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  1. 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.

  2. 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

  3. 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. PMID:26388737

  4. Cell Stress Induces Upregulation of Osteopontin via the ERK Pathway in Type II Alveolar Epithelial Cells

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    Aki Kato; Takafumi Okura; Chizuru Hamada; Seigo Miyoshi; Hitoshi Katayama; Jitsuo Higaki; Ryoji Ito

    2014-01-01

    Osteopontin (OPN) is a multifunctional protein that plays important roles in cell growth, differentiation, migration and tissue fibrosis. In human idiopathic pulmonary fibrosis and murine bleomycin-induced lung fibrosis, OPN is upregulated in type II alveolar epithelial cells (AEC II). However, the mechanism of OPN induction in AEC II is not fully understood. In this study, we demonstrate the molecular mechanism of OPN induction in AEC II and elucidate the functions of OPN in AEC II and lung ...

  5. Metformin inhibits cell growth by upregulating microRNA-26a in renal cancer cells.

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    Yang, Feng-Qiang; Wang, Ji-Jiao; Yan, Jia-Sheng; Huang, Jian-Hua; Li, Wei; Che, Jian-Ping; Wang, Guang-Chun; Liu, Min; Zheng, Jun-Hua

    2014-01-01

    Accumulating evidence suggests that metformin, a biguanide class of anti-diabetic drugs, possesses anti-cancer properties and may reduce cancer risk and improve prognosis. However, the mechanism by which metformin affects various cancers, including renal cancer still unknown. MiR-26a induces cell growth, cell cycle and cell apoptosis progression via direct targeting of Bcl-2, clyclin D1 and PTEN in cancer cells. In the present study, we used 786-O human renal cancer cell lines to study the effects and mechanisms of metformin. Metformin treatment inhibited RCC cells proliferation by increasing expression of miR-26a in 786-O cells (P metformin. Also over-expression of miR-26a can inhibited cell proliferation by down-regulating Bcl-2, cyclin D1 and up-regulating PTEN expression. Therefore, these data for the first time provide novel evidence for a mechanism that the anticancer activities of metformin are due to upregulation of miR-26a and affect its downstream target gene. PMID:25419360

  6. Slug inhibition increases radiosensitivity of oral squamous cell carcinoma cells by upregulating PUMA.

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    Jiang, Fangfang; Zhou, Lijie; Wei, Changbo; Zhao, Wei; Yu, Dongsheng

    2016-08-01

    As a new strategy, radio-gene therapy was widely used for the treatment of cancer patients in recent few years. Slug was involved in the radioresistance of various cancers and has been found to have an anti-apoptotic effect. This study aims to investigate whether the modulation of Slug expression by siRNA affects oral squamous cell carcinoma sensitivity to X-ray irradiation through upregulating PUMA. Two oral squamous cell carcinoma cell lines (HSC3 and HSC6) were transfected with small interfering RNA (siRNA) targeting Slug and subjected to radiotherapy in vitro. After transfection with Slug siRNA, both HSC3 and HSC6 cells showed relatively lower expression of Slug and higher expression of PUMA. The Slug siRNA transfected cells showed decreased survival and proliferation rates, an increased apoptosis rate and enhanced radiosensitivity to X-ray irradiation. Our results revealed that Slug siRNA transfection in combination with radiation increased the expression of PUMA, which contributed to radiosensitivity of oral squamous cell carcinoma cells. Thus, controlling the expression of Slug might contribute to enhance sensitivity of HSC3 and HSC6 cells toward X-ray irradiation in vitro by upregulating PUMA. PMID:27277529

  7. 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.

  8. 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.

    2016-01-01

    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. PMID:27271988

  9. 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...

  10. Nimotuzumab promotes radiosensitivity of EGFR-overexpression esophageal squamous cell carcinoma cells by upregulating IGFBP-3

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    Zhao Lei

    2012-12-01

    Full Text Available Abstract Background Epidermal growth factor receptor (EGFR is suggested to predict the radiosensitivity and/or prognosis of human esophageal squamous cell carcinoma (ESCC. The objective of this study was to investigate the efficacy of Nimotuzumab (an anti-EGFR monoclonal antibody on ESCC radiotherapy (RT and underlying mechanisms. Methods Nimotuzumab was administrated to 2 ESCC cell lines KYSE30 and TE-1 treated with RT. Cell growth, colony formation and apoptosis were used to measure anti-proliferation effects. The method of RNA interference was used to investigate the role of insulin-like growth factor binding protein-3 (IGFBP-3 in ESCC cells radiosensitivity treated with Nimotuzumab. In vivo effect of Nimotuzumab on ESCC radiotherapy was done using a mouse xenograft model. Results Nimotuzumab enhanced radiation response of KYSE30 cells (with high EGFR expression in vitro, as evidenced by increased radiation-inhibited cell growth and colony formation and radiation-mediated apoptosis. Mechanism study revealed that Nimotuzumab inhibited phosphorylated EGFR (p-EGFR induced by EGF in KYSE30 cells. In addition, knockdown of IGFBP-3 by short hairpin RNA significantly reduced KYSE30 cells radiosensitivity (PP>0.05. In KYSE30 cell xenografts, Nimotuzumab combined with radiation led to significant tumor growth delay, compared with that of radiation alone (P=0.029, and also with IGFBP-3 up-regulation in tumor tissue. Conclusions Nimotuzumab could enhance the RT effect of ESCC cells with a functional active EGFR pathway. In particular, the increased ESCC radiosensitivity by Nimotuzumab might be dependent on the up-regulation of IGFBP-3 through EGFR-dependent pathway.

  11. Upregulation of erythropoietin receptor in UT-7/EPO cells inhibits simulated microgravity-induced cell apoptosis

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    Zou, Li-xue; Cui, Shao-yan; Zhong, Jian; Yi, Zong-chun; Sun, Yan; Fan, Yu-bo; Zhuang, Feng-yuan

    2011-07-01

    Hematopoietic progenitor cell proliferation can be altered in either spaceflight or under simulated microgravity experiments on the ground, however, the underlying mechanism remains unknown. Our previous study showed that exposure of the human erythropoietin (EPO)-dependent leukemia cell line UT-7/EPO to conditions of simulated microgravity significantly inhibited the cellular proliferation rate and induced cell apoptosis. We postulated that the downregulation of the erythropoietin receptor (EPOR) expression in UT-7/EPO cells under simulated microgravity may be a possible reason for microgravity triggered apoptosis. In this paper, a human EPOR gene was transferred into UT-7/EPO cells and the resulting expression of EPOR on the surface of UT-7/EPO cells increased approximately 61% ( p < 0.05) as selected by the antibiotic G418. It was also shown through cytometry assays and morphological observations that microgravity-induced apoptosis markedly decreased in these UT-7/EPO-EPOR cells. Thus, we concluded that upregulation of EPOR in UT-7/EPO cells could inhibit the simulated microgravity-induced cell apoptosis in this EPO dependent cell line.

  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. LIF Upregulates Expression of NK-1R in NHBE Cells

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    Cheng-Ping Hu

    2006-01-01

    Full Text Available Leukemia inhibitory factor (LIF, a cytokine at the interface between neurobiology and immunology, is mainly mediated through JAK/STAT pathway and MAPK/ERK pathway. Evidence suggested LIF is related to the higher expression of neurokinin-1 receptor (NK-1R in asthma. In this study, the immunohistochemistry stain showed the expressions of NK-1R, LIF, p-STAT3, and p-ERK1/2 in the lung tissues of allergic rats were increased compared with the controls, and the main positive cell type was airway epithelial cell. Normal human bronchial epithelial cells were treated with LIF in the presence or absence of AG490 (JAK2 inhibitor, PD98059 (MEK inhibitor, and the siRNA against STAT3. Western blot and RT-PCR indicated that LIF induced the expression of NK-1R, which was inhibited by the inhibitors mentioned above. No significant interaction was found between JAK/STAT pathway and MAPK/ERK pathway. In summary, bronchial epithelial cell changes in asthma are induced by LIF which promotes the expression of NK-1R, and JAK/STAT pathway and MAPK/ERK pathway may participate in this process.

  14. Proteins upregulated by mild and severe hypoxia in squamous cell carcinomas in vitro identified by proteomics

    International Nuclear Information System (INIS)

    Background: Solid malignant tumours are characterised by an inadequate vascular system, which can give rise to micro-regional hypoxic areas. As the negative impact of tumour hypoxia is believed largely to depend on dynamic changes in gene expression, it is important to identify the genes regulated by hypoxia to further enlighten the biology behind the cellular response to hypoxia. Previous studies have demonstrated that hypoxia has an impact not only on the gene transcription, but also on gene-specific mRNA translation. Therefore, proteomics is a suitable approach to understand the complexity of gene regulation under hypoxia at protein level. In this in vitro study we have studied the proteome of cells under intermediate hypoxia (1% O2) and anoxia and compared these to normoxic (21% O2) cells to identify proteins upregulated by mild and severe hypoxia. Materials and methods: A human cervix cancer cell line (SiHa) and a human head and neck cancer cell line (FaDuDD) were used. Total cell lysate from hypoxic and normoxic cells was separated by 2-dimensional gel electrophoresis, and images were analysed using Quantity One software. Proteins from significant spots (difference in intensity by more than a factor 2) were identified by Liquid chromatography-mass spectrometry (LC-MS/MS). In order to confirm the hypoxic regulation of the identified proteins, immunoblotting and qPCR were employed when possible. Results: All together 32 spots were found to be upregulated in the hypoxic gels. Of these, 11 different proteins were successfully identified and largely confirmed by Western blotting and qPCR. Amongst these proteins are protein disulfide isomerase family A, member 6 (PDIA6) and dynein light chain roadblock-type 1 (DynLRB1). Both 2D gels and Western blots revealed that PDAI6 exhibited a cell line specific pattern; in FaDuDD there was upregulation at 1% and further upregulated at 0% compared to atmospheric air, whereas there was no upregulation in SiHa cells. DynLRB1 was

  15. Adiponectin Upregulates Ferritin Heavy Chain in Skeletal Muscle Cells

    OpenAIRE

    Ikegami, Yuichi; Inukai, Kouichi; Imai, Kenta; Sakamoto, Yasushi; Katagiri, Hideki; Kurihara, Susumu; Awata, Takuya; Katayama, Shigehiro

    2009-01-01

    OBJECTIVE—Adiponectin is an adipocyte-derived protein that acts to reduce insulin resistance in the liver and muscle and also inhibits atherosclerosis. Although adiponectin reportedly enhances AMP-activated protein kinase and inhibits tumor necrosis factor-α action downstream from the adiponectin signal, the precise physiological mechanisms by which adiponectin acts on skeletal muscles remain unknown. RESEARCH DESIGN AND METHODS—We treated murine primary skeletal muscle cells with recombinant...

  16. 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.

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

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    Yun-Bo Feng

    2016-05-01

    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.

  18. Upregulation of HYAL1 expression in breast cancer promoted tumor cell proliferation, migration, invasion and angiogenesis.

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    Jin-Xiang Tan

    Full Text Available Hyaluronic acid (HA is a component of the Extra-cellular matrix (ECM, it is closely correlated with tumor cell growth, proliferation, metastasis and angiogenesis, etc. Hyaluronidase (HAase is a HA-degrading endoglycosidase, levels of HAase are elevated in many cancers. Hyaluronidase-1 (HYAL1 is the major tumor-derived HAase. We previously demonstrated that HYAL1 were overexpression in human breast cancer. Breast cancer cells with higher HAase expression, exhibited significantly higher invasion ability through matrigel than those cells with lower HAase expression, and knockdown of HYAL1 expression in breast cancer cells resulted in decreased cell growth, adhesion, invasion and angiogenesis. Here, to further elucidate the function of HYAL1 in breast cancer, we investigated the consequences of forcing HYAL1 expression in breast cancer cells by transfection of expression plasmid. Compared with control, HYAL1 up-regulated cells showed increased the HAase activity, and reduced the expression of HA in vitro. Meantime, upregulation of HYAL1 promoted the cell growth, migration, invasion and angiogenesis in vitro. Moreover, in nude mice model, forcing HYAL1 expression induced breast cancer cell xenograft tumor growth and angiogenesis. Interestingly, the HA expression was upregulated by forcing HYAL1 expression in vivo. These findings suggested that HYAL1-HA system is correlated with the malignant behavior of breast cancer.

  19. 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......Mechanical loading of tendon cells results in an upregulation of mechanotransduction signaling pathways, cell-matrix adhesion and collagen synthesis, but whether unloading removes these responses is unclear. We investigated the response to tension release, with regard to matrix proteins, pro...... showed a contrasting response with a clear drop in integrin subunit α11 mRNA and protein expression, and an increase in α2 integrin mRNA and protein levels. Further, specific markers for tendon cell differentiation declined and normal tendon architecture was disturbed, whereas pro-inflammatory molecules...

  20. 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.

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

    International Nuclear Information System (INIS)

    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, H2DCF-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

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

  3. 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.

  4. Elastin fragments induce IL-1beta upregulation via NF-kappaB pathway in melanoma cells.

    Science.gov (United States)

    Debret, Romain; Le Naour, Richard R; Sallenave, Jean-Michel; Deshorgue, Aurelie; Hornebeck, William G; Guenounou, Moncef; Bernard, Philippe; Antonicelli, Frank D

    2006-08-01

    In a previous work, we reported the influence of elastin fragments (EFs) on matrix metalloproteinases-2 and -14 expression and activation in melanoma cells in vitro. We hypothesized that EFs might also modulate expression of other mediators involved during melanoma progression. Therefore we investigated the contribution of EFs on IL-1beta expression, a cytokine playing a key role in melanoma cells activation. Our results evidenced that high tumorigenic melanoma cells (M3Da cells) treated with EFs led to IL-1beta mRNA and protein upregulation. The effects of EFs on M3Da cells were found to be mediated by receptor (spliced galactosidase) occupancy, as being suppressed by lactose and reproduced by cell stimulation with the VGVAPG peptide. Binding of EFs to their receptor induced a rapid activation of extracellular signal-regulated kinase 1/2; and p38 mitogen-activated protein kinase pathways. However, these pathways were not associated with IL-1beta mRNA upregulation by EFs. Concomitantly, we demonstrated that EFs stimulation induced NF-kappaB nuclear translocation and DNA binding on IL-1beta promoter region whereas inhibition of NF-kappaB with the specific chemical inhibitor SN-50 or by overexpression of IkappaB, the endogenous inhibitor of NF-kappaB pathway, totally abolished EFs-mediated IL-1beta mRNA overexpression. These results demonstrate that EFs induce NF-kappaB activation, leading to IL-1beta upregulation in invasive melanoma cells. PMID:16675961

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

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

    International Nuclear Information System (INIS)

    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+-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

  7. Eurycomanone induce apoptosis in HepG2 cells via up-regulation of p53

    Directory of Open Access Journals (Sweden)

    Zakaria Yusmazura

    2009-06-01

    Full Text Available Abstract Background Eurycomanone is a cytotoxic compound found in Eurycoma longifolia Jack. Previous studies had noted the cytotoxic effect against various cancer cell lines. The aim of this study is to investigate the cytotoxicity against human hepato carcinoma cell in vitro and the mode of action. The cytotoxicity of eurycomanone was evaluated using MTT assay and the mode of cell death was detected by Hoechst 33258 nuclear staining and flow cytometry with Annexin-V/propidium iodide double staining. The protein expression Bax, Bcl-2, p53 and cytochrome C were studied by flow cytometry using a spesific antibody conjugated fluorescent dye to confirm the up-regulation of p53 and Bax in cancer cells. Results The findings suggested that eurycomanone was cytotoxic on cancerous liver cell, HepG2 and less toxic on normal cells Chang's liver and WLR-68. Furthermore, various methods proved that apoptosis was the mode of death in eurycomanone-treated HepG2 cells. The characteristics of apoptosis including chromatin condensation, DNA fragmentation and apoptotic bodies were found following eurycomanone treatment. This study also found that apoptotic process triggered by eurycomanone involved the up-regulation of p53 tumor suppressor protein. The up-regulation of p53 was followed by the increasing of pro-apoptotic Bax and decreasing of anti-apoptotic Bcl-2. The increased of cytochrome C levels in cytosol also results in induction of apoptosis. Conclusion The data suggest that eurycomanone was cytotoxic on HepG2 cells by inducing apoptosis through the up-regulation of p53 and Bax, and down-regulation of Bcl-2.

  8. Mu opioid receptor up-regulation and participation in excitability of hippocampal pyramidal cell electrophysiology

    International Nuclear Information System (INIS)

    Chronic administration of opiate antagonists to rats results in up-regulation of their brain opioid receptors. Using subcellular fractionation techniques, brain opioid receptors were resolved into two membrane populations, one associated with synaptic plasma membranes (SPM) and the other enriched in smooth endoplasmic reticulum and Golgi (microsomes). This study addressed in part the question of whether an antagonist induces up-regulation uniformly in these two populations. Rats were administered naltrexone by subcutaneously implanted osmotic minipumps. Forebrain mu receptor levels were determined by homologous displacement of (3H)D-ala2-mePhe4-gly-ol5-enkephalin (DAGO) followed by computer estimation of binding parameters. Receptor levels in crude membranes rose 77% after treatment. Microsomes displayed a 92% increase, a two-fold greater change than in SPMs (51%). These results establish that naltrexone induces up-regulation of both membrane populations; and that microsomal and SPM receptors represent discrete populations of intracellular and cell surface sites, respectively. Binding experiments on isolated hippocampi also demonstrated up-regulation (71%) of mu receptors. To demonstrate up-regulation of opioid receptors electrophysiologically, hippocampal slices were prepared from rats which had been chronically treated with naltrexone. After superfusion with DAGO, these slices showed a 42% greater population spike output than controls in response to the same EPSP input. Hippocampi from animals treated for two weeks showed an additional increase in sensitivity. The results support a disinhibitory role for opioids in pyramidal cell hyper-excitability. More importantly, they demonstrate a significant physiological correlate to opioid receptor up-regulation

  9. Mu opioid receptor up-regulation and participation in excitability of hippocampal pyramidal cell electrophysiology

    Energy Technology Data Exchange (ETDEWEB)

    Moudy, A.M.

    1988-01-01

    Chronic administration of opiate antagonists to rats results in up-regulation of their brain opioid receptors. Using subcellular fractionation techniques, brain opioid receptors were resolved into two membrane populations, one associated with synaptic plasma membranes (SPM) and the other enriched in smooth endoplasmic reticulum and Golgi (microsomes). This study addressed in part the question of whether an antagonist induces up-regulation uniformly in these two populations. Rats were administered naltrexone by subcutaneously implanted osmotic minipumps. Forebrain mu receptor levels were determined by homologous displacement of ({sup 3}H)D-ala{sup 2}-mePhe{sup 4}-gly-ol{sup 5}-enkephalin (DAGO) followed by computer estimation of binding parameters. Receptor levels in crude membranes rose 77% after treatment. Microsomes displayed a 92% increase, a two-fold greater change than in SPMs (51%). These results establish that naltrexone induces up-regulation of both membrane populations; and that microsomal and SPM receptors represent discrete populations of intracellular and cell surface sites, respectively. Binding experiments on isolated hippocampi also demonstrated up-regulation (71%) of mu receptors. To demonstrate up-regulation of opioid receptors electrophysiologically, hippocampal slices were prepared from rats which had been chronically treated with naltrexone. After superfusion with DAGO, these slices showed a 42% greater population spike output than controls in response to the same EPSP input. Hippocampi from animals treated for two weeks showed an additional increase in sensitivity. The results support a disinhibitory role for opioids in pyramidal cell hyper-excitability. More importantly, they demonstrate a significant physiological correlate to opioid receptor up-regulation.

  10. Chronic morphine treatment up-regulates mu opioid receptor binding in cells lacking Filamin A

    OpenAIRE

    Onoprishvili, Irma; Simon, Eric J.

    2007-01-01

    We investigated the effects of morphine and other agonists on the human mu opioid receptor (MOP) expressed in M2 melanoma cells, lacking the actin cytoskeleton protein filamin A and in A7, a sub clone of the M2 melanoma cells, stably transfected with filamin A cDNA. The results of binding experiments showed, that after chronic morphine treatment (24 hr) of A7 cells, MOP binding sites were down-regulated to 63% of control, whereas, unexpectedly, in M2 cells, MOP binding was up-regulated to 188...

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

    Science.gov (United States)

    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.

  12. Curcumin-treated cancer cells show mitotic disturbances leading to growth arrest and induction of senescence phenotype.

    Science.gov (United States)

    Mosieniak, Grażyna; Sliwinska, Małgorzata A; Przybylska, Dorota; Grabowska, Wioleta; Sunderland, Piotr; Bielak-Zmijewska, Anna; Sikora, Ewa

    2016-05-01

    Cellular senescence is recognized as a potent anticancer mechanism that inhibits carcinogenesis. Cancer cells can also undergo senescence upon chemo- or radiotherapy. Curcumin, a natural polyphenol derived from the rhizome of Curcuma longa, shows anticancer properties both in vitro and in vivo. Previously, we have shown that treatment with curcumin leads to senescence of human cancer cells. Now we identified the molecular mechanism underlying this phenomenon. We observed a time-dependent accumulation of mitotic cells upon curcumin treatment. The time-lapse analysis proved that those cells progressed through mitosis for a significantly longer period of time. A fraction of cells managed to divide or undergo mitotic slippage and then enter the next phase of the cell cycle. Cells arrested in mitosis had an improperly formed mitotic spindle and were positive for γH2AX, which shows that they acquired DNA damage during prolonged mitosis. Moreover, the DNA damage response pathway was activated upon curcumin treatment and the components of this pathway remained upregulated while cells were undergoing senescence. Inhibition of the DNA damage response decreased the number of senescent cells. Thus, our studies revealed that the induction of cell senescence upon curcumin treatment resulted from aberrant progression through the cell cycle. Moreover, the DNA damage acquired by cancer cells, due to mitotic disturbances, activates an important molecular mechanism that determines the potential anticancer activity of curcumin. PMID:26916504

  13. 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.

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

    Science.gov (United States)

    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.

  15. 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.

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

    Directory of Open Access Journals (Sweden)

    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.

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

    Directory of Open Access Journals (Sweden)

    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.

  18. 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

  19. 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

  20. 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.

  1. 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.

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

  3. E2F-1 induces melanoma cell apoptosis via PUMA up-regulation and Bax translocation

    International Nuclear Information System (INIS)

    PUMA is a pro-apoptotic Bcl-2 family member that has been shown to be involved in apoptosis in many cell types. We sought to ascertain whether induction of PUMA plays a crucial role in E2F-1-induced apoptosis in melanoma cells. PUMA gene and protein expression levels were detected by real-time PCR and Western blot in SK-MEL-2 and HCT116 cell lines after Ad-E2F-1 infection. Activation of the PUMA promoter by E2F-1 overexpression was detected by dual luciferase reporter assay. E2F-1-induced Bax translocation was shown by immunocytochemistry. The induction of caspase-9 activity was measured by caspase-9 colorimetric assay kit. Up-regulation of the PUMA gene and protein by E2F-1 overexpression was detected by real-time PCR and Western blot analysis in the SK-MEL-2 melanoma cell line. In support of this finding, we found six putative E2F-1 binding sites within the PUMA promoter. Subsequent dual luciferase reporter assay showed that E2F-1 expression could increase the PUMA gene promoter activity 9.3 fold in SK-MEL-2 cells. The role of PUMA in E2F-1-induced apoptosis was further investigated in a PUMA knockout cell line. Cell viability assay showed that the HCT116 PUMA-/- cell line was more resistant to Ad-E2F-1-mediated cell death than the HCT116 PUMA+/+ cell line. Moreover, a 2.2-fold induction of the PUMA promoter was also noted in the HCT116 PUMA+/+ colon cancer cell line after Ad-E2F-1 infection. Overexpression of a truncated E2F-1 protein that lacks the transactivation domain failed to up-regulate PUMA promoter, suggesting that PUMA may be a transcriptional target of E2F-1. E2F-1-induced cancer cell apoptosis was accompanied by Bax translocation from the cytosol to mitochondria and the induction of caspase-9 activity, suggesting that E2F-1-induced apoptosis is mediated by PUMA through the cytochrome C/Apaf-1-dependent pathway. Our studies strongly demonstrated that E2F-1 induces melanoma cell apoptosis via PUMA up-regulation and Bax translocation. The signaling

  4. Porphyromonas gingivalis increases the invasiveness of oral cancer cells by upregulating IL-8 and MMPs.

    Science.gov (United States)

    Ha, Na Hee; Park, Dae Gun; Woo, Bok Hee; Kim, Da Jeong; Choi, Jeom Il; Park, Bong Soo; Kim, Yong Deok; Lee, Ji Hye; Park, Hae Ryoun

    2016-10-01

    Recent studies indicate that chronic inflammation promotes the aggressiveness of cancers. However, the direct molecular mechanisms underlying a functional link between chronic periodontitis, the most common form of oral inflammatory diseases, and the malignancy of oral cancer remain unknown. To elucidate the role of chronic periodontitis in progression of oral cancer, we examined the effect of Porphyromonas gingivalis (P. gingivalis), a major pathogen that causes chronic periodontitis, on the invasiveness of oral squamous cell carcinoma (OSCC) cells, including SCC-25, OSC-20 and SAS cells. Exposures to P. gingivalis promoted the invasive ability of OSC-20 and SAS cells via the upregulation of matrix metalloproteinases (MMPs), specifically MMP-1 and MMP-2. However, P. gingivalis-infected SCC-25 cells did not exhibit changes in their invasive properties or the low expression levels of MMPs. In an effort to delineate the molecular players that control the invasiveness, we first assessed the level of interleukin-8 (IL-8), a well-known inflammatory cytokine, in P. gingivalis-infected OSCC cells. IL-8 secretion was substantially increased in the OSC-20 and SAS cells, but not in the SCC-25 cells, following P. gingivalis infection. When IL-8 was directly applied to SCC-25 cells, their invasive ability and MMP level were significantly increased. Furthermore, the downregulation of IL-8 in P. gingivalis-infected OSC-20 and SAS cells attenuated their invasive potentials and MMP levels. Taken together, our findings strongly suggest that P. gingivalis infection plays an important role in the promotion of the invasive potential of OSCC cells via the upregulation of IL-8 and MMPs. PMID:27468958

  5. 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.

  6. 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.

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

    Directory of Open Access Journals (Sweden)

    Redmond H Paul

    2010-05-01

    Full Text Available 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.

  8. MnSOD upregulation induces autophagic programmed cell death in senescent keratinocytes.

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    Emeric Deruy

    Full Text Available Senescence is a state of growth arrest resulting mainly from telomere attrition and oxidative stress. It ultimately leads to cell death. We have previously shown that, in keratinocytes, senescence is induced by NF-kappaB activation, MnSOD upregulation and H(2O(2 overproduction. We have also shown that senescent keratinocytes do not die by apoptosis but as a result of high macroautophagic activity that targets the primary vital cell components. Here, we investigated the mechanisms that activate this autophagic cell death program. We show that corpses occurring at the senescence plateau display oxidatively-damaged mitochondria and nucleus that colocalize with autophagic vacuoles. The occurrence of such corpses was decreased by specifically reducing the H(2O(2 level with catalase, and, conversely, reproduced by overexpressing MnSOD or applying subtoxic doses of H(2O(2. This H(2O(2-induced cell death did occur through autophagy since it was accompanied by an accumulation of autophagic vesicles as evidenced by Lysotracker staining, LC3 vesiculation and transmission electron microscopy. Most importantly, it was partly abolished by 3-methyladenine, the specific inhibitor of autophagosome formation, and by anti-Atg5 siRNAs. Taken together these results suggest that autophagic cell death is activated in senescent keratinocytes because of the upregulation of MnSOD and the resulting accumulation of oxidative damages to nucleus and mitochondria.

  9. Knockdown of COUP-TFII inhibits cell proliferation and induces apoptosis through upregulating BRCA1 in renal cell carcinoma cells.

    Science.gov (United States)

    Zheng, Jia; Qin, Weijun; Jiao, Dian; Ren, Jing; Wei, Ming; Shi, Shengjia; Xi, Wenjin; Wang, He; Yang, An-Gang; Huan, Yi; Wen, Weihong

    2016-10-01

    COUP-TFII belongs to the nuclear receptor family, which is highly expressed in many kinds of tumors. Previous studies have shown that COUP-TFII can promote tumor progression through regulating tumor angiogenesis and cell proliferation and migration of certain cancer cells. However, the function of COUP-TFII in renal cell carcinoma (RCC) is not clear. Here, we showed that clinical RCC tumor tissues showed much higher COUP-TFII expression level than adjacent normal tissues. When COUP-TFII was knocked down in RCC 769-P and 786-O cells by siRNA or shRNA-expressing lentivirus, the cell proliferation was markedly inhibited, and apoptosis increased. Moreover, the tumor growth of COUP-TFII knockdown 769-P and 786-O xenografts in nude mice was also obviously inhibited. Using qRT-PCR and Western blot, we showed that the expression of the tumor suppressor gene BRCA1 was upregulated in COUP-TFII knockdown cells. Simultaneously knockdown of BRCA1 and COUP-TFII partially rescued the inhibited cell proliferation and increased apoptosis in COUP-TFII single knockdown cells. These results indicate that COUP-TFII may play an oncogenic role in RCC, and COUP-TFII may promote tumor progression through inhibiting BRCA1. PMID:27193872

  10. 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.

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

    Directory of Open Access Journals (Sweden)

    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

  12. 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

  13. 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.

  14. 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.

  15. 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.

  16. Prunella vulgaris L. Upregulates eNOS expression in human endothelial cells.

    Science.gov (United States)

    Xia, Ning; Bollinger, Larissa; Steinkamp-Fenske, Katja; Förstermann, Ulrich; Li, Huige

    2010-01-01

    The purported effects of "circulation-improving" herbs used in traditional Chinese medicine (TCM) show striking similarities with the vascular actions of nitric oxide (NO) produced by the endothelial NO synthase (eNOS). We have previously reported that Salviae miltiorrhizae radix and Zizyphi spinosae semen upregulate eNOS expression. In the present study, we studied the effect on eNOS gene expression of 15 Chinese herbs with potential effects on the vasculature, and identified Prunella vulgaris L. (PVL) (flowering spike) as a potent eNOS-upregulating agent. In EA.hy 926 cells, a cell line derived from human umbilical vein endothelial cells (HUVEC), an aqueous extract of PVL increased eNOS promoter activity, eNOS mRNA and protein expressions, as well as NO production in concentration- and time-dependent manners. We have previously shown that ursolic acid (a constituent of Salviae miltiorrhizae radix), betulinic acid (a compound present in Zizyphi spinosae semen), luteolin and cynaroside (ingredients of artichoke, Cynara scolymus L.) are capable of enhancing eNOS gene expression. These compounds are also present in significant quantities in PVL. Thus, PVL contains active principles that stimulate human eNOS gene expression, and such compounds may have therapeutic potential against cardiovascular diseases. PMID:20503475

  17. PPAR-γ Silencing Inhibits the Apoptosis of A549 Cells by Upregulating Bcl-2

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    Jingyu YANG

    2013-03-01

    Full Text Available Background and objective Drug resistance is the one of primary causes of death in patients with lung cancer, PPAR-γ could induce the apoptosis and reverse drug resistance. The aim of this study is to investigate the expression of PPAR-γ on cisplatin sensitivity and apoptosis response of human lung cancer cell line A549. Methods Reconstruction of PPAR-γ silencing A549 cells (A549/PPAR-γ(- by siRNA. MTT assay was employed to determine the effect of cisplatin on the proliferation of A549/PPAR-γ(-, flow cytometry to determine the effect of cisplatin on the cell apoptosis, Western blot to determine the change of phosphorylation of Akt, caspase-3 and expression of bcl-2/bax. Finally, RT-PCR was employed to determine the transcriptional level of bcl-2. Results Two PPAR-γ silencing A549 cell clones were established successfully, and the expression of PPAR-γ was downregulated significantly as confirmed by RT-PCR and Western blot. After PPAR-γ silencing, the resistance of these two A549 clones to cisplatin was increased by 1.29-fold and 1.60-fold respectively. Flow cytometry showed that the apoptosis rate was decreased, and Western Blot showed that the phosphorylation of Akt and expression of bcl-2/bax were upregulated, caspase-3 was downregulated. Finally, RT-PCR showed that the transcriptional level of bcl-2 was upregulated as well. Conclusion Downregulation of PPAR-γ in A549 cells led to increase of cisplatin resistance. One of the mechanisms was upregulatin of phosphorylation of Akt and expression of bcl-2, which inhibited the apoptosis of cells. The downregulation of PPAR-γ is a possible mechanism that leads to the clinical drug resistance of cancer.

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

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    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

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

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    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.

  20. 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.

  1. 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.

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

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

    International Nuclear Information System (INIS)

    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

  4. ROCK inhibition enhances neurite outgrowth in neural stem cells by upregulating YAP expressionin vitro

    Institute of Scientific and Technical Information of China (English)

    Xu-feng Jia; Fei Ye; Yan-bo Wang; Da-xiong Feng

    2016-01-01

    Spontaneous axonal regeneration of neurons does not occur after spinal cord injury because of inhibition by myelin and other inhibitory factors. Studies have demonstrated that blocking the Rho/Rho-kinase (ROCK) pathway can promote neurite outgrowth in spinal cord injury models. In the present study, we investigated neurite outgrowth and neuronal differentiation in neural stem cells from the mouse subventricular zone after inhibition of ROCK in vitro. Inhibition of ROCK with Y-27632 increased neurite length, enhanced neuronal differentiation, and upregulated the expression of two major signaling pathway effectors, phospho-Akt and phospho-mitogen-activated protein kinase, and the Hippo pathway effector YAP. These results suggest that inhibition of ROCK mediates neurite outgrowth in neural stem cells by activating the Hippo signaling pathway.

  5. ROCK inhibition enhances neurite outgrowth in neural stem cells by upregulating YAP expression in vitro

    Science.gov (United States)

    Jia, Xu-feng; Ye, Fei; Wang, Yan-bo; Feng, Da-xiong

    2016-01-01

    Spontaneous axonal regeneration of neurons does not occur after spinal cord injury because of inhibition by myelin and other inhibitory factors. Studies have demonstrated that blocking the Rho/Rho-kinase (ROCK) pathway can promote neurite outgrowth in spinal cord injury models. In the present study, we investigated neurite outgrowth and neuronal differentiation in neural stem cells from the mouse subventricular zone after inhibition of ROCK in vitro. Inhibition of ROCK with Y-27632 increased neurite length, enhanced neuronal differentiation, and upregulated the expression of two major signaling pathway effectors, phospho-Akt and phospho-mitogen-activated protein kinase, and the Hippo pathway effector YAP. These results suggest that inhibition of ROCK mediates neurite outgrowth in neural stem cells by activating the Hippo signaling pathway. PMID:27482229

  6. 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.

  7. Melatonin enhances arsenic trioxide-induced cell death via sustained upregulation of Redd1 expression in breast cancer cells.

    Science.gov (United States)

    Yun, Sun-Mi; Woo, Sang Hyeok; Oh, Sang Taek; Hong, Sung-Eun; Choe, Tae-Boo; Ye, Sang-Kyu; Kim, Eun-Kyu; Seong, Min Ki; Kim, Hyun-A; Noh, Woo Chul; Lee, Jin Kyung; Jin, Hyeon-Ok; Lee, Yun-Han; Park, In-Chul

    2016-02-15

    Melatonin is implicated in various physiological functions, including anticancer activity. However, the mechanism(s) of its anticancer activity is not well understood. In the present study, we investigated the combined effects of melatonin and arsenic trioxide (ATO) on cell death in human breast cancer cells. Melatonin enhanced the ATO-induced apoptotic cell death via changes in the protein levels of Survivin, Bcl-2, and Bax, thus affecting cytochrome c release from the mitochondria to the cytosol. Interestingly, we found that the cell death induced by co-treatment with melatonin and ATO was mediated by sustained upregulation of Redd1, which was associated with increased production of reactive oxygen species (ROS). Combined treatment with melatonin and ATO induced the phosphorylation of JNK and p38 MAP kinase downstream from Redd1 expression. Rapamycin and S6K1 siRNA enhanced, while activation of mTORC1 by transfection with TSC2 siRNA suppressed the cell death induced by melatonin and ATO treatment. Taken together, our findings suggest that melatonin enhances ATO-induced apoptotic cell death via sustained upregulation of Redd1 expression and inhibition of mTORC1 upstream of the activation of the p38/JNK pathways in human breast cancer cells. PMID:26607805

  8. 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.

  9. 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.

  10. Retinoic acid induces HL-60 cell differentiation via the upregulation of miR-663

    Directory of Open Access Journals (Sweden)

    Zhuan Zhou

    2011-04-01

    Full Text Available Abstract Background Differentiation of the acute myeloid leukemia (AML cell line HL-60 can be induced by all trans-retinoic acid (ATRA; however, the mechanism regulating this process has not been fully characterized. Methods Using bioinformatics and in vitro experiments, we identified the microRNA gene expression profile of HL-60 cells during ATRA induced granulocytic differentiation. Results Six microRNAs were upregulated by ATRA treatment, miR-663, miR-494, miR-145, miR-22, miR-363* and miR-223; and three microRNAs were downregulated, miR-10a, miR-181 and miR-612. Additionally, miR-663 expression was regulated by ATRA. We used a lentivirus (LV backbone incorporating the spleen focus forming virus (SFFV-F promoter to drive miR-663 expression, as the CMV (Cytomegalovirus promoter is ineffective in some lymphocyte cells. Transfection of LV-miR-663 induced significant HL-60 cell differentiation in vitro. Conclusions Our results show miR-663 may play an important role in ATRA induced HL-60 cell differentiation. Lentivirus delivery of miR-663 could potentially be used directly as an anticancer treatment in hematological malignancies

  11. 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

  12. 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.

