calgizzarin by androgen in TM4 mouse Sertoli cells

    Institute of Scientific and Technical Information of China (English)

    Hiroyuki Kasumi; Shinji Komori; Kazuko Sakata; Naoko Yamamoto; Tomohiko Yamasaki; Yonehiro Kanemura; Koji Koyama

    2006-01-01

    Aim: To identify proteins induced by androgen in Sertoli cells during spermatogenesis. Methods: We analyzed protein profiles in TM4 Sertoli cells treated with dihydrotestosterone (DHT) using surface enhanced laser desorption ionization time-of-flight mass spectrometry (SELDI-TOF-MS). Results: We found increases in the expression of a 5.0-kDa protein at 15 min, an 11.3-kDa protein at 24 h and 4.3 kDa, 5.7 kDa, 5.8 kDa, 9.95 kDa and 9.98 kDa proteins at 48 h after the treatment. In contrast, the expression of 6.3 kDa and 8.6 kDa proteins decreased at 30 min,and 4.9 kDa, 5.0 kDa, 12.4 kDa and 19.8 kDa proteins at 48 h after the treatment. The 11.3-kDa protein was identified as macrophage migration inhibitory factor (MIF) known to having various functions. The 9.98-kDa protein was identified as calgizzarin related to calcium channels. The timing of their expression suggests that MIF and calgizzarin are involved in late regulation of spermatogenesis in Sertoli cells by androgen. Conclusion: MIF and calgizzarin are two important androgen-responsive proteins produced by Sertoli cells and they might play a role in regulating spermatogenesis.

  13. MCAK and Stathmin Upregulation in Breast Cancer Cells: Etiology and Response to Pharmacologic Reagents

    Science.gov (United States)

    2004-07-01

    transfection re- transport MCAK along the MT protofilament toward MT agent iMBI Fermentas), Cells were cultured for 24 h after transfertion and ends where...Miki, H. et aL (2001) All kinesin superfamily protein, KIF, genes 2therapies. 65 in mouse and human. Prix . Natl. Acad. Sci, U. S. A. 98, 7004-7011 66

  14. UP-REGULATION OF ANTITHROMBOTIC ECTONUCLEOTIDASES BY ASPIRIN IN HUMAN ENDOTHELIAL-CELLS IN-VITRO

    NARCIS (Netherlands)

    CHEUNG, PK; VISSER, J; BAKKER, WW

    1994-01-01

    Ecto ATP-diphosphohydrolase (apyrase) activity of human endothelial cells following aspirin treatment has been studied in-vitro. It was shown by HPLC analysis of supernatant samples that pre-incubation of the cultures with aspirin resulted in a significantly increased turnover of supplemented ATP in

  15. The proteasome inhibitor bortezomib induces testicular toxicity by upregulation of oxidative stress, AMP-activated protein kinase (AMPK) activation and deregulation of germ cell development in adult murine testis

    Energy Technology Data Exchange (ETDEWEB)

    Li, Wei [Department of Human Anatomy, Histology and Embryology, Fourth Military Medical University, Xi' an 710032 (China); Fu, Jianfang [Department of Endocrinology, Xijing Hospital, Fourth Military Medical University, Xi' an 710032 (China); Zhang, Shun [Reproductive Medicine Center, Department of Gynecology and Obstetrics, Tangdu Hospital, Fourth Military Medical University, Xi' an 710038 (China); Zhao, Jie [Department of Human Anatomy, Histology and Embryology, Fourth Military Medical University, Xi' an 710032 (China); Xie, Nianlin, E-mail: xienianlin@126.com [Department of Thoracic Surgery, Tangdu Hospital, Fourth Military Medical University, Xi' an 710038 (China); Cai, Guoqing, E-mail: firstchair@fmmu.edu.cn [Department of Gynaecology and Obstetrics, Xijing Hospital, Fourth Military Medical University, Xi' an 710032 (China)

    2015-06-01

    Understanding how chemotherapeutic agents mediate testicular toxicity is crucial in light of compelling evidence that male infertility, one of the severe late side effects of intensive cancer treatment, occurs more often than they are expected to. Previous study demonstrated that bortezomib (BTZ), a 26S proteasome inhibitor used to treat refractory multiple myeloma (MM), exerts deleterious impacts on spermatogenesis in pubertal mice via unknown mechanisms. Here, we showed that intermittent treatment with BTZ resulted in fertility impairment in adult mice, evidenced by testicular atrophy, desquamation of immature germ cells and reduced caudal sperm storage. These deleterious effects may originate from the elevated apoptosis in distinct germ cells during the acute phase and the subsequent disruption of Sertoli–germ cell anchoring junctions (AJs) during the late recovery. Mechanistically, balance between AMP-activated protein kinase (AMPK) activation and Akt/ERK pathway appeared to be indispensable for AJ integrity during the late testicular recovery. Of particular interest, the upregulated testicular apoptosis and the following disturbance of Sertoli–germ cell interaction may both stem from the excessive oxidative stress elicited by BTZ exposure. We also provided the in vitro evidence that AMPK-dependent mechanisms counteract follicle-stimulating hormone (FSH) proliferative effects in BTZ-exposed Sertoli cells. Collectively, BTZ appeared to efficiently prevent germ cells from normal development via multiple mechanisms in adult mice. Employment of antioxidants and/or AMPK inhibitor may represent an attractive strategy of fertility preservation in male MM patients exposed to conventional BTZ therapy and warrants further investigation. - Highlights: • Intermittent treatment with BTZ caused fertility impairment in adult mice. • BTZ treatment elicited apoptosis during early phase of testicular recovery. • Up-regulation of oxidative stress by BTZ treatment

  16. The CCAAT/enhancer-binding protein beta-2 isoform (CEBPβ-2 upregulates galectin-7 expression in human breast cancer cells.

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    Carole G Campion

    Full Text Available Galectin-7 is considered a gene under the control of p53. However, elevated expression of galectin-7 has been reported in several forms of cancer harboring an inactive p53 pathway. This is especially true for breast cancer where galectin-7 expression is readily expressed in a high proportion in basal-like breast cancer tissues, conferring cancer cells with increased resistance to cell death and metastatic properties. These observations suggest that other transcription factors are capable of inducing galectin-7 expression. In the present work, we have examined the role of CCAAT/enhancer-binding protein beta (C/EBPβ in inducing expression of galectin-7. C/EBP proteins have been shown to contribute to breast cancer by upregulating pro-metastatic genes. We paid particular attention to C/EBPβ-2 (also known as LAP2, the most transcriptionally active of the C/EBPβ isoforms. Our results showed that ectopic expression of C/EBPβ-2 in human breast cancer cells was sufficient to induce expression of galectin-7 at both the mRNA and protein levels. In silico analysis further revealed the presence of an established CEBP element in the galectin-7 promoter. Mutation of this binding site abolished the transcriptional activity of the galectin-7 promoter. Chromatin immunoprecipitation analysis confirmed that C/EBPβ-2 binds to the endogenous galectin-7 promoter. Analysis of galectin-7 protein expression in normal epithelia and in breast carcinoma by immunohistochemistry further showed the expression pattern of C/EBPβ closely micmicked that of galectin-7, most notably in mammary myoepithelial cells and basal-like breast cancer where galectin-7 is preferentially expressed. Taken together, our findings suggest that C/EBPβ is an important mediator of galectin-7 gene activation in breast cancer cells and highlight the different transcriptional mechanisms controlling galectin-7 in cancer cells.

  17. Fibronectin upregulates cGMP-dependent protein kinase type Iβ through C/EBP transcription factor activation in contractile cells.

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    Chamorro-Jorganes, Aranzazu; Calleros, Laura; Griera, Mercedes; Saura, Marta; Luengo, Alicia; Rodriguez-Puyol, D; Rodriguez-Puyol, M

    2011-03-01

    The nitric oxide (NO)-soluble guanylate cyclase (sGC) pathway exerts most of its cellular actions through the activation of the cGMP-dependent protein kinase (PKG). Accumulation of extracellular matrix is one of the main structural changes in pathological conditions characterized by a decreased activity of this pathway, such as hypertension, diabetes, or aging, and it is a well-known fact that extracellular matrix proteins modulate cell phenotype through the interaction with membrane receptors such as integrins. The objectives of this study were 1) to evaluate whether extracellular matrix proteins, particularly fibronectin (FN), modulate PKG expression in contractile cells, 2) to analyze the mechanisms involved, and 3) to evaluate the functional consequences. FN increased type I PKG (PKG-I) protein content in human mesangial cells, an effect dependent on the interaction with β(1)-integrin. The FN upregulation of PKG-I protein content was due to increased mRNA expression, determined by augmented transcriptional activity of the PKG-I promoter region. Akt and the transcription factor CCAAT enhancer-binding protein (C/EBP) mediated the genesis of these changes. FN also increased PKG-I in another type of contractile cell, rat vascular smooth muscle cells (RVSMC). Tirofiban, a pharmacological analog of FN, increased PKG-I protein content in RVSMC and rat aortic walls and magnified the hypotensive effect of dibutyryl cGMP in conscious Wistar rats. The present results provide evidence of a mechanism able to increase PKG-I protein content in contractile cells. Elucidation of this novel mechanism provides a rationale for future pharmacotherapy in certain vascular diseases.

  18. Disturbed α-Cell Function in Mice with β-Cell Specific Overexpression of Human Islet Amyloid Polypeptide

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    Bo Ahrén

    2008-01-01

    Full Text Available Exogenous administration of islet amyloid polypeptide (IAPP has been shown to inhibit both insulin and glucagon secretion. This study examined α-cell function in mice with β-cell specific overexpression of human IAPP (hIAPP after an oral protein gavage (75 mg whey protein/mouse. Baseline glucagon levels were higher in transgenic mice (41±4.0 pg/mL, n=6 than in wildtype animals (19±5.1 pg/mL, n=5, P=.015. In contrast, the glucagon response to protein was impaired in transgenic animals (21±2.7 pg/mL in transgenic mice versus 38±5.7 pg/mL in wildtype mice at 15 minutes; P=.027. Baseline insulin levels did not differ between the groups, while the insulin response, as the glucagon response, was impaired after protein challenge (P=.018. Glucose levels were not different between the groups and did not change significantly after protein gavage. Acetaminophen was given through gavage to the animals (2 mg/mouse to estimate gastric emptying. The plasma acetaminophen profile was similar in the two groups of mice. We conclude that disturbances in glucagon secretion exist in mice with β-cell specific overexpression of human IAPP, which are not secondary to changes in gastric emptying. The reduced glucagon response to protein challenge may reflect a direct inhibitory influence of hIAPP on glucagon secretion.

  19. Peroxiredoxin 6 promotes upregulation of the prion protein (PrP in neuronal cells of prion-infected mice

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    Wagner Wibke

    2012-12-01

    Full Text Available Abstract Background It has been widely established that the conversion of the cellular prion protein (PrPC into its abnormal isoform (PrPSc is responsible for the development of transmissible spongiform encephalopathies (TSEs. However, the knowledge of the detailed molecular mechanisms and direct functional consequences within the cell is rare. In this study, we aimed at the identification of deregulated proteins which might be involved in prion pathogenesis. Findings Apolipoprotein E and peroxiredoxin 6 (PRDX6 were identified as upregulated proteins in brains of scrapie-infected mice and cultured neuronal cell lines. Downregulation of PrP gene expression using specific siRNA did not result in a decrease of PRDX6 amounts. Interestingly, selective siRNA targeting PRDX6 or overexpression of PRDX6 controlled PrPC and PrPSc protein amounts in neuronal cells. Conclusions Besides its possible function as a novel marker protein in the diagnosis of TSEs, PDRX6 represents an attractive target molecule in putative pharmacological intervention strategies in the future.

  20. Prolonged in vivo administration of testosterone-enanthate, the widely used and abused anabolic androgenic steroid, disturbs prolactin and cAMP signaling in Leydig cells of adult rats.

    Science.gov (United States)

    Bjelic, Maja M; Stojkov, Natasa J; Radovic, Sava M; Baburski, Aleksandar Z; Janjic, Marija M; Kostic, Tatjana S; Andric, Silvana A

    2015-05-01

    This study was designed to systematically analyze and define the effects of 1-day, 2-weeks, 10-weeks intramuscular administration of testosterone-enanthate, widely used and abused anabolic androgenic steroid (AAS), on main regulators of steroidogenesis and steroidogenic genes expression in testosterone-producing Leydig cells of adult rats. The results showed that prolonged (10-weeks) intramuscular administration of testosterone-enanthate, in clinically relevant dose, significantly increased prolactin, but decreased Prlr2 and Gnrhr in pituitary of adult rat. The levels of testosterone, Insl3, cAMP and mitochondrial membrane potential of Leydig cells were significantly reduced. This was followed by decreased expression of some steroidogenic enzymes and regulatory proteins such as Lhcgr, Prlr1/2, Tspo, Star, Cyp11a1, Cyp17a1, Dax1. Oppositely, Hsd3b1/2, Hsd3b5, Hsd17b4, Ar, Arr19 increased. In the same cells, transcriptional milieu of cAMP signaling elements was disturbed with remarkable up-regulation of PRKA (the main regulator of steroidogenesis). Increased prolactin together with stimulated transcription of Jak2/Jak3 could account for increased Hsd3b1/2 and Hsd3b5 in Leydig cells following 10-weeks in vivo treatment with testosterone-enanthate. In vitro studies revealed that testosterone is capable to increase level of Prlr1, Prlr2, Hsd3b1/2, Hsd3b5 in Leydig cells. Accordingly, testosterone-induced changes in prolactin receptor signaling together with up-regulation of PRKA, Hsd3b1/2, Hsd3b5, Ar in Leydig cells, could be the possible mechanism that contribute to the establishment of a new adaptive response to maintain homeostasis and prevent loss of steroidogenic function. Presented data provide new molecular insights into the relationship between disturbed testosterone homeostasis and mammalian reproduction and are important in terms of wide use and abuse of AASs and human reproductive health.

  1. Corticosteroids reduce IL-6 in ASM cells via up-regulation of MKP-1.

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    Quante, Timo; Ng, Yee Ching; Ramsay, Emma E; Henness, Sheridan; Allen, Jodi C; Parmentier, Johannes; Ge, Qi; Ammit, Alaina J

    2008-08-01

    The mechanisms by which corticosteroids reduce airway inflammation are not completely understood. Traditionally, corticosteroids were thought to inhibit cytokines exclusively at the transcriptional level. Our recent evidence, obtained in airway smooth muscle (ASM), no longer supports this view. We have found that corticosteroids do not act at the transcriptional level to reduce TNF-alpha-induced IL-6 gene expression. Rather, corticosteroids inhibit TNF-alpha-induced IL-6 secretion by reducing the stability of the IL-6 mRNA transcript. TNF-alpha-induced IL-6 mRNA decays at a significantly faster rate in ASM cells pretreated with the corticosteroid dexamethasone (t(1/2) = 2.4 h), compared to vehicle (t(1/2) = 9.0 h; P ASM cells.

  2. PD-1 inhibits T cell proliferation by upregulating p27 and p15 and suppressing Cdc25A.

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    Patsoukis, Nikolaos; Sari, Duygu; Boussiotis, Vassiliki A

    2012-12-01

    The programmed cell death-1 (PD)-1 receptor (CD279) is a potent T cell inhibitor with a critical role in peripheral tolerance, but it can also compromise anti-viral and antitumor T cell responses. The effects of PD-1 on the cell cycle leading to inhibition of T cell expansion are poorly understood. Recently, we examined the effects of PD-1 on the molecular control of the cell cycle machinery and on TCR-activated signaling pathways that regulate these downstream outcomes. Our studies showed that PD-1 blocks cell cycle progression in the G 1 phase. PD-1 did not alter the expression of G 1 phase cyclins or cyclin-dependent kinases (Cdks) but, instead, suppressed the transcription of SKP2, the substrate recognition component of the SCF (Skp2) ubiquitin ligase that leads p27 (kip1) to degradation and resulted in accumulation of p27 (kip1) . Subsequently, T cells receiving PD-1 signals displayed impaired Cdk2 activation and failed to phosphorylate two critical Cdk2 substrates, the retinoblastoma gene product (Rb) and the TGFβ-specific transcription factor Smad3, leading to suppression of E2F target genes but enhanced Smad3 transactivation. These events resulted in upregulation of the Cdk4/6 inhibitor p15 (INK4B) and repression of the Cdk-activating phosphatase Cdc25A. The suppressive effect of PD-1 on Skp2 expression was mediated by inhibition of both PI3K/Akt and Ras/MEK/Erk pathways and was only partially reversed by IL-2, which restored activation of MEK/Erk but not Akt. Thus, PD-1 targets Ras and PI3K/Akt signaling to inhibit transcription of Skp2 and to activate Smad3 as an integral component of a pathway that regulates blockade of cell cycle progression in T lymphocytes. Here, we discuss the detailed sequence of these signaling events and their implications in mediating cell-intrinsic and -extrinsic mechanisms that inhibit proliferation of T effector cells in response to PD-1-mediated signaling.

  3. Zinc Protoporphyrin Upregulates Heme Oxygenase-1 in PC-3 Cells via the Stress Response Pathway

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    Simon C. M. Kwok

    2013-01-01

    Full Text Available Zinc protoporphyrin IX (ZnPP, a naturally occurring molecule formed in iron deficiency or lead poisoning, is a potent competitive inhibitor of heme oxygenase-1 (HO-1. It also regulates expression of HO-1 at the transcriptional level. However, the effect of ZnPP on HO-1 expression is controversial. It was shown to induce HO-1 expression in some cells, but suppress it in others. The objective of this study is to investigate the effect of ZnPP on HO-1 expression in prostate cancer PC-3 cells. Incubation of PC-3 cells with 10 μM ZnPP for 4 h showed only a slight induction of HO-1 mRNA and protein, but the induction was high after 16 h and was maintained through 48 h of incubation. Of all the known responsive elements in the HO-1 promoter, ZnPP activated mainly the stress response elements. Of the various protein kinase inhibitors and antioxidant tested, only Ro 31-8220 abrogated ZnPP-induced HO-1 expression, suggesting that activation of HO-1 gene by ZnPP may involve protein kinase C (PKC. The involvement of PKC α, β, δ, η, θ, and ζ isoforms was ruled out by the use of specific inhibitors. The isoform of PKC involved and participation of other transcription factors remain to be studied.

  4. STMN1 Promotes Progesterone Production Via StAR Up-regulation in Mouse Granulosa Cells

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    Dou, Yun-De; Zhao, Han; Huang, Tao; Zhao, Shi-Gang; Liu, Xiao-Man; Yu, Xiao-Chen; Ma, Zeng-Xiang; Zhang, Yu-Chao; Liu, Tao; Gao, Xuan; Li, Lei; Lu, Gang; Chan, Wai-Yee; Gao, Fei; Liu, Hong-Bin; Chen, Zi-Jiang

    2016-01-01

    Stathmin 1 (STMN1) is a biomarker in several types of neoplasms. It plays an important role in cell cycle progression, mitosis, signal transduction and cell migration. In ovaries, STMN1 is predominantly expressed in granulosa cells (GCs). However, little is known about the role of STMN1 in ovary. In this study, we demonstrated that STMN1 is overexpressed in GCs in patients with polycystic ovary syndrome (PCOS). In mouse primary GCs, the overexpression of STMN1 stimulated progesterone production, whereas knockdown of STMN1 decreased progesterone production. We also found that STMN1 positively regulates the expression of Star (steroidogenic acute regulatory protein) and Cyp11a1 (cytochrome P450 family 11 subfamily A member 1). Promoter and ChIP assays indicated that STMN1 increased the transcriptional activity of Star and Cyp11a1 by binding to their promoter regions. The data suggest that STMN1 mediates the progesterone production by modulating the promoter activity of Star and Cyp11a1. Together, our findings provide novel insights into the molecular mechanisms of STMN1 in ovary GC steroidogenesis. A better understanding of this potential interaction between STMN1 and Star in progesterone biosynthesis in GCs will facilitate the discovery of new therapeutic targets in PCOS. PMID:27270953

  5. Glucocorticoids Suppress Mitochondrial Oxidant Production via Upregulation of Uncoupling Protein 2 in Hyperglycemic Endothelial Cells.

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    Domokos Gerö

    Full Text Available Diabetic complications are the leading cause of morbidity and mortality in diabetic patients. Elevated blood glucose contributes to the development of endothelial and vascular dysfunction, and, consequently, to diabetic micro- and macrovascular complications, because it increases the mitochondrial proton gradient and mitochondrial oxidant production. Therapeutic approaches designed to counteract glucose-induced mitochondrial reactive oxygen species (ROS production in the vasculature are expected to show efficacy against all diabetic complications, but direct pharmacological targeting (scavenging of mitochondrial oxidants remains challenging due to the high reactivity of some of these oxidant species. In a recent study, we have conducted a medium-throughput cell-based screening of a focused library of well-annotated pharmacologically active compounds and identified glucocorticoids as inhibitors of mitochondrial superoxide production in microvascular endothelial cells exposed to elevated extracellular glucose. The goal of the current study was to investigate the mechanism of glucocorticoids' action. Our findings show that glucocorticoids induce the expression of the mitochondrial UCP2 protein and decrease the mitochondrial potential. UCP2 silencing prevents the protective effect of the glucocorticoids on ROS production. UCP2 induction also increases the oxygen consumption and the "proton leak" in microvascular endothelial cells. Furthermore, glutamine supplementation augments the effect of glucocorticoids via further enhancing the expression of UCP2 at the translational level. We conclude that UCP2 induction represents a novel experimental therapeutic intervention in diabetic vascular complications. While direct repurposing of glucocorticoids may not be possible for the therapy of diabetic complications due to their significant side effects that develop during chronic administration, the UCP2 pathway may be therapeutically targetable by other

  6. Glucocorticoids Suppress Mitochondrial Oxidant Production via Upregulation of Uncoupling Protein 2 in Hyperglycemic Endothelial Cells

    Science.gov (United States)

    Szabo, Csaba

    2016-01-01

    Diabetic complications are the leading cause of morbidity and mortality in diabetic patients. Elevated blood glucose contributes to the development of endothelial and vascular dysfunction, and, consequently, to diabetic micro- and macrovascular complications, because it increases the mitochondrial proton gradient and mitochondrial oxidant production. Therapeutic approaches designed to counteract glucose-induced mitochondrial reactive oxygen species (ROS) production in the vasculature are expected to show efficacy against all diabetic complications, but direct pharmacological targeting (scavenging) of mitochondrial oxidants remains challenging due to the high reactivity of some of these oxidant species. In a recent study, we have conducted a medium-throughput cell-based screening of a focused library of well-annotated pharmacologically active compounds and identified glucocorticoids as inhibitors of mitochondrial superoxide production in microvascular endothelial cells exposed to elevated extracellular glucose. The goal of the current study was to investigate the mechanism of glucocorticoids' action. Our findings show that glucocorticoids induce the expression of the mitochondrial UCP2 protein and decrease the mitochondrial potential. UCP2 silencing prevents the protective effect of the glucocorticoids on ROS production. UCP2 induction also increases the oxygen consumption and the “proton leak” in microvascular endothelial cells. Furthermore, glutamine supplementation augments the effect of glucocorticoids via further enhancing the expression of UCP2 at the translational level. We conclude that UCP2 induction represents a novel experimental therapeutic intervention in diabetic vascular complications. While direct repurposing of glucocorticoids may not be possible for the therapy of diabetic complications due to their significant side effects that develop during chronic administration, the UCP2 pathway may be therapeutically targetable by other, glucocorticoid

  7. Reactive oxygen and nitrogen species disturb Ca2+ oscillations in insulin-secreting MIN6 β-cells

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    Antonucci, Salvatore; Tagliavini, Alessia; Pedersen, Morten Gram

    2015-01-01

    Disturbances in pulsatile insulin secretion and Ca2+ oscillations in pancreatic β-cells are early markers of diabetes, but the underlying mechanisms are still incompletely understood. Reactive oxygen/nitrogen species (ROS/RNS) are implicated in reduced β-cell function, and ROS/RNS target several Ca2+ pumps and channels. Thus, we hypothesized that ROS/RNS could disturb Ca2+ oscillations and downstream insulin pulsatility. We show that ROS/RNS production by photoactivation of aluminum phthalocyanine chloride (AlClPc) abolish or accelerate Ca2+ oscillations in the MIN6 β-cell line, depending on the amount of ROS/RNS. Application of the sarcoplasmic/endoplasmic reticulum Ca2+ ATPase (SERCA) inhibitor thapsigargin modifies the Ca2+ response to high concentrations of ROS/RNS. Further, thapsigargin produces effects that resemble those elicited by moderate ROS/RNS production. These results indicate that ROS/RNS interfere with endoplasmic reticulum Ca2+ handling. This idea is supported by theoretical studies using a mathematical model of Ca2+ handling adapted to MIN6 cells. Our results suggest a putative link between ROS/RNS and disturbed pulsatile insulin secretion. PMID:26732126

  8. Lipid rafts promote liver cancer cell proliferation and migration by up-regulation of TLR7 expression

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    Liu, Yuan; Guo, Xiaodong; Wu, Liyuan; Yang, Mei; Li, Zhiwei; Gao, Yinjie; Liu, Shuhong; Zhou, Guangde; Zhao, Jingmin

    2016-01-01

    Hepatocellular carcinoma (HCC) occurs predominantly in patients with underlying chronic liver disease and cirrhosis. Toll-like receptors (TLRs) play an important role in innate immune responses and TLR signaling has been associated with various chronic liver diseases. Lipid rafts provide the necessary microenvironment for certain specialized signaling events to take place, such as the innate immune recognition. The purpose of this study was to determine the pattern of TLR7 expression in HCC, how to recruit TLR7 into lipid rafts responded to ligands and whether targeting TLR7 might have beneficial effects. The study group was comprised of 130 human liver tissues: 23 chronic hepatitis B (CHB), 18 liver cirrhosis (LC), 68 HCC and 21 normal livers. The expression of TLR7 was evaluated using immunohistochemistry, western blotting, and flow cytometry. Proliferation and migration of human HepG2 cells were studied following stimulation of TLR7 using the agonist gardiquimod and inhibition with a specific antagonist 20S-protopanaxadiol (aPPD). The activation of lipid raft-associated TLR7 signaling was measured using western blotting, double immunohistochemistry and immunoprecipitation in liver tissues and HepG2 cells. TLR7 expression was up-regulated in human HCC tissues and hepatoma cell line. Proliferation and migration of HepG2 cells in vitro increased significantly in response to stimulation of TLR7. TLR7 inhibition using aPPD significantly reduced HepG2 cell migration in vitro. The lipid raft protein caveolin-1 and flotillin-1 were involved with enhanced TLR7 signaling in HCC. Conclusions The data suggest that inhibiting TLR7 with antagonists, like aPPD, could potentially be used as a novel therapeutic approach for HCC. PMID:27588480

  9. Direct laser machining-induced topographic pattern promotes up-regulation of myogenic markers in human mesenchymal stem cells.

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    Li, Huaqiong; Wen, Feng; Wong, Yee Shan; Boey, Freddy Yin Chiang; Subbu, Venkatraman S; Leong, David Tai; Ng, Kee Woei; Ng, Gary Ka Lai; Tan, Lay Poh

    2012-02-01

    The engineering of tissue is preferably done with stem cells, which can be differentiated into the tissue of interest using biochemical or physical cues. While much effort has been focused on using biological factors to regulate stem cell differentiation, recently interest in the contribution of physical factors has increased. In this work, three-dimensional (3-D) microchannels with topographic micropatterns were fabricated by femtosecond laser machining on a biodegradable polymer (poly(L-lactide-co-ε-caprolactone)) substrate. Two substrates with narrow and wide channels respectively were created. Human mesenchymal stem cells (hMSCs) were cultured on the scaffolds for cell proliferation and cellular organization. Gene expression and the immunostaining of myogenic and neurogenic markers were studied. Both scaffolds improved the cell alignment along the channels as compared to the control group. Microfilaments within hMSCs were more significantly aligned and elongated on the narrower microchannels. The gene expression study revealed significant up-regulation of several hallmark markers associated with myogenesis for hMSCs cultured on the scaffold with narrow microchannels, while osteogenic and neurogenic markers were down-regulated or remained similar to the control at day 14. Immunostaining of myogen- and neurogen-specific differentiation markers were used to further confirm the specific differentiation towards a myogenic lineage. This study demonstrates that femtosecond laser machining is a versatile tool for generating controllable 3-D microchannels with topographic features that can be used to induce specific myogenic differentiation of hMSCs in vitro, even in the absence of biological factors.

  10. beta-TrCP inhibition reduces prostate cancer cell growth via upregulation of the aryl hydrocarbon receptor.

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    Udi Gluschnaider

    Full Text Available BACKGROUND: Prostate cancer is a common and heterogeneous disease, where androgen receptor (AR signaling plays a pivotal role in development and progression. The initial treatment for advanced prostate cancer is suppression of androgen signaling. Later on, essentially all patients develop an androgen independent stage which does not respond to anti hormonal treatment. Thus, alternative strategies targeting novel molecular mechanisms are required. beta-TrCP is an E3 ligase that targets various substrates essential for many aspects of tumorigenesis. METHODOLOGY/PRINCIPAL FINDINGS: Here we show that beta-TrCP depletion suppresses prostate cancer and identify a relevant growth control mechanism. shRNA targeted against beta-TrCP reduced prostate cancer cell growth and cooperated with androgen ablation in vitro and in vivo. We found that beta-TrCP inhibition leads to upregulation of the aryl hydrocarbon receptor (AhR mediating the therapeutic effect. This phenomenon could be ligand independent, as the AhR ligand 2,3,7,8-Tetrachlorodibenzo-p-Dioxin (TCDD did not alter prostate cancer cell growth. We detected high AhR expression and activation in basal cells and atrophic epithelial cells of human cancer bearing prostates. AhR expression and activation is also significantly higher in tumor cells compared to benign glandular epithelium. CONCLUSIONS/SIGNIFICANCE: Together these observations suggest that AhR activation may be a cancer counteracting mechanism in the prostate. We maintain that combining beta-TrCP inhibition with androgen ablation could benefit advanced prostate cancer patients.

  11. Vitamin D Receptor-Mediated Upregulation of CYP3A4 and MDR1 by Quercetin in Caco-2 cells.

    Science.gov (United States)

    Chae, Yoon-Jee; Cho, Kwan Hyung; Yoon, In-Soo; Noh, Chi-Kyoung; Lee, Hyo-Jong; Park, Yohan; Ji, Eunhee; Seo, Min-Duk; Maeng, Han-Joo

    2016-01-01

    To examine whether quercetin interacts with vitamin D receptor, we investigated the effects of quercetin on vitamin D receptor activity in human intestinal Caco-2 cells. The effects of quercetin on the expression of the vitamin D receptor target genes, vitamin D3 24-hydroxylase, cytochrome P450 3A4, multidrug resistance protein 1, and transient receptor potential vanilloid type 6 were measured using quantitative polymerase chain reaction. The vitamin D receptor siRNA was used to assess the involvement of the vitamin D receptor. Vitamin D receptor activation using a vitamin D responsive element-mediated cytochrome P450 3A4 reporter gene assay was investigated in Caco-2 cells transfected with human vitamin D receptor. We also studied the magnitude of the vitamin D receptor activation and/or synergism between 1α,25-dihydroxyvitamin D3 [1,25(OH)2D3] and quercetin-like flavonoids. Slight but significant increases in the mRNA expression of cytochrome P450 3A4, vitamin D3 24-hydroxylase, multidrug resistance protein 1, and transient receptor potential vanilloid type 6 were observed after 3 days of continual quercetin treatment. The silencing effect of vitamin D receptor by vitamin D receptor siRNA in Caco-2 cells significantly attenuated the induction of the vitamin D receptor target genes. Moreover, quercetin significantly enhanced cytochrome P450 3A4 reporter activity in Caco-2 cells in a dose-dependent manner, and the expression of exogenous vitamin D receptor further stimulated the vitamin D receptor activity. Quercetin-like flavonoids such as kaempferol stimulated the vitamin D receptor activity in a manner similar to that seen with quercetin. Taken together, the data indicates that quercetin upregulates cytochrome P450 3A4 and multidrug resistance protein 1 expression in Caco-2 cells likely via a vitamin D receptor-dependent pathway.

  12. Upregulation of APE/ref-1 in recurrence stage I, non small cell lung cancer.

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    Kang, Min-Woong; Kang, Shin Kwang; Choi, Songyi; Lee, Choong Sik; Jeon, Byeong Hwa; Lim, Seung Pyung

    2012-02-01

    Lung cancer, the leading cause of cancer-related death, still lacks reliable biomarkers. Apurinic/apyrimidinic endonuclease 1/Ref-1 is a multifunctional protein involved in the base excision repair of DNA damaged by oxidative stress or alkylating compounds, as well as in the regulation of multiple transcription factors. To validate apurinic/apyrimidinic endonuclease 1/Ref-1 as a biomarker for prediction of lung cancer recurrence, we studied 42 patients who received curative resection and mediastinal lymph node dissection for stage I non-small-cell lung cancer. They were divided into 2 groups based on recurrence, and compared by immunohistochemistry staining of paraffin-embedded tissues and Western blot analysis. Immunohistochemical staining showed a significant difference between the cytoplasm and nucleus in patients who had a recurrence compared to those with nonrecurrent adenocarcinoma. In Western blot analysis, the recurrent adenocarcinoma group showed increased expression of apurinic/apyrimidinic endonuclease 1/Ref-1 in cytoplasm, nucleus, and in total. This indicates that apurinic/apyrimidinic endonuclease 1/Ref-1 is unregulated in recurrent stage I adenocarcinoma. For clinical application as a prognostic marker for non-small-cell lung cancer, further investigation into the role of apurinic/apyrimidinic endonuclease 1/Ref-1 in carcinogenesis is needed in an expanded prospective study.

  13. Cancer metastasis-suppressing peptide metastin upregulates excitatory synaptic transmission in hippocampal dentate granule cells.

    Science.gov (United States)

    Arai, Amy C; Xia, Yan-Fang; Suzuki, Erika; Kessler, Markus; Civelli, Olivier; Nothacker, Hans-Peter

    2005-11-01

    Metastin is an antimetastatic peptide encoded by the KiSS-1 gene in cancer cells. Recent studies found that metastin is a ligand for the orphan G-protein-coupled receptor GPR54, which is highly expressed in specific brain regions such as the hypothalamus and parts of the hippocampus. This study shows that activation of GPR54 by submicromolar concentrations of metastin reversibly enhances excitatory synaptic transmission in hippocampal dentate granule cells in a mitogen-activated protein (MAP) kinase-dependent manner. Synaptic enhancement by metastin was suppressed by intracellular application of the G-protein inhibitor GDP-beta-S and the calcium chelator BAPTA. Analysis of miniature excitatory postsynaptic currents (mEPSCs) revealed an increase in the mean amplitude but no change in event frequency. This indicates that GPR54 and the mechanism responsible for the increase in EPSCs are postsynaptic. Metastin-induced synaptic potentiation was abolished by 50 microM PD98059 and 20 microM U0126, two inhibitors of the MAP kinases ERK1 and ERK2. The effect was also blocked by inhibitors of calcium/calmodulin-dependent kinases and tyrosine kinases. RT-PCR experiments showed that both KiSS-1 and GPR54 are expressed in the hippocampal dentate gyrus. Metastin is thus a novel endogenous factor that modulates synaptic excitability in the dentate gyrus through mechanisms involving MAP kinases, which in turn may be controlled upstream by calcium-activated kinases and tyrosine kinases.

