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Sample records for downregulates p75ntr expression

  1. p75NTR enhances PC12 cell tumor growth by a non-receptor mechanism involving downregulation of cyclin D2

    International Nuclear Information System (INIS)

    Fritz, Melinda D.; Mirnics, Zeljka K.; Nylander, Karen D.; Schor, Nina F.

    2006-01-01

    p75NTR is a member of the tumor necrosis superfamily of proteins which is variably associated with induction of apoptosis and proliferation. Cyclin D2 is one of the mediators of cellular progression through G1 phase of the cell cycle. The present study demonstrates the inverse relationship between expression of cyclin D2 and expression of p75NTR in PC12 cells. Induction of p75NTR expression in p75NTR-negative PC12 cells results in downregulation of cyclin D2; suppression of p75NTR expression with siRNA in native PC12 cells results in upregulation of cyclin D2. The effects of p75NTR on cyclin D2 expression are mimicked in p75NTR-negative cells by transfection with the intracellular domain of p75NTR. Cyclin-D2-positive PC12 cell cultures grow more slowly than cyclin-D2-negative cultures, and induction of expression of cyclin D2 slows the culture growth rate of cyclin-D2-negative cells. Finally, subcutaneous murine xenografts of cyclin-D2-negative, p75NTR-positive PC12 cells more frequently and more rapidly produce tumors than the analogous xenografts of cyclin-D2-positive, p75NTR-negative cells. These results suggest that p75NTR suppresses cyclin D2 expression in PC12 cells by a mechanism distinct from its function as a nerve growth factor receptor and that cyclin D2 expression decreases cell culture and xenografted tumor growth

  2. Changes in expression of BDNF and its receptors TrkB and p75NTR in the hippocampus of a dog model of chronic alcoholism and abstinence

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    Xu, R.; Duan, S.R.; Zhao, J.W.; Wang, C.Y. [Neurology Ward of Internal Medicine, First Affiliated Hospital of Harbin Medical University, Harbin, Heilongjiang Province (China)

    2015-06-23

    Chronic ethanol consumption can produce learning and memory deficits. Brain-derived neurotrophic factor (BDNF) and its receptors affect the pathogenesis of alcoholism. In this study, we examined the expression of BDNF, tropomyosin receptor kinase B (TrkB) and p75 neurotrophin receptor (p75NTR) in the hippocampus of a dog model of chronic alcoholism and abstinence. Twenty domestic dogs (9-10 months old, 15-20 kg; 10 males and 10 females) were obtained from Harbin Medical University. A stable alcoholism model was established through ad libitum feeding, and anti-alcohol drug treatment (Zhong Yao Jie Jiu Ling, the main ingredient was the stems of watermelon; developed in our laboratory), at low- and high-doses, was carried out. The Zhong Yao Jie Jiu Ling was effective for the alcoholism in dogs. The morphology of hippocampal neurons was evaluated using hematoxylin-eosin staining. The number and morphological features of BDNF, TrkB and p75NTR-positive neurons in the dentate gyrus (DG), and the CA1, CA3 and CA4 regions of the hippocampus were observed using immunohistochemistry. One-way ANOVA was used to determine differences in BDNF, TrkB and p75NTR expression. BDNF, TrkB and p75NTR-positive cells were mainly localized in the granular cell layer of the DG and in the pyramidal cell layer of the CA1, CA3 and CA4 regions (DG>CA1>CA3>CA4). Expression levels of both BDNF and TrkB were decreased in chronic alcoholism, and increased after abstinence. The CA4 region appeared to show the greatest differences. Changes in p75NTR expression were the opposite of those of BDNF and TrkB, with the greatest differences observed in the DG and CA4 regions.

  3. Changes in expression of BDNF and its receptors TrkB and p75NTR in the hippocampus of a dog model of chronic alcoholism and abstinence

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    R. Xu

    2015-08-01

    Full Text Available Chronic ethanol consumption can produce learning and memory deficits. Brain-derived neurotrophic factor (BDNF and its receptors affect the pathogenesis of alcoholism. In this study, we examined the expression of BDNF, tropomyosin receptor kinase B (TrkB and p75 neurotrophin receptor (p75NTR in the hippocampus of a dog model of chronic alcoholism and abstinence. Twenty domestic dogs (9-10 months old, 15-20 kg; 10 males and 10 females were obtained from Harbin Medical University. A stable alcoholism model was established through ad libitum feeding, and anti-alcohol drug treatment (Zhong Yao Jie Jiu Ling, the main ingredient was the stems of watermelon; developed in our laboratory, at low- and high-doses, was carried out. The Zhong Yao Jie Jiu Ling was effective for the alcoholism in dogs. The morphology of hippocampal neurons was evaluated using hematoxylin-eosin staining. The number and morphological features of BDNF, TrkB and p75NTR-positive neurons in the dentate gyrus (DG, and the CA1, CA3 and CA4 regions of the hippocampus were observed using immunohistochemistry. One-way ANOVA was used to determine differences in BDNF, TrkB and p75NTR expression. BDNF, TrkB and p75NTR-positive cells were mainly localized in the granular cell layer of the DG and in the pyramidal cell layer of the CA1, CA3 and CA4 regions (DG>CA1>CA3>CA4. Expression levels of both BDNF and TrkB were decreased in chronic alcoholism, and increased after abstinence. The CA4 region appeared to show the greatest differences. Changes in p75NTR expression were the opposite of those of BDNF and TrkB, with the greatest differences observed in the DG and CA4 regions.

  4. Changes in expression of BDNF and its receptors TrkB and p75NTR in the hippocampus of a dog model of chronic alcoholism and abstinence

    International Nuclear Information System (INIS)

    Xu, R.; Duan, S.R.; Zhao, J.W.; Wang, C.Y.

    2015-01-01

    Chronic ethanol consumption can produce learning and memory deficits. Brain-derived neurotrophic factor (BDNF) and its receptors affect the pathogenesis of alcoholism. In this study, we examined the expression of BDNF, tropomyosin receptor kinase B (TrkB) and p75 neurotrophin receptor (p75NTR) in the hippocampus of a dog model of chronic alcoholism and abstinence. Twenty domestic dogs (9-10 months old, 15-20 kg; 10 males and 10 females) were obtained from Harbin Medical University. A stable alcoholism model was established through ad libitum feeding, and anti-alcohol drug treatment (Zhong Yao Jie Jiu Ling, the main ingredient was the stems of watermelon; developed in our laboratory), at low- and high-doses, was carried out. The Zhong Yao Jie Jiu Ling was effective for the alcoholism in dogs. The morphology of hippocampal neurons was evaluated using hematoxylin-eosin staining. The number and morphological features of BDNF, TrkB and p75NTR-positive neurons in the dentate gyrus (DG), and the CA1, CA3 and CA4 regions of the hippocampus were observed using immunohistochemistry. One-way ANOVA was used to determine differences in BDNF, TrkB and p75NTR expression. BDNF, TrkB and p75NTR-positive cells were mainly localized in the granular cell layer of the DG and in the pyramidal cell layer of the CA1, CA3 and CA4 regions (DG>CA1>CA3>CA4). Expression levels of both BDNF and TrkB were decreased in chronic alcoholism, and increased after abstinence. The CA4 region appeared to show the greatest differences. Changes in p75NTR expression were the opposite of those of BDNF and TrkB, with the greatest differences observed in the DG and CA4 regions

  5. Changes in expression of BDNF and its receptors TrkB and p75NTR in the hippocampus of a dog model of chronic alcoholism and abstinence.

    Science.gov (United States)

    Xu, R; Duan, S R; Zhao, J W; Wang, C Y

    2015-08-01

    Chronic ethanol consumption can produce learning and memory deficits. Brain-derived neurotrophic factor (BDNF) and its receptors affect the pathogenesis of alcoholism. In this study, we examined the expression of BDNF, tropomyosin receptor kinase B (TrkB) and p75 neurotrophin receptor (p75NTR) in the hippocampus of a dog model of chronic alcoholism and abstinence. Twenty domestic dogs (9-10 months old, 15-20 kg; 10 males and 10 females) were obtained from Harbin Medical University. A stable alcoholism model was established through ad libitum feeding, and anti-alcohol drug treatment (Zhong Yao Jie Jiu Ling, the main ingredient was the stems of watermelon; developed in our laboratory), at low- and high-doses, was carried out. The Zhong Yao Jie Jiu Ling was effective for the alcoholism in dogs. The morphology of hippocampal neurons was evaluated using hematoxylin-eosin staining. The number and morphological features of BDNF, TrkB and p75NTR-positive neurons in the dentate gyrus (DG), and the CA1, CA3 and CA4 regions of the hippocampus were observed using immunohistochemistry. One-way ANOVA was used to determine differences in BDNF, TrkB and p75NTR expression. BDNF, TrkB and p75NTR-positive cells were mainly localized in the granular cell layer of the DG and in the pyramidal cell layer of the CA1, CA3 and CA4 regions (DG>CA1>CA3>CA4). Expression levels of both BDNF and TrkB were decreased in chronic alcoholism, and increased after abstinence. The CA4 region appeared to show the greatest differences. Changes in p75NTR expression were the opposite of those of BDNF and TrkB, with the greatest differences observed in the DG and CA4 regions.

  6. [NRH2 induces cell apoptosis of cerebral tissues around hematomas after intracerebral hemorrhage through up-regulating proNGF, sortilin and p75NTR expressions].

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    Zeng, Zhiqing; Liu, Hong; Jiang, Di

    2015-04-01

    To observe the expressions of neurotrophin receptor homolog 2 (NRH2), nerve growth factor precursor (proNGF), sortilin and neurotrophin receptor p75 (p75NTR) in cerebral tissues around hematomas in the different periods after intracerebral hemorrhage, and explore their relationships to cell apoptosis. The specimens of cerebral tissues around hematomas were collected from the patients undergoing hematoma removal operation after intracerebral hemorrhage. These specimens were divided into four groups, namely ≤ 6 hours, 6-24 hours(including 24 hours), 24-72 hours (including 72 hours) and over 72 hours according to the time from intracerebral hemorrhage to specimen collection. At the same time, 10 brain tissues distant to hemorrhage that dropped in the operative process were collected as a control group. Apoptosis index (AI) was examined in brain cells by terminal deoxynucleotidyl transferase mediated deoxyuridine triphosphate nick end labeling (TUNEL). The expressions of NRH2, proNGF, sortilin and p75NTR mRNAs and proteins in brain tissues were detected through real-time quantitative PCR and Western blotting, respectively. Also, the expressions of Bcl-2 and Bax in brain tissues were analyzed using Western blotting. In vitro cultured astrocytes of rat cortex were transfected by NRH2 siRNA or scramble siRNA. The expressions of proNGF, sortilin and p75NTR proteins were detected using Western blotting. AI was higher in all groups of hemorrhage for 6 hours or longer than that in control and ≤ 6 hours groups, and AI in the group of 24-72 hours after intracerebral hemorrhage was the highest. However, there was no significant difference in AI between ≤ 6 hours group and control group. With the extension of intracerebral hemorrhage time, the expression levels of proNGF and p75NTR mRNAs and proteins were gradually elevated, reached the peak in 24-72 hours, and maintained a higher level after 72 hours, whereas there were no significant differences in the above indicators

  7. NADE, a p75NTR-associated cell death executor, is involved in signal transduction mediated by the common neurotrophin receptor p75NTR.

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    Mukai, J; Hachiya, T; Shoji-Hoshino, S; Kimura, M T; Nadano, D; Suvanto, P; Hanaoka, T; Li, Y; Irie, S; Greene, L A; Sato, T A

    2000-06-09

    The low affinity neurotrophin receptor p75NTR can mediate cell survival as well as cell death of neural cells by NGF and other neurotrophins. To elucidate p75NTR-mediated signal transduction, we screened p75NTR-associated proteins by a yeast two-hybrid system. We identified one positive clone and named NADE (p75NTR-associated cell death executor). Mouse NADE has marked homology to the human HGR74 protein. NADE specifically binds to the cell-death domain of p75NTR. Co-expression of NADE and p75NTR induced caspase-2 and caspase-3 activities and the fragmentation of nuclear DNA in 293T cells. However, in the absence of p75NTR, NADE failed to induce apoptosis, suggesting that NADE expression is necessary but insufficient for p75NTR-mediated apoptosis. Furthermore, p75NTR/NADE-induced cell death was dependent on NGF but not BDNF, NT-3, or NT-4/5, and the recruitment of NADE to p75NTR (intracellular domain) was dose-dependent. We obtained similar results from PC12 cells, nnr5 cells, and oligodendrocytes. Taken together, NADE is the first signaling adaptor molecule identified in the involvement of p75NTR-mediated apoptosis induced by NGF, and it may play an important role in the pathogenesis of neurogenetic diseases.

  8. Expression and Significance of Stem Cell Markers CK19, Notch3, CD133, P75NTR, STRO-1 and ABCG2 in Pulmonary Squamous Carcinomas

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    Xuyong LIN, , , , ,

    2009-04-01

    Full Text Available Background and objective Increasing reports showed that some tumor stem cells were selfrenewal and multi-lineage differentiated in tumors, similar to the normal stem cells in human body. The aim of this study is to observe the expression of stem cell markers in lung squamous carcinoma tissues. Methods Fifty-four lung cancer specimens from surgery were analyzed for CK19, Notch3, CD133, P75NTR, STRO-1 and ABCG2 expression by using S-P immunohistochemistry. In addition, ten normal lung tissue samples were included as control. Results CK19, Notch3, CD133 and ABCG2 were expressed in 54 Lung cancer tissues, without expression of P75NTR and STRO-1. The expressionrate of CK19, Notch3, CD133 and ABCG2 was 66.67% (36/54, 87.04% (47/54, 50% (27/54, and 61.11% (33/54 respectively. The levels of expression of Notch3, CD133 and ABCG2 were significantly lower in high differentiation group than those in moderate and low differentiation group (P <0.05. The levels of expression of CK19, CD133 and ABCG2 were significantly higher in lymph node metastasis group than those in non-metastasis group (P <0.05. The percentage of total positive cells of four stem cell markers in serial tissue sections was lower than 2%. Conclusion There was expression ofsome stem cell markers in pulmonary squamous carcinomas, and there was relationship between expression degree withdifferentiation degree and lymph node metastasis.

  9. Self-renewal and chemotherapy resistance of p75NTR positive cells in esophageal squamous cell carcinomas

    International Nuclear Information System (INIS)

    Huang, Sheng-Dong; Yuan, Yang; Liu, Xiao-Hong; Gong, De-Jun; Bai, Chen-Guang; Wang, Feng; Luo, Jun-Hui; Xu, Zhi-Yun

    2009-01-01

    p75 NTR has been used to isolate esophageal and corneal epithelial stem cells. In the present study, we investigated the expression of p75 NTR in esophageal squamous cell carcinoma (ESCC) and explored the biological properties of p75 NTR+ cells. p75 NTR expression in ESCC was assessed by immunohistochemistry. p75 NTR+ and p75 NTR- cells of 4 ESCC cell lines were separated by fluorescence-activated cell sorting. Differentially expressed genes between p75 NTR+ and p75 NTR- cells were determined by real-time quantitative reverse transcription-PCR. Sphere formation assay, DDP sensitivity assay, 64 copper accumulation assay and tumorigenicity analysis were performed to determine the capacity of self-renewal, chemotherapy resistance and tumorigenicity of p75 NTR+ cells. In ESCC specimens, p75 NTR was found mainly confined to immature cells and absent in cells undergoing terminal differentiation. The percentage of p75 NTR+ cells was 1.6%–3.7% in Eca109 and 3 newly established ESCC cell lines. The expression of Bmi-1, which is associated with self-renewal of stem cells, was significantly higher in p75 NTR+ cells. p63, a marker identified in keratinocyte stem cells, was confined mainly to p75 NTR+ cells. The expression of CTR1, which is associated with cisplatin (DDP)-resistance, was significantly decreased in p75 NTR+ cells. Expression levels of differentiation markers, such as involucrin, cytokeratin 13, β1-integrin and β4-integrin, were lower in p75 NTR+ cells. In addition, p75 NTR+ cells generated both p75 NTR+ and p75 NTR- cells, and formed nonadherent spherical clusters in serum-free medium supplemented with growth factors. Furthermore, p75 NTR+ cells were found to be more resistant to DDP and exhibited lower 64 copper accumulation than p75 NTR- cells. Our results demonstrated that p75 NTR+ cells possess some characteristics of CSCs, namely, self-renewal and chemotherapy resistance. Chemotherapy resistance of p75 NTR+ cells may probably be attributable to

  10. The p75NTR mediates a bifurcated signal transduction cascade through the NFκB and JNK pathways to inhibit cell survival

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    Allen, Jeffrey; Khwaja, Fatima; Byers, Stephen; Djakiew, Daniel

    2005-01-01

    p75 NTR is most abundantly expressed in the nervous system, but is also widely expressed in many other organs and tissues where it primarily functions as a negative regulator of cell survival. In the prostate, p75 NTR functions as an inhibitory protein capable of slowing proliferation and inducing apoptosis. It has been shown that p75 NTR is expressed in the normal prostate, progressively lost from malignant tumor cells in vivo, and largely absent from prostate cancer cell lines derived from metastases. Although the role of p75 NTR in prostate cancer has been well established, the signal transduction pathway that mediates its inhibitory activity has only been partially elucidated. This study demonstrates that exogenous expression of p75 NTR down-regulates, in a dose-dependent manner, a bifurcated signaling cascade that results in reduced expression of potent transcription effectors. This two-arm signal transduction cascade was directly linked to the upstream receptor by using dominant-negative deletion constructs of p75 NTR that rescued tumor cells from p75 NTR -induced loss of survival and promotion of apoptosis. Furthermore, the dominant negatives rescued alterations in the levels of signal transduction intermediates. Conversely, the use of kinase-inactive intermediates that are downstream of the receptor further reduced expression of involved transcription effectors and reduced survival of the cells. These results provide a definitive link between the proximate p75 NTR and signal transduction intermediates leading to the transcription effectors NFκB and JNK, with associated growth suppression and induction of apoptosis

  11. Reduced anxiety-like behavior and altered hippocampal morphology in female p75NTR exon IV-/- mice.

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    Zoe ePuschban

    2016-06-01

    Full Text Available The presence of the neurotrophin receptor p75NTR in adult basal forebrain cholinergic neurons, precursor cells in the subventricular cell layer and the subgranular cell layer of the hippocampus has been linked to alterations in learning as well as anxiety- and depression- related behaviors. In contrast to previous studies performed in a p75NTR exonIII-/- model still expressing the short isoform of the p75NTR, we focused on locomotor and anxiety–associated behavior in p75NTR exonIV-/- mice lacking both p75NTR isoforms. Comparing p75NTR exonIV-/- and wildtype mice for both male and female animals showed an anxiolytic-like behavior as evidenced by increased central activities in the open field paradigm and flex field activity system as well as higher numbers of open arm entries in the elevated plus maze test in female p75NTR knockout mice.Morphometrical analyses of dorsal and ventral hippocampus revealed a reduction of width of the dentate gyrus and the granular cell layer in the dorsal but not ventral hippocampus in male and female p75NTR exonIV -/- mice. We conclude that germ-line deletion of p75NTR seems to differentially affect morphometry of dorsal and ventral dentate gyrus and that p75NTR may play a role in anxiety-like behavior, specifically in female mice.

  12. The p75NTR tumor suppressor induces cell cycle arrest facilitating caspase mediated apoptosis in prostate tumor cells

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    Khwaja, Fatima; Tabassum, Arshia; Allen, Jeff; Djakiew, Daniel

    2006-01-01

    The p75 neurotrophin receptor (p75 NTR ) is a death receptor which belongs to the tumor necrosis factor receptor super-family of membrane proteins. This study shows that p75 NTR retarded cell cycle progression by induced accumulation of cells in G0/G1 and a reduction in the S phase of the cell cycle. The rescue of tumor cells from cell cycle progression by a death domain deleted (ΔDD) dominant-negative antagonist of p75 NTR showed that the death domain transduced anti-proliferative activity in a ligand-independent manner. Conversely, addition of NGF ligand rescued retardation of cell cycle progression with commensurate changes in components of the cyclin/cdk holoenzyme complex. In the absence of ligand, p75 NTR -dependent cell cycle arrest facilitated an increase in apoptotic nuclear fragmentation of the prostate cancer cells. Apoptosis of p75 NTR expressing cells occurred via the intrinsic mitochondrial pathway leading to a sequential caspase-9 and -7 cascade. Since the death domain deleted dominant-negative antagonist of p75 NTR rescued intrinsic caspase associated apoptosis in PC-3 cells, this shows p75 NTR was integral to ligand independent induction of apoptosis. Moreover, the ability of ligand to ameliorate the p75 NTR -dependent intrinsic apoptotic cascade indicates that NGF functioned as a survival factor for p75 NTR expressing prostate cancer cells

  13. Co-induction of p75NTR and p75NTR-associated death executor in neurons after zinc exposure in cortical culture or transient ischemia in the rat.

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    Park, J A; Lee, J Y; Sato, T A; Koh, J Y

    2000-12-15

    Recently, a 22 kDa protein termed p75(NTR)-associated death executor (NADE) was discovered to be a necessary factor for p75(NTR)-mediated apoptosis in certain cells. However, the possible role for p75(NTR)/NADE in pathological neuronal death has yet been undetermined. In the present study, we have examined this possibility in vivo and in vitro. Exposure of cortical cultures to zinc induced both p75(NTR) and NADE in neurons, whereas exposure to NMDA, ionomycin, iron, or H(2)O(2) induced neither. In addition, zinc exposure increased neuronal NGF expression and its release into the medium. A function-blocking antibody of p75(NTR) (REX) inhibited association between p75(NTR) and NADE as well as neuronal death induced by zinc. Conversely, NGF augmented zinc-induced neuronal death. Caspase inhibitors reduced zinc-induced neuronal death, indicating that caspases were involved. Because reduction of NADE expression with cycloheximide or NADE antisense oligonucleotides attenuated zinc-induced neuronal death, NADE appears to contribute to p75(NTR)-induced cortical neuronal death as shown in other cells. Because zinc neurotoxicity may be a key mechanism of neuronal death after transient forebrain ischemia, we next examined this model. After ischemia, p75(NTR) and NADE were induced in degenerating rat hippocampal CA1 neurons. There was a close correlation between zinc accumulation and p75(NTR)/NADE induction. Suggesting the role of zinc here, injection of a metal chelator, CaEDTA, into the lateral ventricle completely blocked the induction of p75(NTR) and NADE. Our results suggest that co-induction of p75(NTR) and NADE plays a role in zinc-triggered neuronal death in vitro and in vivo.

  14. Carprofen Induction of p75NTR Dependent Apoptosis via the p38 MAPK Pathway in Prostate Cancer Cells

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    Khwaja, Fatima S.; Quann, Emily J.; Pattabiraman, Nagarajan; Wynne, Shehla; Djakiew, Daniel

    2008-01-01

    The p75NTR functions as a tumor suppressor in prostate epithelial cells, where its expression declines with progression to malignant cancer. Previously, we demonstrated that treatment with R-flurbiprofen or ibuprofen induced p75NTR expression in several prostate cancer cell lines leading to p75NTR mediated decreased survival. Utilizing the 2-phenyl propionic acid moiety of these profens as a pharmacophore, we screened an in silico data base of 30 million compounds and identified carprofen as having an order of magnitude greater activity for induction of p75NTR levels and inhibition of cell survival. Prostate (PC-3, DU-145) and bladder (T24) cancer cells were more sensitive to carprofen induction of p75NTR associated loss of survival than breast (MCF7) and fibroblast (3T3) cells. Transfection of prostate cell lines with a dominant negative form of p75NTR prior to carprofen treatment partially rescued cell survival demonstrating a cause and effect relationship between carprofen induction of p75NTR levels and inhibition of survival. Carprofen induced apoptotic nuclear fragmentation in prostate but not in MCF7 and 3T3 cells. Furthermore, siRNA knockdown of the p38 MAPK protein prevented induction of p75NTR by carprofen in both prostate cell lines. Carprofen treatment induced phosphorylation of p38 MAPK as early as within 1 minute. Expression of a dominant negative form of MK2, the kinase downstream of p38 MAPK frequently associated with signaling cascades leading to apoptosis, prevented carprofen induction of the p75NTR protein. Collectively, we identify carprofen as a highly potent profen capable of inducing p75NTR dependent apoptosis via the p38 MAPK pathway in prostate cancer cells. PMID:18974393

  15. Cholinergic Degeneration and Alterations in the TrkA and p75NTR Balance as a Result of Pro-NGF Injection into Aged Rats

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    Ashley M. Fortress

    2011-01-01

    Full Text Available Learning and memory impairments occurring with Alzheimer's disease (AD are associated with degeneration of the basal forebrain cholinergic neurons (BFCNs. BFCNs extend their axons to the hippocampus where they bind nerve growth factor (NGF which is retrogradely transported to the cell body. While NGF is necessary for BFCN survival and function via binding to the high-affinity receptor TrkA, its uncleaved precursor, pro-NGF has been proposed to induce neurodegeneration via binding to the p75NTR and its coreceptor sortilin. Basal forebrain TrkA and NGF are downregulated with aging while pro-NGF is increased. Given these data, the focus of this paper was to determine a mechanism for how pro-NGF accumulation may induce BFCN degeneration. Twenty-four hours after a single injection of pro-NGF into hippocampus, we found increased hippocampal p75NTR levels, decreased hippocampal TrkA levels, and cholinergic degeneration. The data suggest that the increase in p75NTR with AD may be mediated by elevated pro-NGF levels as a result of decreased cleavage, and that pro-NGF may be partially responsible for age-related degenerative changes observed in the basal forebrain. This paper is the first in vivo evidence that pro-NGF can affect BFCNs and may do so by regulating expression of p75NTR neurotrophin receptors.

  16. Neurotrophin Receptor p75NTR Regulates Immune Function of Plasmacytoid Dendritic Cells

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    Joanna Bandoła

    2017-08-01

    Full Text Available Plasmacytoid dendritic cells (pDCs regulate innate and adaptive immunity. Neurotrophins and their receptors control the function of neuronal tissue. In addition, they have been demonstrated to be part of the immune response but little is known about the effector immune cells involved. We report, for the first time, the expression and immune-regulatory function of the low affinity neurotrophin receptor p75 neurotrophin receptor (p75NTR by the antigen-presenting pDCs, mediated by toll-like receptor (TLR 9 activation and differential phosphorylation of interferon regulatory factor 3 and 7. The modulation of p75NTR on pDCs significantly influences disease progression of asthma in an ovalbumin-induced mouse model mediated by the TLR9 signaling pathway. p75NTR activation of pDCs from patients with asthma increased allergen-specific T cell proliferation and cytokine secretion in nerve growth factor concentration-dependent manner. Further, p75NTR activation of pDCs delayed the onset of autoimmune diabetes in RIP-CD80GP mice and aggravated graft-versus-host disease in a xenotransplantation model. Thus, p75NTR signaling on pDCs constitutes a new and critical mechanism connecting neurotrophin signaling and immune response regulation with great therapeutic potential for a variety of immune disorders.

  17. Neurotrophin Receptor p75NTR Regulates Immune Function of Plasmacytoid Dendritic Cells.

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    Bandoła, Joanna; Richter, Cornelia; Ryser, Martin; Jamal, Arshad; Ashton, Michelle P; von Bonin, Malte; Kuhn, Matthias; Dorschner, Benjamin; Alexopoulou, Dimitra; Navratiel, Katrin; Roeder, Ingo; Dahl, Andreas; Hedrich, Christian M; Bonifacio, Ezio; Brenner, Sebastian; Thieme, Sebastian

    2017-01-01

    Plasmacytoid dendritic cells (pDCs) regulate innate and adaptive immunity. Neurotrophins and their receptors control the function of neuronal tissue. In addition, they have been demonstrated to be part of the immune response but little is known about the effector immune cells involved. We report, for the first time, the expression and immune-regulatory function of the low affinity neurotrophin receptor p75 neurotrophin receptor (p75NTR) by the antigen-presenting pDCs, mediated by toll-like receptor (TLR) 9 activation and differential phosphorylation of interferon regulatory factor 3 and 7. The modulation of p75NTR on pDCs significantly influences disease progression of asthma in an ovalbumin-induced mouse model mediated by the TLR9 signaling pathway. p75NTR activation of pDCs from patients with asthma increased allergen-specific T cell proliferation and cytokine secretion in nerve growth factor concentration-dependent manner. Further, p75NTR activation of pDCs delayed the onset of autoimmune diabetes in RIP-CD80GP mice and aggravated graft-versus-host disease in a xenotransplantation model. Thus, p75NTR signaling on pDCs constitutes a new and critical mechanism connecting neurotrophin signaling and immune response regulation with great therapeutic potential for a variety of immune disorders.

  18. Characterization of NGF, trkANGFR, and p75NTR in Retina of Mice Lacking Reelin Glycoprotein

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    Bijorn Omar Balzamino

    2014-01-01

    Full Text Available Both Reelin and Nerve Growth Factor (NGF exert crucial roles in retinal development. Retinogenesis is severely impaired in E-reeler mice, a model of Reelin deficiency showing specific Green Fluorescent Protein expression in Rod Bipolar Cells (RBCs. Since no data are available on Reelin and NGF cross-talk, NGF and trkANGFR/ p75NTR expression was investigated in retinas from E-reeler versus control mice, by confocal microscopy, Western blotting, and real time PCR analysis. A scattered increase of NGF protein was observed in the Ganglion Cell Layer and more pronounced in the Inner Nuclear Layer (INL. A selective increase of p75NTR was detected in most of RBCs and in other cell subtypes of INL. On the contrary, a slight trend towards a decrease was detected for trkANGFR, albeit not significant. Confocal data were validated by Western blot and real time PCR. Finally, the decreased trkANGFR/ p75NTR ratio, representative of p75NTR increase, significantly correlated with E-reeler versus E-control. These data indicate that NGF-trkANGFR/ p75NTR is affected in E-reeler retina and that p75NTR might represent the main NGF receptor involved in the process. This first NGF-trkANGFR/ p75NTR characterization suggests that E-reeler might be suitable for exploring Reelin-NGF cross-talk, representing an additional information source in those pathologies characterized by retinal degeneration.

  19. Carprofen induction of p75NTR-dependent apoptosis via the p38 mitogen-activated protein kinase pathway in prostate cancer cells.

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    Khwaja, Fatima S; Quann, Emily J; Pattabiraman, Nagarajan; Wynne, Shehla; Djakiew, Daniel

    2008-11-01

    The p75 neurotrophin receptor (p75(NTR)) functions as a tumor suppressor in prostate epithelial cells, where its expression declines with progression to malignant cancer. Previously, we showed that treatment with R-flurbiprofen or ibuprofen induced p75(NTR) expression in several prostate cancer cell lines leading to p75(NTR)-mediated decreased survival. Using the 2-phenyl propionic acid moiety of these profens as a pharmacophore, we screened an in silico database of 30 million compounds and identified carprofen as having an order of magnitude greater activity for induction of p75(NTR) levels and inhibition of cell survival. Prostate (PC-3 and DU-145) and bladder (T24) cancer cells were more sensitive to carprofen induction of p75(NTR)-associated loss of survival than breast (MCF-7) and fibroblast (3T3) cells. Transfection of prostate cell lines with a dominant-negative form of p75(NTR) before carprofen treatment partially rescued cell survival, showing a cause-and-effect relationship between carprofen induction of p75(NTR) levels and inhibition of survival. Carprofen induced apoptotic nuclear fragmentation in prostate but not in MCF-7 and 3T3 cells. Furthermore, small interfering RNA knockdown of the p38 mitogen-activated protein kinase (MAPK) protein prevented induction of p75(NTR) by carprofen in both prostate cell lines. Carprofen treatment induced phosphorylation of p38 MAPK as early as within 1 min. Expression of a dominant-negative form of MK2, the kinase downstream of p38 MAPK frequently associated with signaling cascades leading to apoptosis, prevented carprofen induction of the p75(NTR) protein. Collectively, we identify carprofen as a highly potent profen capable of inducing p75(NTR)-dependent apoptosis via the p38 MAPK pathway in prostate cancer cells.

  20. Silencing p75NTR prevents proNGF-induced endothelial cell death and development of acellular capillaries in rat retina

    Directory of Open Access Journals (Sweden)

    Ahmed Y Shanab

    Full Text Available Accumulation of the nerve growth factor precursor (proNGF and its receptor p75NTR have been associated with several neurodegenerative diseases in both brain and retina. However, whether proNGF contributes to microvascular degeneration remain unexplored. This study seeks to investigate the mechanism by which proNGF/p75NTR induce endothelial cell (EC death and development of acellular capillaries, a surrogate marker of retinal ischemia. Stable overexpression of the cleavage-resistant proNGF and molecular silencing of p75NTR were utilized in human retinal EC and rat retinas in vivo. Stable overexpression of proNGF decreased NGF levels and induced retinal vascular cell death evident by 1.9-fold increase in acellular capillaries and activation of JNK and cleaved-PARP that were mitigated by p75NTRshRNA. In vitro, overexpression of proNGF did not alter TNF-α level, reduced NGF, however induced EC apoptosis evident by activation of JNK and p38 MAPK, cleaved-PARP. Silencing p75NTR using siRNA restored expression of NGF and TrkA activation and prevented EC apoptosis. Treatment of EC with human-mutant proNGF induced apoptosis that coincided with marked protein interaction and nuclear translocation of p75NTR and the neurotrophin receptor interacting factor. These effects were abolished by a selective p75NTR antagonist. Therefore, targeting p75NTR represents a potential therapeutic strategy for diseases associated with aberrant expression of proNGF.

  1. Neurotrophin Receptors TrkA, p75NTR, and Sortilin Are Increased and Targetable in Thyroid Cancer.

    Science.gov (United States)

    Faulkner, Sam; Jobling, Philip; Rowe, Christopher W; Rodrigues Oliveira, S M; Roselli, Severine; Thorne, Rick F; Oldmeadow, Christopher; Attia, John; Jiang, Chen Chen; Zhang, Xu Dong; Walker, Marjorie M; Hondermarck, Hubert

    2018-01-01

    Neurotrophin receptors are emerging targets in oncology, but their clinicopathologic significance in thyroid cancer is unclear. In this study, the neurotrophin tyrosine receptor kinase TrkA (also called NTRK1), the common neurotrophin receptor p75 NTR , and the proneurotrophin receptor sortilin were analyzed with immunohistochemistry in a cohort of thyroid cancers (n = 128) and compared with adenomas and normal thyroid tissues (n = 62). TrkA was detected in 20% of thyroid cancers, compared with none of the benign samples (P = 0.0007). TrkA expression was independent of histologic subtypes but associated with lymph node metastasis (P = 0.0148), suggesting the involvement of TrkA in tumor invasiveness. Nerves in the tumor microenvironment were positive for TrkA. p75 NTR was overexpressed in anaplastic thyroid cancers compared with papillary and follicular subtypes (P Neurotrophin receptor expression was confirmed in a panel of thyroid cancer cell lines at the mRNA and protein levels. Functional investigations using the anaplastic thyroid cancer cell line CAL-62 found that siRNA against TrkA, p75 NTR , and sortilin decreased cell survival and cell migration through decreased SRC and ERK activation. Together, these data reveal TrkA, p75 NTR , and sortilin as potential therapeutic targets in thyroid cancer. Copyright © 2018 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

  2. NADE (p75NTR-associated cell death executor) suppresses cellular growth in vivo.

    Science.gov (United States)

    Tong, Xiangjun; Xie, Dong; Roth, Wilfried; Reed, John; Koeffler, H Phillip

    2003-06-01

    NADE, a p75NTR (low-affinity neurotrophin receptor p75) -associated cell death executor, was initially cloned from a human ovarian granulosa cell cDNA library, as an unknown protein with the name, pHGR74. It was reported to mediate nerve growth factor-induced apoptosis. We independently isolated human NADE (pHGR74) from breast cancer cell lines. Expression of NADE in various human cancer cell lines, and human and murine tissues was examined. NADE was highly expressed in human endocrine-related organs and embryotic murine tissues. Forced expression of NADE in CHO (Chinese hamster ovary) cells and MDA-MB-231 human breast cancer cells had little effect on the growth of the cells in vitro, while it dramatically suppressed cellular growth in vivo. We used the yeast two-hybrid system to search for NADE binding protein. Dynactin was identified as a candidate. The p75NTR was not found in this assay and did not co-immunoprecipitate with human NADE. Furthermore, the cells stably transfected with NADE did not respond to NGF or TNF. Thus, human and murine NADE appear to have different functions.

  3. The Role of Neurotrophin Signaling in Gliomagenesis: A Focus on the p75 Neurotrophin Receptor (p75NTR/CD271).

    Science.gov (United States)

    Alshehri, M M; Robbins, S M; Senger, D L

    2017-01-01

    The p75 neurotrophin receptor (p75 NTR , a.k.a. CD271), a transmembrane glycoprotein and a member of the tumor necrosis family (TNF) of receptors, was originally identified as a nerve growth factor receptor in the mid-1980s. While p75 NTR is recognized to have important roles during neural development, its presence in both neural and nonneural tissues clearly supports the potential to mediate a broad range of functions depending on cellular context. Using an unbiased in vivo selection paradigm for genes underlying the invasive behavior of glioma, a critical characteristic that contributes to poor clinical outcome for glioma patients, we identified p75 NTR as a central regulator of glioma invasion. Herein we review the expanding role that p75 NTR plays in glioma progression with an emphasis on how p75 NTR may contribute to the treatment refractory nature of glioma. Based on the observation that p75 NTR is expressed and functional in two critical glioma disease reservoirs, namely, the highly infiltrative cells that evade surgical resection, and the radiation- and chemotherapy-resistant brain tumor-initiating cells (also referred to as brain tumor stem cells), we propose that p75 NTR and its myriad of downstream signaling effectors represent rationale therapeutic targets for this devastating disease. Lastly, we provide the provocative hypothesis that, in addition to the well-documented cell autonomous signaling functions, the neurotrophins, and their respective receptors, contribute in a cell nonautonomous manner to drive the complex cellular and molecular composition of the brain tumor microenvironment, an environment that fuels tumorigenesis. © 2017 Elsevier Inc. All rights reserved.

  4. Neurotrophin receptor p75NTR mediates Huntington’s disease–associated synaptic and memory dysfunction

    Science.gov (United States)

    Brito, Verónica; Giralt, Albert; Enriquez-Barreto, Lilian; Puigdellívol, Mar; Suelves, Nuria; Zamora-Moratalla, Alfonsa; Ballesteros, Jesús J.; Martín, Eduardo D.; Dominguez-Iturza, Nuria; Morales, Miguel; Alberch, Jordi; Ginés, Sílvia

    2014-01-01

    Learning and memory deficits are early clinical manifestations of Huntington’s disease (HD). These cognitive impairments have been mainly associated with frontostriatal HD pathology; however, compelling evidence provided by several HD murine models suggests that the hippocampus may contribute to synaptic deficits and memory dysfunction in HD. The neurotrophin receptor p75NTR negatively regulates spine density, which is associated with learning and memory; therefore, we explored whether disturbed p75NTR function in the hippocampus could contribute to synaptic dysfunction and memory deficits in HD. Here, we determined that levels of p75NTR are markedly increased in the hippocampus of 2 distinct mouse models of HD and in HD patients. Normalization of p75NTR levels in HD mutant mice heterozygous for p75NTR prevented memory and synaptic plasticity deficits and ameliorated dendritic spine abnormalities, likely through normalization of the activity of the GTPase RhoA. Moreover, viral-mediated overexpression of p75NTR in the hippocampus of WT mice reproduced HD learning and memory deficits, while knockdown of p75NTR in the hippocampus of HD mice prevented cognitive decline. Together, these findings provide evidence of hippocampus-associated memory deficits in HD and demonstrate that p75NTR mediates synaptic, learning, and memory dysfunction in HD. PMID:25180603

  5. Induction of the p75NTR by Aryl Propionic Acids in Prostate Cancer Cells

    National Research Council Canada - National Science Library

    Quann, Emily

    2007-01-01

    .... I have found that treatment of prostate cancer cells with the aryl propionic acids R-flurbiprofen and ibuprofen induces reexpression of p75NTR, decreases cell survival, and increases apoptosis...

  6. Contribution of p75NTR to Schwannoma Growth and Therapeutic Responses

    Science.gov (United States)

    2017-05-01

    reducing schwannoma cell growth in culture and in animal models of human schwannoma disease. We find that the NF2 gene product, merlin, regulates p75NTR...treatment period. B. Waterfall plots of change in tumor volume for each tumor. Task 6- Define the ability of simultaneous inhibition of p75NTR and mTOR...mTOR inhibitors in culture, we have not used these compounds in animal models . Changes that had a significant impact on expenditures- None

  7. Neurotrophin p75 receptor (p75NTR) promotes endothelial cell apoptosis and inhibits angiogenesis: implications for diabetes-induced impaired neovascularization in ischemic limb muscles

    NARCIS (Netherlands)

    Caporali, Andrea; Pani, Elisabetta; Horrevoets, Anton J. G.; Kraenkel, Nicolle; Oikawa, Atsuhiko; Sala-Newby, Graciela B.; Meloni, Marco; Cristofaro, Brunella; Graiani, Gallia; Leroyer, Aurelie S.; Boulanger, Chantal M.; Spinetti, Gaia; Yoon, Sung Ok; Madeddu, Paolo; Emanueli, Costanza

    2008-01-01

    Diabetes impairs endothelial function and reparative neovascularization. The p75 receptor of neurotrophins (p75(NTR)), which is scarcely present in healthy endothelial cells (ECs), becomes strongly expressed by capillary ECs after induction of peripheral ischemia in type-1 diabetic mice. Here, we

  8. Subclinical hypothyroidism in pregnant rats impaired learning and memory of their offspring by promoting the p75NTR signal pathway

    Directory of Open Access Journals (Sweden)

    Fan Zhang

    2018-05-01

    Full Text Available Objective: Maternal hypothyroidism during pregnancy can affect the neurodevelopment of their offspring. This study aimed to investigate the effects of maternal subclinical hypothyroidism (SCH on spatial learning and memory, and its relationship with the apoptotic factors in cerebral cortex of the offspring. Methods: Female adult Wistar rats were randomly divided into three groups (n = 15 per group: control (CON group, SCH group and overt hypothyroidism (OH group. Spatial learning and memory in the offspring were evaluated by long-term potentiation (LTP and Morris water-maze (MWM test. The protein expression of the p75 neurotrophin receptor (p75NTR, phospho-c-Jun N-terminal kinase (p-JNK, the pro-apoptotic protein p53 and Bax were detected by Western blotting. Results: The Pups in the SCH and OH groups showed longer escape latencies in the MWM and decreased field-excitatory post synaptic potentials in LTP tests compared with those in the CON group. p75NTR, p-JNK, p53 and Bax expression levels in the cerebral cortex increased in pups in the SCH and OH groups compared with those in the CON group. Conclusions: Maternal SCH during pregnancy may impair spatial learning and memory in the offspring and may be associated with the increased apoptosis in the cerebral cortex.

  9. Localization of brain-derived neurotrophic factor, neurotrophin-4, tropomyosin-related kinase b receptor, and p75 NTR receptor by high-resolution immunohistochemistry on the adult mouse neuromuscular junction.

    Science.gov (United States)

    Garcia, Neus; Tomàs, Marta; Santafe, Manel M; Lanuza, M Angel; Besalduch, Nuria; Tomàs, Josep

    2010-03-01

    Neurotrophins and their receptors, the trk receptor tyrosine kinases (trks) and p75(NTR), are differentially expressed among the cell types that make up synapses. It is important to determine the precise location of these molecules involved in neurotransmission. Here we use immunostaining and Western blotting to study the localization and expression of neurotrophin brain-derived neurotrophic factor (BDNF) and neurotrophin-4 (NT-4) and the receptors tropomyosin-related kinase b (trkB) and p75(NTR) at the adult neuromuscular junction. Our confocal immunofluorescence results on the whole mounts of the mouse Levator auris longus muscle and on semithin cross-sections showed that BDNF, NT-4, trkB, and p75(NTR) were localized on the three cells in the neuromuscular synapse (motor axons, post-synaptic muscle and Schwann cells).

  10. Blocking p75 (NTR) receptors alters polyinnervationz of neuromuscular synapses during development.

    Science.gov (United States)

    Garcia, Neus; Tomàs, Marta; Santafe, Manel M; Lanuza, Maria A; Besalduch, Nuria; Tomàs, Josep

    2011-09-01

    High-resolution immunohistochemistry shows that the receptor protein p75(NTR) is present in the nerve terminal, muscle cell, and glial Schwann cell at the neuromuscular junction (NMJ) of postnatal rats (P4-P6) during the synapse elimination period. Blocking the receptor with the antibody anti-p75-192-IgG (1-5 μg/ml, 1 hr) results in reduced endplate potentials (EPPs) in mono- and polyinnervated synapses ex vivo, but the mean number of functional inputs per NMJ does not change for as long as 3 hr. Incubation with exogenous brain-derived neurotrophic factor (BDNF) for 1 hr (50 nM) resulted in a significant increase in the size of the EPPs in all nerve terminals, and preincubation with anti-p75-192-IgG prevented this potentiation. Long exposure (24 hr) in vivo of the NMJs to the antibody anti-p75-192-IgG (1-2 μg/ml) results in a delay of postnatal synapse elimination and even some regrowth of previously withdrawn axons, but also in some acceleration of the morphologic maturation of the postsynaptic nicotinic acetylcholine receptor (nAChR) clusters. The results indicate that p75(NTR) is involved in both ACh release and axonal retraction during postnatal axonal competition and synapse elimination. Copyright © 2011 Wiley-Liss, Inc.

  11. Essential role for zinc-triggered p75NTR activation in preconditioning neuroprotection.

    Science.gov (United States)

    Lee, Jin-Yeon; Kim, Yu-Jin; Kim, Tae-Youn; Koh, Jae-Young; Kim, Yang-Hee

    2008-10-22

    Ischemic preconditioning (PC) of the brain is a phenomenon by which mild ischemic insults render neurons resistant to subsequent strong insults. Key steps in ischemic PC of the brain include caspase-3 activation and poly(ADP-ribose) polymerase-1 (PARP-1) cleavage, but upstream events have not been clearly elucidated. We have tested whether endogenous zinc is required for ischemic PC of the brain in rats. Mild, transient zinc accumulation was observed in certain neurons after ischemic PC. Moreover, intraventricular administration of CaEDTA during ischemic PC abrogated both zinc accumulation and the protective effect against subsequent full ischemia. To elucidate the mechanism of the zinc-triggered PC (Zn PC) effect, cortical cultures were exposed to sublethal levels of zinc, and 18 h later to lethal levels of zinc or NMDA. Zn PC exhibited the characteristic features of ischemic PC, including caspase-3 activation, PARP-1 cleavage, and HSP70 induction, all of which are crucial for subsequent neuroprotection against NMDA or zinc toxicity. HSP70 induction was necessary for protection, as it halted caspase-3 activation before apoptosis. Interestingly, in both Zn PC in vitro and ischemic PC in vivo, p75(NTR) was necessary for neuroprotection. These results suggest that caspase-3 activation during ischemic PC, a necessary event for subsequent neuroprotection, may result from mild zinc accumulation and the consequent p75(NTR) activation in neurons.

  12. Organotypic culture of normal, dysplastic and squamous cell carcinoma-derived oral cell lines reveals loss of spatial regulation of CD44 and p75 NTR in malignancy.

    Science.gov (United States)

    Dalley, Andrew J; AbdulMajeed, Ahmad A; Upton, Zee; Farah, Camile S

    2013-01-01

    Oral squamous cell carcinomas (OSCC) often arise from dysplastic lesions. The role of cancer stem cells in tumour initiation is widely accepted, yet the potential existence of pre-cancerous stem cells in dysplastic tissue has received little attention. Cell lines from oral diseases ranging in severity from dysplasia to malignancy provide opportunity to investigate the involvement of stem cells in malignant progression from dysplasia. Stem cells are functionally defined by their ability to generate hierarchical tissue structures in consortium with spatial regulation. Organotypic cultures readily display tissue hierarchy in vitro; hence, in this study, we compared hierarchical expression of stem cell-associated markers in dermis-based organotypic cultures of oral epithelial cells from normal tissue (OKF6-TERT2), mild dysplasia (DOK), severe dysplasia (POE-9n) and OSCC (PE/CA P J15). Expression of CD44, p75(NTR), CD24 and ALDH was studied in monolayers by flow cytometry and in organotypic cultures by immunohistochemistry. Spatial regulation of CD44 and p75(NTR) was evident for organotypic cultures of normal (OKF6-TERT2) and dysplasia (DOK and POE-9n) but was lacking for OSCC (PE/CA PJ15)-derived cells. Spatial regulation of CD24 was not evident. All monolayer cultures exhibited CD44, p75(NTR), CD24 antigens and ALDH activity (ALDEFLUOR(®) assay), with a trend towards loss of population heterogeneity that mirrored disease severity. In monolayer, increased FOXA1 and decreased FOXA2 expression correlated with disease severity, but OCT3/4, Sox2 and NANOG did not. We conclude that dermis-based organotypic cultures give opportunity to investigate the mechanisms that underlie loss of spatial regulation of stem cell markers seen with OSCC-derived cells. © 2012 John Wiley & Sons A/S.

  13. Characterization of human dopamine responsive protein DRG-1 that binds to p75NTR-associated cell death executor NADE.

    Science.gov (United States)

    Yu, Yao; Wang, Jiadong; Yuan, Hanying; Qin, Feng; Wang, Jing; Zhang, Nailing; Li, Yu-Yang; Liu, Jianping; Lu, Hong

    2006-07-19

    Expression of human dopamine responsive gene-1 (DRG-1) is up-regulated in response to treatment of dopamine in the rat astrocytes. However, its functions are not clear up to now. In the presented studies, DRG-1 was identified to be a conserved gene in the vertebrate and expressed abundantly in human testis, brain and skeletal muscle. DRG-1 was shown to interact with human p75NTR-associated cell death executor (NADE) in vivo and in vitro, and the interaction occurred in cytoplasm. The regions required for the interaction were subsequently mapped to the N-terminal of DRG-1 and the C-terminal of NADE. Furthermore, MTT assay showed that stable expression of DRG-1 in 293 cells could promote cell proliferation, and this promotion was suppressed by overexpression of NADE. In flow cytometry cell cycle analysis, overexpression of DRG-1 in 293 or PC12 cells increased the population of cells in the S phase with a concomitant decrease in G0/G1 population. These findings suggest that DRG-1 may contribute to the dopamine-induced cell growth, which is negatively regulated by NADE.

  14. Subconjunctival Delivery of p75NTR Antagonists Reduces the Inflammatory, Vascular, and Neurodegenerative Pathologies of Diabetic Retinopathy.

    Science.gov (United States)

    Galan, Alba; Barcelona, Pablo F; Nedev, Hinyu; Sarunic, Marinko V; Jian, Yifan; Saragovi, H Uri

    2017-06-01

    The p75NTR is a novel therapeutic target validated in a streptozotocin mouse model of diabetic retinopathy. Intravitreal (IVT) injection of small molecule p75NTR antagonist THX-B was therapeutic and resolved the inflammatory, vascular, and neurodegenerative phases of the retinal pathology. To simplify clinical translation, we sought a superior drug delivery method that circumvents risks associated with IVT injections. We compared the pharmacokinetics of a single 40 μg subconjunctival (SCJ) depot to the reported effective 5 μg IVT injections of THX-B. We quantified therapeutic efficacy, with endpoints of inflammation, edema, and neuronal death. The subconjunctival depot affords retinal exposure equal to IVT injection, without resulting in detectable drug in circulation. At week 2 of diabetic retinopathy, the SCJ depot provided therapeutic efficacy similar to IVT injections, with reduced inflammation, reduced edema, reduced neuronal death, and a long-lasting protection of the retinal structure. Subconjunctival injections are a safe and effective route for retinal delivery of p75NTR antagonists. The subconjunctival route offers an advantageous, less-invasive, more compliant, and nonsystemic method to deliver p75NTR antagonists for the treatment of retinal diseases.

  15. Co-induction of p75(NTR) and the associated death executor NADE in degenerating hippocampal neurons after kainate-induced seizures in the rat.

    Science.gov (United States)

    Yi, Jung-Sun; Lee, Soon-Keum; Sato, Taka-Aki; Koh, Jae-Young

    2003-08-21

    Zinc induces in cultured cortical neurons both p75(NTR) and p75(NTR)-associated death executor (NADE), which together contribute to caspase-dependent neuronal apoptosis. Since zinc neurotoxicity may contribute to neuronal death following seizures, we examined whether p75(NTR) and NADE are co-induced also in rat hippocampal neurons degenerating after seizures. Staining of brain sections with a zinc-specific fluorescent dye (N-(6-methoxy-8-quinolyl)-p-carboxybenzoylsulphonamide) and acid fuchsin revealed zinc accumulation in degenerating neuronal cell bodies in CA1 and CA3 of hippocampus 24 h after kainate injection. Both anti-p75(NTR) and anti-NADE immunoreactivities appeared in zinc-accumulating/degenerating neurons in both areas. Intraventricular injection of CaEDTA, without altering the severity or time course of kainate-induced seizures, markedly attenuated the induction of p75(NTR)/NADE in hippocampus, which correlated with the decrease of caspase-3 activation and zinc accumulation/cell death. The present study has demonstrated that p75(NTR) and NADE are co-induced in neurons degenerating after kainate-induced seizures in rats, likely in a zinc-dependent manner.

  16. Establishing a cellular FRET-based fluorescence plate reader assay to monitor proNGF-induced cross-linking of sortilin and the neurotrophin receptor p75(NTR)

    DEFF Research Database (Denmark)

    Skeldal, Sune; Kjaergaard, Maj M; Alwasel, Saleh

    2015-01-01

    the vps10p domain receptor sortilin and the neurotrophin receptor p75(NTR). However, proNGF-induced receptor complex formation has been difficult to directly assess other than by western blotting. We here describe a fluorescence resonance energy transfer (FRET) based fluorescence plate reader assay...

  17. Involvement of TrkB- and p75NTR-signaling pathways in two contrasting forms of long-lasting synaptic plasticity

    Science.gov (United States)

    Sakuragi, Shigeo; Tominaga-Yoshino, Keiko; Ogura, Akihiko

    2013-11-01

    The repetition of experience is often necessary to establish long-lasting memory. However, the cellular mechanisms underlying this repetition-dependent consolidation of memory remain unclear. We previously observed in organotypic slice cultures of the rodent hippocampus that repeated inductions of long-term potentiation (LTP) led to a slowly developing long-lasting synaptic enhancement coupled with synaptogenesis. We also reported that repeated inductions of long-term depression (LTD) produced a long-lasting synaptic suppression coupled with synapse elimination. We proposed these phenomena as useful in vitro models for analyzing repetition-dependent consolidation. Here, we hypothesized that the enhancement and suppression are mediated by the brain-derived neurotrophic factor (BDNF)-TrkB signaling pathway and the proBDNF-p75NTR pathway, respectively. When we masked the respective pathways, reversals of the enhancement and suppression resulted. These results suggest the alternative activation of the p75NTR pathway by BDNF under TrkB-masking conditions and of the TrkB pathway by proBDNF under p75NTR-masking conditions, thus supporting the aforementioned hypothesis.

  18. Peptides derived from the solvent-exposed loops 3 and 4 of BDNF bind TrkB and p75(NTR) receptors and stimulate neurite outgrowth and survival

    DEFF Research Database (Denmark)

    Fobian, Kristina; Owczarek, Sylwia; Budtz, Christian

    2010-01-01

    Brain-derived neurotrophic factor (BDNF) is critically involved in modeling the developing nervous system and is an important regulator of a variety of crucial functions in the mature CNS. BDNF exerts its action through interactions with two transmembrane receptors, either separately or in concert....... BDNF has been implicated in several neurological disorders, and irregularities in BDNF function may have severe consequences. Administration of BDNF as a drug has thus far yielded few practicable results, and the potential side effects when using a multifunctional protein are substantial. In an effort...... to produce more specific compounds without side effects, small peptides mimicking protein function have been developed. The present study characterized two mimetic peptides, Betrofin 3 and Betrofin 4, derived from the BDNF sequence. Both Betrofins bound the cognate BDNF receptors, TrkB and p75(NTR...

  19. The genome sequence of the protostome Daphnia pulex encodes respective orthologues of a neurotrophin, a Trk and a p75NTR: Evolution of neurotrophin signaling components and related proteins in the bilateria

    Directory of Open Access Journals (Sweden)

    Wilson Karen HS

    2009-10-01

    Full Text Available Abstract Background Neurotrophins and their Trk and p75NTR receptors play an important role in the nervous system. To date, neurotrophins, Trk and p75NTR have only been found concomitantly in deuterostomes. In protostomes, homologues to either neurotrophin, Trk or p75NTR are reported but their phylogenetic relationship to deuterostome neurotrophin signaling components is unclear. Drosophila has neurotrophin homologues called Spätzles (Spz, some of which were recently renamed neurotrophins, but direct proof that these are deuterostome neurotrophin orthologues is lacking. Trks belong to the receptor tyrosine kinase (RTK family and among RTKs, Trks and RORs are closest related. Flies lack Trks but have ROR and ROR-related proteins called NRKs playing a neurotrophic role. Mollusks have so far the most similar proteins to Trks (Lymnaea Trk and Aplysia Trkl but the exact phylogenetic relationship of mollusk Trks to each other and to vertebrate Trks is unknown. p75NTR belongs to the tumor necrosis factor receptor (TNFR superfamily. The divergence of the TNFR families in vertebrates has been suggested to parallel the emergence of the adaptive immune system. Only one TNFR representative, the Drosophila Wengen, has been found in protostomes. To clarify the evolution of neurotrophin signaling components in bilateria, this work analyzes the genome of the crustacean Daphnia pulex as well as new genetic data from protostomes. Results The Daphnia genome encodes a neurotrophin, p75NTR and Trk orthologue together with Trkl, ROR, and NRK-RTKs. Drosophila Spz1, 2, 3, 5, 6 orthologues as well as two new groups of Spz proteins (Spz7 and 8 are also found in the Daphnia genome. Searching genbank and the genomes of Capitella, Helobdella and Lottia reveals neurotrophin signaling components in other protostomes. Conclusion It appears that a neurotrophin, Trk and p75NTR existed at the protostome/deuterostome split. In protostomes, a "neurotrophin superfamily" includes

  20. p75 neurotrophin receptor cleavage by α- and γ-secretases is required for neurotrophin-mediated proliferation of brain tumor-initiating cells.