  13. 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

  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. MAGI3 Suppresses Glioma Cell Proliferation via Upregulation of PTEN Expression

    Institute of Scientific and Technical Information of China (English)

    MA Qian; ZHAO Ji Zong; HE Jun Qi; ZHANG Yan; MENG Ran; XIE Kun Ming; XIONG Ying; LIN Song; HE Zong Lin K; TAO Tao; YANG Ying

    2015-01-01

    Objective To investigate the role and molecular mechanism of membrane-associated guanylate kinase inverted 3 (MAGI3) in glioma cell proliferation. Methods The expression levels of MAGI3 and PTEN were assessed in glioma samples by Western blotting. MAGI3 was stably transfected into C6 glioma cells to obtain C6-MAGI3 cells. Then, the proliferation, the expression levels of MAGI3 and PTEN, and Akt phosphorylation were evaluated in C6 and C6-MAGI3 cells. Xenograft tumor models were established by subcutaneous injection of C6 and C6-MAGI3 cells into nude mice, and the growth rates of xenografts in the mice were compared. The potential role of MAGI3 expression in PI3K/Akt signaling activation was further investigated by examining the correlation between MAGI3 expression and the expression of PI3K/Akt signaling downstream target genes in a glioma dataset using gene set enrichment analysis (GSEA). Results Expression levels of MAGI3 and PTEN were significantly downregulated in gliomas. Overexpression of MAGI3 in the glioma C6 cell line upregulated PTEN protein expression, inhibited the phosphorylation of Akt, and suppressed cell proliferation. MAGI3 overexpression also inhibited the growth of C6 glioma tumor xenografts in nude mice. Analysis based on the GEO database confirmed the negative correlation between activation of PI3K/Akt pathway and MAGI3 mRNA levels in human glioma samples. Conclusion The loss of MAGI3 expression in glioma may enhance the proliferation of glioma cells via downregulation of PTEN expression, leading to the activation of the PI3K/Akt pathway. MAGI3 is a potential glioma suppressor.

  16. 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.

  17. Glucocorticoid up-regulation of high-affinity interleukin 6 receptors on human epithelial cells

    International Nuclear Information System (INIS)

    Interleukin 6 (IL-6) is a potent pleiotropic cytokine, known, among others, to stimulate immunoglobulin production by B cells and to trigger acute-phase protein synthesis by hepatocytes. Similar to IL-1, it is produced by monocytes and macrophages following an inflammatory challenge. Analysis of IL-6 receptor (IL-6R) expression on different human cell lines indicates that dexamethasone could up-regulate the number of IL-6R on one epithelial cell line (UAC) and on two hepatoma cell lines (HepG2 and Hep3B). This effect was confirmed by Scatchard analysis of binding experiments, using [35S]methionine and [35S]cysteine metabolically labeled IL-6. It was confirmed at the level of mRNA expression by Northern blot analysis. These results provide evidence for a link between IL-6 and glucocorticoids. They could represent an example of a system in which one role of glucocorticoids is to define more accurately the target of cytokines, and they could explain, at least partly, the frequently observed synergy between IL-6 and glucocorticoids, notably in the case of hepatocytes

  18. 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.

  19. 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.

  20. 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. PMID:26506049

  1. 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.

  2. 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

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

    International Nuclear Information System (INIS)

    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

  4. 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.

  5. 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.

  6. 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.

  7. 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. PMID:22298254

  8. Hes1 potentiates T cell lymphomagenesis by up-regulating a subset of notch target genes.

    Directory of Open Access Journals (Sweden)

    Darryll D Dudley

    Full Text Available BACKGROUND: Hairy/Enhancer of Split (Hes proteins are targets of the Notch signaling pathway and make up a class of basic helix-loop-helix (bHLH proteins that function to repress transcription. Data from Hes1 deficient mice suggested that Hes1, like Notch1, is necessary for the progression of early T cell progenitors. Constitutive activation of Notch is known to cause T cell leukemia or lymphoma but whether Hes1 has any oncogenic activity is not known. METHODOLOGY/PRINCIPAL FINDINGS: We generated mice carrying a Hes1 transgene under control of the proximal promote of the lck gene. Hes1 expression led to a reduction in numbers of total thymocytes, concomitant with the increased percentage and number of immature CD8+ (ISP T cells and sustained CD25 expression in CD4+CD8+ double positive (DP thymocytes. Hes1 transgenic mice develop thymic lymphomas at about 20 weeks of age with a low penetrance. However, expression of Hes1 significantly shortens the latency of T cell lymphoma developed in Id1 transgenic mice, where the function of bHLH E proteins is inhibited. Interestingly, Hes1 increased expression of a subset of Notch target genes in pre-malignant ISP and DP thymocytes, which include Notch1, Notch3 and c-myc, thus suggesting a possible mechanism for lymphomagenesis. CONCLUSIONS/SIGNIFICANCE: We have demonstrated for the first time that Hes1 potentiates T cell lymphomagenesis, by up-regulating a subset of Notch target genes and by causing an accumulation of ISP thymocytes particularly vulnerable to oncogenic transformation.

  9. 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.

  10. Metformin inhibits cell growth by upregulating microRNA-26a in renal cancer cells

    OpenAIRE

    Yang, Feng-Qiang; Wang, Ji-Jiao; Yan, Jia-Sheng; Huang, Jian-Hua; Li, Wei; Che, Jian-Ping; Wang, Guang-Chun; Liu, Min; Zheng, Jun-Hua

    2014-01-01

    Accumulating evidence suggests that metformin, a biguanide class of anti-diabetic drugs, possesses anti-cancer properties and may reduce cancer risk and improve prognosis. However, the mechanism by which metformin affects various cancers, including renal cancer still unknown. MiR-26a induces cell growth, cell cycle and cell apoptosis progression via direct targeting of Bcl-2, clyclin D1 and PTEN in cancer cells. In the present study, we used 786-O human renal cancer cell lines to study the ef...

  11. 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.

  12. 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.

  13. PMA/IONO affects diffuse large B-cell lymphoma cell growth through upregulation of A20 expression.

    Science.gov (United States)

    Yang, Wenxiu; Li, Yi; Li, Pinhao; Wang, Lingling

    2016-08-01

    Diffuse large B-cell lymphoma (DLBCL) is a common non-Hodgkin lymphoma. A20 and mucosa-associated lymphoid tissue lymphoma translocation gene 1 (MALT1) are known to be related to DLBCL pathogenesis and progression. This study aimed to assess the effects of phorbol myristate acetate/ionomycin (PMA/IONO) on the growth and apoptosis of the DLBCL cell line OCI-LY1, and their associations with A20, MALT1 and survivin levels. Cell viability was assessed by MTT assay. Cell cycle distribution and apoptosis were evaluated using flow cytometry after incubation with Annexin V-FITC/propidium iodide (PI) and RNase/PI, respectively. Gene and protein expression levels were determined by quantitative real-time PCR and western blotting, respectively. To further determine the role of A20, this gene was silenced in the OCI-LY1 cell line by specific siRNA transfection. A20 protein levels were higher in the OCI-LY1 cells treated with PMA/IONO compared with the controls, and were positively correlated with the concentration and treatment time of IONO, but not with changes of PMA and MALT1. Meanwhile, survivin expression was reduced in the OCI-LY1 cells after PMA/IONO treatment. In addition, OCI-LY1 proliferation was markedly inhibited, with a negative correlation between cell viability and IONO concentration. In concordance, apoptosis rates were higher in the OCI-LY1 cells after PMA + IONO treatment. Cell cycle distribution differed between the OCI-LY1 cells with and without PMA/IONO treatment only at 24 h, with increased cells in the G0/G1 stage after PMA/IONO treatment. These findings indicate that PMA/IONO promotes the apoptosis and inhibits the growth of DLBCL cells, in association with A20 upregulation. Thus, A20 may be a potential therapeutic target for DLBCL. PMID:27349720

  14. Upregulation of PTEN suppresses invasion in Tca8113 tongue cancer cells through repression of epithelial-mesenchymal transition (EMT).

    Science.gov (United States)

    Xie, Siming; Lu, Zhiyuan; Lin, Yanzhu; Shen, Lijia; Yin, Cao

    2016-05-01

    We previously discovered that the expression of the tumor suppressor phosphatase and tensin homolog (PTEN) was downregulated in the majority patients with tongue squamous cell carcinoma (TSCC). The aim of this study was to investigate the role of PTEN overexpression in the regulation of epithelial-mesenchymal transition (EMT) of the tongue squamous carcinoma cell line Tca8113 as well as explore the underlying mechanism. GV230 (containing the PTEN gene) and empty vectors were transfected into Tca8113 cells. After stable transfection, the messenger RNA (mRNA) and protein levels of PTEN were validated using quantitative real-time PCR (qPCR) and Western blot analysis. The growth and cell cycle were analyzed using Cell Counting Kit-8 (CCK-8) and flow cytometry, respectively. The invasion ability was measured with a transwell assay. The effects of PTEN overexpression on EMT and Hedgehog signaling were assessed by comparing Tca8113-PTEN cells with control and negative control cell groups. We found that PTEN expression was significantly upregulated after transfection. Meanwhile, upregulated PTEN inhibited the proliferation and invasion of Tca8113 cells. In addition, we observed changes in the EMT- and Hedgehog-associated proteins. These data demonstrated that PTEN upregulation could reduce invasion by inhibiting the process of EMT in Tca8113 cells, which might be related to the Hedgehog signaling pathway. PMID:26649861

  15. 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.

  16. Hypoxic stress up-regulates Kir2.1 expression and facilitates cell proliferation in brain capillary endothelial cells.

    Science.gov (United States)

    Yamamura, Hideto; Suzuki, Yoshiaki; Yamamura, Hisao; Asai, Kiyofumi; Imaizumi, Yuji

    2016-08-01

    The blood-brain barrier (BBB) is mainly composed of brain capillary endothelial cells (BCECs), astrocytes and pericytes. Brain ischemia causes hypoxic encephalopathy and damages BBB. However, it remains still unclear how hypoxia affects BCECs. In the present study, t-BBEC117 cells, an immortalized bovine brain endothelial cell line, were cultured under hypoxic conditions at 4-5% oxygen for 72 h. This hypoxic stress caused hyperpolarization of resting membrane potential. Patch-clamp recordings revealed a marked increase in Ba(2+)-sensitive inward rectifier K(+) current in t-BBEC117 cells after hypoxic culture. Western blot and real-time PCR analyses showed that Kir2.1 expression was significantly up-regulated at protein level but not at mRNA level after the hypoxic culture. Ca(2+) imaging study revealed that the hypoxic stress enhanced store-operated Ca(2+) (SOC) entry, which was significantly reduced in the presence of 100 μM Ba(2+). On the other hand, the expression of SOC channels such as Orai1, Orai2, and transient receptor potential channels was not affected by hypoxic stress. MTT assay showed that the hypoxic stress significantly enhanced t-BBEC117 cell proliferation, which was inhibited by approximately 60% in the presence of 100 μM Ba(2+). We first show here that moderate cellular stress by cultivation under hypoxic conditions hyperpolarizes membrane potential via the up-regulation of functional Kir2.1 expression and presumably enhances Ca(2+) entry, resulting in the facilitation of BCEC proliferation. These findings suggest potential roles of Kir2.1 expression in functional changes of BCECs in BBB following ischemia. PMID:27235552

  17. Upregulation of cell adhesion through delta Np63 silencing in human 5637 bladder cancer cells

    Institute of Scientific and Technical Information of China (English)

    Yun-Feng He; Dai-Yin Tian; Zheng-Jin Yi; Zhi-Kang Yin; Chun-Li Luo; Wei Tang; Xiao-Hou Wu

    2012-01-01

    Some researchs have demonstrated that the loss of delta Np63 is associated with aggressive phenotypes and poor prognosis.However,other research indicates that delta Np63 is considered to have oncogenic properties,Delta Np63 overexpression is often observed in association with the oncogenic growth of squamous cell carcinomas and bladder cancer.In this study,we investigated the oocogenic role of delta Np63 in regulating cell adhesion in transitional cell carcinoma of the bladder (TCCB).The Cells were stably transfected with the delta Np63 short hairpin RNA (shRNA) plasmid.Immunocytochemistry was performed to determine the knockdown efficiency.Tumour cells were studied for their ability to adhere to vascular endothelial cells.Confocal microscopy was used to analyse the changes in cytoskeletal F-actin.F-actin expression was measured by flow cytometry.Cell invasion ability was assessed using transwell chambers.fhe delta Np63-silenced tumour cells were shown to adhere more tightly than controls to vascular endothelial cells (P<0.05).The content of F-actin in the delta Np63-silenced cells was enhanced (P<0.05),The Matrigel invasion assays showed that human 5637 bladder cancer cells had a lower degree of motility when transfected with pdetta Np63-shRNA ( P< 0.05).In conclusion,silencing of the delta Np63 expression can enhance the adhesiveness of 5637 cells by inducing F-actin cytoskeleton production,and it will possibly inhibit the TCCB invasion and metastasis.

  18. 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.

  19. Upregulation of MiR-1280 Expression in Non-small Cell Lung Cancer Tissues

    Institute of Scientific and Technical Information of China (English)

    Li-Min Xu; Li-Qin Li; Jing Li; Hong-Wei Li; Qi-Bin Shen; Jin-Liang Ping; Zhi-Hong Ma

    2015-01-01

    Background:Non-small cell lung cancer (NSCLC) is a prolific and high-mortality disease with few effective treatments.Although the detection and surgical techniques for NSCLC continue to advance,the survival rate for the patients with NSCLC remains poor.Enhanced predictive biomarkers such as microRNAs (miRNAs) are needed at the time of diagnosis to better tailor therapies for patients.This study focused on the expression ofmiR-1280 in NSCLC tissues and distal normal tissues in order to explore the association between miR-1280 expression and NSCLC.Methods:A total of 72 newly diagnosed primary NSCLC patients were enrolled in this study.Quantitative real-time polymerase chain reaction (PCR) was performed to identify the expression level ofmiR-1280 in the NSCLC tissues and distal normal tissues of these patients.Results:The miR-1280 expression was significantly higher in the NSCLC tissues (0.084 ± 0.099) than distal normal tissues (0.014 ± 0.015,P =0.009).In 54 patients (75%),the miR-1280 expression in the NSCLC tissues was upregulated (2-△△ct > 2),and no case showed a downregulation of miR-1280 expression.Conclusions:The expression level ofmiR-1280 could be regarded as a biomarker for NSCLC.

  20. 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

  1. Effect of up-regulated expression of tumor suppressor gene p14ARF on apoptosis of chronic myeloid leukemia cells

    Institute of Scientific and Technical Information of China (English)

    白元松

    2013-01-01

    Objective To investigate the effect of up-regulated expression of tumor suppressor gene p14ARFon apoptosis of chronic myeloid leukemia (CML) cells and its interaction with imatinib.Methods Tumor suppressor gene p14ARFwas transduced into K562 (K562-p14ARF) and 4blast crisis primary CML cells (CML-BC 1-4) using vesicular stomatitis virus glycoprotein (VSV-G)

  2. 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.

  3. 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.

  4. 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.

  5. 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

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

    Directory of Open Access Journals (Sweden)

    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.

  7. 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.

  8. 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.

  9. Overexpression of GAB2 in ovarian cancer cells promotes tumor growth and angiogenesis by upregulating chemokine expression

    Science.gov (United States)

    Duckworth, C; Zhang, L; Carroll, S L; Ethier, S P; Cheung, H W

    2016-01-01

    We previously found that the scaffold adapter GRB2-associated binding protein 2 (GAB2) is amplified and overexpressed in a subset of primary high-grade serous ovarian cancers and cell lines. Ovarian cancer cells overexpressing GAB2 are dependent on GAB2 for activation of the phosphatidylinositol 3-kinase (PI3K) pathway and are sensitive to PI3K inhibition. In this study, we show an important role of GAB2 overexpression in promoting tumor angiogenesis by upregulating expression of multiple chemokines. Specifically, we found that suppression of GAB2 by inducible small hairpin RNA in ovarian cancer cells inhibited tumor cell proliferation, angiogenesis and peritoneal tumor growth in immunodeficient mice. Overexpression of GAB2 upregulated the secretion of several chemokines from ovarian cancer cells, including CXCL1, CXCL2 and CXCL8. The secreted chemokines not only signal through endothelial CXCR2 receptor in a paracrine manner to promote endothelial tube formation, but also act as autocrine growth factors for GAB2-induced transformation of fallopian tube secretory epithelial cells and clonogenic growth of ovarian cancer cells overexpressing GAB2. Pharmacological inhibition of inhibitor of nuclear factor kappa-B kinase subunit β (IKKβ), but not PI3K, mechanistic target of rapamycin (mTOR) or mitogen-activated protein kinase (MEK), could effectively suppress GAB2-induced chemokine expression. Inhibition of IKKβ augmented the efficacy of PI3K/mTOR inhibition in suppressing clonogenic growth of ovarian cancer cells with GAB2 overexpression. Taken together, these findings suggest that overexpression of GAB2 in ovarian cancer cells promotes tumor growth and angiogenesis by upregulating expression of CXCL1, CXCL2 and CXCL8 that is IKKβ-dependent. Co-targeting IKKβ and PI3K pathways downstream of GAB2 might be a promising therapeutic strategy for ovarian cancer that overexpresses GAB2. PMID:26657155

  10. 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.

  11. Tibolone protects astrocytic cells from glucose deprivation through a mechanism involving estrogen receptor beta and the upregulation of neuroglobin expression.

    Science.gov (United States)

    Avila-Rodriguez, Marco; Garcia-Segura, Luis Miguel; Hidalgo-Lanussa, Oscar; Baez, Eliana; Gonzalez, Janneth; Barreto, George E

    2016-09-15

    Tibolone, a synthetic steroid used for the prevention of osteoporosis and the treatment of climacteric symptoms in post-menopausal women, may exert tissue selective estrogenic actions acting on estrogen receptors (ERs). We previously showed that tibolone protects human T98G astroglial cells against glucose deprivation (GD). In this study we have explored whether the protective effect of tibolone on these cells is mediated by ERs. Experimental studies showed that both ERα and ERβ were involved in the protection by tibolone on GD cells, being ERβ preferentially involved on these actions over ERα. Tibolone increased viability of GD cells by a mechanism fully blocked by an ERβ antagonist and partially blocked by an ERα antagonist. Furthermore, ERβ inhibition prevented the effect of tibolone on nuclear fragmentation, ROS and mitochondrial membrane potential in GD cells. The protective effect of tibolone was mediated by neuroglobin. Tibolone upregulated neuroglobin in T98G cells and primary mouse astrocytes by a mechanism involving ERβ and neuroglobin silencing prevented the protective action of tibolone on GD cells. In summary, tibolone protects T98G cells by a mechanism involving ERβ and the upregulation of neuroglobin. PMID:27250720

  12. 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.

  13. 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.

  14. Discovering up-regulated VEGF–C expression in swine umbilical vein endothelial cells by classical swine fever virus Shimen

    OpenAIRE

    Ning, Pengbo; Zhang, Yanming; Guo, Kangkang; Chen, Ru; Liang, Wulong; Lin, Zhi; Li, Helin

    2014-01-01

    International audience Infection of domestic swine with the highly virulent Shimen strain of classical swine fever virus causes hemorrhagic lymphadenitis and diffuse hemorrhaging in infected swine. We analyzed patterns of gene expression for CSFV Shimen in swine umbilical vein endothelial cells (SUVECs). Transcription of the vascular endothelial growth factor (VEGF) C gene (VEGF-C) and translation of the corresponding protein were significantly up-regulated in SUVECs. Our findings suggest ...

  15. Lysophosphatidic acid stimulates gastric cancer cell proliferation via ERK1-dependent upregulation of sphingosine kinase 1 transcription

    OpenAIRE

    Ramachandran, Subramaniam; Shida, Dai; Nagahashi, Masayuki; Fang, Xianjun; Milstien, Sheldon; Takabe, Kazuaki; Spiegel, Sarah

    2010-01-01

    In MKN1 gastric cancer cells, lysophosphatidic acid (LPA) upregulates expression of sphingosine kinase 1 (SphK1) and its downregulation or inhibition suppresses LPA-mediated proliferation. Although LPA activates numerous signaling pathways downstream of its receptors, including ERK1/2, p38, JNK, and Akt, and the transactivation of the EGF receptor, pharmacological and molecular approaches demonstrated that only activation of ERK1, in addition to the CCAAT/enhancer-binding protein β (C/EBPβ) t...

  16. Upregulation of alpha cell glucagon-like peptide 1 (GLP-1) in Psammomys obesus--an adaptive response to hyperglycaemia?

    DEFF Research Database (Denmark)

    Hansen, A M K; Bödvarsdottir, T B; Nordestgaard, D N E;

    2011-01-01

    of the study was to evaluate if, during the development of diabetes, alpha cells produce GLP-1 that, in turn, might trigger beta cell growth. Methods Beta cell mass, GLP-1 and insulin levels were measured in the gerbil Psammomys obesus (P. obesus), a rodent model of nutritionally induced diabetes. Furthermore......, the presence of biologically active forms of GLP-1 and PC1/3 in alpha cells was demonstrated by immunofluorescence, and the release of GLP-1 from isolated P. obesus, mouse and human islets was investigated. Results During the development of diabetes in P. obesus, a significant increase in GLP-1 was detected...... from alpha cells is upregulated in P. obesus during the development of diabetes. A similar response is seen in islets exposed to high glucose, which supports the hypothesis that GLP-1 released from alpha cells promotes an increase in beta cell mass and function during metabolic challenge...

  17. CD40-TRAF Signaling Upregulates CX3CL1 and TNF-α in Human Aortic Endothelial Cells but Not in Retinal Endothelial Cells.

    Directory of Open Access Journals (Sweden)

    Jennifer A Greene

    Full Text Available CD40, CX3CL1 and TNF-α promote atheroma and neointima formation. CD40 and TNF-α are also central to the development of diabetic retinopathy while CX3CL1 may play a role in the pathogenesis of this retinopathy. The purpose of this study was to examine whether CD40 ligation increases CX3CL1 and TNF-α protein expression in human endothelial cells from the aorta and retina. CD154 (CD40 ligand upregulated membrane-bound and soluble CX3CL1 in human aortic endothelial cells. CD154 triggered TNF-α production by human aortic endothelial cells. TNF Receptor Associated Factors (TRAF are key mediators of CD40 signaling. Compared to human aortic endothelial cells that express wt CD40, cells that express CD40 with a mutation that prevents TRAF2,3 recruitment, or CD40 with a mutation that prevents TRAF6 recruitment exhibited a profound inhibition of CD154-driven upregulation of membrane bound and soluble CX3CL1 as well as of TNF-α secretion. While both CD154 and TNF-α upregulated CX3CL1 in human aortic endothelial cells, these stimuli could act independently of each other. In contrast to human aortic endothelial cells, human retinal endothelial cells did not increase membrane bound or soluble CX3CL1 expression or secrete TNF-α in response to CD154 even though CD40 ligation upregulated ICAM-1 and CCL2 in these cells. Moreover, TNF-α did not upregulate CX3CL1 in retinal endothelial cells. In conclusion, CD40 ligation increases CX3CL1 protein levels and induces TNF-α production in endothelial cells. However, endothelial cells are heterogeneous in regards to these responses. Human aortic but not retinal endothelial cells upregulated CX3CL1 and TNF-α in response to CD40 ligation, as well as upregulated CX3CL1 in response to TNF-α. These dissimilarities may contribute to differences in regulation of inflammation in large vessels versus the retina.

  18. 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.

  19. 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.

  20. Berry anthocyanins reduce proliferation of human colorectal carcinoma cells by inducing caspase-3 activation and p21 upregulation.

    Science.gov (United States)

    Anwar, Sirajudheen; Fratantonio, Deborah; Ferrari, Daniela; Saija, Antonella; Cimino, Francesco; Speciale, Antonio

    2016-08-01

    Colorectal cancer is the fourth most common type of cancer worldwide, and adenocarcinoma cells that form the majority of colorectal tumors are markedly resistant to antineoplastic agents. Epidemiological studies have demonstrated that consumption of fruits and vegetables that are rich in polyphenols, is linked to reduced risk of colorectal cancer. In the present study, the effect of a standardized anthocyanin (ACN)‑rich extract on proliferation, apoptosis and cell cycle in the Caco-2 human colorectal cancer cell line was evaluated by trypan blue and clonogenic assays and western blot analysis of cleaved caspase‑3 and p21Waf/Cif1. The results of the current study demonstrated that the ACN extract markedly decreased Caco‑2 cell proliferation, induced apoptosis by activating caspase‑3 cleavage, and upregulated cyclin‑dependent kinase inhibitor 1 (p21Waf/Cif1) expression in a dose dependent manner. Furthermore, ACN extract was able to produce a dose‑dependent increase of intracellular reactive oxygen species (ROS) in Caco‑2 cells, together with a light increase of the cell total antioxidant status. In conclusion, the present study demonstrated that a standardized berry anthocyanin rich extract inhibited proliferation of Caco‑2 cells by promoting ROS accumulation, inducing caspase‑3 activation, and upregulating the expression of p21Waf/Cif1. PMID:27314273

  1. 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.

  2. Isoreserpine promotes β-catenin degradation via Siah-1 up-regulation in HCT116 colon cancer cells

    International Nuclear Information System (INIS)

    Aberrant accumulation of intracellular β-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 β-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 β-catenin by up-regulation of Siah-1 in HEK293 and HCT116 colon cancer cells. Moreover, isoreserpine repressed the expression of β-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.

  3. 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.

  4. 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.

  5. Cell cycle is disturbed in mucopolysaccharidosis type II fibroblasts, and can be improved by genistein.

    Science.gov (United States)

    Moskot, Marta; Gabig-Cimińska, Magdalena; Jakóbkiewicz-Banecka, Joanna; Węsierska, Magdalena; Bocheńska, Katarzyna; Węgrzyn, Grzegorz

    2016-07-01

    Mucopolysaccharidoses (MPSs) are inherited metabolic diseases caused by mutations resulting in deficiency of one of enzymes involved in degradation of glycosaminoglycans (GAGs). These compounds accumulate in cells causing their dysfunctions. Genistein is a molecule previously found to both modify GAG metabolism and modulate cell cycle. Therefore, we investigated whether the cell cycle is affected in MPS cells and if genistein can influence this process. Fibroblasts derived from patients suffering from MPS types I, II, IIIA and IIIB, as well as normal human fibroblasts (the HDFa cell line) were investigated. MTT assay was used for determination of cell proliferation, and the cell cycle was analyzed by using the MUSE® Cell Analyzer. While effects of genistein on cell proliferation were similar in both normal and MPS fibroblasts, fractions of cells in the G0/G1 phase were higher, and number of cells entering the S and G2/M phases was considerably lower in MPS II fibroblasts relative to control cells. Somewhat similar tendency, though significantly less pronounced, could be noted in MPS I, but only at longer times of incubation. However, this was not observed in MPS IIIA and MPS IIIB fibroblasts. Genistein (5, 7-dihydroxy-3-(4-hydroxyphenyl)-4H-1-benzopyran-4-one) was found to be able to partially correct the disturbances in the MPS II cell cycle, and to some extent in MPS I, at higher concentrations of this compound. The tendency to increase the fractions of cells entering the S and G2/M phases was also observed in MPS IIIA and IIIB fibroblasts treated with genistein. In conclusion, this is the first report indicating that the cell cycle can be impaired in MPS cells. The finding that genistein can improve the MPS II (and to some extent also MPS I) cell cycle provides an input to our knowledge on the molecular mechanisms of action of this compound. PMID:27016302

  6. 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.

  7. 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.

  8. Upregulation of retinoic acid receptor-β reverses drug resistance in cholangiocarcinoma cells by enhancing susceptibility to apoptosis.

    Science.gov (United States)

    Ren, Hong-Yue; Chen, Bo; Huang, Gui-Li; Liu, Yu; Shen, Dong-Yan

    2016-10-01

    Retinoic acid receptor β (RARβ), a known tumor suppressor gene, is frequently silenced in numerous malignant types of tumor. Recent reports have demonstrated that loss of RARβ expression may be responsible, in part, for the drug resistance observed in clinical trials. However, little is known about the role of RARβ in regulating drug sensitivity in patients with cholangiocarcinoma (CCA) with a high risk of mortality and poor outcomes. In the present study, low RARβ expression was observed in the majority of CCA tissues investigated (28/33, 84.8%). In addition, the CCA cell line QBC939, which exhibits low RARβ expression, was found to be significantly resistant to chemotherapeutic agents compared with SK‑ChA‑1, MZ‑ChA‑1 and Hccc9810 CCA cell lines, which exhibit high RARβ expression. Furthermore, upregulation of RARβ significantly enhanced the sensitivity of QBC939 cells to common chemotherapeutic agents both in vitro and in vivo. Upregulation of RARβ was shown to increase the expression of proapoptotic genes bax, bak and bim, in addition to caspase‑3 activity, and decrease the expression of antiapoptotic genes bcl‑2, bcl‑xL and mcl‑1. As a result, CCA cells were more susceptible to caspase‑dependent apoptosis. Taken together, these data suggest that RARβ upregulation rendered CCA cells more sensitive to chemotherapeutic agents by increasing the susceptibility of cells to caspase-dependent apoptosis. These results support the hypothesis that RARβ may be an ideal chemosensitization target for the treatment of patients with drug-resistant CCA. PMID:27599527

  9. Differentiation of mouse erythroleukemia cells is blocked by late up-regulation of a c-myb transgene.

    OpenAIRE

    McClinton, D; Stafford, J; Brents, L; Bender, T. P.; Kuehl, W M

    1990-01-01

    During chemically induced differentiation of Friend virus-infected mouse erythroleukemia (MEL) cell lines, there is a biphasic down-regulation of the c-myb proto-oncogene. A plasmid containing a murine c-myb cDNA controlled by a mouse metallothionein I promoter was transfected into the C19 MEL cell line. For six transfected clones, it was found that expression of the exogenous c-myb mRNA could be up-regulated by the addition of 120 microM ZnCl2 and that the N,N'-hexamethylenebisacetamide-indu...

  10. CD34 cells from patients with trisomy 8 myelodysplastic syndrome (MDS) express early apoptotic markers but avoid programmed cell death by up-regulation of antiapoptotic proteins

    OpenAIRE

    Sloand, Elaine M.; Pfannes, Loretta; Chen, Gubin; Shah, Simant; Solomou, Elena E.; Barrett, John; Neal S Young

    2007-01-01

    CD34 cells from patients with trisomy 8 myelodysplastic syndrome (MDS) are distinguished from other MDS cells and from normal hematopoietic cells by their pronounced expression of apoptotic markers. Paradoxically, trisomy 8 clones can persist in patients with bone marrow failure and expand following immunosuppression. We previously demonstrated up-regulation of c-myc and CD1 by microarray analysis. Here, we confirmed these findings by real-time polymerase chain reaction (PCR), demonstrated up...

  11. Salubrinal-Mediated Upregulation of eIF2α Phosphorylation Increases Doxorubicin Sensitivity in MCF-7/ADR Cells.

    Science.gov (United States)

    Jeon, Yong-Joon; Kim, Jin Hyun; Shin, Jong-Il; Jeong, Mini; Cho, Jaewook; Lee, Kyungho

    2016-02-01

    Eukaryotic translation initiation factor 2 alpha (eIF2α), which is a component of the eukaryotic translation initiation complex, functions in cell death and survival under various stress conditions. In this study, we investigated the roles of eIF2α phosphorylation in cell death using the breast cancer cell lines MCF-7 and MCF-7/ADR. MCF-7/ADR cells are MCF-7-driven cells that have acquired resistance to doxorubicin (ADR). Treatment of doxorubicin reduced the viability and induced apoptosis in both cell lines, although susceptibility to the drug was very different. Treatment with doxorubicin induced phosphorylation of eIF2α in MCF-7 cells but not in MCF-7/ADR cells. Basal expression levels of Growth Arrest and DNA Damage 34 (GADD34), a regulator of eIF2α, were higher in MCF-7/ADR cells compared to MCF-7 cells. Indeed, treatment with salubrinal, an inhibitor of GADD34, resulted in the upregulation of eIF2α phosphorylation and enhanced doxorubicin-mediated apoptosis in MCF-7/ADR cells. However, MCF-7 cells did not show such synergic effects. These results suggest that dephosphorylation of eIF2α by GADD34 plays an important role in doxorubicin resistance in MCF-7/ADR cells. PMID:26743901

  12. 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.

  13. 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.

  14. 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.

  15. 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.

  16. 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.

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

    International Nuclear Information System (INIS)

    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

  18. 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

  19. 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.

  20. 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.

  1. 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.

  2. Epstein-Barr virus Zta upregulates matrix metalloproteinases 3 and 9 that synergistically promote cell invasion in vitro.

    Directory of Open Access Journals (Sweden)

    Yu-Yan Lan

    Full Text Available Zta is a lytic transactivator of Epstein-Barr virus (EBV and has been shown to promote migration and invasion of epithelial cells. Although previous studies indicate that Zta induces expression of matrix metalloproteinase (MMP 9 and MMP1, direct evidence linking the MMPs to Zta-induced cell migration and invasion is still lacking. Here we performed a series of in vitro studies to re-examine the expression profile and biologic functions of Zta-induced MMPs in epithelial cells derived from nasopharyngeal carcinoma. We found that, in addition to MMP9, MMP3 was a new target gene upregulated by Zta. Ectopic Zta expression in EBV-negative cells increased both mRNA and protein production of MMP3. Endogenous Zta also contributed to induction of MMP3 expression, migration and invasion of EBV-infected cells. Zta activated the MMP3 promoter through three AP-1 elements, and its DNA-binding domain was required for the promoter binding and MMP3 induction. We further tested the effects of MMP3 and MMP9 on cell motility and invasiveness in vitro. Zta-promoted cell migration required MMP3 but not MMP9. On the other hand, both MMP3 and MMP9 were essential for Zta-induced cell invasion, and co-expression of the two MMPs synergistically increased cell invasiveness. Therefore, this study provides integrated evidence demonstrating that, at least in the in vitro cell models, Zta drives cell migration and invasion through MMPs.

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

    Science.gov (United States)

    Li, Yanchun; Luo, Qiyu; Yuan, Lei; Miao, Caixia; Mu, Xiaoshuo; Xiao, Wei; Li, Jianchun; Sun, Tiemin; Ma, Enlong

    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. PMID:22668848

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

    International Nuclear Information System (INIS)

    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

  5. 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.

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

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    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.

  7. 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.

  8. NADPH oxidase and lipid raft-associated redox signaling are required for PCB153-induced upregulation of cell adhesion molecules in human brain endothelial cells

    International Nuclear Information System (INIS)

    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.

  9. Upregulation of PTEN in glioma cells by cord blood mesenchymal stem cells inhibits migration via downregulation of the PI3K/Akt pathway.

    Directory of Open Access Journals (Sweden)

    Venkata Ramesh Dasari

    Full Text Available BACKGROUND: PTEN (phosphatase and tensin homologue deleted on chromosome ten is a tumor suppressor gene implicated in a wide variety of human cancers, including glioblastoma. PTEN is a major negative regulator of the PI3K/Akt signaling pathway. Most human gliomas show high levels of activated Akt, whereas less than half of these tumors carry PTEN mutations or homozygous deletions. The unique ability of mesenchymal stem cells to track down tumor cells makes them as potential therapeutic agents. Based on this capability, new therapeutic approaches have been developed using mesenchymal stem cells to cure glioblastoma. However, molecular mechanisms of interactions between glioma cells and stem cells are still unknown. METHODOLOGY/PRINCIPAL FINDINGS: In order to study the mechanisms by which migration of glioma cells can be inhibited by the upregulation of the PTEN gene, we studied two glioma cell lines (SNB19 and U251 and two glioma xenograft cell lines (4910 and 5310 alone and in co-culture with human umbilical cord blood-derived mesenchymal stem cells (hUCBSC. Co-cultures of glioma cells showed increased expression of PTEN as evaluated by immunofluorescence and immunoblotting assays. Upregulation of PTEN gene is correlated with the downregulation of many genes including Akt, JUN, MAPK14, PDK2, PI3K, PTK2, RAS and RAF1 as revealed by cDNA microarray analysis. These results have been confirmed by reverse-transcription based PCR analysis of PTEN and Akt genes. Upregulation of PTEN resulted in the inhibition of migration capability of glioma cells under in vitro conditions. Also, wound healing capability of glioma cells was significantly inhibited in co-culture with hUCBSC. Under in vivo conditions, intracranial tumor growth was inhibited by hUCBSC in nude mice. Further, hUCBSC upregulated PTEN and decreased the levels of XIAP and Akt, which are responsible for the inhibition of tumor growth in the mouse brain. CONCLUSIONS/SIGNIFICANCE: Our studies

  10. Shikonin Induces Apoptosis, Necrosis, and Premature Senescence of Human A549 Lung Cancer Cells through Upregulation of p53 Expression

    Directory of Open Access Journals (Sweden)

    Yueh-Chiao Yeh

    2015-01-01

    Full Text Available Shikonin, a natural naphthoquinone pigment isolated from Lithospermum erythrorhizon, has been reported to suppress growth of various cancer cells. This study was aimed to investigate whether this chemical could also inhibit cell growth of lung cancer cells and, if so, works via what molecular mechanism. To fulfill this, A549 lung cancer cells were treated with shikonin and then subjected to microscopic, biochemical, flow cytometric, and molecular analyses. Compared with the controls, shikonin significantly induced cell apoptosis and reduced proliferation in a dose-dependent manner. Specially, lower concentrations of shikonin (1–2.5 μg/mL cause viability reduction; apoptosis and cellular senescence induction is associated with upregulated expressions of cell cycle- and apoptotic signaling-regulatory proteins, while higher concentrations (5–10 μg/mL precipitate both apoptosis and necrosis. Treatment of cells with pifithrin-α, a specific inhibitor of p53, suppressed shikonin-induced apoptosis and premature senescence, suggesting the role of p53 in mediating the actions of shikonin on regulation of lung cancer cell proliferation. These results indicate the potential and dose-related cytotoxic actions of shikonin on A549 lung cancer cells via p53-mediated cell fate pathways and raise shikonin a promising adjuvant chemotherapeutic agent for treatment of lung cancer in clinical practice.