  14. Upregulated functional expression of Toll like receptor 4 in mesenchymal stem cells induced by lipopolysaccharide

    Institute of Scientific and Technical Information of China (English)

    SHI Liang; WANG Ji-shi; LIU Xing-mei; HU Xiao-yan; FANG Qin

    2007-01-01

    Background The coordinated change of haematopoietic supporting microenvironment in bone marrow (BM) is crucial for innate immunity and inflammation. As the precursors of marrow stroma, BM derived mesenchymal stem cells (MSCs)promote haematopoietic function, but their roles in innate immunity or inflammation have not been investigated. Here we investigated the expression of Toll like receptor 4 (TLR-4) and the effect of lipopolysaccharide (LPS) on its expression in BM MSCs in vitro.Methods MSCs were harvested from adult rat's BM cells by density gradient centrifugation and adhesive culture. The purity of MSCs were identified with the cell morphological feature and osteogenic capacity, the phenotypes were tested by flow cytometry. Cultured MSCs were treated by LPS (1 μg/ml, 10 μg/ml or 100 μg/ml) for 24 hours. The relative expression levels of TLR-4 mRNA were detected by semiquantitative reverse transcription polymerase chain reaction and costimulatory molecules (CD80, CD86 and MHC-Ⅱ) expressed on MSCs were analyzed by flow cytometry. The levels of tumor necrosis factor-α (TNF-α) in supernatants were determined by enzyme linked immunosorbent assay.Results After incubation with LPS, MSCs expressed the higher levels of TLR-4 mRNA, costimulatory molecules and TNF-α than the untreated group: LPS 10 μg/ml was the most effective (P<0.01); the levels of TLR-4 mRNA, costimulatory molecules and TNF-α decreased when MSCs were exposed to 100 μg/ml LPS. Except for MHC-Ⅱ and TNF-α (P>0.05),the levels of CD80, CD86 and TLR-4 mRNA were significantly lower than that in the treated group of 10 μg/ml (P<0.01).Conclusion MSCs expressed TLR-4 mRNA. LPS activated the functional expression levels of TLR-4 in MSCs although the activity may depend on the concentration of LPS.

  15. Loss of tricellular tight junction protein LSR promotes cell invasion and migration via upregulation of TEAD1/AREG in human endometrial cancer

    Science.gov (United States)

    Shimada, Hiroshi; Abe, Shyuetsu; Kohno, Takayuki; Satohisa, Seiro; Konno, Takumi; Takahashi, Syunta; Hatakeyama, Tsubasa; Arimoto, Chihiro; Kakuki, Takuya; Kaneko, Yakuto; Takano, Ken-ichi; Saito, Tsuyoshi; Kojima, Takashi

    2017-01-01

    Lipolysis-stimulated lipoprotein receptor (LSR) is a unique molecule of tricellular contacts of normal and cancer cells. We investigated how the loss of LSR induced cell migration, invasion and proliferation in endometrial cancer cell line Sawano. mRNAs of amphiregulin (AREG) and TEA domain family member 1 (TEAD1) were markedly upregulated by siRNA-LSR. In endometrial cancer tissues, downregulation of LSR and upregulation of AREG were observed together with malignancy, and Yes-associated protein (YAP) was present in the nuclei. siRNA-AREG prevented the cell migration and invasion induced by siRNA-LSR, whereas treatment with AREG induced cell migration and invasion. LSR was colocalized with TRIC, angiomotin (AMOT), Merlin and phosphorylated YAP (pYAP). siRNA-LSR increased expression of pYAP and decreased that of AMOT and Merlin. siRNA-YAP prevented expression of the mRNAs of AREG and TEAD1, and the cell migration and invasion induced by siRNA-LSR. Treatment with dobutamine and 2-deoxy-D-glucose and glucose starvation induced the pYAP expression and prevented the cell migration and invasion induced by siRNA-LSR. siRNA-AMOT decreased the Merlin expression and prevented the cell migration and invasion induced by siRNA-LSR. The loss of LSR promoted cell invasion and migration via upregulation of TEAD1/AREG dependent on YAP/pYAP and AMOT/Merlin in human endometrial cancer cells. PMID:28071680

  16. Synergistic Action of Genistein and Calcitriol in Immature Osteosarcoma MG-63 Cells by SGPL1 Up-Regulation

    Science.gov (United States)

    Engel, Nadja; Adamus, Anna; Schauer, Nicolas; Kühn, Juliane; Nebe, Barbara; Seitz, Guido; Kraft, Karin

    2017-01-01

    Background Phytoestrogens such as genistein, the most prominent isoflavone from soy, show concentration-dependent anti-estrogenic or estrogenic effects. High genistein concentrations (>10 μM) also promote proliferation of bone cancer cells in vitro. On the other hand, the most active component of the vitamin D family, calcitriol, has been shown to be tumor protective in vitro and in vivo. The purpose of this study was to examine a putative synergism of genistein and calcitriol in two osteosarcoma cell lines MG-63 (early osteoblast), Saos-2 (mature osteoblast) and primary osteoblasts. Methods Thus, an initial screening based on cell cycle phase alterations, estrogen (ER) and vitamin D receptor (VDR) expression, live cell metabolic monitoring, and metabolomics were performed. Results Exposure to the combination of 100 μM genistein and 10 nM calcitriol reduced the number of proliferative cells to control levels, increased ERß and VDR expression, and reduced extracellular acidification (40%) as well as respiratory activity (70%), primarily in MG-63 cells. In order to identify the underlying cellular mechanisms in the MG-63 cell line, metabolic profiling via GC/MS technology was conducted. Combined treatment significantly influenced lipids and amino acids preferably, whereas metabolites of the energy metabolism were not altered. The comparative analysis of the log2-ratios revealed that after combined treatment only the metabolite ethanolamine was highly up-regulated. This is the result: a strong overexpression (350%) of the enzyme sphingosine-1-phosphate lyase (SGPL1), which irreversibly degrades sphingosine-1-phosphate (S1P), thereby, generating ethanolamine. S1P production and secretion is associated with an increased capability of migration and invasion of cancer cells. Conclusion From these results can be concluded that the tumor promoting effect of high concentrations of genistein in immature osteosarcoma cells is reduced by the co-administration of calcitriol

  17. Overexpression of PGC‑1α enhances cell proliferation and tumorigenesis of HEK293 cells through the upregulation of Sp1 and Acyl-CoA binding protein.

    Science.gov (United States)

    Shin, Sung-Won; Yun, Seong-Hoon; Park, Eun-Seon; Jeong, Jin-Sook; Kwak, Jong-Young; Park, Joo-In

    2015-03-01

    Peroxisome proliferator-activated receptor γ coactivator-1α (PGC‑1α), a coactivator interacting with multiple transcription factors, regulates several metabolic processes. Although recent studies have focused on the role of PGC‑1α in cancer, the underlying molecular mechanism has not been clarified. Therefore, we evaluated the role of PGC‑1α in cell proliferation and tumorigenesis using human embryonic kidney (HEK)293 cells and colorectal cancer cells. We established stable HEK293 cell lines expressing PGC‑1α and examined cell proliferation, anchorage-independent growth, and oncogenic potential compared to parental HEK293 cells. To identify the molecular PGC‑1α targets for increased cell proliferation and tumorigenesis, the GeneFishing™ DEG (differentially expressed genes) screening system was used. Western blot analysis and immunofluorescence staining were performed for a regulated gene product to confirm the results. Forced expression of PGC‑1α in HEK293 cells promoted cell proliferation and anchorage-independent growth in soft agar. In addition, HEK293 cells that highly expressed PGC‑1α showed enhanced tumor formation when subcutaneously injected into the bilateral flanks of immunodeficient mice. The results of the GeneFishing DEG screening system identified one upregulated gene (Acyl-CoA binding protein; ACBP). Real-time RT-PCR, western blot analysis, and immunofluorescence staining showed that ACBP was markedly increased in HEK293 cells stably overexpressing PGC‑1α (PGC‑1α-HEK293 cells) compared to those expressing an empty vector. In PGC‑1α, ACBP, and specificity protein 1 (Sp1) siRNA knockdown experiments in PGC‑1α-HEK293 and SNU-C4 cells, we also observed inhibition of cell proliferation, reduced expression of antioxidant enzymes, and increased H2O2-induced reactive oxygen species production and apoptosis. These findings suggest that PGC‑1α may promote cell proliferation and tumorigenesis through upregulation of ACBP

  18. ADAM9 up-regulates N-cadherin via miR-218 suppression in lung adenocarcinoma cells.

    Directory of Open Access Journals (Sweden)

    Yuh-Pyng Sher

    Full Text Available Lung cancer is the leading cause of cancer death worldwide, and brain metastasis is a major cause of morbidity and mortality in lung cancer. CDH2 (N-cadherin, a mesenchymal marker of the epithelial-mesenchymal transition and ADAM9 (a type I transmembrane protein are related to lung cancer brain metastasis; however, it is unclear how they interact to mediate this metastasis. Because microRNAs regulate many biological functions and disease processes (e.g., cancer by down-regulating their target genes, microRNA microarrays were used to identify ADAM9-regulated miRNAs that target CDH2 in aggressive lung cancer cells. Luciferase assays and western blot analysis showed that CDH2 is a target gene of miR-218. MiR-218 was generated from pri-mir-218-1, which is located in SLIT2, in non-invasive lung adenocarcinoma cells, whereas its expression was inhibited in aggressive lung adenocarcinoma. The down-regulation of ADAM9 up-regulated SLIT2 and miR-218, thus down-regulating CDH2 expression. This study revealed that ADAM9 activates CDH2 through the release of miR-218 inhibition on CDH2 in lung adenocarcinoma.

  19. Up-regulation of expression of selected genes in human bone cells with specific capacitively coupled electric fields.

    Science.gov (United States)

    Clark, Charles C; Wang, Wei; Brighton, Carl T

    2014-07-01

    The objective of the described experiments was to determine the electrical parameters that lead to optimal expression of a number of bone-related genes in cultured human bone cells exposed to a capacitively coupled electric field. Human calvarial osteoblasts were grown in modified plastic Cooper dishes in which the cells could be exposed to various capacitively coupled electric fields. The optimal duration of stimulation and optimal duration of response to the electrical field, and the optimal amplitude, frequency and duty cycle were all determined for each of the genes analyzed. Results indicated that a capacitively coupled electric field of 60 kHz, 20 mV/cm, 50% duty cycle for 2 h duration per day significantly up-regulated mRNA expression of a number of transforming growth factor (TGF)-β family genes (bone morphogenetic proteins (BMP)-2 and -4, TGF-β1, - β2 and -β3) as well as fibroblast growth factor (FGF)-2, osteocalcin (BGP) and alkaline phosphatase (ALP). Protein levels of BMP-2 and -4, and TGF-β1 and - β2 were also elevated. The clinical relevance of these findings in the context of a noninvasive treatment modality for delayed union and nonunion fracture healing is discussed.

  20. Inhibiting PPARγ by erythropoietin while upregulating TAZ by IGF1 synergistically promote osteogenic differentiation of mesenchymal stem cells.

    Science.gov (United States)

    Zhou, Jianwei; Wei, Fangyuan; Ma, Yuquan

    2016-09-09

    Erythropoietin (EPO) is reported to promote osteogenesis and inhibit adipogenesis of mesenchymal stem cells (MSC) through inhibiting PPARγ, while insulin-like growth factor 1 (IGF1) is able to enhance osteogenesis via upregulating transcriptional coactivator with PDZ-binding motif (TAZ). The different targets of EPO and IGF1 suggested their potential synergism to enhance osteogenesis. In this study, we aimed to determine the potential synergism of EPO and IGF1 and its efficacy on MSC differentiation. Rat adipose-derived mesenchymal stem cells (ADSCs) were separately treated with EPO, IGF1 and EPO/IGF1. It was observed that the co-treatment using EPO and IGF1 was able to potently promote the osteogenic differentiation of rat ADSCs compared with EPO or IGF1 alone, which offered a promising effective option to strengthen bone tissue regeneration for bone defects. Further, we demonstrated that the enhanced osteogenic differentiation by EPO and IGF1 co-treatment was almost counteracted by activating PPARγ through PPARγ agonist, RSG, and blocking TAZ through TAZ silencing RNA, siTAZ. Thus, it could be concluded that EPO and IGF1 possessed a potent synergism in promoting osteogenic differentiation, and the synergism was mainly attributed to co-regulation of different osteogenic regulators PPARγ and TAZ, which were targeted genes of EPO and IGF1 respectively.

  1. Propofol Inhibits Neurogenesis of Rat Neural Stem Cells by Upregulating MicroRNA-141-3p.

    Science.gov (United States)

    Jiang, Qiliang; Wang, Yingwei; Shi, Xueyin

    2017-02-01

    Prolonged or high-dose exposure to anesthetics, such as propofol, can cause brain cell degeneration and subsequent long-term learning or memory deficits, particularly in the developing brain. However, the cellular and molecular mechanisms underlying the deleterious effects of propofol at certain stages of development remain unclear. In this study we found that propofol inhibited the proliferation, neuronal differentiation, and migration of neural stem cells (NSCs) while upregulating miR-141-3p. Silencing of miR-141-3p abrogated the effects of propofol on NSC neurogenesis. Propofol treatment downregulated IGF2BP2, a direct target of miR-141-3p, whereas overexpression of IGF2BP2 attenuated the effects of propofol and miR-141-3p on NSC neurogenesis. In short, propofol inhibits NSC neurogenesis through a mechanism involving the miR-141-3p/IGF2BP2 axis. Our results may provide a potential approach for preventing the neurodegenerative effects of propofol in the developing brain.

  2. Agouti Related Peptide Secreted Via Human Mesenchymal Stem Cells Upregulates Proteasome Activity in an Alzheimer’s Disease Model

    Science.gov (United States)

    Lee, Na Kyung; Park, Sang Eon; Kwon, Soo Jin; Shim, Sangmi; Byeon, Yeji; Kim, Jong-Hwa; Na, Duk L.; Chang, Jong Wook

    2017-01-01

    The activity of the ubiquitin proteasome system (UPS) is downregulated in aggregation diseases such as Alzheimer’s disease (AD). In this study, we investigated the therapeutic potential of the Agouti-related peptide (AgRP), which is secreted by human mesenchymal stem cells (MSCs), in terms of its effect on the regulation of proteasome activity in AD. When SH-SY5Y human neuroblastoma cells were co-cultured with MSCs isolated from human Wharton’s Jelly (WJ-MSC), their proteasome activity was significantly upregulated. Further analysis of the conditioned media after co-culture allowed us to identify significant concentrations of a neuropeptide, called AgRP. The stereotactic delivery of either WJ-MSCs or AgRP into the hippocampi of C57BL6/J and 5XFAD mice induced a significant increase of proteasome activity and suppressed the accumulation of ubiquitin-conjugated proteins. Collectively, these findings suggest strong therapeutic potential for WJ-MSCs and AgRP to enhance proteasome activity, thereby potentially reducing abnormal protein aggregation and delaying the clinical progression of various neurodegenerative diseases. PMID:28051110

  3. Adaptation to acrolein through upregulating the protection by glutathione in human bronchial epithelial cells: the materialization of the hormesis concept.

    Science.gov (United States)

    Sthijns, Mireille M J P E; Randall, Matthew J; Bast, Aalt; Haenen, Guido R M M

    2014-04-18

    Acrolein is a thiol reactive compound present in cigarette smoke and plays a pivotal role in the deleterious effects of smoking. Acrolein causes toxicity in human bronchial epithelial cells in a dose dependent manner. GSH forms the first line of defense against acrolein-induced toxicity. At high doses of acrolein (⩾10 μM) the capacity of the cellular protection by GSH is overwhelmed and GSH is not able to quench all the acrolein, resulting in cytotoxicity. At a relatively low dose of acrolein (3 μM), no cytotoxicity is observed due to protection by GSH. Moreover we found that exposure to a low dose of acrolein protects cells against the toxic effect of a second higher dose of acrolein. The adaptation to acrolein is induced via Nrf2 mediated gene expression of γ-glutamylcysteine synthetase leading to elevated GSH levels. This upregulation of the protection by GSH demonstrates a hormetic response to acrolein. Hormesis is an adaptive or compensatory response induced by a relatively subtle challenge of homeostasis by a toxic compound. Insight into the mechanism of hormesis is mandatory for a more accurate societal regulation of toxic compounds.

  4. Tumor Necrosis Factor Receptor 1 Expression Is Upregulated in Dendritic Cells in Patients with Chronic HCV Who Respond to Therapy

    Directory of Open Access Journals (Sweden)

    Raul Cubillas

    2010-01-01

    Full Text Available The present studies assessed the level of tumor necrosis factor receptor (TNFR expression in peripheral blood mononuclear cells (PBMCs subsets from patients with chronic HCV undergoing interferon /ribavirin-based therapy (Ifn/R. Methods. TNFR family member mRNA expression was determined using quantitative real-time PCR assays (RTPCRs in PBMC from 39 HCV+ patients and 21 control HCV− patients. Further subset analysis of HCV + patients (untreated (U, sustained virological responders (SVR, and nonresponders (NR/relapsers (Rel PBMC was performed via staining with anti-CD123, anti-CD33, anti-TNFR1 or via RTPCR for TNFR1 mRNA. Results. A similar level of TNFR1 mRNA in PBMC from untreated HCV+ genotype 1 patients and controls was noted. TNFR1 and TNFR2 mRNA levels in PBMC from HCV+ patients with SVR were statistically different than levels in HCV(− patients. A significant difference was noted between the peak values of TNFR1 of the CD123+ PBMC isolated from SVR and the NR/Rel. Conclusion. Upregulation of TNFR1 expression, occurring in a specific subset of CD123+ dendritic cells, appeared in HCV+ patients with SVR.

  5. Latency-Associated Nuclear Antigen of Kaposi's Sarcoma-Associated Herpesvirus (KSHV) Upregulates Survivin Expression in KSHV-Associated B-Lymphoma Cells and Contributes to Their Proliferation▿

    OpenAIRE

    Lu, Jie; Verma, Subhash C.; Murakami, Masanao; Cai, Qiliang; KUMAR, Pankaj; Xiao, Bingyi; Robertson, Erle S.

    2009-01-01

    Survivin is a master regulator of cell proliferation and cell viability and is highly expressed in most human tumors. The molecular network linked to survivin expression in tumors has not been completely elucidated. In this study, we show that latency-associated nuclear antigen (LANA), a multifunctional protein of Kaposi's sarcoma-associated herpesvirus (KSHV) that is found in Kaposi's sarcoma tumors, upregulates survivin expression and increases the proliferation of KSHV-infected B cells. An...

  6. The transcription elongation factor ELL2 is specifically upregulated in HTLV-1-infected T-cells and is dependent on the viral oncoprotein Tax

    Energy Technology Data Exchange (ETDEWEB)

    Mann, Melanie C., E-mail: melanie.mann@viro.med.uni-erlangen.de; Strobel, Sarah, E-mail: sarah.strobel@viro.med.uni-erlangen.de; Fleckenstein, Bernhard, E-mail: bernhard.fleckenstein@viro.med.uni-erlangen.de; Kress, Andrea K., E-mail: andrea.kress@viro.med.uni-erlangen.de

    2014-09-15

    The oncoprotein Tax of human T-cell leukemia virus type 1 (HTLV-1) is a potent transactivator of viral and cellular transcription. Here, we identified ELL2 as the sole transcription elongation factor to be specifically upregulated in HTLV-1-/Tax-transformed T-cells. Tax contributes to regulation of ELL2, since transient transfection of Tax increases ELL2 mRNA, Tax transactivates the ELL2 promoter, and repression of Tax results in decrease of ELL2 in transformed T-lymphocytes. However, we also measured upregulation of ELL2 in HTLV-1-transformed cells exhibiting undetectable amounts of Tax, suggesting that ELL2 can still be maintained independent of continuous Tax expression. We further show that Tax and ELL2 synergistically activate the HTLV-1 promoter, indicating that ELL2 cooperates with Tax in viral transactivation. This is supported by our findings that Tax and ELL2 accumulate in nuclear fractions and that they co-precipitate upon co-expression in transiently-transfected cells. Thus, upregulation of ELL2 could contribute to HTLV-1 gene regulation. - Highlights: • ELL2, a transcription elongation factor, is upregulated in HTLV-1-positive T-cells. • Tax transactivates the ELL2 promoter. • Tax and ELL2 synergistically activate the HTLV-1 promoter. • Tax and ELL2 interact in vivo.

  7. ERβ1 inhibits the migration and invasion of breast cancer cells through upregulation of E-cadherin in a Id1-dependent manner

    Energy Technology Data Exchange (ETDEWEB)

    Zhou, Yan [Breast Disease Center, Southwest Hospital, Third Military Medical University, Chongqing (China); Ming, Jia [Department of Breast, Thyroid and Pancreas Surgery, The Second Affiliated Hospital of Chongqing Medical University, Chongqing (China); Xu, Yan [Department of Breast and Thyroid Surgery, Institute of Surgery Research, Daping Hospital, Third Military Medical University, Chongqing (China); Zhang, Yi, E-mail: zy53810@163.com [Breast Disease Center, Southwest Hospital, Third Military Medical University, Chongqing (China); Jiang, Jun, E-mail: Jcbd@medmail.com.cn [Breast Disease Center, Southwest Hospital, Third Military Medical University, Chongqing (China)

    2015-02-06

    Highlights: • Expression of ERβ1 was positively correlated with E-cadherin in breast cancer cell. • ERβ1 upregulates E-cadherin expression in breast cancer cell lines. • ERβ1 upregulates E-cadherin expression in a Id1-dependent manner. - Abstract: ERβ1 is a member of the nuclear receptor superfamily of ligand-regulated transcription factors. It plays an important role in regulating the progression of breast cancer. However, the mechanisms of ERβ1 in tumorigenesis, metastasis and prognosis are still not fully clear. In this study, we showed that the expression of ERβ1 was positively correlated with E-cadherin expression in breast cancer cell lines. In addition, we found that ERβ1 upregulates E-cadherin expression in breast cancer cell lines. Furthermore, we also found that ERβ1 inhibits the migration and invasion of breast cancer cells and upregulated E-cadherin expression in a Id1-dependent manner. Taken together, our study provides further understanding of the molecular mechanism of ERβ1 in tumor metastasis and suggests the feasibility of developing novel therapeutic approaches to target Id1 to inhibit breast cancer metastasis.

  8. Up-regulation of interleukin-8 expressions induced by mast cell tryptase via protease activated receptor-2 in endothelial cell line

    Institute of Scientific and Technical Information of China (English)

    LU Chao; ZHAO Feng-di; LI Xiao-bo; YIN Lian-hua

    2005-01-01

    Background Protease activated receptor-2 is cleaved and activated by trypsin or mast cell tryptase and may play an important role in inflammation. However, it is unknown whetehr PAR-2 can mediate tryptase-induced inflammatory reaction. This study was conduct to investigate wheter PAR-2 could be the activated by mast cell tryptase and medicated the tryptase induced interleukin-8 expression in endothelial cells.Methods Protease activated receptor-2 expression was found in endothelial cell lines ECV304 cell by reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting. Interleukin-8 stimulated by purified human mast cell tryptase was determined by RT-PCR and enzyme linked immunosorbent assay (ELISA). Data were analysed by the S-N-K one-way ANOVA test.Results The present study shows that mRNA and protein of protease activated receptor-2 could be expressed in ECV304 cells, and tryptase upregulated the expression levels of both interleukin-8 mRNA and protein. The increased expression of interleukin-8 was inhibited by an antiprotease activated receptor-2 monoclonal antibody, SAM11. An additional band was observed by Western blotting after the incubation of ECV304 cells with tryptase for 2 hours, which suggested that protease activated receptor-2 was activated. Conclusion Protease activated receptor-2 can mediate the mast cell tryptase stimulated expression of interleukin-8 in ECV304 cell.

  9. A benzimidazole derivative exhibiting antitumor activity blocks EGFR and HER2 activity and upregulates DR5 in breast cancer cells.

    Science.gov (United States)

    Chu, B; Liu, F; Li, L; Ding, C; Chen, K; Sun, Q; Shen, Z; Tan, Y; Tan, C; Jiang, Y

    2015-03-12

    Aberrant expression or function of epidermal growth factor receptor (EGFR) or the closely related human epidermal growth factor receptor 2 (HER2) can promote cell proliferation and survival, thereby contributing to tumorigenesis. Specific antibodies and low-molecular-weight tyrosine kinase inhibitors of both proteins are currently in clinical trials for cancer treatment. Benzimidazole derivatives possess diverse biological activities, including antitumor activity. However, the anticancer mechanism of 5a (a 2-aryl benzimidazole compound; 2-chloro-N-(2-p-tolyl-1H-benzo[d]imidazol-5-yl)acetamide, C(16)H(14)ClN(3)O, MW299), a novel 2-aryl benzimidazole derivative, toward breast cancer is largely unknown. Here, we demonstrate that 5a potently inhibited both EGFR and HER2 activity by reducing EGFR and HER2 tyrosine phosphorylation and preventing downstream activation of PI3K/Akt and MEK/Erk pathways in vitro and in vivo. We also show that 5a inhibited the phosphorylation of FOXO and promoted FOXO translocation from the cytoplasm into the nucleus, resulting in the G1-phase cell cycle arrest and apoptosis. Moreover, 5a potently induced apoptosis via the c-Jun N-terminal kinase (JNK)-mediated death receptor 5 upregulation in breast cancer cells. The antitumor activity of 5a was consistent with additional results demonstrating that 5a significantly reduced tumor volume in nude mice in vivo. Analysis of the primary breast cancer cell lines with HER2 overexpression further confirmed that 5a significantly inhibited Akt Ser473 and Bad Ser136 phosphorylation and reduced cyclin D3 expression. On the basis of our findings, further development of this 2-aryl benzimidazole derivative, a new class of multitarget anticancer agents, is warranted and represents a novel strategy for improving breast cancer treatment.

  10. Stimulatory effects of propylthiouracil on pregnenolone production through upregulation of steroidogenic acute regulatory protein expression in rat granulosa cells.

    Science.gov (United States)

    Chen, Mei-Chih; Wang, Shyi-Wu; Kan, Shu-Fen; Tsai, Shiow-Chwen; Wu, Yu-Ching; Wang, Paulus S

    2010-12-01

    Propylthiouracil (PTU) is a common and effective clinical medicine for the treatment of hyperthyroidism. Our previous study demonstrated that short-term treatment with PTU inhibits progesterone production in rat granulosa cells. However, our present results indicate that a 16-h treatment with PTU was able to stimulate pregnenolone production in rat granulosa cells, although progesterone production was diminished by PTU through inhibition of 3β-hydroxysteroid dehydrogenase. Notably, we found that PTU treatment enhanced the conversion of cholesterol into pregnenolone, whereas the protein level of the cytochrome P450 side-chain cleavage enzyme (P450scc, which is the enzyme responding to this conversion) was not affected. Interestingly, the levels of steroidogenic acute regulatory protein (StAR) in both total cell lysate and the mitochondrial fraction were significantly increased by PTU treatment. Furthermore, the binding of steroidogenic factor-1 (SF-1) to the StAR promoter region was also enhanced by PTU treatment, which suggests that PTU could upregulate StAR gene expression. In addition to SF-1 regulation, we found that mitogen-activated protein (MAP) kinase kinase activation is an important regulator of PTU-stimulated StAR protein expression, based on the effects of the MEK inhibitor PD98059. In conclusion, these results indicate that PTU plays opposite roles in the production of progesterone and its precursor, pregnenolone. The regulation of negative feedback on speeding the cholesterol transportation and pregnenolone conversion after a 16-h PTU treatment may be the mechanism explaining PTU's inhibition of progesterone production in rat granulosa cells.

  11. Nuclear β-catenin and CD44 upregulation characterize invasive cell populations in non-aggressive MCF-7 breast cancer cells

    Directory of Open Access Journals (Sweden)

    Uchino Masahiro

    2010-08-01

    cells as well as MDA-MB-231 cells. Conclusions MCF-7-14 cells are a novel model for breast cancer metastasis without requiring constitutive EMT and are categorized as a "metastable phenotype", which can be distinguished from both epithelial and mesenchymal cells. The alterations and characteristics of MCF-7-14 cells, especially nuclear β-catenin and CD44 upregulation, may characterize invasive cell populations in breast cancer.

  12. Triamcinolone up-regulates GLUT 1 and GLUT 3 expression in cultured human placental endothelial cells.

    Science.gov (United States)

    Kipmen-Korgun, Dijle; Ozmen, Asli; Unek, Gozde; Simsek, Mehmet; Demir, Ramazan; Korgun, Emin Turkay

    2012-01-01

    The placenta is a glucocorticoid target organ, and glucocorticoids (GCs) are essential for the development and maturation of fetal organs. They are widely used for treatment of a variety of diseases during pregnancy. In various tissues, GCs have regulated by glucose transport systems; however, their effects on glucose transporters in the human placental endothelial cells (HPECs) are unknown. In the present study, HPECs were cultured 24 h in the presence or absence of 0.5, 5 and 50 µmol · l(-1) of synthetic GC triamcinolone (TA). The glucose carrier proteins GLUT 1, GLUT 3 and GC receptor (GR) were detected in the HPECs. We showed increased expression of GLUT 1 and GLUT 3 proteins and messenger RNA (mRNA) levels (p GLUT 1 and GLUT 3 expression through GR. Excessive exposure to GCs causes maternal and fetal hypoglycemia and diminished fetal growth. We speculate that to compensate for fetal hypoglycemia and diminished fetal growth, the expression of placental endothelial glucose transporters might be increased.

  13. TTYH2, a human homologue of the Drosophila melanogaster gene tweety, is up-regulated in colon carcinoma and involved in cell proliferation and cell aggregation

    Institute of Scientific and Technical Information of China (English)

    Yuji Toiyama; Akira Mizoguchi; Kazushi Kimura; Junichirou Hiro; Yasuhiro Inoue; Tomonari Tutumi; Chikao Miki; Masato Kusunoki

    2007-01-01

    AIM: To investigate the expression patterns of TTYH2 in the human colon cancer and colon cancer cell lines and to evaluate the inhibitory effect of small interfering RNA (siRIMA) on the expression of TTYH2 in colon cancer cell lines.METHODS: We investigated the expression patterns of TTYH2 in colon cancer, adjacent non-tumorous colon mucosa, and cancer cell lines (DLD-1, caco-2, and Lovo) by RT-PCR. Furthermore, a siRNA plasmid expression vector against TTYH2 was constructed and transfected into DLD-1 and Caco-2 with LipofectamineTM 2000. The down regulation of TTYH2 expression was detected by RT-PCR and the role of siRNA in inducing cell proliferation and cell aggregation was evaluated by MTT and aggregation assay.RESULTS: TTYH2 gene expression in colon cancer tissue was significantly up-regulated compared with normal colonic mucosa (1.23 ± 0.404 vs 0.655 ± 0.373, P=0.0103). Colon cancer derived cell lines including DLD-1, Caco-2, and Lovo also expressed high levels of TTYH2. In contrast, transfection with siRNA-TTYH2 significantly inhibited both proliferation and scattering of these cancer cell lines.CONCLUSION: The present work demonstrates, for the first time, that the TTYH2 gene expression is significantly up-regulated in colon cancer. The TTYH2 gene may play an important role in regulating both proliferating and metastatic potentials of colorectal cancer.

  14. BMP-2 Overexpression Augments Vascular Smooth Muscle Cell Motility by Upregulating Myosin Va via Erk Signaling

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    Ming Zhang

    2014-01-01

    Full Text Available Background. The disruption of physiologic vascular smooth muscle cell (VSMC migration initiates atherosclerosis development. The biochemical mechanisms leading to dysfunctional VSMC motility remain unknown. Recently, cytokine BMP-2 has been implicated in various vascular physiologic and pathologic processes. However, whether BMP-2 has any effect upon VSMC motility, or by what manner, has never been investigated. Methods. VSMCs were adenovirally transfected to genetically overexpress BMP-2. VSMC motility was detected by modified Boyden chamber assay, confocal time-lapse video assay, and a colony wounding assay. Gene chip array and RT-PCR were employed to identify genes potentially regulated by BMP-2. Western blot and real-time PCR detected the expression of myosin Va and the phosphorylation of extracellular signal-regulated kinases 1/2 (Erk1/2. Immunofluorescence analysis revealed myosin Va expression locale. Intracellular Ca2+ oscillations were recorded. Results. VSMC migration was augmented in VSMCs overexpressing BMP-2 in a dose-dependent manner. siRNA-mediated knockdown of myosin Va inhibited VSMC motility. Both myosin Va mRNA and protein expression significantly increased after BMP-2 administration and were inhibited by Erk1/2 inhibitor U0126. BMP-2 induced Ca2+ oscillations, generated largely by a “cytosolic oscillator”. Conclusion. BMP-2 significantly increased VSMCs migration and myosin Va expression, via the Erk signaling pathway and intracellular Ca2+ oscillations. We provide additional insight into the pathophysiology of atherosclerosis, and inhibition of BMP-2-induced myosin Va expression may represent a potential therapeutic strategy.

  15. Glioma Stem Cells but Not Bulk Glioma Cells Upregulate IL-6 Secretion in Microglia/Brain Macrophages via Toll-like Receptor 4 Signaling.

    Science.gov (United States)

    a Dzaye, Omar Dildar; Hu, Feng; Derkow, Katja; Haage, Verena; Euskirchen, Philipp; Harms, Christoph; Lehnardt, Seija; Synowitz, Michael; Wolf, Susanne A; Kettenmann, Helmut

    2016-05-01

    Peripheral macrophages and resident microglia constitute the dominant glioma-infiltrating cells. The tumor induces an immunosuppressive and tumor-supportive phenotype in these glioma-associated microglia/brain macrophages (GAMs). A subpopulation of glioma cells acts as glioma stem cells (GSCs). We explored the interaction between GSCs and GAMs. Using CD133 as a marker of stemness, we enriched for or deprived the mouse glioma cell line GL261 of GSCs by fluorescence-activated cell sorting (FACS). Over the same period of time, 100 CD133(+ )GSCs had the capacity to form a tumor of comparable size to the ones formed by 10,000 CD133(-) GL261 cells. In IL-6(-/-) mice, only tumors formed by CD133(+ )cells were smaller compared with wild type. After stimulation of primary cultured microglia with medium from CD133-enriched GL261 glioma cells, we observed an selective upregulation in microglial IL-6 secretion dependent on Toll-like receptor (TLR) 4. Our results show that GSCs, but not the bulk glioma cells, initiate microglial IL-6 secretion via TLR4 signaling and that IL-6 regulates glioma growth by supporting GSCs. Using human glioma tissue, we could confirm the finding that GAMs are the major source of IL-6 in the tumor context.

  16. Retinal pigment epithelial cells upregulate expression of complement factors after co-culture with activated T cells

    DEFF Research Database (Denmark)

    Juel, Helene Bæk; Kaestel, Charlotte; Folkersen, Lasse

    2011-01-01

    In this study we examined the effect of T cell-derived cytokines on retinal pigment epithelial (RPE) cells with respect to expression of complement components. We used an in vitro co-culture system in which CD3/CD28-activated human T cells were separated from the human RPE cell line (ARPE-19) by ...... of inflammatory ocular diseases such as uveitis and age-related macular degeneration. --------------------------------------------------------------------------------...

  17. INFLUENZA-INDUCED UP-REGULATION OF TLR3 IN RESPIRATORY EPITHELIAL CELLS MAY OCCUR THROUGH A POSITIVE FEEDBACK LOOP INVOLVING TYPE I INTERFERON

    Science.gov (United States)

    Toll-like receptor 3 (TLR3) plays an important role in the host defense responses against viral infections, including Influenza virus infections. Based on our previous observations showing that Influenza infection of respiratory epithelial cells results in an up-regulation of Tol...