    Science.gov (United States)

    Forsyth, Peter A; Krishna, Niveditha; Lawn, Samuel; Valadez, J Gerardo; Qu, Xiaotao; Fenstermacher, David A; Fournier, Michelle; Potthast, Lisa; Chinnaiyan, Prakash; Gibney, Geoffrey T; Zeinieh, Michele; Barker, Philip A; Carter, Bruce D; Cooper, Michael K; Kenchappa, Rajappa S

    2014-03-21

    Malignant gliomas are highly invasive, proliferative, and resistant to treatment. Previously, we have shown that p75 neurotrophin receptor (p75NTR) is a novel mediator of invasion of human glioma cells. However, the role of p75NTR in glioma proliferation is unknown. Here we used brain tumor-initiating cells (BTICs) and show that BTICs express neurotrophin receptors (p75NTR, TrkA, TrkB, and TrkC) and their ligands (NGF, brain-derived neurotrophic factor, and neurotrophin 3) and secrete NGF. Down-regulation of p75NTR significantly decreased proliferation of BTICs. Conversely, exogenouous NGF stimulated BTIC proliferation through α- and γ-secretase-mediated p75NTR cleavage and release of its intracellular domain (ICD). In contrast, overexpression of the p75NTR ICD induced proliferation. Interestingly, inhibition of Trk signaling blocked NGF-stimulated BTIC proliferation and p75NTR cleavage, indicating a role of Trk in p75NTR signaling. Further, blocking p75NTR cleavage attenuated Akt activation in BTICs, suggesting role of Akt in p75NTR-mediated proliferation. We also found that p75NTR, α-secretases, and the four subunits of the γ-secretase enzyme were elevated in glioblastoma multiformes patients. Importantly, the ICD of p75NTR was commonly found in malignant glioma patient specimens, suggesting that the receptor is activated and cleaved in patient tumors. These results suggest that p75NTR proteolysis is required for BTIC proliferation and is a novel potential clinical target.

  1. Biophysical Studies on BEX3, the p75NTR-Associated Cell Death Executor, Reveal a High-Order Oligomer with Partially Folded Regions.

    Directory of Open Access Journals (Sweden)

    Katia M S Cabral

    Full Text Available BEX3 (Brain Expressed X-linked protein 3 is a member of a mammal-specific placental protein family. Several studies have found the BEX proteins to be associated with neurodegeneration, the cell cycle and cancer. BEX3 has been predicted to be intrinsically disordered and also to represent an intracellular hub for cell signaling. The pro-apoptotic activity of BEX3 in association with a number of additional proteins has been widely supported; however, to the best of our knowledge, very limited data are available on the conformation of any of the members of the BEX family. In this study, we structurally characterized BEX3 using biophysical experimental data. Small angle X-ray scattering and atomic force microscopy revealed that BEX3 forms a specific higher-order oligomer that is consistent with a globular molecule. Solution nuclear magnetic resonance, partial proteinase K digestion, circular dichroism spectroscopy, and fluorescence techniques that were performed on the recombinant protein indicated that the structure of BEX3 is composed of approximately 31% α-helix and 20% β-strand, contains partially folded regions near the N- and C-termini, and a core which is proteolysis-resistant around residues 55-120. The self-oligomerization of BEX3 has been previously reported in cell culture and is consistent with our in vitro data.

  2. Biophysical Studies on BEX3, the p75NTR-Associated Cell Death Executor, Reveal a High-Order Oligomer with Partially Folded Regions.

    Science.gov (United States)

    Cabral, Katia M S; Raymundo, Diana P; Silva, Viviane S; Sampaio, Laura A G; Johanson, Laizes; Hill, Luis Fernando; Almeida, Fabio C L; Cordeiro, Yraima; Almeida, Marcius S

    2015-01-01

    BEX3 (Brain Expressed X-linked protein 3) is a member of a mammal-specific placental protein family. Several studies have found the BEX proteins to be associated with neurodegeneration, the cell cycle and cancer. BEX3 has been predicted to be intrinsically disordered and also to represent an intracellular hub for cell signaling. The pro-apoptotic activity of BEX3 in association with a number of additional proteins has been widely supported; however, to the best of our knowledge, very limited data are available on the conformation of any of the members of the BEX family. In this study, we structurally characterized BEX3 using biophysical experimental data. Small angle X-ray scattering and atomic force microscopy revealed that BEX3 forms a specific higher-order oligomer that is consistent with a globular molecule. Solution nuclear magnetic resonance, partial proteinase K digestion, circular dichroism spectroscopy, and fluorescence techniques that were performed on the recombinant protein indicated that the structure of BEX3 is composed of approximately 31% α-helix and 20% β-strand, contains partially folded regions near the N- and C-termini, and a core which is proteolysis-resistant around residues 55-120. The self-oligomerization of BEX3 has been previously reported in cell culture and is consistent with our in vitro data.

  3. Effects of age and insulin-like growth factor-1 on rat neurotrophin receptor expression after nerve injury.

    Science.gov (United States)

    Luo, T David; Alton, Timothy B; Apel, Peter J; Cai, Jiaozhong; Barnwell, Jonathan C; Sonntag, William E; Smith, Thomas L; Li, Zhongyu

    2016-10-01

    Neurotrophin receptors, such as p75(NTR) , direct neuronal response to injury. Insulin-like growth factor-1 receptor (IGF-1R) mediates the increase in p75(NTR) during aging. The aim of this study was to examine the effect of aging and insulin-like growth factor-1 (IGF-1) treatment on recovery after peripheral nerve injury. Young and aged rats underwent tibial nerve transection with either local saline or IGF-1 treatment. Neurotrophin receptor mRNA and protein expression were quantified. Aged rats expressed elevated baseline IGF-1R (34% higher, P = 0.01) and p75(NTR) (68% higher, P < 0.01) compared with young rats. Post-injury, aged animals expressed significantly higher p75(NTR) levels (68.5% above baseline at 4 weeks). IGF-1 treatment suppressed p75(NTR) gene expression at 4 weeks (17.2% above baseline, P = 0.002) post-injury. Local IGF-1 treatment reverses age-related declines in recovery after peripheral nerve injuries by suppressing p75(NTR) upregulation and pro-apoptotic complexes. IGF-1 may be considered a viable adjuvant therapy to current treatment modalities. Muscle Nerve 54: 769-775, 2016. © 2016 Wiley Periodicals, Inc.

  4. p75 Neurotrophin Receptor Suppresses the Proliferation of Human Gastric Cancer Cells

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    Haifeng Jin

    2007-06-01

    Full Text Available Identifying an effective therapeutic target is pivotal in the treatment of gastric cancer. In this study, we investigated the expression of p75 neurotrophin receptor (p75NTR in gastric cancer and the impact of its alteration on tumor growth. p75NTR expression was absent or significantly decreased in 212 cases of gastric cancers compared with the normal gastric mucosa (P < .05. Moreover, p75NTR expression was also lost or significantly decreased in various human gastric cancer cell lines. p75NTR inhibited in vitro growth activities and caused dramatic attenuation of tumor growth in animal models by induction of cell cycle arrest. Upregulation of p75NTR led to downregulation of cyclin A, cyclin D1, cyclin E, cyclin-dependent kinase 2, p-Rb, and PCNA, but to upregulation of Rb and p27 expressions. Conversely, downregulating p75NTR with specific siRNA yielded inverse results. The rescue of tumor cells from cell cycle progression by a death domain-deleted dominant-negative antagonist of p75NTRp75NTR showed that the death domain transduced antiproliferative activity in a ligandindependent manner and further demonstrated the inhibitive effect of p75NTR on growth in gastric cancer. Therefore, we provided evidence that p75NTR was a potential tumor suppressor and may be used as a therapeutic target for gastric cancer.

  5. p75 Neurotrophin Receptor Cleavage by α- and γ-Secretases Is Required for Neurotrophin-mediated Proliferation of Brain Tumor-initiating Cells*

    Science.gov (United States)

    Forsyth, Peter A.; Krishna, Niveditha; Lawn, Samuel; Valadez, J. Gerardo; Qu, Xiaotao; Fenstermacher, David A.; Fournier, Michelle; Potthast, Lisa; Chinnaiyan, Prakash; Gibney, Geoffrey T.; Zeinieh, Michele; Barker, Philip A.; Carter, Bruce D.; Cooper, Michael K.; Kenchappa, Rajappa S.

    2014-01-01

    Malignant gliomas are highly invasive, proliferative, and resistant to treatment. Previously, we have shown that p75 neurotrophin receptor (p75NTR) is a novel mediator of invasion of human glioma cells. However, the role of p75NTR in glioma proliferation is unknown. Here we used brain tumor-initiating cells (BTICs) and show that BTICs express neurotrophin receptors (p75NTR, TrkA, TrkB, and TrkC) and their ligands (NGF, brain-derived neurotrophic factor, and neurotrophin 3) and secrete NGF. Down-regulation of p75NTR significantly decreased proliferation of BTICs. Conversely, exogenouous NGF stimulated BTIC proliferation through α- and γ-secretase-mediated p75NTR cleavage and release of its intracellular domain (ICD). In contrast, overexpression of the p75NTR ICD induced proliferation. Interestingly, inhibition of Trk signaling blocked NGF-stimulated BTIC proliferation and p75NTR cleavage, indicating a role of Trk in p75NTR signaling. Further, blocking p75NTR cleavage attenuated Akt activation in BTICs, suggesting role of Akt in p75NTR-mediated proliferation. We also found that p75NTR, α-secretases, and the four subunits of the γ-secretase enzyme were elevated in glioblastoma multiformes patients. Importantly, the ICD of p75NTR was commonly found in malignant glioma patient specimens, suggesting that the receptor is activated and cleaved in patient tumors. These results suggest that p75NTR proteolysis is required for BTIC proliferation and is a novel potential clinical target. PMID:24519935

  6. Cerebellar Expression of the Neurotrophin Receptor p75 in Naked-Ataxia Mutant Mouse

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    Maryam Rahimi Balaei

    2016-01-01

    Full Text Available Spontaneous mutation in the lysosomal acid phosphatase 2 (Acp2 mouse (nax—naked-ataxia mutant mouse correlates with severe cerebellar defects including ataxia, reduced size and abnormal lobulation as well as Purkinje cell (Pc degeneration. Loss of Pcs in the nax cerebellum is compartmentalized and harmonized to the classic pattern of gene expression of the cerebellum in the wild type mouse. Usually, degeneration starts in the anterior and posterior zones and continues to the central and nodular zones of cerebellum. Studies have suggested that the p75 neurotrophin receptor (NTR plays a role in Pc degeneration; thus, in this study, we investigated the p75NTR pattern and protein expression in the cerebellum of the nax mutant mouse. Despite massive Pc degeneration that was observed in the nax mouse cerebellum, p75NTR pattern expression was similar to the HSP25 pattern in nax mice and comparable with wild type sibling cerebellum. In addition, immunoblot analysis of p75NTR protein expression did not show any significant difference between nax and wild type sibling (p > 0.5. In comparison with wild type counterparts, p75NTR pattern expression is aligned with the fundamental cytoarchitecture organization of the cerebellum and is unchanged in the nax mouse cerebellum despite the severe neurodevelopmental disorder accompanied with Pc degeneration.

  7. Deficient p75 low-affinity neurotrophin receptor expression does alter the composition of cellular infiltrate in experimental autoimmune encephalomyelitis in C57BL/6 mice

    NARCIS (Netherlands)

    Kust, B; Mantingh-Otter, [No Value; Boddeke, E; Copray, S

    We have shown earlier that induction of experimental autoimmune encephalomyelitis (EAE)-a model for the human disease multiple sclerosis-in C5713L/6 wild-type mice resulted in the expression of the p75 low-affinity neurotrophin receptor (p75(NTR)) in endothelial cells in the CNS. In comparison to

  8. Leukomogenic factors downregulate heparanase expression in acute myeloid leukemia cells

    International Nuclear Information System (INIS)

    Eshel, Rinat; Ben-Zaken, Olga; Vainas, Oded; Nadir, Yona; Minucci, Saverio; Polliack, Aaron; Naparstek, Ella; Vlodavsky, Israel; Katz, Ben-Zion

    2005-01-01

    Heparanase is a heparan sulfate-degrading endoglycosidase expressed by mature monocytes and myeloid cells, but not by immature hematopoietic progenitors. Heparanase gene expression is upregulated during differentiation of immature myeloid cells. PML-RARα and PLZF-RARα fusion gene products associated with acute promyelocytic leukemia abrogate myeloid differentiation and heparanase expression. AML-Eto, a translocation product associated with AML FAB M2, also downregulates heparanase gene expression. The common mechanism that underlines the activity of these three fusion gene products involves the recruitment of histone deacetylase complexes to specific locations within the DNA. We found that retinoic acid that dissociates PML-RARα from the DNA, and which is used to treat acute promyelocytic leukemia patients, restores heparanase expression to normal levels in an acute promyelocytic leukemia cell line. The retinoic acid effects were also observed in primary acute promyelocytic leukemia cells and in a retinoic acid-treated acute promyelocytic leukemia patient. Histone deacetylase inhibitor reverses the downregulation of heparanase expression induced by the AML-Eto fusion gene product in M2 type AML. In summary, we have characterized a link between leukomogenic factors and the downregulation of heparanase in myeloid leukemic cells

  9. Neurotrophin receptors expression and JNK pathway activation in human astrocytomas

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    Maraziotis Theodore

    2007-10-01

    Full Text Available Abstract Background Neurotrophins are growth factors that regulate cell growth, differentiation and apoptosis in the nervous system. Their diverse actions are mediated through two different transmembrane – receptor signaling systems: Trk receptor tyrosine kinases (TrkA, TrkB, TrkC and p75NTR neurotrophin receptor. Trk receptors promote cell survival and differentiation while p75NTR induces, in most cases, the activity of JNK-p53-Bax apoptosis pathway or suppresses intracellular survival signaling cascades. Robust Trk activation blocks p75NTR -induced apoptosis by suppressing the JNK-p53-Bax pathway. The aim of this exploratory study was to investigate the expression levels of neurotrophin receptors, Trks and p75NTR, and the activation of JNK pathway in human astrocytomas and in adjacent non-neoplastic brain tissue. Methods Formalin-fixed paraffin-embedded serial sections from 33 supratentorial astrocytomas (5 diffuse fibrillary astrocytomas, WHO grade II; 6 anaplastic astrocytomas, WHO grade III; 22 glioblastomas multiforme, WHO grade IV were immunostained following microwave pretreatment. Polyclonal antibodies against TrkA, TrkB, TrkC and monoclonal antibodies against p75NTR and phosphorylated forms of JNK (pJNK and c-Jun (pc-Jun were used. The labeling index (LI, defined as the percentage of positive (labeled cells out of the total number of tumor cells counted, was determined. Results Moderate to strong, granular cytoplasmic immunoreactivity for TrkA, TrkB and TrkC receptors was detected in greater than or equal to 10% of tumor cells in the majority of tumors independently of grade; on the contrary, p75NTR receptor expression was found in a small percentage of tumor cells (~1% in some tumors. The endothelium of tumor capillaries showed conspicuous immunoreactivity for TrkB receptor. Trk immunoreactivity seemed to be localized in some neurons and astrocytes in non-neoplastic tissue. Phosphorylated forms of JNK (pJNK and c-Jun (pc-Jun were

  10. Neurotrophin receptors expression and JNK pathway activation in human astrocytomas

    International Nuclear Information System (INIS)

    Assimakopoulou, Martha; Kondyli, Maria; Gatzounis, George; Maraziotis, Theodore; Varakis, John

    2007-01-01

    Neurotrophins are growth factors that regulate cell growth, differentiation and apoptosis in the nervous system. Their diverse actions are mediated through two different transmembrane – receptor signaling systems: Trk receptor tyrosine kinases (TrkA, TrkB, TrkC) and p75 NTR neurotrophin receptor. Trk receptors promote cell survival and differentiation while p75 NTR induces, in most cases, the activity of JNK-p53-Bax apoptosis pathway or suppresses intracellular survival signaling cascades. Robust Trk activation blocks p75 NTR -induced apoptosis by suppressing the JNK-p53-Bax pathway. The aim of this exploratory study was to investigate the expression levels of neurotrophin receptors, Trks and p75 NTR , and the activation of JNK pathway in human astrocytomas and in adjacent non-neoplastic brain tissue. Formalin-fixed paraffin-embedded serial sections from 33 supratentorial astrocytomas (5 diffuse fibrillary astrocytomas, WHO grade II; 6 anaplastic astrocytomas, WHO grade III; 22 glioblastomas multiforme, WHO grade IV) were immunostained following microwave pretreatment. Polyclonal antibodies against TrkA, TrkB, TrkC and monoclonal antibodies against p75 NTR and phosphorylated forms of JNK (pJNK) and c-Jun (pc-Jun) were used. The labeling index (LI), defined as the percentage of positive (labeled) cells out of the total number of tumor cells counted, was determined. Moderate to strong, granular cytoplasmic immunoreactivity for TrkA, TrkB and TrkC receptors was detected in greater than or equal to 10% of tumor cells in the majority of tumors independently of grade; on the contrary, p75 NTR receptor expression was found in a small percentage of tumor cells (~1%) in some tumors. The endothelium of tumor capillaries showed conspicuous immunoreactivity for TrkB receptor. Trk immunoreactivity seemed to be localized in some neurons and astrocytes in non-neoplastic tissue. Phosphorylated forms of JNK (pJNK) and c-Jun (pc-Jun) were significantly co-expressed in a tumor

  11. DMBT1 expression is down-regulated in breast cancer

    DEFF Research Database (Denmark)

    Braidotti, P; Nuciforo, P G; Mollenhauer, J

    2004-01-01

    and hyperplastic mammary cells positive with DMBTh12 were also MCM5-positive. CONCLUSIONS: The redistribution and up-regulation of DMBT1 in normal and hyperplastic tissues flanking malignant tumours and its down-regulation in carcinomas suggests a potential role in breast cancer. Moreover, the concomitant......BACKGROUND: We studied the expression of DMBT1 (deleted in malignant brain tumor 1), a putative tumor suppressor gene, in normal, proliferative, and malignant breast epithelium and its possible relation to cell cycle. METHODS: Sections from 17 benign lesions and 55 carcinomas were immunostained...... expression was down-regulated in the cancerous lesions compared to the normal and/or hyperplastic epithelium adjacent to carcinomas (3/55 positive carcinomas versus 33/42 positive normal/hyperplastic epithelia; p = 0.0001). In 72% of cases RT-PCR confirmed immunohistochemical results. Most of normal...

  12. DMBT1 expression is down-regulated in breast cancer

    International Nuclear Information System (INIS)

    Braidotti, P; Pietra, GG; Nuciforo, PG; Mollenhauer, J; Poustka, A; Pellegrini, C; Moro, A; Bulfamante, G; Coggi, G; Bosari, S

    2004-01-01

    We studied the expression of DMBT1 (deleted in malignant brain tumor 1), a putative tumor suppressor gene, in normal, proliferative, and malignant breast epithelium and its possible relation to cell cycle. Sections from 17 benign lesions and 55 carcinomas were immunostained with anti DMBT1 antibody (DMBTh12) and sections from 36 samples, were double-stained also with anti MCM5, one of the 6 pre-replicative complex proteins with cell proliferation-licensing functions. DMBT1 gene expression at mRNA level was assessed by RT-PCR in frozen tissues samples from 39 patients. Normal glands and hyperplastic epithelium in benign lesions displayed a luminal polarized DMBTh12 immunoreactivity. Normal and hyperplastic epithelium adjacent to carcinomas showed a loss of polarization, with immunostaining present in basal and perinuclear cytoplasmic compartments. DMBT1 protein expression was down-regulated in the cancerous lesions compared to the normal and/or hyperplastic epithelium adjacent to carcinomas (3/55 positive carcinomas versus 33/42 positive normal/hyperplastic epithelia; p = 0.0001). In 72% of cases RT-PCR confirmed immunohistochemical results. Most of normal and hyperplastic mammary cells positive with DMBTh12 were also MCM5-positive. The redistribution and up-regulation of DMBT1 in normal and hyperplastic tissues flanking malignant tumours and its down-regulation in carcinomas suggests a potential role in breast cancer. Moreover, the concomitant expression of DMTB1 and MCM5 suggests its possible association with the cell-cycle regulation

  13. Hypoxia downregulates Ku70/80 expression in cervical carcinoma tumors

    International Nuclear Information System (INIS)

    Lara, Pedro Carlos; Lloret, Marta; Clavo, Bernardino; Apolinario, Rosa Maria; Bordon, Elisa; Rey, Agustin; Falcon, Orlando; Alonso, Ana Ruiz; Belka, Claus

    2008-01-01

    Hypoxia may inhibits the NHEJ DNA repair through downregulating Ku70/80 expression and combined with an increased angiogenesis and altered p53 expression would be responsible for tumor progression in cervical carcinoma

  14. Effect of electro-acupuncture on ovarian expression of α (1- and β (2-adrenoceptors, and p75 neurotrophin receptors in rats with steroid-induced polycystic ovaries

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    Holmäng Agneta

    2005-06-01

    Full Text Available Abstract Background Estradiol valerate (EV-induced polycystic ovaries (PCO in rats is associated with an increase in ovarian sympathetic outflow. Low-frequency (2 Hz electro-acupuncture (EA has been shown to modulate sympathetic markers as well as ovarian blood flow as a reflex response via the ovarian sympathetic nerves, in rats with EV-induced PCO. Methods In the present study, we further tested the hypothesis that repeated 2 Hz EA treatments modulate ovarian sympathetic outflow in rats with PCO, induced by a single i.m. injection of EV, by investigating the mRNA expression, the amount and distribution of proteins of α1a-, α1b-, α1d-, and β2-adrenoceptors (ARs, as well as the low-affinity neurotrophin receptor (p75NTR. Results It was found that EV injection results in significantly higher mRNA expression of ovarian α1b- and α1d-AR in PCO rats compared to control rats. The p75NTR and β2-ARs mRNA expression were unchanged in the PCO ovary. Low-frequency EA resulted in a significantly lower expression of β2-ARs mRNA expression in PCO rats. The p75NTR mRNA was unaffected in both PCO and control rats. PCO ovaries displayed significantly higher amount of protein of α1a-, α1b- and α1d-ARs, and of p75NTR, compared to control rats, that were all counteracted by repeated low-frequency EA treatments, except for α1b-AR. Conclusion The present study shows that EA normalizes most of the EV-induced changes in ovarian ARs. Furthermore, EA was able to prevent the EV-induced up regulation of p75NTR, probably by normalizing the sympathetic ovarian response to NGF action. Our data indicate a possible role of EA in the regulation of ovarian responsiveness to sympathetic inputs and depict a possible complementary therapeutic approach to overcoming sympathetic-related anovulation in women with PCOS.

  15. Gene Expression Profiles of Main Olfactory Epithelium in Adenylyl Cyclase 3 Knockout Mice

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    Zhenshan Wang

    2015-11-01

    Full Text Available Adenylyl Cyclase 3 (AC3 plays an important role in the olfactory sensation-signaling pathway in mice. AC3 deficiency leads to defects in olfaction. However, it is still unknown whether AC3 deficiency affects gene expression or olfactory signal transduction pathways within the main olfactory epithelium (MOE. In this study, gene microarrays were used to screen differentially expressed genes in MOE from AC3 knockout (AC3−/− and wild-type (AC3+/+ mice. The differentially expressed genes identified were subjected to bioinformatic analysis and verified by qRT-PCR. Gene expression in the MOE from AC3−/− mice was significantly altered, compared to AC3+/+ mice. Of the 41266 gene probes, 3379 had greater than 2-fold fold change in expression levels between AC3−/− and AC3+/+ mice, accounting for 8% of the total gene probes. Of these genes, 1391 were up regulated, and 1988 were down regulated, including 425 olfactory receptor genes, 99 genes that are specifically expressed in the immature olfactory neurons, 305 genes that are specifically expressed in the mature olfactory neurons, and 155 genes that are involved in epigenetic regulation. Quantitative RT-PCR verification of the differentially expressed epigenetic regulation related genes, olfactory receptors, ion transporter related genes, neuron development and differentiation related genes, lipid metabolism and membrane protein transport etc. related genes showed that P75NTR, Hinfp, Gadd45b, and Tet3 were significantly up-regulated, while Olfr370, Olfr1414, Olfr1208, Golf, Faim2, Tsg101, Mapk10, Actl6b, H2BE, ATF5, Kirrrel2, OMP, Drd2 etc. were significantly down-regulated. In summary, AC3 may play a role in proximal olfactory signaling and play a role in the regulation of differentially expressed genes in mouse MOE.

  16. ADAM15 expression is downregulated in melanoma metastasis compared to primary melanoma

    International Nuclear Information System (INIS)

    Ungerer, Christopher; Doberstein, Kai; Buerger, Claudia; Hardt, Katja; Boehncke, Wolf-Henning; Boehm, Beate; Pfeilschifter, Josef; Dummer, Reinhard; Mihic-Probst, Daniela; Gutwein, Paul

    2010-01-01

    Research highlights: → Strong ADAM15 expression is found in normal melanocytes. → ADAM15 expression is significantly downregulated in patients with melanoma metastasis. → TGF-β can downregulate ADAM15 expression in melanoma cells. → Overexpression of ADAM15 in melanoma cells inhibits migration, proliferation and invasion of melanoma cells. → Conclusion: ADAM15 represents an tumor suppressor protein in melanoma. -- Abstract: In a mouse melanoma metastasis model it has been recently shown that ADAM15 overexpression in melanoma cells significantly reduced the number of metastatic nodules on the lung. Unfortunately, the expression of ADAM15 in human melanoma tissue has not been determined so far. In our study, we characterized the expression of ADAM15 in tissue micro-arrays of patients with primary melanoma with melanoma metastasis. ADAM15 was expressed in melanocytes and endothelial cells of benign nevi and melanoma tissue. Importantly, ADAM15 was significantly downregulated in melanoma metastasis compared to primary melanoma. We further demonstrate that IFN-γ and TGF-β downregulate ADAM15 protein levels in melanoma cells. To investigate the role of ADAM15 in melanoma progression, we overexpressed ADAM15 in melanoma cells. Importantly, overexpression of ADAM15 in melanoma cells reduced the migration, invasion and the anchorage dependent and independent cell growth of melanoma cells. In summary, the downregulation of ADAM15 plays an important role in melanoma progression and ADAM15 act as a tumorsuppressor in melanoma.

  17. ADAM15 expression is downregulated in melanoma metastasis compared to primary melanoma

    Energy Technology Data Exchange (ETDEWEB)

    Ungerer, Christopher; Doberstein, Kai [Pharmazentrum Frankfurt/ZAFES, University Hospital Goethe University Frankfurt, Frankfurt am Main (Germany); Buerger, Claudia; Hardt, Katja; Boehncke, Wolf-Henning [Department of Dermatology, Clinic of the Goethe-University, Theodor-Stern-Kai, Frankfurt (Germany); Boehm, Beate [Division of Rheumatology, Goethe University, Frankfurt am Main (Germany); Pfeilschifter, Josef [Pharmazentrum Frankfurt/ZAFES, University Hospital Goethe University Frankfurt, Frankfurt am Main (Germany); Dummer, Reinhard [Department of Pathology, Institute of Surgical Pathology, University Hospital, Zurich (Switzerland); Mihic-Probst, Daniela [Department of Dermatology, University Hospital Zurich (Switzerland); Gutwein, Paul, E-mail: p.gutwein@med.uni-frankfurt.de [Pharmazentrum Frankfurt/ZAFES, University Hospital Goethe University Frankfurt, Frankfurt am Main (Germany)

    2010-10-22

    Research highlights: {yields} Strong ADAM15 expression is found in normal melanocytes. {yields} ADAM15 expression is significantly downregulated in patients with melanoma metastasis. {yields} TGF-{beta} can downregulate ADAM15 expression in melanoma cells. {yields} Overexpression of ADAM15 in melanoma cells inhibits migration, proliferation and invasion of melanoma cells. {yields} Conclusion: ADAM15 represents an tumor suppressor protein in melanoma. -- Abstract: In a mouse melanoma metastasis model it has been recently shown that ADAM15 overexpression in melanoma cells significantly reduced the number of metastatic nodules on the lung. Unfortunately, the expression of ADAM15 in human melanoma tissue has not been determined so far. In our study, we characterized the expression of ADAM15 in tissue micro-arrays of patients with primary melanoma with melanoma metastasis. ADAM15 was expressed in melanocytes and endothelial cells of benign nevi and melanoma tissue. Importantly, ADAM15 was significantly downregulated in melanoma metastasis compared to primary melanoma. We further demonstrate that IFN-{gamma} and TGF-{beta} downregulate ADAM15 protein levels in melanoma cells. To investigate the role of ADAM15 in melanoma progression, we overexpressed ADAM15 in melanoma cells. Importantly, overexpression of ADAM15 in melanoma cells reduced the migration, invasion and the anchorage dependent and independent cell growth of melanoma cells. In summary, the downregulation of ADAM15 plays an important role in melanoma progression and ADAM15 act as a tumorsuppressor in melanoma.

  18. Downregulation of Clusterin Expression in Human Testicular Seminoma

    Directory of Open Access Journals (Sweden)

    Bianjiang Liu

    2013-11-01

    Full Text Available Background: Clusterin, a heterodimeric glycoprotein of approximately 80 kDa, exists extensively in human body fluids. The abnormal expression of clusterin is closely related to the occurrence, progression, and prognosis of tumors. Up to now, few studies have focused on clusterin in human testicular cancer. This study describes an extensive exploration of the presence and expression of clusterin in testicular seminoma. Methods: Tumor tissues and normal testis tissues were collected from 13 patients with testicular seminoma and 16 patients undergoing surgical castration for prostate cancer. Real-time polymerase chain reaction (PCR was performed to detect the expression difference of clusterin mRNA between testicular seminoma and normal testis. Western blot and immunohistochemical analysis were performed to detect the presence and expression difference of clusterin protein between two groups. Results: Real-time PCR showed the expression of clusterin mRNA in testicular seminoma to be significantly lower than in normal testis (only 13% relative quantification. Western blot analysis indicated marked reductions in the expression of clusterin protein in testicular seminoma. Similar results were observed upon immunohistochemical analysis. Conclusion: In testicular seminoma and normal testis, clusterin exists in its heterodimeric secretory isoform. Clusterin expression is significantly lower in testicular seminoma than in normal testis. This is the first comprehensive study of the presence and expression of clusterin in human testicular cancer.

  19. Use of bacterially expressed dsRNA to downregulate Entamoeba histolytica gene expression.

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    Carlos F Solis

    Full Text Available BACKGROUND: Modern RNA interference (RNAi methodologies using small interfering RNA (siRNA oligonucleotide duplexes or episomally synthesized hairpin RNA are valuable tools for the analysis of gene function in the protozoan parasite Entamoeba histolytica. However, these approaches still require time-consuming procedures including transfection and drug selection, or costly synthetic molecules. PRINCIPAL FINDINGS: Here we report an efficient and handy alternative for E. histolytica gene down-regulation mediated by bacterial double-stranded RNA (dsRNA targeting parasite genes. The Escherichia coli strain HT115 which is unable to degrade dsRNA, was genetically engineered to produce high quantities of long dsRNA segments targeting the genes that encode E. histolytica beta-tubulin and virulence factor KERP1. Trophozoites cultured in vitro were directly fed with dsRNA-expressing bacteria or soaked with purified dsRNA. Both dsRNA delivery methods resulted in significant reduction of protein expression. In vitro host cell-parasite assays showed that efficient downregulation of kerp1 gene expression mediated by bacterial dsRNA resulted in significant reduction of parasite adhesion and lytic capabilities, thus supporting a major role for KERP1 in the pathogenic process. Furthermore, treatment of trophozoites cultured in microtiter plates, with a repertoire of eighty-five distinct bacterial dsRNA segments targeting E. histolytica genes with unknown function, led to the identification of three genes potentially involved in the growth of the parasite. CONCLUSIONS: Our results showed that the use of bacterial dsRNA is a powerful method for the study of gene function in E. histolytica. This dsRNA delivery method is also technically suitable for the study of a large number of genes, thus opening interesting perspectives for the identification of novel drug and vaccine targets.

  20. Downregulation of the expression of HDGF attenuates malignant biological behaviors of hilar cholangiocarcinoma cells.

    Science.gov (United States)

    Liu, Yanfeng; Sun, Jingxian; Yang, Guangyun; Liu, Zhaojian; Guo, Sen; Zhao, Rui; Xu, Kesen; Wu, Xiaopeng; Zhang, Zhaoyang

    2015-09-01

    Hepatoma-derived growth factor (HDGF) has been reported to be a potential predictive and prognostic marker for several types of cancer and important in malignant biological behaviors. However, its role in human hilar cholangiocarcinoma remains to be elucidated. Our previous study demonstrated that high expression levels of HDGF in hilar cholangiocarcinoma tissues correlates with tumor progression and patient outcome. The present study aimed to elucidate the detailed functions of the HDGF protein. This was performed by downregulating the protein expression of HDGF in the FRH0201 hilar cholangiocarcinoma cell line by RNA interference (RNAi) in vitro, and revealed that downregulation of the HDGF protein significantly inhibited the malignant biological behavior of the FRH0201 cells. In addition, further investigation revealed that downregulation of the protein expression of HDGF significantly decreased the secretion of vascular endothelial growth factor, which may be the mechanism partially responsible for the inhibition of malignant biological behaviors. These findings demonstrated that HDGF is important in promoting malignant biological behaviors, including proliferation, migration and invasion of hilar cholangiocarcinoma FRH0201 cells. Inhibition of the expression of HDGF downregulated the malignant biological behaviors, suggesting that downregulation of the protein expression of HDGF by RNAi may be a novel therapeutic approach to inhibit the progression of hilar cholangiocarcinoma.

  1. Downregulation of transferrin receptor surface expression by intracellular antibody

    International Nuclear Information System (INIS)

    Peng Jilin; Wu Sha; Zhao Xiaoping; Wang Min; Li Wenhan; Shen Xin; Liu Jing; Lei Ping; Zhu Huifen; Shen Guanxin

    2007-01-01

    To deplete cellular iron uptake, and consequently inhibit the proliferation of tumor cells, we attempt to block surface expression of transferrin receptor (TfR) by intracellular antibody technology. We constructed two expression plasmids (scFv-HAK and scFv-HA) coding for intracellular single-chain antibody against TfR with or without endoplasmic reticulum (ER) retention signal, respectively. Then they were transfected tumor cells MCF-7 by liposome. Applying RT-PCR, Western blotting, immunofluorescence microscopy and immunoelectron microscope experiments, we insure that scFv-HAK intrabody was successfully expressed and retained in ER contrasted to the secreted expression of scFv-HA. Flow cytometric analysis confirmed that the TfR surface expression was markedly decreased approximately 83.4 ± 2.5% in scFv-HAK transfected cells, while there was not significantly decrease in scFv-HA transfected cells. Further cell growth and apoptosis characteristics were evaluated by cell cycle analysis, nuclei staining and MTT assay. Results indicated that expression of scFv-HAK can dramatically induce cell cycle G1 phase arrest and apoptosis of tumor cells, and consequently significantly suppress proliferation of tumor cells compared with other control groups. For First time this study demonstrates the potential usage of anti-TfR scFv-intrabody as a growth inhibitor of TfR overexpressing tumors

  2. C-reactive protein inhibits survivin expression via Akt/mTOR pathway downregulation by PTEN expression in cardiac myocytes.

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    Beom Seob Lee

    Full Text Available C-reactive protein (CRP is one of the most important biomarkers for arteriosclerosis and cardiovascular disease. Recent studies have shown that CRP affects cell cycle and inflammatory process in cardiac myocytes. Survivin is also involved in cardiac myocytes replication and apoptosis. Reduction of survivin expression is associated with less favorable cardiac remodeling in animal models. However, the effect of CRP on survivin expression and its cellular mechanism has not yet been studied. We demonstrated that treatment of CRP resulted in a significant decrease of survivin protein expression in a concentration-dependent manner in cardiac myocytes. The upstream signaling proteins of survivin, such as Akt, mTOR and p70S6K, were also downregulated by CRP treatment. In addition, CRP increased the protein and mRNA levels of PTEN. The siRNA transfection or specific inhibitor treatment for PTEN restored the CRP-induced downregulation of Akt/mTOR/p70S6K pathway and survivin protein expression. Moreover, pretreatment with a specific p53 inhibitor decreased the CRP-induced PTEN expression. ERK-specific inhibitor also blocked the p53 phosphorylation and PTEN expression induced by CRP. Our study provides a novel insight into CRP-induced downregulation of survivin protein expression in cardiac myocytes through mechanisms that involved in downregulation of Akt/mTOR/p70S6K pathway by expression of PTEN.

  3. High SINE RNA Expression Correlates with Post-Transcriptional Downregulation of BRCA1

    Directory of Open Access Journals (Sweden)

    Giovanni Bosco

    2013-04-01

    Full Text Available Short Interspersed Nuclear Elements (SINEs are non-autonomous retrotransposons that comprise a large fraction of the human genome. SINEs are demethylated in human disease, but whether SINEs become transcriptionally induced and how the resulting transcripts may affect the expression of protein coding genes is unknown. Here, we show that downregulation of the mRNA of the tumor suppressor gene BRCA1 is associated with increased transcription of SINEs and production of sense and antisense SINE small RNAs. We find that BRCA1 mRNA is post-transcriptionally down-regulated in a Dicer and Drosha dependent manner and that expression of a SINE inverted repeat with sequence identity to a BRCA1 intron is sufficient for downregulation of BRCA1 mRNA. These observations suggest that transcriptional activation of SINEs could contribute to a novel mechanism of RNA mediated post-transcriptional silencing of human genes.

  4. Contribution of p75NTR to Schwannoma Growth and Therapeutic Responses

    Science.gov (United States)

    2015-05-01

    in 0.125% typ- sin with EDTA (Life Technology) and 0.2% collagenase (Sigma) in Hank’s balanced salt solution without calcium and magnesium (Life...cells following deafness correlate with cell proliferation. Molecular and cellular neurosciences 47:306-315. Rong R, Surace EI , Haipek CA, Gutmann DH

  5. Inflammation and ER Stress Downregulate BDH2 Expression and Dysregulate Intracellular Iron in Macrophages

    Directory of Open Access Journals (Sweden)

    Susu M. Zughaier

    2014-01-01

    Full Text Available Macrophages play a very important role in host defense and in iron homeostasis by engulfing senescent red blood cells and recycling iron. Hepcidin is the master iron regulating hormone that limits dietary iron absorption from the gut and limits iron egress from macrophages. Upon infection macrophages retain iron to limit its bioavailability which limits bacterial growth. Recently, a short chain butyrate dehydrogenase type 2 (BDH2 protein was reported to contain an iron responsive element and to mediate cellular iron trafficking by catalyzing the synthesis of the mammalian siderophore that binds labile iron; therefore, BDH2 plays a crucial role in intracellular iron homeostasis. However, BDH2 expression and regulation in macrophages have not yet been described. Here we show that LPS-induced inflammation combined with ER stress led to massive BDH2 downregulation, increased the expression of ER stress markers, upregulated hepcidin expression, downregulated ferroportin expression, caused iron retention in macrophages, and dysregulated cytokine release from macrophages. We also show that ER stress combined with inflammation synergistically upregulated the expression of the iron carrier protein NGAL and the stress-inducible heme degrading enzyme heme oxygenase-1 (HO-1 leading to iron liberation. This is the first report to show that inflammation and ER stress downregulate the expression of BDH2 in human THP-1 macrophages.

  6. Downregulation of leptin and resistin expression in blood following bariatric surgery.

    Science.gov (United States)

    Edwards, Claire; Hindle, A Katharine; Fu, Sidney; Brody, Fredrick

    2011-06-01

    Type 2 diabetes (T2D) resolves rapidly after bariatric surgery, even before substantial weight is lost. However, the molecular pathways underlying this phenomenon remain unclear. Microarray data has shown that numerous genes are differentially expressed in blood after bariatric surgery, including resistin and leptin. Resistin and leptin are circulating hormones derived from adipose tissue, which are associated with obesity and insulin resistance. This study examined expression of these genes before and after bariatric surgery in diabetic and nondiabetic obese patients. The study included 16 obese patients who underwent bariatric surgery, either Roux-en-Y gastric bypass (RYGB) or adjustable gastric banding. Eight patients had T2D. Preoperative blood samples were collected in PAXgene tubes to stabilize mRNA. Postoperative samples were collected 3 months after surgery. Total RNA was isolated and cDNA was synthesized. Real-time quantitative PCR was used to quantify mRNA. Results were analyzed using Student's t test with a P<0.05 considered significant. Postoperatively, five diabetic patients had discontinued hypoglycemic medications and one showed improved glycemic control. Both leptin and resistin mRNA levels were elevated in the diabetic group but decreased after surgery to levels near those of the nondiabetic group. Greater downregulation of resistin and leptin expression occurred in patients who lost more excess body weight (EBW), while patients who lost less than 10% EBW had a mean increase in expression of the two genes. Downregulation of both genes was more pronounced after RYGB compared to gastric banding. Downregulation of resistin and leptin gene expression after bariatric surgery may play a role in normalizing obesity-associated insulin resistance. Interestingly, downregulation is greater after RYGB and in patients who lose a greater proportion of EBW. Targeted therapies for obesity and diabetes may be developed by understanding the pathways by which these

  7. Elevated COX2 expression and PGE2 production by downregulation of RXRα in senescent macrophages

    International Nuclear Information System (INIS)

    Chen, Huimin; Ma, Feng; Hu, Xiaona; Jin, Ting; Xiong, Chuhui; Teng, Xiaochun

    2013-01-01

    Highlights: •Downregulation of RXRα in senescent macrophage. •RXRα suppresses NF-κB activity and COX2 expression. •Increased PGE2 production due to downregulation of RXRα. -- Abstract: Increased systemic level of inflammatory cytokines leads to numerous age-related diseases. In senescent macrophages, elevated prostaglandin E2 (PGE2) production contributes to the suppression of T cell function with aging, which increases the susceptibility to infections. However, the regulation of these inflammatory cytokines and PGE2 with aging still remains unclear. We have verified that cyclooxygenase (COX)-2 expression and PGE2 production are higher in LPS-stimulated macrophages from old mice than that from young mice. Downregulation of RXRα, a nuclear receptor that can suppress NF-κB activity, mediates the elevation of COX2 expression and PGE2 production in senescent macrophages. We also have found less induction of ABCA1 and ABCG1 by RXRα agonist in senescent macrophages, which partially accounts for high risk of atherosclerosis in aged population. Systemic treatment with RXRα antagonist HX531 in young mice increases COX2, TNF-α, and IL-6 expression in splenocytes. Our study not only has outlined a mechanism of elevated NF-κB activity and PGE2 production in senescent macrophages, but also provides RXRα as a potential therapeutic target for treating the age-related diseases

  8. Proneurotrophin-3 promotes cell cycle withdrawal of developing cerebellar granule cell progenitors via the p75 neurotrophin receptor.

    Science.gov (United States)

    Zanin, Juan Pablo; Abercrombie, Elizabeth; Friedman, Wilma J

    2016-07-19

    Cerebellar granule cell progenitors (GCP) proliferate extensively in the external granule layer (EGL) of the developing cerebellum prior to differentiating and migrating. Mechanisms that regulate the appropriate timing of cell cycle withdrawal of these neuronal progenitors during brain development are not well defined. The p75 neurotrophin receptor (p75(NTR)) is highly expressed in the proliferating GCPs, but is downregulated once the cells leave the cell cycle. This receptor has primarily been characterized as a death receptor for its ability to induce neuronal apoptosis following injury. Here we demonstrate a novel function for p75(NTR) in regulating proper cell cycle exit of neuronal progenitors in the developing rat and mouse EGL, which is stimulated by proNT3. In the absence of p75(NTR), GCPs continue to proliferate beyond their normal period, resulting in a larger cerebellum that persists into adulthood, with consequent motor deficits.

  9. Downregulation of ATM Gene and Protein Expression in Canine Mammary Tumors.

    Science.gov (United States)

    Raposo-Ferreira, T M M; Bueno, R C; Terra, E M; Avante, M L; Tinucci-Costa, M; Carvalho, M; Cassali, G D; Linde, S D; Rogatto, S R; Laufer-Amorim, R

    2016-11-01

    The ataxia telangiectasia mutated (ATM) gene encodes a protein associated with DNA damage repair and maintenance of genomic integrity. In women, ATM transcript and protein downregulation have been reported in sporadic breast carcinomas, and the absence of ATM protein expression has been associated with poor prognosis. The aim of this study was to evaluate ATM gene and protein expression in canine mammary tumors and their association with clinical outcome. ATM gene and protein expression was evaluated by reverse transcription-quantitative polymerase chain reaction and immunohistochemistry, respectively, in normal mammary gland samples (n = 10), benign mammary tumors (n = 11), nonmetastatic mammary carcinomas (n = 19), and metastatic mammary carcinomas (n = 11). Lower ATM transcript levels were detected in benign mammary tumors and carcinomas compared with normal mammary glands (P = .011). Similarly, lower ATM protein expression was observed in benign tumors (P = .0003), nonmetastatic mammary carcinomas (P ATM gene or protein levels were detected among benign tumors and nonmetastatic and metastatic mammary carcinomas (P > .05). The levels of ATM gene or protein expression were not significantly associated with clinical and pathological features or with survival. Similar to human breast cancer, the data in this study suggest that ATM gene and protein downregulation is involved in canine mammary gland tumorigenesis. © The Author(s) 2016.

  10. Downregulation of telomerase maintenance-related ACD expression in patients undergoing immunosuppresive therapy following kidney transplantation.

    Science.gov (United States)

    Witkowska, Agnieszka; Strzalka-Mrozik, Barbara; Owczarek, Aleksander; Gola, Joanna; Mazurek, Urszula; Grzeszczak, Wladyslaw; Gumprecht, Janusz

    2015-12-01

    Chronic administration of immunosuppressants has been associated with long-term consequences, including a higher risk of neoplasm development. The processes regulating telomere function exert a major influence on human cancer biology. The present study aimed to assess the effect of immunosuppressive therapy on the expression of genes associated with telomere maintenance and protection in patients following renal transplantation. A total of 51 patients that had undergone kidney transplantation and 54 healthy controls were enrolled in the study. The 51 transplant patients received a three-drug immunosuppressive regimen consisting of cyclosporine A, prednisone and mycophenolate mofetil. In stage 1 of the study, the expression profiles of 123 transcripts, which represented 70 genes, were assessed in peripheral mononuclear blood cells using an oligonucleotide microarray technique in 8 transplant recipients and 4 healthy control subjects. Among the analyzed transcripts, the expression levels of 4 differed significantly between the studied groups; however, only the ACD (adrenocortical dysplasia homolog) gene, encoding the telomere-binding protein POT1-interacting protein 1 (TPP1), was sufficiently specific for telomere homeostasis. The expression of ACD was downregulated in transplant recipients (fold change, 2.11; P=0.006). In stage 2 of the study, reverse transcription-quantitative polymerase chain reaction analysis of ACD , DKC1 and hTERT mRNA was conducted for all transplant patients and control subjects. The results confirmed the downregulation of the ACD gene in patients that had received immunosuppressive therapy (P=0.002). The results of the present study indicate that the downregulation of ACD gene transcription, and thus TPP1 protein expression, may enhance the capacity for cell immortalization, despite normal levels of other key telomere maintenance factors, in patients undergoing immunosuppressive therapy. Furthermore, the results indicate that TPP1 has

  11. Protein kinase B/Akt1 inhibits autophagy by down-regulating UVRAG expression

    Energy Technology Data Exchange (ETDEWEB)

    Yang, Wonseok; Ju, Ji-hyun; Lee, Kyung-min; Nam, KeeSoo; Oh, Sunhwa [Department of Life Science, College of Natural Science, Hanyang University, 17 Haengdang-dong, Seongdong-gu, Seoul 133-791 (Korea, Republic of); Shin, Incheol, E-mail: incheol@hanyang.ac.kr [Department of Life Science, College of Natural Science, Hanyang University, 17 Haengdang-dong, Seongdong-gu, Seoul 133-791 (Korea, Republic of)

    2013-02-01

    Autophagy, or autophagocytosis, is a selective intracellular degradative process involving the cell's own lysosomal apparatus. An essential component in cell development, homeostasis, repair and resistance to stress, autophagy may result in either cell death or survival. The targeted region of the cell is sequestered within a membrane structure, the autophagosome, for regulation of the catabolic process. A key factor in both autophagosome formation and autophagosome maturation is a protein encoded by the ultraviolet irradiation resistance-associated gene (UVRAG). Conversely, the serine/threonine-specific protein kinase B (PKB, also known as Akt), which regulates survival in various cancers, inhibits autophagy through mTOR activation. We found that Akt1 may also directly inhibit autophagy by down-regulating UVRAG both in a 293T transient transfection system and breast cancer cells stably expressing Akt1. The UVRAG with mutations at putative Akt1-phosphorylation sites were still inhibited by Akt1, and dominant-negative Akt1 also inhibited UVRAG expression, suggesting that Akt1 down-regulates UVRAG by a kinase activity-independent mechanism. We showed that Akt1 overexpression in MDA-MB-231 breast cancer cells down-regulated UVRAG transcription. Cells over-expressing Akt1 were more resistant than control cells to ultraviolet light-induced autophagy and exhibited the associated reduction in cell viability. Levels of the autophagosome indicator protein LC3B-II and mRFP-GFP-LC3 were reduced in cells that over-expressing Akt1. Inhibiting Akt1 by siRNA or reintroducing UVRAG gene rescued the level of LC3B-II in UV-irradiation. Altogether, these data suggest that Akt1 may inhibit autophagy by decreasing UVRAG expression, which also sensitizes cancer cells to UV irradiation.

  12. Down-regulation of PRKCB1 expression in Han Chinese patients with subsyndromal symptomatic depression.

    Science.gov (United States)

    Guo, Xiaoyun; Li, Zezhi; Zhang, Chen; Yi, Zhenghui; Li, Haozhe; Cao, Lan; Yuan, Chengmei; Hong, Wu; Wu, Zhiguo; Peng, Daihui; Chen, Jun; Xia, Weiping; Zhao, Guoqing; Wang, Fan; Yu, Shunying; Cui, Donghong; Xu, Yifeng; Golam, Chowdhury M I; Smith, Alicia K; Wang, Tong; Fang, Yiru

    2015-10-01

    Subsyndromal symptomatic depression (SSD) is a common disease with significant social dysfunction. However, SSD is still not well understood and the pathophysiology of it remains unclear. We classified 48 candidate genes for SSD according to our previous study into clusters and pathways using DAVID Bioinformatics Functional Annotation Tool. We further replicated the result by using real-time Quantitative PCR (qPCR) studies to examine the expression of identified genes (i.e., STAT5b, PKCB1, ABL1 and NRAS) in another group of Han Chinese patients with SSD (n = 50). We further validated the result by examining PRKCB1 expression collected from MDD patients (n = 20). To test whether a deficit in PRKCB1 expression leads to dysregulation in PRKCB1 dependent transcript networks, we tested mRNA expression levels for the remaining 44 genes out of 48 genes in SSD patients. Finally, the power of discovery was improved by incorporating information from Quantitative Trait (eQTL) analysis. The results showed that the PRCKB1 gene expression in peripheral blood mononuclear cells (PBMC) was 33.3% down-regulated in SSD patients (n = 48, t = 3.202, p = 0.002), and a more dramatic (n = 17, 49%) down-regulation in MDD patients than control (n = 49, t = 2.114, p = 0.001). We also identified 37 genes that displayed a strong correlation with PRKCB1 mRNA expression levels in SSD patients. The expression of PRKCB1 was regulated by multiple single nucleotide polymorphisms (SNPs) both at the transcript level and exon level. In conclusion, we first found a significant decrease of PRCKB1 mRNA expression in SSD, suggesting PRKCB1 might be the candidate gene and biomarker for SSD. Copyright © 2015 Elsevier Ltd. All rights reserved.

  13. Active RNA replication of hepatitis C virus downregulates CD81 expression.

    Science.gov (United States)

    Ke, Po-Yuan; Chen, Steve S-L

    2013-01-01

    So far how hepatitis C virus (HCV) replication modulates subsequent virus growth and propagation still remains largely unknown. Here we determine the impact of HCV replication status on the consequential virus growth by comparing normal and high levels of HCV RNA expression. We first engineered a full-length, HCV genotype 2a JFH1 genome containing a blasticidin-resistant cassette inserted at amino acid residue of 420 in nonstructural (NS) protein 5A, which allowed selection of human hepatoma Huh7 cells stably-expressing HCV. Short-term establishment of HCV stable cells attained a highly-replicating status, judged by higher expressions of viral RNA and protein as well as higher titer of viral infectivity as opposed to cells harboring the same genome without selection. Interestingly, maintenance of highly-replicating HCV stable cells led to decreased susceptibility to HCV pseudotyped particle (HCVpp) infection and downregulated cell surface level of CD81, a critical HCV entry (co)receptor. The decreased CD81 cell surface expression occurred through reduced total expression and cytoplasmic retention of CD81 within an endoplasmic reticulum -associated compartment. Moreover, productive viral RNA replication in cells harboring a JFH1 subgenomic replicon containing a similar blasticidin resistance gene cassette in NS5A and in cells robustly replicating full-length infectious genome also reduced permissiveness to HCVpp infection through decreasing the surface expression of CD81. The downregulation of CD81 surface level in HCV RNA highly-replicating cells thus interfered with reinfection and led to attenuated viral amplification. These findings together indicate that the HCV RNA replication status plays a crucial determinant in HCV growth by modulating the expression and intracellular localization of CD81.

  14. Active RNA replication of hepatitis C virus downregulates CD81 expression.

    Directory of Open Access Journals (Sweden)

    Po-Yuan Ke

    Full Text Available So far how hepatitis C virus (HCV replication modulates subsequent virus growth and propagation still remains largely unknown. Here we determine the impact of HCV replication status on the consequential virus growth by comparing normal and high levels of HCV RNA expression. We first engineered a full-length, HCV genotype 2a JFH1 genome containing a blasticidin-resistant cassette inserted at amino acid residue of 420 in nonstructural (NS protein 5A, which allowed selection of human hepatoma Huh7 cells stably-expressing HCV. Short-term establishment of HCV stable cells attained a highly-replicating status, judged by higher expressions of viral RNA and protein as well as higher titer of viral infectivity as opposed to cells harboring the same genome without selection. Interestingly, maintenance of highly-replicating HCV stable cells led to decreased susceptibility to HCV pseudotyped particle (HCVpp infection and downregulated cell surface level of CD81, a critical HCV entry (coreceptor. The decreased CD81 cell surface expression occurred through reduced total expression and cytoplasmic retention of CD81 within an endoplasmic reticulum -associated compartment. Moreover, productive viral RNA replication in cells harboring a JFH1 subgenomic replicon containing a similar blasticidin resistance gene cassette in NS5A and in cells robustly replicating full-length infectious genome also reduced permissiveness to HCVpp infection through decreasing the surface expression of CD81. The downregulation of CD81 surface level in HCV RNA highly-replicating cells thus interfered with reinfection and led to attenuated viral amplification. These findings together indicate that the HCV RNA replication status plays a crucial determinant in HCV growth by modulating the expression and intracellular localization of CD81.

  15. Mycobacterium leprae downregulates the expression of PHEX in Schwann cells and osteoblasts

    Directory of Open Access Journals (Sweden)

    Sandra R Boiça Silva

    2010-08-01

    Full Text Available Neuropathy and bone deformities, lifelong sequelae of leprosy that persist after treatment, result in significant impairment to patients and compromise their social rehabilitation. Phosphate-regulating gene with homologies to endopeptidase on the X chromosome (PHEX is a Zn-metalloendopeptidase, which is abundantly expressed in osteoblasts and many other cell types, such as Schwann cells, and has been implicated in phosphate metabolism and X-linked rickets. Here, we demonstrate that Mycobacterium leprae stimulation downregulates PHEX transcription and protein expression in a human schwannoma cell line (ST88-14 and human osteoblast lineage. Modulation of PHEX expression was observed to a lesser extent in cells stimulated with other species of mycobacteria, but was not observed in cultures treated with latex beads or with the facultative intracellular bacterium Salmonella typhimurium. Direct downregulation of PHEX by M. leprae could be involved in the bone resorption observed in leprosy patients. This is the first report to describe PHEX modulation by an infectious agent.