  11. Upregulator of cell proliferation predicts poor prognosis in hepatocellular carcinoma and contributes to hepatocarcinogenesis by downregulating FOXO3a.

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    Chan Xie

    Full Text Available OBJECTIVE: The goal of the present study was to investigate the potential correlation between the expression level of upregulator of cell proliferation (URGCP/URG4 and the prognosis of hepatocellular carcinoma (HCC, and to examine the biological function of URGCP/URG4 in the progression of HCC, to better understand its underlying molecular mechanism in hepatic tumorigenesis. DESIGN: URGCP/URG4 expression was analyzed in 15 HCC cell lines, in 278 archived paraffin-embedded HCC sections, and in 10 pairs of fresh HCC tumor and para-tumor non-cancerous tissues using immunohistochemistry (IHC and Western blotting analysis (WB. The effect of URGCP/URG4 on cell proliferation and tumorigenesis was examined in vitro and in vivo. WB and luciferase reporter analyses were performed to identify the effects of URGCP/URG4-overexpression or -knockdown on expression of cell cycle regulators and transcriptional activity of FOXO3a. RESULTS: IHC results revealed an upregulation of URGCP/URG4 in all HCC cell lines and fresh HCC samples as compared with normal liver cells and para-tumor tissues, respectively. URGCP/URG4 was also expressed at a high level in 122 of the 278 (43.8% archived HCC specimens. The expression level of URGCP/URG4 was significantly correlated with clinical staging and poor patient survival of HCC in the study cohort, and in various clinical subgroups. Strikingly, ectopic expression of URGCP/URG4 induced proliferation and anchorage-independent growth of HCC cells, while silencing of URGCP/URG4 had the opposite effect. Furthermore, URGCP/URG4 overexpression in HCC cells increased cellular entry into the G1/S transitional phase, associated with downregulation of p27(Kip1 and p21(Cip1 and upregulation of cyclin D1. These effects were accompanied by enhanced Akt activity and reduced FOXO3a transcriptional activity. CONCLUSIONS: URGCP/URG4 plays an important role in promoting proliferation and tumorigenesis of HCC and may represent a novel prognostic

  12. Fibroin and sericin from Bombyx mori silk stimulate cell migration through upregulation and phosphorylation of c-Jun.

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    Celia Martínez-Mora

    Full Text Available Wound healing is a biological process directed to the restoration of tissue that has suffered an injury. An important phase of wound healing is the generation of a basal epithelium able to wholly replace the epidermis of the wound. A broad range of products derived from fibroin and sericin from Bombyx mori silk are used to stimulate wound healing. However, so far the molecular mechanism underlying this phenomenon has not been elucidated. The aim of this work was to determine the molecular basis underlying wound healing properties of silk proteins using a cell model. For this purpose, we assayed fibroin and sericin in a wound healing scratch assay using MDA-MB-231 and Mv1Lu cells. Both proteins stimulated cell migration. Furthermore, treatment with sericin and fibroin involved key factors of the wound healing process such as upregulation of c-Jun and c-Jun protein phosphorylation. Moreover, fibroin and sericin stimulated the phosphorylation of ERK 1/2 and JNK 1/2 kinases. All these experiments were done in the presence of specific inhibitors for some of the cell signalling pathways referred above. The obtained results revealed that MEK, JNK and PI3K pathways are involved in fibroin and sericin stimulated cells migration. Inhibition of these three kinases prevented c-Jun upregulation and phosphorylation by fibroin or sericin. Fibroin and sericin were tested in the human keratinocyte cell line, HaCaT, with similar results. Altogether, our results showed that fibroin and sericin initiate cell migration by activating the MEK, JNK and PI3K signalling pathways ending in c-Jun activation.

  13. Freezing tolerance of sea urchin embryonic cells: Differentiation commitment and cytoskeletal disturbances in culture.

    Science.gov (United States)

    Odintsova, Nelly A; Ageenko, Natalya V; Kipryushina, Yulia O; Maiorova, Mariia A; Boroda, Andrey V

    2015-08-01

    This study focuses on the freezing tolerance of sea urchin embryonic cells. To significantly reduce the loss of physiological activity of these cells that occurs after cryopreservation and to study the effects of ultra-low temperatures on sea urchin embryonic cells, we tested the ability of the cells to differentiate into spiculogenic or pigment directions in culture, including an evaluation of the expression of some genes involved in pigment differentiation. A morphological analysis of cytoskeletal disturbances after freezing in a combination of penetrating (dimethyl sulfoxide and ethylene glycol) and non-penetrating (trehalose and polyvinylpyrrolidone) cryoprotectants revealed that the distribution pattern of filamentous actin and tubulin was similar to that in the control cultures. In contrast, very rare spreading cells and a small number of cells with filamentous actin and tubulin were detected after freezing in the presence of only non-penetrating cryoprotectants. The largest number of pigment cells was found in cultures frozen with trehalose or trehalose and dimethyl sulfoxide. The ability to induce the spicule formation was lost in the cells frozen only with non-penetrating cryoprotectants, while it was maximal in cultures frozen in a cryoprotective mixture containing both non-penetrating and penetrating cryoprotectants (particularly, when ethylene glycol was present). Using different markers for cell state assessment, an effective cryopreservation protocol for sea urchin cells was developed: three-step freezing with a low cooling rate (1-2°C/min) and a combination of non-penetrating and penetrating cryoprotectants made it possible to obtain a high level of cell viability (up to 65-80%). PMID:26049089

  14. 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.

  15. Gamma (γ) tocopherol upregulates peroxisome proliferator activated receptor (PPAR) gamma (γ) expression in SW 480 human colon cancer cell lines

    International Nuclear Information System (INIS)

    Tocopherols are lipid soluble antioxidants that exist as eight structurally different isoforms. The intake of γ-tocopherol is higher than α-tocopherol in the average US diet. The clinical results of the effects of vitamin E as a cancer preventive agent have been inconsistent. All published clinical trials with vitamin E have used α-tocopherol. Recent epidemiological, experimental and molecular studies suggest that γ-tocopherol may be a more potent chemopreventive form of vitamin E compared to the more-studied α-tocopherol. γ-Tocopherol exhibits differences in its ability to detoxify nitrogen dioxide, growth inhibitory effects on selected cancer cell lines, inhibition of neoplastic transformation in embryonic fibroblasts, and inhibition of cyclooxygenase-2 (COX-2) activity in macrophages and epithelial cells. Peroxisome proliferator activator receptor γ (PPARγ) is a promising molecular target for colon cancer prevention. Upregulation of PPARγ activity is anticarcinogenic through its effects on downstream genes that affect cellular proliferation and apoptosis. The thiazolidine class of drugs are powerful PPARγ ligands. Vitamin E has structural similarity to the thiazolidine, troglitazone. In this investigation, we tested the effects of both α and γ tocopherol on the expression of PPARγ mRNA and protein in SW 480 colon cancer cell lines. We also measured the intracellular concentrations of vitamin E in SW 480 colon cancer cell lines. We have discovered that the α and γ isoforms of vitamin E upregulate PPARγ mRNA and protein expression in the SW480 colon cancer cell lines. γ-Tocopherol is a better modulator of PPARγ expression than α-tocopherol at the concentrations tested. Intracellular concentrations increased as the vitamin E concentration added to the media was increased. Further, γ-tocopherol-treated cells have higher intracellular tocopherol concentrations than those treated with the same concentrations of α-tocopherol. Our data suggest that

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

  17. Upregulation of NAD(PH:Quinone Oxidoreductase By Radiation Potentiates the Effect of Bioreductive β-Lapachone on Cancer Cells

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    Eun K. Choi

    2007-08-01

    Full Text Available We found that (β-Lapachone ((β-Lap, a novel bioreductive drug, caused rapid apoptosis, clonogenic cell death in A549 human lung epithelial cancer cells in vitro in a dose-dependent manner. The clonogenic cell death caused by (β-Lap could be significantly inhibited by dicoumarol, an inhibitor of NAD(PH:quinone oxidoreductase (NO01, also by siRNA for NO01, demonstrating that N001-induced bioreduction of (β-Lap is an essential step in (β-Lap-induced cell death. Irradiation of A549 cells with 4 Gy caused a long-lasting upregulation of N001, thereby increasing N001mediated (β-Lap-induced cell deaths. Although the direct cause of (β-Lap-induced apoptosis is not yet clear, (β-Lap treatment reduced the expression of p53, NF-κB, whereas it increased cytochrome C release, caspase-3 activity, δ2AX foci formation. Importantly, (β-Lap treatment immediately after irradiation enhanced radiation-induced cell death, indicating that (β-Lap sensitizes cancer cells to radiation, in addition to directly killing some of the cells. The growth of A549 tumors induced in immunocompromised mice could be markedly suppressed by local radiation therapy when followed by (β-Lap treatment. This is the first study to demonstrate that combined radiotherapy, (β-Lap treatment can have a significant effect on human tumor xenografts.

  18. 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.

  19. 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.

  20. 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.

  1. 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

    2016-01-01

    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.

  2. 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

  3. Estrogen upregulates MICA/B expression in human non-small cell lung cancer through the regulation of ADAM17.

    Science.gov (United States)

    Ren, Jing; Nie, Yunzhong; Lv, Mingming; Shen, Sunan; Tang, Ruijing; Xu, Yujun; Hou, Yayi; Zhao, Shuli; Wang, Tingting

    2015-11-01

    Estrogen is involved in promoting lung cancer cell division and metastasis. MICA and MICB function as ligands for NKG2D, an important immunoreceptor expressed on natural killer (NK) cells. However, whether estrogen regulates MICA/B expression and affects tumor immune escape remains unknown. In this study, we measured the mRNA levels of MICA, MICB and ADAM17in non-small cell lung cancer (NSCLC) cell lines treated with estrogen. Surface expression of MICA/B on LTEP-a2 and A549 was detected using flow cytometry. We demonstrate that both mRNA and secretory protein levels of MICA/B in lung adenocarcinoma cell lines were upregulated by estradiol. Estradiol enhanced the expression of ADAM17, which was associated with the secretion of MICA/B. This secretion of MICA/B downregulated the NKG2D receptor on the surface of NK92 cells and impaired the cytotoxic activity of NK cells. Estradiol enhanced the expression of ADAM17, which was associated with the secretion of MICA/B. Furthermore, a significant correlation between the concentration of estradiol and the expression of MICA was found in tumor tissues of NSCLC patients. Therefore, we conclude that estrogen can regulate the expression and secretion of MICA/B through ADAM17, which helps lung cancer cells escape NKG2D-mediated immune surveillance.

  4. Inhibition of β-ionone on SGC-7901 cell proliferation and upregulation of metalloproteinases-1 and-2 expression

    Institute of Scientific and Technical Information of China (English)

    Jia-Ren Liu; Bao-Feng Yang; Bing-Qing Chen; Yan-Mei Yang; Hong-Wei Dong; You-Qiang Song

    2004-01-01

    AIM: To observe the effect of β-ionone on the proliferation of human gastric adenocarcinoma cell line SGC-7901 and the inhibition of metalloproteinase.METHODS: Using growth inhibition, Zymograms assays and reverse transcription-polymerase-chain reaction(RT-PCR), we examined cell growth rates, activities of matrix metalloproteinases-2 (MMP-2) and -9 (MMP-9), and expression of metalloproteinases-1 (TIMP-1) and -2 (TIMP-2) in SGC-7901 cells after the treatment with β-ionone for 24h and 48 h, respectively.RESULTS: β-ionone had an inhibitory effect on the growth of SGC-7901 cells. Eight days after the treatment with β-ionone at concentrations of 25, 50, 100 and 200 μmol/L,the inhibition rates were 25.9%, 28.2%, 74.4% and 90.1%,respectively. The IC50 value of β-ionone for SGC-7901 cells was estimated to be 89 μmol/L. The effects of β-ionone on MMP-2 and MMP-9 activities in SGC-7901 cells were not observed. However, the levels of TIMP-1 and TIMP-2 transcripts were elevated in cells treated with β-ionone in a dose-dependent manner.CONCLUSION: β-ionone can inhibit the proliferation of SGC-7901 cells, upregulate the expression of TIMP-1 and TIMP-2 expression, and may influence metastasis of cancer.

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

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    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

  6. Upregulation of CRABP1 in human neuroblastoma cells overproducing the Alzheimer-typical Aβ42 reduces their differentiation potential

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    Weninger Annette

    2008-12-01

    Full Text Available Abstract Background Alzheimer's disease (AD is characterized by neurodegeneration and changes in cellular processes, including neurogenesis. Proteolytic processing of the amyloid precursor protein (APP plays a central role in AD. Owing to varying APP processing, several β-amyloid peptides (Aβ are generated. In contrast to the form with 40 amino acids (Aβ40, the variant with 42 amino acids (Aβ42 is thought to be the pathogenic form triggering the pathological cascade in AD. While total-Aβ effects have been studied extensively, little is known about specific genome-wide effects triggered by Aβ42 or Aβ40 derived from their direct precursor C99. Methods A combined transcriptomics/proteomics analysis was performed to measure the effects of intracellularly generated Aβ peptides in human neuroblastoma cells. Data was validated by real-time polymerase chain reaction (real-time PCR and a functional validation was carried out using RNA interference. Results Here we studied the transcriptomic and proteomic responses to increased or decreased Aβ42 and Aβ40 levels generated in human neuroblastoma cells. Genome-wide expression profiles (Affymetrix and proteomic approaches were combined to analyze the cellular response to the changed Aβ42- and Aβ40-levels. The cells responded to this challenge with significant changes in their expression pattern. We identified several dysregulated genes and proteins, but only the cellular retinoic acid binding protein 1 (CRABP1 was up-regulated exclusively in cells expressing an increased Aβ42/Aβ40 ratio. This consequently reduced all-trans retinoic acid (RA-induced differentiation, validated by CRABP1 knock down, which led to recovery of the cellular response to RA treatment and cellular sprouting under physiological RA concentrations. Importantly, this effect was specific to the AD typical increase in the Aβ42/Aβ40 ratio, whereas a decreased ratio did not result in up-regulation of CRABP1. Conclusion We

  7. Upregulation of CRABP1 in human neuroblastoma cells overproducing the Alzheimer-typical Aβ42 reduces their differentiation potential

    Science.gov (United States)

    Uhrig, Markus; Brechlin, Peter; Jahn, Olaf; Knyazev, Yuri; Weninger, Annette; Busia, Laura; Honarnejad, Kamran; Otto, Markus; Hartmann, Tobias

    2008-01-01

    Background Alzheimer's disease (AD) is characterized by neurodegeneration and changes in cellular processes, including neurogenesis. Proteolytic processing of the amyloid precursor protein (APP) plays a central role in AD. Owing to varying APP processing, several β-amyloid peptides (Aβ) are generated. In contrast to the form with 40 amino acids (Aβ40), the variant with 42 amino acids (Aβ42) is thought to be the pathogenic form triggering the pathological cascade in AD. While total-Aβ effects have been studied extensively, little is known about specific genome-wide effects triggered by Aβ42 or Aβ40 derived from their direct precursor C99. Methods A combined transcriptomics/proteomics analysis was performed to measure the effects of intracellularly generated Aβ peptides in human neuroblastoma cells. Data was validated by real-time polymerase chain reaction (real-time PCR) and a functional validation was carried out using RNA interference. Results Here we studied the transcriptomic and proteomic responses to increased or decreased Aβ42 and Aβ40 levels generated in human neuroblastoma cells. Genome-wide expression profiles (Affymetrix) and proteomic approaches were combined to analyze the cellular response to the changed Aβ42- and Aβ40-levels. The cells responded to this challenge with significant changes in their expression pattern. We identified several dysregulated genes and proteins, but only the cellular retinoic acid binding protein 1 (CRABP1) was up-regulated exclusively in cells expressing an increased Aβ42/Aβ40 ratio. This consequently reduced all-trans retinoic acid (RA)-induced differentiation, validated by CRABP1 knock down, which led to recovery of the cellular response to RA treatment and cellular sprouting under physiological RA concentrations. Importantly, this effect was specific to the AD typical increase in the Aβ42/Aβ40 ratio, whereas a decreased ratio did not result in up-regulation of CRABP1. Conclusion We conclude that increasing

  8. Up-regulation of alpha1-microglobulin by hemoglobin and reactive oxygen species in hepatoma and blood cell lines.

    Science.gov (United States)

    Olsson, Magnus G; Allhorn, Maria; Olofsson, Tor; Akerström, Bo

    2007-03-15

    alpha(1)-Microglobulin is a 26-kDa glycoprotein synthesized in the liver, secreted to the blood, and rapidly distributed to the extravascular compartment of all tissues. Recent results show that alpha(1)-microglobulin has heme-binding and heme-degrading properties and it has been suggested that the protein is involved in the defense against oxidation by heme and reactive oxygen species. In the present study the influence of hemoglobin and reactive oxygen species (ROS) on the cellular expression of alpha(1)-microglobulin was investigated. Oxy- and methemoglobin, free heme, and Fenton reaction-induced hydroxyl radicals induced a dose-dependent up-regulation of alpha(1)-microglobulin on both mRNA and protein levels in hepatoma cells and an increased secretion of alpha(1)-microglobulin. The up-regulation was reversed by the addition of catalase and ascorbate, and by reacting hemoglobin with cyanide which prevents redox reactions. Furthermore, the blood cell lines U937 and K562 expressed alpha(1)-microglobulin at low levels, and this expression increased up to 11-fold by the addition of hemoglobin. These results suggest that alpha(1)-microglobulin expression is induced by ROS, arising from redox reactions of hemoglobin or from other sources and are consistent with the hypothesis that alpha(1)-microglobulin participates in the defense against oxidation by hemoglobin, heme, and reactive oxygen species.

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

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    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.

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

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    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.

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

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    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

  12. 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.

  13. Mesenchymal stem cells improve cardiac conduction by upregulation of connexin 43 through paracrine signaling

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    Mureli, Shwetha; Gans, Christopher P.; Bare, Dan J; Geenen, David L.; Kumar, Nalin M.; Banach, Kathrin

    2012-01-01

    Mesenchymal stem cells (MSCs) were shown to improve cell survival and alleviate cardiac arrhythmias when transplanted into cardiac tissue; however, little is known about the mechanism by which MSCs modify the electrophysiological properties of cardiac tissue. We aimed to distinguish the influence of cell-cell coupling between myocytes and MSCs from that of MSC-derived paracrine factors on the spontaneous activity and conduction velocity (θ) of multicellular cardiomyocyte preparations. HL-1 ce...

  14. Up-regulation of sonic hedgehog contributes to TGF-β1-induced epithelial to mesenchymal transition in NSCLC cells.

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    Ma'in Y Maitah

    Full Text Available BACKGROUND: Lung cancer, especially non-small cell lung cancer (NSCLC is the major cause of cancer-related deaths in the United States. The aggressiveness of NSCLC has been shown to be associated with the acquisition of epithelial-to-mesenchymal transition (EMT. The acquisition of EMT phenotype induced by TGF-β1in several cancer cells has been implicated in tumor aggressiveness and resistance to conventional therapeutics; however, the molecular mechanism of EMT and tumor aggressiveness in NSCLC remains unknown. METHODOLOGY/PRINCIPAL FINDINGS: In this study we found for the first time that the induction of EMT by chronic exposure of A549 NSCLC cells to TGF-β1 (A549-M cells led to the up-regulation of sonic hedgehog (Shh both at the mRNA and protein levels causing activation of hedgehog signaling. These results were also reproduced in another NSCLC cell line (H2030. Induction of EMT was found to be consistent with aggressive characteristics such as increased clonogenic growth, cell motility and invasion. The aggressiveness of these cells was attenuated by the treatment of A549-M cells with pharmacological inhibitors of Hh signaling in addition to Shh knock-down by siRNA. The inhibition of Hh signaling by pharmacological inhibitors led to the reversal of EMT phenotype as confirmed by the reduction of mesenchymal markers such as ZEB1 and Fibronectin, and induction of epithelial marker E-cadherin. In addition, knock-down of Shh by siRNA significantly attenuated EMT induction by TGF-β1. CONCLUSIONS/SIGNIFICANCE: Our results show for the first time the transcriptional up-regulation of Shh by TGF-β1, which is mechanistically associated with TGF-β1 induced EMT phenotype and aggressive behavior of NSCLC cells. Thus the inhibitors of Shh signaling could be useful for the reversal of EMT phenotype, which would inhibit the metastatic potential of NSCLC cells and also make these tumors more sensitive to conventional therapeutics.

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

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    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.

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

    International Nuclear Information System (INIS)

    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.

  17. Up-regulation of phosphoinositide metabolism in tobacco cells constitutively expressing the human type I inositol polyphosphate 5-phosphatase

    Science.gov (United States)

    Perera, Imara Y.; Love, John; Heilmann, Ingo; Thompson, William F.; Boss, Wendy F.; Brown, C. S. (Principal Investigator)

    2002-01-01

    To evaluate the impact of suppressing inositol 1,4,5-trisphosphate (InsP(3)) in plants, tobacco (Nicotiana tabacum) cells were transformed with the human type I inositol polyphosphate 5-phosphatase (InsP 5-ptase), an enzyme which specifically hydrolyzes InsP(3). The transgenic cell lines showed a 12- to 25-fold increase in InsP 5-ptase activity in vitro and a 60% to 80% reduction in basal InsP(3) compared with wild-type cells. Stimulation with Mas-7, a synthetic analog of the wasp venom peptide mastoparan, resulted in an approximately 2-fold increase in InsP(3) in both wild-type and transgenic cells. However, even with stimulation, InsP(3) levels in the transgenic cells did not reach wild-type basal values, suggesting that InsP(3) signaling is compromised. Analysis of whole-cell lipids indicated that phosphatidylinositol 4,5-bisphosphate (PtdInsP(2)), the lipid precursor of InsP(3), was greatly reduced in the transgenic cells. In vitro assays of enzymes involved in PtdInsP(2) metabolism showed that the activity of the PtdInsP(2)-hydrolyzing enzyme phospholipase C was not significantly altered in the transgenic cells. In contrast, the activity of the plasma membrane PtdInsP 5 kinase was increased by approximately 3-fold in the transgenic cells. In vivo labeling studies revealed a greater incorporation of (32)P into PtdInsP(2) in the transgenic cells compared with the wild type, indicating that the rate of PtdInsP(2) synthesis was increased. These studies show that the constitutive expression of the human type I InsP 5-ptase in tobacco cells leads to an up-regulation of the phosphoinositide pathway and highlight the importance of PtdInsP(2) synthesis as a regulatory step in this system.

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

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    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.

  19. Neuroprotective Effect of Osthole on Neuron Synapses in an Alzheimer's Disease Cell Model via Upregulation of MicroRNA-9.

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    Li, Shaoheng; Yan, Yuhui; Jiao, Yanan; Gao, Zhong; Xia, Yang; Kong, Liang; Yao, Yingjia; Tao, Zhenyu; Song, Jie; Yan, Yaping; Zhang, Guangxian; Yang, Jingxian

    2016-09-01

    Accumulation of β-amyloid peptide (Aβ) in the brain plays an important role in the pathogenesis of Alzheimer's disease (AD). It has been reported that osthole exerts its neuroprotective effect on neuronal synapses, but its exact mechanism is obscure. Recently, microRNAs have been demonstrated to play a crucial role in inducing synaptotoxicity by Aβ, implying that targeting microRNAs could be a therapeutic approach of AD. In the present study, we investigated the neuroprotective effects of osthole on a cell model of AD by transducing APP695 Swedish mutant (APP695swe, APP) into mouse cortical neurons and human SH-SY5Y cells. In this study, the cell counting kit CCK-8, apoptosis assay, immunofluorescence analysis, enzyme-linked immunosorbent assay (ELISA), quantitative real-time polymerase chain reaction, and Western blot assay were used. We found that osthole could enhance cell viability, prevent cell death, and reverse the reduction of synaptic proteins (synapsin-1, synaptophysin, and postsynaptic density-95) in APP-overexpressed cells, which was attributed to increases in microRNA-9 (miR-9) expression and subsequent decreases in CAMKK2 and p-AMPKα expressions. These results demonstrated that osthole plays a neuroprotective activity role in part through upregulating miR-9 in AD. PMID:27394443

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

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    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.

  1. 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.

  2. miR-6734 Up-Regulates p21 Gene Expression and Induces Cell Cycle Arrest and Apoptosis in Colon Cancer Cells

    Science.gov (United States)

    Kang, Moo Rim; Park, Ki Hwan; Yang, Jeong-Ook; Lee, Chang Woo; Oh, Soo Jin; Yun, Jieun; Lee, Myeong Youl; Han, Sang-Bae; Kang, Jong Soon

    2016-01-01

    Recently, microRNAs have been implicated in the regulation of gene expression in terms of both gene silencing and gene activation. Here, we investigated the effects of miR-6734, which has a sequence homology with a specific region of p21WAF1/CIP1 (p21) promoter, on cancer cell growth and the mechanisms involved in this effect. miR-6734 up-regulated p21 expression at both mRNA and protein levels and chromatin immunoprecipitation analysis using biotin-labeled miR-6734 confirmed the association of miR-6734 with p21 promoter. Moreover, miR-6734 inhibited cancer cell growth and induced cell cycle arrest and apoptosis in HCT-116 cells, which was abolished by knockdown of p21. The phosphorylation of Rb and the cleavage of caspase 3 and PARP were suppressed by miR-6734 transfection in HCT-116 cells and these effects were also reversed by p21 knockdown. In addition, miR-6734 transfection caused prolonged induction of p21 gene and modification of histones in p21 promoter, which are typical aspects of a phenomenon referred to as RNA activation (RNAa). Collectively, our results demonstrated that miR-6734 inhibits the growth of colon cancer cells by up-regulating p21 gene expression and subsequent induction of cell cycle arrest and apoptosis, suggesting its role as an important endogenous regulator of cancer cell proliferation and survival. PMID:27509128

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

    Directory of Open Access Journals (Sweden)

    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.

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

    Directory of Open Access Journals (Sweden)

    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.

  5. 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

  6. Hiwi Promotes the Proliferation of Colorectal Cancer Cells via Upregulating Global DNA Methylation

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    Lin Yang

    2015-01-01

    Full Text Available Hiwi is well known for its role in stem cell renewal, maintaining the resting stage, and downregulating cell cycle of stem cells via RNA silencing. And Hiwi overexpression has been recognized in several types of cancers. In the present study, we examined the Hiwi expression in colorectal cancer (CRC specimens in both mRNA and protein levels via real-time quantitative PCR, western blot assay, and immunohistochemical staining. Then we explored the role of Hiwi in the cancer cell proliferation and in the DNA methylation in human CRC Caro-2 and HT-29 cell lines. Results demonstrated that both mRNA and protein levels of Hiwi were significantly higher in 38 CRC tissues than in 38 peritumor tissues. Moreover, the Hiwi overexpression with an adenovirus vector significantly promoted the proliferation of Caro-2 and HT-29 cells, associated with significant increase in the global DNA methylation levels. And the chemical inhibition of DNA methylation significantly restrained such proliferation promotion. In summary, we confirmed that Hiwi was overexpressed in CRC tissues and that the forced Hiwi overexpression promoted the proliferation and global DNA methylation of CRC cell lines. Our results imply for the first time that Hiwi promotes the proliferation of CRC cells via promoting global DNA methylation.

  7. Normal T-cell telomerase activity and upregulation in human immunodeficiency virus-1 infection

    NARCIS (Netherlands)

    Wolthers, KC; Otto, SA; Wisman, GBA; Fleury, S; Reiss, P; ten Kate, RW; van der Zee, AGJ; Miedema, F

    1999-01-01

    In human immunodeficiency virus (HIV)-1 infection, decrease of telomere length is mainly found in CD8(+) T cells and not in CD4(+) T cells. Telomerase, a ribonucleoprotein enzyme that can synthesize telomeric sequence onto chromosomal ends, can compensate for telomere loss. Here, we investigated if

  8. Upregulation of RNA Processing Factors in Poorly Differentiated Lung Cancer Cells

    Directory of Open Access Journals (Sweden)

    Kenneth G. Geles

    2016-04-01

    Full Text Available Intratumoral heterogeneity in non–small cell lung cancer (NSCLC has been appreciated at the histological and cellular levels, but the association of less differentiated pathology with poor clinical outcome is not understood at the molecular level. Gene expression profiling of intact human tumors fails to reveal the molecular nature of functionally distinct epithelial cell subpopulations, in particular the tumor cells that fuel tumor growth, metastasis, and disease relapse. We generated primary serum-free cultures of NSCLC and then exposed them to conditions known to promote differentiation: the air-liquid interface (ALI and serum. The transcriptional network of the primary cultures was associated with stem cells, indicating a poorly differentiated state, and worse overall survival of NSCLC patients. Strikingly, the overexpression of RNA splicing and processing factors was a prominent feature of the poorly differentiated cells and was also observed in clinical datasets. A genome-wide analysis of splice isoform expression revealed many alternative splicing events that were specific to the differentiation state of the cells, including an unexpectedly high frequency of events on chromosome 19. The poorly differentiated cells exhibited alternative splicing in many genes associated with tumor progression, as exemplified by the preferential expression of the short isoform of telomeric repeat-binding factor 1 (TERF1, also known as Pin2. Our findings demonstrate the utility of the ALI method for probing the molecular mechanisms that underlie NSCLC pathogenesis and provide novel insight into posttranscriptional mechanisms in poorly differentiated lung cancer cells.

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

    Directory of Open Access Journals (Sweden)

    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

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

    International Nuclear Information System (INIS)

    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

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

  12. Arsenic trioxide up-regulates Fas expression in human osteosarcoma cells

    Institute of Scientific and Technical Information of China (English)

    YANG Guo-fu; LI Xiang-hui; ZHAO Zhe; WANG Wen-bo

    2010-01-01

    Background Osteosarcoma is a common primary malignant tumor of bone with a poor prognosis due to its propensity for metastasis. The prognosis of patients is highly dependent on the presence or absence of lung metastasis and on the effectiveness of treatment against it. It has been reported that low level expression of Fas protein in human osteosarcoma cell is closely associated with lung metastasis. A large number of studies have shown that arsenic trioxide (ATO) can inhibit proliferation and induce apoptosis of many cancer cell lines; however, its effects on human osteosarcoma cells (Saos-2 cell line) remains unknown. The aim of this study was to investigate the effects of ATO on Saos-2 cells and to characterize its mechanism of Fas-expressing.Methods A group of Saos-2 cells was treated with or without 0.5,1,2,4 and 8 urnol/L ATO for three successive days, and the cytotoxicity of ATO was determined by an 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay. Morphological changes in cells were studied by acridine orange/ethidium bromide (AO/EB) double staining. Flow cytometry (FCM) was used to assay cell DNA distribution. Another group of cells was pretreated with 10 nmol/L matrix metalloproteinase 7 (MMP-7) for 3 hours. They were then incubated with or without 2 umol/L ATO for 24, 48 and 72 hours. Cytotoxicity, Fas protein and mRNA levels were systematically studied using MTT, Western blotting and real-time PCR, respectively. Cell proliferation, cell cycle progression and apoptosis were examined in this study. Results Proliferation of Saos-2 cells was inhibited by ATO in both a dose- and time-dependent manner. The IC50 values at 24, 48 and 72 hours were 9.30, 5.54 and 3.49 μmol/L, respectively. The survival rate of Saos-2 cells in the MMP-7 and ATO co-treated group was significantly higher than the ATO group, but it was lower than the control group. ATO induced G1 phase arrest of the cell cycle and very efficiently stimulated apoptosis in Saos

  13. Upregulation of the Tim-3/galectin-9 pathway of T cell exhaustion in chronic hepatitis B virus infection.

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    Gaia Nebbia

    Full Text Available The S-type lectin galectin-9 binds to the negative regulatory molecule Tim-3 on T cells and induces their apoptotic deletion or functional inactivation. We investigated whether galectin-9/Tim-3 interactions contribute to the deletion and exhaustion of the antiviral T cell response in chronic hepatitis B virus infection (CHB. We found Tim-3 to be expressed on a higher percentage of CD4 and CD8 T cells from patients with CHB than healthy controls (p<0.0001 and to be enriched on activated T cells and those infiltrating the HBV-infected liver. Direct ex vivo examination of virus-specific CD8 T cells binding HLA-A2/peptide multimers revealed that Tim-3 was more highly upregulated on HBV-specific CD8 T cells than CMV-specific CD8 T cells or the global CD8 T cell population in patients with CHB (p<0.001 or than on HBV-specific CD8 after resolution of infection. T cells expressing Tim-3 had an impaired ability to produce IFN-γ and TNF-α upon recognition of HBV-peptides and were susceptible to galectin-9-triggered cell death in vitro. Galectin-9 was detectable at increased concentrations in the sera of patients with active CHB-related liver inflammation (p = 0.02 and was strongly expressed by Kupffer cells within the liver sinusoidal network. Tim-3 blockade resulted in enhanced expansion of HBV-specific CD8 T cells able to produce cytokines and mediate cytotoxicity in vitro. Blocking PD-1 in combination with Tim-3 enhanced the number of patients from whom functional antiviral responses could be recovered and/or the strength of responses, indicating that these co-inhibitory molecules play a non-redundant role in driving T cell exhaustion in CHB. Patients taking antivirals able to potently suppress HBV viraemia continued to express Tim-3 on their T cells and respond to Tim-3 blockade. In summary, both Tim-3 and galectin-9 are increased in CHB and may contribute to the inhibition and deletion of T cells as they infiltrate the HBV-infected liver.

  14. Cell-cycle dependent micronucleus formation and mitotic disturbances induced by 5-azacytidine in mammalian cells

    OpenAIRE

    Stopper, Helga; Körber, C.; Schiffmann, D; Caspary, W J

    2012-01-01

    5-Azacytidine was originally developed to treat human myelogenous leukemia. However, interest in this compound has expanded because of reports of its ability to affect cell differentiation and to alter eukaryotic gene expression. In an ongoing attempt to understand the biochemical effects of this compound, we examined the effects of 5-azacytidine on mitosis and on micronucleus formation in mammalian cells. In L5178Y mouse cells, 5-azacytidine induced micronuclei at concentrations at which we ...

  15. Aldehyde dehydrogenase 3A1 is robustly upregulated in gastric cancer stem-like cells and associated with tumorigenesis.

    Science.gov (United States)

    Wu, Di; Mou, Yi-Ping; Chen, Ke; Cai, Jia-Qin; Zhou, Yu-Cheng; Pan, Yu; Xu, Xiao-Wu; Zhou, Wei; Gao, Jia-Qi; Chen, Ding-Wei; Zhang, Ren-Chao

    2016-08-01

    Enhanced aldehyde dehydrogenase (ALDH) activity has been shown to serve as a hallmark for cancer stem cells (CSCs). Recent evidence suggests that its role as a stem cell-related marker has come down to the specific isoform. However, little is known about the specific ALDH isoform contributing to aldefluor activity in gastric cancer. In this study, we isolated ALDHbright cells from 2 human gastric cancer cell lines MKN-45 and SGC‑7901 by using an Aldefluor assay and found elevated self-renewal, differentiation and tumorigenicity, as demonstration of stemness characteristics. We also found that ALDHbright cells expressed decreased levels of E-cadherin but increased levels of Snail and Vimentin, indication of an epithelial-mesenchymal transition (EMT) phenotype which may be responsible for the enhanced metastatic potential. Since further research and prognostic application based on ALDH prevalence require the quantification of the specific ALDH isoform, we characterized the expression of all 19 ALDH isoforms in the sorted gastric cancer cell lines by quantitative real-time polymerase chain reaction (qRT-PCR). Compared with the non-stem counterparts, robust upregulation of ALDH-3A1 was observed in these gastric cancer stem-like cells. Furthermore, we performed immunohistological analysis on 93 fixed patient gastric tumor samples and found that ALDH-3A1 expression correlated well with gastric cancer dysplasia and grades, differentiation, lymph node metastasis and cancer stage. Our data, therefore, provide strong evidence that ALDH-3A1 is a novel gastric cancer stem cell related marker with potential prognostic values and demonstrate a clear association between ALDH-3A1 prevalence and gastric cancer progression. PMID:27279633

  16. Intratumoral regulatory T cells upregulate immunosuppressive molecules in head and neck cancer patients

    OpenAIRE

    Jie, H-B; Gildener-Leapman, N; Li, J.; Srivastava, R. M.; Gibson, S P; Whiteside, T L; Ferris, R. L.

    2013-01-01

    Background: Although regulatory T cells (Treg) are highly enriched in human tumours compared with peripheral blood, expression of the immune-checkpoint receptors, immunosuppressive molecules and function of Treg in these two sites remains undefined. Methods: Tumour-infiltrating lymphocytes and peripheral blood lymphocytes were isolated from a cohort of head and neck squamous cell carcinoma (HNSCC) patients. The immunosuppressive phenotypes and function of intratumoral Treg were compared with ...

  17. Recombinant human decorin suppresses liver HepG2 carcinoma cells by p21 upregulation

    OpenAIRE

    Zhang Y.; Wang Y; Du Z.; Wang Q.; Wu M; Wang X; Wang L.; Cao L; Hamid AS; Zhang G

    2012-01-01

    Yucheng Zhang, Yali Wang,* Zhenwu Du, Qian Wang, Mei Wu, Xiaofeng Wang, Lingling Wang, Linlin Cao, Abdu Selim Hamid, Guizhen Zhang*Central Laboratory, China-Japan Union Hospital, Jilin University, Changchun, People's Republic of China *These authors contributed equally to this workBackground: Decorin is a multifunctional molecule of the extracellular matrix and impedes different kinds of tumor cell growth, but the role and molecular mechanism by which decorin inhibits HepG2 cells is n...