  18. Jagged1 upregulation in prostate epithelial cells promotes formation of reactive stroma in the Pten null mouse model for prostate cancer

    Science.gov (United States)

    Su, Qingtai; Zhang, Boyu; Zhang, Li; Dang, Truong; Rowley, David; Ittmann, Michael; Xin, Li

    2016-01-01

    The role of Notch signaling in prostate cancer has not been defined definitively. Several large scale tissue microarray studies revealed that the expression of some Notch signaling components including the Jagged1 ligand are upregulated in advanced human prostate cancer specimens. Jagged1 expressed by tumor cells may activate Notch signaling in both adjacent tumor cells and cells in tumor microenvironment. However, it remains undetermined whether increased Jagged1 expression reflects a cause for or a consequence of tumor progression in vivo. To address this question, we generated a novel R26-LSL-JAG1 mouse model that enables spatiotemporal Jagged1 expression. Prostate specific upregulation of Jagged1 neither interferes with prostate epithelial homeostasis nor significantly accelerates tumor initiation or progression in the prostate-specific Pten deletion mouse model for prostate cancer. However, Jagged1 upregulation results in increased inflammatory foci in tumors and incidence of intracystic adenocarcinoma. In addition, Jagged1 overexpression upregulates Tgfβ signaling in prostate stromal cells and promotes progression of a reactive stromal microenvironment in the Pten null prostate cancer model. Collectively, Jagged1 overexpression does not significantly accelerate prostate cancer initiation and progression in the context of loss-of-function of Pten, but alters tumor histopathology and microenvironment. Our study also highlights an understudied role of Notch signaling in regulating prostatic stromal homeostasis. PMID:27345403

  19. Upregulation of human immunodeficiency virus (HIV) replication by CD4 cross-linking in peripheral blood mononuclear cells of HIV-infected adults.

    Science.gov (United States)

    Than, S; Oyaizu, N; Tetali, S; Romano, J; Kaplan, M; Pahwa, S

    1997-08-01

    This study was conducted with peripheral blood mononuclear cells from 67 human immunodeficiency virus (HIV)-infected adults. It supports the hypothesis that cross-linking of CD4 molecules by HIV gp120 can result in HIV upregulation and spread of infection. Underlying mechanisms include activation of latent infection by factors in addition to, or other than, tumor necrosis factor alpha.

  20. Up-regulation of DRP-3 long isoform during the induction of neural progenitor cells by glutamate treatment in the ex vivo rat retina

    Energy Technology Data Exchange (ETDEWEB)

    Tokuda, Kazuhiro, E-mail: r502um@yamaguchi-u.ac.jp [Department of Ophthalmology, Yamaguchi University Graduate School of Medicine, Ube, Yamaguchi (Japan); Department of Biochemistry and Functional Proteomics, Yamaguchi University Graduate School of Medicine, Ube, Yamaguchi (Japan); Kuramitsu, Yasuhiro; Byron, Baron; Kitagawa, Takao [Department of Biochemistry and Functional Proteomics, Yamaguchi University Graduate School of Medicine, Ube, Yamaguchi (Japan); Tokuda, Nobuko [Faculty of Health Sciences, Yamaguchi University Graduate School of Medicine, Ube (Japan); Kobayashi, Daiki; Nagayama, Megumi; Araki, Norie [Department of Tumor Genetics and Biology, Graduate School of Medical Sciences, Kumamoto University, Kumamoto (Japan); Sonoda, Koh-Hei [Department of Ophthalmology, Yamaguchi University Graduate School of Medicine, Ube, Yamaguchi (Japan); Nakamura, Kazuyuki [Department of Biochemistry and Functional Proteomics, Yamaguchi University Graduate School of Medicine, Ube, Yamaguchi (Japan)

    2015-08-07

    Glutamate has been shown to induce neural progenitor cells in the adult vertebrate retina. However, protein dynamics during progenitor cell induction by glutamate are not fully understood. To identify specific proteins involved in the process, we employed two-dimensional electrophoresis-based proteomics on glutamate untreated and treated retinal ex vivo sections. Rat retinal tissues were incubated with 1 mM glutamate for 1 h, followed by incubation in glutamate-free media for a total of 24 h. Consistent with prior reports, it was found that mitotic cells appeared in the outer nuclear layer without any histological damage. Immunohistological evaluations and immunoblotting confirmed the emergence of neuronal progenitor cells in the mature retina treated with glutamate. Proteomic analysis revealed the up-regulation of dihydropyrimidinase-related protein 3 (DRP-3), DRP-2 and stress-induced-phosphoprotein 1 (STIP1) during neural progenitor cell induction by glutamate. Moreover, mRNA expression of DRP-3, especially, its long isoform, robustly increased in the treated retina compared to that in the untreated retina. These results may indicate that glutamate induces neural progenitor cells in the mature rat retina by up-regulating the proteins which mediate cell mitosis and neurite growth. - Highlights: • Glutamate induced neuronal progenitor cells in the mature rat retina. • Proteomic analysis revealed the up-regulation of DRP-3, DRP-2 and STIP1. • mRNA expression of DRP-3, especially, its long isoform, robustly increased.

  1. Dose response relationship of disturbed migration of Purkinje cells in the cerebellum due to X-irradiation

    Energy Technology Data Exchange (ETDEWEB)

    Darmanto, W.; Inouye, Minoru; Hayasaka, Shizu; Takagishi, Yoshiko; Aolad, H.; Murata, Yoshiharu [Nagoya Univ. (Japan). Research Inst. of Environmental Medicine

    1998-10-01

    Pregnant rats were exposed to 2.0, 2.25 or 2.5 Gy X-irradiation on gestation day 21. Pups were sacrificed 12 hr after exposure, and on postnatal day 5 (P5), P7 and P9. Their cerebella were observed immunohistochemically using anti-inositol 1,4,5 triphosphate (IP3) receptor antibody to identify Purkinje cells. These cells were disturbed to migrate and remained in the internal granular layer and white matter of the cerebellum. They had short dendrites, and some showed an abnormal direction of dendrites in rats exposed to 2.25 or 2.5 Gy. Alignment of Purkinje cells was also disturbed when examined either on P5, P7 or P9 especially by doses of 2.25 and 2.5 Gy. There was a relationship between X-ray doses and the number of cells piling up in the Purkinje cell layer of the cerebellum. The dose-response relationship with the number of ectopic Purkinje cells was noted in the anterior lobes of the cerebellum. (author)

  2. The role of T-regulatory cells in the pathogenesis of immunological disturbances accompanying obesity and atherosclerosis

    Directory of Open Access Journals (Sweden)

    Włodzimierz Łuczyński

    2010-03-01

    Full Text Available Obesity and atherosclerosis, and their consequences, including cardiovascular disease, are plagues of the 21st century. Chronic inflammation, whose mechanism is not well understood, underlies the pathophysiological bases of both processes. T lymphocytes, macrophages, and the proinflammatory cytokines produced by these cells play key roles in the immunological disturbances accompanying obesity and atherosclerosis. It was recently shown that T-regulatory cells can play a role in these processes. T-regulatory cells are a small subpopulation of T cells which are responsible for inhibition of the immune response. In this review, experiments conducted in mice and human models on the role of diminished number and/or function of T-regulatory lymphocytes in the pathogenesis of immune disturbances accompanying obesity and atherosclerosis are discussed. The results of studies using T-regulatory cells to stabilize and decrease atherosclerotic lesions in blood vessel walls are also summarized. The results of experiments performed so far are encouraging and give some hope for the future use of T-regulatory cells in the therapy of obesity and atherosclerosis.

  3. A critical role of IFNγ in priming MSC-mediated suppression of T cell proliferation through up-regulation of B7-H1

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    Bone-marrow-derived mesenchymal stem cells (MSCs) have been shown to possess immunosuppressive properties, e.g., by inhibiting T cell proliferation. Activated T cells can also enhance the immunosuppression ability of MSCs. The precise mechanisms underlying MSC-mediated immunosuppression remain largely undefined, although both cell-cell contact and soluble factors have been implicated; nor is it clear how the immunosuppressive property of MSCs is modulated by T cells. Using MSCs isolated from mouse bone marrow, we show here that interferon gamma (IFNγ), a well-known proinflammatory cytokine produced by activated T cells, plays an important role in priming the immunosuppressive property of MSCs. Mechanistically, IFNγ acts directly on MSCs and leads to up-regulation of B7-H1, an inhibitory surface molecule in these stem cells. MSCs primed by activated T cells derived from IFNγ-/- mouse exhibited dramatically reduced ability to suppress T cell proliferation, a defect that can be rescued by supplying exogenous IFNy. Moreover, siRNA-mediated knockdown of B7-H1 in MSCs abolished immunosuppression by these cells. Taken together, our results suggest that IFNy plays a critical role in triggering the immunosuppresion by MSCs through upregulating B7-H1 in these cells, and provide evidence supporting the cell-cell contact mechanism in MSC-mediated immunosuppression.

  4. Tumor cell adhesion to endothelial cells is increased by endotoxin via an upregulation of beta-1 integrin expression.

    LENUS (Irish Health Repository)

    Andrews, E J

    2012-02-03

    BACKGROUND: Recent studies have demonstrated that metastatic disease develops from tumor cells that adhere to endothelial cells and proliferate intravascularly. The beta-1 integrin family and its ligand laminin have been shown to be important in tumor-to-endothelial cell adhesion. Lipopolysaccharide (LPS) has been implicated in the increased metastatic tumor growth that is seen postoperatively. We postulated that LPS increases tumor cell expression of beta-1 integrins and that this leads to increased adhesion. METHODS: The human metastatic colon cancer cell line LS174T was labeled with an enhanced green fluorescent protein (eGFP) using retroviral transfection. Cell cultures were treated with LPS for 1, 2, and 4 h (n = 6 each) and were subsequently cocultured for 30 or 120 min with confluent human umbilical vein endothelial cells (HUVECs), to allow adherence. Adherent tumor cells were counted using fluorescence microscopy. These experiments were carried out in the presence or absence of a functional blocking beta-1 integrin monoclonal antibody (4B4). Expression of beta-1 integrin and laminin on tumor and HUVECs was assessed using flow cytometric analysis. Tumor cell NF-kappaB activation after incubation with LPS was measured. RESULTS: Tumor cell and HUVEC beta-1 integrin expression and HUVEC expression of laminin were significantly (P < 0.05) enhanced after incubation with LPS. Tumor cell adhesion to HUVECs was significantly increased. Addition of the beta-1 integrin blocking antibody reduced tumor cell adhesion to control levels. LPS increased tumor cell NF-kappaB activation. CONCLUSIONS: Exposure to LPS increases tumor cell adhesion to the endothelium through a beta-1 integrin-mediated pathway that is NF-kappaB dependent. This may provide a target for immunotherapy directed at reducing postoperative metastatic tumor growth.

  5. PRMT5 Is Upregulated in HTLV-1-Mediated T-Cell Transformation and Selective Inhibition Alters Viral Gene Expression and Infected Cell Survival.

    Science.gov (United States)

    Panfil, Amanda R; Al-Saleem, Jacob; Howard, Cory M; Mates, Jessica M; Kwiek, Jesse J; Baiocchi, Robert A; Green, Patrick L

    2015-12-30

    Human T-cell leukemia virus type-1 (HTLV-1) is a tumorigenic retrovirus responsible for development of adult T-cell leukemia/lymphoma (ATLL). This disease manifests after a long clinical latency period of up to 2-3 decades. Two viral gene products, Tax and HBZ, have transforming properties and play a role in the pathogenic process. Genetic and epigenetic cellular changes also occur in HTLV-1-infected cells, which contribute to transformation and disease development. However, the role of cellular factors in transformation is not completely understood. Herein, we examined the role of protein arginine methyltransferase 5 (PRMT5) on HTLV-1-mediated cellular transformation and viral gene expression. We found PRMT5 expression was upregulated during HTLV-1-mediated T-cell transformation, as well as in established lymphocytic leukemia/lymphoma cell lines and ATLL patient PBMCs. shRNA-mediated reduction in PRMT5 protein levels or its inhibition by a small molecule inhibitor (PRMT5i) in HTLV-1-infected lymphocytes resulted in increased viral gene expression and decreased cellular proliferation. PRMT5i also had selective toxicity in HTLV-1-transformed T-cells. Finally, we demonstrated that PRMT5 and the HTLV-1 p30 protein had an additive inhibitory effect on HTLV-1 gene expression. Our study provides evidence for PRMT5 as a host cell factor important in HTLV-1-mediated T-cell transformation, and a potential target for ATLL treatment.

  6. PRMT5 Is Upregulated in HTLV-1-Mediated T-Cell Transformation and Selective Inhibition Alters Viral Gene Expression and Infected Cell Survival

    Directory of Open Access Journals (Sweden)

    Amanda R. Panfil

    2015-12-01

    Full Text Available Human T-cell leukemia virus type-1 (HTLV-1 is a tumorigenic retrovirus responsible for development of adult T-cell leukemia/lymphoma (ATLL. This disease manifests after a long clinical latency period of up to 2–3 decades. Two viral gene products, Tax and HBZ, have transforming properties and play a role in the pathogenic process. Genetic and epigenetic cellular changes also occur in HTLV-1-infected cells, which contribute to transformation and disease development. However, the role of cellular factors in transformation is not completely understood. Herein, we examined the role of protein arginine methyltransferase 5 (PRMT5 on HTLV-1-mediated cellular transformation and viral gene expression. We found PRMT5 expression was upregulated during HTLV-1-mediated T-cell transformation, as well as in established lymphocytic leukemia/lymphoma cell lines and ATLL patient PBMCs. shRNA-mediated reduction in PRMT5 protein levels or its inhibition by a small molecule inhibitor (PRMT5i in HTLV-1-infected lymphocytes resulted in increased viral gene expression and decreased cellular proliferation. PRMT5i also had selective toxicity in HTLV-1-transformed T-cells. Finally, we demonstrated that PRMT5 and the HTLV-1 p30 protein had an additive inhibitory effect on HTLV-1 gene expression. Our study provides evidence for PRMT5 as a host cell factor important in HTLV-1-mediated T-cell transformation, and a potential target for ATLL treatment.

  7. HIV-1 infection ex vivo accelerates measles virus infection by upregulating signaling lymphocytic activation molecule (SLAM) in CD4+ T cells.

    Science.gov (United States)

    Mitsuki, Yu-ya; Terahara, Kazutaka; Shibusawa, Kentaro; Yamamoto, Takuya; Tsuchiya, Takatsugu; Mizukoshi, Fuminori; Ishige, Masayuki; Okada, Seiji; Kobayashi, Kazuo; Morikawa, Yuko; Nakayama, Tetsuo; Takeda, Makoto; Yanagi, Yusuke; Tsunetsugu-Yokota, Yasuko

    2012-07-01

    Measles virus (MV) infection in children harboring human immunodeficiency virus type 1 (HIV-1) is often fatal, even in the presence of neutralizing antibodies; however, the underlying mechanisms are unclear. Therefore, the aim of the present study was to examine the interaction between HIV-1 and wild-type MV (MVwt) or an MV vaccine strain (MVvac) during dual infection. The results showed that the frequencies of MVwt- and MVvac-infected CD4(+) T cells within the resting peripheral blood mononuclear cells (PBMCs) were increased 3- to 4-fold after HIV-1 infection, and this was associated with a marked upregulation of signaling lymphocytic activation molecule (SLAM) expression on CD4(+) T cells but not on CD8(+) T cells. SLAM upregulation was induced by infection with a replication-competent HIV-1 isolate comprising both the X4 and R5 types and to a lesser extent by a pseudotyped HIV-1 infection. Notably, SLAM upregulation was observed in HIV-infected as well as -uninfected CD4(+) T cells and was abrogated by the removal of HLA-DR(+) cells from the PBMC culture. Furthermore, SLAM upregulation did not occur in uninfected PBMCs cultured together with HIV-infected PBMCs in compartments separated by a permeable membrane, indicating that no soluble factors were involved. Rather, CD4(+) T cell activation mediated through direct contact with dendritic cells via leukocyte function-associated molecule 1 (LFA-1)/intercellular adhesion molecule 1 (ICAM-1) and LFA-3/CD2 was critical. Thus, HIV-1 infection induces a high level of SLAM expression on CD4(+) T cells, which may enhance their susceptibility to MV and exacerbate measles in coinfected individuals.

  8. Exosomes from hypoxic endothelial cells have increased collagen crosslinking activity through up-regulation of lysyl oxidase-like 2.

    Science.gov (United States)

    de Jong, Olivier G; van Balkom, Bas W M; Gremmels, Hendrik; Verhaar, Marianne C

    2016-02-01

    Exosomes are important mediators of intercellular communication. Additionally, they contain a variety of components capable of interacting with the extracellular matrix (ECM), including integrins, matrix metalloproteinases and members of the immunoglobin superfamily. Despite these observations, research on exosome-ECM interactions is limited. Here, we investigate whether the exosome-associated lysyl oxidase family member lysyl oxidase-like 2 (LOXL2) is involved in ECM remodelling. We found that LOXL2 is present on the exterior of endothelial cell (EC)-derived exosomes, placing it in direct vicinity of the ECM. It is up-regulated twofold in EC-derived exosomes cultured under hypoxic conditions. Intact exosomes from hypoxic EC and LOXL2 overexpressing EC show increased activity in a fluorometric lysyl oxidase enzymatic activity assay as well as in a collagen gel contraction assay. Concordantly, knockdown of LOXL2 in exosome-producing EC in both normal and hypoxic conditions reduces activity of exosomes in both assays. Our findings show for the first time that ECM crosslinking by EC-derived exosomes is mediated by LOXL2 under the regulation of hypoxia, and implicate a role for exosomes in hypoxia-regulated focal ECM remodelling, a key process in both fibrosis and wound healing.

  9. Melanoma Development and Progression Are Associated with Rad6 Upregulation and β-Catenin Relocation to the Cell Membrane

    Directory of Open Access Journals (Sweden)

    Karli Rosner

    2014-01-01

    Full Text Available We have previously demonstrated that Rad6 and β-catenin enhance each other's expression through a positive feedback loop to promote breast cancer development/progression. While β-catenin has been implicated in melanoma pathogenesis, Rad6 function has not been investigated. Here, we examined the relationship between Rad6 and β-catenin in melanoma development and progression. Eighty-eight cutaneous tumors, 30 nevi, 29 primary melanoma, and 29 metastatic melanomas, were immunostained with anti-β-catenin and anti-Rad6 antibodies. Strong expression of Rad6 was observed in only 27% of nevi as compared to 100% of primary and 96% of metastatic melanomas. β-Catenin was strongly expressed in 97% of primary and 93% of metastatic melanomas, and unlike Rad6, in 93% of nevi. None of the tumors expressed nuclear β-catenin. β-Catenin was exclusively localized on the cell membrane of 55% of primary, 62% of metastatic melanomas, and only 10% of nevi. Cytoplasmic β-catenin was detected in 90% of nevi, 17% of primary, and 8% of metastatic melanoma, whereas 28% of primary and 30% of metastatic melanomas exhibited β-catenin at both locations. These data suggest that melanoma development and progression are associated with Rad6 upregulation and membranous redistribution of β-catenin and that β-catenin and Rad6 play independent roles in melanoma development.

  10. Synergistic effect of interleukin 1 alpha on nontypeable Haemophilus influenzae-induced up-regulation of human beta-defensin 2 in middle ear epithelial cells

    Directory of Open Access Journals (Sweden)

    Park Raekil

    2006-01-01

    Full Text Available Abstract Background We recently showed that beta-defensins have antimicrobial activity against nontypeable Haemophilus influenzae (NTHi and that interleukin 1 alpha (IL-1 alpha up-regulates the transcription of beta-defensin 2 (DEFB4 according to new nomenclature of the Human Genome Organization in human middle ear epithelial cells via a Src-dependent Raf-MEK1/2-ERK signaling pathway. Based on these observations, we investigated if human middle ear epithelial cells could release IL-1 alpha upon exposure to a lysate of NTHi and if this cytokine could have a synergistic effect on beta-defensin 2 up-regulation by the bacterial components. Methods The studies described herein were carried out using epithelial cell lines as well as a murine model of acute otitis media (OM. Human cytokine macroarray analysis was performed to detect the released cytokines in response to NTHi exposure. Real time quantitative PCR was done to compare the induction of IL-1 alpha or beta-defensin 2 mRNAs and to identify the signaling pathways involved. Direct activation of the beta-defensin 2 promoter was monitored using a beta-defensin 2 promoter-Luciferase construct. An IL-1 alpha blocking antibody was used to demonstrate the direct involvement of this cytokine on DEFB4 induction. Results Middle ear epithelial cells released IL-1 alpha when stimulated by NTHi components and this cytokine acted in an autocrine/paracrine synergistic manner with NTHi to up-regulate beta-defensin 2. This synergistic effect of IL-1 alpha on NTHi-induced beta-defensin 2 up-regulation appeared to be mediated by the p38 MAP kinase pathway. Conclusion We demonstrate that IL-1 alpha is secreted by middle ear epithelial cells upon exposure to NTHi components and that it can synergistically act with certain of these molecules to up-regulate beta-defensin 2 via the p38 MAP kinase pathway.

  11. Ursolic acid attenuates diabetic mesangial cell injury through the up-regulation of autophagy via miRNA-21/PTEN/Akt/mTOR suppression.

    Directory of Open Access Journals (Sweden)

    Xinxing Lu

    Full Text Available To investigate the effect of ursolic acid on autophagy mediated through the miRNA-21-targeted phosphoinositide 3 kinase (PI3K/protein kinase B (Akt/mammalian target of rapamycin (mTOR pathway in rat mesangial cells cultured under high glucose (HG conditions.Rat glomerular mesangial cells were cultured under normal glucose, HG, HG with the PI3K inhibitor LY294002 or HG with ursolic acid conditions. Cell proliferation and hypertrophy were assayed using an MTT assay and the ratio of total protein to cell number, respectively. The miRNA-21 expression was detected using RT-qPCR. The expression of phosphatase and tensin homolog (PTEN/AKT/mTOR signaling signatures, autophagy-associated protein and collagen I was detected by western blotting and RT-qPCR. Autophagosomes were observed using electron microscopy.Compared with mesangial cells cultured under normal glucose conditions, the cells exposed to HG showed up-regulated miRNA-21 expression, down-regulated PTEN protein and mRNA expression, up-regulated p85PI3K, pAkt, pmTOR, p62/SQSTMI, and collagen I expression and down-regulated LC3II expression. Ursolic acid and LY294002 inhibited HG-induced mesangial cell hypertrophy and proliferation, down-regulated p85PI3K, pAkt, pmTOR, p62/SQSTMI, and collagen I expression and up-regulated LC3II expression. However, LY294002 did not affect the expression of miRNA-21 and PTEN. Ursolic acid down-regulated miRNA-21 expression and up-regulated PTEN protein and mRNA expression.Ursolic acid inhibits the glucose-induced up-regulation of mesangial cell miRNA-21 expression, up-regulates PTEN expression, inhibits the activation of PI3K/Akt/mTOR signaling pathway, and enhances autophagy to reduce the accumulation of the extracellular matrix and ameliorate cell hypertrophy and proliferation.

  12. Stress hormone epinephrine enhances adipogenesis in murine embryonic stem cells by up-regulating the neuropeptide Y system.

    Directory of Open Access Journals (Sweden)

    Ruijun Han

    Full Text Available Prenatal stress, psychologically and metabolically, increases the risk of obesity and diabetes in the progeny. However, the mechanisms of the pathogenesis remain unknown. In adult mice, stress activates NPY and its Y2R in a glucocorticoid-dependent manner in the abdominal fat. This increased adipogenesis and angiogenesis, leading to abdominal obesity and metabolic syndrome which were inhibited by intra-fat Y2R inactivation. To determine whether stress elevates NPY system and accelerates adipogenic potential of embryo, here we "stressed" murine embryonic stem cells (mESCs in vitro with epinephrine (EPI during their adipogenic differentiation. EPI was added during the commitment stage together with insulin, and followed by dexamethasone in the standard adipogenic differentiation medium. Undifferentiated embryonic bodies (EBs showed no detectable expression of NPY. EPI markedly up-regulated the expression NPY and the Y1R at the commitment stage, followed by increased Y2R mRNA at the late of the commitment stage and the differentiation stage. EPI significantly increased EB cells proliferation and expression of the preadipocyte marker Pref-1 at the commitment stage. EPI also accelerated and amplified adipogenic differentiation detected by increasing the adipocyte markers FABP4 and PPARγ mRNAs and Oil-red O-staining at the end of the differentiation stage. EPI-induced adipogenesis was completely prevented by antagonists of the NPY receptors (Y1R+Y2R+Y5R, indicating that it was mediated by the NPY system in mESC's. Taken together, these data suggest that stress may play an important role in programming ESCs for accelerated adipogenesis by altering the stress induced hormonal regulation of the NPY system.

  13. IL-6 upregulation contributes to the reduction of miR-26a expression in hepatocellular carcinoma cells

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Yafei [People' s Liberation Army, Department of Oncology, Wuhan General Hospital of Guangzhou Command, Wuhan (China); Third Military Medical University, Department of Gastroenterology, Southwest Hospital, Chongqing (China); Zhang, Bicheng [People' s Liberation Army, Department of Oncology, Wuhan General Hospital of Guangzhou Command, Wuhan (China); Zhang, Anran [Third Military Medical University, Department of Gastroenterology, Southwest Hospital, Chongqing (China); Li, Xiaohua [Fourth Military Medical University, State Key Laboratory of Cancer Biology and Xijing Hospital of Digestive Diseases, Xi' an (China); Liu, Jian; Zhao, Jie; Zhao, Yong; Gao, Jianfei [People' s Liberation Army, Department of Oncology, Wuhan General Hospital of Guangzhou Command, Wuhan (China); Fang, Dianchun [Third Military Medical University, Department of Gastroenterology, Southwest Hospital, Chongqing (China); Rao, Zhiguo [People' s Liberation Army, Department of Oncology, Wuhan General Hospital of Guangzhou Command, Wuhan (China)

    2012-09-28

    A recent study showed that miR-26a is downregulated in hepatocellular carcinoma tissues and that this downregulation is an independent predictor of survival. Interestingly, the same study also reported that miR-26a downregulation causes a concomitant elevation of IL-6 expression. Because miR-26a expression was found to be transcriptionally downregulated by oncogene c-Myc in various cancers, and the expression of c-Myc was increased by IL-6 stimulation, we hypothesized that IL-6 contributes to reduction of miR-26a in hepatocellular carcinoma. Serum IL-6 was measured by ELISA and miR-26a was detected by qRT-PCR. The data of 30 patients with hepatocellular carcinoma who had undergone surgical tumor resection revealed that serum IL-6 could be considered to be a predictor of survival up to 5 years for hepatocellular carcinoma patients (log-rank test, P < 0.05). We observed that the serum IL-6 concentration was inversely correlated with miR-26a expression in cancerous tissues (Pearson correlation test, r = -0.651, P < 0.01). Furthermore, by in vitro experiments with HepG2 cells, we showed that IL-6 stimulation can lead to miR-26a suppression via c-Myc activation, whereas in normal hepatocyte LO2 cells incubation with IL-6 had no significant effect on miR-26a expression. Taken together, these results indicate that miR-26a reduction in hepatocellular carcinoma might be due to IL-6 upregulation.

  14. Repetitive magnetic stimulation promotes neural stem cells proliferation by upregulating MiR-106b in vitro.

    Science.gov (United States)

    Liu, Hua; Han, Xiao-hua; Chen, Hong; Zheng, Cai-xia; Yang, Yi; Huang, Xiao-lin

    2015-10-01

    Neural stem cells (NSCs) proliferation can be influenced by repetitive transcranial magnetic stimulation (rTMS) in vivo via microRNA-106b-25 cluster, but the underlying mechanisms are poorly understood. This study investigated the involvement of microRNA-106b-25 cluster in the proliferation of NSCs after repetitive magnetic stimulation (rMS) in vitro. NSCs were stimulated by rMS (200/400/600/800/1000 pulses per day, with 10 Hz frequency and 50% maximum machine output) over a 3-day period. NSCs proliferation was detected by using ki-67 and EdU staining. Ki-67, p21, p57, cyclinD1, cyclinE, cyclinA, cdk2, cdk4 proteins and miR-106b, miR-93, miR-25 mRNAs were detected by Western blotting and qRT-PCR, respectively. The results showed that rMS could promote NSCs proliferation in a dose-dependent manner. The proportions of ki-67+ and Edu+ cells in 1000 pulses group were 20.65% and 4.00%, respectively, significantly higher than those in control group (9.25%, 2.05%). The expression levels of miR-106b and miR-93 were significantly upregulated in 600-1000 pulses groups compared with control group (Pp21 protein were decreased significantly in 800/1000 pulses groups, and those of cyclinD1, cyclinA, cyclinE, cdk2 and cdk4 were obviously increased after rMS as compared with control group (Pp21/cdks/cyclins pathway was involved in the process.

  15. Up-regulation of Ras/Raf/ERK1/2 signaling in the spinal cord impairs neural cell migration, neurogenesis, synapse formation, and dendritic spine development

    Institute of Scientific and Technical Information of China (English)

    CAO Fu-jiang; ZHANG Xu; LIU Tao; LI Xia-wen; Mazar Malik; FENG Shi-qing

    2013-01-01

    Background The Ras/Raf/ERK1/2 signaling pathway controls many cellular responses such as cell proliferation,migration,differentiation,and death.In the nervous system,emerging evidence also points to a death-promoting role for ERK1/2 in both in vitro and in vivo models of neuronal death.To further investigate how Ras/Raf/ERK1/2 up-regulation may lead to the development of spinal cord injury,we developed a cellular model of Raf/ERK up-regulation by overexpressing c-Raf in cultured spinal cord neurons (SCNs) and dorsal root ganglions (DRGs).Methods DRGs and SCNs were prepared from C57BL/6J mouse pups.DRGs or SCNs were infected with Ad-Raf-1 or Ad-Null adenovirus alone.Cell adhesion assay and cell migration assay were investigated,Dil labeling was employed to examine the effect of the up-regulation of Ras/Raf/ERK1/2 signaling on the dendritic formation of spinal neurons.We used the TO-PRO-3 staining to examine the apoptotic effect of c-Raf on DRGs or SCNs.The effect on the synapse formation of neurons was measured by using immunofluorescence.Results We found that Raf/ERK up-regulation stimulates the migration of both SCNs and DRGs,and impairs the formation of excitatory synapses in SCNs.In addition,we found that Raf/ERK up-regulation inhibits the development of mature dendritic spines in SCNs.Investigating the possible mechanisms through which Raf/ERK up-regulation affects the excitatory synapse formation and dendritic spine development,we discovered that Raf/ERK up-regulation suppresses the development and maturation of SCNs.Conclusion The up-regulation of the Raf/ERK signaling pathway may contribute to the pathogenesis of spinal cord injury through both its impairment of the SCN development and causing neural circuit imbalances.

  16. Up-regulation of JAM-1 in AR42J cells treated with activin A and betacellulin and the diabetic regenerating islets.

    Science.gov (United States)

    Yoshikumi, Yukako; Ohno, Hideki; Suzuki, Junko; Isshiki, Masashi; Morishita, Yasuyuki; Ohnishi, Hirohide; Yasuda, Hiroshi; Omata, Masao; Fujita, Toshiro; Mashima, Hirosato

    2008-08-01

    Pancreatic AR42J cells demonstrate the pluripotency in precursor cells of the gut endoderm and also provide an excellent model system to study the differentiation of the pancreas. Using the mRNA differential display technique, we identified junctional adhesion molecule-1 (JAM-1), a component of the tight junction, was highly up-regulated during the differentiation of AR42J cells, although junctions were not formed. The expression level of JAM-1 showed an up-regulation in the mRNA level after 3 hours and in the protein level after 24 hours in [activin A + betacellulin]-treated AR42J cells. The expressions of its signaling molecules, PAR-3 and atypical PKC lambda, also increased after the addition of activin A + betacellulin. When JAM-1 was over-expressed in [activin A + betacellulin]-treated AR42J cells, tagged-JAM-1 was observed in cytoplasm as vesicular structures and JAM-1 was colocalized with Rab3B and Rab13, members of the Rab family expressed at tight junctions. In streptozotocin-induced regenerating islets, the expression of JAM-1 was also up-regulated in the mRNA level and the protein level. JAM-1 might therefore play an important role in the differentiation of AR42J cells and the regeneration of pancreatic islets.

  17. Coordinated Upregulation of Mitochondrial Biogenesis and Autophagy in Breast Cancer Cells: The Role of Dynamin Related Protein-1 and Implication for Breast Cancer Treatment

    Science.gov (United States)

    Zou, Peng; Liu, Longhua; Zheng, Louise D.; Payne, Kyle K.; Idowu, Michael O.; Zhang, Jinfeng; Schmelz, Eva M.

    2016-01-01

    Overactive mitochondrial fission was shown to promote cell transformation and tumor growth. It remains elusive how mitochondrial quality is regulated in such conditions. Here, we show that upregulation of mitochondrial fission protein, dynamin related protein-1 (Drp1), was accompanied with increased mitochondrial biogenesis markers (PGC1α, NRF1, and Tfam) in breast cancer cells. However, mitochondrial number was reduced, which was associated with lower mitochondrial oxidative capacity in breast cancer cells. This contrast might be owing to enhanced mitochondrial turnover through autophagy, because an increased population of autophagic vacuoles engulfing mitochondria was observed in the cancer cells. Consistently, BNIP3 (a mitochondrial autophagy marker) and autophagic flux were significantly upregulated, indicative of augmented mitochondrial autophagy (mitophagy). The upregulation of Drp1 and BNIP3 was also observed in vivo (human breast carcinomas). Importantly, inhibition of Drp1 significantly suppressed mitochondrial autophagy, metabolic reprogramming, and cancer cell viability. Together, this study reveals coordinated increase of mitochondrial biogenesis and mitophagy in which Drp1 plays a central role regulating breast cancer cell metabolism and survival. Given the emerging evidence of PGC1α contributing to tumor growth, it will be of critical importance to target both mitochondrial biogenesis and mitophagy for effective cancer therapeutics. PMID:27746856

  18. Coordinated Upregulation of Mitochondrial Biogenesis and Autophagy in Breast Cancer Cells: The Role of Dynamin Related Protein-1 and Implication for Breast Cancer Treatment

    Directory of Open Access Journals (Sweden)

    Peng Zou

    2016-01-01

    Full Text Available Overactive mitochondrial fission was shown to promote cell transformation and tumor growth. It remains elusive how mitochondrial quality is regulated in such conditions. Here, we show that upregulation of mitochondrial fission protein, dynamin related protein-1 (Drp1, was accompanied with increased mitochondrial biogenesis markers (PGC1α, NRF1, and Tfam in breast cancer cells. However, mitochondrial number was reduced, which was associated with lower mitochondrial oxidative capacity in breast cancer cells. This contrast might be owing to enhanced mitochondrial turnover through autophagy, because an increased population of autophagic vacuoles engulfing mitochondria was observed in the cancer cells. Consistently, BNIP3 (a mitochondrial autophagy marker and autophagic flux were significantly upregulated, indicative of augmented mitochondrial autophagy (mitophagy. The upregulation of Drp1 and BNIP3 was also observed in vivo (human breast carcinomas. Importantly, inhibition of Drp1 significantly suppressed mitochondrial autophagy, metabolic reprogramming, and cancer cell viability. Together, this study reveals coordinated increase of mitochondrial biogenesis and mitophagy in which Drp1 plays a central role regulating breast cancer cell metabolism and survival. Given the emerging evidence of PGC1α contributing to tumor growth, it will be of critical importance to target both mitochondrial biogenesis and mitophagy for effective cancer therapeutics.

  19. Hispolon from Phellinus linteus induces apoptosis and sensitizes human cancer cells to the tumor necrosis factor-related apoptosis-inducing ligand through upregulation of death receptors.