  16. Cholesterol Down-Regulates BK Channels Stably Expressed in HEK 293 Cells

    Science.gov (United States)

    Deng, Xiu-Ling; Sun, Hai-Ying; Li, Gui-Rong

    2013-01-01

    Cholesterol is one of the major lipid components of the plasma membrane in mammalian cells and is involved in the regulation of a number of ion channels. The present study investigates how large conductance Ca2+-activated K+ (BK) channels are regulated by membrane cholesterol in BK-HEK 293 cells expressing both the α-subunit hKCa1.1 and the auxiliary β1-subunit or in hKCa1.1-HEK 293 cells expressing only the α-subunit hKCa1.1 using approaches of electrophysiology, molecular biology, and immunocytochemistry. Membrane cholesterol was depleted in these cells with methyl-β-cyclodextrin (MβCD), and enriched with cholesterol-saturated MβCD (MβCD-cholesterol) or low-density lipoprotein (LDL). We found that BK current density was decreased by cholesterol enrichment in BK-HEK 293 cells, with a reduced expression of KCa1.1 protein, but not the β1-subunit protein. This effect was fully countered by the proteasome inhibitor lactacystin or the lysosome function inhibitor bafilomycin A1. Interestingly, in hKCa1.1-HEK 293 cells, the current density was not affected by cholesterol enrichment, but directly decreased by MβCD, suggesting that the down-regulation of BK channels by cholesterol depends on the auxiliary β1-subunit. The reduced KCa1.1 channel protein expression was also observed in cultured human coronary artery smooth muscle cells with cholesterol enrichment using MβCD-cholesterol or LDL. These results demonstrate the novel information that cholesterol down-regulates BK channels by reducing KCa1.1 protein expression via increasing the channel protein degradation, and the effect is dependent on the auxiliary β1-subunit. PMID:24260325

  17. Downregulation of CXCR4 Expression and Functionality After Zoledronate Exposure in Canine Osteosarcoma.

    Science.gov (United States)

    Byrum, M L; Pondenis, H C; Fredrickson, R L; Wycislo, K L; Fan, T M

    2016-07-01

    The establishment and progression of metastases remains the life-limiting factor for dogs diagnosed with osteosarcoma (OS). The pattern of metastases is likely regulated through interactions between chemokine receptors and chemokines, and perturbations in these signaling cascades responsible for cytoskeletal organization and directional migration have the potential to alter metastatic cell trafficking behaviors. Zoledronate will impair directional migration of OS cells through downregulation of chemokine (C-X-C motif) receptor 4 (CXCR4) expression and functionality. Nineteen archived tumor specimens and plasma from 20 dogs with OS. Prospectively, the expressions of CXCR4 were studied in OS cell lines and spontaneous tumor samples. The effect of zoledronate on CXCR4 expression and functionality was investigated by characterizing responses in 3 OS cell lines. In 19 OS specimens and 20 dogs with OS, changes in CXCR4 expression and circulating CXCR4 concentrations were characterized in response to zoledronate therapy respectively. All canine OS cells express CXCR4, and zoledronate reduces CXCR4 expression and functionality by 27.7% (P < .0001), through augmented proteasome degradation and reduced prenylation of heterotrimeric G-proteins in 33% of tumor cell lines evaluated. In OS-bearing dogs, zoledronate reduces CXCR4 expressions by 40% within the primary tumor compared to untreated controls (P = .03) and also decreases the circulating concentrations of CXCR4 in 18 of 20 dogs with OS. Zoledronate can alter CXCR4 expression and functionality in OS cells, and consequent perturbations in CXCR4 intracellular signaling cascades might influence patterns of metastases. Copyright © 2016 The Authors. Journal of Veterinary Internal Medicine published by Wiley Periodicals, Inc. on behalf of the American College of Veterinary Internal Medicine.

  18. [The Effect of TALENs-mediated Downregulation Expression of Nanog on Malignant Behavior of Cervical Cancer HeLa Cells].

    Science.gov (United States)

    Yu, Ai-qing; Li, Cheng-lin; Yang, Yi; Yan, Shi-rong

    2016-01-01

    To study the effect of downregulation expression of Nanog on malignant behavior of cervical cancer HeLa cells. Gene editing tool TALENs was employed to induce downregulation expression of Nanog, and Nanog mutation was evaluated by sequencing. RT-PCR and Western blot was used to detect the mRNA and protein expression level, respectively. Colony-formation assay, Transwell invasion assay, and chemotherapy sensibility assay was carried out to assess the capacity of colony-formation, invasion, and chemoresistance, respectively. TALENs successfully induced Nanog mutation and downregulated Nanog expression. Nanog mRNA and protein expression of Nanog-mutated monoclonal HeLa cells downregulated 3 times compared to thoses of wild-type HeLa cells (P HeLa cells were observed when compared to those of wild-type HeLa cells (P HeLa cells. Importantly, downregulation or silencing of Nanog is promising to be a novel strategy for the treatment of cervical carcinoma.

  19. Downregulation of keratin 76 expression during oral carcinogenesis of human, hamster and mouse.

    Directory of Open Access Journals (Sweden)

    Srikant Ambatipudi

    Full Text Available Keratins are structural marker proteins with tissue specific expression; however, recent reports indicate their involvement in cancer progression. Previous study from our lab revealed deregulation of many genes related to structural molecular integrity including KRT76. Here we evaluate the role of KRT76 downregulation in oral precancer and cancer development.We evaluated KRT76 expression by qRT-PCR in normal and tumor tissues of the oral cavity. We also analyzed K76 expression by immunohistochemistry in normal, oral precancerous lesion (OPL, oral squamous cell carcinoma (OSCC and in hamster model of oral carcinogenesis. Further, functional implication of KRT76 loss was confirmed using KRT76-knockout (KO mice.We observed a strong association of reduced K76 expression with increased risk of OPL and OSCC development. The buccal epithelium of DMBA treated hamsters showed a similar trend. Oral cavity of KRT76-KO mice showed preneoplastic changes in the gingivobuccal epithelium while no pathological changes were observed in KRT76 negative tissues such as tongue.The present study demonstrates loss of KRT76 in oral carcinogenesis. The KRT76-KO mice data underlines the potential of KRT76 being an early event although this loss is not sufficient to drive the development of oral cancers. Thus, future studies to investigate the contributing role of KRT76 in light of other tumor driving events are warranted.

  20. Abscisic acid ameliorates experimental IBD by downregulating cellular adhesion molecule expression and suppressing immune cell infiltration.

    Science.gov (United States)

    Guri, Amir J; Hontecillas, Raquel; Bassaganya-Riera, Josep

    2010-12-01

    Abscisic acid (ABA) has shown effectiveness in ameliorating inflammation in obesity, diabetes and cardiovascular disease models. The objective of this study was to determine whether ABA prevents or ameliorates experimental inflammatory bowel disease (IBD). C57BL/6J mice were fed diets with or without ABA (100mg/kg) for 35 days prior to challenge with 2.5% dextran sodium sulfate (DSS). The severity of clinical disease was assessed daily. Colonic mucosal lesions were evaluated by histopathology, and cellular adhesion molecular and inflammatory markers were assayed by real-time quantitative PCR. Flow cytometry was used to quantify leukocyte populations in the blood, spleen, and mesenteric lymph nodes (MLN). The effect of ABA on cytotoxic T-lymphocyte antigen 4 (CTLA-4) expression in splenocytes was also investigated. ABA significantly ameliorated disease activity, colitis and reduced colonic leukocyte infiltration and inflammation. These improvements were associated with downregulation in vascular cell adhesion marker-1 (VCAM-1), E-selectin, and mucosal addressin adhesion marker-1 (MAdCAM-1) expression. ABA also increased CD4(+) and CD8(+) T-lymphocytes in blood and MLN and regulatory T cells in blood. In vitro, ABA increased CTLA-4 expression through a PPAR γ-dependent mechanism. We conclude that ABA ameliorates gut inflammation by modulating T cell distribution and adhesion molecule expression. Copyright © 2010 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism. All rights reserved.

  1. Aryl hydrocarbon receptor downregulates MYCN expression and promotes cell differentiation of neuroblastoma.

    Directory of Open Access Journals (Sweden)

    Pei-Yi Wu

    Full Text Available Neuroblastoma (NB is the most common malignant disease of infancy. MYCN amplification is a prognostic factor for NB and is a sign of highly malignant disease and poor patient prognosis. In this study, we aimed to investigate novel MYCN-related genes and assess how they affect NB cell behavior. The different gene expression found in 10 MYCN amplification NB tumors and 10 tumors with normal MYCN copy number were analyzed using tissue oligonucleotide microarrays. Ingenuity Pathway Analysis was subsequently performed to identify the potential genes involved in MYCN regulation pathways. Aryl hydrocarbon receptor (AHR, a receptor for dioxin-like compounds, was found to be inversely correlated with MYCN expression in NB tissues. This correlation was confirmed in a further 14 human NB samples. Moreover, AHR expression in NB tumors was found to correlate highly with histological grade of differentiation. In vitro studies revealed that AHR overexpression in NB cells induced spontaneous cell differentiation. In addition, it was found that ectopic expression of AHR suppressed MYCN promoter activity resulting in downregulation of MYCN expression. The suppression effect of AHR on the transcription of MYCN was compensated for by E2F1 overexpression, indicating that E2F1 is involved in the AHR-regulating MYCN pathway. Furthermore, AHR shRNA promotes the expression of E2F1 and MYCN in NB cells. These findings suggest that AHR is one of the upstream regulators of MYCN. Through the modulation of E2F1, AHR regulates MYCN gene expression, which may in turn affect NB differentiation.

  2. Mechanical Stress Downregulates MHC Class I Expression on Human Cancer Cell Membrane

    DEFF Research Database (Denmark)

    La Rocca, Rosanna; Tallerico, Rossana; Hassan, Almosawy Talib

    2014-01-01

    In our body, cells are continuously exposed to physical forces that can regulate different cell functions such as cell proliferation, differentiation and death. In this work, we employed two different strategies to mechanically stress cancer cells. The cancer and healthy cell populations were...... treated either with mechanical stress delivered by a micropump (fabricated by deep X-ray nanolithography) or by ultrasound wave stimuli. A specific down-regulation of Major Histocompatibility Complex (MHC) class I molecules expression on cancer cell membrane compared to different kinds of healthy cells...... between 700–1800 cm-1, indicated a relative concentration variation of MHC class I. PCA analysis was also performed to distinguish control and stressed cells within different cell lines. These mechanical induced phenotypic changes increase the tumor immunogenicity, as revealed by the related increased...

  3. Nutlin-3 down-regulates retinoblastoma protein expression and inhibits muscle cell differentiation

    Energy Technology Data Exchange (ETDEWEB)

    Walsh, Erica M. [Department of Biochemistry, Boston University School of Medicine, Boston, MA 02118 (United States); Niu, MengMeng; Bergholz, Johann [Center of Growth, Metabolism and Aging, College of Life Sciences, Sichuan University, Chengdu, 610014 China (China); Jim Xiao, Zhi-Xiong, E-mail: jxiao@bu.edu [Department of Biochemistry, Boston University School of Medicine, Boston, MA 02118 (United States); Center of Growth, Metabolism and Aging, College of Life Sciences, Sichuan University, Chengdu, 610014 China (China)

    2015-05-29

    The p53 tumor suppressor gene plays a critical role in regulation of proliferation, cell death and differentiation. The MDM2 oncoprotein is a major negative regulator for p53 by binding to and targeting p53 for proteasome-mediated degradation. The small molecule inhibitor, nutlin-3, disrupts MDM2-p53 interaction resulting in stabilization and activation of p53 protein. We have previously shown that nutlin-3 activates p53, leading to MDM2 accumulation as concomitant of reduced retinoblastoma (Rb) protein stability. It is well known that Rb is important in muscle development and myoblast differentiation and that rhabdomyosarcoma (RMS), or cancer of the skeletal muscle, typically harbors MDM2 amplification. In this study, we show that nutlin-3 inhibited myoblast proliferation and effectively prevented myoblast differentiation, as evidenced by lack of expression of muscle differentiation markers including myogenin and myosin heavy chain (MyHC), as well as a failure to form multinucleated myotubes, which were associated with dramatic increases in MDM2 expression and decrease in Rb protein levels. These results indicate that nutlin-3 can effectively inhibit muscle cell differentiation. - Highlights: • Nutlin-3 inhibits myoblast proliferation and prevents differentiation into myotubes. • Nutlin-3 increases MDM2 expression and down-regulates Rb protein levels. • This study has implication in nutlin-3 treatment of rhabdomyosarcomas.

  4. Downregulation of SPARC expression inhibits the invasion of human trophoblast cells in vitro.

    Directory of Open Access Journals (Sweden)

    Yahong Jiang

    Full Text Available Successful pregnancy depends on the precise regulation of extravilloustrophoblast (EVT invasion into the uterine decidua. SPARC (secreted protein acidic and rich in cysteine is a matricellular glycoprotein that plays critical roles in the pathologies associated with obesity and diabetes, as well as tumorigenesis. The objective of this study was to investigate the role of SPARC in the process of trophoblast invasion which shares many similarities with tumor cell invasion. By Western blot, higher expression of SPARC was observed in mouse brain, ovary and uterus compared to other mouse tissues. Immunohistochemistry analysis revealed a spatio-temporal expression of SPARC in mouse uterus in the periimplantation period. At the implantation site of d8 pregnancy, SPARC mainly accumulated in the secondary decidua zone (SDZ, trophoblast cells and blastocyst. The expression of SPARC was also detected in human placental villi and trophoblast cell lines. In a Matrigel invasion assay, we found SPARC-specific RNA interference significantly reduced the invasion of human extravilloustrophoblast HTR8/SVneo cells. Microarray analysis revealed that SPARC depletion upregulated the expression of interleukin 11 (IL11, KISS1, insulin-like growth factor binding protein 4 (IGFBP4, collagen type I alpha 1 (COLIA1, matrix metallopeptidase 9 (MMP9, and downregulated the expression of the alpha polypeptide of chorionic gonadotropin (CGA, MMP1, gap junction protein alpha 1 (GJA1, et al. The gene array result was further validated by qRT-PCR and Western blot. The present data indicate that SPARC may play an important role in the regulation of normal placentation by promoting the invasion of trophoblast cells into the uterine decidua.

  5. Tunicamycin promotes apoptosis in leukemia cells through ROS generation and downregulation of survivin expression.

    Science.gov (United States)

    Lim, Eun Jin; Heo, Jeonghoon; Kim, Young-Ho

    2015-08-01

    Tunicamycin (TN), one of the endoplasmic reticulum stress inducers, has been reported to inhibit tumor cell growth and exhibit anticarcinogenic activity. However, the mechanism by which TN initiates apoptosis remains poorly understood. In the present study, we investigated the effect of TN on the apoptotic pathway in U937 cells. We show that TN induces apoptosis in association with caspase-3 activation, generation of reactive oxygen species (ROS), and downregulation of survivin expression. P38 MAPK (mitogen-activated protein kinase) and the generation of ROS signaling pathway play crucial roles in TN-induced apoptosis in U937 cells. We hypothesized that TN-induced activation of p38 MAPK signaling pathway is responsible for cell death. To test this hypothesis, we selectively inhibited MAPK during treatment with TN. Our data demonstrated that inhibitor of p38 (SB), but not ERK (PD) or JNK (SP), partially maintained apoptosis during treatment with TN. Pre-treatment with NAC and GSH markedly prevented cell death, suggesting a role for ROS in this process. Ectopic expression of survivin in U937 cells attenuated TN-induced apoptosis by suppression of caspase-3 cleavage, mitochondrial membrane potential, and cytochrome c release in U937 cells. Taken together, our results show that TN modulates multiple components of the apoptotic response of human leukemia cells and raise the possibility of a novel therapeutic strategy for hematological malignancies.

  6. Znhit1 causes cell cycle arrest and down-regulates CDK6 expression

    International Nuclear Information System (INIS)

    Yang, Zhengmin; Cao, Yonghao; Zhu, Xiaoyan; Huang, Ying; Ding, Yuqiang; Liu, Xiaolong

    2009-01-01

    Cyclin-dependent kinase 6 (CDK6) is the key element of the D-type cyclin holoenzymes which has been found to function in the regulation of G1-phase of the cell cycle and is presumed to play important roles in T cell function. In this study, Znhit1, a member of a new zinc finger protein family defined by a conserved Zf-HIT domain, induced arrest in the G1-phase of the cell cycle in NIH/3T3 cells. Of the G1 cell cycle factors examined, the expression of CDK6 was found to be strongly down-regulated by Znhit1 via transcriptional repression. This effect may have correlations with the decreased acetylation level of histone H4 in the CDK6 promoter region. In addition, considering that CDK6 expression predominates in T cells, the negative regulatory role of Znhit1 in TCR-induced T cell proliferation was validated using transgenic mice. These findings identified Znhit1 as a CDK6 regulator that plays an important role in cell proliferation.

  7. microRNA-7 down-regulation mediates excessive collagen expression in localized scleroderma.

    Science.gov (United States)

    Etoh, Mitsuhiko; Jinnin, Masatoshi; Makino, Katsunari; Yamane, Keitaro; Nakayama, Wakana; Aoi, Jun; Honda, Noritoshi; Kajihara, Ikko; Makino, Takamitsu; Fukushima, Satoshi; Ihn, Hironobu

    2013-01-01

    Localized scleroderma (LSc), a connective tissue disorder restricted to the skin and subcutaneous tissue, is characterized by skin fibrosis due to an excessive deposition of types I collagen. The mechanism of such fibrosis is still unknown, but epigenetics may play some roles in the excessive collagen expression. In the present study, we investigated the mechanism of fibrosis seen in LSc, focusing on microRNA (miRNA). miRNA expression was determined by PCR array, real-time PCR, and in situ hybridization. The function of miRNA was evaluated using specific inhibitor. Immunoblotting was performed to detect α2(I) collagen protein. PCR array analysis using tissue miRNA demonstrated miR-7 level was significantly decreased in LSc skin as well as keloid tissue compared to normal skin in vivo. In situ hybridization also showed miR-7 expression in dermal fibroblasts was decreased in LSc dermis. The transfection of specific inhibitor for miR-7 into cultured normal dermal fibroblasts resulted in the up-regulation of α2(I) collagen protein in vitro. Also, the serum levels of miR-7 were significantly decreased in LSc patients compared with healthy controls, but serum miR-29a levels not. Systemic or local down-regulation of miR-7 may contribute to the pathogenesis of LSc via the overexpression of α2(I) collagen, and serum miR-7 may be useful as a disease marker. Investigation of the regulatory mechanisms of LSc by miRNA may lead to new treatments by the transfection into the lesional skin of this disease.

  8. Hyperglycemia and Diabetes Downregulate the Functional Expression of TRPV4 Channels in Retinal Microvascular Endothelium

    Science.gov (United States)

    Monaghan, Kevin; McNaughten, Jennifer; McGahon, Mary K.; Kelly, Catriona; Kyle, Daniel; Yong, Phaik Har

    2015-01-01

    Retinal endothelial cell dysfunction is believed to play a key role in the etiology and pathogenesis of diabetic retinopathy. Numerous studies have shown that TRPV4 channels are critically involved in maintaining normal endothelial cell function. In the current paper, we demonstrate that TRPV4 is functionally expressed in the endothelium of the retinal microcirculation and that both channel expression and activity is downregulated by hyperglycaemia. Quantitative PCR and immunostaining demonstrated molecular expression of TRPV4 in cultured bovine retinal microvascular endothelial cells (RMECs). Functional TRPV4 activity was assessed in cultured RMECs from endothelial Ca2+-responses recorded using fura-2 microfluorimetry and electrophysiological recordings of membrane currents. The TRPV4 agonist 4α-phorbol 12,13-didecanoate (4-αPDD) increased [Ca2+]i in RMECs and this response was largely abolished using siRNA targeted against TRPV4. These Ca2+-signals were completely inhibited by removal of extracellular Ca2+, confirming their dependence on influx of extracellular Ca2+. The 4-αPDD Ca2+-response recorded in the presence of cyclopiazonic acid (CPA), which depletes the intracellular stores preventing any signal amplification through store release, was used as a measure of Ca2+-influx across the cell membrane. This response was blocked by HC067047, a TRPV4 antagonist. Under voltage clamp conditions, the TRPV4 agonist GSK1016790A stimulated a membrane current, which was again inhibited by HC067047. Following incubation with 25mM D-glucose TRPV4 expression was reduced in comparison with RMECs cultured under control conditions, as were 4αPDD-induced Ca2+-responses in the presence of CPA and ion currents evoked by GSK1016790A. Molecular expression of TRPV4 in the retinal vascular endothelium of 3 months’ streptozotocin-induced diabetic rats was also reduced in comparison with that in age-matched controls. We conclude that hyperglycaemia and diabetes reduce the

  9. SENP1 attenuates the liver fibrosis through down-regulating the expression of SMAD2.

    Science.gov (United States)

    Wu, Linshi; Qiu, Weiqing; Sun, Jianhua; Wang, Jian

    2018-01-01

    To investigate whether SENP1 could play a regulating role in the liver fibrosis process, the Sprague-Dawley (SD) rats were used to establish the liver fibrosis rat models by intraperitoneally injecting with 1 ml/kg of 10% CCl 4 , while the control normal rats were injected with olive oil. Then confirmation experiments to verify the successful establishment of these models were conducted by detecting the cellular and lobular architecture, and liver function indexes using hematoxylin-eosin staining, Masson's trichrome staining and microplate method, respectively. In addition, the expression levels of fibrosis markers including collagen I, collagen III, α-SMA and TGF-β1 were inspected using quantitative real-time PCR (qRT-PCR), as well as SMAD2. Subsequently, the relative mRNA and protein level of SENP1 was also determined via qRT-PCR and western blot analysis. Next, the HSC-T6 cells of SENP1 knock-down were constructed and used to test the relative protein expression levels of α-SMA and SMAD2 in these cells. The results of hematoxylin-eosin staining, Masson's trichrome staining and microplate method turned out that the rat liver fibrosis models were constructed successfully, which was further confirmed by the increased expression of collagen I, collagen III, α-SMA and TGF-β1 in mRNA and protein level, as well as SMAD2. Then the expression of SENP1 was overexpressed in the rat liver fibrosis models induced by CCl 4 and the TGF-β1 treatment could increase the protein expression level of collagen I, collagen III and α-SMA. Lastly, the SENP1 knockdown HSC-T6 cells were successfully constructed, while the silence of SENP1 down-regulated the protein expression of α-SMA and SMAD2. In conclusion, this study provided a new regulation mechanism about the liver fibrosis process. Copyright © 2017 Elsevier Inc. All rights reserved.

  10. Ethanol extracts of black pepper or turmeric down-regulated SIRT1 protein expression in Daudi culture cells.

    Science.gov (United States)

    Nishimura, Yuri; Kitagishi, Yasuko; Yoshida, Hitomi; Okumura, Naoko; Matsuda, Satoru

    2011-01-01

    SIRT1 is a mammalian candidate molecule involved in longevity and diverse metabolic processes. The present study aimed to determine the effects of certain herbs and spices on SIRT1 expression. Human cell lines Daudi, Jurkat, U937 and K562 were cultured in RPMI-1640. Herb and spice powders were prepared and the supernatants were collected. RT-PCR was used to quantify the expression level of the gene. Protein samples were then analyzed by Western blotting. Western blotting revealed the down-regulation of SIRT1 protein expression in Daudi cells treated with extracts of black pepper or turmeric. On the other hand, the effect on the SIRT1 gene expression examined by reverse transcription polymerase chain reaction was unaltered. In conclusion, component(s) of certain herbs and spices may induce the down-regulation of SIRT1 protein.

  11. Down-Regulation of Gene Expression by RNA-Induced Gene Silencing

    Science.gov (United States)

    Travella, Silvia; Keller, Beat

    Down-regulation of endogenous genes via post-transcriptional gene silencing (PTGS) is a key to the characterization of gene function in plants. Many RNA-based silencing mechanisms such as post-transcriptional gene silencing, co-suppression, quelling, and RNA interference (RNAi) have been discovered among species of different kingdoms (plants, fungi, and animals). One of the most interesting discoveries was RNAi, a sequence-specific gene-silencing mechanism initiated by the introduction of double-stranded RNA (dsRNA), homologous in sequence to the silenced gene, which triggers degradation of mRNA. Infection of plants with modified viruses can also induce RNA silencing and is referred to as virus-induced gene silencing (VIGS). In contrast to insertional mutagenesis, these emerging new reverse genetic approaches represent a powerful tool for exploring gene function and for manipulating gene expression experimentally in cereal species such as barley and wheat. We examined how RNAi and VIGS have been used to assess gene function in barley and wheat, including molecular mechanisms involved in the process and available methodological elements, such as vectors, inoculation procedures, and analysis of silenced phenotypes.

  12. Mechanical Stress Downregulates MHC Class I Expression on Human Cancer Cell Membrane

    KAUST Repository

    La Rocca, Rosanna

    2014-12-26

    In our body, cells are continuously exposed to physical forces that can regulate different cell functions such as cell proliferation, differentiation and death. In this work, we employed two different strategies to mechanically stress cancer cells. The cancer and healthy cell populations were treated either with mechanical stress delivered by a micropump (fabricated by deep X-ray nanolithography) or by ultrasound wave stimuli. A specific down-regulation of Major Histocompatibility Complex (MHC) class I molecules expression on cancer cell membrane compared to different kinds of healthy cells (fibroblasts, macrophages, dendritic and lymphocyte cells) was observed, stimulating the cells with forces in the range of nano-newton, and pressures between 1 and 10 bar (1 bar = 100.000 Pascal), depending on the devices used. Moreover, Raman spectroscopy analysis, after mechanical treatment, in the range between 700–1800 cm−1, indicated a relative concentration variation of MHC class I. PCA analysis was also performed to distinguish control and stressed cells within different cell lines. These mechanical induced phenotypic changes increase the tumor immunogenicity, as revealed by the related increased susceptibility to Natural Killer (NK) cells cytotoxic recognition.

  13. Mechanical stress downregulates MHC class I expression on human cancer cell membrane.

    Directory of Open Access Journals (Sweden)

    Rosanna La Rocca

    Full Text Available In our body, cells are continuously exposed to physical forces that can regulate different cell functions such as cell proliferation, differentiation and death. In this work, we employed two different strategies to mechanically stress cancer cells. The cancer and healthy cell populations were treated either with mechanical stress delivered by a micropump (fabricated by deep X-ray nanolithography or by ultrasound wave stimuli. A specific down-regulation of Major Histocompatibility Complex (MHC class I molecules expression on cancer cell membrane compared to different kinds of healthy cells (fibroblasts, macrophages, dendritic and lymphocyte cells was observed, stimulating the cells with forces in the range of nano-newton, and pressures between 1 and 10 bar (1 bar = 100.000 Pascal, depending on the devices used. Moreover, Raman spectroscopy analysis, after mechanical treatment, in the range between 700-1800 cm(-1, indicated a relative concentration variation of MHC class I. PCA analysis was also performed to distinguish control and stressed cells within different cell lines. These mechanical induced phenotypic changes increase the tumor immunogenicity, as revealed by the related increased susceptibility to Natural Killer (NK cells cytotoxic recognition.

  14. Cysteinyl leukotrienes C4 and D4 downregulate human mast cell expression of toll-like receptors 1 through 7.

    Science.gov (United States)

    Karpov, V; Ilarraza, R; Catalli, A; Kulka, M

    2018-01-01

    Cysteinyl leukotrienes (CysLT) are potent inflammatory lipid molecules that mediate some of the pathophysiological responses associated with asthma such as bronchoconstriction, vasodilation and increased microvascular permeability. As a result, CysLT receptor antagonists (LRA), such as montelukast, have been used to effectively treat patients with asthma. We have recently shown that mast cells are necessary modulators of innate immune responses to bacterial infection and an important component of this innate immune response may involve the production of CysLT. However, the effect of LRA on innate immune receptors, particularly on allergic effector cells, is unknown. This study determined the effect of CysLT on toll-like receptor (TLR) expression by the human mast cell line LAD2. Real-time PCR analysis determined that LTC4, LTD4 and LTE4 downregulated mRNA expression of several TLR. Specifically in human CD34+-derived human mast cells (HuMC), LTC4 inhibited expression of TLR1, 2, 4, 5, 6 and 7 while LTD4 inhibited expression of TLR1-7. Montelukast blocked LTC4-mediated downregulation of all TLR, suggesting that these effects were mediated by activation of the CysLT1 receptor (CysLT1R). Flow cytometry analysis confirmed that LTC4 downregulated surface expression of TLR2 which was blocked by montelukast. These data show that CysLT can modulate human mast cell expression of TLR and that montelukast may be beneficial for innate immune responses mediated by mast cells.

  15. RANKL downregulates cell surface CXCR6 expression through JAK2/STAT3 signaling pathway during osteoclastogenesis

    Energy Technology Data Exchange (ETDEWEB)

    Li, Changhong; Zhao, Jinxia; Sun, Lin; Yao, Zhongqiang; Liu, Rui [Department of Rheumatology and Immunology, Peking University Third Hospital, Beijing 100191 (China); Huang, Jiansheng [Department of Pediatrics, Washington University School of Medicine, St. Louis, MO (United States); Liu, Xiangyuan, E-mail: liu-xiangyuan@263.net [Department of Rheumatology and Immunology, Peking University Third Hospital, Beijing 100191 (China)

    2012-12-14

    Highlights: Black-Right-Pointing-Pointer CXCR6 is down-regulated during RANKL-induced osteoclastogenesis in RAW264.7 cells. Black-Right-Pointing-Pointer CXCR6 reduction was nearly reversed by inhibition of JAK2/STAT3 signaling pathway. Black-Right-Pointing-Pointer CXCL16 alone does not positively regulate osteoclastogenesis. -- Abstract: The receptor activator of nuclear factor-{kappa}B ligand (RANKL), as a member of the tumor necrosis factor (TNF) family, plays an essential role in osteoclast differentiation and function. Chemokines and their receptors have recently been shown to play critical roles in osteoclastogenesis, however, whether CXCL16-CXCR6 plays role in RANKL-mediated osteoclastogenesis is unknown. In this study, we first reported that RANKL decreased CXCR6 in a dose-dependent manner, which may be through deactivation of Akt and STAT3 signaling induced by CXCL16. Interestingly, RANKL-mediated CXCR6 reduction may be associated to the activation of STAT3 by phosphorylation. When STAT3 activation was blocked by JAK2/STAT3 inhibitor AG490, RANKL failed to shut down CXCR6 expression during osteoclastogenesis. However, CXCL16 alone did not augment RANKL-mediated osteoclast differentiation and did not alter RANKL-receptor RANK mRNA expression. These results demonstrate that reduction of CXCL16-CXCR6 is critical in RANKL-mediated osteoclastogenesis, which is mainly through the activation of JAK2/STAT3 signaling. CXCL16-CXCR6 axis may become a novel target for the therapeutic intervention of bone resorbing diseases such as rheumatoid arthritis and osteoporosis.

  16. Metformin suppresses CYP1A1 and CYP1B1 expression in breast cancer cells by down-regulating aryl hydrocarbon receptor expression

    Energy Technology Data Exchange (ETDEWEB)

    Do, Minh Truong; Kim, Hyung Gyun; Tran, Thi Thu Phuong; Khanal, Tilak; Choi, Jae Ho [Department of Toxicology, College of Pharmacy, Chungnam National University, Daejeon (Korea, Republic of); Chung, Young Chul [Department of Food Science and Culinary, International University of Korea, Jinju (Korea, Republic of); Jeong, Tae Cheon, E-mail: taecheon@ynu.ac.kr [College of Pharmacy, Yeungnam University, Gyeongsan (Korea, Republic of); Jeong, Hye Gwang, E-mail: hgjeong@cnu.ac.kr [Department of Toxicology, College of Pharmacy, Chungnam National University, Daejeon (Korea, Republic of)

    2014-10-01

    Induction of cytochrome P450 (CYP) 1A1 and CYP1B1 by environmental xenobiotic chemicals or endogenous ligands through the activation of the aryl hydrocarbon receptor (AhR) has been implicated in a variety of cellular processes related to cancer, such as transformation and tumorigenesis. Here, we investigated the effects of the anti-diabetes drug metformin on expression of CYP1A1 and CYP1B1 in breast cancer cells under constitutive and inducible conditions. Our results indicated that metformin down-regulated the expression of CYP1A1 and CYP1B1 in breast cancer cells under constitutive and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced conditions. Down-regulation of AhR expression was required for metformin-mediated decreases in CYP1A1 and CYP1B1 expression, and the metformin-mediated CYP1A1 and CYP1B1 reduction is irrelevant to estrogen receptor α (ERα) signaling. Furthermore, we found that metformin markedly down-regulated Sp1 protein levels in breast cancer cells. The use of genetic and pharmacological tools revealed that metformin-mediated down-regulation of AhR expression was mediated through the reduction of Sp1 protein. Metformin inhibited endogenous AhR ligand-induced CYP1A1 and CYP1B1 expression by suppressing tryptophan-2,3-dioxygenase (TDO) expression in MCF-7 cells. Finally, metformin inhibits TDO expression through a down-regulation of Sp1 and glucocorticoid receptor (GR) protein levels. Our findings demonstrate that metformin reduces CYP1A1 and CYP1B1 expression in breast cancer cells by down-regulating AhR signaling. Metformin would be able to act as a potential chemopreventive agent against CYP1A1 and CYP1B1-mediated carcinogenesis and development of cancer. - Graphical abstract: Schematic of the CYP1A1 and CYP1B1 gene regulation by metformin. - Highlights: • Metformin inhibits CYP1A1 and CYP1B1 expression. • Metformin down-regulates the AhR signaling. • Metformin reduces Sp1 protein expression. • Metformin suppresses TDO expression.

  17. Metformin suppresses CYP1A1 and CYP1B1 expression in breast cancer cells by down-regulating aryl hydrocarbon receptor expression

    International Nuclear Information System (INIS)

    Do, Minh Truong; Kim, Hyung Gyun; Tran, Thi Thu Phuong; Khanal, Tilak; Choi, Jae Ho; Chung, Young Chul; Jeong, Tae Cheon; Jeong, Hye Gwang

    2014-01-01

    Induction of cytochrome P450 (CYP) 1A1 and CYP1B1 by environmental xenobiotic chemicals or endogenous ligands through the activation of the aryl hydrocarbon receptor (AhR) has been implicated in a variety of cellular processes related to cancer, such as transformation and tumorigenesis. Here, we investigated the effects of the anti-diabetes drug metformin on expression of CYP1A1 and CYP1B1 in breast cancer cells under constitutive and inducible conditions. Our results indicated that metformin down-regulated the expression of CYP1A1 and CYP1B1 in breast cancer cells under constitutive and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced conditions. Down-regulation of AhR expression was required for metformin-mediated decreases in CYP1A1 and CYP1B1 expression, and the metformin-mediated CYP1A1 and CYP1B1 reduction is irrelevant to estrogen receptor α (ERα) signaling. Furthermore, we found that metformin markedly down-regulated Sp1 protein levels in breast cancer cells. The use of genetic and pharmacological tools revealed that metformin-mediated down-regulation of AhR expression was mediated through the reduction of Sp1 protein. Metformin inhibited endogenous AhR ligand-induced CYP1A1 and CYP1B1 expression by suppressing tryptophan-2,3-dioxygenase (TDO) expression in MCF-7 cells. Finally, metformin inhibits TDO expression through a down-regulation of Sp1 and glucocorticoid receptor (GR) protein levels. Our findings demonstrate that metformin reduces CYP1A1 and CYP1B1 expression in breast cancer cells by down-regulating AhR signaling. Metformin would be able to act as a potential chemopreventive agent against CYP1A1 and CYP1B1-mediated carcinogenesis and development of cancer. - Graphical abstract: Schematic of the CYP1A1 and CYP1B1 gene regulation by metformin. - Highlights: • Metformin inhibits CYP1A1 and CYP1B1 expression. • Metformin down-regulates the AhR signaling. • Metformin reduces Sp1 protein expression. • Metformin suppresses TDO expression

  18. Chronic ethanol exposure downregulates hepatic expression of pregnane X receptor and P450 3A11 in female ICR mice

    International Nuclear Information System (INIS)

    Wang Jianping; Xu Dexiang; Sun Meifang; Chen Yuanhua; Wang Hua; Wei Wei

    2005-01-01

    Pregnane X receptor (PXR) is a nuclear receptor that regulates cytochrome P450 3A (CYP3A) gene transcription in a ligand-dependent manner. Ethanol has been reported to be either an inducer or an inhibitor of CYP3A expression. In this study, we investigated the effects of chronic ethanol exposure on PXR and P450 3A11 gene expression in mouse liver. Female ICR mice were administered by gavage with different doses (1000, 2000 and 4000 mg/kg) of ethanol for up to 5 weeks. Hepatic PXR and P450 3A11 mRNA levels were measured using RT-PCR. Erythromycin N-demethylase (ERND) activity was used as an indicator of CYP3A protein expression. Results showed that chronic ethanol exposure markedly decreased hepatic PXR and P450 3A11 mRNA levels. Consistent with downregulation of P450 3A11 mRNA, chronic ethanol exposure significantly decreased ERND activity in a dose-dependent manner. Additional experiment showed that chronic ethanol exposure significantly increased plasma endotoxin level and hepatic CD14 and TLR-4 mRNA expression, all of which were blocked by elimination of Gram-negative bacteria and endotoxin with antibiotics. Correspondingly, pretreatment with antibiotics reversed the downregulation of PXR and P450 3A11 mRNA expression and ERND activity in mouse liver. Furthermore, the downregulation of hepatic PXR and P450 3A11 mRNA expression was significantly attenuated in mice pretreated with GdCl 3 , a selective Kupffer cell toxicant. GdCl 3 pretreatment also significantly attenuated chronically ethanol-induced decrease in ERND activity. These results indicated that activation of Kupffer cells by gut-derived endotoxin contributes to downregulation of hepatic PXR and P450 3A11 expression during chronic alcohol intoxication

  19. miR-30a suppresses osteosarcoma proliferation and metastasis by downregulating MEF2D expression

    Directory of Open Access Journals (Sweden)

    Du L

    2018-04-01

    Full Text Available Liuxue Du,* Tianpei Chen,* Kai Zhao,* Dong Yang Department of Orthopedics, the First Affiliated Hospital of Nanchang University, Nanchang, People’s Republic of China *These authors contributed equally to this work Abstract: Many studies have revealed that microRNAs (miRNAs play crucial roles in cancer development and progression. miRNA-30a (miR-30a, as a member of the miR-30 family, has been implicated in various cancers. However, the role of miR-30a in osteosarcoma remains unclear. In the current study, we found that miR-30a was significantly downregulated in osteosarcoma tissues and cell lines by using quantitative real-time polymerase chain reaction (qRT-PCR. In addition, miR-30a could inhibit cancer cell growth, migration, and invasion in vitro. Furthermore, bioinformatics of miRNA target prediction and luciferase reporter assay indicated that MEF2D is a direct target of miR-30a. miR-30a was able to reduce the mRNA and protein expression of MEF2D as assessed using RT-PCR and Western blotting assay. Interestingly, overexpression of MEF2D partially reversed the miR-30a-reduced cell proliferation, migration, and invasion of osteosarcoma cell, indicating that miR-30a suppresses osteosarcoma cell proliferation and metastasis partially mediated by inhibition of MEF2D. Overall, our study demonstrated that miR-30a functions as a tumor suppressor by targeting MEF2D in osteosarcoma, providing a promising prognostic biomarker and a therapeutic strategy for osteosarcoma. Keywords: miR-30a, MEF2D, osteosarcoma, proliferation, invasion, migration

  20. Activation of the complement cascade enhances motility of leukemic cells by downregulating expression of HO-1.

    Science.gov (United States)

    Abdelbaset-Ismail, A; Borkowska-Rzeszotek, S; Kubis, E; Bujko, K; Brzeźniakiewicz-Janus, K; Bolkun, L; Kloczko, J; Moniuszko, M; Basak, G W; Wiktor-Jedrzejczak, W; Ratajczak, M Z

    2017-02-01

    As a crucial arm of innate immunity, the complement cascade (ComC) is involved both in mobilization of normal hematopoietic stem/progenitor cells (HSPCs) from bone marrow (BM) into peripheral blood and in their homing to BM. Despite the fact that ComC cleavage fragments alone do not chemoattract normal HSPCs, we found that leukemia cell lines as well as clonogenic blasts from chronic myeloid leukemia and acute myeloid leukemia patients respond robustly to C3 and C5 cleavage fragments by chemotaxis and increased adhesion. This finding was supported by the detection of C3a and C5a receptors in cells from human malignant hematopoietic cell lines and patient blasts at the mRNA (reverse transcriptase-polymerase chain reaction) and protein level (fluorescence-activated cell sorting), and by the demonstration that these receptors respond to stimulation by C3a and C5a by phosphorylation of p42/44 and p38 mitogen-activated protein kinases (MAPK), and protein kinase B (PKB/AKT). We also found that inducible heme oxygenase 1 (HO-1) is a negative regulator of ComC-mediated trafficking of leukemic cells, and that stimulation of leukemic cells by C3 or C5 cleavage fragments activates p38 MAPK, which downregulates HO-1 expression, rendering cells more mobile. We conclude that activation of the ComC in leukemia/lymphoma patients (for example, as a result of accompanying infections) enhances the motility of malignant cells and contributes to their spread in a p38 MAPK-HO-1-dependent manner. Therefore, inhibition of p38 MAPK or upregulation of HO-1 by small-molecule modulators would have a beneficial effect on ameliorating cell migration-mediated expansion of leukemia/lymphoma cells when the ComC becomes activated.

  1. Activation of the complement cascade enhances motility of leukemic cells by downregulating expression of HO-1

    Science.gov (United States)

    Abdelbaset-Ismail, A; Borkowska-Rzeszotek, S; Kubis, E; Bujko, K; Brzeźniakiewicz-Janus, K; Bolkun, L; Kloczko, J; Moniuszko, M; Basak, G W; Wiktor-Jedrzejczak, W; Ratajczak, M Z

    2017-01-01

    As a crucial arm of innate immunity, the complement cascade (ComC) is involved both in mobilization of normal hematopoietic stem/progenitor cells (HSPCs) from bone marrow (BM) into peripheral blood and in their homing to BM. Despite the fact that ComC cleavage fragments alone do not chemoattract normal HSPCs, we found that leukemia cell lines as well as clonogenic blasts from chronic myeloid leukemia and acute myeloid leukemia patients respond robustly to C3 and C5 cleavage fragments by chemotaxis and increased adhesion. This finding was supported by the detection of C3a and C5a receptors in cells from human malignant hematopoietic cell lines and patient blasts at the mRNA (reverse transcriptase-polymerase chain reaction) and protein level (fluorescence-activated cell sorting), and by the demonstration that these receptors respond to stimulation by C3a and C5a by phosphorylation of p42/44 and p38 mitogen-activated protein kinases (MAPK), and protein kinase B (PKB/AKT). We also found that inducible heme oxygenase 1 (HO-1) is a negative regulator of ComC-mediated trafficking of leukemic cells, and that stimulation of leukemic cells by C3 or C5 cleavage fragments activates p38 MAPK, which downregulates HO-1 expression, rendering cells more mobile. We conclude that activation of the ComC in leukemia/lymphoma patients (for example, as a result of accompanying infections) enhances the motility of malignant cells and contributes to their spread in a p38 MAPK–HO-1-dependent manner. Therefore, inhibition of p38 MAPK or upregulation of HO-1 by small-molecule modulators would have a beneficial effect on ameliorating cell migration-mediated expansion of leukemia/lymphoma cells when the ComC becomes activated. PMID:27451975

  2. Interleukin 6 downregulates p53 expression and activity by stimulating ribosome biogenesis: a new pathway connecting inflammation to cancer

    Science.gov (United States)

    Brighenti, E; Calabrese, C; Liguori, G; Giannone, F A; Trerè, D; Montanaro, L; Derenzini, M

    2014-01-01

    Chronic inflammation is an established risk factor for the onset of cancer, and the inflammatory cytokine IL-6 has a role in tumorigenesis by enhancing proliferation and hindering apoptosis. As factors stimulating proliferation also downregulate p53 expression by enhancing ribosome biogenesis, we hypothesized that IL-6 may cause similar changes in inflamed tissues, thus activating a mechanism that favors neoplastic transformation. Here, we showed that IL-6 downregulated the expression and activity of p53 in transformed and untransformed human cell lines. This was the consequence of IL-6-dependent stimulation of c-MYC mRNA translation, which was responsible for the upregulation of rRNA transcription. The enhanced rRNA transcription stimulated the MDM2-mediated proteasomal degradation of p53, by reducing the availability of ribosome proteins for MDM2 binding. The p53 downregulation induced the acquisition of cellular phenotypic changes characteristic of epithelial–mesenchymal transition, such as a reduced level of E-cadherin expression, increased cell invasiveness and a decreased response to cytotoxic stresses. We found that these changes also occurred in colon epithelial cells of patients with ulcerative colitis, a very representative example of chronic inflammation at high risk for tumor development. Histochemical and immunohistochemical analysis of colon biopsy samples showed an upregulation of ribosome biogenesis, a reduced expression of p53, together with a focal reduction or absence of E-cadherin expression in chronic colitis in comparison with normal mucosa samples. These changes disappeared after treatment with anti-inflammatory drugs. Taken together, the present results highlight a new mechanism that may link chronic inflammation to cancer, based on p53 downregulation, which is activated by the enhancement of rRNA transcription upon IL-6 exposure. PMID:24531714

  3. Agitation down-regulates immunoglobulin binding protein EibG expression in Shiga toxin-producing Escherichia coli (STEC.

    Directory of Open Access Journals (Sweden)

    Thorsten Kuczius

    Full Text Available Shiga toxin (Stx-producing Escherichia coli (STEC carrying eibG synthesize Escherichia coli immunoglobulin binding protein (EibG. EibG nonspecifically binds to immunoglobulins and tends to aggregate in multimers but is poorly expressed in wild-type strains. To study synthesis of the proteins and their regulation in the pathogens, we identified natural growth conditions that increased EibG synthesis. EibG proteins as well as corresponding mRNA were highly expressed under static growth conditions while shearing stress created by agitation during growth repressed protein synthesis. Further regulation effects were driven by reduced oxygen tension, and pH up-regulated EibG expression, but to a lesser extent than growth conditions while decreased temperature down-regulated EibG. Bacteria with increased EibG expression during static growth conditions showed a distinct phenotype with chain formation and biofilm generation, which disappeared with motion. High and low EibG expression was reversible indicating a process with up- and down-regulation of the protein expression. Our findings indicate that shear stress represses EibG expression and might reduce bacterial attachments to cells and surfaces.

  4. Resveratrol via sirtuin-1 downregulates RE1-silencing transcription factor (REST) expression preventing PCB-95-induced neuronal cell death.

    Science.gov (United States)

    Guida, Natascia; Laudati, Giusy; Anzilotti, Serenella; Secondo, Agnese; Montuori, Paolo; Di Renzo, Gianfranco; Canzoniero, Lorella M T; Formisano, Luigi

    2015-11-01

    Resveratrol (3,5,4'-trihydroxystilbene) (RSV), a polyphenol widely present in plants, exerts a neuroprotective function in several neurological conditions; it is an activator of class III histone deacetylase sirtuin1 (SIRT1), a crucial regulator in the pathophysiology of neurodegenerative diseases. By contrast, the RE1-silencing transcription factor (REST) is involved in the neurotoxic effects following exposure to polychlorinated biphenyl (PCB) mixture A1254. The present study investigated the effects of RSV-induced activation of SIRT1 on REST expression in SH-SY5Y cells. Further, we investigated the possible relationship between the non-dioxin-like (NDL) PCB-95 and REST through SIRT1 to regulate neuronal death in rat cortical neurons. Our results revealed that RSV significantly decreased REST gene and protein levels in a dose- and time-dependent manner. Interestingly, overexpression of SIRT1 reduced REST expression, whereas EX-527, an inhibitor of SIRT1, increased REST expression and blocked RSV-induced REST downregulation. These results suggest that RSV downregulates REST through SIRT1. In addition, RSV enhanced activator protein 1 (AP-1) transcription factor c-Jun expression and its binding to the REST promoter gene. Indeed, c-Jun knockdown reverted RSV-induced REST downregulation. Intriguingly, in SH-SY5Y cells and rat cortical neurons the NDL PCB-95 induced necrotic cell death in a concentration-dependent manner by increasing REST mRNA and protein expression. In addition, SIRT1 knockdown blocked RSV-induced neuroprotection in rat cortical neurons treated with PCB-95. Collectively, these results indicate that RSV via SIRT1 activates c-Jun, thereby reducing REST expression in SH-SY5Y cells under physiological conditions and blocks PCB-95-induced neuronal cell death by activating the same SIRT1/c-Jun/REST pathway. Copyright © 2015 Elsevier Inc. All rights reserved.

  5. Downregulation of RBO-PI4KIIIα Facilitates Aβ42 Secretion and Ameliorates Neural Deficits in Aβ42-Expressing Drosophila.

    Science.gov (United States)

    Zhang, Xiao; Wang, Wen-An; Jiang, Li-Xiang; Liu, Hai-Yan; Zhang, Bao-Zhu; Lim, Nastasia; Li, Qing-Yi; Huang, Fu-De

    2017-05-10

    Phosphoinositides and their metabolizing enzymes are involved in Aβ 42 metabolism and Alzheimer's disease pathogenesis. In yeast and mammals, Eighty-five requiring 3 (EFR3), whose Drosophila homolog is Rolling Blackout (RBO), forms a plasma membrane-localized protein complex with phosphatidylinositol-4-kinase Type IIIα (PI4KIIIα) and a scaffold protein to tightly control the level of plasmalemmal phosphatidylinositol-4-phosphate (PI 4 P). Here, we report that RBO binds to Drosophila PI4KIIIα, and that in an Aβ 42 -expressing Drosophila model, separate genetic reduction of PI4KIIIα and RBO, or pharmacological inhibition of PI4KIIIα ameliorated synaptic transmission deficit, climbing ability decline, premature death, and reduced neuronal accumulation of Aβ 42 Moreover, we found that RBO-PI4KIIIa downregulation increased neuronal Aβ 42 release and that PI4P facilitated the assembly or oligomerization of Aβ 42 in/on liposomes. These results indicate that RBO-PI4KIIIa downregulation facilitates neuronal Aβ 42 release and consequently reduces neuronal Aβ 42 accumulation likely via decreasing Aβ 42 assembly in/on plasma membrane. This study suggests the RBO-PI4KIIIα complex as a potential therapeutic target and PI4KIIIα inhibitors as drug candidates for Alzheimer's disease treatment. SIGNIFICANCE STATEMENT Phosphoinositides and their metabolizing enzymes are involved in Aβ 42 metabolism and Alzheimer's disease pathogenesis. Here, in an Aβ 42 -expressing Drosophila model, we discovered and studied the beneficial role of downregulating RBO or its interacting protein PI4KIIIα-a protein that tightly controls the plasmalemmal level of PI 4 P-against the defects caused by Aβ 42 expression. Mechanistically, RBO-PI4KIIIα downregulation reduced neuronal Aβ 42 accumulation, and interestingly increased neuronal Aβ 42 release. This study suggests the RBO-PI4KIIIα complex as a novel therapeutic target, and PI4KIIIα inhibitors as new drug candidates. Copyright

  6. Artichoke, Cynarin and Cyanidin Downregulate the Expression of Inducible Nitric Oxide Synthase in Human Coronary Smooth Muscle Cells

    Directory of Open Access Journals (Sweden)

    Ning Xia

    2014-03-01

    Full Text Available Artichoke (Cynara scolymus L. is one of the world’s oldest medicinal plants with multiple health benefits. We have previously shown that artichoke leaf extracts and artichoke flavonoids upregulate the gene expression of endothelial-type nitric oxide synthase (eNOS in human endothelial cells. Whereas NO produced by the eNOS is a vasoprotective molecule, NO derived from the inducible iNOS plays a pro-inflammatory role in the vasculature. The present study was aimed to investigate the effects of artichoke on iNOS expression in human coronary artery smooth muscle cells (HCASMC. Incubation of HCASMC with a cytokine mixture led to an induction of iNOS mRNA expression. This iNOS induction was concentration- and time-dependently inhibited by an artichoke leaf extract (1–100 µg/mL, 6 h or 24 h. Consistently, the artichoke leaf extract also reduced cytokine-induced iNOS promoter activation and iNOS protein expression. In addition, treatment of HCASMC with four well-known artichoke compounds (cynarin > cyanidin > luteolin ≈ cynaroside led to a downregulation iNOS mRNA and protein expression, with cynarin being the most potent one. In conclusion, artichoke contains both eNOS-upregulating and iNOS-downregulating compounds. Such compounds may contribute to the beneficial effects of artichoke and may per se have therapeutic potentials.

  7. Artichoke, cynarin and cyanidin downregulate the expression of inducible nitric oxide synthase in human coronary smooth muscle cells.

    Science.gov (United States)

    Xia, Ning; Pautz, Andrea; Wollscheid, Ursula; Reifenberg, Gisela; Förstermann, Ulrich; Li, Huige

    2014-03-24

    Artichoke (Cynara scolymus L.) is one of the world's oldest medicinal plants with multiple health benefits. We have previously shown that artichoke leaf extracts and artichoke flavonoids upregulate the gene expression of endothelial-type nitric oxide synthase (eNOS) in human endothelial cells. Whereas NO produced by the eNOS is a vasoprotective molecule, NO derived from the inducible iNOS plays a pro-inflammatory role in the vasculature. The present study was aimed to investigate the effects of artichoke on iNOS expression in human coronary artery smooth muscle cells (HCASMC). Incubation of HCASMC with a cytokine mixture led to an induction of iNOS mRNA expression. This iNOS induction was concentration- and time-dependently inhibited by an artichoke leaf extract (1-100 µg/mL, 6 h or 24 h). Consistently, the artichoke leaf extract also reduced cytokine-induced iNOS promoter activation and iNOS protein expression. In addition, treatment of HCASMC with four well-known artichoke compounds (cynarin > cyanidin > luteolin ≈ cynaroside) led to a downregulation iNOS mRNA and protein expression, with cynarin being the most potent one. In conclusion, artichoke contains both eNOS-upregulating and iNOS-downregulating compounds. Such compounds may contribute to the beneficial effects of artichoke and may per se have therapeutic potentials.

  8. Melittin restores PTEN expression by down-regulating HDAC2 in human hepatocelluar carcinoma HepG2 cells.

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    Hui Zhang

    Full Text Available Melittin is a water-soluble toxic peptide derived from the venom of the bee. Although many studies show the anti-tumor activity of melittin in human cancer including glioma cells, the underlying mechanisms remain elusive. Here the effect of melittin on human hepatocelluar carcinoma HepG2 cell proliferation in vitro and further mechanisms was investigated. We found melittin could inhibit cell proliferation in vitro using Flow cytometry and MTT method. Besides, we discovered that melittin significantly downregulated the expressions of CyclinD1 and CDK4. Results of western Blot and Real-time PCR analysis indicated that melittin was capable to upregulate the expression of PTEN and attenuate histone deacetylase 2 (HDAC2 expression. Further studies demonstrated that knockdown of HDAC2 completely mimicked the effects of melittin on PTEN gene expression. Conversely, it was that the potential utility of melittin on PTEN expression was reversed in cells treated with a recombinant pEGFP-C2-HDAC2 plasmid. In addition, treatment with melittin caused a downregulation of Akt phosphorylation, while overexpression of HDAC2 promoted Akt phosphorylation. These findings suggested that the inhibitory of cell growth by melittin might be led by HDAC2-mediated PTEN upregulation, Akt inactivation, and inhibition of the PI3K/Akt signaling pathways.

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

    Directory of Open Access Journals (Sweden)

    Uchida Daisuke

    2009-08-01

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

  10. Resveratrol via sirtuin-1 downregulates RE1-silencing transcription factor (REST) expression preventing PCB-95-induced neuronal cell death

    Energy Technology Data Exchange (ETDEWEB)

    Guida, Natascia [IRCSS SDN, Naples 80131 (Italy); Laudati, Giusy [Division of Pharmacology, Department of Neuroscience, Reproductive and Dentistry Sciences, School of Medicine, “Federico II” University of Naples, Via Pansini, 5, 80131 Naples (Italy); Anzilotti, Serenella [IRCSS SDN, Naples 80131 (Italy); Secondo, Agnese [Division of Pharmacology, Department of Neuroscience, Reproductive and Dentistry Sciences, School of Medicine, “Federico II” University of Naples, Via Pansini, 5, 80131 Naples (Italy); Montuori, Paolo [Department of Public Health, ‘Federico II’ University of Naples, Naples (Italy); Di Renzo, Gianfranco [Division of Pharmacology, Department of Neuroscience, Reproductive and Dentistry Sciences, School of Medicine, “Federico II” University of Naples, Via Pansini, 5, 80131 Naples (Italy); Canzoniero, Lorella M.T. [Division of Pharmacology, Department of Neuroscience, Reproductive and Dentistry Sciences, School of Medicine, “Federico II” University of Naples, Via Pansini, 5, 80131 Naples (Italy); Division of Pharmacology, Department of Science and Technology, University of Sannio, Via Port' Arsa 11, 82100 Benevento (Italy); Formisano, Luigi, E-mail: cformisa@unisannio.it [Division of Pharmacology, Department of Neuroscience, Reproductive and Dentistry Sciences, School of Medicine, “Federico II” University of Naples, Via Pansini, 5, 80131 Naples (Italy); Division of Pharmacology, Department of Science and Technology, University of Sannio, Via Port' Arsa 11, 82100 Benevento (Italy)

    2015-11-01

    Resveratrol (3,5,4′-trihydroxystilbene) (RSV), a polyphenol widely present in plants, exerts a neuroprotective function in several neurological conditions; it is an activator of class III histone deacetylase sirtuin1 (SIRT1), a crucial regulator in the pathophysiology of neurodegenerative diseases. By contrast, the RE1-silencing transcription factor (REST) is involved in the neurotoxic effects following exposure to polychlorinated biphenyl (PCB) mixture A1254. The present study investigated the effects of RSV-induced activation of SIRT1 on REST expression in SH-SY5Y cells. Further, we investigated the possible relationship between the non-dioxin-like (NDL) PCB-95 and REST through SIRT1 to regulate neuronal death in rat cortical neurons. Our results revealed that RSV significantly decreased REST gene and protein levels in a dose- and time-dependent manner. Interestingly, overexpression of SIRT1 reduced REST expression, whereas EX-527, an inhibitor of SIRT1, increased REST expression and blocked RSV-induced REST downregulation. These results suggest that RSV downregulates REST through SIRT1. In addition, RSV enhanced activator protein 1 (AP-1) transcription factor c-Jun expression and its binding to the REST promoter gene. Indeed, c-Jun knockdown reverted RSV-induced REST downregulation. Intriguingly, in SH-SY5Y cells and rat cortical neurons the NDL PCB-95 induced necrotic cell death in a concentration-dependent manner by increasing REST mRNA and protein expression. In addition, SIRT1 knockdown blocked RSV-induced neuroprotection in rat cortical neurons treated with PCB-95. Collectively, these results indicate that RSV via SIRT1 activates c-Jun, thereby reducing REST expression in SH-SY5Y cells under physiological conditions and blocks PCB-95-induced neuronal cell death by activating the same SIRT1/c-Jun/REST pathway. - Highlights: • Resveratrol via SIRT1/c-Jun downregulates REST mRNA and protein in SH-SY5Y cells. • Non-dioxin-like (NDL) PCB-95 is cytotoxic to

  11. Resveratrol via sirtuin-1 downregulates RE1-silencing transcription factor (REST) expression preventing PCB-95-induced neuronal cell death

    International Nuclear Information System (INIS)

    Guida, Natascia; Laudati, Giusy; Anzilotti, Serenella; Secondo, Agnese; Montuori, Paolo; Di Renzo, Gianfranco; Canzoniero, Lorella M.T.; Formisano, Luigi

    2015-01-01

    Resveratrol (3,5,4′-trihydroxystilbene) (RSV), a polyphenol widely present in plants, exerts a neuroprotective function in several neurological conditions; it is an activator of class III histone deacetylase sirtuin1 (SIRT1), a crucial regulator in the pathophysiology of neurodegenerative diseases. By contrast, the RE1-silencing transcription factor (REST) is involved in the neurotoxic effects following exposure to polychlorinated biphenyl (PCB) mixture A1254. The present study investigated the effects of RSV-induced activation of SIRT1 on REST expression in SH-SY5Y cells. Further, we investigated the possible relationship between the non-dioxin-like (NDL) PCB-95 and REST through SIRT1 to regulate neuronal death in rat cortical neurons. Our results revealed that RSV significantly decreased REST gene and protein levels in a dose- and time-dependent manner. Interestingly, overexpression of SIRT1 reduced REST expression, whereas EX-527, an inhibitor of SIRT1, increased REST expression and blocked RSV-induced REST downregulation. These results suggest that RSV downregulates REST through SIRT1. In addition, RSV enhanced activator protein 1 (AP-1) transcription factor c-Jun expression and its binding to the REST promoter gene. Indeed, c-Jun knockdown reverted RSV-induced REST downregulation. Intriguingly, in SH-SY5Y cells and rat cortical neurons the NDL PCB-95 induced necrotic cell death in a concentration-dependent manner by increasing REST mRNA and protein expression. In addition, SIRT1 knockdown blocked RSV-induced neuroprotection in rat cortical neurons treated with PCB-95. Collectively, these results indicate that RSV via SIRT1 activates c-Jun, thereby reducing REST expression in SH-SY5Y cells under physiological conditions and blocks PCB-95-induced neuronal cell death by activating the same SIRT1/c-Jun/REST pathway. - Highlights: • Resveratrol via SIRT1/c-Jun downregulates REST mRNA and protein in SH-SY5Y cells. • Non-dioxin-like (NDL) PCB-95 is cytotoxic to

  12. Assisted Reproduction Causes Reduced Fetal Growth Associated with Downregulation of Paternally Expressed Imprinted Genes That Enhance Fetal Growth in Mice.