  18. Angiostatin up-regulation in gastric cancer cell SGC7901 inhibits tumorigenesis in nude mice

    Institute of Scientific and Technical Information of China (English)

    Jing Wu; Yong-Quan Shi; Kai-Chun Wu; De-Xin Zhang; Jing-Hua Yang; Dai-Ming Fan

    2003-01-01

    AIM: To explore the influence of angiostatin up-regulationon the biologic behavior of gastric cancer cells in vitro andin vivo, and the potential of angiostatin gene therapy in thetreatment of human gastric cancer.METHODS: Mouse angiostatin cDNA was subcloned intothe eukaryotic expression vector pcDNA3.1(+) and identifiedby restriction endonucleases digestion and sequencing. Therecombinant vector pcDNA3. 1(+)-angio was transfected intohuman gastric cancer cells SGC7901 with liposome andparalleled with the vector control and the mock control.Angiostatin transcription and protein expression wereexamined by RT-PCR and Western blot in the stable celllines selected by G418. Cell proliferation and growth in vitroof the three groups were observed respectively undermicroscope, cell number counting and FACS. The cellsoverexpressing angiostatin, vector transfected and untreatedwere respectively implanted subcutaneously into nude mice.After 30days the size of tumors formed was measured, andmicrovessel density count (MVD) in the tumor tissues wasassessed by immunohistochemistry with the primary anti-vWF antibody.RESULTS: The recombinant vector pcDNA3.1(+)-angio wasconfirmed with the correct sequence of mouse angiostatinunder the promoter CMV. After 30 d of transfection andselection with G418, macroscopic resistant cell clones wereformed in the experimental group transfected with pcDNA3.1(+)-angio and the vector control. But no untreated cellssurvived in the mock control. Angiostatin mRNAtranscription and protein expression were detected in theexperimental group. No significant differences wereobserved among the three groups in cell morphology, cellgrowth curves and cell cycle phase distributions in vitro.However, in nude mice model, markedly inhibitedtumorigenesis and slowed tumor expansion were observedin the experimental group as compared with the controls,which was paralleled with decreased microvessel density inand around tumor tissues (P<0. 05).CONCLUSION: Angiostatin

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

    Science.gov (United States)

    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

  20. 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.

  1. 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....

  2. Cilostazol Upregulates Autophagy via SIRT1 Activation: Reducing Amyloid-β Peptide and APP-CTFβ Levels in Neuronal Cells.

    Directory of Open Access Journals (Sweden)

    Hye Rin Lee

    Full Text Available Autophagy is a vital pathway for the removal of β-amyloid peptide (Aβ and the aggregated proteins that cause Alzheimer's disease (AD. We previously found that cilostazol induced SIRT1 expression and its activity in neuronal cells, and thus, we hypothesized that cilostazol might stimulate clearances of Aβ and C-terminal APP fragment β subunit (APP-CTFβ by up-regulating autophagy.When N2a cells were exposed to soluble Aβ1-42, protein levels of beclin-1, autophagy-related protein5 (Atg5, and SIRT1 decreased significantly. Pretreatment with cilostazol (10-30 μM or resveratrol (20 μM prevented these Aβ1-42 evoked suppressions. LC3-II (a marker of mammalian autophagy levels were significantly increased by cilostazol, and this increase was reduced by 3-methyladenine. To evoke endogenous Aβ overproduction, N2aSwe cells (N2a cells stably expressing human APP containing the Swedish mutation were cultured in medium with or without tetracycline (Tet+ for 48 h and then placed in Tet- condition. Aβ and APP-CTFβ expressions were increased after 12~24 h in Tet- condition, and these increased expressions were significantly reduced by pretreating cilostazol. Cilostazol-induced reductions in the expressions of Aβ and APP-CTFβ were blocked by bafilomycin A1 (a blocker of autophagosome to lysosome fusion. After knockdown of the SIRT1 gene (to ~40% in SIRT1 protein, cilostazol failed to elevate the expressions of beclin-1, Atg5, and LC3-II, indicating that cilostazol increases these expressions by up-regulating SIRT1. Further, decreased cell viability induced by Aβ was prevented by cilostazol, and this inhibition was reversed by 3-methyladenine, indicating that the protective effect of cilostazol against Aβ induced neurotoxicity is, in part, ascribable to the induction of autophagy. In conclusion, cilostazol modulates autophagy by increasing the activation of SIRT1, and thereby enhances Aβ clearance and increases cell viability.

  3. 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

  4. 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.

  5. Fibulin-5 is upregulated in decidualized human endometrial stromal cells and promotes primary human extravillous trophoblast outgrowth.

    Science.gov (United States)

    Winship, Amy; Cuman, Carly; Rainczuk, Katarzyna; Dimitriadis, Evdokia

    2015-12-01

    Interactions between the highly invasive trophoblasts and the maternal uterine decidual extracellular matrix (ECM) are crucial in the determination of a successful pregnancy. Fibulin-5 (FBLN5) is a member of the fibulin family that alters cell adhesive and invasive properties and is expressed in human villous cytotrophoblasts. We aimed to determine the expression and immunolocalization of FBLN5 in human first trimester decidua and examine the effect of FBLN5 in trophoblast invasion in vitro using a first trimester placental villous outgrowth assay. We demonstrated that FBLN5 mRNA expression is upregulated in response to cAMP-mediated decidualization of primary human endometrial stromal cells, although FBLN5 itself does not enhance decidualization. We reported for the first time, FBLN5 protein production in first trimester decidual cells and also co-localization to HLAG-positive EVTs in first trimester decidua. Consequently, we investigated the effects of exogenous FBLN5 on placental villous outgrowth in vitro and demonstrated that FBLN5 promotes EVT migration/invasion. This is the first study to identify FBLN5 in decidualized human endometrial stromal cells, first trimester decidua and EVT and determine a functional role for FBLN5 in human EVTs, suggesting that decidual and or EVT-derived FBLN5 regulates EVT invasion and placentation in women. PMID:26506560

  6. Trigonella foenum (Fenugreek) Induced Apoptosis in Hepatocellular Carcinoma Cell Line, HepG2, Mediated by Upregulation of p53 and Proliferating Cell Nuclear Antigen.

    Science.gov (United States)

    Khalil, Mahmoud I M; Ibrahim, Mohamed M; El-Gaaly, Gehan A; Sultan, Ahmed S

    2015-01-01

    Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide and most current therapies are of limited efficacy. Trigonella foenum (Fenugreek) is a traditional herbal plant with antitumor activity, although the mechanisms of its activity remain unclear. Herein, a crude methanol extract was prepared from Fenugreek seeds (FCE) and its anticancer mechanism was evaluated, using HepG2 cell line. Growth-inhibitory effect and apoptosis induction of HepG2 cells were evidenced by MTT assay, cell morphology alteration, apoptosis enzyme-linked immunosorbent assay, flow cytometric analysis, caspase-3 activity, and expression of p53, proapoptotic protein, Bax, and proliferating cell nuclear antigen (PCNA) after (100 ∼ 500 μg/mL) FCE treatment for 48 h. Furthermore, FCE was analyzed by Chromatography-Mass Spectrometry (GC/MS). Our results revealed that FCE treatment for 48 h showed a cytotoxic effect and apoptosis induction in a dose-dependent manner that was mediated by upregulation of p53, Bax, PCNA, and caspase-3 activation in HepG2 cells. GC-MS analysis of FCE showed the presence of fourteen bioactive compounds such as Terpenoids and Flavonoids, including two main constituents with anticancer activity, Squalene and Naringenin (27.71% and 24.05%), respectively. Our data introduced FCE as a promising nontoxic herbal with therapeutic potential to induce apoptosis in HepG2 cells through p53, Bax, and PCNA upregulation in caspase-3 dependent manner. PMID:26557712

  7. A miRNA upregulated in asthma airway T cells promotes TH2 cytokine production

    Science.gov (United States)

    Simpson, Laura J.; Patel, Sana; Bhakta, Nirav R.; Choy, David F.; Brightbill, Hans D.; Ren, Xin; Wang, Yanli; Pua, Heather H.; Baumjohann, Dirk; Montoya, Misty M.; Panduro, Marisella; Remedios, Kelly A.; Huang, Xiaozhu; Fahy, John V.; Arron, Joseph R.; Woodruff, Prescott G.; Ansel., Karl M.

    2014-01-01

    MicroRNAs (miRNAs) exert powerful effects on immune function by tuning networks of target genes that orchestrate cell behavior. We sought to uncover miRNAs and miRNA-regulated pathways that control the TH2 responses that drive pathogenic inflammation in asthma. Profiling miRNA expression in human airway-infiltrating T cells revealed miR-19a elevation in asthma. Modulating miR-19 activity altered TH2 cytokine production in both human and mouse T cells, and TH2 cell responses were markedly impaired in cells lacking the entire miR-17∼92 cluster. miR-19 promotes TH2 cytokine production and amplifies PI(3)K, JAK-STAT, and NF-κB signaling by direct targeting of PTEN, SOCS1, and A20. Thus, miR-19a up regulation in asthma may be an indicator and a cause of increased TH2 cytokine production in the airways. PMID:25362490

  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. Up-regulation of Hsp27 by ERα/Sp1 facilitates proliferation and confers resistance to apoptosis in human papillary thyroid cancer cells.

    Science.gov (United States)

    Mo, Xiao-Mei; Li, Li; Zhu, Ping; Dai, Yu-Jie; Zhao, Ting-Ting; Liao, Ling-Yao; Chen, George G; Liu, Zhi-Min

    2016-08-15

    17β-estradiol (E2) has been suggested to play a role in the development and progression of papillary thyroid cancer. Heat shock protein 27 (Hsp27) is a member of the Hsp family that is responsible for cell survival under stressful conditions. Previous studies have shown that the 5'-promoter region of Hsp27 gene contains a specificity protein-1 (Spl) and estrogen response element half-site (ERE-half), which contributes to Hsp27 induction by E2 in breast cancer cells. However, it is unclear whether Hsp27 can be up-regulated by E2 and which estrogen receptor (ER) isoform and tethered transcription factor are involved in this regulation in papillary thyroid cancer cells. In the present study, we demonstrated that Hsp27 can be effectively up-regulated by E2 at mRNA and protein levels in human K1 and BCPAP papillary thyroid cancer cells which have more than two times higher level of ERα than that of ERβ. The up-regulation of Hsp27 by E2 is mediated by ERα/Sp1 and ERβ has repressive effect on this ERα/Sp1-mediated up-regulation of Hsp27. Moreover, we showed that the up-regulation of Hsp27 by ERα/Sp1 facilitates proliferation and confers resistance to apoptosis through interaction with procaspase-3. Targeting this pathway may be a potential strategy for therapy of papillary thyroid cancer. PMID:27179757

  10. Tumor necrosis factor-α attenuates starvation-induced apoptosis through upregulation of ferritin heavy chain in hepatocellular carcinoma cells

    International Nuclear Information System (INIS)

    Tumor microenviroment is characteristic of inflammation, ischemia and starvation of nutrient. TNF-α, which is an extraordinarily pleiotropic cytokine, could be an endogenous tumor promoter in some tumor types. The basic objective of this study was to investigate the effects of TNF-α on the cell viability and apoptosis of hepatocellular carcinoma cells under serum starvation, and to identify the molecular mechanisms involved. For this purpose, five different concentrations of TNF-α and two different serum settings (serum-cultured and serum-deprived) were used to investigate the effects of TNF-α on the cell viability and apoptosis of Hep3B and SMMC-7721 cells. TNF-α (10 ng/ml) attenuated serum starvation-induced apoptosis of hepatocellular carcinoma cells, and autophagy conferred this process. BAY11-7082, a specific inhibitor of NF-κB, reversed the suppression of serum starvation-induced apoptosis by TNF-α. Moreover, TNF-α-induced NF-κB transactivation was suppressed by autophagy inhibitor 3-MA. In addition, TNF-α up-regulated Ferritin heavy chain (FHC) transiently by NF-κB activation and FHC levels were correlated with the TNF-α-induced protection against serum starvation-mediated apoptosis of hepatocellular carcinoma cells. Furthermore, FHC-mediated inhibition of apoptosis depended on suppressing ROS accumulation. Our findings suggested that autophagy conferred the TNF-α protection against serum starvation-mediated apoptosis of hepatocellular carcinoma cells, the mechanism involved with the activation of the TNF-α/ NF-κB /FHC signaling pathway

  11. 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.

  12. 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.

  13. 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

  14. 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.

  15. 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.

  16. 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.

  17. Altered efficacy of AT1R-targeted treatment after spontaneous cancer cell-AT1R upregulation

    International Nuclear Information System (INIS)

    Targeting of the renin angiotensin system (RAS) reduces tumour growth in experimental models of cancer. We aimed to establish if combined targeting of the 'classical' and 'alternative' arms of the RAS could result in synergistic inhibition of colorectal cancer (CRC) liver metastases. Immediately following induction of CRC liver metastases through intrasplenic injection of murine CRC cells, treatment with irbesartan (AT1R blocker; 50 mg/kg/day s.c.), captopril (ACE inhibitor; 750 mg/kg/day i.p.), CGP42112A (AT2R agonist; 0.6 μg/kg/hr i.p.), and/or ANG-(1-7) (24 μg/kg/hr i.p.) began and continued for 21 days. Liver to body weight ratio and/or stereology were used as a measure of tumour burden. Immunohistochemistry was used to determine AT1R and VEGF expression as well as proliferation (Ki67), apoptosis (active caspase 3) and angiogenesis (CD34). Combined RAS therapies failed to improve upon single arm therapies. However, while irbesartan previously inhibited tumour growth in this model, in the current experiments irbesartan failed to affect tumour burden. Subsequent analysis showed a cancer-cell specific upregulation of the angiotensin II type I receptor (AT1R) in irbesartan-insensitive compared to irbesartan-sensitive tumours. The upregulation of AT1R was associated with an increase in proliferation and VEGF expression by cancer cells. While animals bearing irbesartan-sensitive tumours showed a marked decrease in the number of proliferating cells in the liver and VEGF-expressing infiltrating cells in the tumour following AT1R treatment, these were unchanged by treatment in animals bearing irbesartan-insensitive (high AT1R expressing) tumours. Although the results do not support increased efficacy of combined treatment, they provide intriguing evidence of the importance of RAS expression in determining patient response and tumour growth potential and suggest that components of the RAS could be used as biomarkers to aid in patient selection

  18. 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.

  19. Steroidogenic enzymes and stem cell markers are upregulated during androgen deprivation in prostate cancer

    NARCIS (Netherlands)

    Pfeiffer, M.J.; Smit, F.P.; Sedelaar, J.P.M.; Schalken, J.A.

    2011-01-01

    Considerable levels of testosterone and dihydrotestosterone (DHT) are found in prostate cancer (PCa) tissue after androgen deprivation therapy. Treatment of surviving cancer-initiating cells and the ability to metabolize steroids from precursors may be the keystones for the appearance of recurrent t

  20. Glutamate Receptor Stimulation Up-Regulates Glutamate Uptake in Human Müller Glia Cells.

    Science.gov (United States)

    López-Colomé, Ana María; López, Edith; Mendez-Flores, Orquidia G; Ortega, Arturo

    2016-07-01

    Glutamate, the main excitatory amino acid in the vertebrate retina, is a well know activator of numerous signal transduction pathways, and has been critically involved in long-term synaptic changes acting through ionotropic and metabotropic glutamate receptors. However, recent findings underlining the importance of intensity and duration of glutamate stimuli for specific neuronal responses, including excitotoxicity, suggest a crucial role for Na(+)-dependent glutamate transporters, responsible for the removal of this neurotransmitter from the synaptic cleft, in the regulation of glutamate-induced signaling. Transporter proteins are expressed in neurons and glia cells, albeit most of glutamate uptake occurs in the glial compartment. Within the retina, Müller glia cells are in close proximity to glutamatergic synapses and participate in the recycling of glutamate through the glutamate/glutamine shuttle. In this context, we decided to investigate a plausible role of glutamate as a regulatory signal for its own transport in human retinal glia cells. To this end, we determined [(3)H]-D-aspartate uptake in cultures of spontaneously immortalized human Müller cells (MIO-M1) exposed to distinct glutamatergic ligands. A time and dose-dependent increase in the transporter activity was detected. This effect was dependent on the activation of the N-methyl D-aspartate subtype of glutamate receptors, due to a dual effect: an increase in affinity and an augmented expression of the transporter at the plasma membrane, as established via biotinylation experiments. Furthermore, a NMDA-dependent association of glutamate transporters with the cystoskeletal proteins ezrin and glial fibrillary acidic protein was also found. These results add a novel mediator of the glutamate transporter modulation and further strengthen the notion of the critical involvement of glia cells in synaptic function. PMID:27017513

  1. The mechanisms of up-regulation of dendritic cell activity by oxidative stress.

    Science.gov (United States)

    Batal, Ibrahim; Azzi, Jamil; Mounayar, Marwan; Abdoli, Rozita; Moore, Robert; Lee, Jack Y; Rosetti, Florencia; Wang, Chang; Fiorina, Paolo; Sackstein, Robert; Ichimura, Takaharu; Abdi, Reza

    2014-08-01

    Whereas DC have increasingly been recognized for their role in activating the inflammatory cascades during IRIs, the mechanisms by which oxidative stress enhances DC activation remain to be explored. We examined the role of oxidative stress on two important features of DC: T cell activation and trafficking. Bone marrow-derived OS-DC were compared with untreated DC. DC exposed to oxidative stress augmented allogeneic T cell proliferation and showed increased migration in a chemotaxis chamber. These results were confirmed by using hypoxanthine and xanthine oxidase as another inducer of oxidative stress. We used OT-II and OT-I mice to assess the effect of oxidative stress on DC activation of OVA-specific CD4(+) and CD8(+) T cells, respectively. Oxidative stress increased DC capacity to promote OVA-specific CD4(+) T cell activity, demonstrated by an increase in their proliferation and production of IFN-γ, IL-6, and IL-2 proinflammatory cytokines. Whereas oxidative stress increased the DC ability to stimulate IFN-γ production by OVA-specific CD8(+) T cells, cellular proliferation and cytotoxicity were not affected. Compared with untreated DC, oxidative stress significantly reduced the capacity of DC to generate T(regs), which were restored by using anti-IL-6. With regard to DC trafficking, whereas oxidative stress increased DC expression of p-Akt and p-NF-κB, targeting PI3Kγ and NF-κB pathways abrogated the observed increase in DC migration. Our data propose novel insights on the activation of DC by oxidative stress and provide rationales for targeted therapies, which can potentially attenuate IRI.

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

    Directory of Open Access Journals (Sweden)

    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.

  3. 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

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

    Directory of Open Access Journals (Sweden)

    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.

  5. The cytoskeleton adaptor protein ankyrin-1 is upregulated by p53 following DNA damage and alters cell migration.

    Science.gov (United States)

    Hall, A E; Lu, W-T; Godfrey, J D; Antonov, A V; Paicu, C; Moxon, S; Dalmay, T; Wilczynska, A; Muller, P A J; Bushell, M

    2016-01-01

    The integrity of the genome is maintained by a host of surveillance and repair mechanisms that are pivotal for cellular function. The tumour suppressor protein p53 is a major component of the DNA damage response pathway and plays a vital role in the maintenance of cell-cycle checkpoints. Here we show that a microRNA, miR-486, and its host gene ankyrin-1 (ANK1) are induced by p53 following DNA damage. Strikingly, the cytoskeleton adaptor protein ankyrin-1 was induced over 80-fold following DNA damage. ANK1 is upregulated in response to a variety of DNA damage agents in a range of cell types. We demonstrate that miR-486-5p is involved in controlling G1/S transition following DNA damage, whereas the induction of the ankyrin-1 protein alters the structure of the actin cytoskeleton and sustains limited cell migration during DNA damage. Importantly, we found that higher ANK1 expression correlates with decreased survival in cancer patients. Thus, these observations highlight ANK1 as an important effector downstream of the p53 pathway. PMID:27054339

  6. A Transporter of Ibuprofen is Upregulated in MDCK I cells under Hyperosmotic Culture Conditions

    DEFF Research Database (Denmark)

    Nielsen, Carsten U; Rasmussen, Rune N; Mo, Junying;

    2016-01-01

    by (R)-(-)- and (S)-(+)-ibuprofen with IC50 values of 19 µM (LogIC50 1.39± 0.34) and 0.47 µM (LogIC50 -0.36 ± 0.41), respectively. Furthermore, the [3H]-ibuprofen uptake rate was increased by decreased extracellular pH, but not dependent on Na+ or Cl- ions. The mRNA of Mct1, 2, 4 and 6 as well as Oat1...... in Madin-Darby Canine Kidney I (MDCK I) renal cells. [3H]-ibuprofen uptake rate was measured in MDCK I cell cultured under normal (300 mOsm) and hyperosmotic (500 mOsm) conditions. Hyperosmotic conditions were obtained by supplementing urea, NaCl, mannitol or raffinose to culture medium. The effect...

  7. Akt1/PKB upregulation leads to vascular smooth muscle cell hypertrophy and polyploidization

    OpenAIRE

    Hixon, Mary L.; Muro-Cacho, Carlos; Wagner, Mark W.; Obejero-Paz, Carlos; Millie, Elise; Fujio, Yasushi; Kureishi, Yasuko; Hassold, Terry; Walsh, Kenneth; Gualberto, Antonio

    2000-01-01

    Vascular smooth muscle cells (VSMCs) at capacitance arteries of hypertensive individuals and animals undergo marked age- and blood pressure–dependent polyploidization and hypertrophy. We show here that VSMCs at capacitance arteries of rat models of hypertension display high levels of Akt1/PKB protein and activity. Gene transfer of Akt1 to VSMCs isolated from a normotensive rat strain was sufficient to abrogate the activity of the mitotic spindle cell–cycle checkpoint, promoting polyploidizati...

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

    Science.gov (United States)

    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

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

    Directory of Open Access Journals (Sweden)

    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.

  10. PDGFRα up-regulation mediated by sonic hedgehog pathway activation leads to BRAF inhibitor resistance in melanoma cells with BRAF mutation

    OpenAIRE

    Sabbatino, Francesco; Wang, Yangyang; Wang, Xinhui; Flaherty, Keith T.; Yu, Ling; Pepin, David; Scognamiglio, Giosue'; Pepe, Stefano; Kirkwood, John M; Cooper, Zachary A; Frederick, Dennie T.; Wargo, Jennifer A.; Ferrone, Soldano; Ferrone, Cristina R.

    2014-01-01

    Control of BRAF(V600E) metastatic melanoma by BRAF inhibitor (BRAF-I) is limited by intrinsic and acquired resistance. Growth factor receptor up-regulation is among the mechanisms underlying BRAF-I resistance of melanoma cells. Here we demonstrate for the first time that PDGFRα up-regulation causes BRAF-I resistance. PDGFRα inhibition by PDGFRα-specific short hairpin (sh)RNA and by PDGFRα inhibitors restores and increases melanoma cells' sensitivity to BRAF-I in vitro and in vivo. This effect...

  11. Inhibition of miR-7 promotes angiogenesis in human umbilical vein endothelial cells by upregulating VEGF via KLF4.

    Science.gov (United States)

    Li, Yi-Ze; Wen, Lei; Wei, Xu; Wang, Qian-Rong; Xu, Long-Wen; Zhang, Hong-Mei; Liu, Wen-Chao

    2016-09-01

    Recent lentiviral-based microRNA (miRNA) library screening has identified miRNA-7 (miR-7) as an anti‑angiogenic miRNA in human umbilical vein endothelial cells (HUVECs). However, the underlying mechanism of miR-7 in the suppression of angiogenesis remains largely unknown. In the present study, we report that miR-7 inhibition promoted angiogenesis by upregulating vascular endothelial growth factor (VEGF) and directly targeting Krüppel-like factor 4 (KLF4). Downregulation of miR-7 promoted tube formation of HUVECs, accompanied by upregulation of mRNA and protein levels of both VEGF and KLF4. miR-7 directly targeted KLF4 as demonstrated by luciferase reporter assay and miR-7 mimics decreased KLF4. Furthermore, bioinformatic analysis revealed the presence of multiple DNA-binding elements of KLF4 in the VEGF promoter. Chromatin immunoprecipitation (ChIP) demonstrated that the KLF4 antibody specifically pulled down the VEGF promoter in the HUVECs. Furthermore, ectopic overexpression of KLF4 induced VEGF mRNA and protein levels. In addition, KLF4 silencing inhibited the angiogenesis induced by the miR-7 inhibitor in the HUVECs. Our results demonstrated that KLF4 is a direct target of miR-7 and a transcription activator of VEGF. These findings indicate that the miR-7-KLF4-VEGF signaling axis plays an important role in the regulation of angiogenesis in HUVECs, suggesting that miR-7 is a potential agent for the development of anti-angiogenic therapeutics in vascular diseases and solid tumors. PMID:27431648

  12. 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

  13. 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

  14. 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

  15. Mouse ES cells overexpressing DNMT1 produce abnormal neurons with upregulated NR1

    OpenAIRE

    D'Aiuto, Leonardo; Di Maio, Roberto; Mohan, K. Naga; Minervini, Crescenzio; Saporiti, Federica; Soreca, Isabella; Greenamyre, J. Timothy; Chaillet, J. Richard

    2011-01-01

    High levels of DNA methyltransferase 1 (DNMT1), hypermethylation, and downregulation of GAD67 and reelin have been described in GABAergic interneurons of patients with schizophrenia (SZ) and bipolar (BP) disorders. However, overexpression of DNMT1 is lethal, making it difficult to assess the direct effect of high levels of DNMT1 on neuronal development in vivo. We therefore used Dnmt1tet/tet mouse ES cells that overexpress DNMT1 as an in vitro model to investigate the impact of high levels of...

  16. Cornus officinalis Methanol Extract Upregulates Melanogenesis in Melan-a Cells.

    Science.gov (United States)

    An, Yun Ah; Hwang, Ji Yeon; Lee, Jae Soon; Kim, Young Chul

    2015-06-01

    Cornus officinalis is widely distributed in Korea, and its fruit has been used to make as herbal drug for traditional medicine in Korea, Japan, and China because of its tonic, analgesic, and diuretic properties. However, the effects of C. officinalis methanol extract (COME) on melanogenesis remain poorly understood. We evaluated the melanogenic capability of COME in melan-a cells, which are immortalized mouse melanocytes. COME increased melanin synthesis in a dose-dependent manner. Treatment with 12.5 μg/mL of COME significantly increased melanin content by 36.1% (p hair graying. PMID:26191383

  17. FAT10, a gene up-regulated in various cancers, is cell-cycle regulated

    OpenAIRE

    Zhang Dongwei; Lim Chuan-Bian; Lee Caroline GL

    2006-01-01

    Abstract Background FAT10 is a member of the ubiquitin-like-modifier family of proteins. Over-expression of the FAT10 gene was observed in the tumors of several epithelial cancers. High FAT10 expression was found to lead to increased chromosome instability via the reduction in the kinetochore localization of MAD2 during the prometaphase stage of the cell-cycle. FAT10 expression was also previously reported to be regulated by cytokines and p53. Results Here, we report that FAT10 expression is ...

  18. The anti-EGFR antibody cetuximab sensitizes human head and neck squamous cell carcinoma cells to radiation in part through inhibiting radiation-induced upregulation of HIF-1α

    OpenAIRE

    Lu, Haiquan; Liang, Ke; Lu, Yang; Fan, Zhen

    2012-01-01

    In this study, we investigated the mechanisms underlying cetuximab-mediated radiosensitization of HNSCC. Irradiation of HNSCC cells upregulated hypoxia-inducible factor-1 alpha (HIF-1α) via a mechanism involving de novo synthesis of HIF-1α protein. Radiation-induced upregulation of HIF-1α was completely abolished by concurrent treatment of HNSCC cells with cetuximab. Experimental elevation of constitutively expressed HIF-1α abolished cetuximab-mediated radiosensitization in HNSCC cells, where...

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

    Science.gov (United States)

    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.

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

    Directory of Open Access Journals (Sweden)

    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.

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

    Science.gov (United States)

    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.

  2. Ginsenoside compound K sensitizes human colon cancer cells to TRAIL-induced apoptosis via autophagy-dependent and -independent DR5 upregulation.

    Science.gov (United States)

    Chen, Lei; Meng, Yue; Sun, Qi; Zhang, Zhongyu; Guo, Xiaoqing; Sheng, Xiaotong; Tai, Guihua; Cheng, Hairong; Zhou, Yifa

    2016-01-01

    Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a potent cancer cell-specific apoptosis-inducing cytokine with little toxicity to most normal cells. However, acquired resistance of cancer cells to TRAIL is a roadblock. Agents that can either potentiate the effect of TRAIL or overcome resistance to TRAIL are urgently needed. This article reports that ginsenoside compound K (CK) potentiates TRAIL-induced apoptosis in HCT116 colon cancer cells and sensitizes TRAIL-resistant colon cancer HT-29 cells to TRAIL. On a cellular mechanistic level, CK downregulated cell survival proteins including Mcl-1, Bcl-2, surviving, X-linked inhibitor of apoptosis protein and Fas-associated death domain-like IL-1-converting enzyme-inhibitory protein, upregulated cell pro-apoptotic proteins including Bax, tBid and cytochrome c, and induced the cell surface expression of TRAIL death receptor DR5. Reduction of DR5 levels by siRNAs significantly decreases CK- and TRAIL-mediated apoptosis. Importantly, our results indicate, for the first time, that DR5 upregulation is mediated by autophagy, as blockade of CK-induced autophagy by 3-MA, LY294002 or Atg7 siRNAs substantially decreases DR5 upregulation and reduces the synergistic effect. Furthermore, CK-stimulated autophagy is mediated by the reactive oxygen species-c-Jun NH2-terminal kinase pathway. Moreover, we found that p53 and the C/EBP homologous (CHOP) protein is also required for DR5 upregulation but not related with autophagy. Our findings contribute significantly to the understanding of the mechanism accounted for the synergistic anticancer activity of CK and TRAIL, and showed a novel mechanism related with DR5 upregulation. PMID:27512955

  3. 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.

  4. Positive regulation of osteoclastic differentiation by growth differentiation factor 15 upregulated in osteocytic cells under hypoxia.

    Science.gov (United States)

    Hinoi, Eiichi; Ochi, Hiroki; Takarada, Takeshi; Nakatani, Eri; Iezaki, Takashi; Nakajima, Hiroko; Fujita, Hiroyuki; Takahata, Yoshifumi; Hidano, Shinya; Kobayashi, Takashi; Takeda, Shu; Yoneda, Yukio

    2012-04-01

    Osteocytes are thought to play a role as a mechanical sensor through their communication network in bone. Although osteocytes are the most abundant cells in bone, little attention has been paid to their physiological and pathological functions in skeletogenesis. Here, we have attempted to delineate the pivotal functional role of osteocytes in regulation of bone remodeling under pathological conditions. We first found markedly increased osteoclastic differentiation by conditioned media (CM) from osteocytic MLO-Y4 cells previously exposed to hypoxia in vitro. Using microarray and real-time PCR analyses, we identified growth differentiation factor 15 (GDF15) as a key candidate factor secreted from osteocytes under hypoxia. Recombinant GDF15 significantly promoted osteoclastic differentiation in a concentration-dependent manner, with concomitant facilitation of phosphorylation of both p65 and inhibitory-κB in the presence of receptor activator of nuclear factor-κB ligand. To examine the possible functional significance of GDF15 in vivo, mice were subjected to ligation of the right femoral artery as a hypoxic model. A significant increase in GDF15 expression was specifically observed in tibias of the ligated limb but not in tibias of the normally perfused limb. Under these experimental conditions, in cancellous bone of proximal tibias in the ligated limb, a significant reduction was observed in bone volume, whereas a significant increase was seen in the extent of osteoclast surface/bone surface when determined by bone histomorphometric analysis. Finally, the anti-GDF15 antibody prevented bone loss through inhibiting osteoclastic activation in tibias from mice with femoral artery ligation in vivo, in addition to suppressing osteoclastic activity enhanced by CM from osteocytes exposed to hypoxia in vitro. These findings suggest that GDF15 could play a pivotal role in the pathogenesis of bone loss relevant to hypoxia through promotion of osteoclastogenesis after

  5. 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

  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. Hyperketonemia (Acetoacetate Upregulates NADPH Oxidase 4 and Elevates Oxidative Stress, ICAM-1, and Monocyte Adhesivity in Endothelial Cells

    Directory of Open Access Journals (Sweden)

    Preeti Kanikarla-Marie

    2015-01-01

    Full Text Available Background/Aims: The incidence of developing microvascular dysfunction is significantly higher in type 1 diabetic (T1D patients. Hyperketonemia (acetoacetate, β-hydroxybutyrate is frequently found along with hyperglycemia in T1D. Whether hyperketonemia per se contributes to the excess oxidative stress and cellular injury observed in T1D is not known. Methods: HUVEC were treated with ketones in the presence or absence of high glucose for 24 h. NOX4 siRNA was used to specifically knockdown NOX4 expression in HUVEC. Results: Ketones alone or in combination with high glucose treatment cause a significant increase in oxidative stress, ICAM-1, and monocyte adhesivity to HUVEC. Using an antisense approach, we show that ketone induced increases in ROS, ICAM-1 expression, and monocyte adhesion in endothelial cells were prevented in NOX4 knockdown cells. Conclusion: This study reports that elevated levels of ketones upregulate NOX, contributing to increased oxidative stress, ICAM-1 levels, and cellular dysfunction. This provides a novel biochemical mechanism that elucidates the role of hyperketonemia in the excess cellular injury in T1D. New drugs targeting inhibition of NOX seems promising in preventing higher risk of complications associated with T1D.

  9. Gmelina arborea Roxb. (Family: Verbenaceae Extract Upregulates the β-Cell Regeneration in STZ Induced Diabetic Rats

    Directory of Open Access Journals (Sweden)

    Anoja Priyadarshani Attanayake

    2016-01-01

    Full Text Available Gmelina arborea Roxb. (common name: Et-demata, Family: Verbenaceae has been used traditionally in Sri Lanka as a remedy against diabetes mellitus. The objective of the present study was to evaluate antidiabetic mechanisms of the aqueous bark extract of G. arborea in streptozotocin induced (STZ diabetic male Wistar rats. Aqueous bark extract of G. arborea (1.00 g/kg and glibenclamide as the standard drug (0.50 mg/kg were administered orally using a gavage to STZ diabetic rats (65 mg/kg, ip for 30 days. The antidiabetic mechanisms of aqueous extract of G. arborea (1.00 g/kg were determined at the end of the experiment. The fasting blood glucose concentration was significantly lowered and the serum insulin and C-peptide concentrations were increased by 57% and 39% in plant extract treated rats on day 30, respectively (p<0.05. The histopathology and immunohistochemistry results of the plant extract treated group showed a regenerative effect on β-cells of the pancreas in diabetic rats. In addition, serum lipid parameters were improved in G. arborea extract treated diabetic rats. The results revealed that the aqueous stem bark extract of G. arborea (1.00 g/kg showed beneficial effects against diabetes mellitus through upregulating the β-cell regeneration and biosynthesis of insulin in diabetic rats.

  10. 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.

  11. 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

    2016-09-01

    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. PMID:27422606

  12. 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.

  13. 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.

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

    International Nuclear Information System (INIS)

    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

  15. Beta-Adrenergic Receptor Population is Up-Regulated in Chicken Skeletal Muscle Cells Treated with Forskolin

    Science.gov (United States)

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

    1998-01-01

    Skeletal muscle hypertrophy is promoted by in vivo administration of beta-adrenergic receptor (betaAR) agonists. These compounds presumably exert their physiological action through the betaAR, and alterations in the population of betaAR could potentially change the ability of the cell to respond to the betaAR agonists. Since the intracellular chemical signal generated by the betaAR is cyclic AMP (cAMP), experiments were initiated in primary chicken muscle cell cultures to determine if artificial elevation of intracellular cAMP by treatment with forskolin would alter the population of functional betaAR expressed on the surface of muscle cells. Chicken skeletal muscle cells after 7 days in culture were employed for the experiments because muscle cells have attained a steady state with respect to muscle protein metabolism at this stage. Cells were treated with 0-10 microM forskolin for a total of three days. At the end of the 1, 2, and 3 day treatment intervals, the concentration of cAMP and the betaAR population were measured. Receptor population was measured in intact muscle cell cultures as the difference between total binding of [H-3]CGP-12177 and non-specific binding of [H-3]CGP-12177 in the presence of 1 microM propranolol. Intracellular cAMP concentration was measured by radioimmunoassay. The concentration of cAMP in forskolin-treated cells increased up to 10-fold in a dose dependent manner. Increasing concentrations of forskolin also led to an increase in betaAR population, with a maximum increase of approximately 50% at 10 microM. This increase in PAR population was apparent after only 1 day of treatment, and the pattern of increase was maintained for all 3 days of the treatment period. Thus, increasing the intracellular concentration of cAMP leads to up-regulation of betaAR population. The effect of forskolin on the quantity and apparent synthesis rate of the heavy chain of myosin (mhc) were also investigated. A maximum increase of 50% in the quantity of mhc

  16. 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.

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

    Directory of Open Access Journals (Sweden)

    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.

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

    Directory of Open Access Journals (Sweden)

    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. 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.