    Science.gov (United States)

    Kim, Ji-Hun; Kim, Yu Chul; Park, Byoungduck

    2016-02-01

    The tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a potent anticancer agent possessing the ability to induce apoptosis in various cancer cells but not in non‑malignant cells. However, certain type of cancer cells are resistant to TRAIL‑induced apoptosis and some acquire resistance after the first treatment. So development of an agent that can reduce or avoid resistance in TRAIL‑induced apoptosis has garnered significant attention. The present study evaluated the anticancer potential of hispolon in TRAIL‑induced apoptosis and indicated hispolon can sensitize cancer cells to TRAIL. As the mechanism of action was examined, hispolon was found to activate caspase‑3, caspase‑8 and caspase‑9, while downregulating the expression of cell survival proteins such as cFLIP, Bcl‑2 and Bcl‑xL and upregulating the expression of Bax and truncated Bid. We also found hispolon induced death receptors in a non‑cell type‑specific manner. Upregulation of death receptors by hispolon was found to be p53-independent but linked to the induction of CAAT enhancer binding protein homologous protein (CHOP). Overall, hispolon was demonstrated to potentiate the apoptotic effects of TRAIL through downregulation of anti‑apoptotic proteins and upregulation of death receptors linked with CHOP and pERK elevation.

  20. Propofol inhibits lung cancer cell viability and induces cell apoptosis by upregulating microRNA-486 expression

    Science.gov (United States)

    Yang, N.; Liang, Y.; Yang, P.; Yang, T.; Jiang, L.

    2017-01-01

    Propofol is a frequently used intravenous anesthetic agent. Recent studies show that propofol exerts a number of non-anesthetic effects. The present study aimed to investigate the effects of propofol on lung cancer cell lines H1299 and H1792 and functional role of microRNA (miR)-486 in these effects. H1299 and/or H1792 cells were treated with or without propofol and transfected or not with miR-486 inhibitor, and then cell viability and apoptosis were analyzed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and flow cytometry. The expression of miR-486 was determined by quantitative real-time polymerase chain reaction (qRT-PCR) with or without propofol treatment. Western blot was performed to analyze the protein expression of Forkhead box, class O (FOXO) 1 and 3, Bcl-2 interacting mediator of cell death (Bim), and pro- and activated caspases-3. Results showed that propofol significantly increased the miR-486 levels in both H1299 and H1792 cells compared to untreated cells in a dose-dependent manner (PBim, and pro- and activated caspases-3; however, miR-486 inhibitor reversed the effects of propofol on cell viability, apoptosis, and protein expression (P<0.05 or P<0.01). In conclusion, propofol might be an ideal anesthetic for lung cancer surgery by effectively inhibiting lung cancer cell viability and inducing cell apoptosis. Modulation of miR-486 might contribute to the anti-tumor activity of propofol. PMID:28076456

  1. Estrogen-Mediated Upregulation of Noxa Is Associated with Cell Cycle Progression in Estrogen Receptor-Positive Breast Cancer Cells

    OpenAIRE

    2011-01-01

    Noxa is a Bcl-2-homology domain (BH3)-only protein reported to be a proapoptotic member of the Bcl-2 family. Estrogen has been well documented to stimulate cell growth and inhibit apoptosis in estrogen receptor (ER)-positive breast cancer cells. Intriguingly, recent reports have shown that 17β-estradiol (E2) induces Noxa expression, although the mechanisms underlying E2-mediated induction of Noxa and its functional significance are unknown. Using MCF7 human breast cancer cells as an experimen...

  2. IFN-γ upregulates survivin and Ifi202 expression to induce survival and proliferation of tumor-specific T cells.

    Directory of Open Access Journals (Sweden)

    Mary Zimmerman

    role of the IFN-γ signaling pathway in regulating T cell proliferation and apoptosis through upregulating survivin and ifi202 expression.

  3. Release of tensile strain on engineered human tendon tissue disturbs cell adhesions, changes matrix architecture, and induces an inflammatory phenotype

    DEFF Research Database (Denmark)

    Bayer, Monika L; Schjerling, Peter; Herchenhan, Andreas

    2014-01-01

    -inflammatory mediators and tendon phenotypic specific molecules, in an in vitro model where tendon-like tissue was engineered from human tendon cells. Tissue sampling was performed 1, 2, 4 and 6 days after surgical de-tensioning of the tendon construct. When tensile stimulus was removed, integrin type collagen receptors...... were upregulated. Stimulation with the cytokine TGF-β1 had distinct effects on some tendon-related genes in both tensioned and de-tensioned tissue. These findings indicate an important role of mechanical loading for cellular and matrix responses in tendon, including that loss of tension leads...

  4. Up-Regulation of P21 Inhibits TRAIL-Mediated Extrinsic Apoptosis, Contributing Resistance to SAHA in Acute Myeloid Leukemia Cells

    Directory of Open Access Journals (Sweden)

    Xing Wu

    2014-08-01

    Full Text Available Background/Aim: P21, a multifunctional cell cycle-regulatory molecule, regulates apoptotic cell death. In this study we examined the effect of altered p21 expression on the sensitivity of acute myeloid leukemia cells in response to HDAC inhibitor SAHA treatment and investigated the underlying mechanism. Methods: Stably transfected HL60 cell lines were established in RPMI-1640 with supplementation of G-418. Cell viability was measured by MTT assay. Western blot was applied to assess the protein expression levels of target genes. Cell apoptosis was monitored by AnnexinV-PE/7AAD assay. Results: We showed HL60 cells that that didn't up-regulate p21 expression were more sensitive to SAHA-mediated apoptosis than NB4 and U937 cells that had increased p21 level. Enforced expression of p21 in HL60 cells reduced sensitivity to SAHA and blocked TRAIL-mediated apoptosis. Conversely, p21 silencing in NB4 cells enhanced SAHA-mediated apoptosis and lethality. Finally, we found that combined treatment with SAHA and rapamycin down-regulated p21 and enhanced apoptosis in AML cells. Conclusion: We conclude that up-regulated p21 expression mediates resistance to SAHA via inhibition of TRAIL apoptotic pathway. P21 may serve as a candidate biomarker to predict responsiveness or resistance to SAHA-based therapy in AML patients. In addition, rapamycin may be an effective agent to override p21-mediated resistance to SAHA in AML patients.

  5. AS1411-Induced Growth Inhibition of Glioma Cells by Up-Regulation of p53 and Down-Regulation of Bcl-2 and Akt1 via Nucleolin

    OpenAIRE

    Cheng, Ye; Zhao, Gang; Zhang, Siwen; Nigim, Fares; Zhou, Guangtong; Yu, Zhiyun; Song, Yang; Chen, Yong; Li, Yunqian

    2016-01-01

    AS1411 binds nucleolin (NCL) and is the first oligodeoxynucleotide aptamer to reach phase I and II clinical trials for the treatment of several cancers. However, the mechanisms by which AS1411 targets and kills glioma cells and tissues remain unclear. Here we report that AS1411 induces cell apoptosis and cycle arrest, and inhibits cell viability by up-regulation of p53 and down-regulation of Bcl-2 and Akt1 in human glioma cells. NCL was overexpressed in both nucleus and cytoplasm in human gli...

  6. Up-regulation of NKX3.1 Expression and Inhibition of LNCaP Cell Proliferation Induced by an Inhibitory Element Decoy

    Institute of Scientific and Technical Information of China (English)

    An-Li JIANG; Xiao-Yan HU; Peng-Ju ZHANG; Mei-Lan HE; Feng KONG; Zhi-Fang LIU; Hui-Qing YUAN; Jian-Ye ZHANG

    2005-01-01

    NKX3.1 is an androgen-regulated prostate-specific homeobox gene that is thought to play an important role in prostate development and cancerogenesis. NKX3.1 acts as a tumor suppressor gene specifically in the prostate. Up-regulation of NKX3.1 gene offers a promising gene therapy for prostate cancer. The decoy strategy has been developed and is considered a useful tool for regulating gene expression and gene therapy. In our previous studies, we identified a 20 bp inhibitory element upstream of the NKX3.1 promoter.In this study, we focused on using the 20 bp inhibitory element decoy to block negative regulation of the NKX3.1 gene and to up-regulate NKX3.1 expression using synthetic double-stranded oligodeoxynucleotides of the 20 bp inhibitory element. We found in an electrophoretic mobility shift assay experiment that the 20 bp inhibitory decoy presented competitive binding to a specific binding protein of the 20 bp inhibitory element in prostate cancer cell line LNCaP. In luciferase reporter gene assays, we found that the 20 bp inhibitory decoy could enhance NKX3.1 promoter activity, and RT-PCR and Western blot analysis revealed that NKX3.1expression was up-regulated effectively by the transfection with the 20 bp inhibitory decoy. Furthermore,cell proliferation was inhibited by up-regulated NKX3.1 expression induced by the 20 bp inhibitory decoy.

  7. Epstein-Barr Virus nuclear antigen 1 (EBNA1) confers resistance to apoptosis in EBV-positive B-lymphoma cells through up-regulation of survivin.

    Science.gov (United States)

    Lu, Jie; Murakami, Masanao; Verma, Subhash C; Cai, Qiliang; Haldar, Sabyasachi; Kaul, Rajeev; Wasik, Mariusz A; Middeldorp, Jaap; Robertson, Erle S

    2011-02-05

    Resistance to apoptosis is an important component of the overall mechanism which drives the tumorigenic process. EBV is a ubiquitous human gamma-herpesvirus which preferentially establishes latent infection in viral infected B-lymphocytes. EBNA1 is typically expressed in most forms of EBV-positive malignancies and is important for replication of the latent episome in concert with replication of the host cells. Here, we investigate the effects of EBNA1 on survivin up-regulation in EBV-infected human B-lymphoma cells. We present evidence which demonstrates that EBNA1 forms a complex with Sp1 or Sp1-like proteins bound to their cis-element at the survivin promoter. This enhances the activity of the complex and up-regulates survivin. Knockdown of survivin and EBNA1 showed enhanced apoptosis in infected cells and thus supports a role for EBNA1 in suppressing apoptosis in EBV-infected cells. Here, we suggest that EBV encoded EBNA1 can contribute to the oncogenic process by up-regulating the apoptosis suppressor protein, survivin in EBV-associated B-lymphoma cells.

  8. Mechanism of sphingosine 1-phosphate- and lysophosphatidic Acid-induced up-regulation of adhesion molecules and eosinophil chemoattractant in nerve cells.

    LENUS (Irish Health Repository)

    Costello, Richard W

    2012-02-01

    The lysophospholipids sphingosine 1-phosphate (S1P) and lysophosphatidic acid (LPA) act via G-protein coupled receptors S1P(1-5) and LPA(1-3) respectively, and are implicated in allergy. Eosinophils accumulate at innervating cholinergic nerves in asthma and adhere to nerve cells via intercellular adhesion molecule-1 (ICAM-1). IMR-32 neuroblastoma cells were used as an in vitro cholinergic nerve cell model. The G(i) coupled receptors S1P(1), S1P(3), LPA(1), LPA(2) and LPA(3) were expressed on IMR-32 cells. Both S1P and LPA induced ERK phosphorylation and ERK- and G(i)-dependent up-regulation of ICAM-1 expression, with differing time courses. LPA also induced ERK- and G(i)-dependent up-regulation of the eosinophil chemoattractant, CCL-26. The eosinophil granule protein eosinophil peroxidase (EPO) induced ERK-dependent up-regulation of transcription of S1P(1), LPA(1), LPA(2) and LPA(3), providing the situation whereby eosinophil granule proteins may enhance S1P- and\\/or LPA- induced eosinophil accumulation at nerve cells in allergic conditions.

  9. Mechanism of sphingosine 1-phosphate- and lysophosphatidic Acid-induced up-regulation of adhesion molecules and eosinophil chemoattractant in nerve cells.

    LENUS (Irish Health Repository)

    Costello, Richard W

    2011-05-01

    The lysophospholipids sphingosine 1-phosphate (S1P) and lysophosphatidic acid (LPA) act via G-protein coupled receptors S1P(1-5) and LPA(1-3) respectively, and are implicated in allergy. Eosinophils accumulate at innervating cholinergic nerves in asthma and adhere to nerve cells via intercellular adhesion molecule-1 (ICAM-1). IMR-32 neuroblastoma cells were used as an in vitro cholinergic nerve cell model. The G(i) coupled receptors S1P(1), S1P(3), LPA(1), LPA(2) and LPA(3) were expressed on IMR-32 cells. Both S1P and LPA induced ERK phosphorylation and ERK- and G(i)-dependent up-regulation of ICAM-1 expression, with differing time courses. LPA also induced ERK- and G(i)-dependent up-regulation of the eosinophil chemoattractant, CCL-26. The eosinophil granule protein eosinophil peroxidase (EPO) induced ERK-dependent up-regulation of transcription of S1P(1), LPA(1), LPA(2) and LPA(3), providing the situation whereby eosinophil granule proteins may enhance S1P- and\\/or LPA- induced eosinophil accumulation at nerve cells in allergic conditions.

  10. CIAPIN1 confers multidrug resistance through up-regulation of MDR-1 and Bcl-L in LoVo/Adr cells and is independent of p53.

    Science.gov (United States)

    Zhang, Ya-Fei; Li, Xiao-Hua; Shi, Yong-Quan; Wu, Yu-Yun; Li, Ning; He, Qiang; Ji, Qing; Wang, Rong-Quan; Yang, Shi-Ming; Fang, Dian-Chun

    2011-04-01

    Recent investigations discovered that CIAPIN1 might be another drug resistance-associated molecule in cancer cells. However, the underlying mechanisms of CIAPIN1-related multidrug resistance (MDR) remain elusive. In the present study, we investigated the role and possible mechanisms of CIAPIN1 in MDR of human colon carcinoma LoVo/Adr cells which express the wild-type p53 gene. By using small interference RNA and gene transfection techniques, we found that knockdown of CIAPIN1 expression re-sensitized LoVo/Adr cells to anti-cancer drugs and up-regulation of CIAPIN1 in sensitive LoVo cells resulted in a distinct MDR phenotype. We further revealed that CIAPIN1 conferred the MDR phenotype in LoVo/Adr cells through up-regulating expression of MDR-1 (P-gp) and Bcl-xL. Finally, by analyzing the effect of inactivation of wild-type p53 on CIAPIN1-induced up-regulation of P-gp and Bcl-xL, we determined that CIAPIN1 could exhibit its MDR-related function independently of the p53 signaling pathway. Overall, the results presented here further suggest that over-expression of CIAPIN1 is an important mechanism of drug resistance in human cancers, even if not the sole one.

  11. Estrogen receptor-related receptor alpha mediates up-regulation of aromatase expression by prostaglandin E2 in prostate stromal cells.

    Science.gov (United States)

    Miao, Lin; Shi, Jiandang; Wang, Chun-Yu; Zhu, Yan; Du, Xiaoling; Jiao, Hongli; Mo, Zengnan; Klocker, Helmut; Lee, Chung; Zhang, Ju

    2010-06-01

    Estrogen receptor-related receptor alpha (ERRalpha) is an orphan member of the nuclear receptor superfamily of transcription factors. ERRalpha is highly expressed in the prostate, especially in prostate stromal cells. However, little is known about the regulation and function of ERRalpha, which may contribute to the progression of prostatic diseases. We previously found that prostaglandin E2 (PGE2) up-regulated the expression of aromatase in prostate stromal cells. Here we show that PGE2 also up-regulates the expression of ERRalpha, which, as a transcription factor, further mediates the regulatory effects of PGE2 on the expression of aromatase. ERRalpha expression was up-regulated by PGE2 in prostate stromal cell line WPMY-1, which was mediated mainly through the protein kinase A signaling pathway by PGE2 receptor EP2. Suppression of ERRalpha activity by chlordane (an antagonist of ERRalpha) or small interfering RNA knockdown of ERRalpha blocked the increase of expression and promoter activity of aromatase induced by PGE2. Overexpression of ERRalpha significantly increased aromatase expression and promoter activity, which were further augmented by PGE2. Chromatin immunoprecipitation assay demonstrated that ERRalpha directly bound to the aromatase promoter in vivo, and PGE2 enhanced the recruitment of ERRalpha and promoted transcriptional regulatory effects on aromatase expression in WPMY-1. 17Beta-estradiol concentration in WPMY-1 medium was up-regulated by ERRalpha expression, and that was further increased by PGE2. Our results provided evidence that ERRalpha contributed to local estrogen production by up-regulating aromatase expression in response to PGE2 and provided further insights into the potential role of ERRalpha in estrogen-related prostatic diseases.

  12. Myocardial injection of apelin-overexpressing bone marrow cells improves cardiac repair via upregulation of Sirt3 after myocardial infarction.

    Directory of Open Access Journals (Sweden)

    Lanfang Li

    Full Text Available Our previous study shows that treatment with apelin increases bone marrow cells (BMCs recruitment and promotes cardiac repair after myocardial infarction (MI. The objective of this study was to investigate whether overexpression of apelin in BMCs improved cell therapy and accelerated cardiac repair and functional recovery in post-MI mice. Mouse myocardial infarction was achieved by coronary artery ligation and BMCs overexpressing apelin (apelin-BMCs or GFP (GFP-BMCs were injected into ischemic area immediately after surgery. In vitro, exposure of cultured BMCs to apelin led to a gradual increase in SDF-1á and CXCR4 expression. Intramyocardial delivery of apelin-BMCs in post-MI mice resulted in a significant increase number of APJ⁺/c-kit⁺/Sca1⁺ cells in the injected area compared to GFP-BMCs treated post-MI mice. Treatment with apelin-BMCs increased expression of VEGF, Ang-1 and Tie-2 in post-MI mice. Apelin-BMCs treatment also significantly increased angiogenesis and attenuated cardiac fibrosis formation in post-MI mice. Most importantly, treatment with apelin-BMCs significantly improved left ventricular (LV systolic function in post-MI mice. Mechanistically, Apelin-BMCs treatment led to a significant increase in Sirtuin3 (Sirt3 expression and reduction of reactive oxygen species (ROS formation. Treatment of cultured BMCs with apelin also increased Notch3 expression and Akt phosphorylation. Apelin treatment further attenuated stress-induced apoptosis whereas knockout of Sirt3 abolished anti-apoptotic effect of apelin in cultured BMCs. Moreover, knockout of Sirt3 significantly attenuated apelin-BMCs-induced VEGF expression and angiogenesis in post-MI mice. Knockout of Sirt3 further blunted apelin-BMCs-mediated improvement of cardiac repair and systolic functional recovery in post-MI mice. These data suggest that apelin improves BMCs therapy on cardiac repair and systolic function in post-MI mice. Upregulation of Sirt3 may contribute to the

  13. AS1411-Induced Growth Inhibition of Glioma Cells by Up-Regulation of p53 and Down-Regulation of Bcl-2 and Akt1 via Nucleolin.

    Science.gov (United States)

    Cheng, Ye; Zhao, Gang; Zhang, Siwen; Nigim, Fares; Zhou, Guangtong; Yu, Zhiyun; Song, Yang; Chen, Yong; Li, Yunqian

    2016-01-01

    AS1411 binds nucleolin (NCL) and is the first oligodeoxynucleotide aptamer to reach phase I and II clinical trials for the treatment of several cancers. However, the mechanisms by which AS1411 targets and kills glioma cells and tissues remain unclear. Here we report that AS1411 induces cell apoptosis and cycle arrest, and inhibits cell viability by up-regulation of p53 and down-regulation of Bcl-2 and Akt1 in human glioma cells. NCL was overexpressed in both nucleus and cytoplasm in human glioma U87, U251 and SHG44 cells compared to normal human astrocytes (NHA). AS1411 bound NCL and inhibited the proliferation of glioma cells but not NHA, which was accompanied with up-regulation of p53 and down-regulation of Bcl-2 and Akt1. Moreover, AS1411 treatment resulted in the G2/M cell cycle arrest in glioma cells, which was however abolished by overexpression of NCL. Further, AS1411 induced cell apoptosis, which was prevented by silencing of p53 and overexpression of Bcl-2. In addition, AS1411 inhibited the migration and invasion of glioma cells in an Akt1-dependent manner. Importantly, AS1411 inhibited the growth of glioma xenograft and prolonged the survival time of glioma tumor-bearing mice. These results revealed a promising treatment of glioma by oligodeoxynucleotide aptamer.

  14. Transforming growth factor-β stimulates human ovarian cancer cell migration by up-regulating connexin43 expression via Smad2/3 signaling.

    Science.gov (United States)

    Qiu, Xin; Cheng, Jung-Chien; Zhao, Jianfang; Chang, Hsun-Ming; Leung, Peter C K

    2015-10-01

    Reduced connexin43 (Cx43) expression is frequently detected in different types of human cancer. Cx43 has been shown to regulate cancer cell migration in a cell-type dependent manner. In both primary and recurrent human ovarian cancer, overexpression of TGF-β ligand and its receptors have been detected. TGF-β can regulate Cx43 expression in other cell types and stimulate human ovarian cancer cell migration. However, whether Cx43 can be regulated by TGF-β and is involved in TGF-β-stimulated cell migration in human ovarian cancer cells remain unknown. In this study, we demonstrate that TGF-β up-regulates Cx43 in two human ovarian cancer cell lines, SKOV3 and OVCAR4. The stimulatory effect of TGF-β on Cx43 expression is blocked by inhibition of TGF-β receptor. Treatment with TGF-β activates Smad2 and Smad3 signaling pathways in both ovarian cancer cell lines. In addition, siRNA-mediated knockdown of Smad2 or Smd3 abolishes TGF-β-induced up-regulation of Cx43 expression. Moreover, knockdown of Cx43 attenuates TGF-β-stimulated cell migration. This study demonstrates an important role for Cx43 in mediating the effects of TGF-β on human ovarian cancer cell migration.

  15. Platelet rich plasma promotes skeletal muscle cell migration in association with up-regulation of FAK, paxillin, and F-Actin formation.

    Science.gov (United States)

    Tsai, Wen-Chung; Yu, Tung-Yang; Lin, Li-Ping; Lin, Mioa-Sui; Tsai, Ting-Ta; Pang, Jong-Hwei S

    2017-02-24

    Platelet rich plasma (PRP) contains various cytokines and growth factors which may be beneficial to the healing process of injured muscle. The aim of this study was to investigate the effect and molecular mechanism of PRP on migration of skeletal muscle cells. Skeletal muscle cells intrinsic to Sprague-Dawley rats were treated with PRP. The cell migration was evaluated by transwell filter migration assay and electric cell-substrate impedance sensing. The spreading of cells was evaluated microscopically. The formation of filamentous actin (F-actin) cytoskeleton was assessed by immunofluorescence staining. The protein expressions of paxillin and focal adhesion kinase (FAK) were assessed by Western blot analysis. Transfection of paxillin small-interfering RNA (siRNAs) to muscle cells was performed to validate the role of paxillin in PRP-mediated promotion of cell migration. Dose-dependently PRP promotes migration of and spreading and muscle cells. Protein expressions of paxillin and FAK were up-regulated dose-dependently. F-actin formation was also enhanced by PRP treatment. Furthermore, the knockdown of paxillin expression impaired the effect of PRP to promote cell migration. It was concluded that PRP promoting migration of muscle cells is associated with up-regulation of proteins expression of paxillin and FAK as well as increasing F-actin formation. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res.

  16. Up-regulation of TRPV1 in mononuclear cells of end-stage kidney disease patients increases susceptibility to N-arachidonoyl-dopamine (NADA)-induced cell death.

    Science.gov (United States)

    Saunders, Cassandra I; Fassett, Robert G; Geraghty, Dominic P

    2009-10-01

    Transient receptor potential vanilloid (TRPV) 1 channels function as sensors for a variety of noxious and inflammatory signals, including capsaicin, heat and protons, and are up-regulated under inflammatory conditions. As end-stage kidney disease (ESKD) is associated with chronic inflammation, impaired immunity and depressed lymphocyte numbers, we sought to determine whether altered TRPV1 (and related TRPV2) expression in immune cells might be a contributing factor. TRPV1 and TRPV2 mRNA expression in peripheral blood mononuclear cells (PBMC) was similar in controls and ESKD patients by quantitative real-time RT-PCR. However, using immunocytochemistry, TRPV1-immunoreactivity was significantly higher and TRPV2-immunoreactivity was significantly lower in PBMC from ESKD patients compared to controls. The plant-derived TRPV1 agonists, capsaicin and resiniferatoxin (RTX) and the putative endovanilloid/endocannabinoids, N-arachidonoyl-dopamine (NADA) and N-oleoyl-dopamine (OLDA), induced concentration-dependent death of PBMC from healthy donors with a rank order of potency of RTX>NADA>OLDA>capsaicin. TRPV1 (5'-iodoresiniferatoxin) and cannabinoid (CB2; AM630) receptor antagonists blocked the cytotoxic effect of NADA. In subsequent experiments, PBMC from ESKD patients exhibited significantly increased susceptibility to NADA-induced death compared to PBMC from controls. The apparent up-regulation of TRPV1 may be a response to the inflammatory milieu in which PBMC exist in ESKD and may be responsible for the increased susceptibility of these cells to NADA-induced death, providing a possible explanation as to why ESKD patients have reduced lymphocyte counts and impaired immune function. Thus, TRPV1 (and possibly CB2) antagonists may have potential for the treatment of immune dysfunction in ESKD.

  17. CCL21/CCR7 up-regulate vascular endothelial growth factor-D expression via ERK pathway in human non-small cell lung cancer cells.

    Science.gov (United States)

    Sun, Limei; Zhang, Qingfu; Li, Yang; Tang, Na; Qiu, Xueshan

    2015-01-01

    Lymphangiogenesis has received considerable attention and become a new research hotspot of tumor metastasis. Recently, C-C chemokine receptor 7 (CCR7) is known to promote metastasis of non-small cell lung cancer (NSCLC) cells into lymph nodes. In this study, we investigated the relationship between CCL21/CCR7 and the lymphangiogenic factor vascular endothelial growth factor (VEGF)-D in human lung cancer cells and its impact on patients' prognosis. We found that CCL21/CCR7 increase the expression of VEGF-D in NSCLC Cell Lines through induced ERK1/2 and Akt phosphorylation. In addition, our study found that the expression levels of CCR7 and CCL21 were correlated with VEGF-D, lymphatic vessels density (LVD), clinical stages, lymph node metastasis, and patient Survival in 90 human non-small cell lung cancer (NSCLC) specimens. Taken together, our results provide evidence that CCL21/CCR7 induce VEGF-D up-regulation and promote lymphangiogenesis via ERK/Akt pathway in lung cancer.

  18. Irradiation-induced up-regulation of HLA-E on macrovascular endothelial cells confers protection against killing by activated natural killer cells.

    Directory of Open Access Journals (Sweden)

    Isabelle Riederer

    -induced, transient up-regulation of HLA-E on macrovascular ECs might confer protection against NK cell-mediated vascular injury.

  19. Transcription factor Ets-1 inhibits glucose-stimulated insulin secretion of pancreatic β-cells partly through up-regulation of COX-2 gene expression.

    Science.gov (United States)

    Zhang, Xiong-Fei; Zhu, Yi; Liang, Wen-Biao; Zhang, Jing-Jing

    2014-08-01

    Increased cyclooxygenase-2 (COX-2) expression is associated with pancreatic β-cell dysfunction. We previously demonstrated that the transcription factor Ets-1 significantly up-regulated COX-2 gene promoter activity. In this report, we used the pancreatic β-cell line INS-1 and isolated rat islets to investigate whether Ets-1 could induce β-cell dysfunction through up-regulating COX-2 gene expression. We investigated the effects of ETS-1 overexpression and the effects of ETS-1 RNA interference on endogenous COX-2 expression in INS-1 cells. We used site-directed mutagenesis and a dual luciferase reporter assay to study putative Ets-1 binding sites in the COX-2 promoter. The effect of ETS-1 1 overexpression on the insulin secretion function of INS-1 cells and rat islets and the potential reversal of these effects by a COX-2 inhibitor were determined in a glucose-stimulated insulin secretion (GSIS) assay. ETS-1 overexpression significantly induces endogenous COX-2 expression, but ETS-1 RNA interference has no effect on basal COX-2 expression in INS-1 cells. Ets-1 protein significantly increases COX-2 promoter activity through the binding site located in the -195/-186 region of the COX-2 promoter. ETS-1 overexpression significantly inhibited the GSIS function of INS-1 cells and islet cells and COX-2 inhibitor treatment partly reversed this effect. These findings indicated that ETS-1 overexpression induces β-cell dysfunction partly through up-regulation of COX-2 gene expression. Moreover, Ets-1, the transcriptional regulator of COX-2 expression, may be a potential target for the prevention of β-cell dysfunction mediated by COX-2.

  20. Expression of SCM-1alpha/lymphotactin and SCM-1beta in natural killer cells is upregulated by IL-2 and IL-12.

    Science.gov (United States)

    Hennemann, B; Tam, Y K; Tonn, T; Klingemann, H G

    1999-07-01

    Recruitment of lymphocytes is an important feature of the host immune response against pathogens. However, the mechanisms by which lymphocytes are attracted are not yet fully understood. Recently, the cDNA of a lymphocyte-specific chemokine, lymphotactin (Lptn), was isolated from murine and human T cells and was also found to be expressed in murine NK cells and human NK cell clones. This study investigated the influence of interleukin (IL)-2 and IL-12 on the expression of Lptn, also known as SCM (single cysteine motif)-1alpha, and SCM-1beta, a 97% homolog of Lptn, in freshly isolated human NK cells and the human NK cell line NK-92. Northern blot analysis and RT-PCR confirmed that nonactivated human NK cells expressed both genes at low level. After activation with IL-2 or IL-12, the expression of both Lptn and SCM-1beta was upregulated within hours. NK-92 cells maintained in medium supplemented with IL-2 constitutively expressed SCM-1 mRNA. However, after 24 h of IL-2 starvation and subsequent culturing at various IL-2 concentrations, the expression of Lptn/SCM-1alpha was upregulated in a dose-dependent manner, whereas the expression of SCM-1beta remained consistently high. These observations indicate that NK cells, in addition to T lymphocytes, express Lptn/SCM-1alpha and SCM-1beta after cytokine activation. The upregulation of these chemokines in NK cells on activation likely acts to increase the number of effector cells reaching the site of an immune response such as inflammation.

  1. Angiotensin II upregulates the expression of placental growth factor in human vascular endothelial cells and smooth muscle cells

    Directory of Open Access Journals (Sweden)

    Guo Yingqiang

    2010-05-01

    Full Text Available Abstract Background Atherosclerosis is now recognized as a chronic inflammatory disease. Angiotensin II (Ang II is a critical factor in inflammatory responses, which promotes the pathogenesis of atherosclerosis. Placental growth factor (PlGF is a member of the vascular endothelial growth factor (VEGF family cytokines and is associated with inflammatory progress of atherosclerosis. However, the potential link between PlGF and Ang II has not been investigated. In the current study, whether Ang II could regulate PlGF expression, and the effect of PlGF on cell proliferation, was investigated in human vascular endothelial cells (VECs and smooth muscle cells (VSMCs. Results In growth-arrested human VECs and VSMCs, Ang II induced PlGF mRNA expression after 4 hour treatment, and peaked at 24 hours. 10-6 mol/L Ang II increased PlGF protein production after 8 hour treatment, and peaked at 24 hours. Stimulation with Ang II also induced mRNA expression of VEGF receptor-1 and -2(VEGFR-1 and -2 in these cells. The Ang II type I receptor (AT1R antagonist blocked Ang II-induced PlGF gene expression and protein production. Several intracellular signals elicited by Ang II were involved in PlGF synthesis, including activation of protein kinase C, extracellular signal-regulated kinase 1/2 (ERK1/2 and PI3-kinase. A neutralizing antibody against PlGF partially inhibited the Ang II-induced proliferation of VECs and VSMCs. However, this antibody showed little effect on the basal proliferation in these cells, whereas blocking antibody of VEGF could suppress both basal and Ang II-induced proliferation in VECs and VSMCs. Conclusion Our results showed for the first time that Ang II could induce the gene expression and protein production of PlGF in VECs and VSMCs, which might play an important role in the pathogenesis of vascular inflammation and atherosclerosis.

  2. Transcriptional activation and cell cycle block are the keys for 5-fluorouracil induced up-regulation of human thymidylate synthase expression.

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    Alessio Ligabue

    Full Text Available BACKGROUND: 5-fluorouracil, a commonly used chemotherapeutic agent, up-regulates expression of human thymidylate synthase (hTS. Several different regulatory mechanisms have been proposed to mediate this up-regulation in distinct cell lines, but their specific contributions in a single cell line have not been investigated to date. We have established the relative contributions of these previously proposed regulatory mechanisms in the ovarian cancer cell line 2008 and the corresponding cisplatin-resistant and 5-FU cross-resistant-subline C13*. METHODOLOGY/PRINCIPAL FINDINGS: Using RNA polymerase II inhibitor DRB treated cell cultures, we showed that 70-80% of up-regulation of hTS results from transcriptional activation of TYMS mRNA. Moreover, we report that 5-FU compromises the cell cycle by blocking the 2008 and C13* cell lines in the S phase. As previous work has established that TYMS mRNA is synthesized in the S and G(1 phase and hTS is localized in the nuclei during S and G(2-M phase, the observed cell cycle changes are also expected to affect the intracellular regulation of hTS. Our data also suggest that the inhibition of the catalytic activity of hTS and the up-regulation of the hTS protein level are not causally linked, as the inactivated ternary complex, formed by hTS, deoxyuridine monophosphate and methylenetetrahydrofolate, was detected already 3 hours after 5-FU exposure, whereas substantial increase in global TS levels was detected only after 24 hours. CONCLUSIONS/SIGNIFICANCE: Altogether, our data indicate that constitutive TYMS mRNA transcription, cell cycle-induced hTS regulation and hTS enzyme stability are the three key mechanisms responsible for 5-fluorouracil induced up-regulation of human thymidylate synthase expression in the two ovarian cancer cell lines studied. As these three independent regulatory phenomena occur in a precise order, our work provides a feasible rationale for earlier observed synergistic combinations of 5

  3. Paracetamol-induced spindle disturbances in V79 cells with and without expression of human CYP1A2

    DEFF Research Database (Denmark)

    Jensen, K G; Poulsen, H E; Doehmer, J

    1996-01-01

    Spindle disturbing effects in terms of c-mitosis and cytotoxicity of paracetamol were investigated in two Chinese hamster V79 cell lines, one of which (V79MZh1A2) was transfected with human CYP1A2. This enzyme catalyses the oxidative formation of the reactive paracetamol metabolite, NAPQI, believed...... to initiate hepatoxicity by covalent binding to proteins after overdose. In the native V79 cell line paracetamol increased c-mitosis frequency in a concentration dependent manner from 8.7 + or - 3.5% (control) to 66 + or - 18% at 20 mM. A significant increase to 13.3 + or - 3.5% was first seen at 2.5 m......M in the native cell line (Pparacetamol. At 5 mM paracetamol the c-mitosis frequency was 14.4 + or - 5.0% and 19.0 + or - 3...

  4. Hepatitis C virus sensitizes host cells to TRAIL-induced apoptosis by up-regulating DR4 and DR5 via a MEK1-dependent pathway.

    Directory of Open Access Journals (Sweden)

    Zhongfan Deng

    Full Text Available BACKGROUND: Hepatitis C virus (HCV is the leading cause of liver fibrosis, cirrhosis and hepatocellular carcinoma. It is believed that continuous liver cell apoptosis contributes to HCV pathogenesis. Recent studies have shown that HCV infection can sensitize host cells to TNF-related apoptosis-inducing ligand (TRAIL induced apoptosis, but the mechanism by which HCV regulates the TRAIL pathway remains unclear. METHODS AND RESULTS: Using a sub-genomic replicon and full length virus, JFH-1, we demonstrate that HCV can sensitize host cells to TRAIL-induced apoptosis by up-regulating two TRAIL receptors, death receptor 4 (DR4 and death receptor 5 (DR5. Furthermore, the HCV replicon enhanced transcription of DR5 via Sp1, and the HCV-mediated up-regulation of DR4 and DR5 required MEK1 activity. HCV infection also stimulated the activity of MEK1, and the inhibition of MEK1 activity or the knockdown of MEK1 increased the replication of HCV. CONCLUSIONS: Our studies demonstrate that HCV replication sensitizes host cells to TRAIL-induced apoptosis by up-regulating DR4 and DR5 via a MEK1 dependent pathway. These findings may help to further understand the pathogenesis of HCV infection and provide a therapeutic target.