    Science.gov (United States)

    Li, Bo; Chen, Shuqiang; Tang, Na; Xiao, Xifeng; Huang, Jianlei; Jiang, Feng; Huang, Xiuying; Sun, Fangzhen; Wang, Xiaohong

    2016-02-01

    Alteration of intrauterine growth trajectory is linked to metabolic diseases in adulthood. In mammalian and, specifically, human species, pregnancies through assisted reproductive technology (ART) are associated with changes in intrauterine growth trajectory. However, it is still unclear how ART alters intrauterine growth trajectory, especially reduced fetal growth in early to midgestation. In this study, using a mouse model, it was found that ART procedures reduce fetal and placental growth at Embryonic Day 10.5. Furthermore, ART leads to decreased methylation levels at H19, KvDMR1, and Snrpn imprinting control regions in the placentae, instead of fetuses. Furthermore, in the placenta, ART downregulated a majority of parentally expressed imprinted genes, which enhance fetal growth, whereas it upregulated a majority of maternally expressed genes which repress fetal growth. Additionally, the expression of genes that regulate placental development was also affected by ART. ART also downregulated a majority of placental nutrient transporters. Disruption of genomic imprinting and abnormal expression of developmentally and functionally relevant genes in placenta may influence the placental development and function, which affect fetal growth and reprogramming. © 2016 by the Society for the Study of Reproduction, Inc.

  13. Nicotine affects rat Leydig cell function in vivo and vitro via down-regulating some key steroidogenic enzyme expressions.

    Science.gov (United States)

    Guo, Xiaoling; Wang, Huang; Wu, Xiaolong; Chen, Xianwu; Chen, Yong; Guo, Jingjing; Li, Xiaoheng; Lian, Qingquan; Ge, Ren-Shan

    2017-12-01

    Nicotine is consumed largely as a component of cigarettes and has a potential effect on pubertal development of Leydig cells in males. To investigate its effects, 49-day-old male Sprague Dawley rats received intraperitoneal injections of nicotine (0.5 or 1 mg/kg/day) for 2 weeks and immature Leydig cells were isolated from the testes of 35-day-old rats and treated with nicotine (0.05-50 μM). Serum hormones, Leydig cell number and related gene expression levels after in vivo treatment were determined and medium androgen levels were measured and cell cycle, apoptosis, mitochondrial membrane potential (△Ψm), and reactive oxygen species (ROS) of Leydig cells after in vitro treatment were measured. In vivo exposure to nicotine lowered serum luteinizing hormone, follicle stimulating hormone, and testosterone levels and reduced Leydig cell number and gene expression levels. Nicotine in vitro inhibited androgen production in Leydig cells by downregulating the expression levels of P450 cholesterol side cleavage enzyme, 3β-hydroxysteroid dehydrogenase 1, and steroidogenic factor 1 at different concentration ranges. In conclusion, nicotine disrupts Leydig cell steroidogenesis during puberty possibly via down-regulating some key steroidogenic enzyme expressions. Copyright © 2017. Published by Elsevier Ltd.

  14. Human rotavirus strain Wa downregulates NHE1 and NHE6 expressions in rotavirus-infected Caco-2 cells.

    Science.gov (United States)

    Chen, Honglang; Song, Lijun; Li, Guixian; Chen, Wenfeng; Zhao, Shumin; Zhou, Ruoxia; Shi, Xiaoying; Peng, Zhenying; Zhao, Wenchang

    2017-06-01

    Rotavirus (RV) is the most common cause of severe gastroenteritis and fatal dehydration in human infants and neonates of different species. However, the pathogenesis of rotavirus-induced diarrhea is poorly understood. Secretory diarrhea caused by rotavirus may lead to a combination of excessive secretion of fluid and electrolytes into the intestinal lumen. Fluid absorption in the small intestine is driven by Na + -coupled transport mechanisms at the luminal membrane, including Na + /H + exchanger (NHE). Here, we performed qRT-PCR to detect the transcription of NHEs. Western blotting was employed for protein detection. Furthermore, immunocytochemistry was used to validate the NHE's protein expression. Finally, intracellular Ca 2+ concentration was detected by confocal laser scanning microscopy. The results demonstrated that the NHE6 mRNA and protein expressed in the human colon adenocarcinoma cell line (Caco-2). Furthermore, RV-Wa induced decreased expression of the NHE1 and NHE6 in Caco-2 cell in a time-dependent manner. In addition, intracellular Ca 2+ concentration in RV-Wa-infected Caco-2 cells was higher than that in the mock-infected cells. Furthermore, RV-Wa also can downregulate the expression of calmodulin (CaM) and calmodulin kinase II (CaMKII) in Caco-2 cells. These findings provides important insights into the mechanisms of rotavirus-induced diarrhea. Further studies on the underlying pathophysiological mechanisms that downregulate NHEs in RV-induced diarrhea are required.

  15. MicroRNA expression is down-regulated and reorganized in prefrontal cortex of depressed suicide subjects.

    Directory of Open Access Journals (Sweden)

    Neil R Smalheiser

    Full Text Available Recent studies suggest that alterations in expression of genes, including those which regulate neural and structural plasticity, may be crucial in the pathogenesis of depression. MicroRNAs (miRNAs are newly discovered regulators of gene expression that have recently been implicated in a variety of human diseases, including neuropsychiatric diseases.The present study was undertaken to examine whether the miRNA network is altered in the brain of depressed suicide subjects. Expression of miRNAs was measured in prefrontal cortex (Brodmann Area 9 of antidepressant-free depressed suicide (n = 18 and well-matched non-psychiatric control subjects (n = 17 using multiplex RT-PCR plates. We found that overall miRNA expression was significantly and globally down-regulated in prefrontal cortex of depressed suicide subjects. Using individual tests of statistical significance, 21 miRNAs were significantly decreased at p = 0.05 or better. Many of the down-regulated miRNAs were encoded at nearby chromosomal loci, shared motifs within the 5'-seeds, and shared putative mRNA targets, several of which have been implicated in depression. In addition, a set of 29 miRNAs, whose expression was not pairwise correlated in the normal controls, showed a high degree of co-regulation across individuals in the depressed suicide group.The findings show widespread changes in miRNA expression that are likely to participate in pathogenesis of major depression and/or suicide. Further studies are needed to identify whether the miRNA changes lead to altered expression of prefrontal cortex mRNAs, either directly (by acting as miRNA targets or indirectly (e.g., by affecting transcription factors.

  16. Senescence-related functional nuclear barrier by down-regulation of nucleo-cytoplasmic trafficking gene expression

    International Nuclear Information System (INIS)

    Kim, Sung Young; Ryu, Sung Jin; Ahn, Hong Ju; Choi, Hae Ri; Kang, Hyun Tae; Park, Sang Chul

    2010-01-01

    One of the characteristic natures of senescent cells is the hypo- or irresponsiveness not only to growth factors but also to apoptotic stress. In the present study, we confirmed the inhibition of nuclear translocation of activated p-ERK1/2 and NF-kB p50 in response to growth stimuli or LPS in the senescent human diploid fibroblasts. In order to elucidate the underlying mechanism for the senescence-associated hypo-responsiveness, we carried out the comparison study for gene expression profiles through microarray analysis. In consequence, we observed the vast reduction in expression of nucleo-cytoplasmic trafficking genes in senescent cells, when compared with those in young cells. Expression levels of several nucleoporins, karyopherin α, karyopherin β, Ran, and Ran-regulating factors were confirmed to be down-regulated in senescent HDFs by using RT-PCR and Western blot methods. Taken together, these data suggest the operation of certain senescence-associated functional nuclear barriers by down-regulation of the nucleo-cytoplasmic trafficking genes in the senescent cells.

  17. Senescence-related functional nuclear barrier by down-regulation of nucleo-cytoplasmic trafficking gene expression

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Sung Young; Ryu, Sung Jin; Ahn, Hong Ju; Choi, Hae Ri; Kang, Hyun Tae [Department of Biochemistry and Molecular Biology, Aging and Apoptosis Research Center, Institute on Aging, Seoul National University College of Medicine, Seoul 110-799 (Korea, Republic of); Park, Sang Chul, E-mail: scpark@snu.ac.kr [Department of Biochemistry and Molecular Biology, Aging and Apoptosis Research Center, Institute on Aging, Seoul National University College of Medicine, Seoul 110-799 (Korea, Republic of)

    2010-01-01

    One of the characteristic natures of senescent cells is the hypo- or irresponsiveness not only to growth factors but also to apoptotic stress. In the present study, we confirmed the inhibition of nuclear translocation of activated p-ERK1/2 and NF-kB p50 in response to growth stimuli or LPS in the senescent human diploid fibroblasts. In order to elucidate the underlying mechanism for the senescence-associated hypo-responsiveness, we carried out the comparison study for gene expression profiles through microarray analysis. In consequence, we observed the vast reduction in expression of nucleo-cytoplasmic trafficking genes in senescent cells, when compared with those in young cells. Expression levels of several nucleoporins, karyopherin {alpha}, karyopherin {beta}, Ran, and Ran-regulating factors were confirmed to be down-regulated in senescent HDFs by using RT-PCR and Western blot methods. Taken together, these data suggest the operation of certain senescence-associated functional nuclear barriers by down-regulation of the nucleo-cytoplasmic trafficking genes in the senescent cells.

  18. Selective lignin downregulation leads to constitutive defense response expression in alfalfa (Medicago sativa L.).

    Science.gov (United States)

    Gallego-Giraldo, Lina; Jikumaru, Yusuke; Kamiya, Yuji; Tang, Yuhong; Dixon, Richard A

    2011-05-01

    Downregulation of hydroxycinnamoyl CoA: shikimate hydroxycinnamoyl transferase (HCT) in alfalfa (Medicago sativa) reduces lignin levels and improves forage quality and saccharification efficiency for bioethanol production. However, the plants have reduced stature. It was previously reported that HCT-down-regulated Arabidopsis have impaired auxin transport, but this has recently been disproved. • To address the basis for the phenotypes of lignin-modified alfalfa, we measured auxin transport, profiled a range of metabolites including flavonoids and hormones, and performed in depth transcriptome analyses. • Auxin transport is unaffected in HCT antisense alfalfa despite increased flavonoid biosynthesis. The plants show increased cytokinin and reduced auxin levels, and gibberellin levels and sensitivity are both reduced. Levels of salicylic, jasmonic and abscisic acids are elevated, associated with massive upregulation of pathogenesis and abiotic stress-related genes and enhanced tolerance to fungal infection and drought. • We suggest that HCT downregulated alfalfa plants exhibit constitutive activation of defense responses, triggered by release of bioactive cell wall fragments and production of hydrogen peroxide as a result of impaired secondary cell wall integrity. © 2011 The Authors. New Phytologist © 2011 New Phytologist Trust.

  19. eEF-2 Phosphorylation Down-Regulates P-Glycoprotein Over-Expression in Rat Brain Microvessel Endothelial Cells.

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    Xing Hua Tang

    Full Text Available We investigated whether glutamate, NMDA receptors, and eukaryote elongation factor-2 kinase (eEF-2K/eEF-2 regulate P-glycoprotein expression, and the effects of the eEF-2K inhibitor NH125 on the expression of P-glycoprotein in rat brain microvessel endothelial cells (RBMECs.Cortex was obtained from newborn Wistar rat brains. After surface vessels and meninges were removed, the pellet containing microvessels was resuspended and incubated at 37°C in culture medium. Cell viability was assessed by the MTT assay. RBMECs were identified by immunohistochemistry with anti-vWF. P-glycoprotein, phospho-eEF-2, and eEF-2 expression were determined by western blot analysis. Mdr1a gene expression was analyzed by RT-PCR.Mdr1a mRNA, P-glycoprotein and phospho-eEF-2 expression increased in L-glutamate stimulated RBMECs. P-glycoprotein and phospho-eEF-2 expression were down-regulated after NH125 treatment in L-glutamate stimulated RBMECs.eEF-2K/eEF-2 should have played an important role in the regulation of P-glycoprotein expression in RBMECs. eEF-2K inhibitor NH125 could serve as an efficacious anti-multidrug resistant agent.

  20. Beta-Amyloid Downregulates MDR1-P-Glycoprotein (Abcb1 Expression at the Blood-Brain Barrier in Mice

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    Anja Brenn

    2011-01-01

    Full Text Available Neurovascular dysfunction is an important component of Alzheimer's disease, leading to reduced clearance across the blood-brain barrier and accumulation of neurotoxic β-amyloid (Aβ peptides in the brain. It has been shown that the ABC transport protein P-glycoprotein (P-gp, ABCB1 is involved in the export of Aβ from the brain into the blood. To determine whether Aβ influences the expression of key Aβ transporters, we studied the effects of 1-day subcutaneous Aβ1-40 and Aβ1-42 administration via Alzet mini-osmotic pumps on P-gp, BCRP, LRP1, and RAGE expression in the brain of 90-day-old male FVB mice. Our results demonstrate significantly reduced P-gp, LRP1, and RAGE mRNA expression in mice treated with Aβ1-42 compared to controls, while BCRP expression was not affected. The expression of the four proteins was unchanged in mice treated with Aβ1-40 or reverse-sequence peptides. These findings indicate that, in addition to the age-related decrease of P-gp expression, Aβ1-42 itself downregulates the expression of P-gp and other Aβ-transporters, which could exacerbate the intracerebral accumulation of Aβ and thereby accelerate neurodegeneration in Alzheimer's disease and cerebral β-amyloid angiopathy.

  1. Salmonella Typhimurium induces SPI-1 and SPI-2 regulated and strain dependent downregulation of MHC II expression on porcine alveolar macrophages

    Directory of Open Access Journals (Sweden)

    Van Parys Alexander

    2012-06-01

    Full Text Available Abstract Foodborne salmonellosis is one of the most important bacterial zoonotic diseases worldwide. Salmonella Typhimurium is the serovar most frequently isolated from persistently infected slaughter pigs in Europe. Circumvention of the host’s immune system by Salmonella might contribute to persistent infection of pigs. In the present study, we found that Salmonella Typhimurium strain 112910a specifically downregulated MHC II, but not MHC I, expression on porcine alveolar macrophages in a Salmonella pathogenicity island (SPI-1 and SPI-2 dependent way. Salmonella induced downregulation of MHC II expression and intracellular proliferation of Salmonella in macrophages were significantly impaired after opsonization with Salmonella specific antibodies prior to inoculation. Furthermore, the capacity to downregulate MHC II expression on macrophages differed significantly among Salmonella strains, independently of strain specific differences in invasion capacity, Salmonella induced cytotoxicity and altered macrophage activation status. The fact that strain specific differences in MHC II downregulation did not correlate with the extent of in vitro SPI-1 or SPI-2 gene expression indicates that other factors are involved in MHC II downregulation as well. Since Salmonella strain dependent interference with the pig’s immune response through downregulation of MHC II expression might indicate that certain Salmonella strains are more likely to escape serological detection, our findings are of major interest for Salmonella monitoring programs primarily based on serology.

  2. IL-10 down-regulates the expression of survival associated gene hspX of Mycobacterium tuberculosis in murine macrophage

    Directory of Open Access Journals (Sweden)

    Babban Jee

    2017-07-01

    Full Text Available Mycobacterium tuberculosis (MTB adopts a special survival strategy to overcome the killing mechanism(s of host immune system. Amongst the many known factors, small heat shock protein 16.3 (sHSP16.3 of MTB encoded by gene hspX has been reported to be critical for the survival of MTB. In the present study, the effect of recombinant murine interferon-gamma (rmIFN-γ and recombinant murine interleukin-10 (rmIL-10 on the expression of gene hspX of MTB in murine macrophage RAW264.7 has been investigated. By real-time RT-PCR, it was observed that three increasing concentrations (5, 25 and 50 ng/ml of rmIFN-γ significantly up-regulated the expression of hspX whereas similar concentrations of rmIL-10 (5, 25 and 50 ng/ml significantly down-regulated the hspX expression. This effect was not only dependent on the concentration of the stimulus but this was time-dependent as well. A contrasting pattern of hspX expression was observed against combinations of two different concentrations of rmIFN-γ and rmIL-10. The study results suggest that rIL-10 mediated down-regulation of hspX expression, in the presence of low concentration of rIFN-γ, could be used as an important strategy to decrease the dormancy of MTB in its host and thus making MTB susceptible to the standard anti-mycobacterial therapy used for treating tuberculosis. However, as these are only preliminary results in the murine cell line model, this hypothesis needs to be first validated in human cell lines and subsequently in animal models mimicking the latent infection using clinical isolates of MTB before considering the development of modified regimens for humans.

  3. Downregulation of CD147 expression alters cytoskeleton architecture and inhibits gelatinase production and SAPK pathway in human hepatocellular carcinoma cells

    Directory of Open Access Journals (Sweden)

    Weng Yuan-Yuan

    2008-10-01

    Full Text Available Abstract Background CD147 plays a critical role in the invasive and metastatic activity of hepatocellular carcinoma (HCC cells by stimulating the surrounding fibroblasts to express matrix metalloproteinases (MMPs. Tumor cells adhesion to extracellular matrix (ECM proteins is the first step to the tumor metastasis. MMPs degrade the ECM to promote tumor metastasis. The aim of this study is to investigate the effects of small interfering RNA (siRNA against CD147 (si-CD147 on hepatocellular carcinoma cells' (SMMC-7721 architecture and functions. Methods Flow cytometry and western blot assays were employed to detect the transfection efficiency of si-CD147. Confocal microscopy was used to determine the effects of si-CD147 on SMMC-7721 cells' cytoskeleton. Invasion assay, gelatin zymography and cell adhesion assay were employed to investigate the effects of si-CD147 on SMMC-7721 cells' invasion, gelatinase production and cell adhesive abilities. Western blot assay was utilized to detect the effects of si-CD147 on focal adhesion kinase (FAK, vinculiln and mitogen-activated protein kinase (MAPK expression in SMMC-7721 cells. Results Downregulation of CD147 gene induced the alteration of SMMC-7721 cell cytoskeleton including actin, microtubule and vimentin filaments, and inhibited gelatinase production and expression, cells invasion, FAK and vinculin expression. si-CD147 also blocked SMMC-7721 cells adhesion to collagen IV and phosphorylation level of SAPK/JNKs. SAPK/JNKs inhibitor SP600125 inhibited gelatinase production and expression. Conclusion CD147 is required for normal tumor cell architecture and cell invasion. Downregulation of CD147 affects HCC cell structure and function. Moreover, the alteration of cell behavior may be related to SAPK/JNK Pathway. siRNA against CD147 may be a possible new approach for HCC gene therapy.

  4. Morphine Preconditioning Downregulates MicroRNA-134 Expression Against Oxygen-Glucose Deprivation Injuries in Cultured Neurons of Mice.

    Science.gov (United States)

    Meng, Fanjun; Li, Yan; Chi, Wenying; Li, Junfa

    2016-07-01

    Brain protection by narcotics such as morphine is clinically relevant due to the extensive use of narcotics in the perioperative period. Morphine preconditioning induces neuroprotection in neurons, but it remains uncertain whether microRNA-134 (miR-134) is involved in morphine preconditioning against oxygen-glucose deprivation-induced injuries in primary cortical neurons of mice. The present study examined this issue. After cortical neurons of mice were cultured in vitro for 6 days, the neurons were transfected by respective virus vector, such as lentiviral vector (LV)-miR-control-GFP, LV-pre-miR-134-GFP, LV-pre-miR-134-inhibitor-GFP for 24 hours; after being normally cultured for 3 days again, morphine preconditioning was performed by incubating the transfected primary neurons with morphine (3 μM) for 1 hour, and then neuronal cells were exposed to oxygen-glucose deprivation (OGD) for 1 hour and oxygen-glucose recovery for 12 hours. The neuronal cells survival rate and the amount of apoptotic neurons were determined by MTT assay or TUNEL staining at designated time; and the expression levels of miR-134 were detected using real-time reverse transcription polymerase chain reaction at the same time. The neuronal cell survival rate was significantly higher, and the amount of apoptotic neurons was significantly decreased in neurons preconditioned with morphine before OGD than that of OGD alone. The neuroprotection induced by morphine preconditioning was partially blocked by upregulating miR-134 expression, and was enhanced by downregulating miR-134 expression. The expression of miR-134 was significantly decreased in morphine-preconditioned neurons alone without transfection. By downregulating miR-134 expression, morphine preconditioning protects primary cortical neurons of mice against injuries induced by OGD.

  5. Niclosamide, an oral antihelmintic drug, exhibits antimetastatic activity in hepatocellular carcinoma cells through downregulating twist-mediated CD10 expression.

    Science.gov (United States)

    Chien, Ming-Hsien; Ho, Yung-Chuan; Yang, Shun-Fa; Yang, Yi-Chieh; Lai, Szu-Yu; Chen, Wan-Shen; Chen, Ming-Jenn; Yeh, Chao-Bin

    2018-02-26

    Hepatocellular carcinoma (HCC) is one of the most common malignancies in the world, especially, in eastern Asia, and its prognosis is poor once metastasis occurs. Niclosamide, a US Food and Drug Administration-approved antihelmintic drug, was shown to inhibit the growth of various cancers including HCC, but the effect of niclosamide on cell motility and the underlying mechanism have not yet been completely defined. The present study demonstrated that niclosamide, at 0-40 nM, concentration-dependently inhibited wound closure and the migratory/invasive capacities of human Huh7 and SK-Hep-1 HCC cells without exhibiting cytotoxicity. A protease array analysis showed that CD10 was dramatically downregulated in Huh7 cells after niclosamide treatment. Western blot and flow cytometric assays further demonstrated that CD10 expression was concentration-dependently downregulated in Huh7 and SK-Hep-1 cells after niclosamide treatment. Mechanistic investigations found that niclosamide suppressed Twist-mediated CD10 transactivation. Moreover, knockdown of CD10 expression by CD10 small interfering RNA in HCC cells suppressed cell migratory/invasive abilities and overexpression of CD10 relieved the migration inhibition induced by niclosamide. Taken together, our results indicated that niclosamide could be a potential agent for inhibiting metastasis of HCC, and CD10 is an important target of niclosamide for suppressing the motility of HCC cells. © 2018 Wiley Periodicals, Inc.

  6. Carbon nanoparticles downregulate expression of basic fibroblast growth factor in the heart during embryogenesis

    DEFF Research Database (Denmark)

    Wierzbicki, Mateusz; Sawosz, Ewa; Grodzik, Marta

    2013-01-01

    indices of the embryos' health. However, vascularization of the heart and the density of branched vessels were significantly reduced after treatment with diamond nanoparticles and, to a lesser extent, graphite nanoparticles. Application of nanoparticles significantly downregulated gene and protein......Carbon nanoparticles, with their high biocompatibility and low toxicity, have recently been considered for biomedical applications, including antiangiogenic therapy. Critical to normal development and tumor formation, angiogenesis is the process of forming capillary blood vessels from preexisting...... vessels. In the present study, we evaluated the effects of diamond and graphite nanoparticles on the development of chicken embryos, as well as vascularization of the chorioallantoic membrane and heart at the morphological and molecular level. Nanoparticles did not affect either body/heart weight or serum...

  7. Controlled hydrostatic pressure stress downregulates the expression of ribosomal genes in preimplantation embryos: a possible protection mechanism?

    Science.gov (United States)

    Bock, I; Raveh-Amit, H; Losonczi, E; Carstea, A C; Feher, A; Mashayekhi, K; Matyas, S; Dinnyes, A; Pribenszky, C

    2016-04-01

    The efficiency of various assisted reproductive techniques can be improved by preconditioning the gametes and embryos with sublethal hydrostatic pressure treatment. However, the underlying molecular mechanism responsible for this protective effect remains unknown and requires further investigation. Here, we studied the effect of optimised hydrostatic pressure treatment on the global gene expression of mouse oocytes after embryonic genome activation. Based on a gene expression microarray analysis, a significant effect of treatment was observed in 4-cell embryos derived from treated oocytes, revealing a transcriptional footprint of hydrostatic pressure-affected genes. Functional analysis identified numerous genes involved in protein synthesis that were downregulated in 4-cell embryos in response to hydrostatic pressure treatment, suggesting that regulation of translation has a major role in optimised hydrostatic pressure-induced stress tolerance. We present a comprehensive microarray analysis and further delineate a potential mechanism responsible for the protective effect of hydrostatic pressure treatment.

  8. MAL Overexpression Leads to Disturbed Expression of Genes That Influence Cytoskeletal Organization and Differentiation of Schwann Cells

    Directory of Open Access Journals (Sweden)

    Daniela Schmid

    2014-09-01

    Full Text Available In the developing peripheral nervous system, a coordinated reciprocal signaling between Schwann cells and axons is crucial for accurate myelination. The myelin and lymphocyte protein MAL is a component of lipid rafts that is important for targeting proteins and lipids to distinct domains. MAL overexpression impedes peripheral myelinogenesis, which is evident by a delayed onset of myelination and reduced expression of the myelin protein zero (Mpz/P0 and the low-affinity neurotrophin receptor p75NTR . This study shows that MAL overexpression leads to a significant reduction of Mpz and p75NTR expression in primary mouse Schwann cell cultures, which was already evident before differentiation, implicating an effect of MAL in early Schwann cell development. Their transcription was robustly reduced, despite normal expression of essential transcription factors and receptors. Further, the cAMP response element-binding protein (CREB and phosphoinositide 3-kinase signaling pathways important for Schwann cell differentiation were correctly induced, highlighting that other so far unknown rate limiting factors do exist. We identified novel genes expressed by Schwann cells in a MAL-dependent manner in vivo and in vitro. A number of those, including S100a4, RhoU and Krt23, are implicated in cytoskeletal organization and plasma membrane dynamics. We showed that S100a4 is predominantly expressed by nonmyelinating Schwann cells, whereas RhoU was localized within myelin membranes, and Krt23 was detected in nonmyelinating as well as in myelinating Schwann cells. Their differential expression during early peripheral nerve development further underlines their possible role in influencing Schwann cell differentiation and myelination.

  9. Nitric oxide is involved in the down-regulation of sost expression induced by mechanical loading

    NARCIS (Netherlands)

    Delgado-Calle, J.; Riancho, J.A.; Klein-Nulend, J.

    2014-01-01

    Mechanical stimulation reduces sclerostin expression in rodents. However, few data are available about the effect of physical stimuli in human systems. Recently we observed that the demethylating agent AzadC induces SOST expression in bone cells. This allowed us in this study to explore the effect

  10. Lycopene Inhibits Metastasis of Human Liver Adenocarcinoma SK-Hep-1 Cells by Downregulation of NADPH Oxidase 4 Protein Expression.

    Science.gov (United States)

    Jhou, Bo-Yi; Song, Tuzz-Ying; Lee, Inn; Hu, Miao-Lin; Yang, Nae-Cherng

    2017-08-16

    NADPH oxidase 4 (NOX4), with the sole function to produce reactive oxygen species (ROS), can be a molecular target for disrupting cancer metastasis. Several studies have indicated that lycopene exhibited anti-metastatic actions in vitro and in vivo. However, the role of NOX4 in the anti-metastatic action of lycopene remains unknown. Herein, we first confirmed the anti-metastatic effect of lycopene (0.1-5 μM) on human liver adenocarcinoma SK-Hep-1 cells. We showed that lycopene significantly inhibited NOX4 protein expression, with the strongest inhibition of 64.3 ± 10.2% (P lycopene. Lycopene also significantly inhibited NOX4 mRNA expression, NOX activity, and intracellular ROS levels in SK-Hep-1 cells. We then determined the effects of lycopene on transforming growth factor β (TGF-β)-induced metastasis. We found that TGF-β (5 ng/mL) significantly increased migration, invasion, and adhesion activity, the intracellular ROS level, matrix metalloproteinase 9 (MMP-9) and MMP-2 activities, the level of NOX4 protein expression, and NOX activity. All these TGF-β-induced effects were antagonized by the incubation of SK-Hep-1 cells with lycopene (2.5 μM). Using transient transfection of siRNA against NOX4, we found that the downregulation of NOX4 could mimic lycopene by inhibiting cell migration and the activities of MMP-9 and MMP-2 during the incubation with or without TGF-β on SK-Hep-1 cells. The results demonstrate that the downregulation of NOX4 plays a crucial role in the anti-metastatic action of lycopene in SK-Hep-1 cells.

  11. Gene Expression Profiling Identifies Downregulation of the Neurotrophin-MAPK Signaling Pathway in Female Diabetic Peripheral Neuropathy Patients.

    Science.gov (United States)

    Luo, Lin; Zhou, Wen-Hua; Cai, Jiang-Jia; Feng, Mei; Zhou, Mi; Hu, Su-Pei; Xu, Jin; Ji, Lin-Dan

    2017-01-01

    Diabetic peripheral neuropathy (DPN) is a common complication of diabetes mellitus (DM). It is not diagnosed or managed properly in the majority of patients because its pathogenesis remains controversial. In this study, human whole genome microarrays identified 2898 and 4493 differentially expressed genes (DEGs) in DM and DPN patients, respectively. A further KEGG pathway analysis indicated that DPN and DM share four pathways, including apoptosis, B cell receptor signaling pathway, endocytosis, and Toll-like receptor signaling pathway. The DEGs identified through comparison of DPN and DM were significantly enriched in MAPK signaling pathway, NOD-like receptor signaling pathway, and neurotrophin signaling pathway, while the "neurotrophin-MAPK signaling pathway" was notably downregulated. Seven DEGs from the neurotrophin-MAPK signaling pathway were validated in additional 78 samples, and the results confirmed the initial microarray findings. These findings demonstrated that downregulation of the neurotrophin-MAPK signaling pathway may be the major mechanism of DPN pathogenesis, thus providing a potential approach for DPN treatment.

  12. Prostate tumor-derived exosomes down-regulate NKG2D expression on natural killer cells and CD8+ T cells: mechanism of immune evasion.

    Directory of Open Access Journals (Sweden)

    Marie Lundholm

    Full Text Available Tumor-derived exosomes, which are nanometer-sized extracellular vesicles of endosomal origin, have emerged as promoters of tumor immune evasion but their role in prostate cancer (PC progression is poorly understood. In this study, we investigated the ability of prostate tumor-derived exosomes to downregulate NKG2D expression on natural killer (NK and CD8+ T cells. NKG2D is an activating cytotoxicity receptor whose aberrant loss in cancer plays an important role in immune suppression. Using flow cytometry, we found that exosomes produced by human PC cells express ligands for NKG2D on their surface. The NKG2D ligand-expressing prostate tumor-derived exosomes selectively induced downregulation of NKG2D on NK and CD8+ T cells in a dose-dependent manner, leading to impaired cytotoxic function in vitro. Consistent with these findings, patients with castration-resistant PC (CRPC showed a significant decrease in surface NKG2D expression on circulating NK and CD8+ T cells compared to healthy individuals. Tumor-derived exosomes are likely involved in this NKG2D downregulation, since incubation of healthy lymphocytes with exosomes isolated from serum or plasma of CRPC patients triggered downregulation of NKG2D expression in effector lymphocytes. These data suggest prostate tumor-derived exosomes as down-regulators of the NKG2D-mediated cytotoxic response in PC patients, thus promoting immune suppression and tumor escape.

  13. Serum amyloid P down-regulates CCL-1 expression, and inhibits ...

    African Journals Online (AJOL)

    Differentially expressed proteins in SAP-Tg and C57BL/6 serum were analyzed, and further determined by enzymelinked immunosorbent assay (ELISA) and ... SAP recombinant protein, ELISA results showed that CCL-1 secretion significantly ...

  14. Down-regulation of GST and CAT gene expression by methanolic ...

    African Journals Online (AJOL)

    DELL

    2013-07-03

    Jul 3, 2013 ... methanolic extract of Nigella sativa seed in human peripheral blood ... expression in human PBMCs. Key words: ... oxidative stress (Sen et al., 2010; Yoruk et al., 2010). NS .... Antioxidant activities of black cumin. (Nigella ...

  15. Aquaporin-6 Expression in the Cochlear Sensory Epithelium Is Downregulated by Salicylates

    Directory of Open Access Journals (Sweden)

    Paola Perin

    2010-01-01

    Full Text Available We characterize the expression pattern of aquaporin-6 in the mouse inner ear by RT-PCR and immunohistochemistry. Our data show that in the inner ear aquaporin-6 is expressed, in both vestibular and acoustic sensory epithelia, by the supporting cells directly contacting hair cells. In particular, in the Organ of Corti, expression was strongest in Deiters' cells, which provide both a mechanical link between outer hair cells (OHCs and the Organ of Corti, and an entry point for ion recycle pathways. Since aquaporin-6 is permeable to both water and anions, these results suggest its possible involvement in regulating OHC motility, directly through modulation of water and chloride flow or by changing mechanical compliance in Deiters' cells. In further support of this role, treating mice with salicylates, which impair OHC electromotility, dramatically reduced aquaporin-6 expression in the inner ear epithelia but not in control tissues, suggesting a role for this protein in modulating OHCs' responses.

  16. Aquaporin-6 expression in the cochlear sensory epithelium is downregulated by salicylates.

    Science.gov (United States)

    Perin, Paola; Tritto, Simona; Botta, Laura; Fontana, Jacopo Maria; Gastaldi, Giulia; Masetto, Sergio; Tosco, Marisa; Laforenza, Umberto

    2010-01-01

    We characterize the expression pattern of aquaporin-6 in the mouse inner ear by RT-PCR and immunohistochemistry. Our data show that in the inner ear aquaporin-6 is expressed, in both vestibular and acoustic sensory epithelia, by the supporting cells directly contacting hair cells. In particular, in the Organ of Corti, expression was strongest in Deiters' cells, which provide both a mechanical link between outer hair cells (OHCs) and the Organ of Corti, and an entry point for ion recycle pathways. Since aquaporin-6 is permeable to both water and anions, these results suggest its possible involvement in regulating OHC motility, directly through modulation of water and chloride flow or by changing mechanical compliance in Deiters' cells. In further support of this role, treating mice with salicylates, which impair OHC electromotility, dramatically reduced aquaporin-6 expression in the inner ear epithelia but not in control tissues, suggesting a role for this protein in modulating OHCs' responses.

  17. A molecular toolbox for rapid generation of viral vectors to up- or down-regulate in vivo neuronal gene expression

    Directory of Open Access Journals (Sweden)

    Melanie D. White

    2011-07-01

    Full Text Available We introduce a molecular toolbox for manipulation of neuronal gene expression in vivo. The toolbox includes promoters, ion channels, optogenetic tools, fluorescent proteins and intronic artificial microRNAs. The components are easily assembled into adeno-associated virus (AAV or lentivirus vectors using recombination cloning. We demonstrate assembly of toolbox components into lentivirus and AAV vectors and use these vectors for in vivo expression of inwardly rectifying potassium channels (Kir2.1, Kir3.1 and Kir3.2 and an artificial microRNA targeted against the ion channel HCN1 (HCN1 miR. We show that AAV assembled to express HCN1 miR produces efficacious and specific in vivo knockdown of HCN1 channels. Comparison of in vivo viral transduction using HCN1 miR with mice containing a germ line deletion of HCN1 reveals similar physiological phenotypes in cerebellar Purkinje cells. The easy assembly and re-usability of the toolbox components, together with the ability to up- or down-regulate neuronal gene expression in vivo, may be useful for applications in many areas of neuroscience.

  18. Lipopolysaccharide inhibits the self-renewal of spermatogonial stem cells in vitro via downregulation of GDNF expression in Sertoli cells.

    Science.gov (United States)

    Zhang, Xiaoli; Shi, Kun; Li, Yi; Zhang, Haiyu; Hao, Jing

    2014-06-01

    Lipopolysaccharide (LPS) can reduce sperm count and sperm quality. The molecular mechanisms underlying this process are not fully understood. In this report, we investigated the effects of LPS-treated Sertoli cells on self-renewal and differentiation of spermatogoinial stem cells (SSCs). Sertoli cell cultures were established and incubated with LPS (10μg/ml) for 1, 2 or 3 days, respectively. The culture media were collected and used as conditioned media (CM) to culture SSCs. The expression of glial cell-derived neurotrophic factor (GDNF), stem cell factor (SCF) and bone morphogenetic protein 4 (BMP4) in Sertoli cells treated with LPS was analyzed by RT-PCR and Western blotting. The results showed that the expression of SSC differentiation markers, c-kit and Sohlh2, was increased, while the expression of SSC self-renewal markers, plzf, oct4, and PCNA, was repressed when cultured in CM from LPS-treated Sertoli cells. GDNF levels in Sertoli cells and CM reduced dramatically after LPS treatments, while SCF and BMP4 levels did not show any significant changes. Moreover, correlated with the GDNF levels in CM, GDNF target genes, Bcl6b and Etv5, were reduced markedly in SSCs. Our results suggest that LPS inhibits the expression of GDNF in Sertoli cells, and might prevent the SSC self-renewal via down-regulation of GDNF target genes. Copyright © 2014 Elsevier Inc. All rights reserved.

  19. Downregulation of TGF-β Receptor-2 Expression and Signaling through Inhibition of Na/K-ATPase.

    Directory of Open Access Journals (Sweden)

    Jennifer La

    Full Text Available Transforming growth factor-beta (TGF-β is a multi-functional cytokine implicated in the control of cell growth and differentiation. TGF-β signals through a complex of TGF-β receptors 1 and 2 (TGFβR1 and TGFβR2 that phosphorylate and activate Smad2/3 transcription factors driving transcription of the Smad-target genes. The Na+/K+-ATPase is an integral plasma membrane protein critical for maintaining the electro-chemical gradient of Na+ and K+ in the cell. We found that inhibition of the Na+/K+ ATPase by ouabain results in a dramatic decrease in the expression of TGFβR2 in human lung fibrobalsts (HLF at the mRNA and protein levels. This was accompanied by inhibition of TGF-β-induced Smad phosphorylation and the expression of TGF-β target genes, such as fibronectin and smooth muscle alpha-actin. Inhibition of Na+/K+ ATPase by an alternative approach (removal of extracellular potassium had a similar effect in HLF. Finally, treatment of lung alveolar epithelial cells (A549 with ouabain also resulted in the downregulation of TGFβR2, the inhibition of TGF-β-induced Smad phosphorylation and of the expression of mesenchymal markers, vimentin and fibronectin. Together, these data demonstrate a critical role of Na+/K+-ATPase in the control of TGFβR2 expression, TGF-β signaling and cell responses to TGF-β.

  20. Galectin-1 is expressed in early-type neural progenitor cells and down-regulates neurogenesis in the adult hippocampus

    Directory of Open Access Journals (Sweden)

    Imaizumi Yoichi

    2011-01-01

    Full Text Available Abstract Background In the adult mammalian brain, neural stem cells (NSCs proliferate in the dentate gyrus (DG of the hippocampus and generate new neurons throughout life. A multimodal protein, Galectin-1, is expressed in neural progenitor cells (NPCs and implicated in the proliferation of the NPCs in the DG. However, little is known about its detailed expression profile in the NPCs and functions in adult neurogenesis in the DG. Results Our immunohistochemical and morphological analysis showed that Galectin-1 was expressed in the type 1 and 2a cells, which are putative NSCs, in the subgranular zone (SGZ of the adult mouse DG. To study Galectin-1's function in adult hippocampal neurogenesis, we made galectin-1 knock-out mice on the C57BL6 background and characterized the effects on neurogenesis. In the SGZ of the galectin-1 knock-out mice, increased numbers of type 1 cells, DCX-positive immature progenitors, and NeuN-positive newborn neurons were observed. Using triple-labeling immunohistochemistry and morphological analyses, we found that the proliferation of the type-1 cells was increased in the SGZ of the galectin-1 knock-out mice, and we propose that this proliferation is the mechanism for the net increase in the adult neurogenesis in these knock-out mice DG. Conclusions Galectin-1 is expressed in the neural stem cells and down-regulates neurogenesis in the adult hippocampus.

  1. Antidepressant effects of abscisic acid mediated by the downregulation of corticotrophin-releasing hormone gene expression in rats.

    Science.gov (United States)

    Qi, Cong-Cong; Zhang, Zhi; Fang, Hui; Liu, Ji; Zhou, Nan; Ge, Jin-Fang; Chen, Fang-Han; Xiang, Cheng-Bin; Zhou, Jiang-Ning

    2014-10-31

    Corticotrophin-releasing hormone (CRH) is considered to be the central driving force of the hypothalamic-pituitary-adrenal axis, which plays a key role in the stress response and depression. Clinical reports have suggested that excess retinoic acid (RA) is associated with depression. Abscisic acid (ABA) and RA are direct derivatives of carotenoids and share a similar molecular structure. Here, we proposed that ABA also plays a role in the regulation of CRH activity sharing with the RA signaling pathway. [3H]-ABA radioimmunoassay demonstrated that the hypothalamus of rats shows the highest concentration of ABA compared with the cortex and the hippocampus under basal conditions. Under acute stress, ABA concentrations increased in the serum, but decreased in the hypothalamus and were accompanied by increased corticosterone in the serum and c-fos expression in the hypothalamus. Moreover, chronic ABA administration increased sucrose intake and decreased the mRNA expression of CRH and retinoic acid receptor alpha (RARα) in the hypothalamus of rats. Furthermore, ABA improved the symptom of chronic unpredictable mild stress in model rats, as indicated by increased sucrose intake, increased swimming in the forced swim test, and reduced mRNA expression of CRH and RARα in the rat hypothalamus. In vitro, CRH expression decreased after ABA treatment across different neural cells. In BE(2)-C cells, ABA inhibited a series of retinoid receptor expression, including RARα, a receptor that could facilitate CRH expression directly. These results suggest that ABA may play a role in the pathogenesis of depression by downregulating CRH mRNA expression shared with the RA signaling pathway. © The Author 2014. Published by Oxford University Press on behalf of CINP.

  2. Niemann-Pick C1 like 1 gene expression is down-regulated by LXR activators in the intestine

    International Nuclear Information System (INIS)

    Duval, Caroline; Touche, Veronique; Tailleux, Anne; Fruchart, Jean-Charles; Fievet, Catherine; Clavey, Veronique; Staels, Bart; Lestavel, Sophie

    2006-01-01

    Niemann-Pick C1 like 1 (NPC1L1) is a protein critical for intestinal cholesterol absorption. The nuclear receptors peroxisome proliferator-activated receptor alpha (PPARα) and liver X receptors (LXRα and LXRβ) are major regulators of cholesterol homeostasis and their activation results in a reduced absorption of intestinal cholesterol. The goal of this study was to define the role of PPARα and LXR nuclear receptors in the regulation of NPC1L1 gene expression. We show that LXR activators down-regulate NPC1L1 mRNA levels in the human enterocyte cell line Caco-2/TC7, whereas PPARα ligands have no effect. Furthermore, NPC1L1 mRNA levels are decreased in vivo, in duodenum of mice treated with the LXR agonist T0901317. In conclusion, the present study identifies NPC1L1 as a novel LXR target gene further supporting a crucial role of LXR in intestinal cholesterol homeostasis

  3. [Effect of down-regulation of HE4 gene expression on biologic behavior of ovarian cancer cells].

    Science.gov (United States)

    Zhou, Lei; Xiao, Ran; Chen, Ying; Zhang, Jing; Lu, Ren-quan; Guo, Lin

    2013-10-01

    To investigate the effects of HE4 gene knockdown on the proliferation, adhesion and invasion of the ovarian cancer cells SKOV3. The knockdown of HE4 gene was performed by RNAi technology. The recombinant plasmids (pSUPER-HE4 shDNAs) were constructed and transfected into human ovarian cancer cells SKOV3. HE4 expression was then identified by real-time PCR and Western blot analysis. The invasion and adhesion ability of transduced cells were determined. In addition, cell proliferation and growth were analyzed by colonies formation assay. Knockdown of HE4 was achieved, and further confirmed by real-time PCR and Western blot. The proliferation of HE4-down-regulated cells was not affected, but the invasion ability of the transfected cells was reduced (P cells.

  4. Pioglitazone reverses down-regulation of cardiac PPARγ expression in Zucker diabetic fatty rats

    International Nuclear Information System (INIS)

    Pelzer, Theo; Jazbutyte, Virginija; Arias-Loza, Paula Anahi; Segerer, Stephan; Lichtenwald, Margit; Law, Marilyn P.; Schaefers, Michael; Ertl, Georg; Neyses, Ludwig

    2005-01-01

    Peroxisome proliferator-activated receptor-γ (PPARγ) plays a critical role in peripheral glucose homeostasis and energy metabolism, and inhibits cardiac hypertrophy in non-diabetic animal models. The functional role of PPARγ in the diabetic heart, however, is not fully understood. Therefore, we analyzed cardiac gene expression, metabolic control, and cardiac glucose uptake in male Zucker diabetic fatty rats (ZDF fa/fa) and lean ZDF rats (+/+) treated with the high affinity PPARγ agonist pioglitazone or placebo from 12 to 24 weeks of age. Hyperglycemia, hyperinsulinemia, and hypertriglyceridemia as well as lower cardiac PPARγ, glucose transporter-4 and α-myosin heavy chain expression levels were detected in diabetic ZDF rats compared to lean animals. Pioglitazone increased body weight and improved metabolic control, cardiac PPARγ, glut-4, and α-MHC expression levels in diabetic ZDF rats. Cardiac [ 18 F]fluorodeoxyglucose uptake was not detectable by micro-PET studies in untreated and pioglitazone treated ZDF fa/fa rats but was observed after administration of insulin to pioglitazone treated ZDF fa/fa rats. PPARγ agonists favorably affect cardiac gene expression in type-2 diabetic rats via activation and up-regulation of cardiac PPARγ expression whereas improvement of impaired cardiac glucose uptake in advanced type-2 diabetes requires co-administration of insulin

  5. Intra-uterine Growth Restriction Downregulates the Hepatic Toll Like Receptor-4 Expression and Function

    Directory of Open Access Journals (Sweden)

    Ozlem Equils

    2005-01-01

    Full Text Available Maternal starvation is a significant cause of intrauterine growth restriction (IUGR in the world and increases the risk of infection in the neonate. We examined the effect of maternal starvation on Toll like receptor (TLR4 expression in hepatic, splenic and intestinal tissues obtained from the adult IUGR offspring of prenatal calorie restricted rats. The hepatic TLR4 protein concentration was undetectable in the IUGR rats that had restricted milk intake during the suckling period (SM/SP; n = 4, p < 0.05 as compared to the normal growth controls (CM/CP; n=4, and access to ad lib milk intake during the sucking period partially corrected the hepatic TLR4 expression (SM/CP; n = 4. IUGR had no effect on the splenic (n = 4 or intestinal (n = 4 TLR4 mRNA levels. In the liver, IUGR led to a 20% increase in baseline tumor necrosis factor (TNF-α mRNA expression ( p < 0.03 and a 70% increase in interleukin-1β (IL-1β mRNA expression ( p < 0.008 as compared to the control rats (CM/CP; n = 7. LPS-induced hepatic TNF-α release was significantly higher in SM/SP as compared to CM/CP. We propose that IUGR dysregulates TLR4 expression and function in the offspring, which may help explain the increased risk of Gram-negative sepsis and inflammatory diseases in this population.

  6. miR-126 is downregulated in cystic fibrosis airway epithelial cells and regulates TOM1 expression.

    LENUS (Irish Health Repository)

    Oglesby, Irene K

    2010-02-15

    Cystic fibrosis (CF) is one of the most common lethal genetic diseases in which the role of microRNAs has yet to be explored. Predicted to be regulated by miR-126, TOM1 (target of Myb1) has been shown to interact with Toll-interacting protein, forming a complex to regulate endosomal trafficking of ubiquitinated proteins. TOM1 has also been proposed as a negative regulator of IL-1beta and TNF-alpha-induced signaling pathways. MiR-126 is highly expressed in the lung, and we now show for the first time differential expression of miR-126 in CF versus non-CF airway epithelial cells both in vitro and in vivo. MiR-126 downregulation in CF bronchial epithelial cells correlated with a significant upregulation of TOM1 mRNA, both in vitro and in vivo when compared with their non-CF counterparts. Introduction of synthetic pre-miR-126 inhibited luciferase activity in a reporter system containing the full length 3\\'-untranslated region of TOM1 and resulted in decreased TOM1 protein production in CF bronchial epithelial cells. Following stimulation with LPS or IL-1beta, overexpression of TOM1 was found to downregulate NF-kappaB luciferase activity. Conversely, TOM1 knockdown resulted in a significant increase in NF-kappaB regulated IL-8 secretion. These data show that miR-126 is differentially regulated in CF versus non-CF airway epithelial cells and that TOM1 is a miR-126 target that may have an important role in regulating innate immune responses in the CF lung. To our knowledge, this study is the first to report of a role for TOM1 in the TLR2\\/4 signaling pathways and the first to describe microRNA involvement in CF.

  7. SOX2 expression is associated with a cancer stem cell state and down-regulation of CDX2 in colorectal cancer

    International Nuclear Information System (INIS)

    Lundberg, Ida V.; Edin, Sofia; Eklöf, Vincy; Öberg, Åke; Palmqvist, Richard; Wikberg, Maria L.

    2016-01-01

    To improve current treatment strategies for patients with aggressive colorectal cancer (CRC), the molecular understanding of subgroups of CRC with poor prognosis is of vast importance. SOX2 positive tumors have been associated with a poor patient outcome, but the functional role of SOX2 in CRC patient prognosis is still unclear. An in vitro cell culture model expressing SOX2 was used to investigate the functional role of SOX2 in CRC. In vitro findings were verified using RNA from fresh frozen tumor tissue or immunohistochemistry on formalin fixed paraffin embedded (FFPE) tumor tissue from a cohort of 445 CRC patients. Using our in vitro model, we found that SOX2 expressing cells displayed several characteristics of cancer stem cells; such as a decreased proliferative rate, a spheroid growth pattern, and increased expression of stem cell markers CD24 and CD44. Cells expressing SOX2 also showed down-regulated expression of the intestinal epithelial marker CDX2. We next evaluated CDX2 expression in our patient cohort. CDX2 down-regulation was more often found in right sided tumors of high grade and high stage. Furthermore, a decreased expression of CDX2 was closely linked to MSI, CIMP-high as well as BRAF mutated tumors. A decreased expression of CDX2 was also, in a stepwise manner, strongly correlated to a poor patient prognosis. When looking at SOX2 expression in relation to CDX2, we found that SOX2 expressing tumors more often displayed a down-regulated expression of CDX2. In addition, SOX2 expressing tumors with a down-regulated CDX2 expression had a worse patient prognosis compared to those with retained CDX2 expression. Our results indicate that SOX2 expression induces a cellular stem cell state in human CRC with a decreased expression of CDX2. Furthermore, a down-regulated expression of CDX2 results in a poor patient prognosis in CRC and at least part of the prognostic importance of SOX2 is mediated through CDX2 down-regulation. The online version of this

  8. Metformin reduces the endotoxin-induced down-regulation of apolipoprotein E gene expression in macrophages

    Energy Technology Data Exchange (ETDEWEB)

    Stavri, Simona; Trusca, Violeta G.; Simionescu, Maya; Gafencu, Anca V., E-mail: anca.gafencu@icbp.ro

    2015-05-29

    The atheroprotective role of macrophage-derived apolipoprotein E (apoE) is well known. Our previous reports demonstrated that inflammatory stress down-regulates apoE expression in macrophages, aggravating atherogenesis. Metformin, extensively used as an anti-diabetic drug, has also anti-inflammatory properties, and thus confers vascular protection. In this study, we questioned whether metformin could have an effect on apoE expression in macrophages in normal conditions or under lipopolysaccharide (LPS)-induced stress. The results showed that metformin slightly increases the apoE expression only at high doses (5–10 mM). Low doses of metformin (1–3 mM) significantly reduce the LPS down-regulatory effect on apoE expression in macrophages. Our experiments demonstrated that LPS-induced NF-κB binds to the macrophage-specific distal regulatory element of apoE gene, namely to the multienhancer 2 (ME.2) and its 5′-deletion fragments. The NF-κB binding on ME.2 and apoE promoter has a down-regulatory effect. In addition, data revealed that metformin impairs NF-κB nuclear translocation, and thus, improves the apoE levels in macrophages under inflammatory stress. The positive effect of metformin in the inflammatory states, its clinical safety and low cost, make this drug a potential adjuvant in the therapeutic strategies for atherosclerosis. - Highlights: • High doses of metformin slightly increase apoE expression in macrophages. • Low doses of metformin up-regulate apoE gene in endotoxin-stressed macrophages. • Metformin reduces the negative effect of LPS on apoE expression by NF-κB inhibition.

  9. Metformin reduces the endotoxin-induced down-regulation of apolipoprotein E gene expression in macrophages

    International Nuclear Information System (INIS)

    Stavri, Simona; Trusca, Violeta G.; Simionescu, Maya; Gafencu, Anca V.

    2015-01-01

    The atheroprotective role of macrophage-derived apolipoprotein E (apoE) is well known. Our previous reports demonstrated that inflammatory stress down-regulates apoE expression in macrophages, aggravating atherogenesis. Metformin, extensively used as an anti-diabetic drug, has also anti-inflammatory properties, and thus confers vascular protection. In this study, we questioned whether metformin could have an effect on apoE expression in macrophages in normal conditions or under lipopolysaccharide (LPS)-induced stress. The results showed that metformin slightly increases the apoE expression only at high doses (5–10 mM). Low doses of metformin (1–3 mM) significantly reduce the LPS down-regulatory effect on apoE expression in macrophages. Our experiments demonstrated that LPS-induced NF-κB binds to the macrophage-specific distal regulatory element of apoE gene, namely to the multienhancer 2 (ME.2) and its 5′-deletion fragments. The NF-κB binding on ME.2 and apoE promoter has a down-regulatory effect. In addition, data revealed that metformin impairs NF-κB nuclear translocation, and thus, improves the apoE levels in macrophages under inflammatory stress. The positive effect of metformin in the inflammatory states, its clinical safety and low cost, make this drug a potential adjuvant in the therapeutic strategies for atherosclerosis. - Highlights: • High doses of metformin slightly increase apoE expression in macrophages. • Low doses of metformin up-regulate apoE gene in endotoxin-stressed macrophages. • Metformin reduces the negative effect of LPS on apoE expression by NF-κB inhibition

  10. Lactobacillus salivarius and L. gasseri down-regulate Aggregatibacter actinomycetemcomitans exotoxins expression.

    Science.gov (United States)

    Nissen, Lorenzo; Sgorbati, Barbara; Biavati, Bruno; Belibasakis, Georgios N

    2014-01-01

    Beneficial microbes, such as lactobacilli establish a symbiosis with the host and confer health-associated effects, by limiting the growth of indigenous pathogens and challenging microbes introduced by altered foods. Nevertheless, there is scarce information on the effects of beneficial microbes on the virulence properties of bacterial species associated with oral diseases, such as periodontitis. Aggregatibacter actinomycetemcomitans is a Gram-negative species highly implicated in the etiology of localized aggressive periodontitis. The objective of this study was to investigate the effect of lactobacilli on the expression of the two major virulence factors of A. actinomycetemcomitans . Lactobacillus salivarius and L. gasseri were selected as beneficial species. The gene expressions of leukotoxin ( LtxA ) and cytolethal distending toxin ( CdtB ) by A. actinomycetemcomitans were analyzed in response to challenge by lactobacilli cell-free supernatants. Neither lactobacilli affected the growth, but strongly attenuated the expressions of both CdtB and LtxA in the two A. actinomycetemcomitans strains tested. This reduction of the expression of these two exotoxins was time-dependent. These fundamental findings may indicate that lactobacilli can reduce the virulence of putative opportunistic oral pathogens, and may provide insights to future therapeutic approaches for the respective diseases.

  11. Rapamycin down-regulates LDL-receptor expression independently of SREBP-2

    International Nuclear Information System (INIS)

    Sharpe, Laura J.; Brown, Andrew J.