  20. Homeobox A7 increases cell proliferation by up-regulation of epidermal growth factor receptor expression in human granulosa cells

    Directory of Open Access Journals (Sweden)

    Yanase Toshihiko

    2010-06-01

    Full Text Available Abstract Background Homeobox (HOX genes encode transcription factors, which regulate cell proliferation, differentiation, adhesion, and migration. The deregulation of HOX genes is frequently associated with human reproductive system disorders. However, knowledge regarding the role of HOX genes in human granulosa cells is limited. Methods To determine the role of HOXA7 in the regulation and associated mechanisms of cell proliferation in human granulosa cells, HOXA7 and epidermal growth factor receptor (EGFR expressions were examined in primary granulosa cells (hGCs, an immortalized human granulosa cell line, SVOG, and a granulosa tumor cell line, KGN, by real-time PCR and Western blotting. To manipulate the expression of HOXA7, the HOXA7 specific siRNA was used to knockdown HOXA7 in KGN. Conversely, HOXA7 was overexpressed in SVOG by transfection with the pcDNA3.1-HOAX7 vector. Cell proliferation was measured by the MTT assay. Results Our results show that HOXA7 and EGFR were overexpressed in KGN cells compared to hGCs and SVOG cells. Knockdown of HOXA7 in KGN cells significantly decreased cell proliferation and EGFR expression. Overexpression of HOXA7 in SVOG cells significantly promoted cell growth and EGFR expression. Moreover, the EGF-induced KGN proliferation was abrogated, and the activation of downstream signaling was diminished when HOXA7 was knocked down. Overexpression of HOXA7 in SVOG cells had an opposite effect. Conclusions Our present study reveals a novel mechanistic role for HOXA7 in modulating granulosa cell proliferation via the regulation of EGFR. This finding contributes to the knowledge of the pro-proliferation effect of HOXA7 in granulosa cell growth and differentiation.

  1. 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 a membrane. Differential gene expression in the RPE cells of complement factor genes was identified using gene arrays, and selected gene transcripts were validated by q-RT-PCR. Protein expression was determined by ELISA and immunoblotting. Co-culture with activated T cells increased RPE mRNA and/or protein...... expression of complement components C3, factors B, H, H-like 1, CD46, CD55, CD59, and clusterin, in a dose-dependent manner. Soluble factors derived from activated T cells are capable of increasing expression of complement components in RPE cells. This is important for the further understanding...

  2. 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...

  3. 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.

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

    International Nuclear Information System (INIS)

    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

  5. 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.

  6. 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.

  7. 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.

  8. Disturbance Rejection

    OpenAIRE

    2005-01-01

    This interactive tutorial reviews the disturbance rejection capabilities of different feedback control schemes. The interactions in this tutorial involve students analyzing 4 cases of step-like disturbance rejection. ME2801 Introduction to Engineering System Dynamics

  9. 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......The microtubule-targeting taxanes are important in breast cancer therapy, but no predictive biomarkers have yet been identified with sufficient scientific evidence to allow clinical routine use. The purposes of the present study were to develop a cell-culture-based discovery platform for docetaxel...... over 15 months. The cell lines were characterized regarding sensitivity to docetaxel and other chemotherapeutics and subjected to transcriptome-wide mRNA microarray profiling. MCF7RES and MDARES exhibited a biphasic growth inhibition pattern at increasing docetaxel concentrations. Gene expression...

  10. Farnesoid X receptor up-regulates expression of Lipid transfer inhibitor protein in liver cells and mice

    Energy Technology Data Exchange (ETDEWEB)

    Li, Liangpeng [Department of Biochemistry and Molecular Biology, College of Basic Medical Science, Third Military Medical University, Chongqing 400038 (China); Liu, Hong [Department of Hematology, Xinqiao Hospital, Third Military Medical University, Chongqing 400037 (China); Peng, Jiahe; Wang, Yongchao; Zhang, Yan; Dong, Jinyu; Liu, Xiaohua; Guo, Dongmei [Department of Biochemistry and Molecular Biology, College of Basic Medical Science, Third Military Medical University, Chongqing 400038 (China); Jiang, Yu, E-mail: yujiang61@gmail.com [Department of Biochemistry and Molecular Biology, College of Basic Medical Science, Third Military Medical University, Chongqing 400038 (China)

    2013-11-29

    Highlights: •FXR up-regulates apoF. •It binds to ER1 element. •It activates apoF gene promoter. -- Abstract: Apolipoprotein F is a component protein mainly secreted by liver and resides on several lipoprotein classes. It can inhibit lipids transfer between different lipoproteins. FXR is a member of the nuclear receptor superfamily which is also highly expressed in the liver. It modulates bile acids synthesis and lipids metabolism by transcriptional regulation. We aimed to determine whether apoF can be regulated by FXR. The FXR agonist Chenodeoxycholic acid (CDCA) and GW4064 both can activate the expression of apoF in liver cell lines and in C57/BL6 mouse liver. This is dependent on the binding of FXR to the FXR element ER1 (−2904 to −2892 bp) in the apoF gene promoter. Taken together, we have identified apoF as likely another target gene of FXR.

  11. Farnesoid X receptor up-regulates expression of Lipid transfer inhibitor protein in liver cells and mice

    International Nuclear Information System (INIS)

    Highlights: •FXR up-regulates apoF. •It binds to ER1 element. •It activates apoF gene promoter. -- Abstract: Apolipoprotein F is a component protein mainly secreted by liver and resides on several lipoprotein classes. It can inhibit lipids transfer between different lipoproteins. FXR is a member of the nuclear receptor superfamily which is also highly expressed in the liver. It modulates bile acids synthesis and lipids metabolism by transcriptional regulation. We aimed to determine whether apoF can be regulated by FXR. The FXR agonist Chenodeoxycholic acid (CDCA) and GW4064 both can activate the expression of apoF in liver cell lines and in C57/BL6 mouse liver. This is dependent on the binding of FXR to the FXR element ER1 (−2904 to −2892 bp) in the apoF gene promoter. Taken together, we have identified apoF as likely another target gene of FXR

  12. Gmelina arborea Roxb. (Family: Verbenaceae) Extract Upregulates the β-Cell Regeneration in STZ Induced Diabetic Rats.

    Science.gov (United States)

    Attanayake, Anoja Priyadarshani; Jayatilaka, Kamani Ayoma Perera Wijewardana; Pathirana, Chitra; Mudduwa, Lakmini Kumari Boralugoda

    2016-01-01

    Gmelina arborea Roxb. (common name: Et-demata, Family: Verbenaceae) has been used traditionally in Sri Lanka as a remedy against diabetes mellitus. The objective of the present study was to evaluate antidiabetic mechanisms of the aqueous bark extract of G. arborea in streptozotocin induced (STZ) diabetic male Wistar rats. Aqueous bark extract of G. arborea (1.00 g/kg) and glibenclamide as the standard drug (0.50 mg/kg) were administered orally using a gavage to STZ diabetic rats (65 mg/kg, ip) for 30 days. The antidiabetic mechanisms of aqueous extract of G. arborea (1.00 g/kg) were determined at the end of the experiment. The fasting blood glucose concentration was significantly lowered and the serum insulin and C-peptide concentrations were increased by 57% and 39% in plant extract treated rats on day 30, respectively (p arborea extract treated diabetic rats. The results revealed that the aqueous stem bark extract of G. arborea (1.00 g/kg) showed beneficial effects against diabetes mellitus through upregulating the β-cell regeneration and biosynthesis of insulin in diabetic rats. PMID:26881248

  13. 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.

  14. 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.

  15. HBx-upregulated lncRNA UCA1 promotes cell growth and tumorigenesis by recruiting EZH2 and repressing p27Kip1/CDK2 signaling.

    Science.gov (United States)

    Hu, Jiao-Jiao; Song, Wei; Zhang, Shao-Dan; Shen, Xiao-Hui; Qiu, Xue-Mei; Wu, Hua-Zhang; Gong, Pi-Hai; Lu, Sen; Zhao, Zhu-Jiang; He, Ming-Liang; Fan, Hong

    2016-01-01

    It is well accepted that HBx plays the major role in hepatocarcinogenesis associated with hepatitis B virus (HBV) infections. However, little was known about its role in regulating long noncoding RNAs (lncRNAs), a large group of transcripts regulating a variety of biological processes including carcinogenesis in mammalian cells. Here we report that HBx upregulates UCA1 genes and downregulates p27 genes in hepatic LO2 cells. Further studies show that the upregulated UCA1 promotes cell growth by facilitating G1/S transition through CDK2 in both hepatic and hepatoma cells. Knock down of UCA1 in HBx-expressing hepatic and hepatoma cells resulted in markedly increased apoptotic cells by elevating the cleaved caspase-3 and caspase-8. More importantly, UCA1 is found to be physically associated with enhancer of zeste homolog 2 (EZH2), which suppresses p27Kip1 through histone methylation (H3K27me3) on p27Kip1 promoter. We also show that knockdown of UCA1 in hepatoma cells inhibits tumorigenesis in nude mice. In a clinic study, UCA1 is found to be frequently up-regulated in HBx positive group tissues in comparison with the HBx negative group, and exhibits an inverse correlation between UCA1 and p27Kip1 levels. Our findings demonstrate an important mechanism of hepatocarcinogenesis through the signaling of HBx-UCA1/EZH2-p27Kip1 axis, and a potential target of HCC. PMID:27009634

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

    OpenAIRE

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

    2015-01-01

    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 (R) copy number assa...

  17. 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.

  18. SKF95365 induces apoptosis and cell-cycle arrest by disturbing oncogenic Ca2+ signaling in nasopharyngeal carcinoma cells

    Directory of Open Access Journals (Sweden)

    Zhang J

    2015-10-01

    Full Text Available Jinyan Zhang,1 Jiazhang Wei,2 Qian He,3 Yan Lin,1 Rong Liang,1 Jiaxiang Ye,1 Zhe Zhang,4 Yongqiang Li1 1Department of Medical Oncology, Affiliated Cancer Hospital of Guangxi Medical University, 2Department of Otolaryngology-Head and Neck Oncology, The People’s Hospital of Guangxi Zhuang Autonomous Region, Nanning, People’s Republic of China; 3Graduate School of Information Science and Technology, Hokkaido University, Sapporo, Japan; 4Department of Otolaryngology-Head & Neck Surgery, First Affiliated Hospital of Guangxi Medical University, Nanning, People’s Republic of China Background: Aberrant modulation of store-operated calcium ions (Ca2+ entry promotes the progression of human malignancies. Previously, we reported that the blockage of store-operated Ca2+ entry inhibited epidermal growth factor (EGF-stimulated migration and distant metastasis in nasopharyngeal carcinoma (NPC cells. However, the effects of pharmacological blocker on other Ca2+ signaling-regulated malignant characteristics in NPC cells remained poorly understood. Methods: We examined the effects of SKF96365, an inhibitor of store-operated Ca2+ channel, on EGF-launched Ca2+ signaling in two NPC cell lines. We determined the effects of SKF96365 on cell proliferation, colony formation, apoptosis, and cell-cycle status in vitro. We further elucidated the antitumor activity of SKF96365 in xenograft-bearing mice. Results: It was found that SKF96365 disturbed the thapsigargin (TG-stimulated Ca2+ release from endoplasmic reticulum and the subsequent Ca2+ influx. SKF96365 alone stimulated Ca2+ responses merely due to endoplasmic reticulum-released Ca2+. SKF96365 promoted cell mortality, inhibited colony formation, and induced apoptosis and cell-cycle arrest, while blunting the EGF-evoked Ca2+ signaling. Furthermore, we confirmed that SKF96365 reduced NPC xenograft growth while activating caspase-7-related apoptotic pathway. Conclusion: SKF96365 exerts multiple antitumor

  19. Cyanide-induced Death of Dopaminergic Cells is Mediated by Uncoupling Protein-2 Up-regulation and Reduced Bcl-2 Expression

    OpenAIRE

    Zhang, X.; Li, L.; Zhang, L.; Borowitz, J.L.; Isom, G.E.

    2009-01-01

    Cyanide is a potent inhibitor of mitochondrial oxidative metabolism and produces mitochondria-mediated death of dopaminergic neurons and sublethal intoxications are associated with a Parkinson-like syndrome. Cyanide toxicity is enhanced when mitochondrial uncoupling is stimulated following up-regulation of uncoupling protein-2 (UCP-2). In this study, the role of a pro-survival protein, Bcl-2, in cyanide-mediated cell death was determined in a rat dopaminergic immortalized mesencephalic cell l...

  20. 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.

  1. MiR-21 up-regulation mediates glioblastoma cancer stem cells apoptosis and proliferation by targeting FASLG.

    Science.gov (United States)

    Shang, Chao; Guo, Yan; Hong, Yang; Liu, Yun-hui; Xue, Yi-xue

    2015-03-01

    To investigate whether miR-21 can affect the apoptosis and proliferation of glioblastoma cancer stem cells (GSCs) from down-regulating FASLG. The expression of miRNA-21 was detected by quantitative real-time PCR in normal brain tissue and glioblastoma samples, and the changes of miRNA-21 expression between GSCs and non-GSCs were also detected. The apoptosis and proliferation ability of miR-21 in GSCs were analyzed by MTT and flow cytometry assay after anti-miR-21 transfection. For the regulation mechanism analysis of miR-21, TargetScan, PicTar and microRNA were selected to predict some potential target genes of miR-21. The predicted gene was identified to be the direct and specific target gene of miR-21 by luciferase activities assay and western blot. RNA interference technology was used to confirm the apoptosis and proliferation effects of miR-21 were directly induced by FASLG. The expression of miR-21 increased significantly in glioblastoma contrast to normal brain tissue, and miR-21 up-regulated in GSCs remarkably. The proliferation of GSCs cell could be inhibited with high-expression of miR-21 and this effect could be restored by miR-21 knocked down. Mechanism analysis revealed that FASLG was a specific and direct target gene of miR-21. The advanced effects of anti-miR-21 on GSCs apoptosis and proliferation were mediated by expression of silenced FASLG. In summary, aberrantly expressed miR-21 regulates GSCs apoptosis and proliferation partly through directly down-regulating FASLG protein expression in GSCs and this might offer a new potential therapeutic stratagem for glioblastoma. PMID:25394756

  2. 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.

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

    International Nuclear Information System (INIS)

    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

  4. 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.

  5. 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.

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

    Directory of Open Access Journals (Sweden)

    Yafei Zhang

    2013-01-01

    Full Text Available 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.

  7. 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.

  8. Hepatopoietin Cn suppresses apoptosis of human hepatocellular carcinoma cells by up-regulating myeloid cell leukemia-1

    Institute of Scientific and Technical Information of China (English)

    Chu-Tse; Wu

    2010-01-01

    AIM:To investigate the role of hepatopoietin Cn(HPPCn) in apoptosis of hepatocellular carcinoma(HCC)cells and its mechanism. METHODS:Two human HCC cell lines,SMMC7721 and HepG2,were used in this study.Immunostaining, Western blotting and enzyme linked immunosorbent assay were conducted to identify the expression of HPPCn and the existence of an autocrine loop of HPPCn/ HPPCn receptor in SMMC7721 and HepG2.Apoptotic cells were detected using fluorescein isothiocyanate (FITC)-conjugated Annexin V and propidiu...

  9. 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.

  10. Up-regulation of the integrin alpha 1/beta 1 in human neuroblastoma cells differentiated by retinoic acid: correlation with increased neurite outgrowth response to laminin.

    OpenAIRE

    Rossino, P; P. Defilippi; Silengo, L; Tarone, G.

    1991-01-01

    Retinoic acid (RA) is known to induce differentiation of neuroblastoma cells in vitro. Here we show that treatment of two human neuroblastoma cell lines, SY5Y and IMR32, with RA resulted in a fivefold increase of the integrin alpha 1/beta 1 expression. The effect was selective because expression of the alpha 3/beta 1 integrin, also present in these cells, was not increased. The up-regulation of the alpha 1/beta 1 differentiated SY5Y cells correlated with increased neurite response to laminin....

  11. Macrophages promote matrix protrusive and invasive function of breast cancer cells via MIP-1β dependent upregulation of MYO3A gene in breast cancer cells.

    Science.gov (United States)

    Baghel, Khemraj Singh; Tewari, Brij Nath; Shrivastava, Richa; Malik, Showkat Ahmad; Lone, Mehraj U-Din; Jain, Nem Kumar; Tripathi, Chakrapani; Kanchan, Ranjana Kumari; Dixit, Sameer; Singh, Kavita; Mitra, Kalyan; Negi, Mahendra Pal Singh; Srivastava, Mukesh; Misra, Sanjeev; Bhatt, Madan Lal Brahma; Bhadauria, Smrati

    2016-07-01

    The potential of a tumor cell to metastasize profoundly depends on its microenvironment, or "niche" interactions with local components. Tumor-associated-macrophages (TAMs) are the most abundant subpopulation of tumor stroma and represent a key component of tumor microenvironment. The dynamic interaction of cancer cells with neighboring TAMs actively drive cancer progression and metastatic transformation through intercellular signaling networks that need better elucidation. Thus, current study was planned for discerning paracrine communication networks operational between TAMs, and breast cancer cells with special reference to cancer cell invasion and dissemination to distant sites. Here, we report role of MIP-1β in enhancing invasive potential of metastatic breast cancer MDA-MB-231 and MDA-MB-468 cells. In addition, the poorly metastatic MCF-7 cells were also rendered invasive by MIP-1β. The MIP-1β-driven cancer cell invasion was dependent on upregulated expression levels of MYO3A gene, which encodes an unconventional myosin super-family protein harboring a kinase domain. Ex ovo study employing Chick-embryo-model and in vivo Syngenic 4T1/BALB/c mice-model further corroborated aforementioned in vitro findings, thereby substantiating their physiological relevance. Concordantly, human breast cancer specimen exhibited significant association between mRNA expression levels of MIP-1β and MYO3A. Both, MIP-1β and MYO3A exhibited positive correlation with MMP9, an established molecular determinant of cancer cell invasion. Higher expression of these genes correlated with poor survival of breast cancer patients. Collectively, these results point toward so far undisclosed MIP-1β/MYO3A axis being operational during metastasis, wherein macrophage-derived MIP-1β potentiated cancer cell invasion and metastasis via up regulation of MYO3A gene within cancer cells. Our study exposes opportunities for devising potential anti-metastatic strategies for efficient clinical

  12. 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.

  13. 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.

  14. The flavonoid casticin enhances TRAIL-induced apoptosis of colon cancer cells through endoplasmic reticulum stress-mediated up-regulation of DR5

    Institute of Scientific and Technical Information of China (English)

    Sanyuan Tang; Guangjin Yuan; Zhengyang Yu; Leilan Yin; Hao Jiang

    2013-01-01

    Objective: The aim of this study was to explore the mechanisms by which the flavonoid casticin enhances tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis in colon cancer cells. Methods: Human colon cancer HT-29 cells were treated with TRAIL or casticin. Cytotoxicity was examined by MTT assay, and apoptosis determined by morphological observation and flow cytometric analysis. Death receptor 5 (DR5), DR4, and endoplasmic reticulum (ER) stress response markers, including glucose regulating protein 78 (GRP78), activating transcription factor 4 (ATF4) and CHOP (CCAAT/enhancer binding protein homologous protein), were examined with western blot. Small interfering RNA (siRNA) transfection was employed to knock down CHOP. Results: HT-29 cells were resistance to TRAIL-induced apoptosis, but casticin, at subtoxic concentrations, potentiated HT-29 cells to TRAIL-induced apoptosis. Casticin up-regulated the expression of DR5 time- and dose-dependent manners, but had no effect on the expression of DR4. Also, casticin increased the levels of ER stress response markers (GRP78, ATF4 and CHOP) in a similar way to DR5. Knockdown of CHOP by specific siRNA, or salubrinal, an ER stress inhibitor, abolished the up-regulation of DR5 and enhancement of TRAIL-induced apoptosis by casticin. Conclusion: Casticin enhances TRAIL-induced apoptosis of colon cancer cells by ER stress-mediated up-regulation of DR5.

  15. 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.

  16. 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

  17. Bee Venom Acupuncture Alleviates Experimental Autoimmune Encephalomyelitis by Upregulating Regulatory T Cells and Suppressing Th1 and Th17 Responses.

    Science.gov (United States)

    Lee, Min Jung; Jang, Minhee; Choi, Jonghee; Lee, Gihyun; Min, Hyun Jung; Chung, Won-Seok; Kim, Jong-In; Jee, Youngheun; Chae, Younbyoung; Kim, Sung-Hoon; Lee, Sung Joong; Cho, Ik-Hyun

    2016-04-01

    The protective and therapeutic mechanism of bee venom acupuncture (BVA) in neurodegenerative disorders is not clear. We investigated whether treatment with BVA (0.25 and 0.8 mg/kg) at the Zusanli (ST36) acupoints, located lateral from the anterior border of the tibia, has a beneficial effect in a myelin basic protein (MBP)(68-82)-induced acute experimental autoimmune encephalomyelitis (EAE) rat model. Pretreatment (every 3 days from 1 h before immunization) with BVA was more effective than posttreatment (daily after immunization) with BVA with respect to clinical signs (neurological impairment and loss of body weight) of acute EAE rats. Treatment with BVA at the ST36 acupoint in normal rats did not induce the clinical signs. Pretreatment with BVA suppressed demyelination, glial activation, expression of cytokines [interferon (IFN)-γ, IL-17, IL-17A, tumor necrosis factor-alpha (TNF-α), and IL-1β], chemokines [RANTES, monocyte chemotactic protein-1 (MCP-1), and macrophage inflammatory protein (MIP)-1α], and inducible nitric oxide synthase (iNOS), and activation of p38 mitogen-activated protein kinase (MAPK) and nuclear factor (NF)-κB (p65 and phospho-IκBα) signaling pathways in the spinal cord of acute EAE rats. Pretreatment with BVA decreased the number of CD4(+), CD4(+)/IFN-γ(+), and CD4(+)/IL-17(+) T cells, but increased the number of CD4(+)/Foxp3(+) T cells in the spinal cord and lymph nodes of acute EAE rats. Treatment with BVA at six placebo acupoints (SP9, GB39, and four non-acupoints) did not have a positive effect in acute EAE rats. Interestingly, onset and posttreatment with BVA at the ST36 acupoint markedly attenuated neurological impairment in myelin oligodendrocyte glycoprotein (MOG)(35-55)-induced chronic EAE mice compared to treatment with BVA at six placebo acupoints. Our findings strongly suggest that treatment with BVA with ST36 acupoint could delay or attenuate the development and progression of EAE by upregulating regulatory T cells and

  18. DJ-1 Is Upregulated in Oral Squamous Cell Carcinoma and Promotes Oral Cancer Cell Proliferation and Invasion

    Science.gov (United States)

    Xu, Shuaimei; Ma, Dandan; Zhuang, Rui; Sun, Wenjuan; Liu, Ying; Wen, Jun; Cui, Li

    2016-01-01

    Background: The development of oral squamous cell carcinoma (OSCC) is a multistep process that involves in both genetic alterations and epigenetic modifications. DJ-1, a negative regulator of tumor suppressor PTEN, functions as an oncogene in many types of cancers. However, its role in OSCC is poorly known. Methods: Immunohistochemical staining and Western blotting were performed to evaluate the expression level of DJ-1 in oral leukoplakia (OLK) and OSCC tissues respectively. Then lentiviral mediated DJ-1 shRNA was constructed and used to infect the OSCC cell lines (Tca8113 and CAL-27). MTT, cell counting, and Matrigel invasion assay were utilized to examine the effects of DJ-1 down-regulation on proliferation and invasion capacity of oral cancer cells. Results: The immunoreactivity and expression level of DJ-1 protein was significantly increased in OLK and OSCC tissues compared with the controls. Lentiviral-delivered shRNA targeting DJ-1 could effectively knock down DJ-1 at mRNA and protein level (P<0.01). The proliferative and invasion ability of OSCC cell lines was significantly suppressed following DJ-1 inhibition (P<0.01). Conclusions: Our study indicated that DJ-1 is over-expressed in both oral precancer and cancer tissues and shRNA inhibition of DJ-1 expression led to decreased proliferation and invasion capability of oral cancer cells. These findings suggest that DJ-1 might be actively involved in the development of OSCC. Future studies will investigate the potential of DJ-1 as a biomarker for early detection of OSCC. PMID:27313793

  19. Gli1 maintains cell survival by up-regulating IGFBP6 and Bcl-2 through promoter regions in parallel manner in pancreatic cancer cells

    Directory of Open Access Journals (Sweden)

    Xu Xuan-Fu

    2009-01-01

    Full Text Available Background: Aberrant activation of Hedgehog (Hh signaling pathway has been reported to be related to malignant biological behavior of pancreatic cancer but its mechanism is unclear yet. Since IGF pathway and Bcl-2 family are involved in proliferation and apoptosis of pancreatic cancer cells, we hypothesize that they are possibly associated with Hh pathway. Materials and Methods: We studied the relationship of Shh-Gli1 signaling pathway with proliferation and apoptosis of pancreatic cancer cells and the regulation of transcription factor Gli1 to insulin-like growth factor binding protein 6 (IGFBP6 and Bcl-2 genes at the level of transcription. Results: Sonic hedgehog (Shh, Smoothened (Smo, patched and Gli1 were expressed in pancreatic cancer cells. Cyclopamine inhibited cell proliferation at low concentration and induced apoptosis at high concentration. Effect of RNA interference (RNAi for Gli1 to cell survival is mainly due to proliferation inhibition though involved in apoptosis. The transcription factor Gli1 bound to promoter regions of Bcl-2 and IGFBP6 genes and the levels of IGFBP6, proliferating cell nuclear antigen (PCNA and Bcl-2 messenger RNA (mRNA were decreased as well as Gli1 mRNA significantly by cyclopamine or RNAi in cultured pancreatic cancer cells (p < 0.01. Finally PCNA, IGFBP6 and Bcl-2 mRNA were upregulated as well as Shh or Gli1 in pancreatic cancer tissues (p < 0.01. Conclusions: Our study reveals that Gli1 maintained cell survival by binding the promoter regions and facilitating transcription of IGFBP6 and Bcl-2 genes in a parallel manner in pancreatic cancer cells and suggests it may be one of the mechanisms of Shh-Gli1 signaling pathway in pancreatic cancer.

  20. Insulin resistance contributes to multidrug resistance in HepG2 cells via activation of the PERK signaling pathway and upregulation of Bcl-2 and P-gp.

    Science.gov (United States)

    Liu, Xinyue; Li, Linjing; Li, Jing; Cheng, Yan; Chen, Jing; Shen, Minghui; Zhang, Shangdi; Wei, Hulai

    2016-05-01

    Liver tumorigenesis frequently causes insulin resistance which may be used as an independent risk factor for evaluation of survival and post-surgery relapse of liver cancer patients. In the present study, HepG2/IR, an insulin resistant HepG2 cell line, was established by exposing HepG2 cells to 0.5 µmol/l of insulin for 72 h, and comparison of HepG2/IR with the parental HepG2 cells indicated that the HepG2/IR cells showed significantly enhanced resistance to the most frequently used chemotherapeutics for solid tumors, such as cisplatin, 5-fluorouracil, vincristine and mitomycin. Flow cytometric analysis of cisplatin-treated HepG2/IR cells showed a significantly decreased hypodiploid peak and a significantly downregulated expression level of pro-apoptotic protein caspase-3 compared with the parental HepG2 cells. Our data further showed swollen endoplasmic reticulum (ER) in the cisplatin-treated HepG2/IR cells with significantly increased levels of glucose-regulated protein 78 (GRP78), phosphorylated protein kinase R-like ER kinase (p-PERK) and P-glycoprotein (P-gp). There was also an upregulated expression of anti-apoptotic protein B-cell lymphoma 2 (Bcl-2) whereas no significant change was observed for CCAAT-enhancer-binding protein homologous protein (CHOP), which is known to be induced by ER stress and to mediate apoptosis. Our results demonstrated that insulin resistance in HepG2 cells promoted a protective unfolded protein response and upregulated the expression of ER chaperone protein GRP78, which resulted in the phosphorylation of PERK kinase to activate the PERK-mediated ER stress signal transduction pathway and the upregulation of Bcl-2 and P-gp, leading to the inhibition of the caspase-3-dependent apoptosis pathway and to the survival of liver tumor cells. PMID:26935266

  1. 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.

  2. Transcriptional activation and cell cycle block are the keys for 5-fluorouracil induced up-regulation of human thymidylate synthase expression.

    Directory of Open Access Journals (Sweden)

    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. 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.

  4. 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.

  5. 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.

  6. 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.

  7. Repeated 0.5 Gy gamma-ray irradiation attenuates autoimmune disease in MRL-lpr/lpr mice with up-regulation of regulatory T cells

    International Nuclear Information System (INIS)

    cells and productions of IL-6 and autoantibodies, and up-regulates regulatory T cells. These results indicate that up-regulation of regulatory T cells would involve in these therapeutic effects induced by irradiation. The up-regulation of regulatory T cells induced by irradiation could be a novel and important observation in low-dose irradiation-mediated therapeutic effects.

  8. Quantification of uncoupling protein 2 reveals its main expression in immune cells and selective up-regulation during T-cell proliferation.

    Directory of Open Access Journals (Sweden)

    Anne Rupprecht

    Full Text Available Uncoupling protein 2 (UCP2 is an inner mitochondrial membrane protein. Although the protein was discovered in 1997, its function and even its tissue distribution are still under debate. Here we present a quantitative analysis of mRNA and protein expression in various mice tissues, revealing that UCP2 is mainly expressed in organs and cells associated with the immune system. Although the UCP2 gene is present in the brain, as demonstrated using quantitative RT-PCR, the protein was not detectable in neurons under physiological conditions. Instead, we could detect UCP2 in microglia, which act in the immune defense of the central nervous system. In lymphocytes, activation led to a ten-fold increase of UCP2 protein expression simultaneously to the increase in levels of other mitochondrial proteins, whereas lymphocyte re-stimulation resulted in the selective increase of UCP2. The highest detected level of UCP2 expression in stimulated T-cells (0.54 ng/(µg total cellular protein was approximately 200 times lower than the level of UCP1 in brown adipose tissue from room temperature acclimated mice. Both the UCP2 expression pattern and the time course of up-regulation in stimulated T-cells imply UCP2's involvement in the immune response, probably by controlling the metabolism during cell proliferation.

  9. Upregulation of S100 calcium-binding protein A9 is required for induction of smooth muscle cell proliferation by a periodontal pathogen.

    Science.gov (United States)

    Inaba, Hiroaki; Hokamura, Kazuya; Nakano, Kazuhiko; Nomura, Ryota; Katayama, Kazufumi; Nakajima, Atsushi; Yoshioka, Hideo; Taniguchi, Kazuhiro; Kamisaki, Yoshinori; Ooshima, Takashi; Umemura, Kazuo; Murad, Ferid; Wada, Koichiro; Amano, Atsuo

    2009-01-01

    We investigated the effect of a periodontal pathogen, Porphyromonas gingivalis, on human aortic smooth muscle cell (hAOSMC) proliferation as mechanisms of atherosclerosis. Cultured hAOSMCs exposed to the supernatant of plasma incubated with P. gingivalis showed a marked transformation from a contractile to proliferative phenotype, resulting in enhancement of cell growth. DNA microarray analysis revealed a P. gingivalis-dependent upregulation of S100A9 in hAOSMCs. Small interference-RNA for S100A9 dramatically attenuated the effect of P. gingivalis on transformation and proliferation of hAOSMCs. Our data suggested that upregulation of S100A9 mediated by P. gingivalis is an important event in the development of aortic intimal hyperplasia.

  10. 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.

  11. Lactobacillus casei Exerts Anti-Proliferative Effects Accompanied by Apoptotic Cell Death and Up-Regulation of TRAIL in Colon Carcinoma Cells.

    Directory of Open Access Journals (Sweden)

    Angeliki Tiptiri-Kourpeti

    Full Text Available Probiotic microorganisms such as lactic acid bacteria (LAB exert a number of strain-specific health-promoting activities attributed to their immunomodulatory, anti-inflammatory and anti-carcinogenic properties. Despite recent attention, our understanding of the biological processes involved in the beneficial effects of LAB strains is still limited. To this end, the present study investigated the growth-inhibitory effects of Lactobacillus casei ATCC 393 against experimental colon cancer. Administration of live Lactobacillus casei (as well as bacterial components thereof on murine (CT26 and human (HT29 colon carcinoma cell lines raised a significant concentration- and time-dependent anti-proliferative effect, determined by cell viability assays. Specifically, a dramatic decrease in viability of colon cancer cells co-incubated with 10(9 CFU/mL L. casei for 24 hours was detected (78% for HT29 and 52% for CT26 cells. In addition, live L. casei induced apoptotic cell death in both cell lines as revealed by annexin V and propidium iodide staining. The significance of the in vitro anti-proliferative effects was further confirmed in an experimental tumor model. Oral daily administration of 10(9 CFU live L. casei for 13 days significantly inhibited in vivo growth of colon carcinoma cells, resulting in approximately 80% reduction in tumor volume of treated mice. Tumor growth inhibition was accompanied by L. casei-driven up-regulation of the TNF-related apoptosis-inducing ligand TRAIL and down-regulation of Survivin. Taken together, these findings provide evidence for beneficial tumor-inhibitory, anti-proliferative and pro-apoptotic effects driven by this probiotic LAB strain.

  12. Lactobacillus casei Exerts Anti-Proliferative Effects Accompanied by Apoptotic Cell Death and Up-Regulation of TRAIL in Colon Carcinoma Cells.

    Science.gov (United States)

    Tiptiri-Kourpeti, Angeliki; Spyridopoulou, Katerina; Santarmaki, Valentina; Aindelis, Georgios; Tompoulidou, Evgenia; Lamprianidou, Eleftheria E; Saxami, Georgia; Ypsilantis, Petros; Lampri, Evangeli S; Simopoulos, Constantinos; Kotsianidis, Ioannis; Galanis, Alex; Kourkoutas, Yiannis; Dimitrellou, Dimitra; Chlichlia, Katerina

    2016-01-01

    Probiotic microorganisms such as lactic acid bacteria (LAB) exert a number of strain-specific health-promoting activities attributed to their immunomodulatory, anti-inflammatory and anti-carcinogenic properties. Despite recent attention, our understanding of the biological processes involved in the beneficial effects of LAB strains is still limited. To this end, the present study investigated the growth-inhibitory effects of Lactobacillus casei ATCC 393 against experimental colon cancer. Administration of live Lactobacillus casei (as well as bacterial components thereof) on murine (CT26) and human (HT29) colon carcinoma cell lines raised a significant concentration- and time-dependent anti-proliferative effect, determined by cell viability assays. Specifically, a dramatic decrease in viability of colon cancer cells co-incubated with 10(9) CFU/mL L. casei for 24 hours was detected (78% for HT29 and 52% for CT26 cells). In addition, live L. casei induced apoptotic cell death in both cell lines as revealed by annexin V and propidium iodide staining. The significance of the in vitro anti-proliferative effects was further confirmed in an experimental tumor model. Oral daily administration of 10(9) CFU live L. casei for 13 days significantly inhibited in vivo growth of colon carcinoma cells, resulting in approximately 80% reduction in tumor volume of treated mice. Tumor growth inhibition was accompanied by L. casei-driven up-regulation of the TNF-related apoptosis-inducing ligand TRAIL and down-regulation of Survivin. Taken together, these findings provide evidence for beneficial tumor-inhibitory, anti-proliferative and pro-apoptotic effects driven by this probiotic LAB strain. PMID:26849051

  13. Up-regulated microRNA-143 in cancer stem cells differentiation promotes prostate cancer cells metastasis by modulating FNDC3B expression

    International Nuclear Information System (INIS)

    Metastatic prostate cancer is a leading cause of cancer-related death in men. Cancer stem cells (CSCs) are involved in tumor progression and metastasis, including in prostate cancer. There is an obvious and urgent need for effective cancer stem cells specific therapies in metastatic prostate cancer. MicroRNAs (miRNAs) are an important class of pervasive genes that are involved in a variety of biological functions, especially in cancer. The goal of this study was to identify miRNAs involved in prostate cancer metastasis and cancer stem cells. A microarray and qRT-PCR were performed to investigate the miRNA expression profiles in PC-3 sphere cells and adherent cells. A transwell assay was used to evaluate the migration of PC-3 sphere cells and adherent cells. MiR-143 was silenced with antisense oligonucleotides in PC-3, PC-3-M and LNCaP cells. The role of miR-143 in prostate cancer metastasis was measured by wound-healing and transwell assays in vitro and bioluminescence imaging in vivo. Bioinformatics and luciferase report assays were used to identify the target of miR-143. The expression of miR-143 and the migration capability were reduced in PC-3 sphere cells and progressively increased during sphere re-adherent culture. Moreover, the down-regulation of miR-143 suppressed prostate cancer cells migration and invasion in vitro and systemically inhibited metastasis in vivo. Fibronectin type III domain containing 3B (FNDC3B), which regulates cell motility, was identified as a target of miR-143. The inhibition of miR-143 increased the expression of FNDC3B protein but not FNDC3B mRNA in vitro and vivo. These data demonstrate for the first time that miR-143 was up-regulated during the differentiation of prostate cancer stem cells and promoted prostate cancer metastasis by repressing FNDC3B expression. This sheds a new insight into the post-transcriptional regulation of cancer stem cells differentiation by miRNAs, a potential approach for the treatment of prostate cancer

  14. Up-regulation of Heme Oxygenase-1 by Korean Red Ginseng Water Extract as a Cytoprotective Effect in Human Endothelial Cells

    OpenAIRE

    Yang, Hana; Lee, Seung Eun; Jeong, Seong Il; Park, Cheung-Seog; Jin, Young-Ho; Park, Yong Seek

    2011-01-01

    Korean red ginseng (KRG) is used worldwide as a popular traditional herbal medicine. KRG has shown beneficial effects on cardiovascular diseases, such as atherosclerosis, diabetes, and hypertension. Up-regulation of a cytoprotective protein, heme oxygenase (HO)-1, is considered to augment the cellular defense against various agents that may induce cytotoxic injury. In the present study, we demonstrate that KRG water extract induces HO-1 expression in human umbilical vein endothelial cells (HU...