  5. NFATC1 promotes cell growth and tumorigenesis in ovarian cancer up-regulating c-Myc through ERK1/2/p38 MAPK signal pathway.

    Science.gov (United States)

    Xu, Wenwen; Gu, Junjie; Ren, Qingling; Shi, Yanqiu; Xia, Qinhua; Wang, Jing; Wang, Suli; Wang, Yingchun; Wang, Jinhua

    2016-04-01

    It has been reported that nuclear factor of activated T cells (NFATC1) was up-regulated in cancers mediating malignant behaviors. However, the role of NFATC1 in ovarian cancer has not been elucidated. In the present study, we undertook to explore the clinicopathological significance of NFATC1 expression and the mechanism by which NFATC1 works in ovarian cancer. Expression status of NFATC1 was examined using immunohistochemistry. Both knockdown and re-expression of NFATC1 on ovarian cancer cells were employed to observe the effect overgrowth. It was found that NFATC1 was significantly overexpressed in ovarian cancer tissues in comparison with paired normal control tissues and that overexpression of NFATC1 was significantly associated with metastasis and poor prognosis on clinical tissue level. In in vitro ovarian cancer cell lines, we found that NFATC1 can promote proliferation up-regulating c-myc through activation of ERK1/2/p38/MAPK signal pathway. Together, the results we obtained demonstrated that NFATC1 played oncogenic role in ovarian cancer. Mechanistically, NFATC1 promoted growth of ovarian cancer cells up-regulating c-myc through activation of ERK1/2/p38/MAPK signal pathway, suggesting that NFATC1 might be used as a therapeutic target for ovarian cancer.

  6. The transcription elongation factor ELL2 is specifically upregulated in HTLV-1-infected T-cells and is dependent on the viral oncoprotein Tax.

    Science.gov (United States)

    Mann, Melanie C; Strobel, Sarah; Fleckenstein, Bernhard; Kress, Andrea K

    2014-09-01

    The oncoprotein Tax of human T-cell leukemia virus type 1 (HTLV-1) is a potent transactivator of viral and cellular transcription. Here, we identified ELL2 as the sole transcription elongation factor to be specifically upregulated in HTLV-1-/Tax-transformed T-cells. Tax contributes to regulation of ELL2, since transient transfection of Tax increases ELL2 mRNA, Tax transactivates the ELL2 promoter, and repression of Tax results in decrease of ELL2 in transformed T-lymphocytes. However, we also measured upregulation of ELL2 in HTLV-1-transformed cells exhibiting undetectable amounts of Tax, suggesting that ELL2 can still be maintained independent of continuous Tax expression. We further show that Tax and ELL2 synergistically activate the HTLV-1 promoter, indicating that ELL2 cooperates with Tax in viral transactivation. This is supported by our findings that Tax and ELL2 accumulate in nuclear fractions and that they co-precipitate upon co-expression in transiently-transfected cells. Thus, upregulation of ELL2 could contribute to HTLV-1 gene regulation.

  7. The shh signaling pathway is upregulated in multiple cell types in cortical ischemia and influences the outcome of stroke in an animal model.

    Science.gov (United States)

    Jin, Yongmin; Raviv, Nataly; Barnett, Austin; Bambakidis, Nicholas C; Filichia, Emily; Luo, Yu

    2015-01-01

    Recently the sonic hedgehog (shh) signaling pathway has been shown to play an important role in regulating repair and regenerative responses after brain injury, including ischemia. However, the precise cellular components that express and upregulate the shh gene and the cellular components that respond to shh signaling remain to be identified. In this study, using a distal MCA occlusion model, our data show that the shh signal is upregulated both at the cortical area near the injury site and in the adjacent striatum. Multiple cell types upregulate shh signaling in ischemic brain, including neurons, reactive astrocytes and nestin-expressing cells. The shh signaling pathway genes are also expressed in the neural stem cells (NSCs) niche in the subventricular zone (SVZ). Conditional deletion of the shh gene in nestin-expressing cells both at the SVZ niche and at the ischemic site lead to significantly more severe behavioral deficits in these shh iKO mice after cortical stroke, measured using an automated open field locomotion apparatus (Student's t-test, pshh signaling agonist (SAG) demonstrated less deficits in behavioral function, compared to vehicle-treated mice. At 7 days after stroke, SAG-treated mice showed higher values in multiple horizontal movement parameters compared to vehicle treated mice (Student's t-test, p0.05). In summary, our data demonstrate that shh signaling plays critical and ongoing roles in response to ischemic injury and modulation of shh signaling in vivo alters the functional outcome after cortical ischemic injury.

  8. Rapamycin ameliorates experimental autoimmune uveoretinitis by inhibiting Th1/Th2/Th17 cells and upregulating CD4+CD25+ Foxp3 regulatory T cells

    Institute of Scientific and Technical Information of China (English)

    Li-Fei; Yuan; Guang-Da; Li; Xin-Jun; Ren; Hong; Nian; Xiao-Rong; Li; Xiao-Min; Zhang

    2015-01-01

    · AIM: To determine the effects of rapamycin on experimental autoimmune uveoretinitis(EAU) and investigate of role of rapamycin on T cell subsets in the disease.·METHODS: EAU was induced in rats using peptides1169 to 1191 of the interphotoreceptor binding protein(IRBP). Rapamycin(0.2 mg/kg/d) was administrated by intraperitoneal injection for a consecutive 7d after immunization. Th1/Th2/Th17 cytokines, TGF-β1, and IL-6produced by lymphocyteswere measured by ELISA, while Th17 cells and CD4 +CD25 + regulatory T cells(Tregs)from rat spleen were detected by flow cytometry.·RESULTS: Intraperitoneal treatment immediately after immunization dramatically ameliorated the clinical course of EAU. Clinical responses were associated with reduced retinal inflammatory cell infiltration and tissue destruction. Rapamycin induced suppression of Th1/Th2/Th17 cytokines, including IFN-γ, IL-2, IL-17, IL-4, and IL-10 release from T lymphocytes of EAU rats, in vitro.Rapamycin also significantly increased TGF-β1production but had no effect on IL-6 productionof T lymphocytes from EAU rats in vitro. Furthermore,rapamycin decreased the ratio of Th17 cells/CD4 +T cells and upregulated Tregs in EAU, as detected by flow cytometry.·CONCLUSION: Rapamycin effectively interferes with T cell mediated autoimmune uveitis by inhibiting antigen-specific T cell functions and enhancing Tregs in EAU.Rapamycin is a promising new alternative as an adjunct corticosteroid-sparing agent for treating uveitis.

  9. Upregulation of ATG3 contributes to autophagy induced by the detachment of intestinal epithelial cells from the extracellular matrix, but promotes autophagy-independent apoptosis of the attached cells.

    Science.gov (United States)

    Yoo, Byong Hoon; Zagryazhskaya, Anna; Li, Yongling; Koomson, Ananda; Khan, Iman Aftab; Sasazuki, Takehiko; Shirasawa, Senji; Rosen, Kirill V

    2015-01-01

    Detachment of nonmalignant intestinal epithelial cells from the extracellular matrix (ECM) triggers their growth arrest and, ultimately, apoptosis. In contrast, colorectal cancer cells can grow without attachment to the ECM. This ability is critical for their malignant potential. We found previously that detachment-induced growth arrest of nonmalignant intestinal epithelial cells is driven by their detachment-triggered autophagy, and that RAS, a major oncogene, promotes growth of detached cells by blocking such autophagy. In an effort to identify the mechanisms of detachment-induced autophagy and growth arrest of nonmalignant cells we found here that detachment of these cells causes upregulation of ATG3 and that ATG3 upregulation contributes to autophagy and growth arrest of detached cells. We also observed that when ATG3 expression is artificially increased in the attached cells, ATG3 promotes neither autophagy nor growth arrest but triggers their apoptosis. ATG3 upregulation likely promotes autophagy of the detached but not that of the attached cells because detachment-dependent autophagy requires other detachment-induced events, such as the upregulation of ATG7. We further observed that those few adherent cells that do not die by apoptosis induced by ATG3 become resistant to apoptosis caused by cell detachment, a property that is critical for the ability of normal epithelial cells to become malignant. We conclude that cell-ECM adhesion can switch ATG3 functions: when upregulated in detached cells in the context of other autophagy-promoting events, ATG3 contributes to autophagy. However, when overexpressed in the adherent cells, in the circumstances not favoring autophagy, ATG3 triggers apoptosis.

  10. Immune-associated proteins with potential in vivo anti-tumor activities are upregulated in lung cancer cells treated with umbelliprenin: A proteomic approach

    Science.gov (United States)

    Khaghanzadeh, Narges; Nakamura, Kazuyuki; Kuramitsu, Yasuhiro; Ghaderi, Abbas; Mojtahedi, Zahra

    2016-01-01

    Umbelliprenin (Umb), a natural coumarin, has demonstrated anti-tumor activities, both in vitro and particularly in vivo, in several types of cancer, including lung cancer. The present study aimed to identify molecular targets of Umb using a high-throughput approach. Lung cancer cell lines, QU-DB (large-cell lung carcinoma) and A549 (adenocarcinoma), were treated with Umb. Differentially-expressed proteins were identified using two-dimensional electrophoresis coupled to mass spectrometry. In the QU-DB cells, differential expression of proteins, including downregulation of the tumorigenic protein heat shock protein 90 kDa and upregulation of the potential anti-tumor proteins Nipsnap1 and glycine-tRNA ligase (GRS), suggested that Umb is a strong anti-tumor compound. In the A549 cells, differential expression of proteins indicated possible contradictory effects of Umbregarding tumorigenesis, which included downregulation of the tumorigenic protein cyclophilin and the tumor suppressor MST, and upregulation of stathmin (tumorigenic) and calreticulin. Calreticulun, in addition to GRS in QU-DB cells, stimulates anti-tumor immune responses in vivo. To the best of our knowledge, the present study is the first to use a high-throughput approach to identify targets of Umb in cancer. These molecular targets suggested that Umb may exhibit stronger in vitro anti-tumor activity against the large-cell carcinoma model than the adenocarcinoma model. Furthermore, it has been reported that Umb exhibits higher cytotoxicity against QU-DB cells than A549 cells in vitro, and significant Umb anti-tumor activity against lung cancer in vivo, which is consistent with previously published literature. In each cell type, immune-associated molecules were upregulated, indicating that this naturally occurring compound exhibits marked anti-tumor activity in vivo. However, further studies that investigate the effect of Umb in different in vitro models of cancer are required. PMID:28105238

  11. Casticin induces caspase-mediated apoptosis via activation of mitochondrial pathway and upregulation of DR5 in human lung cancer cells

    Institute of Scientific and Technical Information of China (English)

    Yuan Zhou; Mei-Fang Quan; Fei Liu; Su-Fang Zhou; Yong-Xiang Zhao; Yi Peng; Qi-Qi Mao; Xia Li; Ming-Wu Chen; Jing Su; Li Tian; Nai-Quan Mao; Ling-Zhi Long

    2013-01-01

    Objective: To assess if casticin induces caspase-mediated apoptosis via activation of mitochondrial pathway and upregulation of DR5 in human lung cancer cells. Methods: Human non-small-cell lung carcinoma cell lines H460, A549 and H157 were cultured in vitro. The cytotoxic activities were determined using MTT assay. The apoptotic cells death was examined by flow cytometry using PI staining and DNA agarose gel electrophoresis. The activities of caspase-3,-8 and -9 were measured via ELISA. Cellular fractionation was determined by flow cytometry to assess release of cytochrome c and the mitochondrial transmembrane potential. Bcl-2/Bcl-XL/XIAP/Bid/ DR5 and DR4 proteins were analyzed using western blot. Results: The concentrations required for a 50% decrease in cell growth (IC50) ranged from 1.8 to 3.2 μM. Casticin induced rapid apoptosis and triggered a series of effects associated with apoptosis by way of mitochondrial pathway, including the depolarization of the mitochondrial membrane, release of cytochrome c from mitochondria, activation of procaspase-9 and -3, and increase of DNA fragments. Moreover, the pan caspase inhibitor zVAD-FMK and the caspase-3 inhibitor zDEVD-FMK suppressed casticin-induced apoptosis. In addition, casticin induced XIAP and Bcl-XL down-regulation, Bax upregulation and Bid clearage. In H157 cell line, casticin increased expression of DR5 at protein levels but not affect the expression of DR4. The pretreatment with DR5/Fc chimera protein effectively attenuated casticin-induced apoptosis in H157 cells. No correlation was found between cell sensitivity to casticin and that to p53 status, suggesting that casticin induce a p53-independent apoptosis. Conclusions: Our results demonstrate that casticin induces caspase-mediated apoptosis via activation of mitochondrial pathway and upregulation of DR5 in human lung cancer cells.

  12. Korean Red Ginseng and Ginsenoside-Rb1/-Rg1 Alleviate Experimental Autoimmune Encephalomyelitis by Suppressing Th1 and Th17 Cells and Upregulating Regulatory T Cells.

    Science.gov (United States)

    Lee, Min Jung; Jang, Minhee; Choi, Jonghee; Chang, Byung Soo; Kim, Do Young; Kim, Sung-Hoon; Kwak, Yi-Seong; Oh, Seikwan; Lee, Jong-Hwan; Chang, Byung-Joon; Nah, Seung-Yeol; Cho, Ik-Hyun

    2016-04-01

    The effects of Korean red ginseng extract (KRGE) on autoimmune disorders of the nervous system are not clear. We investigated whether KRGE has a beneficial effect on acute and chronic experimental autoimmune encephalomyelitis (EAE). Pretreatment (daily from 10 days before immunization with myelin basic protein peptide) with KRGE significantly attenuated clinical signs and loss of body weight and was associated with the suppression of spinal demyelination and glial activation in acute EAE rats, while onset treatment (daily after the appearance of clinical symptoms) did not. The suppressive effect of KRGE corresponded to the messenger RNA (mRNA) expression of proinflammatory cytokines (tumor necrosis factor-α [TNF-α] and interleukin [IL]-1β), chemokines (RANTES, monocyte chemotactic protein-1 [MCP-1], and macrophage inflammatory protein-1α [MIP-1α]), adhesion molecules (intercellular adhesion molecule-1 [ICAM-1], vascular cell adhesion molecule-1 [VCAM-1], and platelet endothelial cell adhesion molecule [PECAM-1]), and inducible nitric oxide synthase in the spinal cord after immunization. Interestingly, in acute EAE rats, pretreatment with KRGE significantly reduced the population of CD4(+), CD4(+)/IFN-γ(+), and CD4(+)/IL-17(+) T cells in the spinal cord and lymph nodes, corresponding to the downregulation of mRNA expression of IFN-γ, IL-17, and IL-23 in the spinal cord. On the other hand, KRGE pretreatment increased the population of CD4(+)/Foxp3(+) T cells in the spinal cord and lymph nodes of these rats, corresponding to the upregulation of mRNA expression of Foxp3 in the spinal cord. Interestingly, intrathecal pretreatment of rats with ginsenosides (Rg1 and Rb1) significantly decreased behavioral impairment. These results strongly indicate that KRGE has a beneficial effect on the development and progression of EAE by suppressing T helper 1 (Th1) and Th17 T cells and upregulating regulatory T cells. Additionally, pre- and onset treatment with KRGE

  13. Emotional Disturbance

    Science.gov (United States)

    ... skills, and increase self-awareness, self-control, and self-esteem. A large body of research exists regarding methods ... not. Back to top Other Considerations Children and adolescents with an emotional disturbance should receive services based ...

  14. Effects of Arg-Gly-Asp-modified elastin-like polypeptide on pseudoislet formation via up-regulation of cell adhesion molecules and extracellular matrix proteins.

    Science.gov (United States)

    Lee, Kyeong-Min; Jung, Gwon-Soo; Park, Jin-Kyu; Choi, Seong-Kyoon; Jeon, Won Bae

    2013-03-01

    Extracellular matrix (ECM) plays an important role in controlling the β-cell morphology, survival and insulin secretary functions. An RGD-modified elastin-like polypeptide (RGD-ELP), TGPG[VGRGD(VGVPG)(6)](20)WPC, has been reported previously as a bioactive matrix. In this study, to investigate whether RGD-ELP affects β-cell growth characteristics and insulin secretion, β-TC6 cells were cultured on the RGD-ELP coatings prepared via thermally induced phase transition. On RGD-ELP, β-TC6 cells clustered into an islet-like architecture with high cell viability. Throughout 7days' culture, the proliferation rate of the cells within a pseudoislet was similar to that of monolayer culture. Under high glucose (25mM), β-TC6 pseudoislets showed up-regulated insulin gene expression and exhibited glucose-stimulated insulin secretion. Importantly, the mRNA and protein abundances of cell adhesion molecules (CAM) E-cadherin and connexin-36 were much higher in pseudoislets than in monolayer cells. The siRNA-mediated inhibition of E-cadherin or connexin-36 expression severely limited pseudoislet formation. In addition, the mRNA levels of collagen types I and IV, fibronectin and laminin were significantly elevated in pseudoislets. The results suggest that RGD-ELP promotes pseudoislet formation via up-regulation of the CAM and ECM components. The functional roles of RGD-ELP are discussed in respect of its molecular composition.

  15. Coordinate up-regulation of low-density lipoprotein receptor and cyclo-oxygenase-2 gene expression in human colorectal cells and in colorectal adenocarcinoma biopsies

    Science.gov (United States)

    Lum, D. F.; McQuaid, K. R.; Gilbertson, V. L.; Hughes-Fulford, M.

    1999-01-01

    Many colorectal cancers have high levels of cyclo-oxygenase 2 (COX-2), an enzyme that metabolizes the essential fatty acids into prostaglandins. Since the low-density lipoprotein receptor (LDLr) is involved in the uptake of essential fatty acids, we studied the effect of LDL on growth and gene regulation in colorectal cancer cells. DiFi cells grown in lipoprotein-deficient sera (LPDS) grew more slowly than cells with LDL. LDLr antibody caused significant inhibition of tumor cell growth but did not affect controls. In addition, LDL uptake did not change in the presence of excess LDL, suggesting that ldlr mRNA lacks normal feedback regulation in some colorectal cancers. Analysis of the ldlr mRNA showed that excess LDL in the medium did not cause down-regulation of the message even after 24 hr. The second portion of the study examined the mRNA expression of ldlr and its co-regulation with cox-2 in normal and tumor specimens from patients with colorectal adenocarcinomas. The ratio of tumor:paired normal mucosa of mRNA expression of ldlr and of cox-2 was measured in specimens taken during colonoscopy. ldlr and cox-2 transcripts were apparent in 11 of 11 carcinomas. There was significant coordinate up-regulation both of ldlr and of cox-2 in 6 of 11 (55%) tumors compared with normal colonic mucosa. There was no up-regulation of cox-2 without concomitant up-regulation of ldlr. These data suggest that the LDLr is abnormally regulated in some colorectal tumors and may play a role in the up-regulation of cox-2. Copyright 1999 Wiley-Liss, Inc.

  16. ERβ-dependent neuroglobin up-regulation impairs 17β-estradiol-induced apoptosis in DLD-1 colon cancer cells upon oxidative stress injury.

    Science.gov (United States)

    Fiocchetti, Marco; Camilli, Giulia; Acconcia, Filippo; Leone, Stefano; Ascenzi, Paolo; Marino, Maria

    2015-05-01

    Besides other mechanism(s) 17β-estradiol (E2) facilitates neuronal survival by increasing, via estrogen receptor β (ERβ), the levels of neuroglobin (NGB) an anti-apoptotic protein. In contrast, E2 could exert protective effects in cancer cells by activating apoptosis when the ERβ level prevails on that of ERα as in colon cancer cell lines. These apparently contrasting results raise the possibility that E2-induced NGB up-regulation could regulate the ERβ activities shunning this receptor subtype to trigger an apoptotic cascade in neurons but not in non-neuronal cells. Here, human colorectal adenocarcinoma cell line (DLD-1) that only expresses ERβ and HeLa cells transiently transfected with ERβ encoding vector has been used to verify this hypothesis. In addition, neuroblastoma SK-N-BE cells were used as positive control. Surprisingly, E2 also induced NGB up-regulation, in a dose- and time-dependent manner, in DLD-1 cells. The ERβ-mediated activation of p38/MAPK was necessary for this E2 effect. E2 induced NGB re-allocation in mitochondria where, subsequently to an oxidative stress injury (i.e., 100μM H2O2), NGB interacted with cytochrome c preventing its release into the cytosol and the activation of an apoptotic cascade. As a whole, these results demonstrate that E2-induced NGB up-regulation could act as an oxidative stress sensor, which does not oppose to the pro-apoptotic E2 effect in ERβ-containing colon cancer cells unless a rise of oxidative stress occurs. These results support the concept that oxidative stress plays a critical role in E2-induced carcinogenesis and further open an important scenario to develop novel therapeutic strategies that target NGB against E2-related cancers.

  17. Up-regulation of intestinal epithelial cell derived IL-7 expression by keratinocyte growth factor through STAT1/IRF-1, IRF-2 pathway.

    Directory of Open Access Journals (Sweden)

    Yu-Jiao Cai

    Full Text Available BACKGROUND: Epithelial cells(EC-derived interleukin-7 (IL-7 plays a crucial role in control of development and homeostasis of neighboring intraepithelial lymphocytes (IEL, and keratinocyte growth factor (KGF exerts protective effects on intestinal epithelial cells and up-regulates EC-derived IL-7 expression through KGFR pathway. This study was to further investigate the molecular mechanism involved in the regulation of IL-7 expression by KGF in the intestine. METHODS: Intestinal epithelial cells (LoVo cells and adult C57BL/6J mice were treated with KGF. Epithelial cell proliferation was studied by flow cytometry for BrdU-incorporation and by immunohistochemistry for PCNA staining. Western blot was used to detect the changes of expression of P-Tyr-STAT1, STAT1, and IL-7 by inhibiting STAT1. Alterations of nuclear extracts and total proteins of IRF-1, IRF-2 and IL-7 following IRF-1 and IRF-2 RNA interference with KGF treatment were also measured with western blot. Moreover, IL-7 mRNA expressions were also detected by Real-time PCR and IL-7 protein level in culture supernatants was measured by enzyme linked immunosorbent assay(ELISA. RESULTS: KGF administration significantly increased LoVo cell proliferation and also increased intestinal wet weight, villus height, crypt depth and crypt cell proliferation in mice. KGF treatment led to increased levels of P-Tyr-STAT1, RAPA and AG490 both blocked P-Tyr-STAT1 and IL-7 expression in LoVo cells. IRF-1 and IRF-2 expression in vivo and in vitro were also up-regulated by KGF, and IL-7 expression was decreased after IRF-1 and IRF-2 expression was silenced by interfering RNA, respectively. CONCLUSION: KGF could up-regulate IL-7 expression through the STAT1/IRF-1, IRF-2 signaling pathway, which is a new insight in potential effects of KGF on the intestinal mucosal immune system.

  18. S-phase-dependent cell cycle disturbances caused by Aleutian mink disease parvovirus

    DEFF Research Database (Denmark)

    Oleksiewicz, M.B.; Alexandersen, Søren

    1997-01-01

    We examined replication of the autonomous parovirus Aleutian mink disease parovirus (ADV) in relation to cell cycle progression of permissive Crandell feline kidney (CRFK) cells. Flow cytometric analysis showed that ADV caused a composite, binary pattern of cell cycle arrest. ADV-induced cell cycle...... with subthreshold levels of ADV products through the late S/G(2) block and, consequently, that the binary pattern of ADV-induced cell cycle arrest may be governed merely by viral replication levels within a single S phase. Flow cytometric analysis of propidium iodide fluorescence and bromodeoxyuridine uptake showed...... that population A cells sustained significantly higher levels of DNA replication than population B cells during the ADV-induced cell cycle arrest. Therefore, the type of ADV-induced cell cycle arrest was not trivial and could have implications for subsequent viral replication in the target cell....

  19. Ligand-responsive upregulation of 3' CITE-mediated translation in a wheat germ cell-free expression system.

    Science.gov (United States)

    Ogawa, Atsushi; Murashige, Yuta; Tabuchi, Junichiro; Omatsu, Taiki

    2017-01-31

    We have rationally constructed a novel regulation-type of artificial riboswitch that ligand-dose dependently upregulates translation initiation mediated by a 3' cap-independent translation element (3' CITE) with no major hybridization switches in a plant expression system (wheat germ extract).

  20. hsa-miR-125a-5p Enhances Invasion in Non-small Cell Lung Carcinoma Cell Lines by Upregulating Rock-1

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    Lili JIANG

    2009-10-01

    Full Text Available Background and objective MicroRNAs (miRNAs are endogenous, non-coding small RNA in eukaryotes. They recognize their target sites by incomplete base pairing and posttranscriptionally regulate gene expression, and function on a lot of complex vital processes of organisms. The objective of this work is to study how hsa-miR-125a-5p enhances the invasive ability of lung cancer cells. Methods The target gene and its target sites of hsa-miR-125a-5p were predicted by microRNA.org. We investigated Rock-1 mRNA and protein expressions by RT-PCR and Western blot according to the result of prediction further. The invasive ability of A549 cells, which were transfected with sense hsa-miR-125a-5p 2’-O-methyl oligonucleotide after being blocked by anti-Rock-1, was observed by Transwell. Results With RT-PCR and Western blot, Rock-1 mRNA and protein were both increased in A549 cells transfected with sense hsa-miR-125a-5p 2’-O-methyl oligonucleotide and were both decreased in the cells which transfected with antisense vs control groups. The invasive ability of A549 cells transfected with sense hsa-miR-125a-5p 2’-O-methyl oligonucleotide were weakened after being blocked by anti-Rock-1, vs non-blocking group by Transwell test. Conclusion hsa-miR-125a-5p could up-regulate Rock-1 and enhance invasion in lung cancer cells.

  1. p172: An alveolar type II and Clara cell specific protein with late developmental expression and upregulation by hyperoxic lung injury.

    Science.gov (United States)

    Girod, C E; Shin, D H; Hershenson, M B; Solway, J; Dahl, R; Miller, Y E

    1996-06-01

    The epithelium of the alveolus and distal airway meets unique requirements, functioning as a gas exchange membrane and barrier to alveolar flooding by vascular contents as well as to bloodstream contamination by airborne toxins and pathogens. Gene products specifically expressed by this epithelium, notably the surfactant apoproteins, have had important clinical application. No cell surface antigen specific for alveolar type II and Clara cells has been described. We report the biochemical characterization, tissue and developmental expression, and upregulation by injury of a 172 kD protein recognized by a monoclonal antibody, 3F9, synthesized in response to immunization with freshly isolated rat alveolar type II cells. p172 is expressed in a polarized fashion by the apical surface of rat alveolar type II and Clara cells. An immunohistochemical survey of various rat tissues and organs reveals lung specificity. p172 is first detectable in rare epithelial cells at 19 days of gestation, a time when the fully differentiated alveolar type II cell is identified by the first detection of lamellar bodies. There is a dramatic increase in p172 expression just prior to birth. Hyperoxic lung injury results in increased expression of p172. The upregulation of p172 by hyperoxia and its cell-specific expression suggests an important adaptive function.

  2. Up-regulation of DRP-3 long isoform during the induction of neural progenitor cells by glutamate treatment in the ex vivo rat retina.

    Science.gov (United States)

    Tokuda, Kazuhiro; Kuramitsu, Yasuhiro; Byron, Baron; Kitagawa, Takao; Tokuda, Nobuko; Kobayashi, Daiki; Nagayama, Megumi; Araki, Norie; Sonoda, Koh-Hei; Nakamura, Kazuyuki

    2015-08-07

    Glutamate has been shown to induce neural progenitor cells in the adult vertebrate retina. However, protein dynamics during progenitor cell induction by glutamate are not fully understood. To identify specific proteins involved in the process, we employed two-dimensional electrophoresis-based proteomics on glutamate untreated and treated retinal ex vivo sections. Rat retinal tissues were incubated with 1 mM glutamate for 1 h, followed by incubation in glutamate-free media for a total of 24 h. Consistent with prior reports, it was found that mitotic cells appeared in the outer nuclear layer without any histological damage. Immunohistological evaluations and immunoblotting confirmed the emergence of neuronal progenitor cells in the mature retina treated with glutamate. Proteomic analysis revealed the up-regulation of dihydropyrimidinase-related protein 3 (DRP-3), DRP-2 and stress-induced-phosphoprotein 1 (STIP1) during neural progenitor cell induction by glutamate. Moreover, mRNA expression of DRP-3, especially, its long isoform, robustly increased in the treated retina compared to that in the untreated retina. These results may indicate that glutamate induces neural progenitor cells in the mature rat retina by up-regulating the proteins which mediate cell mitosis and neurite growth.

  3. Integrin β1A Upregulates p27 Protein Amount at the Post-translational Level in Human Hepatocellular Carcinoma Cell Line SMMC-7721

    Institute of Scientific and Technical Information of China (English)

    Yi FU; Li-Ying WANG; Yu-Long LIANG; Jia-Wei JIN; Zheng-Yu FANG; Xi-Liang ZHA

    2006-01-01

    Integrins mediate many fundamental cellular processes by binding to components of the extracellular matrix. We showed previously that integrin β1A could inhibit cell proliferation. Integrin β1A stimulated the promoter activity of p21cip1 and enhanced its transcription in SMMC-7721 cells. In this study,we demonstrated that integrin β1A upregulated p27kip1 at the post-translational level in SMMC-7721 cells. Our results showed that integrin β1A increased the p27 protein amount, both in cytoplasm and nucleus, but did not affect the p27m RNA amount. Cycloheximide treatment experiment revealed that the half-life of p27 protein was prolonged in integrin β1A overexpressing cells, indicating that integrin β1A inhibited the degradation of p27 protein. Our data also provided evidence that both the proteasome and calpain were involved in the degradation of p27 protein in SMMC-7721 cells. Integrin β1A decreased the Skp2 expression and repressed the activity of calpain during G1 phase in SMMC-7721 cells. Taken together, these results indicated that integrin β1A might upregulate the protein amount of p27 through repressing Skp2-dependent proteasome degradation and calpainmediated proteolysis in SMMC-7721 cells.

  4. Epigenetic regulations in the IFNγ signalling pathway: IFNγ-mediated MHC class I upregulation on tumour cells is associated with DNA demethylation of antigen-presenting machinery genes.

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    Vlková, Veronika; Štěpánek, Ivan; Hrušková, Veronika; Šenigl, Filip; Mayerová, Veronika; Šrámek, Martin; Šímová, Jana; Bieblová, Jana; Indrová, Marie; Hejhal, Tomáš; Dérian, Nicolas; Klatzmann, David; Six, Adrien; Reiniš, Milan

    2014-08-30

    Downregulation of MHC class I expression on tumour cells, a common mechanism by which tumour cells can escape from specific immune responses, can be associated with coordinated silencing of antigen-presenting machinery genes. The expression of these genes can be restored by IFNγ. In this study we documented association of DNA demethylation of selected antigen-presenting machinery genes located in the MHC genomic locus (TAP-1, TAP-2, LMP-2, LMP-7) upon IFNγ treatment with MHC class I upregulation on tumour cells in several MHC class I-deficient murine tumour cell lines (TC-1/A9, TRAMP-C2, MK16 and MC15). Our data also documented higher methylation levels in these genes in TC-1/A9 cells, as compared to their parental MHC class I-positive TC-1 cells. IFNγ-mediated DNA demethylation was relatively fast in comparison with demethylation induced by DNA methyltransferase inhibitor 5-azacytidine, and associated with increased histone H3 acetylation in the promoter regions of APM genes. Comparative transcriptome analysis in distinct MHC class I-deficient cell lines upon their treatment with either IFNγ or epigenetic agents revealed that a set of genes, significantly enriched for the antigen presentation pathway, was regulated in the same manner. Our data demonstrate that IFNγ acts as an epigenetic modifier when upregulating the expression of antigen-presenting machinery genes.

  5. Exercise protects against obesity induced semen abnormalities via downregulating stem cell factor, upregulating Ghrelin and normalizing oxidative stress.

    Science.gov (United States)

    Alhashem, Fahaid; Alkhateeb, Mahmoud; Sakr, Hussein; Alshahrani, Mesfer; Alsunaidi, Mohammad; Elrefaey, Hesham; Alessa, Riyad; Sarhan, Mohammad; Eleawa, Samy M; Khalil, Mohammad A

    2014-01-01

    Increased oxidative stress and hormonal imbalance have been hypothesized to underlie infertility in obese animals. However, recent evidence suggests that Ghrelin and Stem Cell Factor (SCF) play an important role in fertility, in lean individuals. Therefore, this study aimed at investigating whether changes in the levels of Ghrelin and SCF in rat testes underlie semen abnormal parameters observed in obese rats, and secondly, whether endurance exercise or Orlistat can protect against changes in Ghrelin, SCF, and/or semen parameters in diet induced obese rats. Obesity was modelled in male Wistar rats using High Fat Diet (HFD) 12-week protocol. Eight week-old rats (n=40) were divided into four groups, namely, Group I: fed with a standard diet (12 % of calories as fat); Group II: fed HFD (40 % of calories as fat); Group III: fed the HFD with a concomitant dose of Orlistat (200 mg/kg); and Group IV: fed the HFD and underwent 30 min daily swimming exercise. The model was validated by measuring the levels of testosterone, FSH, LH, estradiol, leptin, triglycerides, total, HDL, and LDL cholesterol, and final change in body weight. Levels were consistent with published obesity models (see Results). As predicted, the HFD group had a 76.8 % decrease in sperm count, 44.72 % decrease in sperm motility, as well as 47.09 % increase in abnormal sperm morphology. Unlike the control group, in the HFD group (i.e. obese rats) Ghrelin mRNA and protein were elevated, while SCF mRNA and protein were diminished in the testes. Furthermore, in the HFD group, SOD and GPx activities were significantly reduced, 48.5±5.8 % (P=0.0012) and 45.6±4.6 % (P=0.0019), respectively, while TBARS levels were significantly increased (112.7±8.9 %, P=0.0001). Finally, endurance exercise training and Orlistat administration individually and differentially protected semen parameters in obese rats. The mechanism includes, but is not limited to, normalizing the levels of Ghrelin, SCF, SOD, GPx and TBARS. In rat

  6. Exercise protects against obesity induced semen abnormalities via downregulating stem cell factor, upregulating Ghrelin and normalizing oxidative stress

    Science.gov (United States)

    Alhashem, Fahaid; Alkhateeb, Mahmoud; Sakr, Hussein; Alshahrani, Mesfer; Alsunaidi, Mohammad; Elrefaey, Hesham; Alessa, Riyad; Sarhan, Mohammad; Eleawa, Samy M; Khalil, Mohammad A.

    2014-01-01

    Increased oxidative stress and hormonal imbalance have been hypothesized to underlie infertility in obese animals. However, recent evidence suggests that Ghrelin and Stem Cell Factor (SCF) play an important role in fertility, in lean individuals. Therefore, this study aimed at investigating whether changes in the levels of Ghrelin and SCF in rat testes underlie semen abnormal parameters observed in obese rats, and secondly, whether endurance exercise or Orlistat can protect against changes in Ghrelin, SCF, and/or semen parameters in diet induced obese rats. Obesity was modelled in male Wistar rats using High Fat Diet (HFD) 12-week protocol. Eight week-old rats (n=40) were divided into four groups, namely, Group I: fed with a standard diet (12 % of calories as fat); Group II: fed HFD (40 % of calories as fat); Group III: fed the HFD with a concomitant dose of Orlistat (200 mg/kg); and Group IV: fed the HFD and underwent 30 min daily swimming exercise. The model was validated by measuring the levels of testosterone, FSH, LH, estradiol, leptin, triglycerides, total, HDL, and LDL cholesterol, and final change in body weight. Levels were consistent with published obesity models (see Results). As predicted, the HFD group had a 76.8 % decrease in sperm count, 44.72 % decrease in sperm motility, as well as 47.09 % increase in abnormal sperm morphology. Unlike the control group, in the HFD group (i.e. obese rats) Ghrelin mRNA and protein were elevated, while SCF mRNA and protein were diminished in the testes. Furthermore, in the HFD group, SOD and GPx activities were significantly reduced, 48.5±5.8 % (P=0.0012) and 45.6±4.6 % (P=0.0019), respectively, while TBARS levels were significantly increased (112.7±8.9 %, P=0.0001). Finally, endurance exercise training and Orlistat administration individually and differentially protected semen parameters in obese rats. The mechanism includes, but is not limited to, normalizing the levels of Ghrelin, SCF, SOD, GPx and TBARS. In rat

  7. Slug-dependent upregulation of L1CAM is responsible for the increased invasion potential of pancreatic cancer cells following long-term 5-FU treatment.