    2008-01-01

    As a key regulator of cholesterol homeostasis, sterol-regulatory element binding protein-2 (SREBP-2) up-regulates expression of genes involved in cholesterol synthesis (e.g., 3-hydroxy-3-methyl-glutaryl-coenzyme A (HMG-CoA) Reductase) and uptake (the low density lipoprotein (LDL)-receptor). Previously, we showed that Akt, a critical kinase in cell growth and proliferation, contributes to SREBP-2 activation. However, the specific Akt target involved is unknown. A potential candidate is the mammalian target of rapamycin, mTOR. Rapamycin can cause hyperlipidaemia clinically, and we hypothesised that this may be mediated via an effect of mTOR on SREBP-2. Herein, we found that SREBP-2 activation and HMG-CoA Reductase gene expression were unaffected by rapamycin treatment. However, LDL-receptor gene expression was decreased by rapamycin, suggesting that this may contribute to the hyperlipidaemia observed in rapamycin-treated patients. Rapamycin did not affect mRNA stability, so the decrease in LDL-receptor gene expression is likely to be occurring at the transcriptional level, although independently of SREBP-2

  12. Antimicrobial activity of apple cider vinegar against Escherichia coli, Staphylococcus aureus and Candida albicans; downregulating cytokine and microbial protein expression.

    Science.gov (United States)

    Yagnik, Darshna; Serafin, Vlad; J Shah, Ajit

    2018-01-29

    The global escalation in antibiotic resistance cases means alternative antimicrobials are essential. The aim of this study was to investigate the antimicrobial capacity of apple cider vinegar (ACV) against E. coli, S. aureus and C. albicans. The minimum dilution of ACV required for growth inhibition varied for each microbial species. For C. albicans, a 1/2 ACV had the strongest effect, S. aureus, a 1/25 dilution ACV was required, whereas for E-coli cultures, a 1/50 ACV dilution was required (p < 0.05). Monocyte co-culture with microbes alongside ACV resulted in dose dependent downregulation of inflammatory cytokines (TNFα, IL-6). Results are expressed as percentage decreases in cytokine secretion comparing ACV treated with non-ACV treated monocytes cultured with E-coli (TNFα, 99.2%; IL-6, 98%), S. aureus (TNFα, 90%; IL-6, 83%) and C. albicans (TNFα, 83.3%; IL-6, 90.1%) respectively. Proteomic analyses of microbes demonstrated that ACV impaired cell integrity, organelles and protein expression. ACV treatment resulted in an absence in expression of DNA starvation protein, citrate synthase, isocitrate and malate dehydrogenases in E-coli; chaperone protein DNak and ftsz in S. aureus and pyruvate kinase, 6-phosphogluconate dehydrogenase, fructose bisphosphate were among the enzymes absent in C.albican cultures. The results demonstrate ACV has multiple antimicrobial potential with clinical therapeutic implications.

  13. Down-regulation of procaspase-8 expression by focal adhesion kinase protects HL-60 cells from TRAIL-induced apoptosis

    International Nuclear Information System (INIS)

    Tamagiku, Yuji; Sonoda, Yoshiko; Kunisawa, Mari; Ichikawa, Daiju; Murakami, Yayoi; Aizu-Yokota, Eriko; Kasahara, Tadashi

    2004-01-01

    We have demonstrated that focal adhesion kinase (FAK)-overexpressed (HL-60/FAK) cells have marked resistance against various apoptotic stimuli such as hydrogen peroxide, etoposide, and ionizing radiation compared with the vector-transfected (HL-60/Vect) cells. HL-60/FAK cells are highly resistant to TRAIL-induced apoptosis, while original HL-60 or HL-60/Vect cells were sensitive. TRAIL at 500 ng/ml induced significant DNA fragmentation, activation of caspase-8 and 3, the processing of a proapoptotic BID, and mitochondrial release of cytochrome c in HL-60/Vect cells, whereas no such events were observed in the HL-60/FAK cells. In particular, the expression of procaspase-8 gene and subsequent cleavage of caspase-8 were markedly reduced in HL-60/FAK cells, while expression of TRAIL-receptor 2 and 3, TRADD, and FADD was equivalent in both types of cells. In HL-60/FAK cells, the phosphoinositide 3 (PI3)-kinase/Akt survival pathway was constitutively activated, accompanied by significant induction of inhibitor-of-apoptosis proteins, XIAP, RIP, and Bcl-XL. The introduction of FAK siRNA in HL-60/FAK cells sensitized them against TRAIL-induced apoptosis, confirming that overexpressed FAK downregulates procaspase-8 expression, which subsequently inhibits downstream apoptosis pathway in the HL-60/FAK cells

  14. BMP9 inhibits the bone metastasis of breast cancer cells by downregulating CCN2 (connective tissue growth factor, CTGF) expression.

    Science.gov (United States)

    Ren, Wei; Sun, Xiaoxiao; Wang, Ke; Feng, Honglei; Liu, Yuehong; Fei, Chang; Wan, Shaoheng; Wang, Wei; Luo, Jinyong; Shi, Qiong; Tang, Min; Zuo, Guowei; Weng, Yaguang; He, Tongchuan; Zhang, Yan

    2014-03-01

    Bone morphogenetic proteins (BMPs), which belong to the transforming growth factor-β superfamily, regulate a wide range of cellular responses including cell proliferation, differentiation, adhesion, migration, and apoptosis. BMP9, the latest BMP to be discovered, is reportedly expressed in a variety of human carcinoma cell lines, but the role of BMP9 in breast cancer has not been fully clarified. In a previous study, BMP9 was found to inhibit the growth, migration, and invasiveness of MDA-MB-231 breast cancer cells. In the current study, the effect of BMP9 on the bone metastasis of breast cancer cells was investigated. After absent or low expression of BMP9 was detected in the MDA-MB-231 breast cancer cells and breast non-tumor adjacent tissues using Western blot and immunohistochemistry, In our previous study, BMP9 could inhibit the proliferation and invasiveness of breast cancer cells MDA-MB-231 in vitro and in vivo. This paper shows that BMP9 inhibit the bone metastasis of breast cancer cells by activating the BMP/Smad signaling pathway and downregulating connective tissue growth factor (CTGF); however, when CTGF expression was maintained, the inhibitory effect of BMP9 on the MDA-MB-231 cells was abolished. Together, these observations indicate that BMP9 is an important mediator of breast cancer bone metastasis and a potential therapeutic target for treating this deadly disease.

  15. MicroRNA-205 downregulates mixed-lineage-AF4 oncogene expression in acute lymphoblastic leukemia

    Directory of Open Access Journals (Sweden)

    Dou L

    2013-08-01

    Full Text Available Liping Dou,1,* Jingxin Li,1,* Dehua Zheng,2,* Yonghui Li,1 Xiaoning Gao,1 Chengwang Xu,1 Li Gao,1 Lili Wang,1 Li Yu1 1Department of Hematology, Chinese PLA General Hospital, Beijing, People's Republic of China; 2Department of Hepatobiliary Surgery, Organ Transplant Center, Chinese PLA 309th Hospital, Beijing, People's Republic of China*These authors contributed equally to this workAbstract: Myeloid/lymphoid or mixed-lineage AF4 acute lymphoblastic leukemia (MLL-AF4 ALL is a pediatric leukemia that occurs rarely in adults. MLL-AF4 ALL is typically characterized by the presence of chromosomal translocation (t(4;11(q21;q23, leading to expression of MLL-AF4 fusion protein. Although MLL-AF4 fusion protein triggers a molecular pathogenesis and hematological presentations that are unique to leukemias, the precise role of this oncogene in leukemogenesis remains unclear. Previous studies have indicated that microRNAs (miRs might modulate the expression of MLL-AF4 ALL fusion protein, thereby suggesting the involvement of miR in progression or suppression of MLL-AF4 ALL. We have previously demonstrated that miR-205 negatively regulates transcription of an MLL-AF4 luciferase reporter. Here, we report that exogenous expression of miR-205 in MLL-AF4 human cell lines (RS4;11 and MV4-11 inversely regulates the expression of MLL-AF4 at both messenger RNA (mRNA and protein level. Furthermore, miR-205 significantly induced apoptosis in MLL-AF4 cells as evidenced by Annexin V staining using fluorescence-activated cell sorting (FACS analysis. The proliferative capacity of leukemic cells was suppressed by miR-205. The addition of an miR-205 inhibitor was able to restore the observed effects. In conclusion, these findings demonstrate that miR-205 may have potential value as a novel therapeutic agent in the treatment of MLL-AF4 ALL.Keywords: miR-205, MLL-AF4, leukemia, microRNA, oncogene expression, untranslated regions, proliferation

  16. Cafestol overcomes ABT-737 resistance in Mcl-1-overexpressed renal carcinoma Caki cells through downregulation of Mcl-1 expression and upregulation of Bim expression.

    Science.gov (United States)

    Woo, S M; Min, K-J; Seo, B R; Nam, J-O; Choi, K S; Yoo, Y H; Kwon, T K

    2014-11-06

    Although ABT-737, a small-molecule Bcl-2/Bcl-xL inhibitor, has recently emerged as a novel cancer therapeutic agent, ABT-737-induced apoptosis is often blocked in several types of cancer cells with elevated expression of Mcl-1. Cafestol, one of the major compounds in coffee beans, has been reported to have anti-carcinogenic activity and tumor cell growth-inhibitory activity, and we examined whether cafestol could overcome resistance against ABT-737 in Mcl-1-overexpressed human renal carcinoma Caki cells. ABT-737 alone had no effect on apoptosis, but cafestol markedly enhanced ABT-737-mediated apoptosis in Mcl-1-overexpressed Caki cells, human glioma U251MG cells, and human breast carcinoma MDA-MB231 cells. By contrast, co-treatment with ABT-737 and cafestol did not induce apoptosis in normal human skin fibroblast. Furthermore, combined treatment with cafestol and ABT-737 markedly reduced tumor growth compared with either drug alone in xenograft models. We found that cafestol inhibited Mcl-1 protein expression, which is important for ABT-737 resistance, through promotion of protein degradation. Moreover, cafestol increased Bim expression, and siRNA-mediated suppression of Bim expression reduced the apoptosis induced by cafestol plus ABT-737. Taken together, cafestol may be effectively used to enhance ABT-737 sensitivity in cancer therapy via downregulation of Mcl-1 expression and upregulation of Bim expression.

  17. Istaroxime Inhibits Motility and Down-Regulates Orai1 Expression, SOCE and FAK Phosphorylation in Prostate Cancer Cells

    Directory of Open Access Journals (Sweden)

    Matias Julian Stagno

    2017-07-01

    Full Text Available Background/Aims: Istaroxime is a validated inotropic Na+/K+ ATPase inhibitor currently in development for the treatment of various cardiac conditions. Recent findings established that this steroidal drug exhibits potent apoptotic responses in prostate tumors in vitro and in vivo, by affecting key signaling orchestrating proliferation and apoptosis, such as c-Myc and caspase 3, Rho GTPases and actin cytoskeleton dynamics. In the present study we examined whether istaroxime is affecting cell motility and analyzed the underlying mechanism in prostate tumor cells. Methods: Migration was assessed by transwell and wound healing assays, Orai1 and Stim1 abundance by RT-PCR and confocal immunofluorescence microscopy, Fura-2 fluorescence was utilized to determine intracellular Ca2+ and Western blotting for FAK/pFAK measurements. Results: We observed strong inhibition of cell migration in istaroxime treated DU-145 prostate cancer cells. Istaroxime further decreased Orai1 and Stim1 transcript levels and downregulated Orai1 protein expression. Moreover, SOCE was significantly decreased upon istaroxime treatment. Furthermore, istaroxime strikingly diminished phosphorylated FAK levels. Interestingly, the efficacy of istaroxime on the inhibition of DU-145 cell migration was further enhanced by blocking Orai1 with 2-APB and FAK with the specific inhibitor PF-00562271. These results provide strong evidence that istaroxime prevents cell migration and motility of DU-145 prostate tumor cells, an effect at least partially attributed to Orai1 downregulation and FAK de-activation. Conclusion: Collectively our results indicate that this enzyme inhibitor, besides its pro-apoptotic action, affects motility of cancer cells, supporting its potential role as a strong candidate for further clinical cancer drug development.

  18. Elevated expression of prostate cancer-associated genes is linked to down-regulation of microRNAs

    International Nuclear Information System (INIS)

    Erdmann, Kati; Kaulke, Knut; Thomae, Cathleen; Huebner, Doreen; Sergon, Mildred; Froehner, Michael; Wirth, Manfred P; Fuessel, Susanne

    2014-01-01

    Recent evidence suggests that the prostate cancer (PCa)-specific up-regulation of certain genes such as AMACR, EZH2, PSGR, PSMA and TRPM8 could be associated with an aberrant expression of non-coding microRNAs (miRNA). In silico analyses were used to search for miRNAs being putative regulators of PCa-associated genes. The expression of nine selected miRNAs (hsa-miR-101, -138, -186, -224, -26a, -26b, -374a, -410, -660) as well as of the aforementioned PCa-associated genes was analyzed by quantitative PCR using 50 malignant (Tu) and matched non-malignant (Tf) tissue samples from prostatectomy specimens as well as 30 samples from patients with benign prostatic hyperplasia (BPH). Then, correlations between paired miRNA and target gene expression levels were analyzed. Furthermore, the effect of exogenously administered miR-26a on selected target genes was determined by quantitative PCR and Western Blot in various PCa cell lines. A luciferase reporter assay was used for target validation. The expression of all selected miRNAs was decreased in PCa tissue samples compared to either control group (Tu vs Tf: -1.35 to -5.61-fold; Tu vs BPH: -1.17 to -5.49-fold). The down-regulation of most miRNAs inversely correlated with an up-regulation of their putative target genes with Spearman correlation coefficients ranging from -0.107 to -0.551. MiR-186 showed a significantly diminished expression in patients with non-organ confined PCa and initial metastases. Furthermore, over-expression of miR-26a reduced the mRNA and protein expression of its potential target gene AMACR in vitro. Using the luciferase reporter assay AMACR was validated as new target for miR-26a. The findings of this study indicate that the expression of specific miRNAs is decreased in PCa and inversely correlates with the up-regulation of their putative target genes. Consequently, miRNAs could contribute to oncogenesis and progression of PCa via an altered miRNA-target gene-interaction

  19. Silymarin and caffeine combination ameliorates experimentally-induced hepatic fibrosis through down-regulation of LPAR1 expression.

    Science.gov (United States)

    Eraky, Salma M; El-Mesery, Mohamed; El-Karef, Amro; Eissa, Laila A; El-Gayar, Amal M

    2018-05-01

    Lysophosphatidic acid is a lipid mediator that is supposed to be implicated in hepatic fibrosis. Silymarin and caffeine are natural compounds known for their anti-inflammatory and antioxidant effects. Our study aimed to explore the effect of silymarin, caffeine, and their combination on lysophosphatidic acid receptor 1 (LPAR1) pathway in thioacetamide (TAA)-induced hepatic fibrosis. Hepatic fibrosis was induced in male Sprague-Dawley rats by intraperitoneal injection of 200 mg/kg of TAA twice a week for 8 weeks. Silymarin (50 mg/kg), caffeine (50 mg/kg), and their combination (50 mg/kg silymarin + 50 mg/kg caffeine) were orally given to rats every day for 8 weeks along with TAA injection. Liver functions were measured. Histopathological examination of liver tissues was performed using hematoxylin and eosin and Masson's trichrome staining. mRNA expressions of LPAR1, transforming growth factor beta 1 (TGF-β1), connective tissue growth factor (CTGF), and alpha smooth muscle actin (α-SMA) were measured using RT-PCR. LPAR1 tissue expression was scored using immunohistochemistry. Silymarin, caffeine, and their combination significantly improved liver function. They caused significant decrease in fibrosis and necro-inflammatory scores. Combination of silymain and caffeine caused a significant decrease in the necro-inflammatory score than the single treatment with silymarin or caffeine. In addition, silymarin, caffeine, and their combination significantly decreased hepatic LPAR1, TGF-β1, CTGF, and α-SMA gene expressions and LPAR1 tissue expression. Silymarin, caffeine, and their combination protect against liver fibrosis through down-regulation of LPAR1, TGF-β1, and CTGF. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

  20. Icotinib inhibits the invasion of Tca8113 cells via downregulation of nuclear factor κB-mediated matrix metalloproteinase expression.

    Science.gov (United States)

    Yang, Cailing; Yan, Jianguo; Yuan, Guoyan; Zhang, Yinghua; Lu, Derong; Ren, Mingxin; Cui, Weigang

    2014-09-01

    Icotinib is an epidermal growth factor receptor tyrosine kinase inhibitor, which has been revealed to inhibit proliferation in tumor cells. However, the effect of icotinib on cancer cell metastasis remains to be explained. This study examines the effect of icotinib on the migration and invasion of squamous cells of tongue carcinoma (Tca8113 cells) in vitro . The results of the Boyden chamber invasion assay demonstrated that icotinib reduced cell invasion, suppressed the protein levels of matrix metalloproteinases (MMPs), MMP-2 and MMP-9, and increased the expression of tissue inhibitor of metalloproteinase-1. In addition, icotinib was found to significantly decrease the protein levels of nuclear factor κB (NF-κB) p65, which suggested that icotinib inhibits NF-κB activity. Furthermore, treatment with the NF-κB inhibitor, pyrrolidine dithiocarbamate, suppressed cell invasion and MMP-2 expression. These results suggested that icotinib inhibits the invasion of Tca8113 cells by downregulating MMP via the inactivation of the NF-κB signaling pathways.

  1. Disruption of a cystine transporter downregulates expression of genes involved in sulfur regulation and cellular respiration

    Directory of Open Access Journals (Sweden)

    Jessica A. Simpkins

    2016-06-01

    Full Text Available Cystine and cysteine are important molecules for pathways such as redox signaling and regulation, and thus identifying cellular deficits upon deletion of the Saccharomyces cerevisiae cystine transporter Ers1p allows for a further understanding of cystine homeostasis. Previous complementation studies using the human ortholog suggest yeast Ers1p is a cystine transporter. Human CTNS encodes the protein Cystinosin, a cystine transporter that is embedded in the lysosomal membrane and facilitates the export of cystine from the lysosome. When CTNS is mutated, cystine transport is disrupted, leading to cystine accumulation, the diagnostic hallmark of the lysosomal storage disorder cystinosis. Here, we provide biochemical evidence for Ers1p-dependent cystine transport. However, the accumulation of intracellular cystine is not observed when the ERS1 gene is deleted from ers1-Δ yeast, supporting the existence of modifier genes that provide a mechanism in ers1-Δ yeast that prevents or corrects cystine accumulation. Upon comparison of the transcriptomes of isogenic ERS1+ and ers1-Δ strains of S. cerevisiae by DNA microarray followed by targeted qPCR, sixteen genes were identified as being differentially expressed between the two genotypes. Genes that encode proteins functioning in sulfur regulation, cellular respiration, and general transport were enriched in our screen, demonstrating pleiotropic effects of ers1-Δ. These results give insight into yeast cystine regulation and the multiple, seemingly distal, pathways that involve proper cystine recycling.

  2. RGD-conjugated gold nanorods induce radiosensitization in melanoma cancer cells by downregulating αvβ3 expression

    Directory of Open Access Journals (Sweden)

    Pang B

    2012-02-01

    Full Text Available Wencai Xu1, Teng Luo2, Ping Li1, Chuanqing Zhou2, Daxiang Cui3, Bo Pang4, Qiushi Ren4, Shen Fu11Department of Radiation Oncology, Shanghai Sixth People's Hospital, 2School of Biomedical Engineering, and 3National Key Laboratory of Nano/Micro Fabrication Technology, Key Laboratory for Thin Film and Microfabrication of Ministry of Education, Institute of Micro-Nano Science and Technology, Shanghai Jiao Tong University, Shanghai, 4Department of Biomedical Engineering, College of Engineering, Peking University, Beijing, People's Republic of ChinaBackground: Melanoma is known to be radioresistant and traditional treatments have been intractable. Therefore, novel approaches are required to improve the therapeutic efficacy of melanoma treatment. In our study, gold nanorods conjugated with Arg-Gly-Asp peptides (RGD-GNRs were used as a sensitizer to enhance the response of melanoma cells to 6 mV radiation.Methods and materials: A375 melanoma cells were treated by gold nanorods or RGD-GNRs with or without irradiation. The antiproliferative impact of the treatments was measured by MTT assay. Radiosensitizing effects were determined by colony formation assay. Apoptosis and cell cycle data were measured by flow cytometry. Integrin αvβ3expression was also investigated by flow cytometry.Results: Addition of RGD-GNRs enhanced the radiosensitivity of A375 cells with a dose-modifying factor of 1.35, and enhanced radiation-induced apoptosis. DNA flow cytometric analysis indicated that RGD-GNRs plus irradiation induced significant G2/M phase arrest in A375 cells. Both spontaneous and radiation-induced expressions of integrin αvβ3 were downregulated by RGD-GNRs.Conclusion: Our study indicated that RGD-GNRs could sensitize melanoma A375 cells to irradiation. It was hypothesized that this was mainly through downregulation of radiation-induced αvβ3, in addition to induction of a higher proportion of cells within the G2/M phase. The combination of RGD-GNRs and

  3. Downregulation of hepatic and intestinal ATP-binding-cassette transporters abcg5 and abcg8 expression associated with altered sterol fluxes in rats with streptozotocin-induced diabetes

    NARCIS (Netherlands)

    Bloks, VW; Bakker-van Waarde, WW; Verkade, HJ; Kema, IP; Havinga, R; Wolters, H; Schaap, FG; Sauer, PJJ; Vink, E; Groen, AK; Kuipers, F

    ABSTRACT: P234 Downregulation of Hepatic and Intestinal ATP-Binding-Cassette Transporters Abcg5 and Abcg8 Expression Associated with Altered Sterol Fluxes in Rats with Streptozotocin-Induced Diabetes Vincent W. Bloks, Willie W. Bakker-van Waarde, Henkjan J. Verkade, Ido P. Kema, Rick Havinga, Henk

  4. Vitamin K2 downregulates the expression of fibroblast growth factor receptor 3 in human hepatocellular carcinoma cells.

    Science.gov (United States)

    Cao, Ke; Liu, Weidong; Nakamura, Hideji; Enomoto, Hirayuki; Yamamoto, Teruhisa; Saito, Masaki; Imanishi, Hiroyasu; Shimomura, Soji; Cao, Peiguo; Nishiguchi, Shuhei

    2009-11-01

    Vitamin K2 exerts an antitumor activity on human hepatocellular carcinoma (HCC), however, its inhibitory mechanism has not yet been clarified. This study was designed to identify the attractive target molecule of vitamin K2 and shed some light on its effects on fibroblast growth factor receptor (FGFR)3 in HCC cells. The changes in the gene expression of HuH-7 after vitamin K2 treatment were evaluated by a DNA chip analysis. The mRNA and protein levels of FGFR were evaluated by semiquantitative reverse transcription polymerase chain reaction (RT-PCR), real-time PCR and western blot analysis. The promoter activity of the FGFR3 gene was measured by a dual-luciferase assay. The DNA chip analysis revealed different inhibitory rates of gene expression of FGFR3 (60.6%) and FGFR1 (19.4%) after vitamin K2 treatment. Vitamin K2 suppresses the proliferation of HuH-7 in a dose-dependent manner and its inhibitory rate reached approximately 61.8% at the dose of 30 microM. FGFR3 mRNA was significantly reduced based on semiquantitative RT-PCR and decreased 61.5% by a real-time PCR method after vitamin K2 treatment, but FGFR1 mRNA was not. The level of FGFR3 protein was also reduced by vitamin K2 treatment. The luciferase assay demonstrated that vitamin K2 significantly suppressed the promoter activity of FGFR3. Furthermore, the FGFR3-ERK1/2 signaling pathway was suppressed by vitamin K2 treatment. These findings suggest that vitamin K2 may suppress the proliferation of HCC cells through the downregulation of the FGFR3 expression. The transcriptional suppression of FGFR3 may be a novel mechanism of the vitamin K2 action for HCC cells.

  5. Aloe vera downregulates LPS-induced inflammatory cytokine production and expression of NLRP3 inflammasome in human macrophages.

    Science.gov (United States)

    Budai, Marietta M; Varga, Aliz; Milesz, Sándor; Tőzsér, József; Benkő, Szilvia

    2013-12-01

    Aloe vera has been used in traditional herbal medicine as an immunomodulatory agent inducing anti-inflammatory effects. However, its role on the IL-1β inflammatory cytokine production has not been studied. IL-1β production is strictly regulated both at transcriptional and posttranslational levels through the activity of Nlrp3 inflammasome. In this study we aimed to determine the effect of Aloe vera on the molecular mechanisms of Nlrp3 inflammasome-mediated IL-1β production in LPS-activated human THP-1 cells and monocyte-derived macrophages. Our results show that Aloe vera significantly reduced IL-8, TNFα, IL-6 and IL-1β cytokine production in a dose dependent manner. The inhibitory effect was substantially more pronounced in the primary cells. We found that Aloe vera inhibited the expression of pro-IL-1β, Nlrp3, caspase-1 as well as that of the P2X7 receptor in the LPS-induced primary macrophages. Furthermore, LPS-induced activation of signaling pathways like NF-κB, p38, JNK and ERK were inhibited by Aloe vera in these cells. Altogether, we show for the first time that Aloe vera-mediated strong reduction of IL-1β appears to be the consequence of the reduced expression of both pro-IL-1β as well as Nlrp3 inflammasome components via suppressing specific signal transduction pathways. Furthermore, we show that the expression of the ATP sensor P2X7 receptor is also downregulated by Aloe vera that could also contribute to the attenuated IL-1β cytokine secretion. These results may provide a new therapeutic approach to regulate inflammasome-mediated responses. Copyright © 2013 Elsevier Ltd. All rights reserved.

  6. Calycosin Inhibits the Migration and Invasion of Human Breast Cancer Cells by Down-Regulation of Foxp3 Expression

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    Shuangxi Li

    2017-12-01

    Full Text Available Background/Aims: Calycosin, a phytoestrogenic compound, has recently emerged as a promising antitumor drug. It has been shown that calycosin suppresses growth and induces apoptosis of breast cancer cells. However, the effect of calycosin on migration and invasion of breast cancer cells and the underlying molecular mechanisms have not been elucidated. Methods: Human breast cancer cells MCF-7 and T47D were treated with, or without, different doses (0, 6.25, 12.5, 25, 50, 100 or 150 μM of calycosin, and the viability of different groups was determined by MTT assay. Next, the inhibitory effect of higher doses (50, 100 or 150 μM of calycosin on migration and invasion of the two cell lines was determined by wound healing and transwell assay. The relative expression levels of forkhead box P3 (Foxp3, vascular endothelial growth factor (VEGF and matrix metalloproteinase-9 (MMP-9 in MCF-7 and T47D cells were determined by quantitative RT-PCR and Western blot. Results: Treatment with lower doses (6.25 or 12.5 μM promoted proliferation of breast cancer cells, but with higher doses significantly reduced the viability of MCF-7 and T47D cells. Furthermore, higher doses of calycosin were found to inhibit migration and invasion of the two cell lines in a dose-dependent manner. Additionally, treatment with a higher dose of calycosin significantly reduced the expression levels of Foxp3, followed by down-regulation of VEGF and MMP-9 in both MCF-7 and T47D breast cancer cells. Conclusion: Treatment with a higher dose of calycosin tends to reduce migration and invasion capacity of human breast cancer cells, by targeting Foxp3-mediated VEGF and MMP-9 expression.

  7. Melittin induces PTCH1 expression by down-regulating MeCP2 in human hepatocellular carcinoma SMMC-7721 cells

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    Wu, Xiaoqin; Zhao, Bin; Cheng, Yahui; Yang, Yang; Huang, Cheng; Meng, Xiaoming; Wu, Baoming; Zhang, Lei; Lv, Xiongwen; Li, Jun, E-mail: xqwu01@foxmail.com

    2015-10-01

    Hepatocellular carcinoma (HCC) has a high mortality rate worldwide and still remains to be a noticeable public health problem. Therefore, new remedies are urgently needed. Melittin, a major component of bee venom, is known to suppress cell growth in various cancers including HCC. However, the mechanism of the anticancer effect of melittin on HCC has not been fully elucidated. It has been reported that Methyl-CpG binding protein 2 (MeCP2) plays a key role in tumor proliferation, apoptosis, migration and invasion. In the present study, we found the high expression of MeCP2 in human HCC tissues and in the SMMC-7721 cell line. MeCP2 silencing inhibited cell proliferation, while over-expression of MeCP2 promoted cell growth in SMMC-7721 cells. It indicates that MeCP2 may be an attractive target for human HCC. We further found that melittin could inhibit cell proliferation by reducing MeCP2 expression in vitro. Interestingly, the inhibitory effect of melittin on cell proliferation was due to a delay in G{sub 0}/G{sub 1} cell cycle progression, without influencing cell apoptosis. Next, we investigated the potential molecular mechanisms and found that MeCP2 could modulate Shh signaling in SMMC-7721 cells. Further study indicates that melittin may induce the demethylation of PTCH1 promoter, resulting in the increased expression of PTCH1. Furthermore, the expression of Shh and GLI1 was significantly lowered upon treatment of melittin. These results suggest that melittin can block Shh signaling in vitro. In short, these results indicate that melittin inhibits cell proliferation by down-regulating MeCP2 through Shh signaling in SMMC-7721 cells. - Highlights: • MeCP2 plays a key role in the proliferation of human HCC cells. • Melittin reduces MeCP2 expression in vitro. • Melittin induces G{sub 0}/G{sub 1} cell cycle arrest in SMMC-7721 cells. • MeCP2 modulates the Shh signaling pathway in SMMC-7721 cells. • Melittin blocks the Shh signaling pathway in SMMC-7721 cells.

  8. Melittin induces PTCH1 expression by down-regulating MeCP2 in human hepatocellular carcinoma SMMC-7721 cells

    International Nuclear Information System (INIS)

    Wu, Xiaoqin; Zhao, Bin; Cheng, Yahui; Yang, Yang; Huang, Cheng; Meng, Xiaoming; Wu, Baoming; Zhang, Lei; Lv, Xiongwen; Li, Jun

    2015-01-01

    Hepatocellular carcinoma (HCC) has a high mortality rate worldwide and still remains to be a noticeable public health problem. Therefore, new remedies are urgently needed. Melittin, a major component of bee venom, is known to suppress cell growth in various cancers including HCC. However, the mechanism of the anticancer effect of melittin on HCC has not been fully elucidated. It has been reported that Methyl-CpG binding protein 2 (MeCP2) plays a key role in tumor proliferation, apoptosis, migration and invasion. In the present study, we found the high expression of MeCP2 in human HCC tissues and in the SMMC-7721 cell line. MeCP2 silencing inhibited cell proliferation, while over-expression of MeCP2 promoted cell growth in SMMC-7721 cells. It indicates that MeCP2 may be an attractive target for human HCC. We further found that melittin could inhibit cell proliferation by reducing MeCP2 expression in vitro. Interestingly, the inhibitory effect of melittin on cell proliferation was due to a delay in G 0 /G 1 cell cycle progression, without influencing cell apoptosis. Next, we investigated the potential molecular mechanisms and found that MeCP2 could modulate Shh signaling in SMMC-7721 cells. Further study indicates that melittin may induce the demethylation of PTCH1 promoter, resulting in the increased expression of PTCH1. Furthermore, the expression of Shh and GLI1 was significantly lowered upon treatment of melittin. These results suggest that melittin can block Shh signaling in vitro. In short, these results indicate that melittin inhibits cell proliferation by down-regulating MeCP2 through Shh signaling in SMMC-7721 cells. - Highlights: • MeCP2 plays a key role in the proliferation of human HCC cells. • Melittin reduces MeCP2 expression in vitro. • Melittin induces G 0 /G 1 cell cycle arrest in SMMC-7721 cells. • MeCP2 modulates the Shh signaling pathway in SMMC-7721 cells. • Melittin blocks the Shh signaling pathway in SMMC-7721 cells.

  9. Cyclic mechanical stretch down-regulates cathelicidin antimicrobial peptide expression and activates a pro-inflammatory response in human bronchial epithelial cells

    Directory of Open Access Journals (Sweden)

    Harpa Karadottir

    2015-12-01

    Full Text Available Mechanical ventilation (MV of patients can cause damage to bronchoalveolar epithelium, leading to a sterile inflammatory response, infection and in severe cases sepsis. Limited knowledge is available on the effects of MV on the innate immune defense system in the human lung. In this study, we demonstrate that cyclic stretch of the human bronchial epithelial cell lines VA10 and BCi NS 1.1 leads to down-regulation of cathelicidin antimicrobial peptide (CAMP gene expression. We show that treatment of VA10 cells with vitamin D3 and/or 4-phenyl butyric acid counteracted cyclic stretch mediated down-regulation of CAMP mRNA and protein expression (LL-37. Further, we observed an increase in pro-inflammatory responses in the VA10 cell line subjected to cyclic stretch. The mRNA expression of the genes encoding pro-inflammatory cytokines IL-8 and IL-1β was increased after cyclic stretching, where as a decrease in gene expression of chemokines IP-10 and RANTES was observed. Cyclic stretch enhanced oxidative stress in the VA10 cells. The mRNA expression of toll-like receptor (TLR 3, TLR5 and TLR8 was reduced, while the gene expression of TLR2 was increased in VA10 cells after cyclic stretch. In conclusion, our in vitro results indicate that cyclic stretch may differentially modulate innate immunity by down-regulation of antimicrobial peptide expression and increase in pro-inflammatory responses.

  10. HER2 expression in breast cancer cells is downregulated upon active targeting by antibody-engineered multifunctional nanoparticles in mice.

    Science.gov (United States)

    Corsi, Fabio; Fiandra, Luisa; De Palma, Clara; Colombo, Miriam; Mazzucchelli, Serena; Verderio, Paolo; Allevi, Raffaele; Tosoni, Antonella; Nebuloni, Manuela; Clementi, Emilio; Prosperi, Davide

    2011-08-23

    Subcellular destiny of targeted nanoparticles in cancer cells within living organisms is still an open matter of debate. By in vivo and ex vivo experiments on tumor-bearing mice treated with antibody-engineered magnetofluorescent nanocrystals, in which we combined fluorescence imaging, magnetic relaxation, and trasmission electron microscopy approaches, we provide evidence that nanoparticles are effectively delivered to the tumor by active targeting. These nanocrystals were demonstrated to enable contrast enhancement of the tumor in magnetic resonance imaging. In addition, we were able to discriminate between the fate of the organic corona and the metallic core upon cell internalization. Accurate immunohistochemical analysis confirmed that hybrid nanoparticle endocytosis is mediated by the complex formation with HER2 receptor, leading to a substantial downregulation of HER2 protein expression on the cell surface. These results provide a direct insight into the pathway of internalization and degradation of targeted hybrid nanoparticles in cancer cells in vivo and suggest a potential application of this immunotheranostic nanoagent in neoadjuvant therapy of cancer. © 2011 American Chemical Society

  11. LIN28 expression in malignant germ cell tumors down-regulates let-7 and increases oncogene levels

    Science.gov (United States)

    Murray, Matthew J.; Saini, Harpreet K.; Siegler, Charlotte A.; Hanning, Jennifer E.; Barker, Emily M.; van Dongen, Stijn; Ward, Dawn M.; Raby, Katie L.; Groves, Ian J.; Scarpini, Cinzia G.; Pett, Mark R.; Thornton, Claire M.; Enright, Anton J.; Nicholson, James C.; Coleman, Nicholas

    2013-01-01

    Despite their clinico-pathologic heterogeneity, malignant germ-cell-tumors (GCTs) share molecular abnormalities that are likely to be functionally important. In this study, we investigated the potential significance of down-regulation of the let-7 family of tumor-suppressor microRNAs in malignant-GCTs. Microarray results from pediatric and adult samples (n=45) showed that LIN28, the negative-regulator of let-7 biogenesis, was abundant in malignant-GCTs, regardless of patient age, tumor site or histologic subtype. Indeed, a strong negative-correlation existed between LIN28 and let-7 levels in specimens with matched datasets. Low let-7 levels were biologically significant, since the sequence complementary to the 2-7nt common let-7 seed ‘GAGGUA’ was enriched in the 3′untranslated regions of mRNAs up-regulated in pediatric and adult malignant-GCTs, compared with normal gonads (a mixture of germ cells and somatic cells). We identified 27 mRNA targets of let-7 that were up-regulated in malignant-GCT cells, confirming significant negative-correlations with let-7 levels. Among 16 mRNAs examined in a largely independent set of specimens by qRT-PCR, we defined negative-associations with let-7e levels for six oncogenes, including MYCN, AURKB, CCNF, RRM2, MKI67 and C12orf5 (when including normal control tissues). Importantly, LIN28 depletion in malignant-GCT cells restored let-7 levels and repressed all of these oncogenic let-7 mRNA targets, with LIN28 levels correlating with cell proliferation and MYCN levels. Conversely, ectopic expression of let-7e was sufficient to reduce proliferation and down-regulate MYCN, AURKB and LIN28, the latter via a double-negative feedback loop. We concluded that the LIN28/let-7 pathway has a critical pathobiological role in malignant-GCTs and therefore offers a promising target for therapeutic intervention. PMID:23774216

  12. Andrographolide Ameliorates Abdominal Aortic Aneurysm Progression by Inhibiting Inflammatory Cell Infiltration through Downregulation of Cytokine and Integrin Expression

    Science.gov (United States)

    Ren, Jun; Liu, Zhenjie; Wang, Qiwei; Giles, Jasmine; Greenberg, Jason; Sheibani, Nader; Kent, K. Craig

    2016-01-01

    Abdominal aortic aneurysm (AAA), characterized by exuberant inflammation and tissue deterioration, is a common aortic disease associated with a high mortality rate. There is currently no established pharmacological therapy to treat this progressive disease. Andrographolide (Andro), a major bioactive component of the herbaceous plant Andrographis paniculata, has been found to exhibit potent anti-inflammatory properties by inhibiting nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) activity in several disease models. In this study, we investigated the ability of Andro to suppress inflammation associated with aneurysms, and whether it may be used to block the progression of AAA. Whereas diseased aortae continued to expand in the solvent-treated group, daily administration of Andro to mice with small aneurysms significantly attenuated aneurysm growth, as measured by the diminished expansion of aortic diameter (165.68 ± 15.85% vs. 90.62 ± 22.91%, P < 0.05). Immunohistochemistry analyses revealed that Andro decreased infiltration of monocytes/macrophages and T cells. Mechanistically, Andro inhibited arterial NF-κB activation and reduced the production of proinflammatory cytokines [CCL2, CXCL10, tumor necrosis factor α, and interferon-γ] in the treated aortae. Furthermore, Andro suppressed α4 integrin expression and attenuated the ability of monocytes/macrophages to adhere to activated endothelial cells. These results indicate that Andro suppresses progression of AAA, likely through inhibition of inflammatory cell infiltration via downregulation of NF-κB–mediated cytokine production and α4 integrin expression. Thus, Andro may offer a pharmacological therapy to slow disease progression in patients with small aneurysms. PMID:26483397

  13. Down-regulated βIII-tubulin Expression Can Reverse Paclitaxel Resistance in A549/Taxol Cells Lines

    Directory of Open Access Journals (Sweden)

    Yinling ZHUO

    2014-08-01

    Full Text Available Background and objective Chemotherapy drug resistance is the primary causes of death in patients with pulmonary carcinoma which make tumor recurrence or metastasis. β-tubulin is the main cell targets of anti-microtubule drug. Increased expression of βIII-tubulin has been implicated in non-small cell lung cancer (NSCLC cell lines. To explore the relationship among the expression level of βIII-tubulin and the sensitivity of A549/Taxolcell lines to Taxol and cell cycles and cell apoptosis by RNA interference-mediated inhibition of βIII-tubulin in A549/Taxol cells. Methods Three pairs of siRNA targetd βIII-tubulin were designed and prepared, which were transfected into A549/Taxol cells using LipofectamineTM 2000. We detected the expression of βIII-tubulin mRNA using Real-time fluorescence qRT-PCR. Tedhen we selected the most efficient siRNA by the expression of βIII-tubulin mRNA in transfected group. βIII-tubulin protein level were mesured by Western blot. The taxol sensitivity in transfected group were evaluated by MTT assay. And the cell apoptosis and cell cycles were determined by flow cytometry. Results βIII-tubulin mRNA levels in A549/Taxol cells were significantly decreased in transfected grop by Real-time qRT-PCR than control groups. And βIII-tubulin siRNA-1 sequence showed the highest transfection efficiency, which was (87.73±4.87% (P<0.01; Western blot results showed that the expressional level of BIII tublin protein was significantly down-reulated in the transfectant cells than thant in the control cells. By MTT assay, we showed that the inhibition ratio of Taxol to A549/Taxol cells transfeced was higher than that of control group (51.77±4.60% (P<0.01. The early apoptosis rate of A549/Taxol cells in transfected group were significantly higher than that of control group (P<0.01; G2-M content in taxol group obviously increased than untreated samples by the cell cycle (P<0.05. Conclusion βIII-tubulin down-regulated significantly

  14. Type-I interferon receptor expression: its circadian rhythm and downregulation after interferon-alpha administration in peripheral blood cells from renal cancer patients.

    Science.gov (United States)

    Shiba, Masahiro; Nonomura, Norio; Nakai, Yasutomo; Nakayama, Masashi; Takayama, Hitoshi; Inoue, Hitoshi; Tsujimura, Akira; Nishimura, Kazuo; Okuyama, Akihiko

    2009-04-01

    To investigate the regulation of interferon-alpha (IFN-alpha) receptor expression in metastatic renal cell carcinoma (RCC) after IFN-alpha administration. Blood sampling was carried out in eight patients with metastatic RCC and six healthy volunteers. Flow-cytometric analysis using a monoclonal antibody against the active subunit of the type-I IFN-alpha receptor (IFNAR2) was carried out to examine the circadian rhythm of IFNAR2 expression in peripheral blood mononuclear cells (PBMC) as well as its downregulation after IFN-alpha administration. According to its circadian rhythm IFNAR2 in PBMC had a peak expression at night. Once IFN-alpha is administered, IFNAR2 levels in PBMC showed downregulation within 48 h and recovered within another 48 h. Our findings might support the establishment of an optimal schedule for IFN-alpha administration.

  15. Downregulation of miR-210 expression inhibits proliferation, induces apoptosis and enhances radiosensitivity in hypoxic human hepatoma cells in vitro

    International Nuclear Information System (INIS)

    Yang, Wei; Sun, Ting; Cao, Jianping; Liu, Fenju; Tian, Ye; Zhu, Wei

    2012-01-01

    Hypoxia is a common feature of solid tumors and an important contributor to tumor radioresistance. miR-210 is the most consistently and robustly induced microRNA under hypoxia in different types of tumor cells and normal cells. In the present study, to explore the feasibility of miR-210 as an effective therapeutic target, lentiviral-mediated anti-sense miR-210 gene transfer technique was employed to downregulate miR-210 expression in hypoxic human hepatoma SMMC-7721, HepG2 and HuH7 cells, and phenotypic changes of which were analyzed. Hypoxia led to an increased hypoxia inducible factor-1α (HIF-1α) and miR-210 expression and cell arrest in the G 0 /G 1 phase in all cell lines. miR-210 downregulation significantly suppressed cell viability, induced cell arrest in the G 0 /G 1 phase, increased apoptotic rate and enhanced radiosensitivity in hypoxic human hepatoma cells. Moreover, apoptosis-inducing factor, mitochondrion-associated, 3 (AIFM3) was identified as a direct target gene of miR-210. AIFM3 downregulation by siRNA attenuated radiation induced apoptosis in miR-210 downregulated hypoxic human hepatoma cells. Taken together, these data suggest that miR-210 might be a potential therapeutic target and specific inhibition of miR-210 expression in combination with radiotherapy might be expected to exert strong anti-tumor effect on hypoxic human hepatoma cells. -- Highlights: ► miR-210 downregulation radiosensitized hypoxic hepatoma. ► AIFM3 was identified as a direct target gene of miR-210. ► miR-210 might be a therapeutic target to hypoxic hepatoma.

  16. Downregulation of miR-210 expression inhibits proliferation, induces apoptosis and enhances radiosensitivity in hypoxic human hepatoma cells in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Yang, Wei, E-mail: detachedy@yahoo.com.cn [Department of Radiobiology, School of Radiological Medicine and Protection, Soochow University, Suzhou (China); Sun, Ting [Brain and Nerve Research Laboratory, The First Affiliated Hospital, Soochow University, Suzhou (China); Cao, Jianping; Liu, Fenju [Department of Radiobiology, School of Radiological Medicine and Protection, Soochow University, Suzhou (China); Tian, Ye [Department of Radiotherapy and Oncology, The Second Affiliated Hospital, Soochow University, Suzhou (China); Zhu, Wei [Department of Radiobiology, School of Radiological Medicine and Protection, Soochow University, Suzhou (China)

    2012-05-01

    Hypoxia is a common feature of solid tumors and an important contributor to tumor radioresistance. miR-210 is the most consistently and robustly induced microRNA under hypoxia in different types of tumor cells and normal cells. In the present study, to explore the feasibility of miR-210 as an effective therapeutic target, lentiviral-mediated anti-sense miR-210 gene transfer technique was employed to downregulate miR-210 expression in hypoxic human hepatoma SMMC-7721, HepG2 and HuH7 cells, and phenotypic changes of which were analyzed. Hypoxia led to an increased hypoxia inducible factor-1{alpha} (HIF-1{alpha}) and miR-210 expression and cell arrest in the G{sub 0}/G{sub 1} phase in all cell lines. miR-210 downregulation significantly suppressed cell viability, induced cell arrest in the G{sub 0}/G{sub 1} phase, increased apoptotic rate and enhanced radiosensitivity in hypoxic human hepatoma cells. Moreover, apoptosis-inducing factor, mitochondrion-associated, 3 (AIFM3) was identified as a direct target gene of miR-210. AIFM3 downregulation by siRNA attenuated radiation induced apoptosis in miR-210 downregulated hypoxic human hepatoma cells. Taken together, these data suggest that miR-210 might be a potential therapeutic target and specific inhibition of miR-210 expression in combination with radiotherapy might be expected to exert strong anti-tumor effect on hypoxic human hepatoma cells. -- Highlights: Black-Right-Pointing-Pointer miR-210 downregulation radiosensitized hypoxic hepatoma. Black-Right-Pointing-Pointer AIFM3 was identified as a direct target gene of miR-210. Black-Right-Pointing-Pointer miR-210 might be a therapeutic target to hypoxic hepatoma.

  17. The Downregulation of the Expression of CD147 by Tumor Suppressor REIC/Dkk-3, and Its Implication in Human Prostate Cancer Cell Growth Inhibition.

    Science.gov (United States)

    Mori, Akihiro; Watanabe, Masami; Sadahira, Takuya; Kobayashi, Yasuyuki; Ariyoshi, Yuichi; Ueki, Hideo; Wada, Koichiro; Ochiai, Kazuhiko; Li, Shun-Ai; Nasu, Yasutomo

    2017-04-01

    The cluster of differentiation 147 (CD147), also known as EMMPRIN, is a key molecule that promotes cancer progression. We previously developed an adenoviral vector encoding a tumor suppressor REIC/Dkk-3 gene (Ad-REIC) for cancer gene therapy. The therapeutic effects are based on suppressing the growth of cancer cells, but, the underlying molecular mechanism has not been fully clarified. To elucidate this mechanism, we investigated the effects of Ad-REIC on the expression of CD147 in LNCaP prostate cancer cells. Western blotting revealed that the expression of CD147 was significantly suppressed by Ad-REIC. Ad-REIC also suppressed the cell growth of LNCaP cells. Since other researchers have demonstrated that phosphorylated mitogen-activated protein kinases (MAPKs) and c-Myc protein positively regulate the expression of CD147, we investigated the correlation between the CD147 level and the activation of MAPK and c-Myc expression. Unexpectedly, no positive correlation was observed between CD147 and its possible regulators, suggesting that another signaling pathway was involved in the downregulation of CD147. This is the first study to show the downregulation of CD147 by Ad-REIC in prostate cancer cells. At least some of the therapeutic effects of Ad-REIC may be due to the downregulation of the cancer-progression factor, CD147.

  18. p75 Neurotrophin Receptor in the Skin: Beyond Its Neurotrophic Function.

    Science.gov (United States)

    Pincelli, Carlo

    2017-01-01

    p75 neurotrophin receptor (p75 NTR ), also known as CD271, is the low-affinity receptor that, together with the tyrosine kinase receptor tropomyosin-receptor kinase (Trk), mediate neurotrophin (NT) functions. Beside their classic role in skin innervation, NT and their receptors constitute a complex cutaneous network associated with a number of autocrine and paracrine activities. In this context, the role of p75 NTR is becoming more and more important. This review will focus on the intriguing functions of p75 NTR in healthy and diseased skin. First, p75 NTR counterbalances the proliferative and survival activities of its cognate receptor Trk by inducing keratinocyte apoptosis. In addition, p75 NTR identifies an early transit-amplifying (TA) keratinocyte population and plays a critical role in keratinocyte stem cell transition to its progeny as well as in epidermal differentiation. p75 NTR is absent in psoriatic TA cells, thus rendering these cells resistant to apoptosis. On the other hand, p75 NTR infection restores NT-induced apoptosis in psoriatic keratinocytes. Taken together, these results provide evidence for a critical role of p75 NTR in epidermal homeostasis, while its lack may account for the TA defect in psoriasis. While the issue of p75 NTR as a marker of melanoma initiating cells is still to be solved, there is strong evidence that downregulation of this receptor is a precondition to melanoma invasion and metastasis in vitro and in vivo . All in all, this review points to p75 NTR as a major actor in both physiologic and pathologic conditions at the skin level.

  19. TGF-β1 downregulates StAR expression and decreases progesterone production through Smad3 and ERK1/2 signaling pathways in human granulosa cells.

    Science.gov (United States)

    Fang, Lanlan; Chang, Hsun-Ming; Cheng, Jung-Chien; Leung, Peter C K; Sun, Ying-Pu

    2014-11-01

    Regulation of progesterone production in granulosa cells is important for normal reproductive functions. Steroidogenic acute regulatory protein (StAR) is recognized as the key regulatory protein involved in the rate-limiting step of steroidogenesis. TGF-β1 protein is detected in human follicular fluid, and TGF-β1 and its receptors are expressed in human granulosa cells. However, the functional role of TGF-β1 in the regulation of StAR expression and progesterone production in human granulosa cells remains unknown. Our objective was to investigate the effects of TGF-β1 on StAR expression and progesterone production in human granulosa cells. SVOG cells are human granulosa cells that were obtained from women undergoing in vitro fertilization and immortalized with SV40 large T antigen. SVOG cells were used to investigate the effects of TGF-β1 on StAR expression and progesterone production at an academic research center. Levels of mRNA and protein were examined by RT-qPCR and western blotting, respectively. The accumulation levels of progesterone were measured by enzyme-linked immunosorbent assay (ELISA). TGF-β1 treatment downregulated StAR expression and decreased progesterone production. The suppressive effects of TGF-β1 on StAR expression and progesterone production were abolished by the inhibition of TGF-β type I receptor. In addition, treatment with TGF-β1 activated the Smad2/3 and ERK1/2 signaling pathways. The inhibition of the Smad3 and ERK1/2 signaling pathways attenuated the TGF-β1-induced downregulation of StAR expression and progesterone production. TGF-β1 downregulated StAR expression and decreased progesterone production by activating the Smad3 and ERK1/2 signaling pathways in human granulosa cells.

  20. Dual inhibition of STAT1 and STAT3 activation downregulates expression of PD-L1 in human breast cancer cells.

    Science.gov (United States)

    Sasidharan Nair, Varun; Toor, Salman M; Ali, Bassam R; Elkord, Eyad

    2018-05-02

    Breast cancer is the most commonly diagnosed cancer, and it is a leading cause of cancer-related deaths in females worldwide. Triple-negative breast cancer (TNBC) constitutes 15% of breast cancer and shows distinct metastasis profiles with poor prognosis. Strong PD-L1 expression has been observed in some tumors, supporting their escape from immune surveillance. Targeting PD-L1 could be a promising therapeutic approach in breast cancer patients. We investigated potential molecular mechanisms for constitutive expression of PD-L1 by inhibiting upstream STAT1 and STAT3 signals. PD-L1 expression in three breast cancer cell lines was measured using quantitative PCR and western blotting. Activation of STAT1 and STAT3 was blocked using pharmacological inhibitors and siRNA. The mechanism underlying the constitutive expression of PD-L1 was investigated using ChIP and co-immunoprecipitation assays. We found that individual inhibition of STAT1 and STAT3 activation partially downregulated PD-L1, while combined inhibition completely downregulated PD-L1 expression. Moreover, our results suggest that pSTAT1-pSTAT3 dimerize in cytosol and translocate to the nucleus, where they bind to PD-L1 promoter and induce PD-L1 expression. These findings provide a rationale for combined targeting of STAT1 and STAT3 for the development of immune-based cancer therapies for down regulation of PD-L1 expression.

  1. Francisella tularensis elicits IL-10 via a PGE₂-inducible factor, to drive macrophage MARCH1 expression and class II down-regulation.

    Directory of Open Access Journals (Sweden)

    Danielle Hunt

    Full Text Available Francisella tularensis is a bacterial pathogen that uses host-derived PGE₂ to subvert the host's adaptive immune responses in multiple ways. Francisella-induced PGE₂ acts directly on CD4 T cells to blunt production of IFN-γ. Francisella-induced PGE₂ can also elicit production of a >10 kDa soluble host factor termed FTMØSN (F. tularensismacrophage supernatant, which acts on IFN-γ pre-activated MØ to down-regulate MHC class II expression via a ubiquitin-dependent mechanism, blocking antigen presentation to CD4 T cells. Here, we report that FTMØSN-induced down-regulation of MØ class II is the result of the induction of MARCH1, and that MØ expressing MARCH1 "resistant" class II molecules are resistant to FTMØSN-induced class II down-regulation. Since PGE₂ can induce IL-10 production and IL-10 is the only reported cytokine able to induce MARCH1 expression in monocytes and dendritic cells, these findings suggested that IL-10 is the active factor in FTMØSN. However, use of IL-10 knockout MØ established that IL-10 is not the active factor in FTMØSN, but rather that Francisella-elicited PGE₂ drives production of a >10 kDa host factor distinct from IL-10. This factor then drives MØ IL-10 production to induce MARCH1 expression and the resultant class II down-regulation. Since many human pathogens such as Salmonella typhi, Mycobacterium tuberculosis and Legionella pneumophila also induce production of host PGE₂, these results suggest that a yet-to-be-identified PGE₂-inducible host factor capable of inducing IL-10 is central to the immune evasion mechanisms of multiple important human pathogens.

  2. BMP6 down-regulates GDNF expression through SMAD1/5 and ERK1/2 signaling pathways in human granulosa-lutein cells.

    Science.gov (United States)

    Zhang, Xin-Yue; Chang, Hsun-Ming; Taylor, Elizabeth L; Leung, Peter C K; Liu, Rui-Zhi

    2018-05-09

    Bone morphogenetic protein 6 (BMP6) is a critical regulator of follicular development that is expressed in mammalian oocytes and granulosa cells. Glial cell line-derived neurotrophic factor (GDNF) is an intraovarian neurotrophic factor that plays an essential role in regulating mammalian oocyte maturation. The aim of this study was to investigate the effect of BMP6 on the regulation of GDNF expression and the potential underlying mechanisms. We used an established immortalized human granulosa cell line (SVOG cells) and primary human granulosa-lutein cells as in vitro cell models. Our results showed that BMP6 significantly down-regulated the expression of GDNF in both SVOG and primary human granulosa-lutein cells. Using dual inhibition approaches (kinase receptor inhibitor and small interfering RNA knockdown), our results showed that both ALK2 and ALK3 are involved in BMP6-induced down-regulation of GDNF. In addition, BMP6 induced the phosphorylation of SMAD1/5/8 and ERK1/2 but not AKT or p38. Among three downstream mediators, both SMAD1 and SMAD5 are involved in BMP6-induced down-regulation of GDNF. Moreover, concomitant knockdown of endogenous SMAD4 and inhibition of ERK1/2 activity completely reversed BMP6-induced down-regulation of GDNF, indicating that both SMAD and ERK1/2 signaling pathways are required for the regulatory effect of BMP6 on GDNF expression. Our findings suggest an additional role for an intrafollicular growth factor in regulating follicular function through their paracrine interactions in human granulosa cells.

  3. A Rhodium(III) Complex as an Inhibitor of Neural Precursor Cell Expressed, Developmentally Down-Regulated 8-Activating Enzyme with in Vivo Activity against Inflammatory Bowel Disease.

    Science.gov (United States)

    Zhong, Hai-Jing; Wang, Wanhe; Kang, Tian-Shu; Yan, Hui; Yang, Yali; Xu, Lipeng; Wang, Yuqiang; Ma, Dik-Lung; Leung, Chung-Hang

    2017-01-12

    We report herein the identification of the rhodium(III) complex [Rh(phq) 2 (MOPIP)] + (1) as a potent and selective ATP-competitive neural precursor cell expressed, developmentally down-regulated 8 (NEDD8)-activating enzyme (NAE) inhibitor. Structure-activity relationship analysis indicated that the overall organometallic design of complex 1 was important for anti-inflammatory activity. Complex 1 showed promising anti-inflammatory activity in vivo for the potential treatment of inflammatory bowel disease.

  4. CCL5 promotes vascular endothelial growth factor expression and induces angiogenesis by down-regulating miR-199a in human chondrosarcoma cells.

    Science.gov (United States)

    Liu, Guan-Ting; Huang, Yuan-Li; Tzeng, Huey-En; Tsai, Chun-Hao; Wang, Shih-Wei; Tang, Chih-Hsin

    2015-02-28

    Chondrosarcoma is a primary malignant bone cancer, with a potent capacity to invade locally and cause distant metastasis. Angiogenesis is a critical step in tumor growth and metastasis. Chemokine CCL5 (previously called RANTES) has been shown to facilitate tumor progression and metastasis. However, the relationship of CCL5 with vascular endothelial growth factor (VEGF) expression and angiogenesis in human chondrosarcoma is mostly unknown. In this study, CCL5 increased VEGF expression and also promoted chondrosarcoma medium-mediated angiogenesis in vitro as well as angiogenesis effects in the chick chorioallantoic membrane and Matrigel plug nude mice model in vivo. MicroRNA analysis was performed in CCL5-treated chondrosarcoma cells versus control cells to investigate the mechanism of CCL5-mediated promotion of chondrosarcoma angiogenesis. Among the miRNAs regulated by CCL5, miR-199a was the most downregulated miRNA after CCL5 treatment. In addition, co-transfection with miR-199a mimic reversed the CCL5-mediated VEGF expression and angiogenesis in vitro and in vivo. Moreover, overexpression of CCL5 increased tumor-associated angiogenesis and tumor growth by downregulating miR-199a in the xenograft tumor angiogenesis model. Taken together, these results demonstrated that CCL5 promotes VEGF-dependent angiogenesis in human chondrosarcoma cells by downregulating miR-199a. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  5. SKP2 siRNA inhibits the degradation of P27kip1 and down-regulates the expression of MRP in HL-60/A cells.