  15. 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.

  16. 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.

    Science.gov (United States)

    Li, Wei; Fu, Jianfang; Zhang, Shun; Zhao, Jie; Xie, Nianlin; Cai, Guoqing

    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. PMID:25886977

  17. 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.

  18. 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.

  19. 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.

    Science.gov (United States)

    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.

  20. hsa-miR-125a-5p Enhances Invasion in Non-small Cell Lung Carcinoma Cell Lines by Upregulating Rock-1

    Directory of Open Access Journals (Sweden)

    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. PPAR-γ activation increases insulin secretion through the up-regulation of the free fatty acid receptor GPR40 in pancreatic β-cells.

    Directory of Open Access Journals (Sweden)

    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.

  2. 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.

  3. 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.

  4. 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.

  5. Induction of cellular senescence by doxorubicin is associated with upregulated miR-375 and induction of autophagy in K562 cells.

    Directory of Open Access Journals (Sweden)

    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

  6. 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 (Pmitosis frequency increased to 12.1 + or - 2.6% (Pmitosis frequency was 14.4 + or - 5.0% and 19.0 + or - 3...

  7. 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.

  8. Higher Matrix Stiffness Upregulates Osteopontin Expression in Hepatocellular Carcinoma Cells Mediated by Integrin β1/GSK3β/β-Catenin Signaling Pathway.

    Directory of Open Access Journals (Sweden)

    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.

  9. 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.

  10. Upregulation of Id3 inhibits cell proliferation and induces apoptosis in A549/DDP human lung cancer cells in vitro.

    Science.gov (United States)

    Chen, Fangfang; Zhao, Qinfei; Wang, Shuxia; Wang, Haiyong; Li, Xiaojun

    2016-07-01

    Inhibitor of DNA binding (Id)3 is a member of the Id multigene family of dominant‑negative helix‑loop-helix transcription factors, which function as oncogenes or tumor suppressors in human cancers. Its upregulation was recently shown to have inhibitory effects on lung cancer, which is the leading cause of cancer‑associated mortality worldwide. As drug resistance represents a major bottleneck of cancer therapy, the present study assessed the ability of Id3 to inhibit cisplatin‑resistant A549 lung adenocarcinoma cells (A549/DDP). A549/DPP cells were transiently transfected with enhanced green fluorescence protein overexpression plasmid (pEGFP) or Id3 overexpression plasmid (Id3/pEGFP), which was confirmed by confocal fluorescence microscopy, PCR and western blot analysis. The effects of Id3 on the viability and apoptosis of A549/DDP were determined using an MTT assay, fluorescence microscopy with Hoechst 33258 staining and flow cytometry following Annexin V/propidium iodide double staining. The results revealed that overexpression of Id3 significantly inhibited the proliferation and viability of A549/DDP cells in a time‑dependent manner. Furthermore, overexpression of Id3 significantly increased the apoptotic rate of A549/DDP cells from 2.73 to 16.92%, confirming the implication of Id3 in the negative control of tumour growth. The results of the present study revealed that overexpression of Id3 may serve as a novel strategy for inhibiting cisplatin‑sensitive lung cancer. Further experiments will be performed to determine whether Id3 overexpression could enhance the sensitivity of lung cancer cells to DDP. PMID:27176047

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

    Energy Technology Data Exchange (ETDEWEB)

    Bagge, Annika [Department of Science, Systems and Models, Roskilde University, Roskilde (Denmark); Clausen, Trine R. [Diabetes Biology, Novo Nordisk, Maaloev (Denmark); Larsen, Sylvester [Department of Science, Systems and Models, Roskilde University, Roskilde (Denmark); Ladefoged, Mette [Diabetes Biology, Novo Nordisk, Maaloev (Denmark); Rosenstierne, Maiken W. [Department of Science, Systems and Models, Roskilde University, Roskilde (Denmark); Department of Virology, Statens Serum Institut (Denmark); Larsen, Louise [Department of Biomedical Sciences, University of Copenhagen, Copenhagen (Denmark); Vang, Ole [Department of Science, Systems and Models, Roskilde University, Roskilde (Denmark); Nielsen, Jens H. [Department of Biomedical Sciences, University of Copenhagen, Copenhagen (Denmark); Dalgaard, Louise T., E-mail: ltd@ruc.dk [Department of Science, Systems and Models, Roskilde University, Roskilde (Denmark)

    2012-09-21

    Highlights: Black-Right-Pointing-Pointer MicroRNA-29a (miR-29a) levels are increased by glucose in human and rat islets and INS-1E cells. Black-Right-Pointing-Pointer miR-29a increases proliferation of INS-1E beta-cells. Black-Right-Pointing-Pointer Forced expression of miR-29a decreases glucose-stimulated insulin secretion (GSIS). Black-Right-Pointing-Pointer Depletion of beta-cell miR-29a improves GSIS. Black-Right-Pointing-Pointer miR-29a may be a mediator of glucose toxicity in beta-cells. -- Abstract: Chronically elevated levels of glucose impair pancreatic beta-cell function while inducing beta-cell proliferation. MicroRNA-29a (miR-29a) levels are increased in several tissues in diabetic animals and mediate decreased insulin-stimulated glucose-transport of adipocytes. The aim was to investigate the impact of glucose on miR-29a levels in INS-1E beta-cells and in human islets of Langerhans and furthermore to evaluate the impact of miR-29a on beta-cell function and proliferation. Increased glucose levels up-regulated miR-29a in beta-cells and human and rat islets of Langerhans. Glucose-stimulated insulin-secretion (GSIS) of INS-1E beta-cells was decreased by forced expression of miR-29a, while depletion of endogenous miR-29a improved GSIS. Over-expression of miR-29a increased INS-1E proliferation. Thus, miR-29a up-regulation is involved in glucose-induced proliferation of beta-cells. Furthermore, as depletion of miR-29a improves beta-cell function, miR-29a is a mediator of glucose-induced beta-cell dysfunction. Glucose-induced up-regulation of miR-29a in beta-cells could be implicated in progression from impaired glucose tolerance to type 2 diabetes.

  12. Pistacia chinensis inhibits NO production and upregulates HO-1 induction via PI-3K/Akt pathway in LPS stimulated macrophage cells.

    Science.gov (United States)

    Yayeh, Taddesse; Hong, Mei; Jia, Qi; Lee, Young-Chul; Kim, Hyun-Jin; Hyun, Eujin; Kim, Tae-Wan; Rhee, Man Hee

    2012-01-01

    Pistacia chinensis has been used for various purposes in China including as an understock for grafting Pistacia vera. However, little attention was given to its health promoting effects. Therefore, in this study, we investigated the effect of Pistacia chinensis methanolic extract (PCME) containing resorcinol class of phenolic lipids on pro-inflammatory mediators and heme oxygenase-1(HO-1) in lipopolysaccharide stimulated RAW264.7 cells. While PCME (2.5-10 μg/ml) inhibited mRNA expressions of inducible nitric oxide synthase (iNOS), cyclooxygenase 2 (COX-2), tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and interleukin (IL)-6, it up-regulated HO-1 expression. Likewise, PCME inhibited iNOS protein expression, but not COX-2, and reduced nitric oxide (NO) release. Moreover, Phosphorylated c-Jun N-terminal Kinase (JNK) was attenuated dose-dependently in PCME pre-treated RAW264.7 cells. In addition, PCME up-regulated HO-1 protein expression was diminished by pre-treatment of PI-3K inhibitor. Furthermore, nuclear factor erythroid 2 related factor 2 (Nrf2) repressor was attenuated time-dependently during PCME treatment. Taken together, our study showed (for the first time) that PCME inhibited NO production and up-regulated HO-1 induction via PI-3K/Akt pathway, suggesting the role of Pistacia chinensis as potential sources of anti-inflammatory and antioxidant natural compounds. PMID:22928837

  13. 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 cyc...

  14. Soluble MICA-NKG2D interaction upregulates IFN-γ production by activated CD3-CD56+ NK cells: potential impact on chronic graft versus host disease.

    Science.gov (United States)

    Boukouaci, Wahid; Al-Daccak, Reem; Dulphy, Nicolas; Lauden, Laura; Amokrane, Kahina; Fortier, Catherine; Marzais, François; Bennabi, Meriem; Peffault de Latour, Regis; Socie, Gerard; Toubert, Antoine; Charron, Dominique; Krishnamoorthy, Rajagopal; Tamouza, Ryad

    2013-12-01

    A soluble isoform of MHC class I chain-related molecule A (soluble MICA), generated by proteolytic shedding from the membrane-bound MICA of various tumor cells, has been shown to downregulate both the expression of natural killer group 2-member D receptor and the cytotoxic function of effectors cells and was postulated as a mechanism for tumor immune evasion. Its effect on the expression of cytokines by the effector cells remained unexplored. Here we demonstrate that the sMICA molecules upregulate interferon gamma expression by interleukin-12/interleukin-18-activated CD3(-)CD56(+) natural killer cells, witnessing the pro-inflammatory effect of soluble MICA. Overall, these data are in line with our previous observations that the raised serum levels of soluble MICA, following allogeneic hematopoietic stem cell transplantation, confer susceptibility to and the presence of pre-transplantation anti-MICA antibodies in the patient's serum confer protection against chronic graft versus host disease.

  15. 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.

  16. Up-regulation of cyclooxygenase-2-derived prostaglandin E2 in colon cancer cells resistant to 5-fluorouracil

    OpenAIRE

    Choi, Cheol Hee; Lee, Tae Bum; Lee, Yeon Ah; Choi, Suk; Kim, Kyung Jong

    2011-01-01

    Purpose It has been suggested that constitutive up-regulation of cyclooxygenase (COX)-2 is associated with resistance to apoptosis, increased angiogenesis, and increased tumor invasiveness in various cancers including colon cancer. There are many factors involved in the resistance to 5-fluorouracil (5-FU) in colon cancer. However, little is known about the role of COX-2 in acquired resistance to 5-FU in colon cancer. Methods Hence we investigated whether COX-2 contribute to acquired resistanc...

  17. Girdin/GIV is upregulated by cyclic tension, propagates mechanical signal transduction, and is required for the cellular proliferation and migration of MG-63 cells

    International Nuclear Information System (INIS)

    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

  18. 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.

  19. Up-regulation of SOX9 in sertoli cells from testiculopathic patients accounts for increasing anti-mullerian hormone expression via impaired androgen receptor signaling.

    Directory of Open Access Journals (Sweden)

    Kuo-Chung Lan

    Full Text Available BACKGROUND: Testosterone provokes Sertoli cell maturation and represses AMH production. In adult patients with Sertoli-cells-only syndrome (SCOS and androgen insensitivity syndrome (AIS, high level of AMH expression is detected in Sertoli cells due to defect of androgen/AR signaling. OBJECTIVE: We postulated that up-regulation of SOX9 due to impairment of androgen/AR signaling in Sertoli cells might explain why high level of anti-Mullerian hormone (AMH expression occur in these testiculopathic patients. METHODS: Biological research of testicular specimens from men with azoospermia or mouse. The serum hormone levels were studied in 23 men with obstructive azoospermia, 33 men with SCOS azoospermia and 21 volunteers with normal seminograms during a period of 4 years. Immunohistochemical staining and reverse-transcription PCR were used to examine the relationships among AR, SOX9 and AMH expression in adult human and mouse testes. The ability of AR to repress the expression of SOX9 and AMH was evaluated in vitro in TM4 Sertoli cells and C3H10T1/2 cells. RESULTS: SCOS specimens showed up-regulation of SOX9 and AMH proteins but down-regulation of AR proteins in Sertoli cells. The mRNA levels of AR were significantly lower and the SOX9, AMH mRNA levels higher in all SCOS patients compared to controls (P< 0.05. The testosterone levels in the SCOS patients were within the normal range, but most were below the median of the controls. Furthermore, our in vitro cell line experiments demonstrated that androgen/AR signaling suppressed the gene and protein levels of AMH via repression of SOX9. CONCLUSIONS: Our data show that the functional androgen/AR signaling to repress SOX9 and AMH expression is essential for Sertoli cell maturation. Impairment of androgen/AR signaling promotes SOX9-mediated AMH production, accounts for impairments of Sertoli cells in SCOS azoospermic patients.

  20. 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.

  1. Pancreatic adenocarcinoma upregulated factor (PAUF) confers resistance to pancreatic cancer cells against oncolytic parvovirus H-1 infection through IFNA receptor-mediated signaling

    International Nuclear Information System (INIS)

    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

  2. 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

  3. Upregulation of a novel eukaryotic translation initiation factor 5A (eIF5A in dengue 2 virus-infected mosquito cells

    Directory of Open Access Journals (Sweden)

    Shih Yu-Tzu

    2010-09-01

    Full Text Available Abstract Background Dengue virus, a mosquito-borne flavivirus, is the etiological agent of dengue fever, dengue hemorrhagic fever, and dengue shock syndrome. It generally induces apoptosis in mammalian cells, but frequently results in persistent infection in mosquito cells. That mechanism remains to be explored. In turn, a genomic survey through subtractive hybridization (PCR-select cDNA subtraction was conducted in order to find gene(s that may play a role in interactions between the virus and its host cells. Results Through this technique, we identified a novel eukaryotic translation initiation factor 5A (eIF5A which is upregulated in Aedes albopictus-derived C6/36 cells infected by the type 2 dengue (Den-2 virus. The full-length of the identified eIF5A gene consisted of 1498 bp of nucleotides with a 41.39% G+C content, and it possessed a higher similarity and shorter evolutionary distance with insects than with other organisms. Upregulation of eIF5A in response to Den-2 virus infection was validated at both the RNA and protein levels. This phenomenon was also observed by confocal microscopy. In addition, cell death obviously occurred when eIF5A activity was inhibited in C6/36 cells even when they were infected by the virus. However, viral multiplication was not obviously affected in infected C6/36 cells when eIF5A activity was reduced. Conclusions Taken together, we postulated that eIF5A plays a role in preventing mosquito cells from death in response to Den-2 viral infection, thus facilitating continued viral growth and potential persistent infection in mosquito cells. It would be worthwhile to further investigate how its downstream factors or cofactors contribute to this phenomenon of dengue infection.

  4. Osthole suppresses the proliferation and accelerates the apoptosis of human glioma cells via the upregulation of microRNA-16 and downregulation of MMP-9.

    Science.gov (United States)

    Lin, Kai; Gao, Zhiyu; Shang, Bin; Sui, Shaohua; Fu, Qiang

    2015-09-01

    Osthole (7-methoxy-8-isoamyl alkenyl coumarin) has been reported to exhibit marked anticancer effects on several types of cancer. The expression levels of matrix metalloproteinase-9 (MMP-9) are closely associated with the pathogenesis of glioma. Furthermore, it is reported that the upregulation of microRNA‑16 (miR‑16) by the MMP‑9 signaling pathway can restrain the proliferation of cancer cells. To examine whether osthole increases the anticancer effect on human glioma cells in the present study, the common glioma cell line, U87, was treated with osthole at concentrations of 0, 50, 100 and 200 µΜ. The effects of osthole on cell viability were determined using a 3‑(4,5‑dimethylthiazol‑2‑thiazolyl)‑2,5‑diphenyl‑tetrazolium bromide assay. The rate of cellular apoptosis was analyzed by measuring the activity of caspase‑3 and using flow cytometry. The expression of MMP‑9 was determined using gelatin zymography assays and the expression of miR‑16 was determined using reverse transcription‑quantitative polymerase chain reaction. The results demonstrated that osthole significantly suppressed the proliferation and accelerated the apoptosis of the U87 cells. Furthermore, increased expression levels of miR‑16 and reduced protein expression levels of MMP‑9 were found in the U87 cells. In addition, miR‑16 was found to regulate the expression of MMP‑9 in the U87 cells through transfection of miR‑16 precursor and anti‑miR‑16 into the U87 cells. In conclusion, these observations indicated that osthole suppressed the proliferation and accelerated the apoptosis of human glioma cells through upregulation of the expression of miR‑16 and downregulation of the expression of MMP-9. PMID:26082082

  5. The negative regulators Foxj1 and Foxo3a are up-regulated by a peptide that inhibits systemic lupus erythematosus-associated T cell responses.

    Science.gov (United States)

    Sela, Uri; Dayan, Molly; Hershkoviz, Rami; Cahalon, Liora; Lider, Ofer; Mozes, Edna

    2006-11-01

    A peptide (hCDR1) based on the complementarity determining region-1 of an anti-DNA antibody ameliorates systemic lupus erythematosus (SLE) in induced and spontaneous lupus models. Our objectives were to determine the effects of hCDR1 on TCR signaling and on its negative regulators, Foxj1 and Foxo3a. BALB/c mice were immunized with the SLE-inducing anti-DNA antibody, designated 16/6Id, and treated with hCDR1. hCDR1 treatment specifically inhibited IFN-gamma secretion by T cells in association with down-regulated T-bet expression and NF-kappaB activation; however, GATA-3 expression was not affected. Furthermore, TCR signaling (ZAP-70 phosphorylation) was inhibited, and the mRNA expression of the two modulators of Th1 activation, Foxj1 and Foxo3a, was significantly up-regulated. The latter were also elevated in SLE-afflicted (NZBxNZW)F1 mice that were treated with hCDR1. Addition of TGF-beta, which was elevated following treatment with hCDR1, to T cells from 16/6Id immunized mice, up-regulated Foxj1 and Foxo3a mRNA expression, similarly to hCDR1. In contrast, anti-TGF-beta antibodies added to hCDR1-treated T cells abrogated its effect. Thus, hCDR1 elevates TGF-beta, which contributes to the up-regulation of T cell Foxj1 and Foxo3a expression, leading to inhibition of NF-kappaB activation and IFN-gamma secretion, which is required for the maintenance of SLE. PMID:17051618

  6. 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.

  7. 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

  8. 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.

  9. 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)

    Cho, Yeon Sook; Kim, Byung Soo; Sim, Chan Kyu; Kim, Inki; Lee, Myeong Sup

    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

  10. Melatonin-mediated Bim up-regulation and cyclooxygenase-2 (COX-2) down-regulation enhances tunicamycin-induced apoptosis in MDA-MB-231 cells.

    Science.gov (United States)

    Woo, Seon Min; Min, Kyoung-jin; Kwon, Taeg Kyu

    2015-04-01

    Melatonin is involved in many physiological functions, and it has differential effects on apoptosis in normal and cancer cells. However, the mechanism of its antitumor roles is not well understood. In this study, we show that melatonin enhances tunicamycin-induced apoptosis in human breast carcinoma MDA-MB-231 cells. Melatonin up-regulates pro-apoptotic protein Bim expression at the transcriptional levels in the presence of tunicamycin. Melatonin inhibits tunicamycin-induced COX-2 expression in MDA-MB-231 cells. Furthermore, inhibition of COX-2 activity using the COX-2 inhibitor, NS398, increases tunicamycin-induced apoptosis. Interestingly, these effects were not associated with melatonin receptor signal pathways. Pertussis toxin (a general Gi protein inhibitor) or luzindole (a nonspecific melatonin receptor antagonist) did not reverse the effect of melatonin. In addition, melatonin blocked tunicamycin-induced NF-κB transcriptional activity, p65 nuclear translocation, and p38 MAPK activation. Melatonin-mediated p38 MAPK inhibition contributed to decreased COX-2 mRNA stability. Taken together, our results suggest that melatonin enhances antitumor function through up-regulation of Bim expression and down-regulation of COX-2 expression in tunicamycin-treated MDA-MB-231 cells. PMID:25711465

  11. Substance P primes lipoteichoic acid- and Pam3CysSerLys4-mediated activation of human mast cells by up-regulating Toll-like receptor 2.

    Science.gov (United States)

    Tancowny, Brian P; Karpov, Victor; Schleimer, Robert P; Kulka, Marianna

    2010-10-01

    Substance P (SP) is a neuropeptide with neuroimmunoregulatory activity that may play a role in susceptibility to infection. Human mast cells, which are important in innate immune responses, were analysed for their responses to pathogen-associated molecules via Toll-like receptors (TLRs) in the presence of SP. Human cultured mast cells (LAD2) were activated by SP and TLR ligands including lipopolysaccharide (LPS), Pam3CysSerLys4 (Pam3CSK4) and lipoteichoic acid (LTA), and mast cell leukotriene and chemokine production was assessed by enzyme-linked immunosorbent assay (ELISA) and gene expression by quantitative PCR (qPCR). Mast cell degranulation was determined using a β-hexosaminidase (β-hex) assay. SP treatment of LAD2 up-regulated mRNA for TLR2, TLR4, TLR8 and TLR9 while anti-immunoglobulin E (IgE) stimulation up-regulated expression of TLR4 only. Flow cytometry and western blot confirmed up-regulation of TLR2 and TLR8. Pretreatment of LAD2 with SP followed by stimulation with Pam3CSK4 or LTA increased production of leukotriene C4 (LTC(4) ) and interleukin (IL)-8 compared with treatment with Pam3CSK4 or LTA alone (>2-fold; P<0·01). SP alone activated 5-lipoxygenase (5-LO) nuclear translocation but also augmented Pam3CSK4 and LTA-mediated 5-LO translocation. Pam3CSK4, LPS and LTA did not induce LAD2 degranulation. SP primed LTA and Pam3CSK4-mediated activation of JNK, p38 and extracellular-signal-regulated kinase (ERK) and activated the nuclear translocation of c-Jun, nuclear factor (NF)-κB, activating transcription factor 2 (ATF-2) and cyclic-AMP-responsive element binding protein (CREB) transcription factors. Pretreatment with SP followed by LTA stimulation synergistically induced production of chemokine (C-X-C motif) ligand 8 (CXCL8)/IL-8, chemokine (C-C motif) ligand 2 (CCL2)/monocyte chemotactic protein 1 (MCP-1), tumour necrosis factor (TNF) and IL-6 protein. SP primes TLR2-mediated activation of human mast cells by up-regulating TLR expression and

  12. 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.

  13. 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. PMID:25727013

  14. Elevated STAT3 Signaling-Mediated Upregulation of MMP-2/9 Confers Enhanced Invasion Ability in Multidrug-Resistant Breast Cancer Cells

    Directory of Open Access Journals (Sweden)

    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.

  15. C/EBPα creates elite cells for iPSC reprogramming by upregulating Klf4 and increasing the levels of Lsd1 and Brd4.

    Science.gov (United States)

    Di Stefano, Bruno; Collombet, Samuel; Jakobsen, Janus Schou; Wierer, Michael; Sardina, Jose Luis; Lackner, Andreas; Stadhouders, Ralph; Segura-Morales, Carolina; Francesconi, Mirko; Limone, Francesco; Mann, Matthias; Porse, Bo; Thieffry, Denis; Graf, Thomas

    2016-04-01

    Reprogramming somatic cells into induced pluripotent stem cells (iPSCs) is typically inefficient and has been explained by elite-cell and stochastic models. We recently reported that B cells exposed to a pulse of C/EBPα (Bα' cells) behave as elite cells, in that they can be rapidly and efficiently reprogrammed into iPSCs by the Yamanaka factors OSKM. Here we show that C/EBPα post-transcriptionally increases the abundance of several hundred proteins, including Lsd1, Hdac1, Brd4, Med1 and Cdk9, components of chromatin-modifying complexes present at super-enhancers. Lsd1 was found to be required for B cell gene silencing and Brd4 for the activation of the pluripotency program. C/EBPα also promotes chromatin accessibility in pluripotent cells and upregulates Klf4 by binding to two haematopoietic enhancers. Bα' cells share many properties with granulocyte/macrophage progenitors, naturally occurring elite cells that are obligate targets for leukaemic transformation, whose formation strictly requires C/EBPα. PMID:26974661

  16. 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. PMID:26472159

  17. 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.

  18. Expression of ABCA3, a causative gene for fatal surfactant deficiency, is up-regulated by glucocorticoids in lung alveolar type II cells

    International Nuclear Information System (INIS)

    We have shown previously that the ATP-binding cassette transporter ABCA3 is expressed predominantly at the limiting membrane of the lamellar bodies in lung alveolar type II cells. Very recently, an ABCA3 gene mutation was reported in human newborns with fatal surfactant deficiency. In the present study, we have shown in rat lung that expression of the ABCA3 protein is dramatically increased after embryonic day (E) 20.5 just before birth. Expression was also markedly induced even at E18.5 when dexamethasone (Dex), which is known to accelerate surfactant formation, was administered to pregnant female rats for 3 days from E15.5. Since Dex increased the ABCA3 mRNA expression level in human alveolar type II cell line A549 cells 4-fold, we cloned and characterized the promoter region of the human ABCA3 gene. Promoter activity of the 5'-flanking region of the ABCA3 gene, which contains a potential glucocorticoid-responsive element (GRE), was up-regulated about 2-fold. Up-regulation by Dex was not observed when the GRE-containing region was deleted or when a point mutation was introduced into the GRE, and electrophoretic mobility shift assay using Dex-treated A549 nuclear extracts demonstrated specific binding of the glucocorticoid receptor to the GRE. These findings demonstrate that glucocorticoid-induced up-regulation of ABCA3 expression in vivo is mediated by transcriptional activation through the GRE in the promoter, and suggest that ABCA3 plays an important role in the formation of pulmonary surfactant, probably by transporting lipids such as cholesterol

  19. 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

  20. HER2 induces cell proliferation and invasion of non-small-cell lung cancer by upregulating COX-2 expression via MEK/ERK signaling pathway

    Directory of Open Access Journals (Sweden)

    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

  1. Long non-coding RNA TUG1 is up-regulated in hepatocellular carcinoma and promotes cell growth and apoptosis by epigenetically silencing of KLF2

    OpenAIRE

    Huang, Ming-De; Chen, Wen-ming; Qi, Fu-zhen; Sun, Ming; Xu, Tong-peng; Ma, Pei; Shu, Yong-qian

    2015-01-01

    Background Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related death worldwide, and the biology of this cancer remains poorly understood. Recent evidence indicates that long non-coding RNAs (lncRNAs) are found to be dysregulated in a variety of cancers, including HCC. Taurine Up-regulated Gene 1 (TUG1), a 7.1-kb lncRNA, recruiting and binding to polycomb repressive complex 2 (PRC2), is found to be disregulated in non-small cell lung carcinoma (NSCLC) and esophageal s...

  2. Cisplatin Restores TRAIL apoptotic pathway in Glioblastoma-Derived Stem Cells through Up-regulation of DR5 and Down-regulation of c-FLIP

    OpenAIRE

    Ding, Lijuan; Yuan, Changji; Wei, Feng; Wang, Guangyi; Zhang, Jing; Bellail, Anita C.; Zhang, Zhaobin; Olson, Jeffrey J.; Hao, Chunhai

    2011-01-01

    Glioblastoma-derived stem cells (GSCs) are responsible for the cancer resistance to therapies. We show here that GSC-enriched neurospheres are resistant to the treatment of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) due to the insufficient expression of the death receptor DR4 and DR5 and the overexpression of cellular Fas-associated death domain-like interleukin-1β-converting enzyme-inhibitory protein (c-FLIP). However, treatment with cisplatin leads to the upregulation o...

  3. Exposure to 9,10-phenanthrenequinone accelerates malignant progression of lung cancer cells through up-regulation of aldo-keto reductase 1B10

    International Nuclear Information System (INIS)

    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

  4. 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.

  5. Upregulation of miR-21 by Ghrelin Ameliorates Ischemia/Reperfusion-Induced Acute Kidney Injury by Inhibiting Inflammation and Cell Apoptosis.

    Science.gov (United States)

    Zhang, Wanzhe; Shu, Liliang

    2016-08-01

    Renal ischemia-reperfusion (I/R) injury can be caused by cardiac surgery, renal vascular obstruction, and kidney transplantation, mainly leading to acute kidney injury (AKI), which is complicated by lack of effective preventative and therapeutic strategies. Ghrelin has recently been reported to possess anti-inflammatory properties in several types of cells; however, little attention has been given to the role of ghrelin in I/R-induced AKI. The aim of this study is to explore the role of ghrelin in I/R-induced AKI. In this study, an I/R-induced rat AKI model and a hypoxia-induced NRK-52E cell I/R model were successfully constructed. Ghrelin expression was increased significantly in these rat and cell models. After enhancing ghrelin level by injecting exogenous ghrelin into rats or transfecting a ghrelin-pcDNA3.1 vector into renal tubular epithelial cells, we observed that I/R-induced AKI can be ameliorated by ghrelin, as shown by alterations in histology, as well as changes in serum creatinine (SCr) level, cell apoptosis, and the levels of inflammatory factors. Based on the importance of microRNA-21 (miR-21) in renal disease and the modulation effect of ghrelin on miR-21 in gastric epithelial cells, we tested whether miR-21 participates in the protective effect of ghrelin on I/R-induced AKI. Ghrelin could upregulate the PI3K/AKT signaling pathway by increasing the miR-21 level, which led to the protective effect of ghrelin on I/R-induced AKI by inhibiting the inflammatory response and renal tubular epithelial cell apoptosis. Our research identifies that ghrelin can ameliorate I/R-induced AKI by upregulating miR-21, which advances the understanding of mechanisms by which ghrelin ameliorates I/R-induced AKI. PMID:27152763

  6. Weight Loss Upregulates the Small GTPase DIRAS3 in Human White Adipose Progenitor Cells, Which Negatively Regulates Adipogenesis and Activates Autophagy via Akt-mTOR Inhibition.

    Science.gov (United States)

    Ejaz, Asim; Mitterberger, Maria C; Lu, Zhen; Mattesich, Monika; Zwierzina, Marit E; Hörl, Susanne; Kaiser, Andreas; Viertler, Hans-Peter; Rostek, Ursula; Meryk, Andreas; Khalid, Sana; Pierer, Gerhard; Bast, Robert C; Zwerschke, Werner

    2016-04-01

    Long-term weight-loss (WL) interventions reduce insulin serum levels, protect from obesity, and postpone age-associated diseases. The impact of long-term WL on adipose-derived stromal/progenitor cells (ASCs) is unknown. We identified DIRAS3 and IGF-1 as long-term WL target genes up-regulated in ASCs in subcutaneous white adipose tissue of formerly obese donors (WLDs). We show that DIRAS3 negatively regulates Akt, mTOR and ERK1/2 signaling in ASCs undergoing adipogenesis and acts as a negative regulator of this pathway and an activator of autophagy. Studying the IGF-1-DIRAS3 interaction in ASCs of WLDs, we demonstrate that IGF-1, although strongly up-regulated in these cells, hardly activates Akt, while ERK1/2 and S6K1 phosphorylation is activated by IGF-1. Overexpression of DIRAS3 in WLD ASCs completely inhibits Akt phosphorylation also in the presence of IGF-1. Phosphorylation of ERK1/2 and S6K1 is lesser reduced under these conditions. In conclusion, our key findings are that DIRAS3 down-regulates Akt-mTOR signaling in ASCs of WLDs. Moreover, DIRAS3 inhibits adipogenesis and activates autophagy in these cells. PMID:27211557

  7. Hepatitis C virus core upregulates the methylation status of the RASSF1A promoter through regulation of SMYD3 in hilar cholangiocarcinoma cells

    Institute of Scientific and Technical Information of China (English)

    Ning Guo; Rufu Chen; Zhihua Li; Yonggang Liu; Di Cheng; Quanbo Zhou; Jiajia Zhou; Qing Lin

    2011-01-01

    Increasing evidence has been accumulated indicating the important role of epigenetic regulation in tumor genesis.Previously, we observed that the transfection of hepatitis C virus core (HCVc) protein led to malignant transformation in normal biliary cells, and that tumor suppressor gene RASSFIA was downregulated in many hilar cholangiocarcinoma patients by hypermethylation in the promoter region. In the present study, we found SET and MYND domain-containing protein 3 (SMYD3), a novel histone methyltransferase, was overexpressed in cholangiocarcinoma patients especially in those with HCV infection. Transfection of HCVc into hilar cholangiocarcinoma cell lines QBC939 and FRH0201 could upregulate the expression of SMYD3 and promote cell growth, which was consistent with the results of our clinical research.This phenomenon indicated that SMYD3 was related to the epigenetic regulation of cholangiocarcinoma genesis with HCV infection. Overexpression of SMYD3 could inhibit RASSFIA expression, whereas inhibition of SMYD3 by siRNA improved its expression. Methylationspecific polymerase chain reaction (MS-PCR) results showed the methylation status of RASSFIA promoter was regulated by SMYD3. In conclusion, HCVc could upregulate the methylation status of the RASSFIA promoter through regulation of SMYD3, and histone methylation may affect the DNA methylation of downstream gene by an unknown mechanism.

  8. 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

  9. 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

    2013-01-01

    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. PMID:24025424

  10. 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.

  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. 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.

  13. ER stress upregulated PGE2/IFNγ-induced IL-6 expression and down-regulated iNOS expression in glial cells

    Science.gov (United States)

    Hosoi, Toru; Honda, Miya; Oba, Tatsuya; Ozawa, Koichiro

    2013-12-01

    The disruption of endoplasmic reticulum (ER) function can lead to neurodegenerative disorders, in which inflammation has also been implicated. We investigated the possible correlation between ER stress and immune function using glial cells. We demonstrated that ER stress synergistically enhanced prostaglandin (PG) E2 + interferon (IFN) γ-induced interleukin (IL)-6 production. This effect was mediated through cAMP. Immune-activated glial cells produced inducible nitric oxide synthase (iNOS). Interestingly, ER stress inhibited PGE2 + IFNγ-induced iNOS expression. Similar results were obtained when cells were treated with dbcAMP + IFNγ. Thus, cAMP has a dual effect on immune reactions; cAMP up-regulated IL-6 expression, but down-regulated iNOS expression under ER stress. Therefore, our results suggest a link between ER stress and immune reactions in neurodegenerative diseases.

  14. Suppressors of cytokine signaling 1 and 3 are up-regulated in brain resident cells in response to virus induced inflammation of the CNS via at least two distinctive pathways

    DEFF Research Database (Denmark)

    Steffensen, Maria Abildgaard; Fenger, Christina; Christensen, Jeanette Erbo;

    2014-01-01

    Suppressors of cytokine signaling (SOCS) proteins are intracellular proteins that inhibit cytokine signaling in a variety of cell types. A number of viral infections have been associated with SOCS up-regulation; however, not much is known about the mechanisms regulating SOCS expression during viral......-cytolytic and a cytolytic virus induce marked up-regulation of SOCS1 and-3 expression. Notably, the kinetics of the observed up-regulation follows that of activity within pro-inflammatory signalling pathways and, interestingly, type II IFN, which is also a key inducer of inflammatory mediators, seems to be essential...... underlie a virus induced up-regulation of SOCS in the CNS. We found that i.c. infection with either lymphocytic choriomeningitis virus (LCMV) or yellow fever virus (YF) results in gradual up-regulation of SOCS1/3 mRNA expression peaking at day 7 post infection (p.i.). In the LCMV model, SOCS m...

  15. Upregulation of miR-27a contributes to the malignant transformation of human bronchial epithelial cells induced by SV40 small T antigen.

    Science.gov (United States)

    Wang, Q; Li, D-C; Li, Z-F; Liu, C-X; Xiao, Y-M; Zhang, B; Li, X-D; Zhao, J; Chen, L-P; Xing, X-M; Tang, S-F; Lin, Y-C; Lai, Y-D; Yang, P; Zeng, J-L; Xiao, Q; Zeng, X-W; Lin, Z-N; Zhuang, Z-X; Zhuang, S-M; Chen, W

    2011-09-01

    The introduction of the Simian virus 40 (SV40) early region, the telomerase catalytic subunit (hTERT) and an oncogenic allele of H-Ras directly transforms primary human cells. SV40 small T antigen (ST), which forms a complex with protein phosphatase 2A (PP2A) and inhibits PP2A activity, is believed to have a critical role in the malignant transformation of human cells. Recent evidence has shown that aberrant microRNA (miRNA) expression patterns are correlated with cancer development. Here, we identified miR-27a as a differentially expressed miRNA in SV40 ST-expressing cells. miR-27a is upregulated in SV40 ST-transformed human bronchial epithelial cells (HBERST). Suppression of miR-27a expression in HBERST cells or lung cancer cell lines (NCI-H226 and SK-MES-1) that exhibited high levels of miR-27a expression lead to cell growth arrested in the G(0)-G(1) phase. In addition, suppression of miR-27a in HBERST cells attenuated the capacity of such cells to grow in an anchorage-independent manner. We also found that suppression of the PP2A B56γ expression resulted in upregulation of miR-27a similar to that achieved by the introduction of ST, indicating that dysregulation of miR-27a expression in ST-expressing cells was mediated by the ST-PP2A interaction. Moreover, we discovered that Fbxw7 gene encoding F-box/WD repeat-containing protein 7 was a potential miR-27a target validated by dual-luciferase reporter system analysis. The inverse correlation between miR-27a expression levels and Fbxw7 protein expression was further confirmed in both cell models and human tumor samples. Fbxw7 regulates cell-cycle progression through the ubiquitin-dependent proteolysis of a set of substrates, including c-Myc, c-Jun, cyclin E1 and Notch 1. Thus, promotion of cell growth arising from the suppression of Fbxw7 by miR-27a overexpression might be responsible for the viral oncoprotein ST-induced malignant transformation. These observations demonstrate that miR-27a functions as an oncogene

  16. 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.

  17. 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.

  18. 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.

  19. 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

  20. Thrombomodulin reduces tumorigenic and metastatic potential of lung cancer cells by up-regulation of E-cadherin and down-regulation of N-cadherin expression.