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    Kaja Lund

    Full Text Available Pancreatic adenocarcinoma is a lethal disease with 5-year survival of less than 5%. 5-fluorouracil (5-FU is a principal first-line therapy, but treatment only extends survival modestly and is seldom curative. Drug resistance and disease recurrence is typical and there is a pressing need to overcome this. To investigate acquired 5-FU resistance in pancreatic adenocarcinoma, we established chemoresistant monoclonal cell lines from the Panc 03.27 cell line by long-term exposure to increasing doses of 5-FU.5-FU-resistant cell lines exhibited increased expression of markers associated with multidrug resistance explaining their reduced sensitivity to 5-FU. In addition, 5-FU-resistant cell lines showed alterations typical for an epithelial-to-mesenchymal transition (EMT, including upregulation of mesenchymal markers and increased invasiveness. Microarray analysis revealed the L1CAM pathway as one of the most upregulated pathways in the chemoresistant clones, and a significant upregulation of L1CAM was seen on the RNA and protein level. In pancreatic cancer, expression of L1CAM is associated with a chemoresistant and migratory phenotype. Using esiRNA targeting L1CAM, or by blocking the extracellular part of L1CAM with antibodies, we show that the increased invasiveness observed in the chemoresistant cells functionally depends on L1CAM. Using esiRNA targeting β-catenin and/or Slug, we demonstrate that in the chemoresistant cell lines, L1CAM expression depends on Slug rather than β-catenin.Our findings establish Slug-induced L1CAM expression as a mediator of a chemoresistant and migratory phenotype in pancreatic adenocarcinoma cells.

  8. PPAR-γ activation increases insulin secretion through the up-regulation of the free fatty acid receptor GPR40 in pancreatic β-cells.

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    Hyo-Sup Kim

    Full Text Available BACKGROUND: It has been reported that peroxisome proliferator-activated receptor (PPAR-γ and their synthetic ligands have direct effects on pancreatic β-cells. We investigated whether PPAR-γ activation stimulates insulin secretion through the up-regulation of GPR40 in pancreatic β-cells. METHODS: Rat insulinoma INS-1 cells and primary rat islets were treated with rosiglitazone (RGZ and/or adenoviral PPAR-γ overexpression. OLETF rats were treated with RGZ. RESULTS: PPAR-γ activation with RGZ and/or adenoviral PPAR-γ overexpression increased free fatty acid (FFA receptor GPR40 expression, and increased insulin secretion and intracellular calcium mobilization, and was blocked by the PLC inhibitors, GPR40 RNA interference, and GLUT2 RNA interference. As a downstream signaling pathway of intracellular calcium mobilization, the phosphorylated levels of CaMKII and CREB, and the downstream IRS-2 and phospho-Akt were significantly increased. Despite of insulin receptor RNA interference, the levels of IRS-2 and phospho-Akt was still maintained with PPAR-γ activation. In addition, the β-cell specific gene expression, including Pdx-1 and FoxA2, increased in a GPR40- and GLUT2-dependent manner. The levels of GPR40, phosphorylated CaMKII and CREB, and β-cell specific genes induced by RGZ were blocked by GW9662, a PPAR-γ antagonist. Finally, PPAR-γ activation up-regulated β-cell gene expressions through FoxO1 nuclear exclusion, independent of the insulin signaling pathway. Based on immunohistochemical staining, the GLUT2, IRS-2, Pdx-1, and GPR40 were more strongly expressed in islets from RGZ-treated OLETF rats compared to control islets. CONCLUSION: These observations suggest that PPAR-γ activation with RGZ and/or adenoviral overexpression increased intracellular calcium mobilization, insulin secretion, and β-cell gene expression through GPR40 and GLUT2 gene up-regulation.

  9. Neurokinin-1 receptor directly mediates glioma cell migration by up-regulation of matrix metalloproteinase-2 (MMP-2) and membrane type 1-matrix metalloproteinase (MT1-MMP).

    Science.gov (United States)

    Mou, Lingyun; Kang, Yawei; Zhou, Ying; Zeng, Qian; Song, Hongjing; Wang, Rui

    2013-01-04

    Neurokinin-1 receptor (NK1R) occurs naturally on human glioblastomas. Its activation mediates glioma cell proliferation. However, it is unknown whether NK1R is directly involved in tumor cell migration. In this study, we found human hemokinin-1 (hHK-1), via NK1R, dose-dependently promoted the migration of U-251 and U-87 cells. In addition, we showed that hHK-1 enhanced the activity of MMP-2 and the expression of MMP-2 and MT1-matrix metalloproteinase (MMP), which were responsible for cell migration, because neutralizing the MMPs with antibodies decreased cell migration. The involved mechanisms were then investigated. In U-251, hHK-1 induced significant calcium efflux; phospholipase C inhibitor U-73122 reduced the calcium mobilization, the up-regulation of MMP-2 and MT1-MMP, and the cell migration induced by hHK-1, which meant the migration effect of NK1R was mainly mediated through the G(q)-PLC pathway. We further demonstrated that hHK-1 boosted rapid phosphorylation of ERK, JNK, and Akt; inhibition of ERK and Akt effectively reduced MMP-2 induction by hHK-1. Meanwhile, inhibition of ERK, JNK, and Akt reduced the MT1-MMP induction. hHK-1 stimulated significant phosphorylation of p65 and c-JUN in U-251. Reporter gene assays indicated hHK-1 enhanced both AP-1 and NF-κB activity; inhibition of ERK, JNK, and Akt dose-dependently suppressed the NF-κB activity; only the inhibition of ERK significantly suppressed the AP-1 activity. Treatment with specific inhibitors for AP-1 or NF-κB strongly blocked the MMP up-regulation by hHK-1. Taken together, our data suggested NK1R was a potential regulator of human glioma cell migration by the up-regulation of MMP-2 and MT1-MMP.

  10. Acute morphine activates satellite glial cells and up-regulates IL-1β in dorsal root ganglia in mice via matrix metalloprotease-9

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    Berta Temugin

    2012-03-01

    Full Text Available Abstract Background Activation of spinal cord glial cells such as microglia and astrocytes has been shown to regulate chronic opioid-induced antinociceptive tolerance and hyperalgesia, due to spinal up-regulation of the proinflammatory cytokines such as interleukin-1 beta (IL-1β. Matrix metalloprotease-9 (MMP-9 has been implicated in IL-1β activation in neuropathic pain. However, it is unclear whether acute opioid treatment can activate glial cells in the peripheral nervous system. We examined acute morphine-induced activation of satellite glial cells (SGCs and up-regulation of IL-1β in dorsal root ganglia (DRGs, and further investigated the involvement of MMP-9 in these opioid-induced peripheral changes. Results Subcutaneous morphine injection (10 mg/kg induced robust peripheral glial responses, as evidenced by increased GFAP expression in DRGs but not in spinal cords. The acute morphine-induced GFAP expression is transient, peaking at 2 h and declining after 3 h. Acute morphine treatment also increased IL-1β immunoreactivity in SGCs and IL-1β activation in DRGs. MMP-9 and GFAP are expressed in DRG neurons and SGCs, respectively. Confocal analysis revealed a close proximity of MMP-9 and GFAP immunostaining. Importantly, morphine-induced DRG up-regulation of GFAP expression and IL-1β activation was abolished after Mmp9 deletion or naloxone pre-treatment. Finally, intrathecal injections of IL-1β-selective siRNA not only reduced DRG IL-1β expression but also prolonged acute morphine-induced analgesia. Conclusions Acute morphine induces opioid receptors- and MMP-9-dependent up-regulation of GFAP expression and IL-1β activation in SGCs of DRGs. MMP-9 could mask and shorten morphine analgesia via peripheral neuron-glial interactions. Targeting peripheral glial activation might prolong acute opioid analgesia.

  11. Identiifcation of genes upregulated by recombinant inter feron-alpha in HepG2 cells by suppressive subtractive hybridization analysis

    Institute of Scientific and Technical Information of China (English)

    Jian-Hui Qu; Jun Cheng; Ling-Xia Zhang; Li-Ying Zhang; Yan-Wei Zhong; Yan Liu; Lin Wang; Jiu-Zeng Dai; Dong-Ping Xu

    2007-01-01

    BACKGROUND:Interferon-alpha (IFN-α) is an important cytokine with multiple functions, but the target genes transactivated by IFN-αremain largely unknown. A study of such genes will help to understand the mechanism of function of IFN-α. To isolate the gene transcripts speciifcally upregulated by IFN-α in HepG2 cells, we conducted suppressive subtractive hybridization (SSH) analysis. METHODS: SSH was used to analyze the target genes transactivated by recombinant IFN-α protein, and a subtractive cDNA library was constructed from HepG2 cells treated with recombinant IFN-α (rIFN-α, 2000 IU/ml) for 16 hours as tester, and cells not treated with rIFN-αas driver. The SSH PCR products from the library were cloned into pGEM-T easy vector and with BLASTX, the positive clones were randomly selected, sequenced and compared to the database in GenBank of the 35 differentially expressed gene fragments from the library, 6 clones showed signiifcant homology to other known proteins. RESULTS: The subtractive cDNA library of genes upregulated by IFN-α was constructed successfully. rIFN-α upregulated the expression of the RAN binding protein 5 (RANBP5), NADH dehydrogenase, exosome component 3 (EXOSC3), zinc ifnger RNA binding protein, Dickkopf homolog 1 (DKK1) and acetyl-coenzyme A acetyltransferase 2 (ACAT2). CONCLUSIONS: These results suggest that rIFN-α can upregulate the expression of important genes to exert its functions, and provide new clues for discovering the molecular mechanisms of action of IFN-α.

  12. Arsenic augments the uptake of oxidized LDL by upregulating the expression of lectin-like oxidized LDL receptor in mouse aortic endothelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Hossain, Ekhtear [Department of Biochemistry, Aichi Medical University School of Medicine, Nagakute, Aichi (Japan); Ota, Akinobu, E-mail: aota@aichi-med-u.ac.jp [Department of Biochemistry, Aichi Medical University School of Medicine, Nagakute, Aichi (Japan); Karnan, Sivasundaram; Damdindorj, Lkhagvasuren [Department of Biochemistry, Aichi Medical University School of Medicine, Nagakute, Aichi (Japan); Takahashi, Miyuki [Department of Biochemistry, Aichi Medical University School of Medicine, Nagakute, Aichi (Japan); Division of Hematology, Department of Internal Medicine, Aichi Medical University School of Medicine, Nagakute, Aichi (Japan); Konishi, Yuko; Konishi, Hiroyuki; Hosokawa, Yoshitaka [Department of Biochemistry, Aichi Medical University School of Medicine, Nagakute, Aichi (Japan)

    2013-12-15

    Although chronic arsenic exposure is a well-known risk factor for cardiovascular diseases, including atherosclerosis, the molecular mechanism underlying arsenic-induced atherosclerosis remains obscure. Therefore, this study aimed to elucidate this molecular mechanism. We examined changes in the mRNA level of the lectin-like oxidized LDL (oxLDL) receptor (LOX-1) in a mouse aortic endothelial cell line, END-D, after sodium arsenite (SA) treatment. SA treatment significantly upregulated LOX-1 mRNA expression; this finding was also verified at the protein expression level. Flow cytometry and fluorescence microscopy analyses showed that the cellular uptake of fluorescence (Dil)-labeled oxLDL was significantly augmented with SA treatment. In addition, an anti-LOX-1 antibody completely abrogated the augmented uptake of Dil-oxLDL. We observed that SA increased the levels of the phosphorylated forms of nuclear factor of kappa light polypeptide gene enhancer in B cells (NF-κB)/p65. SA-induced upregulation of LOX-1 protein expression was clearly prevented by treatment with an antioxidant, N-acetylcysteine (NAC), or an NF-κB inhibitor, caffeic acid phenethylester (CAPE). Furthermore, SA-augmented uptake of Dil-oxLDL was also prevented by treatment with NAC or CAPE. Taken together, our results indicate that arsenic upregulates LOX-1 expression through the reactive oxygen species-mediated NF-κB signaling pathway, followed by augmented cellular oxLDL uptake, thus highlighting a critical role of the aberrant LOX-1 signaling pathway in the pathogenesis of arsenic-induced atherosclerosis. - Highlights: • Sodium arsenite (SA) increases LOX-1 expression in mouse aortic endothelial cells. • SA enhances cellular uptake of oxidized LDL in dose-dependent manner. • SA-induced ROS generation enhances phosphorylation of NF-κB. • SA upregulates LOX-1 expression through ROS-activated NF-κB signaling pathway.

  13. Gambogic acid induces growth inhibition and differentiation via upregulation of p21waf1/cip1 expression in acute myeloid leukemia cells.

    Science.gov (United States)

    Chen, Yan; Hui, Hui; Li, Zheng; Wang, Hong-Mei; You, Qi-Dong; Lu, Na

    2014-10-01

    Gambogic acid (GA) is the major active ingredient of gamboges, a brownish to orange resin product from Garcinia hanburyi tree in Southeast Asia. This compound exhibits anti-cancer effect on solid tumors. In this study, we investigated the effects of GA on the growth and differentiation of acute myeloid leukemia cells by growth-inhibition detection, morphological changes observation, nitroblue tetrazolium reduction, and the expression of the relative cell-surface differentiation markers. The results showed that GA could inhibit cell growth and promote differentiation in U937 and HL-60 cells. In addition, GA upregulated the expression of p21waf1/cip1 in the two cell lines. Finally, downregulating the p21waf1/cip1 expression with small interfering RNA partially blocked GA-induced cell growth inhibition and differentiation. These results of this study revealed that GA may be used as one of the investigational drugs for acute myeloid leukemia.

  14. Induction of cellular senescence by doxorubicin is associated with upregulated miR-375 and induction of autophagy in K562 cells.

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    Ming-Yu Yang

    Full Text Available BACKGROUND: Cellular senescence is a specialized form of growth arrest that is generally irreversible. Upregulated p16, p53, and p21 expression and silencing of E2F target genes have been characterized to promote the establishment of senescence. It can be further aided by the transcriptional repression of proliferation-associated genes by the action of HP1γ, HMGA, and DNMT proteins to produce a repressive chromatin environment. Therefore, senescence has been suggested to functions as a natural brake for tumor development and plays a critical role in tumor suppression and aging. METHODOLOGY/PRINCIPAL FINDINGS: An in vitro senescence model has been established by using K562 cells treated with 50 nM doxorubicin (DOX. Since p53 and p16 are homozygously deleted in the K562 cells, the DOX-induced senescence in K562 cells ought to be independent of p53 and p16-pRb pathways. Indeed, no change in the expression of the typical senescence-associated premalignant cell markers in the DOX-induced senescent K562 cells was found. MicroRNA profiling revealed upregulated miR-375 in DOX-induced senescent K562 cells. Treatment with miR-375 inhibitor was able to reverse the proliferation ability suppressed by DOX (p<0.05 and overexpression of miR-375 suppressed the normal proliferation of K562 cells. Upregulated miR-375 expression was associated with downregulated expression of 14-3-3zeta and SP1 genes. Autophagy was also investigated since DOX treatment was able to induce cells entering senescence and eventually lead to cell death. Among the 24 human autophagy-related genes examined, a 12-fold increase of ATG9B at day 4 and a 20-fold increase of ATG18 at day 2 after DOX treatment were noted. CONCLUSIONS/SIGNIFICANCE: This study has demonstrated that in the absence of p53 and p16, the induction of senescence by DOX was associated with upregulation of miR-375 and autophagy initiation. The anti-proliferative function of miR-375 is possibly exerted, at least in part

  15. β-catenin accumulation in nuclei of hepatocellular carcinoma cells up-regulates glutathione-s-transferase M3 mRNA

    Institute of Scientific and Technical Information of China (English)

    Yu-Sang Li; Min Liu; Yoshihiro Nakata; He-Bin Tang

    2011-01-01

    AIM: To identify the differentially over-expressed genes associated with β-catenin accumulation in nuclei of hepatocellular carcinoma (HCC) cells.METHODS: Differentially expressed genes were identified in radiation-induced B6C3 F1 mouse HCC cells by mRNA differential display, Northern blot and RT-PCR,respectively. Total glutathione-s-transferase (GST) activity was measured by GST activity assay and β-catenin localization was detected with immunostaining in radiation-induced mouse HCC cells and in HepG2 cell lines.RESULTS: Two up-regulated genes, glutamine synthetase and glutathione-s-transferase M3 (GSTM3), were identified in radiation-induced mouse HCC cells. Influence of β-catenin accumulation in nuclei of HCC cells on upregulation of GSTM3 mRNA was investigated. The nearby upstream domain of GSTM3 contained the β-catenin/Tcf-Lef consensus binding site sequences [5'-(A/T)(A/T)CAAAG-3'], and the total GST activity ratio was considerably higher in B6C3F1 mouse HCC cells with β-cateninaccumulation in nuclei of HCC cells than in those withoutβ-catenin accumulation (0.353 ± 0.117 vs 0.071 ± 0.064,P < 0.001). The TWS119 (a distinct GSK-3β inhibitor)-induced total GST activity was significantly higher in HepG2 cells with β-catenin accumulation than in those withoutβ-catenin accumulation in nuclei of HCC cells. Additionally,the GSTM3 mRNA level was significantly higher at 24 h than at 12 h in TWS119-treated HepG2 cells.CONCLUSION: β-catenin accumulation increases GST activity in nuclei of HCC cells, and GSTM3 may be a novel target gene of the β-catenin/Tcf-Lef complex.

  16. Higher Matrix Stiffness Upregulates Osteopontin Expression in Hepatocellular Carcinoma Cells Mediated by Integrin β1/GSK3β/β-Catenin Signaling Pathway.

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    Yang You

    Full Text Available Increased stromal stiffness is associated with hepatocellular carcinoma (HCC development and progression. However, the molecular mechanism by which matrix stiffness stimuli modulate HCC progress is largely unknown. In this study, we explored whether matrix stiffness-mediated effects on osteopontin (OPN expression occur in HCC cells. We used a previously reported in vitro culture system with tunable matrix stiffness and found that OPN expression was remarkably upregulated in HCC cells with increasing matrix stiffness. Furthermore, the phosphorylation level of GSK3β and the expression of nuclear β-catenin were also elevated, indicating that GSK3β/β-catenin pathway might be involved in OPN regulation. Knock-down analysis of integrin β1 showed that OPN expression and p-GSK3β level were downregulated in HCC cells grown on high stiffness substrate compared with controls. Simultaneously, inhibition of GSK-3β led to accumulation of β-catenin in the cytoplasm and its enhanced nuclear translocation, further triggered the rescue of OPN expression, suggesting that the integrin β1/GSK-3β/β-catenin pathway is specifically activated for matrix stiffness-mediated OPN upregulation in HCC cells. Tissue microarray analysis confirmed that OPN expression was positively correlated with the expression of LOX and COL1. Taken together, high matrix stiffness upregulated OPN expression in HCC cells via the integrin β1/GSK-3β/β-catenin signaling pathway. It highlights a new insight into a pathway involving physical mechanical signal and biochemical signal molecules which contributes to OPN expression in HCC cells.

  17. Higher Matrix Stiffness Upregulates Osteopontin Expression in Hepatocellular Carcinoma Cells Mediated by Integrin β1/GSK3β/β-Catenin Signaling Pathway.

    Science.gov (United States)

    You, Yang; Zheng, Qiongdan; Dong, Yinying; Wang, Yaohui; Zhang, Lan; Xue, Tongchun; Xie, Xiaoying; Hu, Chao; Wang, Zhiming; Chen, Rongxin; Wang, Yanhong; Cui, Jiefeng; Ren, Zhenggang

    2015-01-01

    Increased stromal stiffness is associated with hepatocellular carcinoma (HCC) development and progression. However, the molecular mechanism by which matrix stiffness stimuli modulate HCC progress is largely unknown. In this study, we explored whether matrix stiffness-mediated effects on osteopontin (OPN) expression occur in HCC cells. We used a previously reported in vitro culture system with tunable matrix stiffness and found that OPN expression was remarkably upregulated in HCC cells with increasing matrix stiffness. Furthermore, the phosphorylation level of GSK3β and the expression of nuclear β-catenin were also elevated, indicating that GSK3β/β-catenin pathway might be involved in OPN regulation. Knock-down analysis of integrin β1 showed that OPN expression and p-GSK3β level were downregulated in HCC cells grown on high stiffness substrate compared with controls. Simultaneously, inhibition of GSK-3β led to accumulation of β-catenin in the cytoplasm and its enhanced nuclear translocation, further triggered the rescue of OPN expression, suggesting that the integrin β1/GSK-3β/β-catenin pathway is specifically activated for matrix stiffness-mediated OPN upregulation in HCC cells. Tissue microarray analysis confirmed that OPN expression was positively correlated with the expression of LOX and COL1. Taken together, high matrix stiffness upregulated OPN expression in HCC cells via the integrin β1/GSK-3β/β-catenin signaling pathway. It highlights a new insight into a pathway involving physical mechanical signal and biochemical signal molecules which contributes to OPN expression in HCC cells.

  18. Selective upregulation of the expression of plasma membrane calcium ATPase isoforms upon differentiation and 1,25(OH)2D3-vitamin treatment of colon cancer cells.

    Science.gov (United States)

    Ribiczey, Polett; Papp, Béla; Homolya, László; Enyedi, Ágnes; Kovács, Tünde

    2015-08-14

    We have previously presented co-expression of the plasma membrane calcium ATPase isoforms 4b (PMCA4b) and 1b (PMCA1b) in colon carcinoma cells, and selective upregulation of PMCA4b during differentiation initiated by short chain fatty acids or post-confluent growth. Here we show that the induction of PMCA4b expression is a characteristic feature of the post-confluency-induced differentiation of both enterocyte-type and goblet cell-type colon cancer cells. Vitamin D3 (1,25(OH)2D3) is a well-known regulator of intestinal Ca(2+) absorption and of basic cell functions such as growth and differentiation in various cell types. As PMCA proteins are involved both in intestinal Ca(2+) absorption and adenocarcinoma cell differentiation, we investigated the effect of 1,25(OH)2D3 on PMCA expression in enterocyte-like colon carcinoma cells, and monitored its effect on the expression of various differentiation markers. 1,25(OH)2D3 stimulated PMCA1b, but not PMCA4b expression without modulating the expression of the majority of the differentiation markers examined. Caco-2 cells differentiated in post-confluent cultures present normal enterocyte-like intestinal epithelial phenotype. To better understand the role of PMCA proteins in vectorial Ca(2+) transport by enterocytes, we also studied their subcellular localization in mature polarized Caco-2 cells. Both PMCA isoforms were located to the basolateral membrane, and the PMCA-specific immunofluorescent signal was significantly higher in vitamin D3-treated cells, underlining the 1,25(OH)2D3-induced upregulation of PMCA (presumably 1b isoform) expression in differentiated Caco-2 cells. We suggest that while PMCA1b has a housekeeping function in colon cancer cells, PMCA4b participates in the reorganization of the Ca(2+) signalling machinery during cell differentiation. The subcellular localization of PMCA1b and its selective 1,25(OH)2D3-dependent upregulation indicate that this isoform may have a specific role in 1,25(OH)2D3

  19. Disturbance of the bacterial cell wall specifically interferes with biofilm formation.

    Science.gov (United States)

    Bucher, Tabitha; Oppenheimer-Shaanan, Yaara; Savidor, Alon; Bloom-Ackermann, Zohar; Kolodkin-Gal, Ilana

    2015-12-01

    In nature, bacteria communicate via chemical cues and establish complex communities referred to as biofilms, wherein cells are held together by an extracellular matrix. Much research is focusing on small molecules that manipulate and prevent biofilm assembly by modifying cellular signalling pathways. However, the bacterial cell envelope, presenting the interface between bacterial cells and their surroundings, is largely overlooked. In our study, we identified specific targets within the biosynthesis pathways of the different cell wall components (peptidoglycan, wall teichoic acids and teichuronic acids) hampering biofilm formation and the anchoring of the extracellular matrix with a minimal effect on planktonic growth. In addition, we provide convincing evidence that biofilm hampering by transglycosylation inhibitors and D-Leucine triggers a highly specific response without changing the overall protein levels within the biofilm cells or the overall levels of the extracellular matrix components. The presented results emphasize the central role of the Gram-positive cell wall in biofilm development, resistance and sustainment.

  20. NF1 deficiency causes Bcl-xL upregulation in Schwann cells derived from neurofibromatosis type 1-associated malignant peripheral nerve sheath tumors.

    Science.gov (United States)

    Park, Ho-Jin; Lee, Su-Jin; Sohn, Young Bae; Jin, Hyun-Seok; Han, Jae-Ho; Kim, Young-Bae; Yim, Hyunee; Jeong, Seon-Yong

    2013-02-01

    Since the bi-allelic inactivation of both neurofibromin 1 (NF1) gene alleles (NF1(-/-)) in Schwann cells (SCs) is common in both benign plexiform neurofibromas (PNs) and malignant peripheral nerve sheath tumors (MPNSTs) in patients with neurofibromatosis type 1 (NF1), other genetic alterations in SCs may be required for tumor progression of PNs to MPNSTs. We found that the anti-apoptotic Bcl-xL protein is upregulated in MPNST tissues compared to PN tissues from patients with NF1 by immunohistological staining. In addition, we investigated whether Bcl-xL is upregulated in SCs derived from MPNSTs and found a significantly higher Bcl-xL expression level in sNF96.2 MPNST-derived SCs compared to normal human SCs (HSCs). We also discovered that the increased Bcl-xL expression caused an increase in drug resistance to doxorubicin in MPNST-derived SCs. Manipulation of NF1 gene expression levels by treatment with small interfering RNA (siRNA) and overexpression of the neurofibromin GAP-related domain (NF1-GRD) demonstrated that upregulated Bcl-xL expression in MPNST-derived SCs was caused by NF1 deficiency. Treatment with the Erk1/2 inhibitor, PD98059, resulted in a slight increase in Bcl-xL levels in neurofibromin-depleted normal HSCs, indicating that Bcl-xL upregulation in MPNST-derived SCs is mediated by activated Erk1/2, which is a Ras downstream protein regulated by neurofibromin. As the reduction of Bcl-xL expression restored sensitivity to doxorubicin-induced apoptosis in sNF96.2 cells, we examined the effect of the small molecule Bcl-xL inhibitor ABT-737 on sNF96.2 cells. A very low dose of ABT-737 combined with doxorubicin synergistically enhanced sensitivity to doxorubicin-induced apoptosis in sNF96.2 cells, suggesting that ABT-737 and doxorubicin may be a good combination to effectively treat NF1-associated MPNSTs with minimal side-effects. Collectively, our results suggest that upregulation of Bcl-xL in MPNST-derived SCs may be caused by the NF1 deficiency

  1. Phospholipase C δ-4 overexpression upregulates ErbB1/2 expression, Erk signaling pathway, and proliferation in MCF-7 cells

    Directory of Open Access Journals (Sweden)

    Morris Valerie

    2004-05-01

    Full Text Available Abstract Background The expression of the rodent phosphoinositide-specific phospholipase C δ-4 (PLCδ4 has been found to be elevated upon mitogenic stimulation and expression analysis have linked the upregulation of PLCδ4 expression with rapid proliferation in certain rat transformed cell lines. The human homologue of PLCδ4 has not been extensively characterized. Accordingly, we investigate the effects of overexpression of human PLCδ4 on cell signaling and proliferation in this study. Results The cDNA for human PLCδ4 has been isolated and expressed ectopically in breast cancer MCF-7 cells. Overexpression of PLCδ4 selectively activates protein kinase C-φ and upregulates the expression of epidermal growth factor receptors EGFR/erbB1 and HER2/erbB2, leading to constitutive activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2 pathway in MCF-7 cells. MCF-7 cells stably expressing PLCδ4 demonstrates several phenotypes of transformation, such as rapid proliferation in low serum, formation of colonies in soft agar, and capacity to form densely packed spheroids in low-attachment plates. The growth signaling responses induced by PLCδ4 are not reversible by siRNA. Conclusion Overexpression or dysregulated expression of PLCδ4 may initiate oncogenesis in certain tissues through upregulation of ErbB expression and activation of ERK pathway. Since the growth responses induced by PLCδ4 are not reversible, PLCδ4 itself is not a suitable drug target, but enzymes in pathways activated by PLCδ4 are potential therapeutic targets for oncogenic intervention.

  2. Irradiation with narrowband-ultraviolet B suppresses phorbol ester-induced up-regulation of H1 receptor mRNA in HeLa cells.

    Science.gov (United States)

    Kitamura, Yoshiaki; Mizuguchi, Hiroyuki; Okamoto, Kentaro; Kitayama, Mika; Fujii, Tatsuya; Fujioka, Akira; Matsushita, Toshio; Mukai, Takashi; Kubo, Yoshiaki; Kubo, Nobuo; Fukui, Hiroyuki; Takeda, Noriaki

    2016-01-01

    Conclusion These findings suggest that low dose irradiation with 310 nm NB-UVB specifically suppressed the up-regulation of H1R gene expression without inducing apoptosis and that UVB of shorter or longer wavelength than 310 nm NB-UVB had no such effects. Objective To develop a narrowband-ultraviolet B(NB-UVB) phototherapy for allergic rhinitis, this study investigated the effects of irradiation with NB-UVB at wavelength of 310 nm on phorbol-12-myristate-13-acetate (PMA)-induced up-regulation of histamine H1 receptor (H1R) mRNA in HeLa cells. Methods The mRNA levels of H1R in HeLa cells were measured using real-time RT-PCR. Apoptosis were evaluated with DNA fragmentation assay. Results PMA induced a significant increase in H1R mRNA expression in HeLa cells. Irradiation with 305 nm UVB and 310 nm NB-UVB, but not with 315 nm UVB at doses of 200 and 300 mJ/cm(2) significantly suppressed PMA-induced up-regulation of H1R mRNA. At a dose of 200 mJ/cm(2), irradiation with 305 nm UVB, but not with 310 nm NB-UVB, induced apoptosis, although exposure of the cells to both 305 and 310 nm UVB induced apoptosis at a dose of 300 mJ/cm(2) after PMA treatment in HeLa cells. Conversely, irradiation with 315 nm UVB at doses of 200 and 300 mJ/cm(2) did not induce apoptosis.

  3. Increased oxidative stress in AOA3 cells disturbs ATM-dependent DNA damage responses.

    Science.gov (United States)

    Kobayashi, Junya; Saito, Yuichiro; Okui, Michiyo; Miwa, Noriko; Komatsu, Kenshi

    2015-04-01

    Ataxia telangiectasia (AT) is caused by a mutation in the ataxia-telangiectasia-mutated (ATM) gene; the condition is associated with hyper-radiosensitivity, abnormal cell-cycle checkpoints, and genomic instability. AT patients also show cerebellar ataxia, possibly due to reactive oxygen species (ROS) sensitivity in neural cells. The ATM protein is a key regulator of the DNA damage response. Recently, several AT-like disorders have been reported. The genes responsible for them are predicted to encode proteins that interact with ATM in the DNA-damage response. Ataxia with oculomotor apraxia types 1-3 (AOA1, 2, and 3) result in a neurodegenerative and cellular phenotype similar to AT; however, the basis of this phenotypic similarity is unclear. Here, we show that the cells of AOA3 patients display aberrant ATM-dependent phosphorylation and apoptosis following γ-irradiation. The ATM-dependent response to H2O2 treatment was abrogated in AOA3 cells. Furthermore, AOA3 cells had reduced ATM activity. Our results suggest that the attenuated ATM-related response is caused by an increase in endogenous ROS in AOA3 cells. Pretreatment of cells with pyocyanin, which induces endogenous ROS production, abolished the ATM-dependent response. Moreover, AOA3 cells had decreased homologous recombination (HR) activity, and pyocyanin pretreatment reduced HR activity in HeLa cells. These results indicate that excess endogenous ROS represses the ATM-dependent cellular response and HR repair in AOA3 cells. Since the ATM-dependent cell-cycle checkpoint is an important block to carcinogenesis, such inactivation of ATM may lead to tumorigenesis as well as neurodegeneration.

  4. Girdin/GIV is upregulated by cyclic tension, propagates mechanical signal transduction, and is required for the cellular proliferation and migration of MG-63 cells

    Energy Technology Data Exchange (ETDEWEB)

    Hu, Jiang-Tian; Li, Yan; Yu, Bing; Gao, Guo-Jie; Zhou, Ting; Li, Song, E-mail: song_li59@126.com

    2015-08-21

    To explore how Girdin/GIV is regulated by cyclic tension and propagates downstream signals to affect cell proliferation and migration. Human osteoblast-like MG-63 cells were exposed to cyclic tension force at 4000 μstrain and 0.5 Hz for 6 h, produced by a four-point bending system. Cyclic tension force upregulated Girdin and Akt expression and phosphorylation in cultured MG-63 cells. Girdin and Akt each promoted the phosphorylation of the other under stimulated tension. In vitro MTT and transwell assays showed that Girdin and Akt are required for cell proliferation and migration during cellular quiescence. Moreover, STAT3 was determined to be essential for Girdin expression under stimulated tension force in the physiological condition, as well as for osteoblast proliferation and migration during quiescence. These findings suggest that the STAT3/Girdin/Akt pathway activates in osteoblasts in response to mechanical stimulation and may play a significant role in triggering osteoblast proliferation and migration during orthodontic treatment. - Highlights: • Tension force upregulates Girdin and Akt expression and phosphorylation. • Girdin and Akt promotes the phosphorylation of each other under tension stimulation. • Girdin and Akt are required for MG-63 cell proliferation and migration. • STAT3 is essential for Girdin expression after application of the tension forces.

  5. Aging disturbs the balance between effector and regulatory CD4+T cells

    NARCIS (Netherlands)

    van der Geest, Kornelis S. M.; Abdulahad, Wayel H.; Tete, Sarah M.; Lorencetti, Pedro G.; Horst, Gerda; Bos, Nicolaas A.; Kroesen, Bart-Jan; Brouwer, Elisabeth; Boots, Annemieke M. H.

    2014-01-01

    Healthy aging requires an optimal balance between pro-inflammatory and anti-inflammatory immune responses. Although CD4+ T cells play an essential role in many immune responses, few studies have directly assessed the effect of aging on the balance between effector T (Teff) cells and regulatory T (Tr

  6. β Integrin-like protein-mediated adhesion and its disturbances during cell cultivation of the mussel Mytilus trossulus.

    Science.gov (United States)

    Maiorova, Mariia A; Odintsova, Nelly A

    2015-08-01

    In this study, we focus on the specific contribution of β integrin-like protein to adhesion-mediated events in molluscan larval cells in culture that could not have been investigated within the whole animal. An analysis of disturbances to cell-substratum adhesion, caused by the integrin receptor inhibiting Arg-Gly-Asp-Ser (RGDS)-peptide, the Ca(2+)/Mg(2+)-chelators and the stress influence of freezing-thawing, reveals that all these factors resulted in the partial destruction of the integrin-extracellular matrix (ECM) interaction in culture and, in particular, changes in the distribution and relative abundance of β integrin-positive cells. The experiments, carried out on selected substrates, found that β integrin-positive cells demonstrate different affinities for the substrates. This finding further supports the assumption that epithelial differentiation in cultivated cells of larval Mytilus may be mediated by β integrin-like proteins via binding to laminin; direct binding to other components of the ECM could not be demonstrated. The mussel β integrin-positive cells are not involved in myogenic or neuronal differentiation on any of the substrates but part of them has tubulin-positive cilia, forming some epithelia-like structures. Our data indicate that β integrin-positive cells are able to proliferate in vitro which suggests that they could participate in renewing the digestive epithelium in larvae. The findings provide evidence that the distribution pattern of β integrin-like protein depends on the cell type and the factors influencing the adhesion.