    Science.gov (United States)

    Xiao, Jie; Yin, Songmei; Li, Yiqing; Xie, Shuangfeng; Nie, Danian; Ma, Liping; Wang, Xiuju; Wu, Yudan; Feng, Jianhong

    2009-08-01

    S-phase kinase-associated protein 2 (SKP2) gene is a tumor suppressor gene, and is involved in the ubiquitin-mediated degradation of P27kip1. SKP2 and P27kip1 affect the proceeding and prognosis of leukemia through regulating the proliferation, apoptosis and differentiation of leukemia cells. In this study, we explored the mechanism of reversing of HL-60/A drug resistance through SKP2 down-regulation. HL-60/A cells were nucleofected by Amaxa Nucleofector System with SKP2 siRNA. The gene and protein expression levels of Skp2, P27kip1, and multi-drug resistance associated protein (MRP) were determined by reverse transcription-polymerase chain reaction and western blot analysis, respectively. The cell cycle was analyzed by flow cytometry. The 50% inhibitory concentration value was calculated using cytotoxic analysis according to the death rate of these two kinds of cells under different concentrations of chemotherapeutics to compare the sensitivity of the cells. HL-60/A cells showed multi-drug resistance phenotype characteristic by cross-resistance to adriamycin, daunorubicin, and arabinosylcytosine, due to the expression of MRP. We found that the expression of SKP2 was higher in HL-60/A cells than in HL-60 cells, but the expression of P27kip1 was lower. The expression of SKP2 in HL-60/A cells nucleofected by SKP2 siRNA was down-regulated whereas the protein level of P27kip1 was up-regulated. Compared with the MRP expression level in the control group (nucleofected by control siRNA), the mRNA and protein expression levels of MRP in HL-60/A cells nucleofected by SKP2 siRNA were lower, and the latter cells were more sensitive to adriamycin, daunorubicin, and arabinosylcytosine. Down-regulating the SKP2 expression and arresting cells in the G0/G1 phase improve drug sensitivity of leukemia cells with down-regulated MRP expression.

  6. DHA down-regulates phenobarbital-induced cytochrome P450 2B1 gene expression in rat primary hepatocytes by attenuating CAR translocation

    International Nuclear Information System (INIS)

    Li, C.-C.; Lii, C.-K.; Liu, K.-L.; Yang, J.-J.; Chen, H.-W.

    2007-01-01

    The constitutive androstane receptor (CAR) plays an important role in regulating the expression of detoxifying enzymes, including cytochrome P450 2B (CYP 2B). Phenobarbital (PB) induction of human CYP 2B6 and mouse CYP 2b10 has been shown to be mediated by CAR. Our previous study showed that PB-induced CYP 2B1 expression in rat primary hepatocytes is down-regulated by both n-6 and n-3 polyunsaturated fatty acids (PUFAs), especially docosahexaenoic acid (DHA); however, the mechanism for this down-regulation by DHA was previously unknown. The objective of the present study was to determine whether change in CAR translocation is involved in the down-regulation by n-6 and n-3 PUFAs of PB-induced CYP 2B1 expression in rat primary hepatocytes. We used 100 μM arachidonic acid, linoleic acid, eicosapentaenoic acid, and DHA to test this hypothesis. PB triggered the translocation of CAR from the cytosol into the nucleus in a dose-dependent and time-dependent manner in our hepatocyte system, and the CAR distribution in rat primary hepatocytes was significantly affected by DHA. DHA treatment decreased PB-inducible accumulation of CAR in the nuclear fraction and increased it in the cytosolic fraction in a dose-dependent manner. The down-regulation of CYP 2B1 expression by DHA occurred in a dose-dependent manner, and a similar pattern was found for the nuclear accumulation of CAR. The results of immunoprecipitation showed a CAR/RXR heterodimer bound to nuclear receptor binding site 1 (NR-1) of the PB-responsive enhancer module (PBREM) of the CYP 2B1gene. The EMSA results showed that PB-induced CAR binding to NR-1 was attenuated by DHA. Taken together, these results suggest that attenuation of CAR translocation and decreased subsequent binding to NR-1 are involved in DHA's down-regulation of PB-induced CYP 2B1 expression

  7. Overall major histocompatibility complex class I expression is not downregulated in cervix cancer, as detected by immunoelectron microscopy

    NARCIS (Netherlands)

    van Eijkeren, MA; Roovers, JP; Oorschot, [No Value; Geuze, HJ

    2004-01-01

    Downregulation of major histocompatibility complex (MHC) class I molecules in cervix cancer has been proposed as a mechanism for cancer cells to escape immunodetection. By means of light microscopic immunohistochemistry, it has been shown that in 20-70% of cervix cancers MHC class I is

  8. Antisense down-regulation of 4CL expression alters lignification, tree growth, and saccharification potential of field-grown poplar

    Science.gov (United States)

    Steven L. Voelker; Barbara Lachenbruch; Frederick C. Meinzer; Michael Jourdes; Chanyoung Ki; Ann M. Patten; Laurence B. Davin; Norman G. Lewis; Gerald A. Tuskan; Lee Gunter; Stephen R. Decker; Michael J. Selig; Robert Sykes; Michael E. Himmel; Peter Kitin; Olga Shevchenko; Steven H. Strauss

    2010-01-01

    Transgenic down-regulation of the Pt4CL1 gene family encoding 4-coumarate:coenzyme A ligase (4CL) has been reported as a means for reducing lignin content in cell walls and increasing overall growth rates, thereby improving feedstock quality for paper and bioethanol production. Using hybrid poplar (Populus tremula...

  9. Salinomycin enhances cisplatin-induced cytotoxicity in human lung cancer cells via down-regulation of AKT-dependent thymidylate synthase expression.

    Science.gov (United States)

    Ko, Jen-Chung; Zheng, Hao-Yu; Chen, Wen-Ching; Peng, Yi-Shuan; Wu, Chia-Hung; Wei, Chia-Li; Chen, Jyh-Cheng; Lin, Yun-Wei

    2016-12-15

    Salinomycin, a polyether antibiotic, acts as a highly selective potassium ionophore and has anticancer activity on various cancer cell lines. Cisplatin has been proved as chemotherapy drug for advanced human non-small cell lung cancer (NSCLC). Thymidylate synthase (TS) is a key enzyme in the pyrimidine salvage pathway, and increased expression of TS is thought to be associated with resistance to cisplatin. In this study, we showed that salinomycin (0.5-2μg/mL) treatment down-regulating of TS expression in an AKT inactivation manner in two NSCLC cell lines, human lung adenocarcinoma A549 and squamous cell carcinoma H1703 cells. Knockdown of TS using small interfering RNA (siRNA) or inhibiting AKT activity with PI3K inhibitor LY294002 enhanced the cytotoxicity and cell growth inhibition of salinomycin. A combination of cisplatin and salinomycin resulted in synergistic enhancement of cytotoxicity and cell growth inhibition in NSCLC cells, accompanied with reduced activation of phospho-AKT, and TS expression. Overexpression of a constitutive active AKT (AKT-CA) expression vector reversed the salinomycin and cisplatin-induced synergistic cytotoxicity. In contrast, pretreatment with LY294002 further decreased the cell viability in salinomycin and cisplatin cotreated cells. Our findings suggested that the down-regulation of AKT-mediated TS expression by salinomycin enhanced the cisplatin-induced cytotoxicity in NSCLC cells. These results may provide a rationale to combine salinomycin with cisplatin for lung cancer treatment. Copyright © 2016 Elsevier Inc. All rights reserved.

  10. Steroidogenesis and early response gene expression in MA-10 Leydig tumor cells following heterologous receptor down-regulation and cellular desensitization

    Directory of Open Access Journals (Sweden)

    Tsuey-Ming Chen

    2016-03-01

    Full Text Available The Leydig tumor cell line, MA-10, expresses the luteinizing hormone receptor, a G protein-coupled receptor that, when activated with luteinizing hormone or chorionic gonadotropin (CG, stimulates cAMP production and subsequent steroidogenesis, notably progesterone. These cells also respond to epidermal growth factor (EGF and phorbol esters with increased steroid biosynthesis. In order to probe the intracellular pathways along with heterologous receptor down-regulation and cellular desensitization, cells were preincubated with EGF or phorbol esters and then challenged with CG, EGF, dibutryl-cyclic AMP, and a phorbol ester. Relative receptor numbers, steroid biosynthesis, and expression of the early response genes, JUNB and c-FOS, were measured. It was found that in all cases but one receptor down-regulation and decreased progesterone production were closely coupled under the conditions used; the exception involved preincubation of the cells with EGF followed by addition of CG where the CG-mediated stimulation of steroidogenesis was considerably lower than the level of receptor down-regulation. In a number of instances JUNB and c-FOS expression paralleled the decreases in receptor number and progesterone production, while in some cases these early response genes were affected little if at all by the changes in receptor number. This finding may indicate that even low levels of activated signaling kinases, e.g. protein kinase A, protein kinase C, or receptor tyrosine kinase, may suffice to yield good expression of JUNB and c-FOS, or it may suggest alternative pathways for regulating expression of these two early response genes.

  11. Thrombospondin-2 promotes prostate cancer bone metastasis by the up-regulation of matrix metalloproteinase-2 through down-regulating miR-376c expression

    Directory of Open Access Journals (Sweden)

    Po-Chun Chen

    2017-01-01

    Full Text Available Abstract Background Thrombospondin-2 (TSP-2 is a secreted matricellular glycoprotein that is found to mediate cell-to-extracellular matrix attachment and participates in many physiological and pathological processes. The expression profile of TSP-2 on tumors is controversial, and it up-regulates in some cancers, whereas it down-regulates in others, suggesting that the functional role of TSP-2 on tumors is still uncertain. Methods The expression of TSP-2 on prostate cancer progression was determined in the tissue array by the immunohistochemistry. The molecular mechanism of TSP-2 on prostate cancer (PCa metastasis was investigated through pharmaceutical inhibitors, siRNAs, and miRNAs analyses. The role of TSP-2 on PCa metastasis in vivo was verified through xenograft in vivo imaging system. Results Based on the gene expression omnibus database and immunohistochemistry, we found that TSP-2 increased with the progression of PCa, especially in metastatic PCa and is correlated with the matrix metalloproteinase-2 (MMP-2 expression. Additionally, through binding to CD36 and integrin ανβ3, TSP-2 increased cell migration and MMP-2 expression. With inhibition of p38, ERK, and JNK, the TSP-2-induced cell migration and MMP-2 expression were abolished, indicating that the TSP-2’s effect on PCa is MAPK dependent. Moreover, the microRNA-376c (miR-376c was significantly decreased by the TSP-2 treatment. Furthermore, the TSP-2-induced MMP-2 expression and the subsequent cell motility were suppressed upon miR-376c mimic stimulation. On the other hand, the animal studies revealed that the bone metastasis was abolished when TSP-2 was stably knocked down in PCa cells. Conclusions Taken together, our results indicate that TSP-2 enhances the migration of PCa cells by increasing MMP-2 expression through down-regulation of miR-376c expression. Therefore, TSP-2 may represent a promising new target for treating PCa.

  12. Frequent downregulation of BTB and CNC homology 2 expression in Epstein-Barr virus-positive diffuse large B-cell lymphoma.

    Science.gov (United States)

    Noujima-Harada, Mai; Takata, Katsuyoshi; Miyata-Takata, Tomoko; Sakurai, Hiroaki; Igarashi, Kazuhiko; Ito, Etsuro; Nagakita, Keina; Taniguchi, Kohei; Ohnishi, Nobuhiko; Omote, Shizuma; Tabata, Tetsuya; Sato, Yasuharu; Yoshino, Tadashi

    2017-05-01

    Diffuse large B-cell lymphoma (DLBCL) is the most common B-cell lymphoma subtype, and the Epstein-Barr virus (EBV)-positive subtype of DLBCL is known to show a more aggressive clinical behavior than the EBV-negative one. BTB and CNC homology 2 (BACH2) has been highlighted as a tumor suppressor in hematopoietic malignancies; however, the role of BACH2 in EBV-positive DLBCL is unclear. In the present study, BACH2 expression and its significance were studied in 23 EBV-positive and 43 EBV-negative patient samples. Immunohistochemistry revealed BACH2 downregulation in EBV-positive cases (P < 0.0001), although biallelic deletion of BACH2 was not detected by FISH. Next, we analyzed the contribution of BACH2 negativity to aggressiveness in EBV-positive B-cell lymphomas using FL-18 (EBV-negative) and FL-18-EB cells (FL-18 sister cell line, EBV-positive). In BACH2-transfected FL-18-EB cells, downregulation of phosphorylated transforming growth factor-β-activated kinase 1 (pTAK1) and suppression in p65 nuclear fractions were observed by Western blot analysis contrary to non-transfected FL-18-EB cells. In patient samples, pTAK1 expression and significant nuclear p65, p50, and p52 localization were detected immunohistochemically in BACH2-negative DLBCL (P < 0.0001, P = 0.006, and P = 0.001, respectively), suggesting that BACH2 downregulation contributes to constitutive activation of the nuclear factor-κB pathway through TAK1 phosphorylation in BACH2-negative DLBCL (most EBV-positive cases). Although further molecular and pathological studies are warranted to clarify the detailed mechanisms, downregulation of BACH2 may contribute to constitutive activation of the nuclear factor-κB pathway through TAK1 activation. © 2017 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.

  13. Restoration of type 1 iodothyronine deiodinase expression in renal cancer cells downregulates oncoproteins and affects key metabolic pathways as well as anti-oxidative system.

    Science.gov (United States)

    Popławski, Piotr; Wiśniewski, Jacek R; Rijntjes, Eddy; Richards, Keith; Rybicka, Beata; Köhrle, Josef; Piekiełko-Witkowska, Agnieszka

    2017-01-01

    Type 1 iodothyronine deiodinase (DIO1) contributes to deiodination of 3,5,3',5'-tetraiodo-L-thyronine (thyroxine, T4) yielding of 3,5,3'-triiodothyronine (T3), a powerful regulator of cell differentiation, proliferation, and metabolism. Our previous work showed that loss of DIO1 enhances proliferation and migration of renal cancer cells. However, the global effects of DIO1 expression in various tissues affected by cancer remain unknown. Here, the effects of stable DIO1 re-expression were analyzed on the proteome of renal cancer cells, followed by quantitative real-time PCR validation in two renal cancer-derived cell lines. DIO1-induced changes in intracellular concentrations of thyroid hormones were quantified by L-MS/MS and correlations between expression of DIO1 and potential target genes were determined in tissue samples from renal cancer patients. Stable re-expression of DIO1, resulted in 26 downregulated proteins while 59 proteins were overexpressed in renal cancer cells. The 'downregulated' group consisted mainly of oncoproteins (e.g. STAT3, ANPEP, TGFBI, TGM2) that promote proliferation, migration and invasion. Furthermore, DIO1 re-expression enhanced concentrations of two subunits of thyroid hormone transporter (SLC7A5, SLC3A2), enzymes of key pathways of cellular energy metabolism (e.g. TKT, NAMPT, IDH2), sex steroid metabolism and anti-oxidative response (AKR1C2, AKR1B10). DIO1 expression resulted in elevated intracellular concentration of T4. Expression of DIO1-affected genes strongly correlated with DIO1 transcript levels in tissue samples from renal cancer patients as well as with their poor survival. This first study addressing effects of deiodinase re-expression on proteome of cancer cells demonstrates that induced DIO1 re-expression in renal cancer robustly downregulates oncoproteins, affects key metabolic pathways, and triggers proteins involved in anti-oxidative protection. This data supports the notion that suppressed DIO1 expression and changes

  14. Restoration of type 1 iodothyronine deiodinase expression in renal cancer cells downregulates oncoproteins and affects key metabolic pathways as well as anti-oxidative system.

    Directory of Open Access Journals (Sweden)

    Piotr Popławski

    Full Text Available Type 1 iodothyronine deiodinase (DIO1 contributes to deiodination of 3,5,3',5'-tetraiodo-L-thyronine (thyroxine, T4 yielding of 3,5,3'-triiodothyronine (T3, a powerful regulator of cell differentiation, proliferation, and metabolism. Our previous work showed that loss of DIO1 enhances proliferation and migration of renal cancer cells. However, the global effects of DIO1 expression in various tissues affected by cancer remain unknown. Here, the effects of stable DIO1 re-expression were analyzed on the proteome of renal cancer cells, followed by quantitative real-time PCR validation in two renal cancer-derived cell lines. DIO1-induced changes in intracellular concentrations of thyroid hormones were quantified by L-MS/MS and correlations between expression of DIO1 and potential target genes were determined in tissue samples from renal cancer patients. Stable re-expression of DIO1, resulted in 26 downregulated proteins while 59 proteins were overexpressed in renal cancer cells. The 'downregulated' group consisted mainly of oncoproteins (e.g. STAT3, ANPEP, TGFBI, TGM2 that promote proliferation, migration and invasion. Furthermore, DIO1 re-expression enhanced concentrations of two subunits of thyroid hormone transporter (SLC7A5, SLC3A2, enzymes of key pathways of cellular energy metabolism (e.g. TKT, NAMPT, IDH2, sex steroid metabolism and anti-oxidative response (AKR1C2, AKR1B10. DIO1 expression resulted in elevated intracellular concentration of T4. Expression of DIO1-affected genes strongly correlated with DIO1 transcript levels in tissue samples from renal cancer patients as well as with their poor survival. This first study addressing effects of deiodinase re-expression on proteome of cancer cells demonstrates that induced DIO1 re-expression in renal cancer robustly downregulates oncoproteins, affects key metabolic pathways, and triggers proteins involved in anti-oxidative protection. This data supports the notion that suppressed DIO1 expression

  15. PCI-24781 down-regulates EZH2 expression and then promotes glioma apoptosis by suppressing the PIK3K/Akt/mTOR pathway.

    Science.gov (United States)

    Zhang, Wei; Lv, Shengqing; Liu, Jun; Zang, Zhenle; Yin, Junyi; An, Ning; Yang, Hui; Song, Yechun

    2014-10-01

    PCI-24781 is a novel histone deacetylase inhibitor that inhibits tumor proliferation and promotes cell apoptosis. However, it is unclear whether PCI-24781 inhibits Enhancer of Zeste 2 (EZH2) expression in malignant gliomas. In this work, three glioma cell lines were incubated with various concentrations of PCI-24781 (0, 0.25, 0.5, 1, 2.5 and 5 μM) and analyzed for cell proliferation by the MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] assay and colony formation, and cell cycle and apoptosis were assessed by flow cytometry. The expression of EZH2 and apoptosis-related proteins was assessed by western blotting. Malignant glioma cells were also transfected with EZH2 siRNA to examine how PCI-24781 suppresses tumor cells. EZH2 was highly expressed in the three glioma cell lines. Incubation with PCI-24781 reduced cell proliferation and increased cell apoptosis by down-regulating EZH2 in a concentration-dependent manner. These effects were simulated by EZH2 siRNA. In addition, PCI-24781 or EZH2 siRNA accelerated cell apoptosis by down-regulating the expression of AKT, mTOR, p70 ribosomal protein S6 kinase (p70s6k), glycogen synthase kinase 3A and B (GSK3a/b) and eukaryotic initiation factor 4E binding protein 1 (4E-BP1). These data suggest that PCI-24781 may be a promising therapeutic agent for treating gliomas by down-regulating EZH2 which promotes cell apoptosis by suppressing the phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of the rapamycin (mTOR) pathway.

  16. Static compression down-regulates N-cadherin expression and facilitates loss of cell phenotype of nucleus pulposus cells in a disc perfusion culture.

    Science.gov (United States)

    Zhou, Haibo; Shi, Jianmin; Zhang, Chao; Li, Pei

    2018-02-28

    Mechanical compression often induces degenerative changes of disc nucleus pulposus (NP) tissue. It has been indicated that N-cadherin (N-CDH)-mediated signaling helps to preserve the NP cell phenotype. However, N-CDH expression and the resulting NP-specific phenotype alteration under the static compression and dynamic compression remain unclear. To study the effects of static compression and dynamic compression on N-CDH expression and NP-specific phenotype in an in vitro disc organ culture. Porcine discs were organ cultured in a self-developed mechanically active bioreactor for 7 days and subjected to static or dynamic compression (0.4 MPa for 2 h once per day). The noncompressed discs were used as controls. Compared with the dynamic compression, static compression significantly down-regulated the expression of N-CDH and NP-specific markers (laminin, brachyury, and keratin 19); decreased the Alcian Blue staining intensity, glycosaminoglycan and hydroxyproline contents; and declined the matrix macromolecule (aggrecan and collagen II) expression. Compared with the dynamic compression, static compression causes N-CDH down-regulation, loss of NP-specific phenotype, and the resulting decrease in NP matrix synthesis. © 2018 The Author(s).

  17. Benfotiamine increases glucose oxidation and downregulates NADPH oxidase 4 expression in cultured human myotubes exposed to both normal and high glucose concentrations.

    Science.gov (United States)

    Fraser, D A; Hessvik, N P; Nikolić, N; Aas, V; Hanssen, K F; Bøhn, S K; Thoresen, G H; Rustan, A C

    2012-07-01

    The aim of the present work was to study the effects of benfotiamine (S-benzoylthiamine O-monophosphate) on glucose and lipid metabolism and gene expression in differentiated human skeletal muscle cells (myotubes) incubated for 4 days under normal (5.5 mM glucose) and hyperglycemic (20 mM glucose) conditions. Myotubes established from lean, healthy volunteers were treated with benfotiamine for 4 days. Glucose and lipid metabolism were studied with labeled precursors. Gene expression was measured using real-time polymerase chain reaction (qPCR) and microarray technology. Benfotiamine significantly increased glucose oxidation under normoglycemic (35 and 49% increase at 100 and 200 μM benfotiamine, respectively) as well as hyperglycemic conditions (70% increase at 200 μM benfotiamine). Benfotiamine also increased glucose uptake. In comparison, thiamine (200 μM) increased overall glucose metabolism but did not change glucose oxidation. In contrast to glucose, mitochondrial lipid oxidation and overall lipid metabolism were unchanged by benfotiamine. The expression of NADPH oxidase 4 (NOX4) was significantly downregulated by benfotiamine treatment under both normo- and hyperglycemic conditions. Gene set enrichment analysis (GSEA) showed that befotiamine increased peroxisomal lipid oxidation and organelle (mitochondrial) membrane function. In conclusion, benfotiamine increases mitochondrial glucose oxidation in myotubes and downregulates NOX4 expression. These findings may be of relevance to type 2 diabetes where reversal of reduced glucose oxidation and mitochondrial capacity is a desirable goal.

  18. BAFF induces spleen CD4+ T cell proliferation by down-regulating phosphorylation of FOXO3A and activates cyclin D2 and D3 expression

    International Nuclear Information System (INIS)

    Ji, Fang; Chen, Rongjing; Liu, Baojun; Zhang, Xiaoping; Han, Junli; Wang, Haining; Shen, Gang; Tao, Jiang

    2012-01-01

    Highlights: ► Firstly analyze the mechanism of BAFF and anti-CD3 co-stimulation on purified mouse splenic CD4 + T cells. ► Carrying out siRNA technology to study FOXO3A protein function. ► Helpful to understand the T cell especially CD4 + T cell‘s role in immunological reaction. -- Abstract: The TNF ligand family member “B cell-activating factor belonging to the TNF family” (BAFF, also called BLyS, TALL-1, zTNF-4, and THANK) is an important survival factor for B and T cells. In this study, we show that BAFF is able to induce CD4 + spleen T cell proliferation when co-stimulated with anti-CD3. Expression of phosphorylated FOXO3A was notably down-regulated and cyclins D2 and D3 were up-regulated and higher in the CD4 + T cells when treated with BAFF and anti-CD3, as assessed by Western blotting. Furthermore, after FOXO3A was knocked down, expression of cyclin D1 was unchanged, compared with control group levels, but the expression of cyclins D2 and D3 increased, compared with the control group. In conclusion, our results suggest that BAFF induced CD4 + spleen T cell proliferation by down-regulating the phosphorylation of FOXO3A and then activating cyclin D2 and D3 expression, leading to CD4 + T cell proliferation.

  19. MiR-29b inhibits collagen maturation in hepatic stellate cells through down-regulating the expression of HSP47 and lysyl oxidase

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Yifei; Ghazwani, Mohammed; Li, Jiang [Center for Pharmacogenetics, Department of Pharmaceutical Sciences, School of Pharmacy, University of Pittsburgh, Pittsburgh, PA 15261 (United States); Sun, Ming; Stolz, Donna B. [Department of Cell Biology and Physiology, School of Medicine, University of Pittsburgh, Pittsburgh, PA 15261 (United States); He, Fengtian [Department of Biochemistry and Molecular Biology, College of Basic Medical Sciences, Third Military Medical University, Chongqing 400038 (China); Fan, Jie [Department of Surgery, University of Pittsburgh, Pittsburgh, PA 15261 (United States); Xie, Wen [Center for Pharmacogenetics, Department of Pharmaceutical Sciences, School of Pharmacy, University of Pittsburgh, Pittsburgh, PA 15261 (United States); Li, Song, E-mail: sol4@pitt.edu [Center for Pharmacogenetics, Department of Pharmaceutical Sciences, School of Pharmacy, University of Pittsburgh, Pittsburgh, PA 15261 (United States)

    2014-04-18

    Highlights: • Enhanced HSP47 and LOX expression is associated with decreased miR-29b level in liver fibrosis. • miR-29b down-regulates HSP47 and LOX expression. • The suppression of HSP47 and LOX by miR-29b is mediated by putative sites at their 3′-UTRs. • miR-29b inhibits extracellular LOX activity and collagen maturation. - Abstract: Altered expression of miR-29b is implicated in the pathogenesis and progression of liver fibrosis. We and others previously demonstrated that miR-29b down-regulates the expression of several extracellular-matrix (ECM) genes including Col 1A1, Col 3A1 and Elastin via directly targeting their 3′-UTRs. However, whether or not miR-29b plays a role in the post-translational regulation of ECM biosynthesis has not been reported. Heat shock protein 47 (HSP47) and lysyl oxidase (LOX) are known to be essential for ECM maturation. In this study we have demonstrated that expression of HSP47 and LOX was significantly up-regulated in culture-activated primary rat hepatic stellate cells (HSCs), TGF-β stimulated LX-2 cells and liver tissue of CCl{sub 4}-treated mice, which was accompanied by a decrease of miR-29b level. In addition, over-expression of miR-29b in LX-2 cells resulted in significant inhibition on HSP47 and LOX expression. Mechanistically, miR-29b inhibited the expression of a reporter gene that contains the respective full-length 3′-UTR from HSP47 and LOX gene, and this inhibitory effect was abolished by the deletion of a putative miR-29b targeting sequence from the 3′-UTRs. Transfection of LX-2 cells with miR-29b led to abnormal collagen structure as shown by electron-microscopy, presumably through down-regulation of the expression of molecules involved in ECM maturation including HSP47 and LOX. These results demonstrated that miR-29b is involved in regulating the post-translational processing of ECM and fibril formation.

  20. MiR-29b inhibits collagen maturation in hepatic stellate cells through down-regulating the expression of HSP47 and lysyl oxidase

    International Nuclear Information System (INIS)

    Zhang, Yifei; Ghazwani, Mohammed; Li, Jiang; Sun, Ming; Stolz, Donna B.; He, Fengtian; Fan, Jie; Xie, Wen; Li, Song

    2014-01-01

    Highlights: • Enhanced HSP47 and LOX expression is associated with decreased miR-29b level in liver fibrosis. • miR-29b down-regulates HSP47 and LOX expression. • The suppression of HSP47 and LOX by miR-29b is mediated by putative sites at their 3′-UTRs. • miR-29b inhibits extracellular LOX activity and collagen maturation. - Abstract: Altered expression of miR-29b is implicated in the pathogenesis and progression of liver fibrosis. We and others previously demonstrated that miR-29b down-regulates the expression of several extracellular-matrix (ECM) genes including Col 1A1, Col 3A1 and Elastin via directly targeting their 3′-UTRs. However, whether or not miR-29b plays a role in the post-translational regulation of ECM biosynthesis has not been reported. Heat shock protein 47 (HSP47) and lysyl oxidase (LOX) are known to be essential for ECM maturation. In this study we have demonstrated that expression of HSP47 and LOX was significantly up-regulated in culture-activated primary rat hepatic stellate cells (HSCs), TGF-β stimulated LX-2 cells and liver tissue of CCl 4 -treated mice, which was accompanied by a decrease of miR-29b level. In addition, over-expression of miR-29b in LX-2 cells resulted in significant inhibition on HSP47 and LOX expression. Mechanistically, miR-29b inhibited the expression of a reporter gene that contains the respective full-length 3′-UTR from HSP47 and LOX gene, and this inhibitory effect was abolished by the deletion of a putative miR-29b targeting sequence from the 3′-UTRs. Transfection of LX-2 cells with miR-29b led to abnormal collagen structure as shown by electron-microscopy, presumably through down-regulation of the expression of molecules involved in ECM maturation including HSP47 and LOX. These results demonstrated that miR-29b is involved in regulating the post-translational processing of ECM and fibril formation

  1. Curcumin induces down-regulation of EZH2 expression through the MAPK pathway in MDA-MB-435 human breast cancer cells.

    Science.gov (United States)

    Hua, Wen-Feng; Fu, Yong-Shui; Liao, Yi-Ji; Xia, Wen-Jie; Chen, Yang-Chao; Zeng, Yi-Xin; Kung, Hsiang-Fu; Xie, Dan

    2010-07-10

    Curcumin, a natural compound isolated from turmeric, may inhibit cell proliferation in various tumor cells through a mechanism that is not fully understood. The enhancer of zeste homolog 2 (EZH2) gene is overexpressed in human breast cancers with poor prognosis. In this study, we observed a dose- and time-dependent down-regulation of expression of EZH2 by curcumin that correlates with decreased proliferation in the MDA-MB-435 breast cancer cell line. The curcumin treatment resulted in an accumulation of cells in the G(1) phase of the cell cycle. Further investigation revealed that curcumin-induced down-regulation of EZH2 through stimulation of three major members of the mitogen-activated protein kinase (MAPK) pathway: c-Jun NH2-terminal kinase (JNK), extracellular signal-regulated kinase (ERK) and p38 kinase. These data suggest that an underlying mechanism of the MAPK pathway mediates the down-regulation of EZH2, thus contributing to the anti-proliferative effects of curcumin against breast cancer. Copyright 2010 Elsevier B.V. All rights reserved.

  2. D-Serine exposure resulted in gene expression changes indicative of activation of fibrogenic pathways and down-regulation of energy metabolism and oxidative stress response

    International Nuclear Information System (INIS)

    Soto, Armando; DelRaso, Nicholas J.; Schlager, John J.; Chan, Victor T.

    2008-01-01

    Renal toxicity can commonly occur after exposure to xenobiotics, pharmaceutical agents or environmental pollutants. Changes in the gene expression in kidney parenchymal cells that precede and/or accompany renal injury may be hallmark critical events in the onset of pathologic changes of renal functions. Over the last several years, transcriptomic analysis has evolved to enable simultaneous analysis of the expression profiles of tens of thousands of genes in response to various endogenous and exogenous stimuli. In this study, we investigated gene expression changes in the kidney after acute exposure to a nephrotoxin, D-serine, which targets the proximal tubule of the kidney. Male F-344 rats injected intraperitoneally with a single dose of D-serine (5, 20, 50, 200 or 500 mg/kg), and gene expression profiles in the kidney were determined using the Affymetrix RAE230A gene arrays at 96 h post-dosing. D-Serine treatment resulted in the up- and down-regulation of 1158 and 749 genes, respectively, over the entire dose range based on the intersection of the results of t-test, p < 0.01 over two consecutive doses, and ANOVA with Bonferonni correction for multiple testing. Interestingly, both the up-and down-regulated genes show a unified dose response pattern as revealed in the self-organized map clustering analysis using the expression profiles of the 1907 differentially expressed genes as input data. There appears to be minimal changes in the expression level of these genes in the dose range of 5-50 mg/kg, while the most prominent changes were observed at the highest doses tested, i.e. 200 and 500 mg/kg. Pathway analysis of the differentially expressed genes showed perturbation of a large number of biological processes/pathways after D-serine exposure. Among the up-regulated pathways are actin cytoskeleton biogenesis and organization, apoptosis, cell cycle regulation, chromatin assembly, excision repair of damaged DNA, DNA replication and packaging, protein biosynthesis

  3. Progesterone receptor activates Msx2 expression by downregulating TNAP/Akp2 and activating the Bmp pathway in EpH4 mouse mammary epithelial cells.

    Directory of Open Access Journals (Sweden)

    Jodie M Fleming

    Full Text Available Previously we demonstrated that EpH4 mouse mammary epithelial cells induced the homeobox transcription factor Msx2 either when transfected with the progesterone receptor (PR or when treated with Bmp2/4. Msx2 upregulation was unaffected by Wnt inhibitors s-FRP or Dkk1, but was inhibited by the Bmp antagonist Noggin. We therefore hypothesized that PR signaling to Msx2 acts through the Bmp receptor pathway. Herein, we confirm that transcripts for Alk2/ActR1A, a non-canonical BmpR Type I, are upregulated in mammary epithelial cells overexpressing PR (EpH4-PR. Increased phosphorylation of Smads 1,5, 8, known substrates for Alk2 and other BmpR Type I proteins, was observed as was their translocation to the nucleus in EpH4-PR cells. Analysis also showed that Tissue Non-Specific Alkaline Phosphatase (TNAP/Akp2 was also found to be downregulated in EpH4-PR cells. When an Akp2 promoter-reporter construct containing a ½PRE site was transfected into EpH4-PR cells, its expression was downregulated. Moreover, siRNA mediated knockdown of Akp2 increased both Alk2 and Msx2 expression. Collectively these data suggest that PR inhibition of Akp2 results in increased Alk2 activity, increased phosphorylation of Smads 1,5,8, and ultimately upregulation of Msx2. These studies imply that re-activation of the Akp2 gene could be helpful in downregulating aberrant Msx2 expression in PR+ breast cancers.

  4. mRNA-binding protein TIA-1 reduces cytokine expression in human endometrial stromal cells and is down-regulated in ectopic endometrium.

    Science.gov (United States)

    Karalok, Hakan Mete; Aydin, Ebru; Saglam, Ozlen; Torun, Aysenur; Guzeloglu-Kayisli, Ozlem; Lalioti, Maria D; Kristiansson, Helena; Duke, Cindy M P; Choe, Gina; Flannery, Clare; Kallen, Caleb B; Seli, Emre

    2014-12-01

    Cytokines and growth factors play important roles in endometrial function and the pathogenesis of endometriosis. mRNAs encoding cytokines and growth factors undergo rapid turnover; primarily mediated by adenosine- and uridine-rich elements (AREs) located in their 3'-untranslated regions. T-cell intracellular antigen (TIA-1), an mRNA-binding protein, binds to AREs in target transcripts, leading to decreased gene expression. The purpose of this article was to determine whether TIA-1 plays a role in the regulation of endometrial cytokine and growth factor expression during the normal menstrual cycle and whether TIA-1 expression is altered in women with endometriosis. Eutopic endometrial tissue obtained from women without endometriosis (n = 30) and eutopic and ectopic endometrial tissues from women with endometriosis (n = 17) were immunostained for TIA-1. Staining intensities were evaluated by histological scores (HSCOREs). The regulation of endometrial TIA-1 expression by immune factors and steroid hormones was studied by treating primary cultured human endometrial stromal cells (HESCs) with vehicle, lipopolysaccharide, TNF-α, IL-6, estradiol, or progesterone, followed by protein blot analyses. HESCs were engineered to over- or underexpress TIA-1 to test whether TIA-1 regulates IL-6 or TNF-α expression in these cells. We found that TIA-1 is expressed in endometrial stromal and glandular cells throughout the menstrual cycle and that this expression is significantly higher in the perimenstrual phase. In women with endometriosis, TIA-1 expression in eutopic and ectopic endometrium was reduced compared with TIA-1 expression in eutopic endometrium of unaffected control women. Lipopolysaccharide and TNF-α increased TIA-1 expression in HESCs in vitro, whereas IL-6 or steroid hormones had no effect. In HESCs, down-regulation of TIA-1 resulted in elevated IL-6 and TNF-α expression, whereas TIA-1 overexpression resulted in decreased IL-6 and TNF-α expression. Endometrial

  5. Escin Ia suppresses the metastasis of triple-negative breast cancer by inhibiting epithelial-mesenchymal transition via down-regulating LOXL2 expression.

    Science.gov (United States)

    Wang, Yuhui; Xu, Xiaotian; Zhao, Peng; Tong, Bei; Wei, Zhifeng; Dai, Yue

    2016-04-26

    The saponin fraction of Aesculus chinensis Bunge fruits (SFAC) could inhibit the invasion and migration of MDA-MB-231 cells. Among which, escin Ia showed more potent inhibition of the invasion than other five main saponin constituents. It selectively reduced the expression of LOXL2 mRNA and promoted the expression of E-cadherin mRNA, and prevented the EMT process of MDA-MB-231 cells and TNF-α/TGF-β-stimulated MCF-7 cells. Moreover, it reduced the LOXL2 level in MDA-MB-231 cells but not in MCF-7 cells. When MCF-7 cells were stimulated with TNF-α/TGF-β, transfected with LOXL2 or treated with hypoxia, escin Ia down-regulated the level of LOXL2 in MCF-7 cells. Meanwhile, escin Ia suppressed the EMT process in LOXL2-transfected or hypoxia-treated MCF-7 cells. Of interest, escin Ia did not alter the level of HIF-1α in hypoxia-induced MCF-7 cells. In TNBC xenograft mice, the metastasis and EMT of MDA-MB-231 cells were suppressed by escin Ia. In conclusion, escin Ia was the main active ingredient of SFAC for the anti-TNBC metastasis activity, and its action mechanisms involved inhibition of EMT process by down-regulating LOXL2 expression.

  6. NOX4-mediated ROS production induces apoptotic cell death via down-regulation of c-FLIP and Mcl-1 expression in combined treatment with thioridazine and curcumin.

    Science.gov (United States)

    Seo, Seung Un; Kim, Tae Hwan; Kim, Dong Eun; Min, Kyoung-Jin; Kwon, Taeg Kyu

    2017-10-01

    Thioridazine is known to have anti-tumor effects by inhibiting PI3K/Akt signaling, which is an important signaling pathway in cell survival. However, thioridazine alone does not induce apoptosis in head and neck squamous cell carcinoma (AMC-HN4), human breast carcinoma (MDA-MB231), and human glioma (U87MG) cells. Therefore, we investigated whether combined treatment with thioridazine and curcumin induces apoptosis. Combined treatment with thioridazine and curcumin markedly induced apoptosis in cancer cells without inducing apoptosis in human normal mesangial cells and human normal umbilical vein cells (EA.hy926). We found that combined treatment with thioridazine and curcumin had synergistic effects in AMC-HN4 cells. Among apoptosis-related proteins, thioridazine plus curcumin induced down-regulation of c-FLIP and Mcl-1 expression at the post-translational levels in a proteasome-dependent manner. Augmentation of proteasome activity was related to the up-regulation of proteasome subunit alpha 5 (PSMA5) expression in curcumin plus thioridazine-treated cells. Combined treatment with curcumin and thioridazine produced intracellular ROS in a NOX4-dependent manner, and ROS-mediated activation of Nrf2/ARE signaling played a critical role in the up-regulation of PSMA5 expression. Furthermore, ectopic expression of c-FLIP and Mcl-1 inhibited apoptosis in thioridazine and curcumin-treated cells. Therefore, we demonstrated that thioridazine plus curcumin induces proteasome activity by up-regulating PSMA5 expression via NOX4-mediated ROS production and that down-regulation of c-FLIP and Mcl-1 expression post-translationally is involved in apoptosis. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

  7. TGF-β1 Downregulates the Expression of CX3CR1 by Inducing miR-27a-5p in Primary Human NK Cells.

    Science.gov (United States)

    Regis, Stefano; Caliendo, Fabio; Dondero, Alessandra; Casu, Beatrice; Romano, Filomena; Loiacono, Fabrizio; Moretta, Alessandro; Bottino, Cristina; Castriconi, Roberta

    2017-01-01

    Activity of human natural killer (NK) cells against cancer cells is deeply suppressed by TGF-β1, an immunomodulatory cytokine that is released and activated in the tumor microenvironment. Moreover, our previous data showed that TGF-β1 modifies the chemokine receptor repertoire of NK cells. In particular, it decreases the expression of CX 3 CR1 that drives these effectors toward peripheral tissues, including tumor sites. To identify possible mechanisms mediating chemokine receptors modulation, we analyzed the microRNA profile of TGF-β1-treated primary NK cells. The analysis pointed out miR-27a-5p as a possible modulator of CX 3 CR1. We demonstrated the functional interaction of miR-27a-5p with the 3' untranslated region (3'UTR) of CX 3 CR1 mRNA by two different experimental approaches: by the use of a luciferase assay based on a reporter construct containing the CX 3 CR1 3'UTR and by transfection of primary NK cells with a miR-27a-5p inhibitor. We also showed that the TGF-β1-mediated increase of miR-27a-5p expression is a consequence of miR-23a-27a-24-2 cluster induction. Moreover, we demonstrated that miR-27a-5p downregulates the surface expression of CX 3 CR1. Finally, we showed that neuroblastoma cells induced in resting NK cells a downregulation of the CX 3 CR1 expression that was paralleled by a significant increase of miR-27a-5p expression. Therefore, the present study highlights miR-27a-5p as a pivotal TGF-β1-induced regulator of CX 3 CR1 expression.

  8. NOX4-mediated ROS production induces apoptotic cell death via down-regulation of c-FLIP and Mcl-1 expression in combined treatment with thioridazine and curcumin

    Directory of Open Access Journals (Sweden)

    Seung Un Seo

    2017-10-01

    Full Text Available Thioridazine is known to have anti-tumor effects by inhibiting PI3K/Akt signaling, which is an important signaling pathway in cell survival. However, thioridazine alone does not induce apoptosis in head and neck squamous cell carcinoma (AMC-HN4, human breast carcinoma (MDA-MB231, and human glioma (U87MG cells. Therefore, we investigated whether combined treatment with thioridazine and curcumin induces apoptosis. Combined treatment with thioridazine and curcumin markedly induced apoptosis in cancer cells without inducing apoptosis in human normal mesangial cells and human normal umbilical vein cells (EA.hy926. We found that combined treatment with thioridazine and curcumin had synergistic effects in AMC-HN4 cells. Among apoptosis-related proteins, thioridazine plus curcumin induced down-regulation of c-FLIP and Mcl-1 expression at the post-translational levels in a proteasome-dependent manner. Augmentation of proteasome activity was related to the up-regulation of proteasome subunit alpha 5 (PSMA5 expression in curcumin plus thioridazine-treated cells. Combined treatment with curcumin and thioridazine produced intracellular ROS in a NOX4-dependent manner, and ROS-mediated activation of Nrf2/ARE signaling played a critical role in the up-regulation of PSMA5 expression. Furthermore, ectopic expression of c-FLIP and Mcl-1 inhibited apoptosis in thioridazine and curcumin-treated cells. Therefore, we demonstrated that thioridazine plus curcumin induces proteasome activity by up-regulating PSMA5 expression via NOX4-mediated ROS production and that down-regulation of c-FLIP and Mcl-1 expression post-translationally is involved in apoptosis.

  9. The Liver X Receptor Ligand T0901317 Down-regulates APOA5 GeneExpression through Activation of SREBP-1c

    Energy Technology Data Exchange (ETDEWEB)

    Jakel, Heidelinde; Nowak, Maxime; Moitrot, Emanuelle; Dehondt, Helene; Hum, Dean W.; Pennacchio, Len A.; Fruchart-Najib, Jamila; Fruchart,Jean-Charles

    2004-07-23

    Alterations in the expression of the recently discovered apolipoprotein A5 gene strongly affect plasma triglyceride levels. In this study, we investigated the contribution of APOA5 to the liver X-receptor (LXR) ligand mediated effect on plasma triglyceride levels.Following treatment with the LXR ligand T0901317, we found that APOA5mRNA levels were decreased in hepatoma cell lines. The observation that no down-regulation of APOA5 promoter activity was obtained by LXR-retinoid X receptor (RXR) co-transfection prompted us to explore the possible involvement of the known LXR target gene SREBP-1c (sterol regulatory element-binding protein 1c). In fact, we found that co-transfection with the active form of SREBP-1c down-regulated APOA5promoter activity in a dose-dependent manner. We then scanned the human APOA5 promoter sequence and identified two putative E-box elements that were able to bind specifically SREBP-1c in gel-shift assays and were shown to be functional by mutation analysis. Subsequent suppression of SREBP-1 mRNA through small interfering RNA interference abolished the decrease of APOA5 mRNA in response to T0901317. Finally, administration of T0901317 to hAPOA5 transgenic mice revealed a significant decrease OF APOA5 mRNA in liver tissue and circulating apolipoprotein AV protein in plasma, confirming that the described down-regulation also occurs in vivo. Taken together, our results demonstrate that APOA5 gene expression is regulated by the LXR ligand T0901317 in a negative manner through SREBP-1c. These findings may provide a new mechanism responsible for the elevation of plasma triglyceride levels by LXR ligands and support the development of selective LXR agonists, not affecting SREBP-1c, as beneficial modulators of lipid metabolism.

  10. Celecoxib Down-Regulates the Hypoxia-Induced Expression of HIF-1α and VEGF Through the PI3K/AKT Pathway in Retinal Pigment Epithelial Cells

    Directory of Open Access Journals (Sweden)

    Yi-zhou Sun

    2017-12-01

    Full Text Available Background/Aims: The goal of this study was to detect the expression of hypoxia-inducible factor 1α (HIF-1α and vascular endothelial growth factor (VEGF in human retinal pigmented epithelial (RPE cells treated with celecoxib, a selective cyclooxygenase-2 (COX-2 inhibitor, under hypoxic and normoxic conditions and to explore the signaling mechanism involved in regulating the hypoxia-induced expression of HIF-1α and VEGF in RPE cells. Methods: D407 cells were cultured in normoxic or hypoxic conditions, with or without celecoxib or a PI3K inhibitor (LY294002. The anti-proliferative effect of celecoxib was assessed using the MTT assay. RT-PCR, Western blotting and ELISA were performed to detect the levels of PI3K, phosphorylated AKT (p-AKT, HIF-1α, VEGF and COX-2. Results: Celecoxib inhibited the proliferation of RPE cells in a dose-dependent manner. Celecoxib suppressed the expression of VEGF at both the mRNA and protein levels and decreased HIF-1α protein expression. HIF-1α activation was regulated by the PI3K/AKT pathway. The celecoxib-induced down-regulation of HIF-1α and VEGF required the suppression of the hypoxia-induced PI3K/AKT pathway. However, the down-regulation of COX-2 did not occur in cells treated with celecoxib. Conclusions: The antiangiogenic effects of celecoxib in RPE cells under hypoxic conditions resulted from the inhibition of HIF-1α and VEGF expression, which may be partly mediated by a COX-2-independent, PI3K/AKT-dependent pathway.

  11. Differential Regulation of cGMP Signaling in Human Melanoma Cells at Altered Gravity: Simulated Microgravity Down-Regulates Cancer-Related Gene Expression and Motility

    Science.gov (United States)

    Ivanova, Krassimira; Eiermann, Peter; Tsiockas, Wasiliki; Hemmersbach, Ruth; Gerzer, Rupert

    2018-03-01

    Altered gravity is known to affect cellular function by changes in gene expression and cellular signaling. The intracellular signaling molecule cyclic guanosine-3',5'-monophosphate (cGMP), a product of guanylyl cyclases (GC), e.g., the nitric oxide (NO)-sensitive soluble GC (sGC) or natriuretic peptide-activated GC (GC-A/GC-B), is involved in melanocyte response to environmental stress. NO-sGC-cGMP signaling is operational in human melanocytes and non-metastatic melanoma cells, whereas up-regulated expression of GC-A/GC-B and inducible NO synthase (iNOS) are found in metastatic melanoma cells, the deadliest skin cancer. Here, we investigated the effects of altered gravity on the mRNA expression of NOS isoforms, sGC, GC-A/GC-B and multidrug resistance-associated proteins 4/5 (MRP4/MRP5) as selective cGMP exporters in human melanoma cells with different metastatic potential and pigmentation. A specific centrifuge (DLR, Cologne Germany) was used to generate hypergravity (5 g for 24 h) and a fast-rotating 2-D clinostat (60 rpm) to simulate microgravity values ≤ 0.012 g for 24 h. The results demonstrate that hypergravity up-regulates the endothelial NOS-sGC-MRP4/MRP5 pathway in non-metastatic melanoma cells, but down-regulates it in simulated microgravity when compared to 1 g. Additionally, the suppression of sGC expression and activity has been suggested to correlate inversely to tumor aggressiveness. Finally, hypergravity is ineffective in highly metastatic melanoma cells, whereas simulated microgravity down-regulates predominantly the expression of the cancer-related genes iNOS and GC-A/GC-B (shown additionally on protein levels) as well as motility in comparison to 1 g. The results suggest that future studies in real microgravity can benefit from considering GC-cGMP signaling as possible factor for melanocyte transformation.

  12. Down-regulated expression of the protein-tyrosine phosphatase 1B (PTP1B) is associated with aggressive clinicopathologic features and poor prognosis in hepatocellular carcinoma

    International Nuclear Information System (INIS)

    Zheng, Long-Yi; Zhou, Dong-Xun; Lu, Jin; Zhang, Wen-Jun; Zou, Da-Jin

    2012-01-01

    Highlights: ► PTP1B protein showed decreased expression in 67.79% of the HCC patients. ► Low PTP1B expression predicts poor prognosis of HCC. ► Low PTP1B expression is correlated with expansion of OV6 + tumor-initiating cells. ► Down-regulation of PTP1B is associated with activation of Wnt/β-Catenin signaling. -- Abstract: The protein-tyrosine phosphatase 1B (PTP1B) is a classical non-transmembrane protein tyrosine phosphatase that plays a key role in metabolic signaling and can exert both tumor suppressing and tumor promoting effects in different cancers depending on the substrate involved and the cellular context. However, the expression level and function of PTP1B in hepatocellular carcinoma (HCC) remain unclear. In this study, PTP1B expression was detected by immunohistochemistry in normal liver tissue (n = 16) and hepatocellular carcinoma (n = 169). The correlations between PTP1B expression level and clinicopathologic features and patient survival were also analyzed. One hundred and eleven of 169 HCC patients (65.7%) had negative or low PTP1B expression in tumorous tissues, whereas normal tissues always expressed strong PTP1B. Decreased PTP1B expression was significantly associated with aggressive clinicopathologic features and poor prognosis. Immunohistochemistry also showed that low PTP1B expression level was correlated with high percentage of OV6 + tumor-initiating cells (T-ICs) and high frequency of nuclear β-Catenin expression in HCC specimens. Our findings demonstrate for the first time that the loss of inhibitory effect of PTP1B may contribute to progression and invasion of HCC through activation of Wnt/β-Catenin signaling and expansion of liver T-ICs. PTP1B may serve as a valuable prognostic biomarker and potential therapeutic target in HCC.

  13. Down-regulated expression of the protein-tyrosine phosphatase 1B (PTP1B) is associated with aggressive clinicopathologic features and poor prognosis in hepatocellular carcinoma

    Energy Technology Data Exchange (ETDEWEB)

    Zheng, Long-Yi [Department of Endocrinology, Changhai Hospital, 168 Changhai Road, Shanghai 200433 (China); Zhou, Dong-Xun [Department of Comprehensive Treatment II, Eastern Hepatobiliary Surgery Hospital, 225 Changhai Road, Shanghai 200438 (China); Lu, Jin [Department of Endocrinology, Changhai Hospital, 168 Changhai Road, Shanghai 200433 (China); Zhang, Wen-Jun [Department of Emergency, Changhai Hospital, 168 Changhai Road, Shanghai 200433 (China); Zou, Da-Jin, E-mail: dajinzou@hotmail.com [Department of Endocrinology, Changhai Hospital, 168 Changhai Road, Shanghai 200433 (China)

    2012-04-13

    Highlights: Black-Right-Pointing-Pointer PTP1B protein showed decreased expression in 67.79% of the HCC patients. Black-Right-Pointing-Pointer Low PTP1B expression predicts poor prognosis of HCC. Black-Right-Pointing-Pointer Low PTP1B expression is correlated with expansion of OV6{sup +} tumor-initiating cells. Black-Right-Pointing-Pointer Down-regulation of PTP1B is associated with activation of Wnt/{beta}-Catenin signaling. -- Abstract: The protein-tyrosine phosphatase 1B (PTP1B) is a classical non-transmembrane protein tyrosine phosphatase that plays a key role in metabolic signaling and can exert both tumor suppressing and tumor promoting effects in different cancers depending on the substrate involved and the cellular context. However, the expression level and function of PTP1B in hepatocellular carcinoma (HCC) remain unclear. In this study, PTP1B expression was detected by immunohistochemistry in normal liver tissue (n = 16) and hepatocellular carcinoma (n = 169). The correlations between PTP1B expression level and clinicopathologic features and patient survival were also analyzed. One hundred and eleven of 169 HCC patients (65.7%) had negative or low PTP1B expression in tumorous tissues, whereas normal tissues always expressed strong PTP1B. Decreased PTP1B expression was significantly associated with aggressive clinicopathologic features and poor prognosis. Immunohistochemistry also showed that low PTP1B expression level was correlated with high percentage of OV6{sup +} tumor-initiating cells (T-ICs) and high frequency of nuclear {beta}-Catenin expression in HCC specimens. Our findings demonstrate for the first time that the loss of inhibitory effect of PTP1B may contribute to progression and invasion of HCC through activation of Wnt/{beta}-Catenin signaling and expansion of liver T-ICs. PTP1B may serve as a valuable prognostic biomarker and potential therapeutic target in HCC.

  14. Experimental study of inhibitory effects of diallyl trisulfide on the growth of human osteosarcoma Saos-2 cells by downregulating expression of glucose-regulated protein 78

    Directory of Open Access Journals (Sweden)

    Zhang Y

    2018-01-01

    Full Text Available Yue Zhang,1,* Wen-Peng Xie,1,* Yong-Kui Zhang,2 Yi-Qiang Chen,3 Dong-Li Wang,2 Gang Li,2 Dong-Hui Guan2 1First Clinical Medical College, Shandong University of Traditional Chinese Medicine, Jinan, People’s Republic of China; 2Department of Orthopedics, Affiliated Hospital of Shandong University of Traditional Chinese Medicine, Jinan, People’s Republic of China; 3Department of Orthopedics, The First People’s Hospital of Taian City, Taian, People’s Republic of China *These authors contributed equally to the paper Background: Diallyl trisulfide (DATS is a natural organic sulfur compound isolated from garlic that has good anticancer activity according to many previous reports. There are many studies pointing out that DATS can downregulate expression of the glucose-regulated protein 78 (GRP78, which is associated with poor prognosis and drug resistance in various types of human cancers. However, it remains unknown whether DATS has the same effect on human osteosarcoma cells. This study attempted to clarify the potential molecular mechanisms of the action of DATS in human osteosarcoma Saos-2 cells.Methods: We used an inverted phase microscope and immunofluorescent staining to observe the morphological changes of Saos-2 cells after being cultured in different concentrations of DATS (0, 25, 50, and 100 µM for 24 h, or for four time periods (24, 48, 72, and 96 h in the same DATS concentration (50 µM. Quantitative real-time polymerase chain reaction and Western blot were used to detect the expression level of GRP78 mRNA and proteins in Saos-2 cells. GRP78 expression was suppressed in Saos-2 cells by utilizing small-interfering RNA, and the cells were subsequently used to study the anti-proliferative effects of DATS treatment.Results: The expression level of GRP78 mRNA and proteins was significantly downregulated due to the increased concentration and effective times of DATS (P<0.05. In addition, there were significant associations between GRP78

  15. Thrombin-induced, TNFR-dependent miR-181c downregulation promotes MLL1 and NF-κB target gene expression in human microglia.

    Science.gov (United States)

    Yin, Min; Chen, Zhiying; Ouyang, Yetong; Zhang, Huiyan; Wan, Zhigang; Wang, Han; Wu, Wei; Yin, Xiaoping

    2017-06-29

    Controlling thrombin-driven microglial activation may serve as a therapeutic target for intracerebral hemorrhage (ICH). Here, we investigated microRNA (miRNA)-based regulation of thrombin-driven microglial activation using an in vitro thrombin toxicity model applied to primary human microglia. A miRNA array identified 22 differential miRNA candidates. Quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) identified miR-181c as the most significantly downregulated miRNA. TargetScan analysis identified mixed lineage leukemia-1 (MLL1) as a putative gene target for miR-181c. qRT-PCR was applied to assess tumor necrosis factor-alpha (TNF-α), miR-181c, and MLL1 levels following thrombin or proteinase-activated receptor-4-specific activating peptide (PAR4AP) exposure. Anti-TNF-α antibodies and tumor necrosis factor receptor (TNFR) silencing were employed to test TNF-α/TNFR dependence. A dual-luciferase reporter system and miR-181c mimic transfection assessed whether mir-181c directly binds to and negatively regulates MLL1. Nuclear factor kappa-B (NF-κB)-dependent luciferase reporter assays and NF-κB target gene expression were assessed in wild-type (MLL1+) and MLL1-silenced cells. Thrombin or PAR4AP-induced miR-181c downregulation (p < 0.05) and MLL1 upregulation (p < 0.05) that were dependent upon TNF-α/TNFR. miR-181c decreased wild-type MLL1 3'-UTR luciferase reporter activity (p < 0.05), and a miR-181c mimic suppressed MLL1 expression (p < 0.05). Thrombin treatment increased, while miR-181c reduced, NF-κB activity and NF-κB target gene expression in both wild-type (MLL1+) and MLL1-silenced cells (p < 0.05). Thrombin-induced, TNF-α/TNFR-dependent miR-181c downregulation promotes MLL1 expression, increases NF-κB activity, and upregulates NF-κB target gene expression. As miR-181c opposes thrombin's stimulation of pro-inflammatory NF-κB activity, miR-181c mimic therapy may show promise in controlling thrombin

  16. Estrogen-Induced Maldevelopment of the Penis Involves Down-Regulation of Myosin Heavy Chain 11 (MYH11) Expression, a Biomarker for Smooth Muscle Cell Differentiation1

    Science.gov (United States)

    Okumu, L.A.; Bruinton, Sequoia; Braden, Tim D.; Simon, Liz; Goyal, Hari O.