    Science.gov (United States)

    Zheng, Nana; Huo, Zihe; Zhang, Bin; Meng, Mei; Cao, Zhifei; Wang, Zhiwei; Zhou, Quansheng

    2016-08-01

    Thrombomodulin (TM) is an endothelial cell membrane protein and plays critical roles in anti-thrombosis, anti-inflammation, vascular endothelial protection, and is traditionally regarded as a "vascular protection god". In recent years, although TM has been reported to be down-regulated in a variety of malignant tumors including lung cancer, the role and mechanism of TM in lung cancer are enigmatic. In this study, we found that induction of TM overexpression by cholesterol-reducing drug atorvastatin significantly diminished the tumorigenic capability of the lung cancer cells. Moreover, we demonstrated that TM overexpression caused G0/G1 phase arrest and markedly reduced the colony forming capability of the cells. Furthermore, overexpression of TM inhibited cell migration and invasion. Consistently, depletion of TM promoted cell growth, reduced the cell population at the G0/G1 phase, and enhanced cell migratory ability. Mechanistic study revealed that TM up-regulated E-cadherin but down-regulated N-cadherin expression, resulting in reversal of epithelial-mesenchymal transition (EMT) in the lung cancer cells. Moreover, silencing TM expression led to decreased E-cadherin and increased N-cadherin. Taken together, our study suggests that TM functions as a tumor suppressive protein, providing a conceptual framework for inducing TM overexpression as a sensible strategy and approach for novel anti-lung cancer drug discovery. PMID:27223053

  1. 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.

  2. Upregulation of long noncoding RNA SPRY4-IT1 promotes metastasis of esophageal squamous cell carcinoma via induction of epithelial-mesenchymal transition.

    Science.gov (United States)

    Zhang, Chun-Yang; Li, Ren-Ke; Qi, Yu; Li, Xiang-Nan; Yang, Yang; Liu, Dong-Lei; Zhao, Jia; Zhu, Deng-Yan; Wu, Kai; Zhou, Xu-Dong; Zhao, Song

    2016-10-01

    Esophageal squamous cell carcinoma (ESCC) is one of the prevalent and deadly cancers worldwide, especially in Eastern Asia. Recent studies show that long noncoding RNAs (lncRNAs) have critical roles in diverse biological processes, including tumorigenesis. In the present study, we find that the expression of lncRNA SPRY4-IT1 is significantly upregulated in ESCC cell lines as compared with human esophageal epithelial cell line HEEC. Overexpression of SPRY4-IT1 can increase in vitro motility of ESCC cells via induction of epithelial-mesenchymal transition (EMT), which is characterized by increasing the expression of vimentin (Vim) and fibronectin (FN) with a concomitant decrease of E-cadherin (E-Cad) and ZO-1, while silencing of SPRY4-IT1 significantly inhibits the in vitro motility of ESCC cells. Further, the knockdown of SPRY4-IT1 also significantly attenuates TFG-β-induced EMT of ESCC cells. Further, lncRNA SPRY4-IT1 can directly increase the transcription, expression, and nuclear localization of Snail, one key transcription factor during the EMT processes of cancer cells, while siRNA-mediated specific knockdown of Snail can significantly attenuate SPRY4-IT1-induced EMT of ESCC cells. Our results suggest that lncRNA SPRY4-IT1 might be considered as a novel oncogene involved in ESCC progression.

  3. Taxol-induced growth arrest and apoptosis is associated with the upregulation of the Cdk inhibitor, p21WAF1/CIP1, in human breast cancer cells.

    Science.gov (United States)

    Choi, Yung Hyun; Yoo, Young Hyun

    2012-12-01

    The anticancer agent, taxol, stabilizes tubulin polymerization, resulting in arrest at the G2/M phase of the cell cycle and apoptotic cell death. However, the molecular mechanism of this growth inhibition and apoptosis is poorly understood. In this study, we used MCF-7 and MDA-MB-231 human breast carcinoma cells which have different estrogen receptor (ER) and tumor suppressor p53 statuses to examine the mechanisms of taxol-induced growth inhibition and apoptosis. Treatment of the cells with taxol resulted in a time-dependent inhibition of cell viability, which was accompanied by an accumulation of cells at G2/M and the sub-G1 apoptotic region, determined by flow cytometric analysis. Furthermore, chromatin condensation, DNA ladder formation and proteolytic cleavage of poly(ADP-ribose) polymerase (PARP) in both cell lines were observed following treatment with taxol, indicating the occurrence of apoptotic cell death. Western blot analysis using whole cell lysates from MCF-7 and MDA-MB-231 cells treated with taxol demonstrated that taxol treatment inhibited expression of cyclin A and cyclin B1 proteins in a time-dependent manner. The inhibitory effects of taxol on cell growth and apoptosis induced by taxol were also associated with the downregulation of Wee1 kinase expression and a marked induction in the activity of the cyclin-dependent kinase inhibitor, p21WAF/CIP1. Furthermore, taxol elevated p21 promoter activity in both cell lines. These findings suggest that taxol-induced G2/M arrest and apoptosis in human breast carcinoma cells is mediated through the ER- and p53-independent upregulation of p21. PMID:23023313

  4. Advanced Glycation End Products Induce Endothelial-to-Mesenchymal Transition via Downregulating Sirt 1 and Upregulating TGF-β in Human Endothelial Cells

    Directory of Open Access Journals (Sweden)

    Wei He

    2015-01-01

    Full Text Available In the present study, we examined the advanced glycation end products- (AGEs- induced endothelial-to-mesenchymal transition (EndMT in human umbilical vein endothelial cells (HUVECs. Results demonstrated that AGE-BSAs significantly reduced the cluster of differentiation 31 (CD 31 expression, whereas they promoted the expression of fibroblast-specific protein-1 (FSP-1, α-smooth muscle antibody (α-SMA, and collagen I at both mRNA and protein levels in HUVECs. And the AGE-BSAs also promoted the receptors for AGEs (RAGEs and receptor I for TGF-β (TGFR I markedly with a dose dependence, whereas the Sirt 1 was significantly downregulated by the AGE-BSA at both mRNA and protein levels. Moreover, the Sirt 1 activity manipulation with its activator, resveratrol (RSV, or its inhibitor, EX527, markedly inhibited or ameliorated the AGE-mediated TGF-β upregulation. And the manipulated Sirt 1 activity positively regulated the AGE-induced CD31, whereas it negatively regulated the AGE-induced FSP-1. Thus, Sirt 1 was confirmed to regulate the AGE-induced EndMT via TGF-β. In summary, we found that AGE-BSA induced EndMT in HUVECs via upregulating TGF-β and downregulating Sirt 1, which also negatively regulated TGF-β in the cell. This study implied the EndMT probably as an important mechanism of AGE-induced cardiovascular injury.

  5. Hypoxia-Induced Upregulation of miR-132 Promotes Schwann Cell Migration After Sciatic Nerve Injury by Targeting PRKAG3.

    Science.gov (United States)

    Yao, Chun; Shi, Xiangxiang; Zhang, Zhanhu; Zhou, Songlin; Qian, Tianmei; Wang, Yaxian; Ding, Fei; Gu, Xiaosong; Yu, Bin

    2016-10-01

    Following peripheral nerve injury, hypoxia is formed as a result of defects in blood supply at the injury site. Despite accumulating evidence on the effects of microRNAs (miRNAs) on phenotype modulation of Schwann cells (SCs) after peripheral nerve injury, the impact of hypoxia on SC behaviors through miRNAs during peripheral nerve regeneration has not been estimated. In this study, we confirmed our previous microarray data on the upregulation of miR-132 after sciatic nerve injury in rats and observed that overexpression of miR-132 significantly promoted cell migration of primary cultured SCs. Interestingly, hypoxia-increased expression of miR-132 also enhanced SC migration while inhibition of miR-132 suppressed hypoxia-induced increase in SC migration. miR-132 downregulated PRKAG3 through binding to its 3'-UTR, and PRKAG3 knockdown compromised the reducing effect of miR-132 inhibition on SC migration under normal or hypoxia condition. Moreover, in vivo injection of miR-132 agomir into rats with sciatic nerve transection accelerated SC migration from the proximal to distal stump. Overall, our results suggest that the hypoxia-induced upregulation of miR-132 could promote SC migration and facilitate peripheral nerve regeneration.

  6. Naringin promotes differentiation of bone marrow stem cells into osteoblasts by upregulating the expression levels of microRNA-20a and downregulating the expression levels of PPARγ.

    Science.gov (United States)

    Fan, Jifeng; Li, Jie; Fan, Qinbo

    2015-09-01

    Naringin is a dihydrotestosterone flavonoid compound that significantly inhibits bone loss, improves bone density, and enhances biomechanical anti‑compression performance. Previous studies have demonstrated that naringin improves the activity levels of osteocalcin (OC) and alkaline phosphatase (ALP) in MC3T3‑E1 osteoblast precursor cells. The present study investigated the effects of naringin on osteoblastic differentiation and inhibition of adipocyte formation in bone marrow stem cells (BMSCs). The levels of osteogenesis were modulated via upregulation of the expression levels of microRNA (miR)‑20a, and downregulation of the expression levels of peroxisome proliferator‑activated receptor γ (PPARγ). The results indicated that naringin significantly enhanced BMSC proliferation in a dose‑dependent manner. In addition, naringin significantly increased the mRNA expression levels of OC, ALP, and collagen type I. Furthermore, naringin decreased the protein expression levels of PPARγ, and increased the expression levels of miR‑20a in the BMSCs. These results suggested that miR‑20a may regulate the expression of PPARγ in BMSCs. To our knowledge, this is the first study to report naringin‑induced osteogenesis via upregulation of the expression levels of miR‑20a, and downregulation of the expression levels of PPARγ. These results indicated the important role of naringin in BMSC differentiation. PMID:26126997

  7. Phosphatidylinositol 3-Kinase/AKT Pathway Inhibition by Doxazosin Promotes Glioblastoma Cells Death, Upregulation of p53 and Triggers Low Neurotoxicity.

    Directory of Open Access Journals (Sweden)

    Mariana Maier Gaelzer

    Full Text Available Glioblastoma is the most frequent and malignant brain tumor. Treatment includes chemotherapy with temozolomide concomitant with surgical resection and/or irradiation. However, a number of cases are resistant to temozolomide, as well as the human glioblastoma cell line U138-MG. We investigated doxazosin's (an antihypertensive drug activity against glioblastoma cells (C6 and U138-MG and its neurotoxicity on primary astrocytes and organoptypic hippocampal cultures. For this study, the following methods were used: citotoxicity assays, flow cytometry, western-blotting and confocal microscopy. We showed that doxazosin induces cell death on C6 and U138-MG cells. We observed that doxazosin's effects on the PI3K/Akt pathway were similar as LY294002 (PI3K specific inhibitor. In glioblastoma cells treated with doxasozin, Akt levels were greatly reduced. Upon examination of activities of proteins downstream of Akt we observed upregulation of GSK-3β and p53. This led to cell proliferation inhibition, cell death induction via caspase-3 activation and cell cycle arrest at G0/G1 phase in glioblastoma cells. We used in this study Lapatinib, a tyrosine kinase inhibitor, as a comparison with doxazosin because they present similar chemical structure. We also tested the neurocitotoxicity of doxazosin in primary astrocytes and organotypic cultures and observed that doxazosin induced cell death on a small percentage of non-tumor cells. Aggressiveness of glioblastoma tumors and dismal prognosis require development of new treatment agents. This includes less toxic drugs, more selective towards tumor cells, causing less damage to the patient. Therefore, our results confirm the potential of doxazosin as an attractive therapeutic antiglioma agent.

  8. Inhibition of radiation-induced up-regulation of leukocyte adhesion to endothelial cells with the platelet-activating factor inhibitor, BN52021

    International Nuclear Information System (INIS)

    Purpose: The inflammatory process is likely involved in normal tissue damage after radiation exposure, yet few studies have directly evaluated the factors that might be involved in the regulation of inflammation after irradiation in vivo. We tested the hypothesis that platelet-activating factor, a neutrophil agonist synthesized by endothelial cells, is involved in the upregulation of radiation-induced leukocyte-endothelial cell interactions by using an inhibitor of its receptor, BN52021. Methods and Materials: Fischer-344 rats with dorsal skin-fold window chambers were randomized to three experimental groups: control (sham irradiation); 6 Gy radiation; and 6 Gy + BN52021. BN52021 (0.5 mg/kg) was administered 5 min prior to 6 Gy radiation. Leukocytes were stained in vivo with i.v. acridine orange for visualization with fluorescent microscopy. Venous vessel diameters were measured and numbers of rolling leukocytes were counted per 30-s period. The number of adhering leukocytes per unit surface area was also determined. Differences among the three experimental groups for rolling and adhering leukocytes were analyzed using a mixed-effects linear model with vessel shear rate used as a covariate. Results are reported as means ± standard errors. Results: Irradiation caused upregulation of leukocyte rolling, as compared with sham-treated controls (p = 0.04): the BN compound in addition to radiation did not downregulate this effect. Irradiation also upregulated leukocyte adhesion (p < 0.001), but the addition of BN52021 prior to irradiation blocked this effect. The drug did not affect heart rate or blood pressure. Conclusions: These results support the hypothesis that radiation-induced upregulation of leukocyte adhesion is mediated by platelet-activating factor. These results are consistent with prior reports that platelet-activating factor is not involved in leukocyte rolling, which involves separate families of adhesion molecules from those that regulate adhesion. BN

  9. SIRT1 inhibits proliferation of pancreatic cancer cells expressing pancreatic adenocarcinoma up-regulated factor (PAUF), a novel oncogene, by suppression of {beta}-catenin

    Energy Technology Data Exchange (ETDEWEB)

    Cho, Il-Rae [WCU, Department of Cogno-Mechatronics Engineering, Pusan National University, Busan 609-735 (Korea, Republic of); Koh, Sang Seok [Immunotherapy Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon 305-333 (Korea, Republic of); Department of Functional Genomics, University of Science and Technology, Daejeon 305-333 (Korea, Republic of); Malilas, Waraporn; Srisuttee, Ratakorn; Moon, Jeong [WCU, Department of Cogno-Mechatronics Engineering, Pusan National University, Busan 609-735 (Korea, Republic of); Choi, Young-Whan [Department of Horticultural Bioscience, Pusan National University, Miryang 627-706 (Korea, Republic of); Horio, Yoshiyuki [Department of Pharmacology, Sapporo Medical University, Sapporo 060-8556 (Japan); Oh, Sangtaek [Department of Advanced Fermentation Fusion Science and Technology, Kookmin University, Seoul 136-702 (Korea, Republic of); Chung, Young-Hwa, E-mail: younghc@pusan.ac.kr [WCU, Department of Cogno-Mechatronics Engineering, Pusan National University, Busan 609-735 (Korea, Republic of)

    2012-06-29

    Highlights: Black-Right-Pointing-Pointer SIRT1 inhibits protein levels of {beta}-catenin and its transcriptional activity. Black-Right-Pointing-Pointer Nuclear localization of SIRT1 is not required for the decrease of {beta}-catenin expression. Black-Right-Pointing-Pointer SIRT1-mediated degradation of {beta}-catenin is not required for GSK-3{beta} and Siah-1 but for proteosome. Black-Right-Pointing-Pointer SIRT1 activation inhibits proliferation of pancreatic cancer cells expressing PAUF. -- Abstract: Because we found in a recent study that pancreatic adenocarcinoma up-regulated factor (PAUF), a novel oncogene, induces a rapid proliferation of pancreatic cells by up-regulation of {beta}-catenin, we postulated that {beta}-catenin might be a target molecule for pancreatic cancer treatment. We thus speculated whether SIRT1, known to target {beta}-catenin in a colon cancer model, suppresses {beta}-catenin in those pancreatic cancer cells that express PAUF (Panc-PAUF). We further evaluated whether such suppression would lead to inhibition of the proliferation of these cells. The ectopic expression of either SIRT1 or resveratrol (an activator of SIRT1) suppressed levels of {beta}-catenin protein and its transcriptional activity in Panc-PAUF cells. Conversely, suppression of SIRT1 expression by siRNA enhanced {beta}-catenin expression and transcriptional activity. SIRT1 mutant analysis showed that nuclear localization of SIRT1 is not required for reduction of {beta}-catenin. Treatment with MG132, a proteasomal inhibitor, restored {beta}-catenin protein levels, suggesting that SIRT1-mediated degradation of {beta}-catenin requires proteasomal activity. It was reported that inhibition of GSK-3{beta} or Siah-1 stabilizes {beta}-catenin in colon cancer cells, but suppression of GSK-3{beta} or Siah-1 using siRNA in the presence of resveratrol instead diminished {beta}-catenin protein levels in Panc-PAUF cells. This suggests that GSK-3{beta} and Siah-1 are not involved in SIRT1

  10. SIRT1 inhibits proliferation of pancreatic cancer cells expressing pancreatic adenocarcinoma up-regulated factor (PAUF), a novel oncogene, by suppression of β-catenin

    International Nuclear Information System (INIS)

    Highlights: ► SIRT1 inhibits protein levels of β-catenin and its transcriptional activity. ► Nuclear localization of SIRT1 is not required for the decrease of β-catenin expression. ► SIRT1-mediated degradation of β-catenin is not required for GSK-3β and Siah-1 but for proteosome. ► SIRT1 activation inhibits proliferation of pancreatic cancer cells expressing PAUF. -- Abstract: Because we found in a recent study that pancreatic adenocarcinoma up-regulated factor (PAUF), a novel oncogene, induces a rapid proliferation of pancreatic cells by up-regulation of β-catenin, we postulated that β-catenin might be a target molecule for pancreatic cancer treatment. We thus speculated whether SIRT1, known to target β-catenin in a colon cancer model, suppresses β-catenin in those pancreatic cancer cells that express PAUF (Panc-PAUF). We further evaluated whether such suppression would lead to inhibition of the proliferation of these cells. The ectopic expression of either SIRT1 or resveratrol (an activator of SIRT1) suppressed levels of β-catenin protein and its transcriptional activity in Panc-PAUF cells. Conversely, suppression of SIRT1 expression by siRNA enhanced β-catenin expression and transcriptional activity. SIRT1 mutant analysis showed that nuclear localization of SIRT1 is not required for reduction of β-catenin. Treatment with MG132, a proteasomal inhibitor, restored β-catenin protein levels, suggesting that SIRT1-mediated degradation of β-catenin requires proteasomal activity. It was reported that inhibition of GSK-3β or Siah-1 stabilizes β-catenin in colon cancer cells, but suppression of GSK-3β or Siah-1 using siRNA in the presence of resveratrol instead diminished β-catenin protein levels in Panc-PAUF cells. This suggests that GSK-3β and Siah-1 are not involved in SIRT1-mediated degradation of β-catenin in the cells. Finally, activation of SIRT1 inhibited the proliferation of Panc-PAUF cells by down-regulation of cyclin-D1, a target

  11. Upregulated baseline plasma CCL19 and CCR7 cell-surface expression on monocytes in early rheumatoid arthritis normalized during treatment and CCL19 correlated with radiographic progression

    DEFF Research Database (Denmark)

    Ellingsen, T; Hansen, I; Thorsen, J;

    2014-01-01

    OBJECTIVES: The aim of this study was to measure, in early rheumatoid arthritis (RA) patients, the concentration of CC-chemokine ligand 19 (CCL19) in plasma and the cell-surface expression of CC-chemokine receptor 7 (CCR7) on circulating monocytes and CD4+ T lymphocytes and to analyse correlations...... with disease activity and 5-year radiographic progression. METHOD: In disease-modifying anti-rheumatic drug (DMARD)-naïve RA patients (disease duration < 6 months), we measured plasma CCL19 by enzyme-linked immunosorbent assay (ELISA) (n = 160) and CCR7 cell-surface expression on monocytes and CD4+ T......-naïve RA patients, CCL19 plasma level and CCR7 surface expression on monocytes were upregulated and normalized after 1 year of treatment. Increased baseline plasma CCL19 level, anti-CCP antibody status, and TSS > 0 at baseline correlated independently with 5-year radiographic progression....

  12. Up-regulating the abscisic acid inactivation gene ZmABA8ox1b contributes to seed germination heterosis by promoting cell expansion.

    Science.gov (United States)

    Li, Yangyang; Wang, Cheng; Liu, Xinye; Song, Jian; Li, Hongjian; Sui, Zhipeng; Zhang, Ming; Fang, Shuang; Chu, Jinfang; Xin, Mingming; Xie, Chaojie; Zhang, Yirong; Sun, Qixin; Ni, Zhongfu

    2016-04-01

    Heterosis has been widely used in agriculture, but the underlying molecular principles are still largely unknown. During seed germination, we observed that maize (Zea mays) hybrid B73/Mo17 was less sensitive than its parental inbred lines to exogenous abscisic acid (ABA), and endogenous ABA content in hybrid embryos decreased more rapidly than in the parental inbred lines. ZmABA8ox1b, an ABA inactivation gene, was consistently more highly up-regulated in hybrid B73/Mo17 than in its parental inbred lines at early stages of seed germination. Moreover, ectopic expression of ZmABA8ox1b obviously promoted seed germination in Arabidopsis Remarkably, microscopic observation revealed that cell expansion played a major role in the ABA-mediated maize seed germination heterosis, which could be attributed to the altered expression of cell wall-related genes. PMID:27034328

  13. Epithelium-specific ets transcription factor 2 upregulates cytokeratin 18 expression in pulmonary epithelial cells through an interaction with cytokeratin 18 intron 1

    Institute of Scientific and Technical Information of China (English)

    Deanna YANIW; Jim HU

    2005-01-01

    The role of Ese-2, an Ets family transcription factor, in gene regulation is not known. In this study, the interaction between Ese-2 and cytokeratin 18 (K18) intron 1 was characterized in lung epithelial cells. Reporter gene assays showed Ese-2 was able to upregulate K18 intron 1 enhanced reporter gene expression by approximately 2-fold. We found that full length Ese-2 did not bind DNA strongly, therefore truncated versions of the protein, containing the ETS domain or Pointed domain, were created and tested in electrophoresis mobility shift assays. Multiple interactions between the ETS domain and putative DNA binding sites within K18 intron 1 were observed, which led to the determination of a possible Ese-2 DNA binding consensus sequence. These experiments suggest that Ese-2 could play a role in the regulation of K18 expression in lung epithelial cells.

  14. Low-dose levodopa protects nerve cells from oxidative stress and up-regulates expression of pCREB and CD39.

    Directory of Open Access Journals (Sweden)

    Shi-Ying Zhong

    Full Text Available OBJECTIVE: This study aimed to investigate the influence of low-dose levodopa (L-DOPA on neuronal cell death under oxidative stress. METHODS: PC12 cells were treated with L-DOPA at different concentrations. We detected the L-DOPA induced reactive oxygen species (ROS. Meanwhile, MTT and LDH assay were performed to determine the proliferation and growth of PC12 cells with or without ROS scavenger. In addition, after pretreatment with L-DOPA at different concentrations alone or in combination with CD39 inhibitor, PC12 cells were incubated with hydrogen peroxide (H2O2 and the cell viability was evaluated by MTT and LDH assay. In addition, the expression of pCREB and CD39 was detected by immunofluorescence staining and Western blot assay in both cells and rat's brain after L-DOPA treatment. RESULTS: After treatment with L-DOPA for 3 days, the cell proliferation and growth were promoted when the L-DOPA concentration was 30 µM. Low dose L-DOPA could protect the PC12 cells from H2O2 induced oxidative stress, which was compromised by CD39 inhibitor. In addition, the expression of CD39 and pCREB increased in both PC12 cells and rats' brain after L-DOPA treatment. CONCLUSIONS: L-DOPA at different concentrations has distinct influence on proliferation and growth of PC12 cells, and low dose (<30 µM L-DOPA protects PC12 cells against oxidative stress which might be related to the up-regulation of CD39 and pCREB expression.

  15. Upregulation of human telomerase reverse transcriptase mRNA expression by in vitro transfection of hepatitis B virus X gene into human hepatocarcinoma and cholangiocarcinoma cells

    Institute of Scientific and Technical Information of China (English)

    Zhen-Liang Qu; Sheng-Quan Zou; Nai-Qiang Cui; Xian-Zhong Wu; Ming-Fang Qin; Di Kong; Zhen-Li Zhou

    2005-01-01

    AIM: To study the changes of human telomerase reverse transcriptase (hTERT) mRNA expression in human hepatocarcinoma cell lines (HepG2) and cholangiocarcinoma cell lines (QBC939) after HBx gene transfection and to illustrate the significance of transcriptional regulation of hTERT gene by HBx gene in the carcinogenesis.METHODS: HepG2 and QBC939 cell lines were cultured and co-transfected with eukaryotic expression vector containing the HBx coding region and cloning vector containing enhanced green fluorescent protein (EGFP) coding sequence using lipid-mediated gene transduction technique. Thirty-six hours after transfection, EGFP expression in cells was used as the indicator of successful transfection. Flow cytometry was performed to determine the transfection efficiency.Cells were harvested and total RNA was extracted using TRIzol() reagent. The expression of hTERT mRNA in HepG2and QBC939 cell lines was assayed by reverse transcriptionpolymerase chain reaction. The expression of HBx protein in both cell lines was detected by immunocytochemical staining and Western blotting.RESULTS: Flow cytometry showed that the transfection efficiency was 46.4% in HepG2 cells and 29.6% in QBC939cells for both HBx gene expression vector and blank vector. The expression of hTERT mRNA was meaningfully increased in HepG2 and QBC939 cell lines when transfected with HBx gene expression vector compared to those transfected with OPTI-MEM medium and blank vector.Immunocytochemical staining and Western blotting revealed HBx protein expression in HepG2 and QBC939cells only when transfected with HBx gene.CONCLUSION: HBx gene transfection can upregulate the transcriptional expression of hTERT mRNA. The transactivation of hTERT gene by HBx gene is a newfound mechanism for pathogenesis of hepatocarcinomas and cholangiocarcinomas after HBV infection.

  16. Advanced glycation end-products induce apoptosis in pancreatic islet endothelial cells via NF-κB-activated cyclooxygenase-2/prostaglandin E2 up-regulation.

    Directory of Open Access Journals (Sweden)

    Kuo-Cheng Lan

    Full Text Available Microvascular complications eventually affect nearly all patients with diabetes. Advanced glycation end-products (AGEs resulting from hyperglycemia are a complex and heterogeneous group of compounds that accumulate in the plasma and tissues in diabetic patients. They are responsible for both endothelial dysfunction and diabetic vasculopathy. The aim of this study was to investigate the cytotoxicity of AGEs on pancreatic islet microvascular endothelial cells. The mechanism underlying the apoptotic effect of AGEs in pancreatic islet endothelial cell line MS1 was explored. The results showed that AGEs significantly decreased MS1 cell viability and induced MS1 cell apoptosis in a dose-dependent manner. AGEs dose-dependently increased the expressions of cleaved caspase-3, and cleaved poly (ADP-ribose polymerase in MS1 cells. Treatment of MS1 cells with AGEs also resulted in increased nuclear factor (NF-κB-p65 phosphorylation and cyclooxygenase (COX-2 expression. However, AGEs did not affect the expressions of endoplasmic reticulum (ER stress-related molecules in MS1 cells. Pretreatment with NS398 (a COX-2 inhibitor to inhibit prostaglandin E2 (PGE2 production reversed the induction of cleaved caspase-3, cleaved PARP, and MS1 cell viability. Moreover, AGEs significantly increased the receptor for AGEs (RAGE protein expression in MS1 cells, which could be reversed by RAGE neutralizing antibody. RAGE Neutralizing antibody could also reverse the induction of cleaved caspase-3 and cleaved PARP and decreased cell viability induced by AGEs. These results implicate the involvement of NF-κB-activated COX-2/PGE2 up-regulation in AGEs/RAGE-induced islet endothelial cell apoptosis and cytotoxicity. These findings may provide insight into the pathological processes within the pancreatic islet microvasculature induced by AGEs accumulation.

  17. Salubrinal-Mediated Upregulation of eIF2α Phosphorylation Increases Doxorubicin Sensitivity in MCF-7/ADR Cells

    OpenAIRE

    Jeon, Yong-Joon; Kim, Jin Hyun; Shin, Jong-Il; Jeong, Mini; Cho, Jaewook; Lee, Kyungho

    2016-01-01

    Eukaryotic translation initiation factor 2 alpha (eIF2α), which is a component of the eukaryotic translation initiation complex, functions in cell death and survival under various stress conditions. In this study, we investigated the roles of eIF2α phosphorylation in cell death using the breast cancer cell lines MCF-7 and MCF-7/ADR. MCF-7/ADR cells are MCF-7-driven cells that have acquired resistance to doxorubicin (ADR). Treatment of doxorubicin reduced the viability and induced apoptosis in...

  18. Loss of let-7 up-regulates EZH2 in prostate cancer consistent with the acquisition of cancer stem cell signatures that are attenuated by BR-DIM.

    Directory of Open Access Journals (Sweden)

    Dejuan Kong

    Full Text Available The emergence of castrate-resistant prostate cancer (CRPC contributes to the high mortality of patients diagnosed with prostate cancer (PCa, which in part could be attributed to the existence and the emergence of cancer stem cells (CSCs. Recent studies have shown that deregulated expression of microRNAs (miRNAs contributes to the initiation and progression of PCa. Among several known miRNAs, let-7 family appears to play a key role in the recurrence and progression of PCa by regulating CSCs; however, the mechanism by which let-7 family contributes to PCa aggressiveness is unclear. Enhancer of Zeste homolog 2 (EZH2, a putative target of let-7 family, was demonstrated to control stem cell function. In this study, we found loss of let-7 family with corresponding over-expression of EZH2 in human PCa tissue specimens, especially in higher Gleason grade tumors. Overexpression of let-7 by transfection of let-7 precursors decreased EZH2 expression and repressed clonogenic ability and sphere-forming capacity of PCa cells, which was consistent with inhibition of EZH2 3'UTR luciferase activity. We also found that the treatment of PCa cells with BR-DIM (formulated DIM: 3,3'-diindolylmethane by Bio Response, Boulder, CO, abbreviated as BR-DIM up-regulated let-7 and down-regulated EZH2 expression, consistent with inhibition of self-renewal and clonogenic capacity. Moreover, BR-DIM intervention in our on-going phase II clinical trial in patients prior to radical prostatectomy showed upregulation of let-7 consistent with down-regulation of EZH2 expression in PCa tissue specimens after BR-DIM intervention. These results suggest that the loss of let-7 mediated increased expression of EZH2 contributes to PCa aggressiveness, which could be attenuated by BR-DIM treatment, and thus BR-DIM is likely to have clinical impact.

  19. Plumbagin reduces chronic lymphocytic leukemia cell survival by downregulation of Bcl-2 but upregulation of the Bax protein level.

    Science.gov (United States)

    Fu, Chunling; Gong, Yanqing; Shi, Xuanxuan; Sun, Zengtian; Niu, Mingshan; Sang, Wei; Xu, Linyan; Zhu, Feng; Wang, Ying; Xu, Kailin

    2016-09-01

    Chronic lymphocytic leukemia (CLL) is the most common leukemia in Western countries, and mainly originates from an accumulation of abnormal B cells caused by the dysregulation of cell proliferation and apoptosis rates. The aberration of apoptosis-related genes in CLL cells results in defective apoptosis of CLL cells in response to traditional therapeutic medicine. Plumbagin (5-hydroxy-2-methyl-1, 4-naphthoquinone), a natural compound from Plumbago zeylinica, has been shown to exhibit pro-apoptotic activities in tumor cells. In the present study, we report that plumbagin effectively inhibited CLL cell viability with a lower dose compared to fludarabine, and inhibited cell proliferation in a dose-dependent manner. In addition, plumbagin promoted accumulation of MEC-1 cells in the S phase, and blocked cell cycle transition of HG3 cells from G0/G1 to S phase. Molecularly, plumbagin markedly induced CLL cell apoptosis through reduction of Bcl-2, but through an increase in the Bax protein level. These results suggest that plumbagin may be considered as a potential anticancer agent for CLL therapy. PMID:27461100

  20. Nitric oxide preferentially induces type 1 T cell differentiation by selectively up-regulating IL-12 receptor β2 expression via cGMP

    Science.gov (United States)

    Niedbala, Wanda; Wei, Xiao-qing; Campbell, Carol; Thomson, Duncan; Komai-Koma, Mousa; Liew, Foo Y.

    2002-01-01

    Nitric oxide plays an important role in immune regulation. We have shown that although high concentrations of NO generally were immune-suppressive, low concentrations of NO selectively enhanced the differentiation of T helper (Th)1 cells but not Th2 cells. This finding provided an explanation for the crucial role of NO in defense against intracellular pathogens. However, the mechanism for the selective induction of Th1 cells was unknown. We report here that at low concentrations, NO activates soluble guanylyl cyclase, leading to the up-regulation of cGMP, which selectively induces the expression of IL-12 receptor β2 but has no effect on IL-4 receptor. Because IL-12 and IL-4 are the key cytokines for induction of Th1 and Th2 cells, respectively, these results, therefore, provide the mechanism for the selective action of NO on T cell subset differentiation. Furthermore, this selectivity also applies to CD8+ cytotoxic and human T cells and, thus, demonstrates the general implication of this observation in immune regulation. Our results also provide an example of the regulation of cytokine receptor expression by NO. The selectivity of such action via cGMP suggests that it is amenable to therapeutic intervention. PMID:12451176

  1. Enhanced human bone marrow stromal cell affinity for modified poly(L-lactide) surfaces by the upregulation of adhesion molecular genes.

    Science.gov (United States)

    Mao, Xueli; Peng, Hui; Ling, Junqi; Friis, Thor; Whittaker, Andrew K; Crawford, Ross; Xiao, Yin

    2009-12-01

    To enhance and regulate cell affinity for poly (L-lactic acid) (PLLA) based materials, two hydrophilic ligands, poly (ethylene glycol) (PEG) and poly (L-lysine) (PLL), were used to develop triblock copolymers: methoxy-terminated poly (ethylene glycol)-block-poly (L-lactide)-block-poly (L-lysine) (MPEG-b-PLLA-b-PLL) in order to regulate protein absorption and cell adhesion. Bone marrow stromal cells (BMSCs) were cultured on different composition of MPEG-b-PLLA-b-PLL copolymer films to determine the effect of modified polymer surfaces on BMSC attachment. To understand the molecular mechanism governing the initial cell adhesion on difference polymer surfaces, the mRNA expression of 84 human extracellular matrix (ECM) and adhesion molecules was analysed using quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). It was found that down regulation of adhesion molecules was responsible for the impaired BMSC attachment on PLLA surface. MPEG-b-PLLA-b-PLL copolymer films improved significantly the cell adhesion and cytoskeleton expression by upregulation of relevant molecule genes significantly. Six adhesion genes (CDH1, ITGL, NCAM1, SGCE, COL16A1, and LAMA3) were most significantly influenced by the modified PLLA surfaces. In summary, polymer surfaces altered adhesion molecule gene expression of BMSCs, which consequently regulated cell initial attachment on modified PLLA surfaces. PMID:19796804

  2. Depletion of CABYR-a/b sensitizes lung cancer cells to TRAIL-induced apoptosis through YAP/p73-mediated DR5 upregulation.

    Science.gov (United States)

    Xiao, Qianqian; Qian, Zunlei; Zhang, Weiqing; Liu, Jin; Hu, Enze; Zhang, Jinsan; Li, Mingying; Wang, Junhao; Kong, Fei; Li, Yunguang; Wang, Rui; Tan, Xiaohua; He, Dacheng; Xiao, Xueyuan

    2016-02-23

    Our previous study revealed that knockdown of CABYR-a/b increases the chemosensitivity of lung cancer cells through inactivation of Akt. Here, we demonstrated that depletion of CABYR-a/b significantly increased DR5 expression and sensitized lung cancer cells to TRAIL-induced apoptosis in vitro and/or in vivo. Importantly, treatment with AD5-10, a DR5-specific agonistic monoclonal antibody, was able to mimic TRAIL-induced apoptosis in CABYR-a/b-silenced cells. Strikingly, we identified that depletion of CABYR-a/b not only increased the expressions of p73 and DR5 but also decreased the phosphorylation of YAP S127. Loss- or gain-of-function studies of YAP and p73 revealed that double deletions of YAP and p73 effectively decreased the expression of DR5 and abolished TRAIL-induced apoptosis in CABYR-a/b knockdown cells. Conversely, the co-overexpression of YAP and p73 promoted the expression of DR5 and sensitized cells to TRAIL-induced apoptosis. Taken together, our results demonstrate that depletion of CABYR-a/b sensitizes lung cancer cells to TRAIL-induced apoptosis through YAP/p73-mediated DR5 upregulation.

  3. Gene transfer-applied BNCT (g-BNCT) for amelanotic melanoma in brain. Further upregulation of 10B uptake by cell modulation

    International Nuclear Information System (INIS)

    Our success in eradicating melanoma by single BNCT with BPA led to the next urgent theme, i.e. application of such BNCT for currently uncurable melanoma metastasis in brain. In order to establish 10B-BPA-BNCT for melanoma in brain, we have investigated the pharmacokinetics of BPA which is most critical factor for successful BNCT, in melanotic and amelanotic and further tyrosinase gene-transfected amelanotic melanoma proliferating in brain having blood-brain-barrier, as compared to melanoma proliferating in skin. We have established three implanted models for melanoma in brain: 1) A1059 cells, amelanotic melanoma, 2) B16B15b cells, melanotic melanoma cells, highly metastatic to brain, and 3) TA1059 cells, with active melanogenesis induced by tyrosinase gene transfection. We would like to report the results of comparative analysis of the BPA uptake ability in these melanoma cells in both brain and skin. Based on these findings, we are further investigating to enhance 10B-BPA uptake by not only g-BNCT but also by additional melanogenesis upregulating cell modulation. (author)

  4. Methylglyoxal Impairs Insulin Secretion of Pancreatic β-Cells through Increased Production of ROS and Mitochondrial Dysfunction Mediated by Upregulation of UCP2 and MAPKs.