  7. Pancreatic adenocarcinoma upregulated factor (PAUF) confers resistance to pancreatic cancer cells against oncolytic parvovirus H-1 infection through IFNA receptor-mediated signaling

    Energy Technology Data Exchange (ETDEWEB)

    Kaowinn, Sirichat; Cho, Il-Rae; Moon, Jeong; Jun, Seung Won; Kim, Chang Seok [BK21+, Department of Cogno-Mechatronics Engineering, Pusan National University, Busan 609-736 (Korea, Republic of); Kang, Ho Young [Department of Microbiology, Pusan National University, Busan 609-736 (Korea, Republic of); Kim, Manbok [Department of Medical Science, Dankook University College of Medicine, Cheonan 330-714 (Korea, Republic of); Koh, Sang Seok [Department of Biological Sciences, Dong-A University, Busan 604-714 (Korea, Republic of); Chung, Young-Hwa, E-mail: younghc@pusan.ac.kr [BK21+, Department of Cogno-Mechatronics Engineering, Pusan National University, Busan 609-736 (Korea, Republic of)

    2015-04-03

    Pancreatic adenocarcinoma upregulated factor (PAUF), a novel oncogene, plays a crucial role in the development of pancreatic cancer, including its metastasis and proliferation. Therefore, PAUF-expressing pancreatic cancer cells could be important targets for oncolytic virus-mediated treatment. Panc-1 cells expressing PAUF (Panc-PAUF) showed relative resistance to parvovirus H-1 infection compared with Panc-1 cells expressing an empty vector (Panc-Vec). Of interest, expression of type I IFN-α receptor (IFNAR) was higher in Panc-PAUF cells than in Panc-Vec cells. Increased expression of IFNAR in turn increased the activation of Stat1 and Tyk2 in Panc-PAUF cells compared with that in Panc-Vec cells. Suppression of Tyk2 and Stat1, which are important downstream molecules for IFN-α signaling, sensitized pancreatic cancer cells to parvovirus H-1-mediated apoptosis. Further, constitutive suppression of PAUF sensitized Bxpc3 pancreatic cancer cells to parvovirus H-1 infection. Taken together, these results suggested that PAUF conferred resistance to pancreatic cancer cells against oncolytic parvovirus H-1 infection through IFNAR-mediated signaling. - Highlights: • PAUF confers resistance against oncolytic parvovirus H-1 infection. • PAUF enhances the expression of IFNAR in Panc-1 cells. • Increased activation of Tyk2 or Stat1 by PAUF provides resistance to parvovirus H-1-mediated apoptosis. • Constitutive inhibition of PAUF enhances parvovirus H-1-mediated oncolysis of Bxpc3 pancreatic cancer cells.

  8. IL-1β is upregulated in the diabetic retina and retinal vessels: cell-specific effect of high glucose and IL-1β autostimulation.

    Science.gov (United States)

    Liu, Yang; Biarnés Costa, Montserrat; Gerhardinger, Chiara

    2012-01-01

    Many molecular and cellular abnormalities detected in the diabetic retina support a role for IL-1β-driven neuroinflammation in the pathogenesis of diabetic retinopathy. IL-1β is well known for its role in the induction and, through autostimulation, amplification of neuroinflammation. Upregulation of IL-1β has been consistently detected in the diabetic retina; however, the mechanisms and cellular source of IL-1β overexpression are poorly understood. The aim of this study was to investigate the effect of high glucose and IL-1β itself on IL-1β expression in microglial, macroglial (astrocytes and Müller cells) and retinal vascular endothelial cells; and to study the effect of diabetes on the expression of IL-1β in isolated retinal vessels and on the temporal pattern of IL-1β upregulation and glial reactivity in the retina of streptozotocin-diabetic rats. IL-1β was quantified by RealTime RT-PCR and ELISA, glial fibrillar acidic protein, α2-macroglobulin, and ceruloplasmin by immunoblotting. We found that high glucose induced a 3-fold increase of IL-1β expression in retinal endothelial cells but not in macroglia and microglia. IL-1β induced its own synthesis in endothelial and macroglial cells but not in microglia. In retinal endothelial cells, the high glucose-induced IL-1β overexpression was prevented by calphostin C, a protein kinase C inhibitor. The retinal vessels of diabetic rats showed increased IL-1β expression as compared to non-diabetic rats. Retinal expression of IL-1β increased early after the induction of diabetes, continued to increase with progression of the disease, and was temporally associated with upregulation of markers of glial activation. These findings point to hyperglycemia as the trigger and to the endothelium as the origin of the initial retinal upregulation of IL-1β in diabetes; and to IL-1β itself, via autostimulation in endothelial and macroglial cells, as the mechanism of sustained IL-1β overexpression. Interrupting the

  9. IL-1β is upregulated in the diabetic retina and retinal vessels: cell-specific effect of high glucose and IL-1β autostimulation.

    Directory of Open Access Journals (Sweden)

    Yang Liu

    Full Text Available Many molecular and cellular abnormalities detected in the diabetic retina support a role for IL-1β-driven neuroinflammation in the pathogenesis of diabetic retinopathy. IL-1β is well known for its role in the induction and, through autostimulation, amplification of neuroinflammation. Upregulation of IL-1β has been consistently detected in the diabetic retina; however, the mechanisms and cellular source of IL-1β overexpression are poorly understood. The aim of this study was to investigate the effect of high glucose and IL-1β itself on IL-1β expression in microglial, macroglial (astrocytes and Müller cells and retinal vascular endothelial cells; and to study the effect of diabetes on the expression of IL-1β in isolated retinal vessels and on the temporal pattern of IL-1β upregulation and glial reactivity in the retina of streptozotocin-diabetic rats. IL-1β was quantified by RealTime RT-PCR and ELISA, glial fibrillar acidic protein, α2-macroglobulin, and ceruloplasmin by immunoblotting. We found that high glucose induced a 3-fold increase of IL-1β expression in retinal endothelial cells but not in macroglia and microglia. IL-1β induced its own synthesis in endothelial and macroglial cells but not in microglia. In retinal endothelial cells, the high glucose-induced IL-1β overexpression was prevented by calphostin C, a protein kinase C inhibitor. The retinal vessels of diabetic rats showed increased IL-1β expression as compared to non-diabetic rats. Retinal expression of IL-1β increased early after the induction of diabetes, continued to increase with progression of the disease, and was temporally associated with upregulation of markers of glial activation. These findings point to hyperglycemia as the trigger and to the endothelium as the origin of the initial retinal upregulation of IL-1β in diabetes; and to IL-1β itself, via autostimulation in endothelial and macroglial cells, as the mechanism of sustained IL-1β overexpression

  10. Up-regulation of ribosomal protein S13 and L23 are associated with multidrug-resistant phenotype of gastric cancer cells

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    Aims:Prevous study using differential display-PCR had found up-regulation of ribosomal protein S13(RPS13) and L23(RPL23) in vincristine-resistant gastric cancer cells.The aim of this study was to explore the association of RPS13 and RPL23 with multidrug-resistant phenotype of gastric cancer cells.Methods:Northern blot analysis was used to determine the expression of RPS13 and RPL23 in vincristine-resistant gastric cancer cells SGC7901/VCR and its parental cells SGC7901.The full-length cDNAs encoding RPS13 and RPL23 were amplified from SGC7901/VCR cells using RT-PCR.Their sense and antisense expression vectors were constructed by DNA recombinant technique,and transferred into SGC7901 cells(sense vectors) or SGC7901/VCR cells(antisense vectors)by means of Lipofactamine.Drug sensitivity of gastric cancer cells was evalu-ated using MTT assay.Cell cycle analysis was performed using flow cytometry and proliferous index(PI) was calculated.Results:As Northem blot analysis indicated,RNA from SGC7902/VCR cells exhibited moderate signals of PRL23 and RPS13,while RNA from SGC7901 cells exhibited no signal of RPL23 and very weak signal of RPS13.RNA dot blot analysis indicated that RPS13 or RPL23 upregulated SGC7901 cells(SGC7901-RPS13,SGC7901-RPL23)and RPS13 or RPL23 down-regulated SGC7901/VCR cells(SGC7901/VCR-anRPS13,SGC7901/VCR-anRPL23) were successfully prepared by gene transduction.The results of MTT assay indicated that,comparing with non-transfected and empty vector transfected cells,SGC7901-RPL23 cells showed significantly increased IC50 values and resistance index(RI) of vincristine(VCR),adriamycin(ADR),5-fludrouracil(5-Fu) and mitomycin(MMC);SGC7901-RPS12 cells showed significantly increased IC50 values and RI of VCR,ADR and 5-Fu;SGC7901/VCR-anRPL23 cells showed significantly decreased IC50 values and RI of MMC and cisplatin(DDP);SGC7901/VCR-anRPS13 cells showed significantly decreased IC50 values and RI of VCR and MMC.Cell cycle analysis indicated that,comparing with

  11. Carbon nanoparticle induced cytotoxicity in human mesenchymal stem cells through upregulation of TNF3, NFKBIA and BCL2L1 genes.

    Science.gov (United States)

    Periasamy, Vaiyapuri S; Athinarayanan, Jegan; Alfawaz, Mohammed A; Alshatwi, Ali A

    2016-02-01

    Carbon based nanomaterials, including carbon nanotubes, graphene, nanodiamond and carbon nanoparticles, have emerged as potential candidates for a wide variety of applications because of their unusual electrical, mechanical, thermal and optical properties. However, our understanding of how increased usage of carbon based nanomaterials could lead to harmful effects in humans and other biological systems is inadequate. Our present investigation is focused on the cellular toxicity of carbon nanoparticles (CNPs) on human mesenchymal stem cells (hMSCs). Following exposure to CNPs, cell viability, nuclear morphological changes, apoptosis and cell cycle progression were monitored. Furthermore, the expression of genes involved in both cell death (e.g., P53, TNF3, CDKN1A, TNFRSF1A, TNFSF10, NFKBIA, BCL2L1) and cell cycle regulation (e.g., PCNA, EGR1, E2F1, CCNG1, CCND1, CCNC, CYCD3) were assessed using qPCR. Our results indicated that CNPs reduce cell viability and cause chromatin condensation and DNA fragmentation. Cell cycle analysis indicated that CNPs affect the cell cycle progression. However, the gene expression measurements confirmed that CNPs significantly upregulated the P53, TNF3, CDKNIA, and NFKBIA genes and downregulated the EGR1 gene in hMSCs. Our findings suggest that CNPs reduce cell viability by disrupting the expression of cell death genes in human mesenchymal stem cell (hMSC). The results of this investigation revealed that CNPs exhibited moderate toxicity on hMSCs.

  12. Upregulation of vascular endothelial growth factor by cobalt chloride-simulated hypoxia is mediated by persistent induction of cyclooxygenase-2 in a metastatic human prostate cancer cell line.

    Science.gov (United States)

    Liu, X H; Kirschenbaum, A; Yao, S; Stearns, M E; Holland, J F; Claffey, K; Levine, A C

    1999-01-01

    Upregulation of vascular endothelial growth factor (VEGF) expression induced by hypoxia is crucial event leading to neovascularization. Cyclooxygenase-2, an inducible enzyme that catalyzes the formation of prostaglandins (PGs) from arachidonic acid, has been demonstrated to be induced by hypoxia and play role in angiogenesis and metastasis. To investigate the potential effect of COX-2 on hypoxia-induced VEGF expression in prostate cancer. We examined the relationship between COX-2 expression and VEGF induction in response to cobalt chloride (CoCl2)-simulated hypoxia in three human prostate cancer cell lines with differing biological phenotypes. Northern blotting and ELISA revealed that all three tested cell lines constitutively expressed VEGF mRNA, and secreted VEGF protein to different degrees (LNCaP > PC-3 > PC3ML). However, these cell lines differed in the ability to produce VEGF in the presence of CoCl2-simulated hypoxia. CoCl2 treatment resulted in 40% and 75% increases in VEGF mRNA, and 50% and 95% in protein secretion by LNCaP and PC-3 cell lines, respectively. In contrast, PC-3ML cell line, a PC-3 subline with highly invasive, metastatic phenotype, exhibits a dramatic upregulation of VEGF, 5.6-fold in mRNA and 6.3-fold in protein secretion after treatment with CoCl2. The upregulation of VEGF in PC-3ML cells is accompanied by a persistent induction of COX-2 mRNA (6.5-fold) and protein (5-fold). Whereas COX-2 expression is only transiently induced in PC-3 cells and not affected by CoCl2 in LNCaP cells. Moreover, the increases in VEGF mRNA and protein secretion induced by CoCl2 in PC-3ML cells were significantly suppressed following exposure to NS398, a selective COX-2 inhibitor. Finally, the effect of COX-2 inhibition on CoCl2-induced VEGF production was reversed by the treatment with exogenous PGE2. Our data demonstrate that VEGF induction by cobalt chloride-simulated hypoxia is maintained by a concomitant, persistent induction of COX-2 expression and

  13. Establishment of IL-7 Expression Reporter Human Cell Lines, and Their Feasibility for High-Throughput Screening of IL-7-Upregulating Chemicals

    Science.gov (United States)

    Sim, Chan Kyu; Kim, Inki

    2016-01-01

    Interleukin-7 (IL-7) is a cytokine essential for T cell homeostasis, and is clinically important. However, the regulatory mechanism of IL-7 gene expression is not well known, and a systematic approach to screen chemicals that regulate IL-7 expression has not yet been developed. In this study, we attempted to develop human reporter cell lines using CRISPR/Cas9-mediated genome editing technology. For this purpose, we designed donor DNA that contains an enhanced green fluorescent protein (eGFP) gene, drug selection cassette, and modified homologous arms which are considered to enhance the translation of the eGFP reporter transcript, and also a highly efficient single-guide RNA with a minimal off-target effect to target the IL-7 start codon region. By applying this system, we established IL-7 eGFP reporter cell lines that could report IL-7 gene transcription based on the eGFP protein signal. Furthermore, we utilized the cells to run a pilot screen campaign for IL-7-upregulating chemicals in a high-throughput format, and identified a chemical that can up-regulate IL-7 gene transcription. Collectively, these results suggest that our IL-7 reporter system can be utilized in large-scale chemical library screening to reveal novel IL-7 regulatory pathways and to identify potential drugs for development of new treatments in immunodeficiency disease. PMID:27589392

  14. SDF-1/CXCR4 Axis Regulates Cell Cycle Progression and Epithelial-Mesenchymal Transition via Up-regulation of Survivin in Glioblastoma.

    Science.gov (United States)

    Liao, Anyan; Shi, Ranran; Jiang, Yuliang; Tian, Suqing; Li, Panpan; Song, Fuxi; Qu, Yalan; Li, Jinna; Yun, Haiqin; Yang, Xiangshan

    2016-01-01

    Stromal cell-derived factor 1 (SDF-1)/CXCR4 ligand-receptor axis is widely recommended as an attractive target for cancer therapy. Meanwhile, epithelial-mesenchymal transition (EMT) process is linked to disease pathophysiology. As one of inhibitors of apoptosis proteins, survivin is implicated in the onset and development of cancer. In the present study, we tried to determine the cause-effect associations between SDF-1/CXCR4 axis and survivin expression in glioblastoma U-251 cell line. Survivin activation and inhibition were induced with exogenous SDF-1 and survivin small interfering RNA (survivin siRNA), respectively. Western blot was used to detect relevant proteins in SDF-1/CXCR4 axis. Western blot analysis revealed that survivin expression in U-251 increased in a dose- and time-dependent manner in response to SDF-1 treatment. However, the interference with MEK/ERK and PI3K/AKT pathway prohibited SDF-1-induced survivin up-regulation. Importantly, survivin knockdown abrogated cell cycle progression and the expression of snail and N-cadherin, compared with non-transfectants. In conclusion, the present study shows that SDF-1 up-regulates survivin via MEK/ERK and PI3K/AKT pathway, leading to cell cycle progression and EMT occurrence dependent on survivin. The blockade of survivin will allow for the treatment of glioblastoma.

  15. Safrole oxide induces apoptosis by up-regulating Fas and FasL instead of integrin beta4 in A549 human lung cancer cells.

    Science.gov (United States)

    Du, AiYing; Zhao, BaoXiang; Miao, JunYing; Yin, DeLing; Zhang, ShangLi

    2006-04-01

    Previously, we found that 3,4-(methylenedioxy)-1-(2',3'-epoxypropyl)-benzene (safrole oxide) induced a typical apoptosis in A549 human lung cancer cells by activating caspase-3, -8, and -9. In this study, we further investigated which upstream pathways were activated by safrole oxide during the apoptosis. Immunofluorescence assay combined with laser scanning confocal microscopy revealed that both Fas and Fas ligand (FasL) were up-regulated by the small molecule. In addition, Fas protein distribution was altered, showing a clustering distribution instead of a homogeneous one. Subsequently, Western blot analysis confirmed the up-regulations of Fas and its membrane-binding form of FasL (m-FasL), as well as P53 protein. Conversely, safrole oxide hardly affected integrin beta4 subunit expression or distribution, which was reflected from the data obtained by immunofluorescence assay combined with laser scanning confocal microscopy. The results suggested that Fas/FasL pathway might be involved in safrole oxide-induced apoptosis of A549 cells, while integrin beta4 might be irrelevant to the apoptosis. Nevertheless, we first found the strong expression of integrin beta4 in A549 cells. The study first suggested that safrole oxide might be used as a small molecular promoter of Fas/FasL pathway to elicit apoptosis in A549 cells, which would lay the foundation for us to insight into the new strategies for lung cancer therapy.

  16. An in vitro embryotoxicity assay based on the disturbance of the differentiation of murine embryonic stem cells into endothelial cells. II. Testing of compounds.

    Science.gov (United States)

    Festag, Matthias; Viertel, Bruno; Steinberg, Pablo; Sehner, Claudia

    2007-12-01

    The embryonic stem cell test (EST) developed by Spielmann et al. [Spielmann, H., Pohl, I., Doering, B., Liebsch, M., Moldenhauer, F., 1997. The embryonic stem cell test, an in vitro embryotoxicity test using two permanent mouse cell lines: 3T3 fibroblasts and embryonic stem cells. In Vitro. Toxicol. 10, 119-127] is currently the most promising in vitro assay to predict the embryotoxic potential of compounds. In this assay the disturbance of the differentiation of embryonic stem (ES) cells into contracting cardiomyocytes by test compounds as well as the direct cytotoxicity of the test compounds on ES cells and 3T3 fibroblasts is analyzed. On the basis of these results and by applying a biostatistical prediction model (PM) [Genschow, E., Scholz, G., Brown, N., Piersma, A., Brady, M., Clemann, N., Huuskonen, H., Paillard, F., Bremer, S., Becker, K., Spielmann, H., 2000. Development of prediction models for three in vitro embryotoxicity tests in an ECVAM validation study. In Vitr. Mol. Toxicol. 13, 51-66; Genschow, E., Spielmann, H., Scholz, G., Pohl, I., Seiler, A., Clemann, N., Bremer, S., Becker, K., 2004. Validation of the embryonic stem cell test in the international ECVAM validation study on three in vitro embryotoxicity tests. Altern. Lab. Anim. 32, 209-244; Genschow, E., Spielmann, H., Scholz, G., Seiler, A., Brown, N., Piersma, A., Brady, M., Clemann, N., Huuskonen, H., Paillard, F., Bremer, S., Becker, K., 2002. The ECVAM international validation study on in vitro embryotoxicity tests: results of the definitive phase and evaluation of prediction models. European Centre for the Validation of Alternative Methods. Altern. Lab. Anim. 30, 151-176] test compounds can be classified as non-embryotoxic, weakly or strongly embryotoxic. In order to introduce a further endpoint into the EST, the disturbance of vasculogenesis and/or angiogenesis, a protocol to differentiate ES cells into endothelial cells, was established in the accompanying paper. PECAM-1 and VE

  17. Elevated STAT3 Signaling-Mediated Upregulation of MMP-2/9 Confers Enhanced Invasion Ability in Multidrug-Resistant Breast Cancer Cells

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    Fei Zhang

    2015-10-01

    Full Text Available The development of multidrug resistance greatly impedes effective cancer therapy. Recent advances in cancer research have demonstrated that acquisition of multidrug resistance by cancer cells is usually accompanied by enhanced cell invasiveness. Several lines of evidence indicated that cross activation of other signaling pathways during development of drug resistance may increase invasive potential of multidrug-resistant (MDR cancer cells. However, the accurate mechanism of this process is largely undefined. In this study, to better understand the associated molecular pathways responsible for cancer progression induced by drug resistance, a MDR human breast cancer cell line SK-BR-3/EPR with P-glycoprotein overexpression was established using stepwise long-term exposure to increasing concentration of epirubicin. The SK-BR-3/EPR cell line exhibited decreased cell proliferative activity, but enhanced cell invasive capacity. We showed that the expression of metastasis-related matrix metalloproteinase (MMP-2/9 was elevated in SK-BR-3/EPR cells. Moreover, SK-BR-3/EPR cells showed elevated activation of STAT3. Activation of STAT3 signaling is responsible for enhanced invasiveness of SK-BR-3/EPR cells through upregulation of MMP-2/9. STAT3 is a well-known oncogene and is frequently implicated in tumorigenesis and chemotherapeutic resistance. Our findings augment insight into the mechanism underlying the functional association between MDR and cancer invasiveness.

  18. Elevated STAT3 Signaling-Mediated Upregulation of MMP-2/9 Confers Enhanced Invasion Ability in Multidrug-Resistant Breast Cancer Cells.

    Science.gov (United States)

    Zhang, Fei; Wang, Zhiyong; Fan, Yanling; Xu, Qiao; Ji, Wei; Tian, Ran; Niu, Ruifang

    2015-10-16

    The development of multidrug resistance greatly impedes effective cancer therapy. Recent advances in cancer research have demonstrated that acquisition of multidrug resistance by cancer cells is usually accompanied by enhanced cell invasiveness. Several lines of evidence indicated that cross activation of other signaling pathways during development of drug resistance may increase invasive potential of multidrug-resistant (MDR) cancer cells. However, the accurate mechanism of this process is largely undefined. In this study, to better understand the associated molecular pathways responsible for cancer progression induced by drug resistance, a MDR human breast cancer cell line SK-BR-3/EPR with P-glycoprotein overexpression was established using stepwise long-term exposure to increasing concentration of epirubicin. The SK-BR-3/EPR cell line exhibited decreased cell proliferative activity, but enhanced cell invasive capacity. We showed that the expression of metastasis-related matrix metalloproteinase (MMP)-2/9 was elevated in SK-BR-3/EPR cells. Moreover, SK-BR-3/EPR cells showed elevated activation of STAT3. Activation of STAT3 signaling is responsible for enhanced invasiveness of SK-BR-3/EPR cells through upregulation of MMP-2/9. STAT3 is a well-known oncogene and is frequently implicated in tumorigenesis and chemotherapeutic resistance. Our findings augment insight into the mechanism underlying the functional association between MDR and cancer invasiveness.

  19. Pancreatic adenocarcinoma upregulated factor (PAUF) confers resistance to pancreatic cancer cells against oncolytic parvovirus H-1 infection through IFNA receptor-mediated signaling.

    Science.gov (United States)

    Kaowinn, Sirichat; Cho, Il-Rae; Moon, Jeong; Jun, Seung Won; Kim, Chang Seok; Kang, Ho Young; Kim, Manbok; Koh, Sang Seok; Chung, Young-Hwa

    2015-04-01

    Pancreatic adenocarcinoma upregulated factor (PAUF), a novel oncogene, plays a crucial role in the development of pancreatic cancer, including its metastasis and proliferation. Therefore, PAUF-expressing pancreatic cancer cells could be important targets for oncolytic virus-mediated treatment. Panc-1 cells expressing PAUF (Panc-PAUF) showed relative resistance to parvovirus H-1 infection compared with Panc-1 cells expressing an empty vector (Panc-Vec). Of interest, expression of type I IFN-α receptor (IFNAR) was higher in Panc-PAUF cells than in Panc-Vec cells. Increased expression of IFNAR in turn increased the activation of Stat1 and Tyk2 in Panc-PAUF cells compared with that in Panc-Vec cells. Suppression of Tyk2 and Stat1, which are important downstream molecules for IFN-α signaling, sensitized pancreatic cancer cells to parvovirus H-1-mediated apoptosis. Further, constitutive suppression of PAUF sensitized Bxpc3 pancreatic cancer cells to parvovirus H-1 infection. Taken together, these results suggested that PAUF conferred resistance to pancreatic cancer cells against oncolytic parvovirus H-1 infection through IFNAR-mediated signaling.

  20. Copper-induced root growth inhibition of Allium cepa var. agrogarum L. involves disturbances in cell division and DNA damage.

    Science.gov (United States)

    Qin, Rong; Wang, Congyue; Chen, Da; Björn, Lars O; Li, Shaoshan

    2015-05-01

    Copper (Cu) is considered to be an indispensable microelement for plants. Excessive Cu, however, is toxic and disturbs several processes in the plant. The present study addressed the effects of ionic Cu (2.0 µM and 8.0 µM) on mitosis, the microtubule cytoskeleton, and DNA in root tip cells of Allium cepa var. agrogarum L. to better understand Cu toxicity on plant root systems. The results indicated that Cu accumulated in roots and that root growth was inhibited dramatically in Cu treatment groups. Chromosomal aberrations (for example, C-mitosis, chromosome bridges, chromosome stickiness, and micronucleus) were observed, and the mitotic index decreased during Cu treatments at different concentrations. Microtubules were one of the target sites of Cu toxicity in root tip meristematic cells, and Cu exposure substantially impaired microtubule arrangements. The content of α-tubulin decreased following 36 h of exposure to 2.0 µM or 8.0 µM of Cu in comparison with the control group. Copper increased DNA damage and suppressed cell cycle progression. The above toxic effects became more serious with increasing Cu concentration and prolonged exposure time.

  1. P2 receptors in human heart: upregulation of P2X6 in patients undergoing heart transplantation, interaction with TNFalpha and potential role in myocardial cell death.

    Science.gov (United States)

    Banfi, Cristina; Ferrario, Silvia; De Vincenti, Ombretta; Ceruti, Stefania; Fumagalli, Marta; Mazzola, Alessia; D' Ambrosi, Nadia; Volontè, Cinzia; Fratto, Pasquale; Vitali, Ettore; Burnstock, Geoffrey; Beltrami, Elena; Parolari, Alessandro; Polvani, GianLuca; Biglioli, Paolo; Tremoli, Elena; Abbracchio, Maria P

    2005-12-01

    ATP acts as a neurotransmitter via seven P2X receptor-channels for Na(+) and Ca(2+), and eight G-protein-coupled P2Y receptors. Despite evidence suggesting roles in human heart, the map of myocardial P2 receptors is incomplete, and their involvement in chronic heart failure (CHF) has never received adequate attention. In left myocardia from five to nine control and 5-12 CHF subjects undergoing heart transplantation, we analyzed the full repertoire of P2 receptors and of 10 "orphan" P2Y-like receptors. All known P2Y receptors (i.e. P2Y(1,2,4,6,11,12,13,14)) and two P2Y-like receptors (GPR91 and GPR17) were detected in all subjects. All known P2X(1-7) receptors were also detected; of these, only P2X(6) was upregulated in CHF, as confirmed by quantitative real time-PCR. The potential significance of this change was studied in primary cardiac fibroblasts freshly isolated from young pigs. Exposure of cardiac fibroblasts to ATP or its hydrolysis-resistant-analog benzoylATP induced apoptosis. TNFalpha (a cytokine implicated in CHF progression) exacerbated cell death. Similar effects were induced by ATP and TNFalpha in a murine cardiomyocytic cell line. In cardiac fibroblasts, TNFalpha inhibited the downregulation of P2X(6) mRNA associated to prolonged agonist exposure, suggesting that, by preventing ATP-induced P2X(6) desensitization, TNFalpha may abolish a defense mechanism meant at avoiding Ca(2+) overload and, ultimately, Ca(2+)-dependent cell death. This may provide a basis for P2X(6) upregulation in CHF. In conclusion, we provide the first characterization of P2 receptors in the human heart and suggest that the interaction between TNFalpha and the upregulated P2X(6) receptor may represent a novel pathogenic mechanism in CHF.

  2. A new 2-aminosteroid induces cellular differentiation and upregulates the expression of MafB and Egr-1 genes respectively in HL-60 and K562 leukemia cells

    Institute of Scientific and Technical Information of China (English)

    HE Qun; LI Qiong; YUAN Lin-bo; HE Jun

    2005-01-01

    Background In previous work, we suggested that some 2-aminosteroids inhibited proliferation and induced differentiation of both human and murine leukemia cells. Here, we reported the actions of another new 2-aminosteroid designated as H89712 on human leukemia cells. Methods Cell colony counting and MTT assay were used to determine proliferation. Cell morphology, histochemical staining, UV detection and cytometry were used to determine differentiation. RT-PCR was used to detect gene expression. Standard statistical method was used to analyze data.Results H89712 inhibited proliferation of HL-60 leukemia cells and the inhibition percentage in MTT assay was 18% at the dose of 10-8 mol/L and 65% at the dose of 10-5 mol/L, respectively. The inhibition for HL-60 in colony assay was 23% at the dose of 10-8 mol/L and 96% at the dose of 10-5 mol/L, respectively. H89712 also induced HL-60 cells toward macrophage-like differentiation. It was verified by flow cytometry that the percentage of positive CD14 expression in differentiated HL-60 cells was about 9 times higher than that of the control at the dose of 10-8 mol/L and 20 times higher than that of the control at the dose of 10-5 mol/L respectively, and this action involved upregulation of MafB gene in HL-60 leukemia cells. On the other hand, H89712 inhibited proliferation of K562 leukemia cells and the inhibition of K562 leukemia cells in MTT assay was shown by 34% at the dose of 10-8 mol/L and 88% at the dose of 10-5 mol/L respectively. The inhibition of K562 leukemia cells in colony assay was 53% at the dose of 10-8 mol/L and 100% at the dose of 10-5 mol/L respectively. H89712 also induced K562 cells toward erythroid-like differentiation and it was verified by flow cytometry that the percentage of positive CD71 expression in differentiated K562 cells was about 9 times higher than that of the control at the dose of 10-8 mol/L and 16 times higher than that of the control at the dose of 10-5 mol/L respectively. This action

  3. HER2 induces cell proliferation and invasion of non-small-cell lung cancer by upregulating COX-2 expression via MEK/ERK signaling pathway

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    Chi F

    2016-05-01

    Full Text Available Feng Chi, Rong Wu, Xueying Jin, Min Jiang, Xike Zhu Department of Medical Oncology, Shengjing Hospital of China Medical University, Shenyang, People’s Republic of China Abstract: HER2 positivity has been well studied in various cancers, but its importance in non-small-cell lung cancer (NSCLC is still being explored. In this study, quantitative reverse transcription polymerase chain reaction (qRT-PCR was performed to detect HER2 and COX-2 expression in NSCLC tissues. Then, pcDNA3.1-HER2 was used to overexpress HER2, while HER2 siRNA and COX-2 siRNA were used to silence HER2 and COX-2 expression. MTT assay and invasion assay were used to detect the effects of HER2 on cell proliferation and invasion. Our study revealed that HER2 and COX-2 expression were upregulated in NSCLC tissues and HER2 exhibited a significant positive correlation with the levels of COX-2 expression. Overexpression of HER2 evidently elevated COX-2 expression, while silencing of HER2 evidently decreased COX-2 expression. Furthermore, overexpressed HER2 induced the ERK phosphorylation, and this was abolished by the treatment with U0126, a pharmacological inhibitor of MEK, an upstream kinase of ERK. HER2-induced expression and promoter activity of COX-2 were also suppressed by U0126, suggesting that the MEK/ERK signaling pathway regulates COX-2 expression. In addition, HER2 induced activation of AKT signaling pathway, which was reversed by pretreatment with U0126 and COX-2 siRNA. MTT and invasion assays revealed that HER2 induced cell proliferation and invasion that were reversed by pretreatment with U0126 and COX-2 siRNA. In this study, our results demonstrated for the first time that HER2 elevated COX-2 expression through the activation of MEK/ERK pathway, which subsequently induced cell proliferation and invasion via AKT pathway in NSCLC tissues. Keywords: HER2, MEK/ERK, COX-2, AKT signaling pathway, non-small-cell lung cancer

  4. Azole fungicides disturb intracellular Ca2+ in an additive manner in dopaminergic PC12 cells.

    Science.gov (United States)

    Heusinkveld, Harm J; Molendijk, Jeffrey; van den Berg, Martin; Westerink, Remco H S

    2013-08-01

    Humans are exposed to complex mixtures of pesticides and other compounds, mainly via food. Azole fungicides are broad spectrum antifungal compounds used in agriculture and in human and veterinary medicine. The mechanism of antifungal action relies on inhibition of CYP51, resulting in inhibition of fungal cell growth. Known adverse health effects of azole fungicides are mainly linked to CYP inhibition. Additionally, azole fungicide-induced neurotoxicity has been reported, though the underlying mechanism(s) are largely unknown. We therefore investigated the effects of a group of six azole fungicides (imazalil, flusilazole, fluconazole, tebuconazole, triadimefon, and cyproconazole) on cell viability using a combined alamar Blue/CFDA-AM assay and on oxidative stress using a H2-DCFDA fluorescent assay. As calcium plays a pivotal role in neuronal survival and functioning, effects of these six azole fungicides and binary and quaternary mixtures of azole fungicides on the intracellular calcium concentration ([Ca(2+)]i) were investigated using single-cell fluorescence microscopy in dopaminergic PC12 cells loaded with the calcium-sensitive fluorescent dye Fura-2. Only modest changes in cell viability and ROS production were observed. However, five out of six azole fungicides induced a nonspecific inhibition of voltage-gated calcium channels (VGCCs), though with varying potency. Experiments using binary IC20 and quaternary IC10 mixtures indicated that the inhibitory effects on VGCCs are additive. The combined findings demonstrate modulation of intracellular Ca(2+) via inhibition of VGCCs as a novel mode of action of azole fungicides. Furthermore, mixtures of azole fungicides display additivity, illustrating the need to take mixture effects into account in human risk assessment.

  5. Malignant progressive tumor cell clone exhibits significant up-regulation of cofilin-2 and 27-kDa modified form of cofilin-1 compared to regressive clone.

    Science.gov (United States)

    Kuramitsu, Yasuhiro; Wang, Yufeng; Okada, Futoshi; Baron, Byron; Tokuda, Kazuhiro; Kitagawa, Takao; Akada, Junko; Nakamura, Kazuyuki

    2013-09-01

    QR-32 is a regressive murine fibrosarcoma cell clone which cannot grow when they are transplanted in mice; QRsP-11 is a progressive malignant tumor cell clone derived from QR-32 which shows strong tumorigenicity. A recent study showed there to be differentially expressed up-regulated and down-regulated proteins in these cells, which were identified by proteomic differential display analyses by using two-dimensional gel electrophoresis and mass spectrometry. Cofilins are small proteins of less than 20 kDa. Their function is the regulation of actin assembly. Cofilin-1 is a small ubiquitous protein, and regulates actin dynamics by means of binding to actin filaments. Cofilin-1 plays roles in cell migration, proliferation and phagocytosis. Cofilin-2 is also a small protein, but it is mainly expressed in skeletal and cardiac muscles. There are many reports showing the positive correlation between the level of cofilin-1 and cancer progression. We have also reported an increased expression of cofilin-1 in pancreatic cancer tissues compared to adjacent paired normal tissues. On the other hand, cofilin-2 was significantly less expressed in pancreatic cancer tissues. Therefore, the present study investigated the comparison of the levels of cofilin-1 and cofilin-2 in regressive QR-32 and progressive QRsP-11cells by western blotting. Cofilin-2 was significantly up-regulated in QRsP-11 compared to QR-32 cells (p<0.001). On the other hand, the difference of the intensities of the bands of cofilin-1 (18 kDa) in QR-32 and QRsP-11 was not significant. However, bands of 27 kDa showed a quite different intensity between QR-32 and QRsP-11, with much higher intensities in QRsP-11 compared to QR-32 (p<0.001). These results suggested that the 27-kDa protein recognized by the antibody against cofilin-1 is a possible biomarker for progressive tumor cells.