    2012-01-01

    ABSTRACT Cavernous smooth muscle cells are essential components in penile erection. In this study, we investigated effects of estrogen exposure on biomarkers for smooth muscle cell differentiation in the penis. Neonatal rats received diethylstilbestrol (DES), with or without the estrogen receptor (ESR) antagonist ICI 182,780 (ICI) or the androgen receptor (AR) agonist dihydrotestosterone (DHT), from Postnatal Days 1 to 6. Tissues were collected at 7, 10, or 21 days of age. The smooth muscle cell biomarker MYH11 was studied in depth because microarray data showed it was significantly down-regulated, along with other biomarkers, in DES treatment. Quantitative real time-PCR and Western blot analyses showed 50%–80% reduction (P ≤ 0.05) in Myh11 expression in DES-treated rats compared to that in controls; and ICI and DHT coadministration mitigated the decrease. Temporally, from 7 to 21 days of age, Myh11 expression was onefold increased (P ≥ 0.05) in DES-treated rats versus threefold increased (P ≤ 0.001) in controls, implying the long-lasting inhibitory effect of DES on smooth muscle cell differentiation. Immunohistochemical localization of smooth muscle alpha actin, another biomarker for smooth muscle cell differentiation, showed fewer cavernous smooth muscle cells in DES-treated animals than in controls. Additionally, DES treatment significantly up-regulated Esr1 mRNA expression and suppressed the neonatal testosterone surge by 90%, which was mitigated by ICI coadministration but not by DHT coadministration. Collectively, results provided evidence that DES treatment in neonatal rats inhibited cavernous smooth muscle cell differentiation, as shown by down-regulation of MYH11 expression at the mRNA and protein levels and by reduced immunohistochemical staining of smooth muscle alpha actin. Both the ESR and the AR pathways probably mediate this effect. PMID:22976277

  17. Expression of DIAPH1 is up-regulated in colorectal cancer and its down-regulation strongly reduces the metastatic capacity of colon carcinoma cells.

    Science.gov (United States)

    Lin, Yuan-Na; Izbicki, Jakob R; König, Alexandra; Habermann, Jens K; Blechner, Christine; Lange, Tobias; Schumacher, Udo; Windhorst, Sabine

    2014-04-01

    In most cases, metastatic colorectal cancer is not curable, thus new approaches are necessary to identify novel targets for colorectal cancer therapy. Actin-binding-proteins (ABPs) directly regulate motility of metastasising tumor cells, and for cortactin an association with colon cancer metastasis has been already shown. However, as its depletion only incompletely inhibits metastasis, additional, more suitable cellular targets have to be identified. Here we analyzed expression of the ABPs, DIAPH1, VASP, N-WASP, and fascin in comparison with cortactin and found that, besides cortactin, DIAPH1 was expressed with the highest frequency (63%) in colorectal cancer. As well as cortactin, DIAPH1 was not detectable in normal colon tissue and expression of both proteins was positively correlated with metastasis of colorectal cancer. To analyse the mechanistic role of DIAPH1 for metastasis of colon carcinoma cells in comparison with cortactin, expression of the proteins was stably down-regulated in the human colon carcinoma cell lines HT-29, HROC-24 and HCT-116. Analysis of metastasis of colon carcinoma cells in SCID mice revealed that depletion of DIAPH1 reduced metastasis 60-fold and depletion of cortactin 16-fold as compared with control cells. Most likely the stronger effect of DIAPH1 depletion on colon cancer metastasis is due to the fact that in vitro knock down of DIAPH1 impaired all steps of metastasis; adhesion, invasion and migration while down-regulation of cortactin only reduced adhesion and invasion. This very strong reducing effect of DIAPH1 depletion on colon carcinoma cell metastasis makes the protein a promising therapeutic target for individualized colorectal cancer therapy. © 2013 UICC.

  18. Prolonged calorie restriction downregulates skeletal muscle mTORC1 signaling independent of dietary protein intake and associated microRNA expression

    Directory of Open Access Journals (Sweden)

    Lee M Margolis

    2016-10-01

    Full Text Available Short-term (5-10 days calorie restriction (CR downregulates muscle protein synthesis, with consumption of a high protein-based diet attenuating this decline. Benefit of increase protein intake is believed to be due to maintenance of amino acid-mediated anabolic signaling through the mechanistic target of rapamycin complex 1 (mTORC1, however, there is limited evidence to support this contention. The purpose of this investigation was to determine the effects of prolonged CR and high protein diets on skeletal muscle mTORC1 signaling and expression of associated microRNA (miR. 12-wk old male Sprague Dawley rats consumed ad libitum (AL or calorie restricted (CR; 40% adequate (10%, AIN-93M or high (32% protein milk-based diets for 16 weeks. Body composition was determined using dual energy X-ray absorptiometry and muscle protein content was calculated from muscle homogenate protein concentrations expressed relative to fat-free mass to estimate protein content. Western blot and RT-qPCR were used to determine mTORC1 signaling and mRNA and miR expression in fasted mixed gastrocnemius. Independent of dietary protein intake, muscle protein content was 38% lower (P < 0.05 in CR compared to AL. Phosphorylation and total Akt, mTOR, rpS6 and p70S6K were lower (P < 0.05 in CR versus AL, and total rpS6 was associated with muscle protein content (r = 0.64, r2 = 0.36. Skeletal muscle miR expression was not altered by either energy or protein intake. This study provides evidence that chronic CR attenuates muscle protein content by downregulating mTORC1 signaling. This response is independent of skeletal muscle miR and dietary protein.

  19. BAFF induces spleen CD4{sup +} T cell proliferation by down-regulating phosphorylation of FOXO3A and activates cyclin D2 and D3 expression

    Energy Technology Data Exchange (ETDEWEB)

    Ji, Fang; Chen, Rongjing [Department of Orthodontics, Ninth People' s Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai Key Laboratory of Stomatology, Shanghai (China); Liu, Baojun [Laboratory of Lung, Inflammation and Cancers, Huashan Hospital, Fudan University, Shanghai (China); Zhang, Xiaoping [Department of Nuclear Medicine, Shanghai 10th People' s Hospital, Tongji University School of Medicine, Shanghai 200072 (China); Han, Junli; Wang, Haining [Department of General Dentistry, Ninth People' s Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai Key Laboratory of Stomatology, Shanghai (China); Shen, Gang [Department of Orthodontics, Ninth People' s Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai Key Laboratory of Stomatology, Shanghai (China); Tao, Jiang, E-mail: taojiang2012@yahoo.cn [Department of General Dentistry, Ninth People' s Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai Key Laboratory of Stomatology, Shanghai (China)

    2012-09-07

    Highlights: Black-Right-Pointing-Pointer Firstly analyze the mechanism of BAFF and anti-CD3 co-stimulation on purified mouse splenic CD4{sup +} T cells. Black-Right-Pointing-Pointer Carrying out siRNA technology to study FOXO3A protein function. Black-Right-Pointing-Pointer Helpful to understand the T cell especially CD4{sup +} T cell's role in immunological reaction. -- Abstract: The TNF ligand family member 'B cell-activating factor belonging to the TNF family' (BAFF, also called BLyS, TALL-1, zTNF-4, and THANK) is an important survival factor for B and T cells. In this study, we show that BAFF is able to induce CD4{sup +} spleen T cell proliferation when co-stimulated with anti-CD3. Expression of phosphorylated FOXO3A was notably down-regulated and cyclins D2 and D3 were up-regulated and higher in the CD4{sup +} T cells when treated with BAFF and anti-CD3, as assessed by Western blotting. Furthermore, after FOXO3A was knocked down, expression of cyclin D1 was unchanged, compared with control group levels, but the expression of cyclins D2 and D3 increased, compared with the control group. In conclusion, our results suggest that BAFF induced CD4{sup +} spleen T cell proliferation by down-regulating the phosphorylation of FOXO3A and then activating cyclin D2 and D3 expression, leading to CD4{sup +} T cell proliferation.

  20. Downregulation of TIM-3 mRNA expression in peripheral blood mononuclear cells from patients with systemic lupus erythematosus

    Energy Technology Data Exchange (ETDEWEB)

    Cai, X.Z. [Central Laboratory, First Affiliated Hospital, China Medical University, Shenyang (China); Department of Immunology, College of Basic Medical Sciences, China Medical University, Shenyang (China); Huang, W.Y.; Qiao, Y.; Chen, Y.; Du, S.Y.; Chen, D.; Yu, S. [Central Laboratory, First Affiliated Hospital, China Medical University, Shenyang (China); Liu, N. [Department of Nephrology, First Affiliated Hospital, China Medical University, Shenyang (China); Dou, L.Y. [Central Laboratory, First Affiliated Hospital, China Medical University, Shenyang (China); Jiang, Y. [Central Laboratory, First Affiliated Hospital, China Medical University, Shenyang (China); Department of Immunology, College of Basic Medical Sciences, China Medical University, Shenyang (China); Department of Dermatology, First Affiliated Hospital, China Medical University, Shenyang (China)

    2014-10-17

    The T-cell immunoglobulin and mucin domain (TIM) family is associated with autoimmune diseases, but its expression level in the immune cells of systemic lupus erythematosus (SLE) patients is not known. The aim of this study was to investigate whether the expression of TIM-3 mRNA is associated with pathogenesis of SLE. Quantitative real-time reverse transcription-polymerase chain reaction analysis (qRT-PCR) was used to determine TIM-1, TIM-3, and TIM-4 mRNA expression in peripheral blood mononuclear cells (PBMCs) from 132 patients with SLE and 62 healthy controls. The PBMC surface protein expression of TIMs in PBMCs from 20 SLE patients and 15 healthy controls was assayed by flow cytometry. Only TIM-3 mRNA expression decreased significantly in SLE patients compared with healthy controls (P<0.001). No significant differences in TIM family protein expression were observed in leukocytes from SLE patients and healthy controls (P>0.05). SLE patients with lupus nephritis (LN) had a significantly lower expression of TIM-3 mRNA than those without LN (P=0.001). There was no significant difference in the expression of TIM-3 mRNA within different classes of LN (P>0.05). Correlation of TIM-3 mRNA expression with serum IgA was highly significant (r=0.425, P=0.004), but was weakly correlated with total serum protein (r{sub s}=0.283, P=0.049) and serum albumin (r{sub s}=0.297, P=0.047). TIM-3 mRNA expression was weakly correlated with the Systemic Lupus Erythematosus Disease Activity Index (SLEDAI; r{sub s}=-0.272, P=0.032). Our results suggest that below-normal expression of TIM-3 mRNA in PBMC may be involved in the pathogenesis of SLE.

  1. Downregulation of TIM-3 mRNA expression in peripheral blood mononuclear cells from patients with systemic lupus erythematosus

    International Nuclear Information System (INIS)

    Cai, X.Z.; Huang, W.Y.; Qiao, Y.; Chen, Y.; Du, S.Y.; Chen, D.; Yu, S.; Liu, N.; Dou, L.Y.; Jiang, Y.

    2014-01-01

    The T-cell immunoglobulin and mucin domain (TIM) family is associated with autoimmune diseases, but its expression level in the immune cells of systemic lupus erythematosus (SLE) patients is not known. The aim of this study was to investigate whether the expression of TIM-3 mRNA is associated with pathogenesis of SLE. Quantitative real-time reverse transcription-polymerase chain reaction analysis (qRT-PCR) was used to determine TIM-1, TIM-3, and TIM-4 mRNA expression in peripheral blood mononuclear cells (PBMCs) from 132 patients with SLE and 62 healthy controls. The PBMC surface protein expression of TIMs in PBMCs from 20 SLE patients and 15 healthy controls was assayed by flow cytometry. Only TIM-3 mRNA expression decreased significantly in SLE patients compared with healthy controls (P<0.001). No significant differences in TIM family protein expression were observed in leukocytes from SLE patients and healthy controls (P>0.05). SLE patients with lupus nephritis (LN) had a significantly lower expression of TIM-3 mRNA than those without LN (P=0.001). There was no significant difference in the expression of TIM-3 mRNA within different classes of LN (P>0.05). Correlation of TIM-3 mRNA expression with serum IgA was highly significant (r=0.425, P=0.004), but was weakly correlated with total serum protein (r s =0.283, P=0.049) and serum albumin (r s =0.297, P=0.047). TIM-3 mRNA expression was weakly correlated with the Systemic Lupus Erythematosus Disease Activity Index (SLEDAI; r s =-0.272, P=0.032). Our results suggest that below-normal expression of TIM-3 mRNA in PBMC may be involved in the pathogenesis of SLE

  2. Comparative proteomics reveals that YK51, a 4-Hydroxypandurantin-A analogue, downregulates the expression of proteins associated with dengue virus infection

    Directory of Open Access Journals (Sweden)

    Wei-Lian Tan

    2018-01-01

    Full Text Available Dengue is endemic throughout tropical and subtropical regions of the world. Currently, there is no clinically approved therapeutic drug available for this acute viral infection. Although the first dengue vaccine Dengvaxia has been approved for use in certain countries, it is limited to those without a previous dengue infection while the safety and efficacy of the vaccine in those elderly and younger children still need to be identified. Therefore, it is becoming increasingly important to develop therapeutics/drugs to combat dengue virus (DENV infection. YK51 is a synthetic analogue of 4-Hydroxypandurantin A (a compound found in the crude extract of the rhizomes of Boesenbergia rotunda that has been extensively studied by our research group. It has been shown to possess outstanding antiviral activity due to its inhibitory activity against NS2B/NS3 DENV2 protease. However, it is not known how YK51 affects the proteome of DENV infected cells. Therefore, we performed a comparative proteomics analysis to identify changes in protein expression in DENV infected HepG2 cells treated with YK51. Classical two-dimensional gel electrophoresis followed by protein identification using tandem mass spectrometry was employed in this study. Thirty proteins were found to be down-regulated with YK51 treatment. In silico analysis predicted that the down-regulation of eight of these proteins may inhibit viral infection. Our results suggested that apart from inhibiting the NS2B/NS3 DENV2 protease, YK51 may also be causing the down-regulation of a number of proteins that may be responsible in, and/or essential to virus infection. However, functional characterization of these proteins will be necessary before we can conclusively determine their roles in DENV infection.

  3. Comparative proteomics reveals that YK51, a 4-Hydroxypandurantin-A analogue, downregulates the expression of proteins associated with dengue virus infection.

    Science.gov (United States)

    Tan, Wei-Lian; Lee, Yean Kee; Ho, Yen Fong; Yusof, Rohana; Abdul Rahman, Noorsaadah; Karsani, Saiful Anuar

    2018-01-01

    Dengue is endemic throughout tropical and subtropical regions of the world. Currently, there is no clinically approved therapeutic drug available for this acute viral infection. Although the first dengue vaccine Dengvaxia has been approved for use in certain countries, it is limited to those without a previous dengue infection while the safety and efficacy of the vaccine in those elderly and younger children still need to be identified. Therefore, it is becoming increasingly important to develop therapeutics/drugs to combat dengue virus (DENV) infection. YK51 is a synthetic analogue of 4-Hydroxypandurantin A (a compound found in the crude extract of the rhizomes of Boesenbergia rotunda ) that has been extensively studied by our research group. It has been shown to possess outstanding antiviral activity due to its inhibitory activity against NS2B/NS3 DENV2 protease. However, it is not known how YK51 affects the proteome of DENV infected cells. Therefore, we performed a comparative proteomics analysis to identify changes in protein expression in DENV infected HepG2 cells treated with YK51. Classical two-dimensional gel electrophoresis followed by protein identification using tandem mass spectrometry was employed in this study. Thirty proteins were found to be down-regulated with YK51 treatment. In silico analysis predicted that the down-regulation of eight of these proteins may inhibit viral infection. Our results suggested that apart from inhibiting the NS2B/NS3 DENV2 protease, YK51 may also be causing the down-regulation of a number of proteins that may be responsible in, and/or essential to virus infection. However, functional characterization of these proteins will be necessary before we can conclusively determine their roles in DENV infection.

  4. Downregulation of HMGA2 by the pan-deacetylase inhibitor panobinostat is dependent on hsa-let-7b expression in liver cancer cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Di Fazio, Pietro, E-mail: difazio@med.uni-marburg.de [Institute for Surgical Research, Philipps University of Marburg, Baldingerstrasse, 35043 Marburg (Germany); Montalbano, Roberta [Institute for Surgical Research, Philipps University of Marburg, Baldingerstrasse, 35043 Marburg (Germany); Neureiter, Daniel; Alinger, Beate [Institute of Pathology, Paracelsus Private Medical University of Salzburg, Salzburg (Austria); Schmidt, Ansgar [Institute for Pathology, Philipps University of Marburg, Marburg (Germany); Merkel, Anna Lena; Quint, Karl; Ocker, Matthias [Institute for Surgical Research, Philipps University of Marburg, Baldingerstrasse, 35043 Marburg (Germany)

    2012-09-10

    Inhibitors of protein deacetylases represent a novel therapeutic option for cancer diseases due to their effects on transcriptional regulation by interfering with histones acetylation and on several other cellular pathways. Recently, their ability to modulate several transcription factors and, interestingly, also co-factors, which actively participate in formation and modulation of transcription complexes was shown. We here investigate whether HMGA2 (High Mobility Group AT-2 hook), a nuclear non-histone transcriptional co-factor with known oncogenic properties, can be influenced by the novel pan-deacetylase inhibitor panobinostat (LBH589) in human hepatocellular carcinoma models. Panobinostat strongly downregulated HMGA2 in HepG2 and Hep3B cells; this effect was mediated by transcriptional upregulation and promotion of the maturation of the tumorsuppressor miRNA hsa-let-7b, which could inhibit HMGA2 expression via RNA interference pathways. siRNA knockdown of HMGA2 or transfection of hsa-let-7b mimicking oligonucleotides confirmed the role of HMGA2 in regulating cell proliferation and apoptosis in liver cancer cell lines. Co-incubation with panobinostat showed an additive effect on inhibition of cell proliferation using an impedance-based real-time cell analyzer. Treatment of HepG2 xenografts with panobinostat also led to a downregulation of HMGA2 in vivo. These findings show that pan-deacetylase inhibitors also modulate other signaling pathways and networks than histone modifications to influence cell fate. -- Highlights: Black-Right-Pointing-Pointer Panobinostat for the treatment of liver cancer. Black-Right-Pointing-Pointer Panobinostat meddles with miRNAs-dependent transcriptional and translational control. Black-Right-Pointing-Pointer Tumorsuppressor miRNA hsa-let-7b upregulation. Black-Right-Pointing-Pointer HMGA2 is downregulated via RNA interference pathways mediated by hsa-let-7b. Black-Right-Pointing-Pointer Panobinostat determines inhibition of

  5. Transcutaneous electrical nerve stimulation on Yongquan acupoint reduces CFA-induced thermal hyperalgesia of rats via down-regulation of ERK2 phosphorylation and c-Fos expression.

    Science.gov (United States)

    Yang, Lin; Yang, Lianxue; Gao, Xiulai

    2010-07-01

    Activation of extracellular signal-regulated kinase-1/2 (ERK1/2) and its involvement in regulating gene expression in spinal dorsal horn, cortical and subcortical neurons by peripheral noxious stimulation contribute to pain hypersensitivity. Transcutaneous electrical nerve stimulation (TENS) is a treatment used in physiotherapy practice to promote analgesia in acute and chronic inflammatory conditions. In this study, a total number of 114 rats were used for three experiments. Effects of complete Freund's adjuvant (CFA)-induced inflammatory pain hypersensitivity and TENS analgesia on ERK1/2 phosphorylation and c-Fos protein expression were examined by using behavioral test, Western blot, and immunostaining methods. We found that CFA injection caused an area of localized swelling, erythema, hypersensitivity to thermal stimuli, the decreased response time of hind paw licking (HPL), as well as upregulation of c-Fos protein expression and ERK2 phosphorylation in the ipsilateral spinal dorsal horn and the contralateral primary somatosensory area of cortex and the amygdala of rats. TENS on Yongquan acupoint for 20 min produced obvious analgesic effects as demonstrated with increased HPL to thermal stimuli of CFA-treated rats. In addition, TENS application suppressed the CFA-induced ERK2 activation and c-Fos protein expression. These results suggest that down-regulation of ERK2 phosphorylation and c-Fos expression were involved in TENS inhibition on CFA-induced thermal hyperalgesia of rats.

  6. Protective effect of naringenin in experimental ischemic stroke: down-regulated NOD2, RIP2, NF-κB, MMP-9 and up-regulated claudin-5 expression.

    Science.gov (United States)

    Bai, Xue; Zhang, Xiangjian; Chen, Linyu; Zhang, Jian; Zhang, Lan; Zhao, Xumeng; Zhao, Ting; Zhao, Yuan

    2014-08-01

    Inflammatory damage plays a pivotal, mainly detrimental role in cerebral ischemic pathogenesis and may represent a promising target for treatment. Naringenin (NG) has gained growing appreciation for its beneficial biological effects through its anti-inflammatory property. Whether this protective effect applies to cerebral ischemic injury, we therefore investigate the potential neuroprotective role of NG and the underlying mechanisms. Focal cerebral ischemia in male Sprague-Dawley rats was induced by permanent middle cerebral artery occlusion (pMCAO) and NG was pre-administered intragastrically once daily for four consecutive days before surgery. Neurological deficit, brain water content and infarct volume were measured at 24 h after stroke. Immunohistochemistry, Western blot and RT-qPCR were used to explore the anti-inflammatory potential of NG in the regulation of NOD2, RIP2 and NF-κB in ischemic cerebral cortex. Additionally, the activities of MMP-9 and claudin-5 were analyzed to detect NG's influence on blood-brain barrier. Compared with pMCAO and Vehicle groups, NG noticeably improved neurological deficit, decreased infarct volume and edema at 24 h after ischemic insult. Consistent with these results, our data also indicated that NG significantly downregulated the expression of NOD2, RIP2, NF-κB and MMP-9, and upregulated the expression of claudin-5 (P < 0.05). The results provided a neuroprotective profile of NG in cerebral ischemia, this effect was likely exerted by down-regulated NOD2, RIP2, NF-κB, MMP-9 and up-regulated claudin-5 expression.

  7. Sodium arsenite accelerates TRAIL-mediated apoptosis in melanoma cells through upregulation of TRAIL-R1/R2 surface levels and downregulation of cFLIP expression

    International Nuclear Information System (INIS)

    Ivanov, Vladimir N.; Hei, Tom K.

    2006-01-01

    AP-1/cJun, NF-κB and STAT3 transcription factors control expression of numerous genes, which regulate critical cell functions including proliferation, survival and apoptosis. Sodium arsenite is known to suppress both the IKK-NF-κB and JAK2-STAT3 signaling pathways and to activate the MAPK/JNK-cJun pathways, thereby committing some cancers to undergo apoptosis. Indeed, sodium arsenite is an effective drug for the treatment of acute promyelocytic leukemia with little nonspecific toxicity. Malignant melanoma is highly refractory to conventional radio- and chemotherapy. In the present study, we observed strong effects of sodium arsenite treatment on upregulation of TRAIL-mediated apoptosis in human and mouse melanomas. Arsenite treatment upregulated surface levels of death receptors, TRAIL-R1 and TRAIL-R2, through increased translocation of these proteins from cytoplasm to the cell surface. Furthermore, activation of cJun and suppression of NF-κB by sodium arsenite resulted in upregulation of the endogenous TRAIL and downregulation of the cFLIP gene expression (which encodes one of the main anti-apoptotic proteins in melanomas) followed by cFLIP protein degradation and, finally, by acceleration of TRAIL-induced apoptosis. Direct suppression of cFLIP expression by cFLIP RNAi also accelerated TRAIL-induced apoptosis in these melanomas, while COX-2 suppression substantially increased levels of both TRAIL-induced and arsenite-induced apoptosis. In contrast, overexpression of permanently active AKTmyr inhibited TRAIL-mediated apoptosis via downregulation of TRAIL-R1 levels. Finally, AKT overactivation increased melanoma survival in cell culture and dramatically accelerated growth of melanoma transplant in vivo, highlighting a role of AKT suppression for effective anticancer treatment

  8. Interleukin-4 inhibits RANKL-induced expression of NFATc1 and c-Fos: A possible mechanism for downregulation of osteoclastogenesis

    International Nuclear Information System (INIS)

    Kamel Mohamed, Saad Gad; Sugiyama, Eiji; Shinoda, Kouichiro; Hounoki, Hiroyuki; Taki, Hirofumi; Maruyama, Muneharu; Miyahara, Tatsuro; Kobayashi, Masashi

    2005-01-01

    Interleukin-4 (IL-4), an anti-inflammatory cytokine, has been shown to inhibit osteoclast differentiation. Therefore, this cytokine is considered to be a promising therapeutic applicant for bone-resorbing diseases such as rheumatoid arthritis (RA). Recently NFATc1, a transcription factor, has been shown to play critical roles in osteoclastogenesis. The aim of this study was to clarify the role of IL-4 on the intracellular signaling of NFATc1. A RAW264.7 monocyte/macrophage cell line and murine bone marrow precursors were differentiated into osteoclasts in the presence of receptor activator of nuclear factor κB ligand (RANKL) and/or macrophage colony-stimulating factor. Tartrate-resistant acid phosphatase (TRAP) staining and a pit assay using dentine were used for the identification of activated osteoclasts. The protein expression of IL-4 receptor, NFATc1, and c-Fos was determined by Western blot analysis. In addition, the gene expression of NFATc1 and c-Fos was determined by reverse transcription and polymerase chain reaction. The IL-4 receptor was constitutively expressed in RAW264.7 cells. RANKL induced osteoclast generation, as determined by TRAP staining and pit assay. IL-4 inhibited RANKL-induced osteoclastogenesis at low concentrations of 10 ng/ml and more. Interestingly, IL-4 potently inhibited RANKL-induced expression of NFATc1 at mRNA level. Furthermore, IL-4 inhibited c-Fos expression, which is shown to be responsible for NFATc1 expression, in time- and dose-dependent manners. In addition, IL-4 inhibited the RANKL-induced expression of NFATc1 and c-Fos in murine bone marrow cells. Thus, we suggest that IL-4 may downregulate osteoclastogenesis in part through inhibition of the expression of transcription factors, NFATc1 and c-Fos. These findings provide new insight into development of new medication for osteoporosis and RA

  9. Aflatoxin B1 inhibition in Aspergillus flavus by Aspergillus niger through down-regulating expression of major biosynthetic genes and AFB1 degradation by atoxigenic A. flavus.

    Science.gov (United States)

    Xing, Fuguo; Wang, Limin; Liu, Xiao; Selvaraj, Jonathan Nimal; Wang, Yan; Zhao, Yueju; Liu, Yang

    2017-09-01

    Twenty Aspergillus niger strains were isolated from peanuts and 14 strains were able to completely inhibit AFB 1 production with co-cultivation. By using a Spin-X centrifuge system, it was confirmed that there are some soluble signal molecules or antibiotics involved in the inhibition by A. niger, although they are absent during the initial 24h of A. flavus growth when it is sensitive to inhibition. In A. flavus, 19 of 20 aflatoxin biosynthetic genes were down-regulated by A. niger. Importantly, the expression of aflS was significantly down-regulated, resulting in a reduction of AflS/AflR ratio. The results suggest that A. niger could directly inhibit AFB 1 biosynthesis through reducing the abundance of aflS to aflR mRNAs. Interestingly, atoxigenic A. flavus JZ2 and GZ15 effectively degrade AFB 1 . Two new metabolites were identified and the key toxic lactone and furofuran rings both were destroyed and hydrogenated, meaning that lactonase and reductase might be involved in the degradation process. Copyright © 2017 Elsevier B.V. All rights reserved.

  10. Asclepiasterol, a novel C21 steroidal glycoside derived from Asclepias curassavica, reverses tumor multidrug resistance by down-regulating P-glycoprotein expression.

    Science.gov (United States)

    Yuan, Wei-Qi; Zhang, Rong-Rong; Wang, Jun; Ma, Yan; Li, Wen-Xue; Jiang, Ren-Wang; Cai, Shao-Hui

    2016-05-24

    Multidrug resistance (MDR) mediated by P-glycoprotein (P-gp) is a major cause of cancer therapy failure. In this study, we identified a novel C21 steroidal glycoside, asclepiasterol, capable of reversing P-gp-mediated MDR. Asclepiasterol (2.5 and 5.0μM) enhanced the cytotoxity of P-gp substrate anticancer drugs in MCF-7/ADR and HepG-2/ADM cells. MDR cells were more responsive to paclitaxel in the presence of asclepiasterol, and colony formation of MDR cells was only reduced upon treatment with a combination of asclepiasterol and doxorubicin. Consistent with these findings, asclepiasterol treatment increased the intracellular accumulation of doxorubicin and rhodamine 123 (Rh123) in MDR cells. Asclepiasterol decreased expression of P-gp protein without stimulating or suppressing MDR1 mRNA levels. Asclepiasterol-mediated P-gp suppression caused inhibition of ERK1/2 phosphorylation in two MDR cell types, and EGF, an activator of the MAPK/ERK pathway, reversed the P-gp down-regulation, implicating the MAPK/ERK pathway in asclepiasterol-mediated P-gp down-regulation. These results suggest that asclepiasterol could be developed as a modulator for reversing P-gp-mediated MDR in P-gp-overexpressing cancer variants.

  11. TSA-induced DNMT1 down-regulation represses hTERT expression via recruiting CTCF into demethylated core promoter region of hTERT in HCT116.

    Science.gov (United States)

    Choi, Jee-Hye; Min, Na Young; Park, Jina; Kim, Jin Hong; Park, Soo Hyun; Ko, Young Jong; Kang, Yoonsung; Moon, Young Joon; Rhee, Sangmyung; Ham, Seung Wook; Park, Ae Ja; Lee, Kwang-Ho

    2010-01-01

    Trichostatin A (TSA), an inhibitor of histone deacetylase, is a well-known antitumor agent that effectively and selectively induces tumor growth arrest and apoptosis. Recently, it was reported that hTERT is one of the primary targets for TSA-induced apoptosis in cancer cells but the mechanism of which has not yet been elucidated. In the present study, to better understand the epigenetic regulation mechanism responsible for the repression of hTERT by TSA, we examined expression of hTERT in the HCT116 colon cancer cell line after treatment with TSA and performed site-specific CpG methylation analysis of the hTERT promoter. We found that TSA-induced the demethylation of site-specific CpGs on the promoter of hTERT, which was caused by down-regulation of DNA methyltransferase 1 (DNMT1). Among the demethylated region, the 31st-33rd CpGs contained a binding site for CTCF, an inhibitor of hTERT transcription. ChIP analysis revealed that TSA-induced demethylation of the 31st-33rd CpGs promoted CTCF binding on hTERT promoter, leading to repression of hTERT. Taken together, down-regulation of DNMT1 by TSA caused demethylation of a CTCF binding site on the hTERT promoter, the result of which was repression of hTERT via recruitment of CTCF to the promoter. Copyright 2009 Elsevier Inc. All rights reserved.

  12. Neurotrophin responsiveness of sympathetic neurons is regulated by rapid mobilization of the p75 receptor to the cell surface through TrkA activation of Arf6.

    Science.gov (United States)

    Edward Hickman, F; Stanley, Emily M; Carter, Bruce D

    2018-05-22

    The p75 neurotrophin receptor (p75NTR) plays an integral role in patterning the sympathetic nervous system during development. Initially, p75NTR is expressed at low levels as sympathetic axons project toward their targets, which enables neurotrophin-3 (NT3) to activate TrkA receptors and promote growth. Upon reaching nerve growth factor (NGF) producing tissues, p75NTR is up regulated resulting in formation of TrkA-p75 complexes, which are high affinity binding sites selective for NGF, thereby blunting NT3 signaling. The level of p75NTR expressed on the neuron surface is instrumental in regulating trophic factor response; however, the mechanisms by which p75NTR expression is regulated are poorly understood. Here, we demonstrate a rapid, translation independent increase in surface expression of p75NTR in response to NGF in rat sympathetic neurons. p75NTR was mobilized to the neuron surface from GGA3-postitive vesicles through activation of the GTPase Arf6, which was stimulated by NGF, but not NT3 binding to TrkA. Arf6 activation required PI3 kinase activity and was prevented by an inhibitor of the cytohesin family of Arf6 GEFs. Overexpression of a constitutively active Arf6 mutant (Q67L) was sufficient to significantly increase surface expression of p75NTR even in the absence of NGF. Functionally, expression of active Arf6 markedly attenuated the ability of NT3 to promote neuronal survival and neurite outgrowth while the NGF response was unaltered. These data suggest that NGF activation of Arf6 through TrkA is critical for the increase in p75NTR surface expression that enables the switch in neurotrophin responsiveness during development in the sympathetic nervous system. SIGNIFICANCE STATEMENT p75NTR is instrumental in the regulation of neuronal survival and apoptosis during development and is also implicated as a contributor to aberrant neurodegeneration in numerous conditions. Therefore, a better understanding of the mechanisms that mediate p75NTR surface

  13. Promoter-region hypermethylation and expression downregulation of Yy1 (Yin yang 1) in preneoplastic liver lesions in a thioacetamide rat hepatocarcinogenesis model

    International Nuclear Information System (INIS)

    Abe, Hajime; Ogawa, Takashi; Wang, Liyun; Kimura, Masayuki; Tanaka, Takeshi; Morita, Reiko; Yoshida, Toshinori; Shibutani, Makoto

    2014-01-01

    Thioacetamide (TAA) has been used to develop a rodent model for hepatocarcinogenesis. To determine the genes with epigenetic modifications in early hepatocarcinogenesis, we did a genome-wide scan for hypermethylated promoter regions using CpG island microarrays in TAA-promoted rat liver tissue. Eight genes were selected based on the microarray profile; of these, Yy1 and Wdr45b were confirmed to be hypermethylated by methylation-specific polymerase chain reaction (PCR) and pyrosequencing and downregulated by real-time reverse transcription PCR. Non-neoplastic liver cells had nuclear Yy1 immunoreactivity, while preneoplastic foci with glutathione S-transferase placental form (GST-P) immunoreactivity had decreased Yy1 immunoreactivity. The incidence of these foci was proportional to the dose of TAA administered. Co-expression analysis of gene products downstream of Yy1 revealed increased nuclear phospho-c-Myc + foci as well as nuclear and cytoplasmic p21 Cip1+ foci in Yy1 − or GST-P + foci in response to TAA-promotion dose. Although the absolute number of cells was low, the incidence of death receptor 5 − foci was increased in Yy1 − foci in proportion to the TAA dose. Yy1 − /GST-P + foci revealed a higher number of proliferating cell nuclear antigen (PCNA)-immunoreactive cells than Yy1 + /GST-P + foci, while cleaved caspase-3 + cells were unchanged between Yy1 – /GST-P + and Yy1 + /GST-P + foci. In the case of Wdr45b, most GST-P + foci were Wdr45b – and were not increased by TAA promotion. These results suggest involvement of Yy1 in the epigenetic gene regulation at the early stages of TAA promoted cell proliferation and concomitant cell cycle arrest in preneoplastic lesions. - Highlights: • Epigenetically downregulated genes were searched in TAA-promnoted rat livers. • Yy1 and Wdr45b showed promoter-region hypermethylation and mRNA downregulation. • TAA promoted increase of preneoplastic Yy1 – /GST-P + foci showing high proliferation. • TAA

  14. Peroxireduxin-4 is Over-Expressed in Colon Cancer and its Down-Regulation Leads to Apoptosis

    Directory of Open Access Journals (Sweden)

    Sandra M. Leydold

    2011-01-01

    Full Text Available The objective of this study was to gain insight into the biological basis of colon cancer progression by characterizing gene expression differences between normal colon epithelium, corresponding colorectal primary tumors and metastases. We found a close similarity in gene expression patterns between primary tumors and metastases, indicating a correlation between gene expression and morphological characteristics. PRDX4 was identified as highly expressed both in primary colon tumors and metastases, and selected for further characterization. Our study revealed that “Prdx4” (PrxIV, AOE372 shows functional similarities to other Prx family members by negatively affecting apoptosis induction in tumor cells. In addition, our study links Prdx4 with Hif-1α, a key regulatory factor of angiogenesis. Targeting Prdx4 may be an attractive approach in cancer therapy, as its inhibition is expected to lead to induction of apoptosis and blockage of Hif-1α-mediated tumor angiogenesis.

  15. Peroxisome proliferator-activated receptor α agonist-induced down-regulation of hepatic glucocorticoid receptor expression in SD rats

    International Nuclear Information System (INIS)

    Chen Xiang; Li Ming; Sun Weiping; Bi Yan; Cai Mengyin; Liang Hua; Yu Qiuqiong; He Xiaoying; Weng Jianping

    2008-01-01

    It was reported that glucocorticoid production was inhibited by fenofibrate through suppression of type-1 11β-hydroxysteroid dehydrogenase gene expression in liver. The inhibition might be a negative-feedback regulation of glucocorticoid receptor (GR) activity by peroxisome proliferator-activated receptor alpha (PPARα), which is quickly induced by glucocorticoid in the liver. However, it is not clear if GR expression is changed by fenofibrate-induced PPARα activation. In this study, we tested this possibility in the liver of Sprague-Dawley rats. GR expression was reduced by fenofibrate in a time- and does-dependent manner. The inhibition was observed in liver, but not in fat and muscle. The corticosterone level in the blood was increased significantly by fenofibrate. These effects of fenofibrate were abolished by PPARα inhibitor MK886, suggesting that fenofibrate activated through PPARα. In conclusion, inhibition of GR expression may represent a new molecular mechanism for the negative feedback regulation of GR activity by PPARα

  16. Induced ICER Iγ down-regulates cyclin A expression and cell proliferation in insulin-producing β cells

    International Nuclear Information System (INIS)

    Inada, Akari; Weir, Gordon C.; Bonner-Weir, Susan

    2005-01-01

    We have previously found that cyclin A expression is markedly reduced in pancreatic β-cells by cell-specific overexpression of repressor inducible cyclic AMP early repressor (ICER Iγ) in transgenic mice. Here we further examined regulatory effects of ICER Iγ on cyclin A gene expression using Min6 cells, an insulin-producing cell line. The cyclin A promoter luciferase assay showed that ICER Iγ directly repressed cyclin A gene transcription. In addition, upon ICER Iγ overexpression, cyclin A mRNA levels markedly decreased, thereby confirming an inhibitory effect of ICER Iγ on cyclin A expression. Suppression of cyclin A results in inhibition of BrdU incorporation. Under normal culture conditions endogenous cyclin A is abundant in these cells, whereas ICER is hardly detectable. However, serum starvation of Min6 cells induces ICER Iγ expression with a concomitant very low expression level of cyclin A. Cyclin A protein is not expressed unless the cells are in active DNA replication. These results indicate a potentially important anti-proliferative effect of ICER Iγ in pancreatic β cells. Since ICER Iγ is greatly increased in diabetes as well as in FFA- or high glucose-treated islets, this effect may in part exacerbate diabetes by limiting β-cell proliferation

  17. Leishmania donovani resides in modified early endosomes by upregulating Rab5a expression via the downregulation of miR-494

    Science.gov (United States)

    Verma, Jitender Kumar; Rastogi, Ruchir

    2017-01-01

    Several intracellular pathogens arrest the phagosome maturation in the host cells to avoid transport to lysosomes. In contrast, the Leishmania containing parasitophorous vacuole (PV) is shown to recruit lysosomal markers and thus Leishmania is postulated to be residing in the phagolysosomes in macrophages. Here, we report that Leishmania donovani specifically upregulates the expression of Rab5a by degrading c-Jun via their metalloprotease gp63 to downregulate the expression of miR-494 in THP-1 differentiated human macrophages. Our results also show that miR-494 negatively regulates the expression of Rab5a in cells. Subsequently, L. donovani recruits and retains Rab5a and EEA1 on PV to reside in early endosomes and inhibits transport to lysosomes in human macrophages. Similarly, we have also observed that Leishmania PV also recruits Rab5a by upregulating its expression in human PBMC differentiated macrophages. However, the parasite modulates the endosome by recruiting Lamp1 and inactive pro-CathepsinD on PV via the overexpression of Rab5a in infected cells. Furthermore, siRNA knockdown of Rab5a or overexpression of miR-494 in human macrophages significantly inhibits the survival of the parasites. These results provide the first mechanistic insights of parasite-mediated remodeling of endo-lysosomal trafficking to reside in a specialized early endocytic compartment. PMID:28650977

  18. Jolkinolide B inhibits glycolysis by downregulating hexokinase 2 expression through inactivating the Akt/mTOR pathway in non-small cell lung cancer cells.

    Science.gov (United States)

    Gao, Xiang; Han, Han

    2018-06-01

    Jolkinolide B (JB), a bioactive compound isolated from herbal medicine, has been found to inhibit tumor growth by altering glycolysis. However, whether glycolysis is influenced by JB in non-small cell lung cancer (NSCLC) cells and the mechanism remain unknown. The aim of the present study was to evaluate the effect of JB on the glycolysis in NSCLC cells and the underlying molecular mechanism. The results showed that JB treatment inhibited cell viability of A549 and H1299 cells in a concentration-dependent manner. JB reduced the glucose consumption, lactate production, and HK2 expression. The expressions of p-Akt and p-mTOR were also decreased by JB treatment. Knockdown of HK2 reduced glucose consumption and lactate production. Inhibition of the Akt/mTOR pathway decreased HK2 expression and inhibited glycolysis. In conclusion, the results indicated that JB inhibits glycolysis by down-regulating HK2 expression through inactivating the Akt/mTOR pathway in NSCLC cells, suggesting that JB might be a potential therapeutic agent for the treatment of NSCLC. © 2018 Wiley Periodicals, Inc.

  19. Overexpression of LncRNA AC067945.2 Down-Regulates Collagen Expression in Skin Fibroblasts and Possibly Correlates with the VEGF and Wnt Signalling Pathways.

    Science.gov (United States)

    Chen, Ling; Li, Jingyun; Li, Qian; Li, Xue; Gao, Yanli; Hua, Xiangdong; Zhou, Bei; Li, Jun

    2018-01-01

    Long non-coding RNAs (lncRNAs) are thought to play crucial roles in human diseases. However, the function of lncRNAs in hypertrophic scar formation remains poorly understood. Utilizing qRT-PCR, we explored the expression changes of AC067945.2. Overexpression of AC067945.2 in normal skin fibroblasts was performed by transient plasmid transfection. Western blot was used to check the proteins' expression changes. Cell Counting Kit-8 (CCK-8) assay and Annexin V/7-AAD staining were used to examine cell proliferation and apoptosis, respectively. mRNA-seq was applied to dissect the differentially expressed mRNAs in AC067945.2 overexpressed cells. We also performed ELISA to detect the VEGF secretion. AC067945.2 was down-regulated in hypertrophic scar tissues. Overexpression of AC067945.2 did not affect cell proliferation, but it mildly promoted early apoptosis in normal skin fibroblasts. Furthermore, AC067945.2 overexpression inhibited the expression of COL1A1, COL1A2, COL3A1 and α-SMA proteins. Transforming growth factor-β1 (TGF-β1) could inhibit the expression of AC067945.2. Based on mRNA-seq data, compared with mRNAs in the control group, 138 mRNAs were differentially expressed, including 14 up-regulated and 124 down-regulated transcripts, in the AC067945.2 overexpression group. Gene ontology and pathway analyses revealed that AC067945.2 overexpression was correlated with developmental processes, binding, extracellular region, and the vascular endothelial cell growth factor (VEGF) and Wnt signalling pathways. ELISA confirmed that AC067945.2 overexpression could repress VEGF secretion. Taken together, our data uncovered the functions of a novel lncRNA AC067945.2, which might help us understand the mechanisms regulated by AC067945.2 in the pathogenesis of hypertrophic scar formation. © 2018 The Author(s). Published by S. Karger AG, Basel.

  20. Matrine suppresses airway inflammation by downregulating SOCS3 expression via inhibition of NF-κB signaling in airway epithelial cells and asthmatic mice

    Energy Technology Data Exchange (ETDEWEB)

    Sun, Daqing [Department of Respiration, Xi’an Children’s Hospital, Xi’an 710003 (China); Wang, Jing [Department of Neonatology, Xi’an Children’s Hospital, Xi’an 710003 (China); Yang, Niandi [Outpatient Department, School of Aerospace Engineering, Air Force Engineering University, Xi’an 710038 (China); Ma, Haixin, E-mail: drhaixinma@163.com [Department of Quality Control, Xi’an Children’s Hospital, Xi’an 710003 (China)

    2016-08-12

    Matrine has been demonstrated to attenuate allergic airway inflammation. Elevated suppressor of cytokine signaling 3 (SOCS3) was correlated with the severity of asthma. The aim of this study was to investigate the effect of matrine on SOCS3 expression in airway inflammation. In this study, we found that matrine significantly inhibited OVA-induced AHR, inflammatory cell infiltration, goblet cell differentiation, and mucous production in a dose-dependent manner in mice. Matrine also abrogated the level of interleukin (IL)-4 and IL-13, but enhanced interferon (IFN)-γ expression, both in BALF and in lung homogenates. Furthermore, matrine impeded TNF-α-induced the expression of IL-6 and adhesion molecules in airway epithelial cells (BEAS-2B and MLE-12). Additionally, we found that matrine inhibited SOCS3 expression, both in asthmatic mice and TNF-α-stimulated epithelial cells via suppression of the NF-κB signaling pathway by using pcDNA3.1-SOCS3 plasmid, SOCS3 siRNA, or nuclear factor kappa-B (NF-κB) inhibitor PDTC. Conclusions: Matrine suppresses airway inflammation by downregulating SOCS3 expression via inhibition of NF-κB signaling in airway epithelial cells and asthmatic mice. - Highlights: • Matrine attenuates asthmatic symptoms and regulates Th1/Th2 balance in vivo. • Matrine suppresses inflammation responses in vitro. • Matrine decreases SOCS3 expression both in vivo and in vitro. • Matrine inhibits SOCS3 expression by suppressing NF-κB signaling.

  1. Downregulation of cytochrome c oxidase subunit 7A1 expression is important in enhancing cell proliferation in adenocarcinoma cells

    International Nuclear Information System (INIS)

    Mishra, Nawneet; Timilsina, Uddhav; Ghimire, Dibya; Dubey, Ravi C.; Gaur, Ritu

    2017-01-01

    Mitochondrial Dysfunction has been implicated in multiple human diseases, including cancer. Among all cancer, lung cancer is the most common type of cancer worldwide with low survival rates. Mammals possess multiple subunits of the mitochondrial enzyme Cytochrome C oxidase (COX). The COX subunits are expressed in a tissue specific manner and have been implicated in cancer cell metabolism although their molecular and regulatory mechanisms are not clearly understood. In this study, we aimed at identifying novel gene signatures in lung cancer. We performed extensive analysis of seven different Gene Expression Omnibus (GEO) datasets pertaining to different stages of lung adenocarcinoma and identified that multiple subunits of COX genes are differentially expressed in these patients. Amongst all COX genes, the expression of COX7A1 gene was observed to be highly down regulated in these patients. In order to validate the GEO datasets, we looked at the expression of multiple COX genes using quantitative real time PCR (qPCR) using human lung adenocarcinoma cell line A549. Our results confirmed that COX 7A1 gene expression was indeed highly reduced in these cells. Overexpression of COX7A1 in human lung cancer cells led to inhibition of cell proliferation and increase in cell death via apoptosis. These results indicated that low level of COX7A1 gene expression is essential to regulate cell viability and inhibit cell death in lung adenocarcinoma. Our study has identified COX7A1 as a novel gene that might play a crucial role in the etiology of lung adenocarcinoma and can serve as a biomarker for lung cancer disease progression.

  2. Down-regulation of LncRNA TUG1 enhances radiosensitivity in bladder cancer via suppressing HMGB1 expression

    OpenAIRE

    Jiang, Huijuan; Hu, Xigang; Zhang, Hongzhi; Li, Wenbo

    2017-01-01

    Background Long non-coding RNAs (lncRNAs) have been reported to regulate the sensitivity of different cancer cells to chemoradiotherapy. Aberrant expression of lncRNA Taurine-upregulated gene 1 (TUG1) has been found to be involved in the development of bladder cancer, however, its function and underlying mechanism in the radioresistance of bladder cancer remains unclear. Methods Quantitative real-time PCR (qRT-PCR) was conducted to measure the expression of TUG1 and HMGB1 mRNA in bladder canc...

  3. SU-F-T-675: Down-Regulating the Expression of Cdc42 and Inhibition of Migration of A549 with Combined Treatment of Ionizing Radiation and Sevoflurane

    International Nuclear Information System (INIS)

    Feng, Y; Feng, J; Huang, Z

    2016-01-01

    Purpose: Cdc42 is involved in cell transformation, proliferation, invasion and metastasis of human cancer cells. Cdc42 overexpression has been reported in several types of cancers. This study investigated the combined treatment effects of ionizing radiation and sevoflurane on down-regulating Cdc42 expression and suppressing migration of human adenocarcinoma cell line A549. Methods: Samples of A549 cells with Cdc42 overexpression were created and Cdc42 expression was determined by Western blotting. Increase of migration speed by Cdc42-HA overexpression was confirmed with an initial in-vitro scratch assay. The cells grown in culture media were separated into 2 groups of 6 samples: one for the control and the other was treated with 4% sevoflurane for 5hrs prior to a single-fraction radiation of 4Gy using a 6MV beam. Cell migration speeds of the 2 groups were measured with an initial in-vitro scratch assay. The scratch was created with a pipette tip immediately after treatment and images at 4 post-treatment time points (0h, 3h, 6h, 12h) were acquired. The distance between the two separated sides at 0h was used as reference and subsequent changes of the distance over time was defined as the cell migration speed. Image processing and measurement were performed with an in-house software. The experiment was repeated three times independently to evaluate the repeatability and reliability. Statistical analysis was performed with SPSS 19.0. Results: Western blotting showed the treatment down-regulated Cdc42 overexpression. Quantitative analysis and two-tailed t-test showed that cell migration speed of the treated group was higher than the control group at all time points after treatment (p < 0.02). Conclusion: Combined treatment of 6MV photon and sevoflurane can cause the effects of down-regulating Cdc42 overexpression and decrease of migration speed of A549 cells which provides potential of clinical benefit for the cancer therapy. More investigation is needed to further

  4. SU-F-T-675: Down-Regulating the Expression of Cdc42 and Inhibition of Migration of A549 with Combined Treatment of Ionizing Radiation and Sevoflurane

    Energy Technology Data Exchange (ETDEWEB)

    Feng, Y [East Carolina University, Greenville, NC (United States); Feng, J [Tianjin University, Tianjin (China); Huang, Z [East Carolina University, Greenville, NC (United States)

    2016-06-15

    Purpose: Cdc42 is involved in cell transformation, proliferation, invasion and metastasis of human cancer cells. Cdc42 overexpression has been reported in several types of cancers. This study investigated the combined treatment effects of ionizing radiation and sevoflurane on down-regulating Cdc42 expression and suppressing migration of human adenocarcinoma cell line A549. Methods: Samples of A549 cells with Cdc42 overexpression were created and Cdc42 expression was determined by Western blotting. Increase of migration speed by Cdc42-HA overexpression was confirmed with an initial in-vitro scratch assay. The cells grown in culture media were separated into 2 groups of 6 samples: one for the control and the other was treated with 4% sevoflurane for 5hrs prior to a single-fraction radiation of 4Gy using a 6MV beam. Cell migration speeds of the 2 groups were measured with an initial in-vitro scratch assay. The scratch was created with a pipette tip immediately after treatment and images at 4 post-treatment time points (0h, 3h, 6h, 12h) were acquired. The distance between the two separated sides at 0h was used as reference and subsequent changes of the distance over time was defined as the cell migration speed. Image processing and measurement were performed with an in-house software. The experiment was repeated three times independently to evaluate the repeatability and reliability. Statistical analysis was performed with SPSS 19.0. Results: Western blotting showed the treatment down-regulated Cdc42 overexpression. Quantitative analysis and two-tailed t-test showed that cell migration speed of the treated group was higher than the control group at all time points after treatment (p < 0.02). Conclusion: Combined treatment of 6MV photon and sevoflurane can cause the effects of down-regulating Cdc42 overexpression and decrease of migration speed of A549 cells which provides potential of clinical benefit for the cancer therapy. More investigation is needed to further

  5. Indomethacin treatment prior to pentylenetetrazole-induced seizures downregulates the expression of il1b and cox2 and decreases seizure-like behavior in zebrafish larvae.

    Science.gov (United States)

    Barbalho, Patrícia Gonçalves; Lopes-Cendes, Iscia; Maurer-Morelli, Claudia Vianna

    2016-03-09

    It has been demonstrated that the zebrafish model of pentylenetetrazole (PTZ)-evoked seizures and the well-established rodent models of epilepsy are similar pertaining to behavior, electrographic features, and c-fos expression. Although this zebrafish model is suitable for studying seizures, to date, inflammatory response after seizures has not been investigated using this model. Because a relationship between epilepsy and inflammation has been established, in the present study we investigated the transcript levels of the proinflammatory cytokines interleukin-1 beta (il1b) and cyclooxygenase-2 (cox2a and cox2b) after PTZ-induced seizures in the brain of zebrafish 7 days post fertilization. Furthermore, we exposed the fish to the nonsteroidal anti-inflammatory drug indomethacin prior to PTZ, and we measured its effect on seizure latency, number of seizure behaviors, and mRNA expression of il1b, cox2b, and c-fos. We used quantitative real-time PCR to assess the mRNA expression of il1b, cox2a, cox2b, and c-fos, and visual inspection was used to monitor seizure latency and the number of seizure-like behaviors. We found a short-term upregulation of il1b, and we revealed that cox2b, but not cox2a, was induced after seizures. Indomethacin treatment prior to PTZ-induced seizures downregulated the mRNA expression of il1b, cox2b, and c-fos. Moreover, we observed that in larvae exposed to indomethacin, seizure latency increased and the number of seizure-like behaviors decreased. This is the first study showing that il1b and cox-2 transcripts are upregulated following PTZ-induced seizures in zebrafish. In addition, we demonstrated the anticonvulsant effect of indomethacin based on (1) the inhibition of PTZ-induced c-fos transcription, (2) increase in seizure latency, and (3) decrease in the number of seizure-like behaviors. Furthermore, anti-inflammatory effect of indomethacin is clearly demonstrated by the downregulation of the mRNA expression of il1b and cox2b. Our results

  6. Understanding Selective Downregulation of c-Myc Expression through Inhibition of General Transcription Regulators in Multiple Myeloma

    Science.gov (United States)

    2014-06-01

    87 MG cells were cultured in Eagle’s minimum essential medium (EMEM) modi - fied to contain Earle’s Balanced Salt Solution, nonessential amino acids, 2...engineered to ectopically express firefly luciferase and mCherry, allowing surrogate measurement of tumor growth in vivo. Nonobese diabetic severe combined

  7. Phenobarbital reduces blood glucose and gluconeogenesis through down-regulation of phosphoenolpyruvate carboxykinase (GTP) gene expression in rats.

    Science.gov (United States)

    Oda, Hiroaki; Okuda, Yuji; Yoshida, Yukiko; Kimura, Noriko; Kakinuma, Atsushi

    2015-10-23

    The regulatory mechanism of phosphoenolpyruvate carboykinase (GTP) (EC 4.1.1.32) (PEPCK) gene expression and gluconeogenesis by phenobarbital (PB), which is known to induce drug-metabolizing enzymes, was investigated. Higher level of PEPCK mRNA was observed in spherical rat primary hepatocytes on EHS-gel than monolayer hepatocytes on TIC (type I collagen). We found that PB directly suppressed PEPCK gene expression in spherical hepatocytes on EHS-gel, but not in those on TIC. PB strongly suppressed cAMP-dependent induction of PEPCK gene expression. Tyrosine aminotransferase (TAT), another gluconeogenic enzyme, was induced by cAMP, but not suppressed by PB. Chronic administration of PB reduced hepatic PEPCK mRNA in streptozotocin-induced diabetic and nondiabetic rats, and PB reduced blood glucose level in diabetic rats. Increased TAT mRNA in diabetic rats was not suppressed by PB. These results indicated that PB-dependent reduction is specific to PEPCK. From pyrvate challenge test, PB suppressed the increased gluconeogenesis in diabetic rats. PEPCK gene promoter activity was suppressed by PB in HepG2 cells. In conclusion, we found that spherical hepatocytes cultured on EHS-gel are capable to respond to PB to suppress PEPCK gene expression. Moreover, our results indicate that hypoglycemic action of PB result from transcriptional repression of PEPCK gene and subsequent suppression of gluconeogenesis. Copyright © 2015. Published by Elsevier Inc.

  8. TNF-a-induced down-regulation of CDX2 suppresses MEP1A expression in colitis

    DEFF Research Database (Denmark)

    Coskun, Mehmet; Olsen, Anders Krüger; Holm, Thomas Lindebo

    2012-01-01

    was investigated in colonic biopsies of ulcerative colitis (UC) patients and in dextran sodium sulfate (DSS)-induced colitis. CDX2 protein expression was investigated by immunoblotting and immunohistochemical procedures. CDX2 and MEP1A regulation was examined in TNF-a-treated Caco-2 cells by reverse transcription...

  9. c-Fos downregulation positively regulates EphA5 expression in a congenital hypothyroidism rat model.