    Science.gov (United States)

    Bo, Jinshuang; Xie, Shiya; Guo, Yi; Zhang, Chunli; Guan, Yanming; Li, Chunmei; Lu, Jianxin; Meng, Qing H

    2016-01-01

    Methylglyoxal (MG) is a highly reactive glucose metabolic intermediate and a major precursor of advanced glycation end products. MG level is elevated in hyperglycemic disorders such as diabetes mellitus. Substantial evidence has shown that MG is involved in the pathogenesis of diabetes and diabetic complications. We investigated the impact of MG on insulin secretion by MIN6 and INS-1 cells and the potential mechanisms of this effect. Our study demonstrates that MG impaired insulin secretion by MIN6 or ISN-1 cells in a dose-dependent manner. It increased reactive oxygen species (ROS) production and apoptosis rate in MIN6 or ISN-1 cells and inhibited mitochondrial membrane potential (MMP) and ATP production. Furthermore, the expression of UCP2, JNK, and P38 as well as the phosphorylation JNK and P38 was increased by MG. These effects of MG were attenuated by MG scavenger N-acetyl cysteine. Collectively, these data indicate that MG impairs insulin secretion of pancreatic β-cells through increasing ROS production. High levels of ROS can damage β-cells directly via JNK/P38 upregulation and through activation of UCP2 resulting in reduced MMP and ATP production, leading to β-cell dysfunction and impairment of insulin production.

  5. Vascular endothelial growth factor up-regulates the expression of intracellular adhesion molecule-1 in retinal endothelial cells via reactive oxygen species, but not nitric oxide

    Institute of Scientific and Technical Information of China (English)

    ZHANG Xiao-ling; WEN Liang; CHEN Yan-jiong; ZHU Yi

    2009-01-01

    Background The vascular endothelial growth factor (VEGF) is involved in the initiation of retinal vascular leakage and nonperfusion in diabetes. The intracellular adhesion molecule-1 (ICAM-1) is the key mediator of the effect of VEGFs on retinal leukostasis. Although the VEGF is expressed in an early-stage diabetic retina, whether it directly up-regulates ICAM-1 in retinal endothelial cells (ECs) is unknown. In this study, we provided a new mechanism to explain that VEGF does up-regulate the expression of ICAM-1 in retinal ECs.Methods Bovine retinal ECs (BRECs) were isolated and cultured. Immunohistochemical staining was performed to identify BRECs. The cultured cells were divided into corresponding groups. Then, VEGF (100 ng/ml) and other inhibitors were used to treat the cells. Cell lysate and the cultured supernatant were collected, and then, the protein level of ICAM-1 and phosphorylation of the endothelial nitric oxide synthase (eNOS) were detected using Western blotting. Griess reaction was used to detect nitric oxide (NO).Results Western blotting showed that the VEGF up-regulated the expression of ICAM-1 protein and increased phosphorylation of the eNOS in retinal ECs. Neither the block of NO nor protein kinase C (PKC) altered the expression of ICAM-1 or the phosphorylation of eNOS. The result of the Western blotting also showed that inhibition of phosphatidylinositol 3-kinase (PI3K) or reactive oxygen species (ROS) significantly reduced the expression of ICAM-1. Inhibition of PI3K also reduced phosphorylation of eNOS. Griess reaction showed that VEGF significantly increased during NO production. When eNOS was blocked by L-NAME or PI3K was blocked by LY294002, the basal level of NO production and the increment of NO caused by VEGF could be significantly decreased.Conclusion ROS-NO coupling in the retinal endothelium may be a new mechanism that could help to explain why VEGF induces ICAM-1 expression and the resulting leukostasis in diabetic retinopathy.

  6. Control of insulin receptor level in 3T3 cells: effect of insulin-induced down-regulation and dexamethasone-induced up-regulation on rate of receptor inactivation.

    OpenAIRE

    Knutson, V P; Ronnett, G V; Lane, M D

    1982-01-01

    Chronic exposure of 3T3 mouse fibroblasts to insulin or to the glucocorticoid dexamethasone induces down-regulation and up-regulation, respectively, of cell-surface and total cellular insulin binding capacity. Both processes are reversed upon withdrawal of the inducer. Scatchard analysis of insulin binding for receptors in the down- and up-regulated states indicates that the changes in binding capacity result primarily from alterations in insulin receptor level. That these alterations in tota...

  7. Up-regulation of TIMP-2 expression promotes SHI-1 leukemic cells proliferation and infiltration in immunodeficiency mice

    Institute of Scientific and Technical Information of China (English)

    Li Zhenjiang; Chen Zixing; Cen Jiannong; He Jun; Qiu Qiaocheng; Xue Yongquan

    2014-01-01

    Background MMPs and TIMPs play important roles in tumor angiogenesis and invasion.Studies have shown that TIMP-2 has two roles in tumor invasion.However,its role in leukemic infiltration has not been well investigated.This study explored the roles of TIMP-2 in extramedullary infiltration of acute monocytic leukemic SHI-1 cells both in vitro and in vitro.Methods A retroviral vector carrying the human TIMP-2 cDNA was constructed and transfected into the monocytic leukemic cell line SHI-1.The expression of TIMP-2 in the positive clones was determined.The proliferation of SHI-1 cells was examined by MTT assay.Trans-Matrigel invasion assays were used to investigate the infiltration ability in vitro.SHI-1 cells were intravenously injected into pre-treated nu/nu mice to investigate the infiltration ability feature in vitro.Results The expression of TIMP-2 on the cell membrane was significantly elevated in SHI-1/TIMP-2 cells.Over-expression of TIMP-2 promoted the cells proliferation and the invasions in vitro.The SHI-1/TIMP-2 cells demonstrated higher infiltration ability when intravenously injected into nu/nu mice.Conclusion Over-expression of TIMP-2,especially on the cell membrane,may play important roles in promoting the proliferation and infiltration of SHI-1 leukemic cells.

  8. Selection of resistant acute myeloid leukemia SKM-1 and MOLM-13 cells by vincristine-, mitoxantrone- and lenalidomide-induced upregulation of P-glycoprotein activity and downregulation of CD33 cell surface exposure.

    Science.gov (United States)

    Imrichova, D; Messingerova, L; Seres, M; Kavcova, H; Pavlikova, L; Coculova, M; Breier, A; Sulova, Z

    2015-09-18

    Bone marrow cells and peripheral blood mononuclear cells obtained from both acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) patients contain upregulated levels of cell surface antigen CD33 compared with healthy controls. This difference enables the use of humanized anti-CD33 antibody conjugated to cytotoxic agents for CD33 targeted immunotherapy. However, the expression of the membrane-bound drug transporter P-glycoprotein (P-gp) has been shown to be critical for resistance against the cytotoxicity of a humanized anti-CD33 antibody conjugated to maytansine-derivative DM4. The aim of the present study was to examine whether the expression of P-gp in AML cell lines is associated with changes in CD33 expression. For this purpose, we established drug resistant variants of SKM-1 and MOLM-13 AML cell lines via the selection of parental cells for resistance to vincristine, mitoxantrone and lenalidomide. All three substances induced a multidrug resistance (MDR) phenotype in SKM-1 cells associated with strong upregulation of P-gp and downregulation of CD33. However, in MOLM-13 cells, the upregulation of P-gp and downregulation of CD33 were present only in cells selected for resistance to vincristine and mitoxantrone but not lenalidomide. Inverse expression of P-gp and CD33 were observed in all resistant variants of SKM-1 and MOLM-13 cells. The MDR phenotype of resistant variants of SKM-1 and MOLM-13 cells was associated with alterations in apoptotic regulatory proteins and downregulation of the multidrug resistance associated protein 1 and breast cancer resistance protein.

  9. Up-regulation of p21 and TNF-α is mediated in lycorine-induced death of HL-60 cells

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    He Yan

    2010-08-01

    Full Text Available Abstract Background Leukemia is one of the most life-threatening cancers today, and acute promyelogenous leukemia (APL is a common type of leukemia. Many natural compounds have already been found to exhibit significant anti-tumor effects. Lycorine, a natural alkaloid extracted from Amaryllidaceae, exhibited anti-leukemia effects in vitro and in vivo. The survival rate of HL-60 cells exposed to lycorine was decreased, cell growth was slowed down, and cell regeneration potential was inhibited. HL-60 cells exhibited typical apoptotic characteristic. Lycorine can suppress leukemia growth and reduce cell survival and inducing apoptosis of tumor cells. The purpose of this work is to elucidate the mechanism by which lycorine induces APL cells. Results When HL-60 cells were treated with different concentration of lycorine, the expression of p21 and TNF-α was up-regulated in a concentration-dependent manner as shown by real-time quantitative reverse transcriptase-polymerase chain reaction and Western blotting. Lycorine also down-regulated p21-related gene expression, including Cdc2, Cyclin B, Cdk2 and Cyclin E, promoted Bid truncation, decreased IκB phosphorylation and blocked NF-κB nuclear import. Cytochrome c was released from mitochondria as observed with confocal laser microscopy. Conclusions The TNF-α signal transduction pathway and p21-mediated cell-cycle inhibition were involved in the apoptosis of HL-60 cells induced by lycorine. These results contribute to the development of new lycorine-based anti-leukemia drugs.

  10. Genistein inhibits the proliferation of human multiple myeloma cells through suppression of nuclear factor-κB and upregulation of microRNA-29b.

    Science.gov (United States)

    Xie, Jie; Wang, Jianchao; Zhu, Bo

    2016-02-01

    Multiple myeloma (MM) is a malignant tumor and is the most common primary tumor of the bone marrow in the USA. Genistein is predominantly found in Leguminosae and various lines of evidence have indicated that it suppresses cell growth, induces programmed cell death and inhibits angiogenesis. As a result of these capabilities, genistein presents as a promising cancer chemopreventive agent. However, the effect of genistein on MM remains to be elucidated. The present study investigated the effect of genistein on the proliferation and apoptosis of MM cells through the regulation of nuclear factor-κB (NF-κB) and microRNA-29b (miR-29b). In the present study, cell proliferation was examined using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. In addition, apoptosis was detected using an Annexin V-fluorescein isothiocyanate/propidium iodide apoptosis assay and caspase-3 activation assay. The expression of NF-κB and miR-29b was analyzed using western blotting and reverse transcription quantitative polymerase chain reaction, respectively. Finally, miR-29b and anti-miR-29b plasmids were transfected into U266 cells to determine the effect of genistein on MM. In the present study, the results demonstrated that genistein could significantly reduce cell proliferation, induce apoptosis and increase the activity of caspase-3 in U266 cells. Furthermore, it was found that genistein could suppress the protein level of NF-κB and promote the expression of miR-29b in U266 cells. The results also indicated that miR-29b could alter the expression of NF-κB in U266 cells. These findings suggest that genistein inhibits the proliferation of human MM cells by upregulating miR-29b resulting in suppression of NF-κB.

  11. Bystin-like protein is upregulated in hepatocellular carci-noma and required for nucleologenesis in cancer cell pro-liferation

    Institute of Scientific and Technical Information of China (English)

    Hanzhi Wang; Wei Xiao; Qinbo Zhou; Yun Chen; Shuo Yang; Jiansong Sheng; Yanqing Yin; Jia Fan; Jia-wei Zhou

    2009-01-01

    The bystin-like (BYSL) gene was previously characterized to encode an accessory protein for cell adhesion that participates in early embryo implantation. It is also involved in 40S ribosomal subunit biogenesis and is found to be expressed in rapidly growing embryo and cancer cell lines. In order to explore the role of BYSL in cancer cell prolif-eration and growth, we used hepatocellular carcinoma (HCC) as a model. Here, we report that BYSL is crucial for HCC cell growth both in vitro and in vivo. Expression levels of BYSL mRNA and protein in human HCC specimens were markedly increased compared with those seen in adjacent non-cancerous tissue. In vitro, inhibition of BYSL by short hairpin RNA decreased HCC cell proliferation, induced apoptosis and partially arrested the cell cycle in the G2/M phase. In vivo, HCC cells treated with BYSL siRNA failed to form tumors in nude mice after subcutaneous im-plantation. To determine the cellular basis for BYSL RNAi-induced cell growth arrest, BYSL subcellular localization in mitotic and interphase HepG2 cells was examined. BYSL was present at multiple stages during nucleologenesis, including in nucleolus-derived foci (NDF), perichromosomal regions and the prenucleolar body (PNB) during mito-sis. BYSL depletion remarkably suppressed NDF and PNB formation, and disrupted nucleoli assembly after mitosis, resulting in increased apoptosis and reduced tolerance of HCC cells to serum starvation. Taken together, our studies indicate that upregulated BYSL expression plays a role in hepatocarcinogenesis.

  12. Fibroblast growth factor-2 up-regulates the expression of nestin through the Ras–Raf–ERK–Sp1 signaling axis in C6 glioma cells

    Energy Technology Data Exchange (ETDEWEB)

    Chang, Kai-Wei [Institute of Molecular and Cellular Biology, National Taiwan University, Taipei 106, Taiwan (China); Huang, Yuan-Li [Department of Biotechnology, College of Health Science, Asia University, Taichung 413, Taiwan (China); Wong, Zong-Ruei; Su, Peng-Han [Institute of Molecular and Cellular Biology, National Taiwan University, Taipei 106, Taiwan (China); Huang, Bu-Miin [Department of Cell Biology and Anatomy, National Cheng-Kung University, Tainan 701, Taiwan (China); Ju, Tsai-Kai [Instrumentation Center, National Taiwan University, Taipei 106, Taiwan (China); Technology Commons, College of Life Science, National Taiwan University, Taipei 106, Taiwan (China); Yang, Hsi-Yuan, E-mail: hyhy@ntu.edu.tw [Institute of Molecular and Cellular Biology, National Taiwan University, Taipei 106, Taiwan (China)

    2013-05-17

    Highlights: •Nestin expression in C6 glioma cells is induced by FGF-2. •Nestin expression is induced by FGF-2 via de novo RNA and protein synthesis. •The FGFR inhibitor SU5402 blocks the FGF-2-induced nestin expression. •The mRNA of FGFR1 and 3 are detected in C6 glioma cells. •Ras–Raf–ERK–Sp1 signaling pathway is responsibe for FGF-2-induced nestin expression. -- Abstract: Nestin is a 240-kDa intermediate filament protein expressed mainly in neural and myogenic stem cells. Although a substantial number of studies have focused on the expression of nestin during development of the central nervous system, little is known about the factors that induce and regulate its expression. Fibroblast growth factor-2 (FGF-2) is an effective mitogen and stimulates the proliferation and differentiation of a subset of nestin-expressing cells, including neural progenitor cells, glial precursor cells, and smooth muscle cells. To assess whether FGF-2 is a potent factor that induces the expression of nestin, C6 glioma cells were used. The results showed that nestin expression was up-regulated by FGF-2 via de novo RNA and protein synthesis. Our RT-PCR results showed that C6 glioma cells express FGFR1/3, and FGFRs is required for FGF-2-induced nestin expression. Further signaling analysis also revealed that FGF-2-induced nestin expression is mediated through FGFR–MAPK–ERK signaling axis and the transcriptional factor Sp1. These findings provide new insight into the regulation of nestin in glial system and enable the further studies on the function of nestin in glial cells.

  13. Slit2-Robo1 signaling promotes the adhesion, invasion and migration of tongue carcinoma cells via upregulating matrix metalloproteinases 2 and 9, and downregulating E-cadherin

    Science.gov (United States)

    Zhao, Yuan; Zhou, Feng-Li; Li, Wei-Ping; Wang, Jing; Wang, Li-Jing

    2016-01-01

    Whether Slit homologue 2 (Slit2) inhibits or promotes tumor cell migration remains controversial, and the role of Slit2-Roundabout 1 (Robo1) signaling in oral cancer remains to be fully elucidated. The aim of the present study was to investigate the role of Slit2-Robo1 signaling in the adhesion, invasion and migration of tongue carcinoma cells, and the mechanism by which Slit2-Robo1 signaling inhibits or promotes tumor cell migration. Tca8113 tongue carcinoma cells were treated with the monoclonal anti-human Robo1 antibody, R5, to inhibit the Slit2-Robo1 signaling pathway, with immunoglobulin (Ig)G2b treatment as a negative control. The expression levels of Slit2 and Robo1 were determined using flow cytometry. The effects of R5 on the adhesion, invasion and migration of Tca8113 tongue carcinoma cells were investigated. Gelatin zymography was used to investigate the activity of matrix metalloproteinase 2 (MMP2) and MMP9. Western blot analysis was used to evaluate the expression levels of E-cadherin in Tca8113 cells treated with 10 µg/ml of either R5 or IgG2b. Slit2 and Robo1 proteins were found to be expressed in the Tca8113 cells. R5 significantly inhibited the adhesion, invasion and migration of Tca8113 cells in vitro. R5 also inhibited the activities of MMP2 and MMP9, and increased the expression of E-cadherin in the Tca8113 cells. These results suggested that Slit2-Robo1 signaling promoted the adhesion, invasion and migration of tongue carcinoma cells by upregulating the expression levels of MMP2 and MMP9 and, downregulating the expression of E-cadherin. PMID:27431199

  14. Up-regulation of hypoxia inducible factor-1α by cobalt chloride correlates with proliferation and apoptosis in PC-2 cells

    Directory of Open Access Journals (Sweden)

    Dai Zhi-Jun

    2012-03-01

    Full Text Available Abstract Background The exact mechanism of the effects of hypoxia on the proliferation and apoptosis in carcinoma cells is still conflicting. This study investigated the variation of hypoxia-inducible factor-1α(HIF-1α expression and the apoptosis effect of hypoxia stimulated by cobalt chloride (CoCl2 in pancreatic cancer PC-2 cells. Methods PC-2 cells were cultured with different concentration (50-200 μmol/L of CoCl2 after 24-120 hours to simulate hypoxia in vitro. The proliferation of PC-2 cells was examined by MTT assay. The cellular morphology of PC-2 cells were observed by light inverted microscope and transmission electron microscope(EM. The expression of HIF-1α on mRNA and protein level was measured by semi-quantitive RT-PCR and Western blot analysis. Apoptosis of PC-2 cells were demonstrated by flow cytometry with Annexin V-FITC/PI double staining. Results MTT assay showed that the proliferation of PC-2 cells were stimulated in the first 72 h, while after treated over 72 h, a dose- dependent inhibition of cell growth could be observed. By using transmission electron microscope, swollen chondrosomes, accumulated chromatin under the nuclear membrane and apoptosis bodies were observed. Flow cytometer(FCM analysis showed the apoptosis rate was correlated with the dosage of CoCl2. RT-PCR and Western blot analysis indicated that hypoxia could up-regulate the expression of HIF-1α on both mRNA and protein levels. Conclusion Hypoxic microenvironment stimulated by CoCl2 could effectively induce apoptosis and influence cell proliferation in PC-2 cells, the mechanism could be related to up-expression of HIF-1α.

  15. Inhibiting effect of CaMK Ⅱ N up-regulation on leukemia cells growth and its mechanism

    Institute of Scientific and Technical Information of China (English)

    侯军

    2014-01-01

    Objective To investigate the inhibitory effects of CaMKⅡN on acute myeloid leukemia cell line HL-60to explore a novel therapeutic target of leukemia.Methods Human CaMKⅡN gene expression vector pcDNA3.1/hCaMKⅡN or empty vector pcDNA3.1/myc-His(-)B was transfected into HL-60 cells by Lipofectamine2000.Human CaMKⅡN proteins of transfected cells were detected by Westem blot.Cell proliferation affected by human CaMKⅡN was determined by MTT.Colonyforming assay was performed by soft agar

  16. Cadmium induces cytotoxicity in human bronchial epithelial cells through upregulation of eIF5A1 and NF-kappaB

    Energy Technology Data Exchange (ETDEWEB)

    Chen, De-Ju; Xu, Yan-Ming; Du, Ji-Ying [Laboratory of Cancer Biology and Epigenetics, Shantou University Medical College, Shantou, Guangdong 515041 (China); Department of Cell Biology and Genetics, Shantou University Medical College, Shantou, Guangdong 515041 (China); Huang, Dong-Yang [Department of Cell Biology and Genetics, Shantou University Medical College, Shantou, Guangdong 515041 (China); Lau, Andy T.Y., E-mail: andytylau@stu.edu.cn [Laboratory of Cancer Biology and Epigenetics, Shantou University Medical College, Shantou, Guangdong 515041 (China); Department of Cell Biology and Genetics, Shantou University Medical College, Shantou, Guangdong 515041 (China)

    2014-02-28

    Highlights: • Normal human bronchial epithelial cells (BEAS-2B) were dosed with cadmium (Cd). • A low level (2 μM) of Cd treatment for 36 h elicited negligible cytotoxicity. • High levels (20 or 30 μM) of Cd treatment for 36 h induced cell death. • High levels of Cd can upregulate the protein levels of eIF5A1 and NF-κB p65. • We suggest that eIF5A1 level is possibly modulated by NF-κB. - Abstract: Cadmium (Cd) and Cd compounds are widely-distributed in the environment and well-known carcinogens. Here, we report that in CdCl{sub 2}-exposed human bronchial epithelial cells (BEAS-2B), the level of p53 is dramatically decreased in a time- and dose-dependent manner, suggesting that the observed Cd-induced cytotoxicity is not likely due to the pro-apoptotic function of p53. Therefore, this prompted us to further study the responsive pro-apoptotic factors by proteomic approaches. Interestingly, we identified that high levels (20 or 30 μM) of Cd can significantly upregulate the protein levels of eukaryotic translation initiation factor 5A1 (eIF5A1) and redox-sensitive transcription factor NF-κB p65. Moreover, there is an enhanced NF-κB nuclear translocation as well as chromatin-binding in Cd-treated BEAS-2B cells. We also show that small interfering RNA-specific knockdown of eIF5A1 in Cd-exposed cells attenuated the Cd cytotoxicity, indicating the potential role of eIF5A1 in Cd cytotoxicity. As eIF5A1 is reported to be related with cell apoptosis but little is known about its transcriptional control, we hypothesize that NF-κB might likely modulate eIF5A1 gene expression. Notably, by bioinformatic analysis, several potential NF-κB binding sites on the upstream promoter region of eIF5A1 gene can be found. Subsequent chromatin immunoprecipitation assay revealed that indeed there is enhanced NF-κB binding on eIF5A1 promoter region of Cd-treated BEAS-2B cells. Taken together, our findings suggest for the first time a regulatory mechanism for the pro

  17. Translational up-regulation and high-level protein expression from plasmid vectors by mTOR activation via different pathways in PC3 and 293T cells.

    Directory of Open Access Journals (Sweden)

    Prashanthi Karyala

    Full Text Available BACKGROUND: Though 293T cells are widely used for expression of proteins from transfected plasmid vectors, the molecular basis for the high-level expression is yet to be understood. We recently identified the prostate carcinoma cell line PC3 to be as efficient as 293T in protein expression. This study was undertaken to decipher the molecular basis of high-level expression in these two cell lines. METHODOLOGY/PRINCIPAL FINDINGS: In a survey of different cell lines for efficient expression of platelet-derived growth factor-B (PDGF-B, β-galactosidase (β-gal and green fluorescent protein (GFP from plasmid vectors, PC3 was found to express at 5-50-fold higher levels compared to the bone metastatic prostate carcinoma cell line PC3BM and many other cell lines. Further, the efficiency of transfection and level of expression of the reporters in PC3 were comparable to that in 293T. Comparative analyses revealed that the high level expression of the reporters in the two cell lines was due to increased translational efficiency. While phosphatidic acid (PA-mediated activation of mTOR, as revealed by drastic reduction in reporter expression by n-butanol, primarily contributed to the high level expression in PC3, multiple pathways involving PA, PI3K/Akt and ERK1/2 appear to contribute to the abundant reporter expression in 293T. Thus the extent of translational up-regulation attained through the concerted activation of mTOR by multiple pathways in 293T could be achieved through its activation primarily by the PA pathway in PC3. CONCLUSIONS/SIGNIFICANCE: Our studies reveal that the high-level expression of proteins from plasmid vectors is effected by translational up-regulation through mTOR activation via different signaling pathways in the two cell lines and that PC3 is as efficient as 293T for recombinant protein expression. Further, PC3 offers an advantage in that the level of expression of the protein can be regulated by simple addition of n-butanol to

  18. Integrated genomic approaches identify upregulation of SCRN1 as a novel mechanism associated with acquired resistance to erlotinib in PC9 cells harboring oncogenic EGFR mutation

    Science.gov (United States)

    Kim, Nayoung; Cho, Ahye; Watanabe, Hideo; Choi, Yoon-La; Aziz, Meraj; Kassner, Michelle; Joung, Je-Gun; Park, Angela KJ; Francis, Joshua M.; Bae, Joon Seol; Ahn, Soo-min; Kim, Kyoung-Mee; Park, Joon Oh; Park, Woong-Yang; Ahn, Myung-Ju; Park, Keunchil; Koo, Jaehyung; Yin, Hongwei Holly; Cho, Jeonghee

    2016-01-01

    Therapies targeting the tyrosine kinase activity of Epidermal Growth Factor Receptor (EGFR) have been proven to be effective in treating a subset of non-small cell lung cancer (NSCLC) patients harboring activating EGFR mutations. Inevitably these patients develop resistance to the EGFR-targeted tyrosine kinase inhibitors (TKIs). Here, we performed integrated genomic analyses using an in vitro system to uncover alternative genomic mechanisms responsible for acquired resistance to EGFR-TKIs. Specifically, we identified 80 genes whose expression is significantly increased in the erlotinib-resistant clones. RNAi-based systematic synthetic lethal screening of these candidate genes revealed that suppression of one upregulated transcript, SCRN1, a secernin family member, restores sensitivity to erlotinib by enhancing inhibition of PI3K/AKT signaling pathway. Furthermore, immunohistochemical analysis revealed increased levels of SCRN1 in 5 of 11 lung tumor specimens from EGFR-TKIs resistant patients. Taken together, we propose that upregulation of SCRN1 is an additional mechanism associated with acquired resistance to EGFR-TKIs and that its suppression serves as a novel therapeutic strategy to overcome drug resistance in these patients. PMID:26883194

  19. Acute inhibition of PI3K-PDK1-Akt pathway potentiates insulin secretion through upregulation of newcomer granule fusions in pancreatic β-cells.

    Directory of Open Access Journals (Sweden)

    Kyota Aoyagi

    Full Text Available In glucose-induced insulin secretion from pancreatic β-cells, a population of insulin granules fuses with the plasma membrane without the typical docking process (newcomer granule fusions, however, its mechanism is unclear. In this study, we investigated the PI3K signaling pathways involved in the upregulation of newcomer granule fusions. Acute treatment with the class IA-selective PI3K inhibitors, PIK-75 and PI-103, enhanced the glucose-induced insulin secretion. Total internal reflection fluorescent microscopy revealed that the PI3K inhibitors increased the fusion events from newcomer granules. We developed a new system for transfection into pancreatic islets and demonstrated the usefulness of this system in order for evaluating the effect of transfected genes on the glucose-induced secretion in primary cultured pancreatic islets. Using this transfection system together with a series of constitutive active mutants, we showed that the PI3K-3-phosphoinositide dependent kinase-1 (PDK1-Akt pathway mediated the potentiation of insulin secretion. The Akt inhibitor also enhanced the glucose-induced insulin secretion in parallel with the upregulation of newcomer granule fusions, probably via increased motility of intracellular insulin granules. These data suggest that the PI3K-PDK1-Akt pathway plays a significant role in newcomer granule fusions, probably through an alteration of the dynamics of the intracellular insulin granules.

  20. Ultrasound-Targeted Microbubble Destruction Improves the Migration and Homing of Mesenchymal Stem Cells after Myocardial Infarction by Upregulating SDF-1/CXCR4: A Pilot Study

    Directory of Open Access Journals (Sweden)

    Lu Li

    2015-01-01

    Full Text Available Mesenchymal stem cell (MSC therapy shows considerable promise for the treatment of myocardial infarction (MI. However, the inefficient migration and homing of MSCs after systemic infusion have limited their therapeutic applications. Ultrasound-targeted microbubble destruction (UTMD has proven to be promising to improve the homing of MSCs to the ischemic myocardium, but the concrete mechanism remains unclear. We hypothesize that UTMD promotes MSC homing by upregulating SDF-1/CXCR4, and this study was aimed at exploring this potential mechanism. We analyzed SDF-1/CXCR4 expression after UTMD treatment in vitro and in vivo and counted the number of homing MSCs in MI areas. The in vitro results demonstrated that UTMD not only led to elevated secretion of SDF-1 but also resulted in an increased proportion of MSCs that expressed surface CXCR4. The in vivo findings show an increase in the number of homing MSCs and higher expression of SDF-1/CXCR4 in the UTMD combined with MSCs infusion group compared to other groups. In conclusion, UTMD can increase SDF-1 expression in the ischemic myocardium and upregulate the expression of surface CXCR4 on MSCs, which provides a molecular mechanism for the homing of MSCs assisted by UTMD via SDF-1/CXCR4 axis.

  1. T-cell factor-4 and MHC upregulation in pigs receiving a live attenuated classical swine fever virus (CSFV) vaccine strain with interferon-gamma adjuvant.

    Science.gov (United States)

    Fan, Y-H; Lin, Y-L; Hwang, Y-C; Yang, H-C; Chiu, H-C; Chiou, S-H; Jong, M-H; Chow, K-C; Lin, C-C

    2016-10-01

    The effect of co-administration of interferon (IFN)-γ in pigs undergoing vaccination with an attenuated strain (LPC) of classical swine fever virus (CSFV) was investigated. Unvaccinated pigs demonstrated pyrexia and died 7-9 days after challenge with virulent CSFV. Pigs receiving the attenuated vaccine remained healthy after virus challenge, except for mild, transient pyrexia, whereas pigs receiving IFN-γ simultaneously with the vaccine demonstrated normal body temperatures after virus challenge. Examination by nested RT-PCR revealed greater viral load in the spleens of the pigs vaccinated with the attenuated CSFV, compared with those that had additionally received IFN-γ. Expression of major histocompatibility complex (MHC) class I and MHC class II molecules was upregulated in the spleens of the IFN-γ treated vaccinated pigs, demonstrated by immunohistochemistry. Based on Western blot analysis, anti-CSFV IgG2 antibodies were elevated in vaccinated pigs by co-administration of IFN-γ (IFN-γ(Hi): P < 0.01; IFN-γ(Lo): P <0.05). By employing the suppression subtractive hybridization technique, RT-PCR, in situ hybridization, and immunohistochemistry, T-cell factor-4 (Tcf-4) mRNA and protein expression were found to be upregulated in the spleens of vaccinated pigs that had received IFN-γ. This study suggests involvement of Tcf-4 in IFN-γ-mediated immune regulation following CSFV vaccination. PMID:27687943

  2. Up-regulation of METCAM/MUC18 promotes motility, invasion, and tumorigenesis of human breast cancer cells

    Directory of Open Access Journals (Sweden)

    Cai Shao-xi

    2011-03-01

    Full Text Available Abstract Background Conflicting research has identified METCAM/MUC18, an integral membrane cell adhesion molecule (CAM in the Ig-like gene super-family, as both a tumor promoter and a tumor suppressor in the development of breast cancer. To resolve this, we have re-investigated the role of this CAM in the progression of human breast cancer cells. Methods Three breast cancer cell lines were used for the tests: one luminal-like breast cancer cell line, MCF7, which did not express any METCAM/MUC18, and two basal-like breast cancer cell lines, MDA-MB-231 and MDA-MB-468, which expressed moderate levels of the protein. MCF7 cells were transfected with the human METCAM/MUC18 cDNA to obtain G418-resistant clones which expressed the protein and were used for testing effects of human METCAM/MUC18 expression on in vitro motility and invasiveness, and in vitro and in vivo tumorigenesis. Both MDA-MB-231 and MDA-MB-468 cells already expressed METCAM/MUC18. They were directly used for in vitro tests in the presence and absence of an anti-METCAM/MUC18 antibody. Results In MCF7 cells, enforced METCAM/MUC18 expression increased in vitro motility, invasiveness, anchorage-independent colony formation (in vitro tumorigenesis, and in vivo tumorigenesis. In both MDA-MB-231 and MDA-MB-468 cells, the anti-METCAM/MUC18 antibody inhibited both motility and invasiveness. Though both MDA-MB-231 and MDA-MB-468 cells established a disorganized growth in 3D basement membrane culture assay, the introduction of the anti-METCAM/MUC18 antibody completely destroyed their growth in the 3D culture. Conclusion These findings support the notion that human METCAM/MUC18 expression promotes the progression of human breast cancer cells by increasing their motility, invasiveness and tumorigenesis.

  3. Up-regulation of the transient A-type K+ current (IA) in the differentiation of neural stem cells of the early postnatal rat hippocampus

    Institute of Scientific and Technical Information of China (English)

    GUO Hong-bo; HUANG Lian-yan; ZOU Yu-xi; ZOU Fei

    2010-01-01

    Background Neural stem cells (NSCs) not only are essential to cell replacement therapy and transplantation in clinical settings, but also provide a unique model for the research into neurogenesis and epigenesis. However, little attention has been paid to the electrophysiological characterization of NSC development. This work aimed to identify whether the morphological neuronal differentiation process in NSCs included changes in the electrophysiological properties of transient A-type K+ currents (IA).Methods NSCs were isolated from early postnatal rat hippocampus and were multiplied in basic serum-free medium containing basic fibroblast growth factor. Potassium currents were investigated and compared using whole-cell patch-clamp techniques and one-way analysis of variance (ANOVA), respectively.Results Compared with NSC-derived neurons, cloned NSCs (cNSCs) had a more positive resting membrane potential, a higher input resistance, and a lower membrane capacitance. Part of cNSCs and NSC-derived neurons possessed both delayed-rectifier K+ currents (IDR) and IA, steady-state activation of IA in cNSCs (half-maximal activation at (21.34±4.37) mV) occurred at a more positive voltage than in NSC-derived neurons at 1-6 days in vitro (half-maximal activation at (12.85±4.19) mV).Conclusions Our research revealed a developmental up-regulation of the IA component during differentiation of postnatal NSCs. Together with the marked developmental up-regulation of IDR in vitro neuronal differentiation we have previously found, the voltage-gated potassium channels may participate in neuronal maturation process.

  4. FOXM1 upregulation is an early event in human squamous cell carcinoma and it is enhanced by nicotine during malignant transformation.

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    Emilios Gemenetzidis

    Full Text Available BACKGROUND: Cancer associated with smoking and drinking remains a serious health problem worldwide. The survival of patients is very poor due to the lack of effective early biomarkers. FOXM1 overexpression is linked to the majority of human cancers but its mechanism remains unclear in head and neck squamous cell carcinoma (HNSCC. METHODOLOGY/PRINCIPAL FINDINGS: FOXM1 mRNA and protein expressions were investigated in four independent cohorts (total 75 patients consisting of normal, premalignant and HNSCC tissues and cells using quantitative PCR (qPCR, expression microarray, immunohistochemistry and immunocytochemistry. Effect of putative oral carcinogens on FOXM1 transcriptional activity was dose-dependently assayed and confirmed using a FOXM1-specific luciferase reporter system, qPCR, immunoblotting and short-hairpin RNA interference. Genome-wide single nucleotide polymorphism (SNP array was used to 'trace' the genomic instability signature pattern in 8 clonal lines of FOXM1-induced malignant human oral keratinocytes. Furthermore, acute FOXM1 upregulation in primary oral keratinocytes directly induced genomic instability. We have shown for the first time that overexpression of FOXM1 precedes HNSCC malignancy. Screening putative carcinogens in human oral keratinocytes surprisingly showed that nicotine, which is not perceived to be a human carcinogen, directly induced FOXM1 mRNA, protein stabilisation and transcriptional activity at concentrations relevant to tobacco chewers. Importantly, nicotine also augmented FOXM1-induced transformation of human oral keratinocytes. A centrosomal protein CEP55 and a DNA helicase/putative stem cell marker HELLS, both located within a consensus loci (10q23, were found to be novel targets of FOXM1 and their expression correlated tightly with HNSCC progression. CONCLUSIONS/SIGNIFICANCE: This study cautions the potential co-carcinogenic effect of nicotine in tobacco replacement therapies. We hypothesise that

  5. Transgenic expression of BRCA1 disturbs hematopoietic stem and progenitor cells quiescence and function

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    Bai, Lin; Shi, Guiying; Zhang, Xu; Dong, Wei; Zhang, Lianfeng, E-mail: zhanglf@cnilas.org

    2013-10-15

    The balance between quiescence and proliferation of HSCs is an important regulator of hematopoiesis. Loss of quiescence frequently results in HSCs exhaustion, which underscores the importance of tight regulation of proliferation in these cells. Studies have indicated that cyclin-dependent kinases are involved in the regulation of quiescence in HSCs. BRCA1 plays an important role in the repair of DNA double-stranded breaks, cell cycle, apoptosis and transcription. BRCA1 is expressed in the bone marrow. However, the function of BRCA1 in HSCs is unknown. In our study, we generated BRCA1 transgenic mice to investigate the effects of BRCA1 on the mechanisms of quiescence and differentiation in HSCs. The results demonstrate that over-expression of BRCA1 in the bone marrow impairs the development of B lymphocytes. Furthermore, BRCA1 induced an increase in the number of LSKs, LT-HSCs, ST-HSCs and MPPs. A competitive transplantation assay found that BRCA1 transgenic mice failed to reconstitute hematopoiesis. Moreover, BRCA1 regulates the expression of p21{sup waf1}/cip1 and p57{sup kip2}, which results in a loss of quiescence in LSKs. Together, over-expression of BRCA1 in bone marrow disrupted the quiescent of LSKs, induced excessive accumulation of LSKs, and disrupted differentiation of the HSCs, which acts through the down-regulated of p21{sup waf1}/cip1 and p57{sup kip2}. - Highlights: • Over-expression of BRCA1 results in imp