  6. Up-regulation of COX-2/PGE2 by endothelin-1 via MAPK-dependent NF-κB pathway in mouse brain microvascular endothelial cells

    Directory of Open Access Journals (Sweden)

    Lin Chih-Chung

    2013-01-01

    Full Text Available Abstract Background Endothelin-1 (ET-1 is a proinflammatory mediator and elevated in the regions of several brain injury and inflammatory diseases. The deleterious effects of ET-1 on endothelial cells may aggravate brain inflammation mediated through the regulation of cyclooxygenase-2 (COX-2/prostaglandin E2 (PGE2 system in various cell types. However, the signaling mechanisms underlying ET-1-induced COX-2 expression in brain microvascular endothelial cells remain unclear. Herein we investigated the effects of ET-1 in COX-2 regulation in mouse brain microvascular endothelial (bEnd.3 cells. Results The data obtained with Western blotting, RT-PCR, and immunofluorescent staining analyses showed that ET-1-induced COX-2 expression was mediated through an ETB-dependent transcriptional activation. Engagement of Gi- and Gq-protein-coupled ETB receptors by ET-1 led to phosphorylation of ERK1/2, p38 MAPK, and JNK1/2 and then activated transcription factor NF-κB. Moreover, the data of chromatin immunoprecipitation (ChIP and promoter reporter assay demonstrated that the activated NF-κB was translocated into nucleus and bound to its corresponding binding sites in COX-2 promoter, thereby turning on COX-2 gene transcription. Finally, up-regulation of COX-2 by ET-1 promoted PGE2 release in these cells. Conclusions These results suggested that in mouse bEnd.3 cells, activation of NF-κB by ETB-dependent MAPK cascades is essential for ET-1-induced up-regulation of COX-2/PGE2 system. Understanding the mechanisms of COX-2 expression and PGE2 release regulated by ET-1/ETB system on brain microvascular endothelial cells may provide rationally therapeutic interventions for brain injury or inflammatory diseases.

  7. Exposure to 9,10-phenanthrenequinone accelerates malignant progression of lung cancer cells through up-regulation of aldo-keto reductase 1B10

    Energy Technology Data Exchange (ETDEWEB)

    Matsunaga, Toshiyuki, E-mail: matsunagat@gifu-pu.ac.jp [Laboratory of Biochemistry, Gifu Pharmaceutical University, Gifu 501-1196 (Japan); Morikawa, Yoshifumi; Haga, Mariko; Endo, Satoshi [Laboratory of Biochemistry, Gifu Pharmaceutical University, Gifu 501-1196 (Japan); Soda, Midori; Yamamura, Keiko [Laboratory of Clinical Pharmacy, School of Pharmacy, Aichi Gakuin University, Nagoya 464-8650 (Japan); El-Kabbani, Ossama [Monash Institute of Pharmaceutical Sciences, Monash University, Victoria 3052 (Australia); Tajima, Kazuo [Faculty of Pharmaceutical Sciences, Hokuriku University, Kanazawa 920-1181 (Japan); Ikari, Akira [Laboratory of Biochemistry, Gifu Pharmaceutical University, Gifu 501-1196 (Japan); Hara, Akira [Faculty of Engineering, Gifu University, Gifu 501-1193 (Japan)

    2014-07-15

    Inhalation of 9,10-phenanthrenequinone (9,10-PQ), a major quinone in diesel exhaust, exerts fatal damage against a variety of cells involved in respiratory function. Here, we show that treatment with high concentrations of 9,10-PQ evokes apoptosis of lung cancer A549 cells through production of reactive oxygen species (ROS). In contrast, 9,10-PQ at its concentrations of 2 and 5 μM elevated the potentials for proliferation, invasion, metastasis and tumorigenesis, all of which were almost completely inhibited by addition of an antioxidant N-acetyl-L-cysteine, inferring a crucial role of ROS in the overgrowth and malignant progression of lung cancer cells. Comparison of mRNA expression levels of six aldo-keto reductases (AKRs) in the 9,10-PQ-treated cells advocated up-regulation of AKR1B10 as a major cause contributing to the lung cancer malignancy. In support of this, the elevation of invasive, metastatic and tumorigenic activities in the 9,10-PQ-treated cells was significantly abolished by the addition of a selective AKR1B10 inhibitor oleanolic acid. Intriguingly, zymographic and real-time PCR analyses revealed remarkable increases in secretion and expression, respectively, of matrix metalloproteinase 2 during the 9,10-PQ treatment, and suggested that the AKR1B10 up-regulation and resultant activation of mitogen-activated protein kinase cascade are predominant mechanisms underlying the metalloproteinase induction. In addition, HPLC analysis and cytochrome c reduction assay in in vitro 9,10-PQ reduction by AKR1B10 demonstrated that the enzyme catalyzes redox-cycling of this quinone, by which ROS are produced. Collectively, these results suggest that AKR1B10 is a key regulator involved in overgrowth and malignant progression of the lung cancer cells through ROS production due to 9,10-PQ redox-cycling. - Highlights: • 9,10-PQ promotes invasion, metastasis and tumorigenicity in lung cancer cells. • The 9,10-PQ-elicited promotion is possibly due to AKR1B10 up-regulation

  8. Proteomic identification of 14-3-3ϵ as a linker protein between pERK1/2 inhibition and BIM upregulation in human osteosarcoma cells.

    Science.gov (United States)

    Kim, Kyung Ok; Hsu, Anny C; Lee, Heon Goo; Patel, Neel; Chandhanayingyong, Chandhanarat; Hickernell, Thomas; Lee, Francis Young-In

    2014-06-01

    Despite advancements in multimodality chemotherapy, conventional cytotoxic treatments still remain ineffective for a subset of patients with aggressive metastatic or multifocal osteosarcoma. It has been shown that pERK1/2 inhibition enhances chemosensitivity to doxorubicin and promotes osteosarcoma cell death in vivo and in vitro. One of the pro-apoptotic mechanisms is upregulation of Bim by pERK1/2 inhibitors. To this end, we examined proteomic changes of 143B human osteosarcoma cells with and without treatment of PD98059, pERK1/2 inhibitor. Specifically, we identified 14-3-3ϵ protein as a potential mediator of Bim expression in response to inhibition of pERK1/2. We hypothesized that 14-3-3ϵ mediates upregulation of Bim expression after pERK1/2 inhibition. We examined the expression of Bim after silencing 14-3-3ϵ using siRNA. The 14-3-3ϵ gene silencing resulted in downregulation of Bim expression after PD98059 treatment. These data indicate that 14-3-3ϵ is required for Bim expression and that it has an anti-cancer effect under pERK1/2 inhibition in 143B cells. By playing an essential role upstream of Bim, 14-3-3ϵ may potentially be a coadjuvant factor synergizing the effect of pERK1/2 inhibitors in addition to conventional cytotoxic agents for more effective osteosarcoma treatments.

  9. Helicobacter pylori promotes invasion and metastasis of gastric cancer cells through activation of AP-1 and up-regulation of CACUL1.

    Science.gov (United States)

    Kong, Ying; Ma, Li-qing; Bai, Pei-song; Da, Rong; Sun, Hong; Qi, Xiao-gai; Ma, Jie-qun; Zhao, Ru-ming; Chen, Nan-zheng; Nan, Ke-jun

    2013-11-01

    Infection with Helicobacter pylori is important in the development and progression of gastric cancer. However, the mechanisms that regulate this activation in gastric tumors remain elusive. CACUL1 has been cloned and identified as a novel gene that is expressed in many types of cancer and is involved in cell cycle regulation and tumor growth. The current study aimed to examine the expression of CACUL1 in gastric cancer samples and analyze its correlation with H. pylori infection. We found that CACUL1 was highly expressed in gastric cancer tissues and negatively correlated with gastric cancer differentiation and TNM stage. In addition, CACUL1 expression was high in H. pylori-infected tissues compared with H. pylori non-infected tissue. We found that H. pylori could up-regulate CACUL1 expression through activating protein 1. The up-regulation of CACUL1 expression could promote matrix metalloproteinase 9 and Slug expression to increase invasion and metastasis of tumor cells. These results suggested that H. pylori-triggered CACUL1 production occurred in an activating protein 1-dependent manner and regulated matrix metalloproteinase 9 and Slug expression to affect the invasion and metastasis of tumor cells. Therefore, CACUL1 is a potential therapeutic target for the treatment of aggressive gastric cancer.

  10. Neuroprotective effect of arctigenin via upregulation of P-CREB in mouse primary neurons and human SH-SY5Y neuroblastoma cells.

    Science.gov (United States)

    Zhang, Nan; Wen, Qingping; Ren, Lu; Liang, Wenbo; Xia, Yang; Zhang, Xiaodan; Zhao, Dan; Sun, Dong; Hu, Yv; Hao, Haiguang; Yan, Yaping; Zhang, Guangxian; Yang, Jingxian; Kang, Tingguo

    2013-09-10

    Arctigenin (Arc) has been shown to act on scopolamine-induced memory deficit mice and to provide a neuroprotective effect on cultured cortical neurons from glutamate-induced neurodegeneration through mechanisms not completely defined. Here, we investigated the neuroprotective effect of Arc on H89-induced cell damage and its potential mechanisms in mouse cortical neurons and human SH-SY5Y neuroblastoma cells. We found that Arc prevented cell viability loss induced by H89 in human SH-SY5Y cells. Moreover, Arc reduced intracellular beta amyloid (Aβ) production induced by H89 in neurons and human SH-SY5Y cells, and Arc also inhibited the presenilin 1(PS1) protein level in neurons. In addition, neural apoptosis in both types of cells, inhibition of neurite outgrowth in human SH-SY5Y cells and reduction of synaptic marker synaptophysin (SYN) expression in neurons were also observed after H89 exposure. All these effects induced by H89 were markedly reversed by Arc treatment. Arc also significantly attenuated downregulation of the phosphorylation of CREB (p-CREB) induced by H89, which may contribute to the neuroprotective effects of Arc. These results demonstrated that Arc exerted the ability to protect neurons and SH-SY5Y cells against H89-induced cell injury via upregulation of p-CREB.

  11. Neuroprotective Effect of Arctigenin via Upregulation of P-CREB in Mouse Primary Neurons and Human SH-SY5Y Neuroblastoma Cells

    Directory of Open Access Journals (Sweden)

    Tingguo Kang

    2013-09-01

    Full Text Available Arctigenin (Arc has been shown to act on scopolamine-induced memory deficit mice and to provide a neuroprotective effect on cultured cortical neurons from glutamate-induced neurodegeneration through mechanisms not completely defined. Here, we investigated the neuroprotective effect of Arc on H89-induced cell damage and its potential mechanisms in mouse cortical neurons and human SH-SY5Y neuroblastoma cells. We found that Arc prevented cell viability loss induced by H89 in human SH-SY5Y cells. Moreover, Arc reduced intracellular beta amyloid (Aβ production induced by H89 in neurons and human SH-SY5Y cells, and Arc also inhibited the presenilin 1(PS1 protein level in neurons. In addition, neural apoptosis in both types of cells, inhibition of neurite outgrowth in human SH-SY5Y cells and reduction of synaptic marker synaptophysin (SYN expression in neurons were also observed after H89 exposure. All these effects induced by H89 were markedly reversed by Arc treatment. Arc also significantly attenuated downregulation of the phosphorylation of CREB (p-CREB induced by H89, which may contribute to the neuroprotective effects of Arc. These results demonstrated that Arc exerted the ability to protect neurons and SH-SY5Y cells against H89-induced cell injury via upregulation of p-CREB.

  12. Glatiramer Acetate, Dimethyl Fumarate, and Monomethyl Fumarate Upregulate the Expression of CCR10 on the Surface of Natural Killer Cells and Enhance Their Chemotaxis and Cytotoxicity

    Science.gov (United States)

    Maghazachi, Azzam A.; Sand, Kristin L.; Al-Jaderi, Zaidoon

    2016-01-01

    In vitro harnessing of immune cells is the most important advance in the field of cancer immunotherapy. Results shown in the current paper may be used to harness natural killer (NK) cells in vitro. It is observed that drugs used to treat multiple sclerosis such as glatiramer acetate, dimethyl fumarate, and monomethyl fumarate upregulate the expression of chemokines receptor 10 (CCR10) on the surface of human IL-2-activated NK cells. This is corroborated with increased chemotaxis of these cells toward the concentration gradients of the ligands for CCR10, namely CCL27 and CCL28. It is also demonstrated that these three drugs enhance NK cell cytotoxicity against tumor target cells, an activity that is abrogated by prior incubation of the cells with anti-CCR10 antibody. Because CCL27 and CCL28 are secreted by selective tumor types such as malignant melanoma, squamous cell carcinomas, and colorectal cancer, respectively, it is hypothesized that activated NK cells may be harnessed in vitro with any of these drugs before utilizing them as a therapeutic modality for cancer.

  13. Hepatitis B virus X protein mutant HBxΔ127 promotes proliferation of hepatoma cells through up-regulating miR-215 targeting PTPRT

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Fabao [Department of Cancer Research, College of Life Sciences, Nankai University, Tianjin 300071 (China); Department of Biochemistry, College of Life Sciences, Nankai University, Tianjin 300071 (China); You, Xiaona [Department of Cancer Research, College of Life Sciences, Nankai University, Tianjin 300071 (China); Chi, Xiumei [Department of Hepatology, The First Hospital, Jilin University, Changchun 130021 (China); Wang, Tao [Department of Cancer Research, College of Life Sciences, Nankai University, Tianjin 300071 (China); Ye, Lihong [Department of Biochemistry, College of Life Sciences, Nankai University, Tianjin 300071 (China); Niu, Junqi, E-mail: junqiniu@yahoo.com.cn [Department of Hepatology, The First Hospital, Jilin University, Changchun 130021 (China); Zhang, Xiaodong, E-mail: zhangxd@nankai.edu.cn [Department of Cancer Research, College of Life Sciences, Nankai University, Tianjin 300071 (China)

    2014-02-07

    Highlights: • Relative to wild type HBx, HBX mutant HBxΔ127 strongly enhances cell proliferation. • Relative to wild type HBx, HBxΔ127 remarkably up-regulates miR-215 in hepatoma cells. • HBxΔ127-elevated miR-215 promotes cell proliferation via targeting PTPRT mRNA. - Abstract: The mutant of virus is a frequent event. Hepatitis B virus X protein (HBx) plays a vital role in the development of hepatocellular carcinoma (HCC). Therefore, the identification of potent mutant of HBx in hepatocarcinogenesis is significant. Previously, we identified a natural mutant of the HBx gene (termed HBxΔ127). Relative to wild type HBx, HBxΔ127 strongly enhanced cell proliferation and migration in HCC. In this study, we aim to explore the mechanism of HBxΔ127 in promotion of proliferation of hepatoma cells. Our data showed that both wild type HBx and HBxΔ127 could increase the expression of miR-215 in hepatoma HepG2 and H7402 cells. However, HBxΔ127 was able to significantly increase miR-215 expression relative to wild type HBx in the cells. We identified that protein tyrosine phosphatase, receptor type T (PTPRT) was one of the target genes of miR-215 through targeting 3′UTR of PTPRT mRNA. In function, miR-215 was able to promote the proliferation of hepatoma cells. Meanwhile anti-miR-215 could partially abolish the enhancement of cell proliferation mediated by HBxΔ127 in vitro. Knockdown of PTPRT by siRNA could distinctly suppress the decrease of cell proliferation mediated by anti-miR-215 in HepG2-XΔ127/H7402-XΔ127 cells. Moreover, we found that anti-miR-215 remarkably inhibited the tumor growth of hepatoma cells in nude mice. Collectively, relative to wild type HBx, HBxΔ127 strongly enhances proliferation of hepatoma cells through up-regulating miR-215 targeting PTPRT. Our finding provides new insights into the mechanism of HBx mutant HBxΔ127 in promotion of proliferation of hepatoma cells.

  14. Intermediate-Conductance-Ca2-Activated K Channel Intermediate-Conductance Calcium-Activated Potassium Channel (IKCa1) is Upregulated and Promotes Cell Proliferation in Cervical Cancer

    Science.gov (United States)

    Liu, Ling; Zhan, Ping; Nie, Dan; Fan, Lingye; Lin, Hairui; Gao, Lanyang; Mao, Xiguang

    2017-01-01

    Background Accumulating data point to intermediate-conductance calcium-activated potassium channel (IKCa1) as a key player in controlling cell cycle progression and proliferation of human cancer cells. However, the role that IKCa1 plays in the growth of human cervical cancer cells is largely unexplored. Material/Methods In this study, Western blot analysis, immunohistochemical staining, and RT-PCR were first used for IKCa1protein and gene expression assays in cervical cancer tissues and HeLa cells. Then, IKCa1 channel blocker and siRNA were employed to inhibit the functionality of IKCa1 and downregulate gene expression in HeLa cells, respectively. After these treatments, we examined the level of cell proliferation by MTT method and measured IKCa1 currents by conventional whole-cell patch clamp technique. Cell apoptosis was assessed using the Annexin V-FITC/Propidium Iodide (PI) double-staining apoptosis detection kit. Results We demonstrated that IKCa1 mRNA and protein are preferentially expressed in cervical cancer tissues and HeLa cells. We also showed that the IKCa1 channel blocker, clotrimazole, and IKCa1 channel siRNA can be used to suppress cervical cancer cell proliferation and decrease IKCa1 channel current. IKCa1 downregulation by specific siRNAs induced a significant increase in the proportion of apoptotic cells in HeLa cells. Conclusions IKCa1 is overexpressed in cervical cancer tissues, and IKCa1 upregulation in cervical cancer cell linea enhances cell proliferation, partly by reducing the proportion of apoptotic cells. PMID:28280257

  15. Upregulation of CD54 and downregulation of HLA‑ABC contribute to the novel enhancement of the susceptibility of HL-60 cells to NK cell-mediated cytolysis induced by ATRA plus VPA.

    Science.gov (United States)

    Zou, Huijuan; Li, Lianlian; Han, Yang; Ma, Ruiping; Liao, Qiong; Tian, Jing; Zhang, Xiaoyu; Ren, Xia; Song, Guanhua; Guo, Qiang; Li, Xia; Ding, Huifang; Jiang, Guosheng

    2017-01-01

    Enhancement of the susceptibility of HL-60 cells to NK cell-mediated cytolysis induced by all-trans-retinoic acid (ATRA) plus valproate (VPA) was evaluated. In addition to the synergistic effect of ATRA plus VPA on HL-60 cells, the optimal concentration of 1 mM VPA plus 0.5 µM ATRA increased the cytotoxic sensitivity of HL-60 cells to NK cells. The expression of the activated receptors NKp30 and NKG2D on NK-92 cells was higher compared with the levels noted for the other receptors, and the expression of NKG2D ligands MICA/B on HL-60 cells was not significantly upregulated in the ATRA plus VPA goup compared with the control. Moreover, it was observed that the ligands of NKp30 on HL-60 cells presented the same variation trend. As to the co-stimulatory and adhesion molecules on NK-92 and their ligands on HL-60 cells post exposure to ATRA and VPA alone or their combination, there was no obvious change in the expression of CD112, CD48 and CD70 on the HL-60 cells. However, the expression of CD54 on HL-60 cells was significantly upregulated. In contrast, the expression of NKG2A ligands HLA-ABC on HL-60 cells was obviously downregulated. In addition, the expression of HLA-E on the HL-60 cells in the group treated with ATRA plus VPA was not significantly increased. In conclusion, the combination of VPA and ATRA not only induced the differentiation of HL-60 cells, but also induced enhancement of the sensitivity of HL-60 cells to NK cells by downregulating the expression of HLA-ABC and upregulating the expression of CD54, but not MICA/MICB. The results provide experimental and theoretical basis for the clinical combination of a low-dose of ATRA plus VPA for the treatment of leukemia.

  16. Expression of cytosolic and membrane associated tissue transglutaminase in rat hepatic stellate cells and its upregulation during transdifferentiation to myofibroblasts in culture.

    Science.gov (United States)

    Schnabel, Claudia; Sawitza, Iris; Tag, Carmen G.; Lahme, Birgit; Gressner, Axel M.; Breitkopf, Katja

    2004-03-01

    Transdifferentiation of hepatic stellate cells (HSC) to collagen producing myofibroblasts (MFB) is a principal event in liver fibrogenesis. In our studies we investigated if tissue transglutaminase (tTG) from these cell types may play a role in liver fibrosis. Separation of cytosol and membrane components showed membrane associated tTG and during transdifferentiation an upregulation of total tTG on mRNA and protein level was found, but no modulation during stimulation with TGF-beta1. In HSC and fully differentiated MFB a significant amount of the total tTG synthesised during transdifferentiation is found to be membrane-associated whereas the remaining portion is cytosol-associated and only very little is found within the extracellular matrix (ECM). The data implicate that tTG in this cell type seems to play an important role in liver fibrogenesis.

  17. Addition of interferon-alpha to a standard maturation cocktail induces CD38 up-regulation and increases dendritic cell function

    DEFF Research Database (Denmark)

    Trepiakas, Redas; Pedersen, Anders Elm; Met, Ozcan;

    2009-01-01

    differs from sDC maturation: The major phenotypic change after IFN-alpha maturation was dose-dependent up-regulation of CD38 but not CD83, while sDCs expressed the opposite profile with low CD38 and high CD83 expression. Similarly, maturation by Poly I:C leads to CD38high, CD83low DCs indicating...... other changes in DC phenotype and cytokine secretion. Our observations suggest that IFN-alpha could be included in maturation protocols for clinical grade DCs used for immunotherapy against cancer and should be included if DCs are used for CD8+ T-cell stimulation in vitro.......Monocyte-derived dendritic cells (DCs) are used as adjuvant cells in cancer immunotherapy and have shown promising results. In order to obtain full functional capacity, these DCs need to be maturated, and the current "gold standard" for this process is maturation with TNF-alpha, IL-1beta, IL-6...

  18. Leptin increases HER2 protein levels through a STAT3-mediated up-regulation of Hsp90 in breast cancer cells.

    Science.gov (United States)

    Giordano, Cinzia; Vizza, Donatella; Panza, Salvatore; Barone, Ines; Bonofiglio, Daniela; Lanzino, Marilena; Sisci, Diego; De Amicis, Francesca; Fuqua, Suzanne A W; Catalano, Stefania; Andò, Sebastiano

    2013-06-01

    Obesity condition confers risks to breast cancer development and progression, and several reports indicate that the adipokine leptin, whose synthesis and plasma levels increase with obesity, might play an important role in modulating breast cancer cell phenotype. Functional crosstalk occurring between leptin and different signaling molecules contribute to breast carcinogenesis. In this study, we show, in different human breast cancer cell lines, that leptin enhanced the expression of a chaperone protein Hsp90 resulting in increased HER2 protein levels. Silencing of Hsp90 gene expression by RNA interference abrogated leptin-mediated HER2 up-regulation. Leptin effects were dependent on JAK2/STAT3 activation, since inhibition of this signaling cascade by AG490 or ectopic expression of a STAT3 dominant negative abrogated leptin-induced HER2 and Hsp90 expressions. Functional experiments showed that leptin treatment significantly up-regulated human Hsp90 promoter activity. This occurred through an enhanced STAT3 transcription factor binding to its specific responsive element located in the Hsp90 promoter region as revealed by electrophoretic mobility shift assay and chromatin immunoprecipitation assay. Analysis of HER2, Akt and MAPK phosphorylation levels revealed that leptin treatment amplified the responsiveness of breast cancer cells to growth factor stimulation. Furthermore, we found that long-term leptin exposure reduced sensitivity of breast cancer cells to the antiestrogen tamoxifen. In the same experimental conditions, the combined treatment of tamoxifen with the Hsp90 inhibitor 17-AAG completely abrogated leptin-induced anchorage-independent breast cancer cell growth. In conclusion, our results highlight, for the first time, the ability of the adipocyte-secreted factor leptin to modulate Hsp90/HER2 expressions in breast cancer cells providing novel insights into the molecular mechanism linking obesity to breast cancer growth and progression.

  19. RUNX3-mediated up-regulation of miR-29b suppresses the proliferation and migration of gastric cancer cells by targeting KDM2A.

    Science.gov (United States)

    Kong, Ye; Zou, Shuiyan; Yang, Fenghua; Xu, Xia; Bu, Wenhong; Jia, Jihui; Liu, Zhifang

    2016-10-10

    RUNX3 is a transcriptional factor that has been shown to regulate protein-coding gene expression at the transcriptional level. However, the regulation of RUNX3 on miRNAs is not fully understood. In this study, we used miRNA microarray to identify the miRNAs that are regulated by RUNX3 and found that miR-29b showed the most up-regulation in RUNX3 over-expressed cells compared with the control cells. We used qRT-PCR to confirm the miRNA microarray results in several gastric cancer cells and found that RUNX3 could bind to the miR-29b promoter directly and cooperate with Smad3 to increase the promoter activity of miR-29b. In the clinical setting, both RUNX3 and miR-29b are down-regulated significantly in human gastric cancer tissues. A positive correlation between miR-29b and RUNX3 was found in the gastric cancer tissues. Additionally, we found that miR-29b suppressed the proliferation and metastasis of gastric cancer cells by directly targeting KDM2A. The miR-29b/KDM2A axis was involved in the RUNX3-mediated inhibition of gastric cancer cell proliferation and metastasis. Taken together, our results suggested that RUNX3-mediated up-regulation of miR-29b inhibited the proliferation and migration of gastric cancer cells by targeting KDM2A, representing a novel molecular mechanism for the tumor suppression action of RUNX3.

  20. Activation of the aryl hydrocarbon receptor pathway enhances cancer cell invasion by upregulating the MMP expression and is associated with poor prognosis in upper urinary tract urothelial cancer.

    Science.gov (United States)

    Ishida, Masaru; Mikami, Shuji; Kikuchi, Eiji; Kosaka, Takeo; Miyajima, Akira; Nakagawa, Ken; Mukai, Makio; Okada, Yasunori; Oya, Mototsugu

    2010-02-01

    Aryl hydrocarbon receptor (AhR) and the activation of the AhR pathway are involved in xenobiotic-induced toxicity and carcinogenesis. Although xenobiotics, such as cigarette smoke, contribute to the development of urothelial carcinoma (UC), the relationship between AhR and UC is unclear. In the present study, we investigated AhR expression in 209 patients with upper urinary tract UC. The nuclear expression of AhR was significantly associated with histological grade, pathological T stage, lymphovascular invasion and lymph node involvement. A multivariate Cox analysis revealed that nuclear AhR expression was a significant and independent predictor for disease-specific survival (hazard ratio = 2.469, P = 0.013). To determine whether the AhR pathway can be activated in the T24 UC cell line, we examined the expression of cytochrome P450 (CYP) 1A1 and CYP1B1, which are target genes of the AhR pathway, following exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a ligand of AhR. TCDD treatment upregulated the expression levels of AhR, CYP1A1 and CYP1B1. TCDD enhanced T24 cell invasion associated with the upregulation of matrix metalloproteinase (MMP)-1 and MMP-9. Furthermore, targeting AhR messenger RNA (mRNA) expression in T24 cells with small interfering RNA (siRNA) downregulated the mRNA expression of AhR, CYP1A1, CYP1B1, MMP-1, MMP-2 and MMP-9; furthermore, the cells transfected with siRNA for AhR showed decreased invasion activity in comparison with the cells transfected with a non-targeting siRNA. Our results therefore suggest that AhR plays a role in the invasiveness of UC cells and can serve as a marker for the prognosis of upper urinary tract UC.

  1. Galectin-3 facilitates cell motility in gastric cancer by up-regulating protease-activated receptor-1 (PAR-1 and matrix metalloproteinase-1 (MMP-1.

    Directory of Open Access Journals (Sweden)

    Seok-Jun Kim

    Full Text Available BACKGROUND: Galectin-3 is known to regulate cancer metastasis. However, the underlying mechanism has not been defined. Through the DNA microarray studies after galectin-3 silencing, we demonstrated here that galectin-3 plays a key role in up-regulating the expressions of protease-activated receptor-1 (PAR-1 and matrix metalloproteinase-1 (MMP-1 PAR-1 thereby promoting gastric cancer metastasis. METHODOLOGY/PRINCIPAL FINDINGS: We examined the expression levels of Galectin-3, PAR-1, and MMP-1 in gastric cancer patient tissues and also the effects of silencing these proteins with specific siRNAs and of over-expressing them using specific lenti-viral constructs. We also employed zebrafish embryo model for analysis of in vivo gastric cancer cell invasion. These studies demonstrated that: a galectin-3 silencing decreases the expression of PAR-1. b galectin-3 over-expression increases cell migration and invasion and this increase can be reversed by PAR-1 silencing, indicating that galectin-3 increases cell migration and invasion via PAR-1 up-regulation. c galectin-3 directly interacts with AP-1 transcriptional factor, and this complex binds to PAR-1 promoter and drives PAR-1 transcription. d galectin-3 also amplifies phospho-paxillin, a PAR-1 downstream target, by increasing MMP-1 expression. MMP-1 silencing blocks phospho-paxillin amplification and cell invasion caused by galectin-3 over-expression. e Silencing of either galectin-3, PAR-1 or MMP-1 significantly reduced cell migration into the vessels in zebrafish embryo model. f Galectin-3, PAR-1, and MMP-1 are highly expressed and co-localized in malignant tissues from gastric cancer patients. CONCLUSIONS/SIGNIFICANCE: Galectin-3 plays the key role of activating cell surface receptor through production of protease and boosts gastric cancer metastasis. Galectin-3 has the potential to serve as a useful pharmacological target for prevention of gastric cancer metastasis.

  2. TNF-α is upregulated in T2DM patients with fracture and promotes the apoptosis of osteoblast cells in vitro in the presence of high glucose.

    Science.gov (United States)

    Sun, Mingqi; Yang, Junli; Wang, Jianzhong; Hao, Ting; Jiang, Dianming; Bao, Guoyu; Liu, Guanghui

    2016-04-01

    Fracture healing is regulated by proinflammatory mediators such as tumor necrosis factor-α (TNF-α), which poses influence on the balance between bone formation and remodeling. And the diabetes is thought to contribute to the delayed diabetic fracture healing. In the present study, we examined the promotion to proinflammatory cytokines and chemokines in type 2 diabetes mellitus (T2DM) patients with bone fractures, and then evaluated the promotion to TNF-α by the high glucose treatment in human osteoblast-like MG-63 cells and the regulatory role of the promoted TNF-α on the MG-63 cell apoptosis. It was demonstrated that there were significantly-upregulated high-sensitivity C-reactive protein (hsCRP) TNF-α, IL-1β, IL-6, IFN-γ-inducible protein 10 (IP-10) and RANTES in T2DM patients with bone fracture. And the promotion to TNF-α and IL-1β was confirmed in vitro in both mRNA and protein levels in high glucose-treated MG-63 cells. And either TNF-α or high glucose reduced the viability of MG-63 cells, promoted apoptosis and upregulated apoptosis-associated markers, such as released cytochrome c, cleaved caspase 3 and lyzed PARP. Moreover, there was a synergistic effect between TNF-α and high glucose. The viability reduction and the apoptosis induction of MG-63 cells were significantly higher in the group with both TNF-α and high glucose treatments, than in the group with singular TNF-α treatment. In conclusion, our study demonstrated that proinflammatory cytokines and chemokines were promoted in T2DM patients with bone fracture or in osteoblasts by the high glucose stimulation. TNF-α and high glucose synergistically reduced the viability and induced the apoptosis in the osteoblast-like MG-63 cells in vitro. It implies the significant regulatory role of TNF-α in the delayed fracture healing in T2DM.

  3. Phosphorylation of the PDH complex precedes HIF-1-mediated effects and PDK1 upregulation during the first hours of hypoxic treatment in HCC cells

    Directory of Open Access Journals (Sweden)

    Zimmer AD

    2016-08-01

    Full Text Available Andreas David Zimmer, Geoffroy Walbrecq, Ines Kozar, Iris Behrmann, Claude Haan Life Sciences Research Unit, University of Luxembourg, Belvaux, Luxembourg Abstract: The pyruvate dehydrogenase complex (PDC is an important gatekeeper enzyme connecting glycolysis to the tricarboxylic acid (TCA cycle and oxidative phosphorylation (OXPHOS. Thereby, it has a strong impact on the glycolytic flux as well as the metabolic phenotype of a cell. PDC activity is regulated via reversible phosphorylation of three serine residues on the pyruvate dehydrogenase (PDH E1α subunit. Phosphorylation of any of these residues by the PDH kinases (PDKs leads to a strong decrease in PDC activity. Under hypoxia, the inactivation of the PDC has been described to be dependent on the hypoxia-inducible factor 1 (HIF-1-induced PDK1 protein upregulation. In this study, we show in two hepatocellular carcinoma cell lines (HepG2 and JHH-4 that, during the adaptation to hypoxia, PDH is already phosphorylated at time points preceding HIF-1-mediated transcriptional events and PDK1 protein upregulation. Using siRNAs and small molecule inhibitor approaches, we show that this inactivation of PDC is independent of HIF-1α expression but that the PDKs need to be expressed and active. Furthermore, we show that reactive oxygen species might be important for the induction of this PDH phosphorylation since it correlates with the appearance of an altered redox state in the mitochondria and is also inducible by H2O2 treatment under normoxic conditions. Overall, these results show that neither HIF-1 expression nor PDK1 upregulation is necessary for the phosphorylation of PDH during the first hours of the adaptation to hypoxia. Keywords: pyruvate dehydrogenase complex, pyruvate dehydrogenase kinase, hypoxia metabolism, glycolytic switch, radical oxygen species

  4. Engagement of major histocompatibility complex class I and class II molecules up-regulates intercellular adhesion of human B cells via a CD11/CD18-independent mechanism.

    Science.gov (United States)

    Alcover, A; Juillard, V; Acuto, O

    1992-02-01

    We have studied the role of major histocompatibility complex (MHC) molecules in the regulation of intercellular adhesion of human B cells. We found that molecules able to bind to MHC class II molecules, such as monoclonal antibodies or staphylococcal enterotoxins, induced rapid and sustained homotypic adhesion of Epstein-Barr virus (EBV)-transformed B cell lines as well as peripheral blood B lymphocytes. Moreover, anti-MHC class I monoclonal antibodies also stimulated intercellular adherence. Adhesion induced upon MHC engagement was faster and stronger than that triggered by phorbol esters. It needed active metabolism, but divalent cations were not required. Monoclonal antibodies directed against LFA-1 (CD11a/CD18) or its ligand ICAM-1 (CD54) did not inhibit MHC class II-induced homotypic adhesion of various EBV-transformed B cell lines, nor of a variant of the B cell line Raji expressing very low LFA-1 surface levels. Moreover, EBV-transformed B cells from a severe lymphocyte adhesion deficiency patient, lacking surface CD11/CD18, also aggregated in response to anti-MHC class I or class II monoclonal antibodies. Together these data indicate that engagement of MHC molecules may transduce signals to B cells resulting in up-regulation of intercellular adhesion, via an LFA-1-independent mechanism. This may play a role in the stabilization of T cell/antigen-presenting cell conjugates at the moment of antigen recognition.

  5. Enhancement of Cisplatin-Mediated Apoptosis in Ovarian Cancer Cells through Potentiating G2/M Arrest and p21 Upregulation by Combinatorial Epigallocatechin Gallate and Sulforaphane