    Science.gov (United States)

    Song, Honghua; Zheng, Yuqin; Cai, Fuying; Ma, Yanyan; Yang, Jingyue; Wu, Youjia

    2018-04-01

    The EphA5 receptor is well established as an axon guidance molecule during neural system development and plays an important role in dendritic spine formation and synaptogenesis. Our previous study has showed that EphA5 is decreased in the developing brain of congenital hypothyroidism (CH) and the EphA5 promoter methylation modification participates in its decrease. c-Fos, a well-kown transcription factor, has been considered in association with brain development. Bioinformatics analysis showed that the EphA5 promoter region contained five putative c-fos binding sites. The chromatin immunoprecipitation (ChIP) assays were used to assess the direct binding of c-fos to the EphA5 promoter. Furthermore, dual-luciferase assays showed that these three c-fos protein binding sites were positive regulatory elements for EphA5 expression in PC12 cells. Moreover, We verified c-fos positively regulation for EphA5 expression in CH model. Q-PCR and Western blot showed that c-fos overexpression could upregulate EphA5 expression in hippocampal neurons of rats with CH. Our results suggest that c-fos positively regulates EphA5 expression in CH rat model.

  10. Understanding Selective Downregulation of c-Myc Expression through Inhibition of General Transcription Regulators in Multiple Myeloma

    Science.gov (United States)

    2015-06-01

    We next tested whether BET bromodomain inhibition mitigated the acti- vation of proadhesion pathways in aortic endothelium, which oc- curs during the...tinuum of activity as Myc flickers on and off of weakly bound, weakly expressed promoters, but stays longer or more frequently at high output promoters

  11. BNN27, a 17-Spiroepoxy Steroid Derivative, Interacts With and Activates p75 Neurotrophin Receptor, Rescuing Cerebellar Granule Neurons from Apoptosis.

    Science.gov (United States)

    Pediaditakis, Iosif; Kourgiantaki, Alexandra; Prousis, Kyriakos C; Potamitis, Constantinos; Xanthopoulos, Kleanthis P; Zervou, Maria; Calogeropoulou, Theodora; Charalampopoulos, Ioannis; Gravanis, Achille

    2016-01-01

    Neurotrophin receptors mediate a plethora of signals affecting neuronal survival. The p75 pan-neurotrophin receptor controls neuronal cell fate after its selective activation by immature and mature isoforms of all neurotrophins. It also exerts pleiotropic effects interacting with a variety of ligands in different neuronal or non-neuronal cells. In the present study, we explored the biophysical and functional interactions of a blood-brain-barrier (BBB) permeable, C17-spiroepoxy steroid derivative, BNN27, with p75 NTR receptor. BNN27 was recently shown to bind to NGF high-affinity receptor, TrkA. We now tested the p75 NTR -mediated effects of BNN27 in mouse Cerebellar Granule Neurons (CGNs), expressing p75 NTR , but not TrkA receptors. Our findings show that BNN27 physically interacts with p75 NTR receptors in specific amino-residues of its extracellular domain, inducing the recruitment of p75 NTR receptor to its effector protein RIP2 and the simultaneous release of RhoGDI in primary neuronal cells. Activation of the p75 NTR receptor by BNN27 reverses serum deprivation-induced apoptosis of CGNs resulting in the decrease of the phosphorylation of pro-apoptotic JNK kinase and of the cleavage of Caspase-3, effects completely abolished in CGNs, isolated from p75 NTR null mice. In conclusion, BNN27 represents a lead molecule for the development of novel p75 NTR ligands, controlling specific p75 NTR -mediated signaling of neuronal cell fate, with potential applications in therapeutics of neurodegenerative diseases and brain trauma.

  12. Down-regulation of the expression of CCAAT/enhancer binding protein α gene in cervical squamous cell carcinoma

    International Nuclear Information System (INIS)

    Pan, Zemin; Shao, Renfu; Zheng, Weinan; Zhang, Jinli; Gao, Rui; Li, Dongmei; Guo, Xiaoqing; Han, Hu; Li, Feng; Qu, Shen

    2014-01-01

    Cervical carcinoma is the second most common cancer and is an important cause of death in women worldwide. CCAAT/enhancer binding proteins (C/EBPs) are a family of transcription factors that regulate cellular differentiation and proliferation in a variety of tissues. However, the role of C/EBPα gene in cervical cancer is still not clear. We investigated the expression of C/EBPα gene in cervical squamous cell carcinoma. C/EBPα mRNA level was measured by real-time quantitative RT-PCR in cervical cancer tissues and their adjacent normal tissues. C/EBPα protein level was measured by immunohistochemistry. Methylation in the promoter of C/EBPα gene was detected by MALDI TOF MassARRAY. We transfected HeLa cells with C/EBPα expression vector. C/EBPα expression in HeLa cells was examined and HeLa cell proliferation was measured by MTT assay and HeLa cells migration was analyzed by matrigel-coated transwell migration assays. There were significant difference in C/EBPα protein expression between chronic cervicitis and cervical carcinoma (P < 0.001). CEBPα mRNA level was significantly lower in cervical cancer tissues than in normal cervical tissues (P < 0.01). Methylation of the promoter of CEBPα gene in CpG 5, CpG-14.15, CpG-19.20 were significantly higher in cervical cancer than in normal cervical tissues (P < 0.05, P < 0.01, P < 0.05, respectively). CEBPα pcDNA3.1 construct transfected into HeLa cells inhibited cell proliferation and decreased cell migration. Our results indicate that reduced C/EBPα gene expression may play a role in the development of cervical squamous cell carcinoma

  13. Aversive odorant causing appetite decrease downregulates tyrosine decarboxylase gene expression in the olfactory receptor neuron of the blowfly, Phormia regina

    Science.gov (United States)

    Ishida, Yuko; Ozaki, Mamiko

    2012-01-01

    In the blowfly Phormia regina, exposure to d-limonene for 5 days during feeding inhibits proboscis extension reflex behavior due to decreasing tyramine (TA) titer in the brain. TA is synthesized by tyrosine decarboxylase (Tdc) and catalyzed into octopamine (OA) by TA ß-hydroxylase (Tbh). To address the mechanisms of TA titer regulation in the blowfly, we cloned Tdc and Tbh cDNAs from P. regina (PregTdc and PregTbh). The deduced amino acid sequences of both proteins showed high identity to those of the corresponding proteins from Drosophila melanogaster at the amino acid level. PregTdc was expressed in the antenna, labellum, and tarsus whereas PregTbh was expressed in the head, indicating that TA is mainly synthesized in the sensory organs whereas OA is primarily synthesized in the brain. d-Limonene exposure significantly decreased PregTdc expression in the antenna but not in the labellum and the tarsus, indicating that PregTdc expressed in the antenna is responsible for decreasing TA titer. PregTdc-like immunoreactive material was localized in the thin-walled sensillum. In contrast, the OA/TA receptor (PregOAR/TAR) was localized to the thick-walled sensillum. The results indicated that d-limonene inhibits PregTdc expression in the olfactory receptor neurons in the thin-walled sensilla, likely resulting in reduced TA levels in the receptor neurons in the antenna. TA may be transferred from the receptor neuron to the specific synaptic junction in the antennal lobe of the brain through the projection neurons and play a role in conveying the aversive odorant information to the projection and local neurons.

  14. DJ1 Expression Downregulates in Neuroblastoma Cells (SK-N-MC Chronically Exposed to HIV-1 and Cocaine.

    Directory of Open Access Journals (Sweden)

    Upal eRoy

    2015-07-01

    Full Text Available Background: HIV-associated neurological disorder (HAND has long been recognized as a consequence of Human Immunodeficiency Virus (HIV infection in the brain. The pathology of HAND gets more complicated with the recreational drug use such as cocaine. Recent studies have suggested multiple genetic influences involved in the pathology of addiction and HAND but only a fraction of the entire genetic risk has been investigated so far. In this regard, role of DJ1 protein (a gene linked to autosomal recessive early-onset Parkinson’s disease in regulating dopamine transmission and reactive oxygen species (ROS production in neuronal cells will be worth investigating in HIV-1 and cocaine exposed microenvironment. Being a very abundant protein in the brain, DJ1 could serve as a potential marker for early detection of HIV-1 and/or cocaine related neurological disorder.Methods: In vitro analysis was done to observe the effect of HIV-1 and/or cocaine on DJ1 protein expression in neuroblastoma cells (SK-N-MC. Gene expression and protein analysis of DJ1 was done on the HIV infected and/or cocaine treated SK-N-MC and compared to untreated cells using real time PCR, Western Blot and flow cytometry.Results: Gene expression and protein analysis indicated that there was a significant decrease in DJ1 expression in SK-N-MC chronically exposed to HIV-1 and/or cocaine.Conclusion: This is the first study to establish that DJ1 expression level in the neuronal cells significantly decreased in presence of HIV-1and/or cocaine indicating oxidative stress level of dopamine neurons.

  15. Down-regulation of SMT3A gene expression in association with DNA synthesis induction after X-ray irradiation in nevoid basal cell carcinoma syndrome (NBCCS) cells

    International Nuclear Information System (INIS)

    Sugaya, Shigeru; Nakanishi, Hiroshi; Tanzawa, Hideki; Sugita, Katsuo; Kita, Kazuko; Suzuki, Nobuo

    2005-01-01

    Fibroblast cells derived from nevoid basal carcinoma syndrome (NBCCS) patients show increased levels of DNA synthesis after X-ray irradiation. Genes, whose expression is modulated in association with the DNA synthesis induction, were searched by using PCR-based mRNA differential display analysis in one of the NBCCS cell lines, NBCCS1 cells. Decreased levels of SMT3A gene expression were found in X-ray-irradiated NBCCS1 cells. This decrease was also shown by RT-PCR analysis in another cell line, NBCCS3 cells. In addition to NBCCS cells, normal fibroblast cells showed the DNA synthesis induction after X-ray irradiation when they were treated with antisense oligonucleotides (AO) for SMT3A. However, treatment of normal fibroblasts with the random oligonucleotides (RO) resulted in decreased levels of DNA synthesis after X-ray irradiation. Thus, down-regulation of SMT3A gene expression may be involved in the DNA synthesis induction after X-ray irradiation in the NBCCS cells at least tested

  16. Down-regulation of SMT3A gene expression in association with DNA synthesis induction after X-ray irradiation in nevoid basal cell carcinoma syndrome (NBCCS) cells

    Energy Technology Data Exchange (ETDEWEB)

    Sugaya, Shigeru [Department of Environmental Biochemistry, Graduate School of Medicine, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 260-8670 (Japan); Nakanishi, Hiroshi [Department of Clinical Molecular Biology, Graduate School of Medicine, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 260-8670 (Japan); Tanzawa, Hideki [Department of Clinical Molecular Biology, Graduate School of Medicine, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 260-8670 (Japan); Sugita, Katsuo [Department of Clinical Medicine, Faculty of Education, Chiba University, 1-33 Yayoi, Inage-ku, Chiba 263-8522 (Japan); Kita, Kazuko [Department of Environmental Biochemistry, Graduate School of Medicine, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 260-8670 (Japan); Suzuki, Nobuo [Department of Environmental Biochemistry, Graduate School of Medicine, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 260-8670 (Japan)]. E-mail: nobuo@faculty.chiba-u.jp

    2005-10-15

    Fibroblast cells derived from nevoid basal carcinoma syndrome (NBCCS) patients show increased levels of DNA synthesis after X-ray irradiation. Genes, whose expression is modulated in association with the DNA synthesis induction, were searched by using PCR-based mRNA differential display analysis in one of the NBCCS cell lines, NBCCS1 cells. Decreased levels of SMT3A gene expression were found in X-ray-irradiated NBCCS1 cells. This decrease was also shown by RT-PCR analysis in another cell line, NBCCS3 cells. In addition to NBCCS cells, normal fibroblast cells showed the DNA synthesis induction after X-ray irradiation when they were treated with antisense oligonucleotides (AO) for SMT3A. However, treatment of normal fibroblasts with the random oligonucleotides (RO) resulted in decreased levels of DNA synthesis after X-ray irradiation. Thus, down-regulation of SMT3A gene expression may be involved in the DNA synthesis induction after X-ray irradiation in the NBCCS cells at least tested.

  17. Down-regulation of LncRNA TUG1 enhances radiosensitivity in bladder cancer via suppressing HMGB1 expression.

    Science.gov (United States)

    Jiang, Huijuan; Hu, Xigang; Zhang, Hongzhi; Li, Wenbo

    2017-04-04

    Long non-coding RNAs (lncRNAs) have been reported to regulate the sensitivity of different cancer cells to chemoradiotherapy. Aberrant expression of lncRNA Taurine-upregulated gene 1 (TUG1) has been found to be involved in the development of bladder cancer, however, its function and underlying mechanism in the radioresistance of bladder cancer remains unclear. Quantitative real-time PCR (qRT-PCR) was conducted to measure the expression of TUG1 and HMGB1 mRNA in bladder cancer tissues and cell lines. HMGB1 protein levels were tested by western blot assays. Different doses of X-ray were used for radiation treatment of bladder cancer cells. Colony survival and cell viability were detected by clonogenic assay and CCK-8 Kit, respectively. Cell apoptosis was determined by flow cytometry. A xenograft mouse model was constructed to observe the effect of TUG1 on tumor growth in vivo. The levels of TUG1 and HMGB1 were remarkably increased in bladder cancer tissues and cell lines. Radiation treatment markedly elevated the expression of TUG1 and HMGB1. TUG1 knockdown inhibited cell proliferation, promoted cell apoptosis and decreased colony survival in SW780 and BIU87 cells under radiation. Moreover, TUG1 depletion suppressed the HMGB1 mRNA and protein levels. Furthermore, overexpression of HMGB1 reversed TUG1 knockdown-induced effect in bladder cancer cells. Radiation treatment dramatically reduced the tumor volume and weight in xenograft model, and this effect was more obvious when combined with TUG1 silencing. LncRNA TUG1 knockdown enhances radiosensitivity of bladder cancer by suppressing HMGB1 expression. TUG1 acts as a potential regulator of radioresistance of bladder cancer, and it may represent a promising therapeutic target for bladder cancer patients.

  18. Prenatal stress down-regulates Reelin expression by methylation of its promoter and induces adult behavioral impairments in rats.

    Directory of Open Access Journals (Sweden)

    Ismael Palacios-García

    Full Text Available Prenatal stress causes predisposition to cognitive and emotional disturbances and is a risk factor towards the development of neuropsychiatric conditions like depression, bipolar disorders and schizophrenia. The extracellular protein Reelin, expressed by Cajal-Retzius cells during cortical development, plays critical roles on cortical lamination and synaptic maturation, and its deregulation has been associated with maladaptive conditions. In the present study, we address the effect of prenatal restraint stress (PNS upon Reelin expression and signaling in pregnant rats during the last 10 days of pregnancy. Animals from one group, including control and PNS exposed fetuses, were sacrificed and analyzed using immunohistochemical, biochemical, cell biology and molecular biology approaches. We scored changes in the expression of Reelin, its signaling pathway and in the methylation of its promoter. A second group included control and PNS exposed animals maintained until young adulthood for behavioral studies. Using the optical dissector, we show decreased numbers of Reelin-positive neurons in cortical layer I of PNS exposed animals. In addition, neurons from PNS exposed animals display decreased Reelin expression that is paralleled by changes in components of the Reelin-signaling cascade, both in vivo and in vitro. Furthermore, PNS induced changes in the DNA methylation levels of the Reelin promoter in culture and in histological samples. PNS adult rats display excessive spontaneous locomotor activity, high anxiety levels and problems of learning and memory consolidation. No significant visuo-spatial memory impairment was detected on the Morris water maze. These results highlight the effects of prenatal stress on the Cajal-Retzius neuronal population, and the persistence of behavioral consequences using this treatment in adults, thereby supporting a relevant role of PNS in the genesis of neuropsychiatric diseases. We also propose an in vitro model that

  19. Tissue-specific posttranscriptional downregulation of expression of the S100A4(mts1) gene in transgenic animals

    DEFF Research Database (Denmark)

    Ambartsumian, N; Klingelhöfer, Jörg; Grigorian, M

    1998-01-01

    The S100A4(mts1) is a gene associated with generation of metastatic disease. In order to analyze the consequences of alteration of the pattern of expression of the S100A4(mts1) gene we obtained strains of transgenic mice bearing the S100A4(mts1) gene under the control of a ubiquitous and constitu....../or posttranslational degradation....

  20. P02.04MICRORNA-MEDIATED DOWN-REGULATION OF NKG2D LIGAND EXPRESSION REDUCES GLIOMA CELL IMMUNOGENICITY

    Science.gov (United States)

    Codo, P.; Weller, M.; Meister, G.; Szabo, E.; Steinle, A.; Wolter, M.; Reifenberger, G.; Roth, P.

    2014-01-01

    Glioblastoma is a primary brain tumor with a dismal prognosis despite comprehensive therapeutic regimens. It is characterized by diffuse infiltration of the surrounding healthy brain tissue, well-adapted to hypoxic conditions and regarded as paradigmatic for tumor-associated immunosuppression. One of the major activating receptors of natural killer (NK) cells is NKG2D. It binds to at least 8 ligands (NKG2DL) which are induced after malignant transformation and cellular stress. Regulation of NKG2DL expression may be affected by endogenous RNA molecules known as microRNA (miRNA). Here, we aimed at characterizing the role of miRNA in the control of NKG2DL expression in glioma cells. We selected 6 miRNA that were described or predicted to target NKG2DL. Three of the miRNA candidates, miR-20a, miR-93 and miR-106b, were expressed in glioma cell lines and were also detected in glioblastoma tissue specimens. Silencing of these miRNA with locked nucleic acid (LNA) molecules resulted in an up-regulation of NKG2DL cell surface levels which translated into increased sensitivity to immune cell killing. This effect was reversed by neutralizing NKG2D antibodies, confirming that enhanced immune cell lysis upon miRNA silencing was mediated through the NKG2D system. We conclude that the expression of several miRNA may contribute to the immune escape of glioma cells at the level of the NKG2D system. Therapeutic targeting of miRNA that regulate NKG2DL levels may therefore represent a promising approach to allow for more potent immune responses against glioblastoma.

  1. Downregulated kynurenine 3-monooxygenase gene expression and enzyme activity in schizophrenia and genetic association with schizophrenia endophenotypes.

    Science.gov (United States)

    Wonodi, Ikwunga; Stine, O Colin; Sathyasaikumar, Korrapati V; Roberts, Rosalinda C; Mitchell, Braxton D; Hong, L Elliot; Kajii, Yasushi; Thaker, Gunvant K; Schwarcz, Robert

    2011-07-01

    Kynurenic acid, a metabolite of the kynurenine pathway of tryptophan degradation, is an antagonist at N-methyl-d-aspartate and α7 nicotinic acetylcholine receptors and modulates glutamate, dopamine, and acetylcholine signaling. Cortical kynurenic acid concentrations are elevated in the brain and cerebrospinal fluid of schizophrenia patients. The proximal cause may be an impairment of kynurenine 3-monooxygenase (KMO), a rate-limiting enzyme at the branching point of the kynurenine pathway. To examine KMO messenger RNA expression and KMO enzyme activity in postmortem tissue from the frontal eye field (FEF; Brodmann area 6) obtained from schizophrenia individuals compared with healthy control individuals and to explore the relationship between KMO single-nucleotide polymorphisms and schizophrenia oculomotor endophenotypes. Case-control postmortem and clinical study. Maryland Brain Collection, outpatient clinics. Postmortem specimens from schizophrenia patients (n = 32) and control donors (n = 32) and a clinical sample of schizophrenia patients (n = 248) and healthy controls (n = 228). Comparison of quantitative KMO messenger RNA expression and KMO enzyme activity in postmortem FEF tissue between schizophrenia patients and controls and association of KMO single-nucleotide polymorphisms with messenger RNA expression in postmortem FEF and schizophrenia and oculomotor endophenotypes (ie, smooth pursuit eye movements and oculomotor delayed response). In postmortem tissue, we found a significant and correlated reduction in KMO gene expression and KMO enzyme activity in the FEF in schizophrenia patients. In the clinical sample, KMO rs2275163 was not associated with a diagnosis of schizophrenia but showed modest effects on predictive pursuit and visuospatial working memory endophenotypes. Our results provide converging lines of evidence implicating reduced KMO activity in the etiopathophysiology of schizophrenia and related neurocognitive deficits.

  2. Candida krusei and Candida glabrata reduce the filamentation of Candida albicans by downregulating expression of HWP1 gene.

    Science.gov (United States)

    de Barros, Patrícia Pimentel; Freire, Fernanda; Rossoni, Rodnei Dennis; Junqueira, Juliana Campos; Jorge, Antonio Olavo Cardoso

    2017-07-01

    Pathogenicity of Candida albicans is associated with its capacity switch from yeast-like to hyphal growth. The hyphal form is capable to penetrate the epithelial surfaces and to damage the host tissues. Therefore, many investigations have focused on mechanisms that control the morphological transitions of C. albicans. Recently, certain studies have showed that non-albicans Candida species can reduce the capacity of C. albicans to form biofilms and to develop candidiasis in animal models. Then, the objective of this study was to evaluate the effects of Candida krusei and Candida glabrata on the morphogenesis of C. albicans. Firstly, the capacity of reference and clinical strains of C. albicans in forming hyphae was tested in vitro. After that, the expression of HWP1 (hyphal wall protein 1) gene was determined by quantitative real-time PCR (polymerase chain reaction) assay. For both reference and clinical strains, a significant inhibition of the hyphae formation was observed when C. albicans was incubated in the presence of C. krusei or C. glabrata compared to the control group composed only by C. albicans. In addition, the culture mixed of C. albicans-C. krusei or C. albicans-C. glabrata reduced significantly the expression of HWP1 gene of C. albicans in relation to single cultures of this specie. In both filamentation and gene expression assays, C. krusei showed the higher inhibitory activity on the morphogenesis of C. albicans compared to C. glabrata. C. krusei and C. glabrata are capable to reduce the filamentation of C. albicans and consequently decrease the expression of the HWP1 gene.

  3. Dietary sodium deprivation evokes activation of brain regional neurons and down-regulation of angiotensin II type 1 receptor and angiotensin-convertion enzyme mRNA expression.

    Science.gov (United States)

    Lu, B; Yang, X J; Chen, K; Yang, D J; Yan, J Q

    2009-12-15

    Previous studies have indicated that the renin-angiotensin-aldosterone system (RAAS) is implicated in the induction of sodium appetite in rats and that different dietary sodium intakes influence the mRNA expression of central and peripheral RAAS components. To determine whether dietary sodium deprivation activates regional brain neurons related to sodium appetite, and changes their gene expression of RAAS components of rats, the present study examined the c-Fos expression after chronic exposure to low sodium diet, and determined the relationship between plasma and brain angiotensin I (ANG I), angiotensin II (ANG II) and aldosterone (ALD) levels and the sodium ingestive behavior variations, as well as the effects of prolonged dietary sodium deprivation on ANG II type 1 (AT1) and ANG II type 2 (AT2) receptors and angiotensin-convertion enzyme (ACE) mRNA levels in the involved brain regions using the method of real-time polymerase chain reaction (PCR). Results showed that the Fos immunoreactivity (Fos-ir) expression in forebrain areas such as subfornical organ (SFO), paraventricular hypothalamic nuclei (PVN), supraoptic nucleus (SON) and organum vasculosum laminae terminalis (OVLT) all increased significantly and that the levels of ANG I, ANG II and ALD also increased in plasma and forebrain in rats fed with low sodium diet. In contrast, AT1, ACE mRNA in PVN, SON and OVLT decreased significantly in dietary sodium depleted rats, while AT2 mRNA expression did not change in the examined areas. These results suggest that many brain areas are activated by increased levels of plasma and/or brain ANG II and ALD, which underlies the elevated preference for hypertonic salt solution after prolonged exposure to low sodium diet, and that the regional AT1 and ACE mRNA are down-regulated after dietary sodium deprivation, which may be mediated by increased ANG II in plasma and/or brain tissue.

  4. The downregulation of microRNA let-7a contributes to the excessive expression of type I collagen in systemic and localized scleroderma.

    Science.gov (United States)

    Makino, Katsunari; Jinnin, Masatoshi; Hirano, Ayaka; Yamane, Keitaro; Eto, Mitsuhiko; Kusano, Takamitsu; Honda, Noritoshi; Kajihara, Ikko; Makino, Takamitsu; Sakai, Keisuke; Masuguchi, Shinichi; Fukushima, Satoshi; Ihn, Hironobu

    2013-04-15

    Systemic and localized scleroderma (SSc and LSc) is characterized by excessive deposition of collagen and tissue fibrosis in the skin. Although they have fundamental common characteristics including autoimmunity, little is known about the exact mechanism that mediates the excessive collagen expression in these disorders. In the current study, we tried to evaluate the possibility that microRNAs (miRNAs) play some roles in the pathogenesis of fibrosis seen in these diseases. miRNA expression patterns were evaluated by miRNA array analysis, real-time PCR, and in situ hybridization. The function of miRNAs in dermal fibroblasts was assessed using miRNA inhibitors, precursors, or protectors. In the mouse model of bleomycin-induced dermal sclerosis, the overexpression of miRNAs was performed by i.p. miRNA injection. We demonstrated let-7a expression was downregulated in SSc and LSc skin both in vivo and in vitro, compared with normal or keloid skin. The inhibition or overexpression of let-7a in human or mouse skin fibroblasts affected the protein expression of type I collagen or luciferase activity of collagen 3'-untranslated region. Also, we found let-7a was detectable and quantitative in the serum and investigated serum let-7a levels in patients with SSc or LSc. let-7a concentration was significantly decreased in these patients, especially in LSc patients. Moreover, we revealed that the intermittent overexpression of let-7a in the skin by i.p. miRNA injection improved the skin fibrosis induced by bleomycin in mice. Investigation of more detailed mechanisms of miRNA-mediated regulation of collagen expression may lead to new therapeutic approaches against SSc and LSc.

  5. Epstein-Barr virus-encoded EBNA2 alters immune checkpoint PD-L1 expression by downregulating miR-34a in B-cell lymphomas.

    Science.gov (United States)

    Anastasiadou, Eleni; Stroopinsky, Dina; Alimperti, Stella; Jiao, Alan L; Pyzer, Athalia R; Cippitelli, Claudia; Pepe, Giuseppina; Severa, Martina; Rosenblatt, Jacalyn; Etna, Marilena P; Rieger, Simone; Kempkes, Bettina; Coccia, Eliana M; Sui, Shannan J Ho; Chen, Christopher S; Uccini, Stefania; Avigan, David; Faggioni, Alberto; Trivedi, Pankaj; Slack, Frank J

    2018-06-26

    Cancer cells subvert host immune surveillance by altering immune checkpoint (IC) proteins. Some Epstein-Barr virus (EBV)-associated tumors have higher Programmed Cell Death Ligand, PD-L1 expression. However, it is not known how EBV alters ICs in the context of its preferred host, the B lymphocyte and in derived lymphomas. Here, we found that latency III-expressing Burkitt lymphoma (BL), diffuse large B-cell lymphomas (DLBCL) or their EBNA2-transfected derivatives express high PD-L1. In a DLBCL model, EBNA2 but not LMP1 is sufficient to induce PD-L1. Latency III-expressing DLBCL biopsies showed high levels of PD-L1. The PD-L1 targeting oncosuppressor microRNA miR-34a was downregulated in EBNA2-transfected lymphoma cells. We identified early B-cell factor 1 (EBF1) as a repressor of miR-34a transcription. Short hairpin RNA (shRNA)-mediated knockdown of EBF1 was sufficient to induce miR-34a transcription, which in turn reduced PD-L1. MiR-34a reconstitution in EBNA2-transfected DLBCL reduced PD-L1 expression and increased its immunogenicity in mixed lymphocyte reactions (MLR) and in three-dimensional biomimetic microfluidic chips. Given the importance of PD-L1 inhibition in immunotherapy and miR-34a dysregulation in cancers, our findings may have important implications for combinatorial immunotherapy, which include IC inhibiting antibodies and miR-34a, for EBV-associated cancers.

  6. Triptolide Suppresses Alkali Burn-Induced Corneal Angiogenesis Along with a Downregulation of VEGFA and VEGFC Expression.

    Science.gov (United States)

    Wang, Geng; Li, Na; Lv, Xiaohong; Ahmed, Naila; Li, Xinlei; Liu, Huidong; Ma, Jing; Zhang, Yafang

    2017-07-01

    Triptolide (TPL) is an active compound extracted from a Chinese herbal medicine tripterygium wilfordii Hook. f. (Celastraceae), which has been used as an anti-inflammatory drug for years. It also inhibits the growth and proliferation of different types of cancer cells. The inhibitory effect of TPL on angiogenesis after chemical-induced corneal inflammation was studied in vivo. The effects of TPL on the proliferation, apoptosis, migration, and tube formation of rat aortic endothelial cells (RAECs) were studied in vitro. Cell proliferation and apoptosis were measured by MTT assay and flow cytometry, respectively. Migration was analyzed using the scratch wound healing assay and transwell assay. Tube formation assay was used to examine angiogenesis. Real-time PCR and Western blot were used to determine the expression of vascular endothelial growth factor A (VEGFA) and VEGFC. To study the in vivo effects of TPL, the mouse model of alkali burn-induced corneal angiogenesis was used. The angiogenesis was analyzed by determining the density of the newly generated blood vessels in corneas. We found that TPL induced apoptosis and inhibited the proliferation of RAECs in a dose-dependent manner. TPL inhibited migration and tube formation of RAECs and decreased the expression of VEGFA and VEGFC in vitro. Furthermore, TPL suppressed alkali burn-induced corneal angiogenesis and inhibited the expression of VEGFA and VEGFC in corneas in vivo. In conclusion, topical TPL as a pharmacological agent has the ability to reduce angiogenesis in cornea and may have clinical indications for the treatment of corneal angiogenesis diseases which have to be further explored. Anat Rec, 300:1348-1355, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  7. Artichoke, Cynarin and Cyanidin Downregulate the Expression of Inducible Nitric Oxide Synthase in Human Coronary Smooth Muscle Cells

    OpenAIRE

    Ning Xia; Andrea Pautz; Ursula Wollscheid; Gisela Reifenberg; Ulrich Förstermann; Huige Li

    2014-01-01

    Artichoke (Cynara scolymus L.) is one of the world’s oldest medicinal plants with multiple health benefits. We have previously shown that artichoke leaf extracts and artichoke flavonoids upregulate the gene expression of endothelial-type nitric oxide synthase (eNOS) in human endothelial cells. Whereas NO produced by the eNOS is a vasoprotective molecule, NO derived from the inducible iNOS plays a pro-inflammatory role in the vasculature. The present study was aimed to investigate the effects ...

  8. WAFs lead molting retardation of naupliar stages with down-regulated expression profiles of chitin metabolic pathway and related genes in the copepod Tigriopus japonicus.

    Science.gov (United States)

    Hwang, Dae-Sik; Lee, Min-Chul; Kyung, Do-Hyun; Kim, Hui-Su; Han, Jeonghoon; Kim, Il-Chan; Puthumana, Jayesh; Lee, Jae-Seong

    2017-03-01

    Oil pollution is considered being disastrous to marine organisms and ecosystems. As molting is critical in the developmental process of arthropods in general and copepods, in particular, the impact will be adverse if the target of spilled oil is on molting. Thus, we investigated the harmful effects of water accommodated fractions (WAFs) of crude oil with an emphasis on inhibition of chitin metabolic pathways related genes and developmental retardation in the copepod Tigriopus japonicus. Also, we analysed the ontology and domain of chitin metabolic pathway genes and mRNA expression patterns of developmental stage-specific genes. Further, the developmental retardation followed by transcriptional modulations in nuclear receptor genes (NR) and chitin metabolic pathway-related genes were observed in the WAFs-exposed T. japonicus. As a result, the developmental time was found significantly (P<0.05) delayed in response to 40% WAFs in comparison with that of control. Moreover, the NR gene, HR3 and chitinases (CHT9 and CHT10) were up-regulated in N4-5 stages, while chitin synthase genes (CHS-1, CHS-2-1, and CHS-2-2) down-regulated in response to WAFs. In brief, a high concentration of WAFs repressed nuclear receptor genes but elicited activation of some of the transcription factors at low concentration of WAFs, resulting in suppression of chitin synthesis. Thus, we suggest that WAF can lead molting retardation of naupliar stages in T. japonicus through down-regulations of chitin metabolism. These findings will provide a better understanding of the mode of action of chitin biosynthesis associated with molting mechanism in WAF-exposed T. japonicus. Copyright © 2016 Elsevier Inc. All rights reserved.

  9. [Curcumin down-regulates CX3CR1 expression in spinal cord dorsal horn and DRG in neuropathic pain rats].

    Science.gov (United States)

    Zheng, Jinwei; Zheng, Changjian; Cao, Hong; Li, Jun; Lian, Qingquan

    2011-09-01

    To investigate the effects of curcumin on the behavior of chronic constrictive injury (CCI) rats and the CX3CR1 expression in spinal cord dorsal horn and dorsal root ganglia (DRG). Seventy-two male SD rats were randomly divided into 4 groups: 1) Sham operation group (Sham); 2) Chronic constrictive injury group (CCI); 3) Curcumin treated group (Cur), administrated with curcumin 100 mg x kg(-1) x d(-1) ip for 14 days after CCI; 4) Solvent contrast group (SC), administrated with an equal volume of solvent for 14 days after CCI. Paw thermal withdrawal (PTWL) and paw mechanical withdrawal threshold (PMWT) were measured on 2 pre-operative and 1, 3, 5, 7, 10, 14 post-operative days respectively. The lumbar segments L4-5 of the spinal cord and the L4, L5 DRG were removed at 3, 7, 14 days after surgery. The expression of CX3CR1 was determined by immunohistochemical staining. Compared with Sham group, PTWL and PMWT in CCI group were significantly lower on each post-operative day (PDRG. In Cur group, PTWL were higher than in CCI group on 7, 10, 14 post-operative day (Pdorsal root ganglia.

  10. Thymosin From Bombyx mori Is Down-Regulated in Expression by BmNPV Exhibiting Antiviral Activity.

    Science.gov (United States)

    Zhang, Chen; Wang, Yongdi; Fang, Qiang; Xu, Minlin; Lv, Mengyuan; Liao, Jinxu; Li, Si; Nie, Zuoming; Zhang, Wenping

    2016-01-01

    Thymosins have been highly conserved during evolution. These hormones exist in many animal species and play an essential role in many biological events. However, little is known regarding the physiological function of silkworm Bombyx mori thymosin (BmTHY). In this study, we investigated the expression pattern of BmTHY in a Bombyx mori larval ovarian cell line (BmN) challenged with Bombyx mori nuclear polyhydrosis virus (BmNPV) and the antiviral effect of recombinant BmTHY (rBmTHY) for Bombyx mori against BmNPV. Western-blot assay and qRT-PCR analysis revealed that the level of BmTHY protein expression and transcription decreased over time when BmN cells were infected by BmNPV. Treatment with endotoxin-free rBmTHY led to a significant reduction in viral titer in the supernatant of BmN cells challenged with BmNPV. The results from antiviral tests performed in vitro and in vivo showed that endotoxin-free rBmTHY improved the survival rate of Bombyx mori infected with BmNPV. These findings suggest that BmTHY exerts immunomodulatory effects on Bombyx mori, rendering them resistant to viral infection. © The Authors 2016. Published by Oxford University Press on behalf of Entomological Society of America.

  11. Down-regulation of hypothalamic pro-opiomelanocortin (POMC) expression after weaning is associated with hyperphagia-induced obesity in JCR rats overexpressing neuropeptide Y.

    Science.gov (United States)

    Diané, Abdoulaye; Pierce, W David; Russell, James C; Heth, C Donald; Vine, Donna F; Richard, Denis; Proctor, Spencer D

    2014-03-14

    We hypothesised that hypothalamic feeding-related neuropeptides are differentially expressed in obese-prone and lean-prone rats and trigger overeating-induced obesity. To test this hypothesis, in the present study, we measured energy balance and hypothalamic neuropeptide Y (NPY) and pro-opiomelanocortin (POMC) mRNA expressions in male JCR:LA-cp rats. We compared, in independent cohorts, free-feeding obese-prone (Obese-FF) and lean-prone (Lean-FF) rats at pre-weaning (10 d old), weaning (21-25 d old) and early adulthood (8-12 weeks). A group of Obese-pair-feeding (PF) rats pair-fed to the Lean-FF rats was included in the adult cohort. The body weights of 10-d-old Obese-FF and Lean-FF pups were not significantly different. However, when the pups were shifted from dams' milk to solid food (weaning), the obese-prone rats exhibited more energy intake over the days than the lean-prone rats and higher body and fat pad weights and fasting plasma glucose, leptin, insulin and lipid levels. These differences were consistent with higher energy consumption and lower energy expenditure. In the young adult cohort, the differences between the Obese-FF and Lean-FF rats became more pronounced, yielding significant age effects on most of the parameters of the metabolic syndrome, which were reduced in the Obese-PF rats. The obese-prone rats displayed higher NPY expression than the lean-prone rats at pre-weaning and weaning, and the expression levels did not differ by age. In contrast, POMC expression exhibited significant age-by-genotype differences. At pre-weaning, there was no genotype difference in POMC expression, but in the weanling cohort, obese-prone pups exhibited lower POMC expression than the lean-prone rats. This genotype difference became more pronounced at adulthood. Overall, the development of hyperphagia-induced obesity in obese-prone JCR rats is related to POMC expression down-regulation in the presence of established NPY overexpression.

  12. Downregulation of TLX induces TET3 expression and inhibits glioblastoma stem cell self-renewal and tumorigenesis.

    Science.gov (United States)

    Cui, Qi; Yang, Su; Ye, Peng; Tian, E; Sun, Guoqiang; Zhou, Jiehua; Sun, Guihua; Liu, Xiaoxuan; Chen, Chao; Murai, Kiyohito; Zhao, Chunnian; Azizian, Krist T; Yang, Lu; Warden, Charles; Wu, Xiwei; D'Apuzzo, Massimo; Brown, Christine; Badie, Behnam; Peng, Ling; Riggs, Arthur D; Rossi, John J; Shi, Yanhong

    2016-02-03

    Glioblastomas have been proposed to be maintained by highly tumorigenic glioblastoma stem cells (GSCs) that are resistant to current therapy. Therefore, targeting GSCs is critical for developing effective therapies for glioblastoma. In this study, we identify the regulatory cascade of the nuclear receptor TLX and the DNA hydroxylase Ten eleven translocation 3 (TET3) as a target for human GSCs. We show that knockdown of TLX expression inhibits human GSC tumorigenicity in mice. Treatment of human GSC-grafted mice with viral vector-delivered TLX shRNA or nanovector-delivered TLX siRNA inhibits tumour development and prolongs survival. Moreover, we identify TET3 as a potent tumour suppressor downstream of TLX to regulate the growth and self-renewal in GSCs. This study identifies the TLX-TET3 axis as a potential therapeutic target for glioblastoma.

  13. Arctigenin enhances chemosensitivity to cisplatin in human nonsmall lung cancer H460 cells through downregulation of survivin expression.

    Science.gov (United States)

    Wang, Huan-qin; Jin, Jian-jun; Wang, Jing

    2014-01-01

    Arctigenin, a dibenzylbutyrolactone lignan, enhances cisplatin-mediated cell apoptosis in cancer cells. Here, we sought to investigate the effects of arctigenin on cisplatin-treated non-small-cell lung cancer (NSCLC) H460 cells. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and annexin-V/propidium iodide staining were performed to analyze the proliferation and apoptosis of H460 cells. Arctigenin dose-dependently suppressed cell proliferation and potentiated cell apoptosis, coupled with increased cleavage of caspase-3 and poly(ADP-ribose) polymerase. Moreover, arctigenin sensitized H460 cells to cisplatin-induced proliferation inhibition and apoptosis. Arctigenin alone or in combination with cisplatin had a significantly lower amount of survivin. Ectopic expression of survivin decreased cell apoptosis induced by arctigenin (P arctigenin (P arctigenin has a therapeutic potential in combina-tion with chemotherapeutic agents for NSLC. © 2013 Wiley Periodicals, Inc.

  14. miR-671-5p inhibits epithelial-to-mesenchymal transition by downregulating FOXM1 expression in breast cancer

    Science.gov (United States)

    Tan, Xiaohui; Fu, Yebo; Chen, Liang; Lee, Woojin; Lai, Yinglei; Rezaei, Katayoon; Tabbara, Sana; Latham, Patricia; Teal, Christine B.; Man, Yan-Gao; Siegel, Robert S.; Brem, Rachel F.; Fu, Sidney W.

    2016-01-01

    MicroRNA (miRNA) dysfunction is associated with a variety of human diseases, including cancer. Our previous study showed that miR-671-5p was deregulated throughout breast cancer progression. Here, we report for the first time that miR-671-5p is a tumor-suppressor miRNA in breast tumorigenesis. We found that expression of miR-671-5p was decreased significantly in invasive ductal carcinoma (IDC) compared to normal in microdissected formalin-fixed, paraffin-embedded (FFPE) tissues. Forkhead Box M1 (FOXM1), an oncogenic transcription factor, was predicted as one of the direct targets of miR-671-5p, which was subsequently confirmed by luciferase assays. Forced expression of miR-671-5p in breast cancer cell lines downregulated FOXM1 expression, and attenuated the proliferation and invasion in breast cancer cell lines. Notably, overexpression of miR-671-5p resulted in a shift from epithelial-to-mesenchymal transition (EMT) to mesenchymal-to-epithelial transition (MET) phenotypes in MDA-MB-231 breast cancer cells and induced S-phase arrest. Moreover, miR-671-5p sensitized breast cancer cells to cisplatin, 5-fluorouracil (5-FU) and epirubicin exposure. Host cell reactivation (HCR) assays showed that miR-671-5p reduces DNA repair capability in post-drug exposed breast cancer cells. cDNA microarray data revealed that differentially expressed genes when miR-671-5p was transfected are associated with cell proliferation, invasion, cell cycle, and EMT. These data indicate that miR-671-5p functions as a tumor suppressor miRNA in breast cancer by directly targeting FOXM1. Hence, miR-671-5p may serve as a novel therapeutic target for breast cancer management. PMID:26588055

  15. Triptolide inhibits proliferation of Epstein–Barr virus-positive B lymphocytes by down-regulating expression of a viral protein LMP1

    International Nuclear Information System (INIS)

    Zhou, Heng; Guo, Wei; Long, Cong; Wang, Huan; Wang, Jingchao; Sun, Xiaoping

    2015-01-01

    Highlights: • Triptolide inhibits proliferation of EBV-positive lymphoma cells in vitro and in vivo. • Triptolide reduces expression of LMP1 by decreasing its transcription level. • Triptolide inhibits ED-L1 promoter activity. - Abstract: Epstein–Barr virus (EBV) infects various types of cells and mainly establishes latent infection in B lymphocytes. The viral latent membrane protein 1 (LMP1) plays important roles in transformation and proliferation of B lymphocytes infected with EBV. Triptolide is a compound of Tripterygium extracts, showing anti-inflammatory, immunosuppressive, and anti-cancer activities. In this study, it is determined whether triptolide inhibits proliferation of Epstein–Barr virus-positive B lymphocytes. The CCK-8 assays were performed to examine cell viabilities of EBV-positive B95-8 and P3HR-1 cells treated by triptolide. The mRNA and protein levels of LMP1 were examined by real time-PCR and Western blotting, respectively. The activities of two LMP1 promoters (ED-L1 and TR-L1) were determined by Dual luciferase reportor assay. The results showed that triptolide inhibited the cell viability of EBV-positive B lymphocytes, and the over-expression of LMP1 attenuated this inhibitory effect. Triptolide decreased the LMP1 expression and transcriptional levels in EBV-positive B cells. The activity of LMP1 promoter ED-L1 in type III latent infection was strongly suppressed by triptolide treatment. In addition, triptolide strongly reduced growth of B95-8 induced B lymphoma in BALB/c nude mice. These results suggest that triptolide decreases proliferation of EBV-induced B lymphocytes possibly by a mechanism related to down-regulation of the LMP1 expression

  16. Celecoxib Alleviates Memory Deficits by Downregulation of COX-2 Expression and Upregulation of the BDNF-TrkB Signaling Pathway in a Diabetic Rat Model.

    Science.gov (United States)

    Yang, Ying; Gao, Ling

    2017-06-01

    Previous studies conveyed that diabetes causes learning and memory deficits. Data also suggest that celecoxib exerts an anti-hyperalgesic, anti-allodynic, and a plethora of other beneficial effects in diabetic rats. However, whether celecoxib could alleviate memory deficit in diabetic rat is unknown. In the present study, we aimed to examine the potential of celecoxib to counter memory deficits in diabetes. Experimental diabetes was induced by streptozotocin (STZ, 60 mg/kg) in male SD rats. Rats were divided into three groups (n = 16/group): normal control group injected with normal saline, diabetes group injected with STZ, and diabetes + celecoxib group in which diabetic rats were administered with celecoxib by gavage in drinking water (10 mg/kg) for 10 days in terms of which memory performance in animals was measured, hippocampal tissue harvested, and long-term potentiation assessed. Western blotting and immunohistochemical staining were performed to determine cyclooxygenase 2 (COX-2) expression in hippocampus. The results showed that a rat model of STZ-induced diabetes was successfully established and that celecoxib treatment significantly improved the associated nephropathy and inflammation. Moreover, spatial memory and hippocampal long-term potentiation (LTP) were impaired in diabetic model (P memory deficit and hippocampal LTP in the diabetic rats. To understand the underlying mechanisms, the expression of some important pathways involved in memory impairment was determined. We found that brain-derived neurotrophic factor (BDNF) and phosphorylated tropomyosin-related kinase (p-TrkB) were decreased in diabetic rats but were effectively reversed by celecoxib treatment. As evidenced by western blotting and immunohistochemical staining, the expression of COX-2 in hippocampus was significantly upregulated in diabetic rat (P memory deficits via probable downregulation of hippocampal COX-2 expression and upregulation of the BDNF-TrkB signaling pathway in a

  17. Brain Metastases from Lung Cancer Show Increased Expression of DVL1, DVL3 and Beta-Catenin and Down-Regulation of E-Cadherin

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    Anja Kafka

    2014-06-01

    Full Text Available The susceptibility of brain to secondary formation from lung cancer primaries is a well-known phenomenon. In contrast, the molecular basis for invasion and metastasis to the brain is largely unknown. In the present study, 31 brain metastases that originated from primary lung carcinomas were analyzed regarding over expression of Dishevelled-1 (DVL1, Dishevelled-3 (DVL3, E-cadherin (CDH1 and beta-catenin (CTNNB1. Protein expressions and localizations were analyzed by immunohistochemistry. Genetic alterations of E-cadherin were tested by polymerase chain reaction (PCR/loss of heterozygosity (LOH. Heteroduplex was used to investigate mutations in beta-catenin. DVL1 and DVL3 showed over expression in brain metastasis in 87.1% and 90.3% of samples respectively. Nuclear staining was observed in 54.8% of cases for DVL1 and 53.3% for DVL3. The main effector of the Wnt signaling, beta-catenin, was up-regulated in 56%, and transferred to the nucleus in 36% of metastases. When DVL1 and DVL3 were up-regulated the number of cases with nuclear beta-catenin significantly increased (p = 0.0001. Down-regulation of E-cadherin was observed in 80% of samples. Genetic analysis showed 36% of samples with LOH of the CDH1. In comparison to other lung cancer pathologies, the diagnoses adenocarcinoma and small cell lung cancer (SCLC were significantly associated to CDH1 LOH (p = 0.001. Microsatellite instability was detected in one metastasis from adenocarcinoma. Exon 3 of beta-catenin was not targeted. Altered expression of Dishevelled-1, Dishevelled-3, E-cadherin and beta-catenin were present in brain metastases which indicates that Wnt signaling is important and may contribute to better understanding of genetic profile conditioning lung cancer metastasis to the brain.

  18. Down-regulation of Connexin43 expression reveals the involvement of caveolin-1 containing lipid rafts in human U251 glioblastoma cell invasion.

    Science.gov (United States)

    Strale, Pierre-Olivier; Clarhaut, Jonathan; Lamiche, Coralie; Cronier, Laurent; Mesnil, Marc; Defamie, Norah

    2012-11-01

    Glioblastoma cells are characterized by high proliferation and invasive capacities. Tumor development has been associated with a decrease of gap-junctional intercellular communication, but the concrete involvement of gap junction proteins, connexins, remains elusive since they are also suspected to promote cell invasion. In order to better understand how connexins control the glioma cell phenotype, we studied the consequences of inhibiting the intrinsic expression of the major astrocytic connexin, Connexin43, in human U251 glioblastoma cells by the shRNA strategy. The induced down-regulation of Cx43 expression has various effects on the U251 cells such as increased clonogenicity, angiogenesis and decreased adhesion on specific extracellular matrix proteins. We demonstrate that the invasion capacity measured in vitro and ex vivo correlates with Cx43 expression level. For the first time in a cancer cell context, our work demonstrates that Cx43 cofractionates, colocalizes and coimmunoprecipitates with a lipid raft marker, caveolin-1 and that this interaction is inversely correlated to the level of Cx43. This localization of Cx43 in these lipid raft microdomains regulates both homo- and heterocellular gap junctional communications (respectively between U251 cells, or between U251 cells and astrocytes). Moreover, the adhesive and invasive capacities are not dependent, in our model, on Cav-1 expression level. Our results tend to show that heterocellular gap junctional communication between cancer and stroma cells may affect the behavior of the tumor cells. Altogether, our data demonstrate that Cx43 controls the tumor phenotype of glioblastoma U251 cells and in particular, invasion capacity, through its localization in lipid rafts containing Cav-1. Copyright © 2011 Wiley Periodicals, Inc.

  19. Lamprey Prohibitin2 Arrest G2/M Phase Transition of HeLa Cells through Down-regulating Expression and Phosphorylation Level of Cell Cycle Proteins.

    Science.gov (United States)

    Shi, Ying; Guo, Sicheng; Wang, Ying; Liu, Xin; Li, Qingwei; Li, Tiesong

    2018-03-02

    Prohibitin 2(PHB2) is a member of the SFPH trans-membrane family proteins. It is a highly conserved and functionally diverse protein that plays an important role in preserving the structure and function of the mitochondria. In this study, the lamprey PHB2 gene was expressed in HeLa cells to investigate its effect on cell proliferation. The effect of Lm-PHB2 on the proliferation of HeLa cells was determined by treating the cells with pure Lm-PHB2 protein followed by MTT assay. Using the synchronization method with APC-BrdU and PI double staining revealed rLm-PHB2 treatment induced the decrease of both S phase and G0/G1 phase and then increase of G2/M phase. Similarly, cells transfected with pEGFP-N1-Lm-PHB2 also exhibited remarkable reduction in proliferation. Western blot and quantitative real-time PCR(qRT-PCR) assays suggested that Lm-PHB2 caused cell cycle arrest in HeLa cells through inhibition of CDC25C and CCNB1 expression. According to our western blot analysis, Lm-PHB2 was also found to reduce the expression level of Wee1 and PLK1 and the phosphorylation level of CCNB1, CDC25C and CDK1 in HeLa cells. Lamprey prohibitin 2 could arrest G2/M phase transition of HeLa cells through down-regulating expression and phosphorylation level of cell cycle proteins.

  20. Triptolide inhibits proliferation of Epstein–Barr virus-positive B lymphocytes by down-regulating expression of a viral protein LMP1

    Energy Technology Data Exchange (ETDEWEB)

    Zhou, Heng [Department of Pathogen Biology, School of Basic Medical Sciences, Wuhan University, Wuhan 430071 (China); Guo, Wei [Department of Pathology and Physiology, School of Basic Medical Sciences, Wuhan University, Wuhan 430071 (China); Long, Cong; Wang, Huan; Wang, Jingchao [Department of Pathogen Biology, School of Basic Medical Sciences, Wuhan University, Wuhan 430071 (China); Sun, Xiaoping, E-mail: xsun6@whu.edu.cn [Department of Pathogen Biology, School of Basic Medical Sciences, Wuhan University, Wuhan 430071 (China); State Key Laboratory of Virology, Wuhan University, Wuhan 430072 (China)

    2015-01-16

    Highlights: • Triptolide inhibits proliferation of EBV-positive lymphoma cells in vitro and in vivo. • Triptolide reduces expression of LMP1 by decreasing its transcription level. • Triptolide inhibits ED-L1 promoter activity. - Abstract: Epstein–Barr virus (EBV) infects various types of cells and mainly establishes latent infection in B lymphocytes. The viral latent membrane protein 1 (LMP1) plays important roles in transformation and proliferation of B lymphocytes infected with EBV. Triptolide is a compound of Tripterygium extracts, showing anti-inflammatory, immunosuppressive, and anti-cancer activities. In this study, it is determined whether triptolide inhibits proliferation of Epstein–Barr virus-positive B lymphocytes. The CCK-8 assays were performed to examine cell viabilities of EBV-positive B95-8 and P3HR-1 cells treated by triptolide. The mRNA and protein levels of LMP1 were examined by real time-PCR and Western blotting, respectively. The activities of two LMP1 promoters (ED-L1 and TR-L1) were determined by Dual luciferase reportor assay. The results showed that triptolide inhibited the cell viability of EBV-positive B lymphocytes, and the over-expression of LMP1 attenuated this inhibitory effect. Triptolide decreased the LMP1 expression and transcriptional levels in EBV-positive B cells. The activity of LMP1 promoter ED-L1 in type III latent infection was strongly suppressed by triptolide treatment. In addition, triptolide strongly reduced growth of B95-8 induced B lymphoma in BALB/c nude mice. These results suggest that triptolide decreases proliferation of EBV-induced B lymphocytes possibly by a mechanism related to down-regulation of the LMP1 expression.

  1. Ehrlichia chaffeensis TRP120 Activates Canonical Notch Signaling To Downregulate TLR2/4 Expression and Promote Intracellular Survival

    Directory of Open Access Journals (Sweden)

    Taslima T. Lina

    2016-07-01

    Full Text Available Ehrlichia chaffeensis preferentially targets mononuclear phagocytes and survives through a strategy of subverting innate immune defenses, but the mechanisms are unknown. We have shown E. chaffeensis type 1 secreted tandem repeat protein (TRP effectors are involved in diverse molecular pathogen-host interactions, such as the TRP120 interaction with the Notch receptor-cleaving metalloprotease ADAM17. In the present study, we demonstrate E. chaffeensis, via the TRP120 effector, activates the canonical Notch signaling pathway to promote intracellular survival. We found that nuclear translocation of the transcriptionally active Notch intracellular domain (NICD occurs in response to E. chaffeensis or recombinant TRP120, resulting in upregulation of Notch signaling pathway components and target genes notch1, adam17, hes, and hey. Significant differences in canonical Notch signaling gene expression levels (>40% were observed during early and late stages of infection, indicating activation of the Notch pathway. We linked Notch pathway activation specifically to the TRP120 effector, which directly interacts with the Notch metalloprotease ADAM17. Using pharmacological inhibitors and small interfering RNAs (siRNAs against γ-secretase enzyme, Notch transcription factor complex, Notch1, and ADAM17, we demonstrated that Notch signaling is required for ehrlichial survival. We studied the downstream effects and found that E. chaffeensis TRP120-mediated activation of the Notch pathway causes inhibition of the extracellular signal-regulated kinase 1/2 (ERK1/2 and p38 mitogen-activated protein kinase (MAPK pathways required for PU.1 and subsequent Toll-like receptor 2/4 (TLR2/4 expression. This investigation reveals a novel mechanism whereby E. chaffeensis exploits the Notch pathway to evade the host innate immune response for intracellular survival.

  2. Curcumin inhibits cholesterol uptake in Caco-2 cells by down-regulation of NPC1L1 expression

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    Duan Rui-Dong

    2010-04-01

    Full Text Available Abstract Background Curcumin is a polyphenol and the one of the principle curcuminoids of the spice turmeric. Its antioxidant, anti-cancer and anti-inflammatory effects have been intensively studied. Previous in vivo studies showed that administration of curcumin also decreased cholesterol levels in the blood, and the effects were considered to be related to upregulation of LDL receptor. However, since plasma cholesterol levels are also influenced by the uptake of cholesterol in the gut, which is mediated by a specific transporter Niemann-Pick Cl-like 1 (NPC1L1 protein, the present study is to investigate whether curcumin affects cholesterol uptake in the intestinal Caco-2 cells. Methods Caco-2 cells were cultured to confluence. The micelles composed of bile salt, monoolein, and 14C-cholesterol were prepared. We first incubated the cells with the micelles in the presence and absence of ezetimibe, the specific inhibitor of NPC1L1, to see whether the uptake of the cholesterol in the cells was mediated by NPC1L1. We then pretreated the cells with curcumin at different concentrations for 24 h followed by examination of the changes of cholesterol uptake in these curcumin-treated cells. Finally we determined whether curcumin affects the expression of NPC1L1 by both Western blot analysis and qPCR quantification. Results We found that the uptake of radioactive cholesterol in Caco-2 cells was inhibited by ezetimibe in a dose-dependent manner. The results indicate that the uptake of cholesterol in this study was mediated by NPC1L1. We then pretreated the cells with 25-100 μM curcumin for 24 h and found that such a treatment dose-dependently inhibited cholesterol uptake with 40% inhibition obtained by 100 μM curcumin. In addition, we found that the curcumin-induced inhibition of cholesterol uptake was associated with significant decrease in the levels of NPC1L1 protein and NPC1L1 mRNA, as analyzed by Western blot and qPCR, respectively. Conclusion

  3. Curcumin inhibits cholesterol uptake in Caco-2 cells by down-regulation of NPC1L1 expression.

    Science.gov (United States)

    Feng, Dan; Ohlsson, Lena; Duan, Rui-Dong

    2010-04-19

    Curcumin is a polyphenol and the one of the principle curcuminoids of the spice turmeric. Its antioxidant, anti-cancer and anti-inflammatory effects have been intensively studied. Previous in vivo studies showed that administration of curcumin also decreased cholesterol levels in the blood, and the effects were considered to be related to upregulation of LDL receptor. However, since plasma cholesterol levels are also influenced by the uptake of cholesterol in the gut, which is mediated by a specific transporter Niemann-Pick Cl-like 1 (NPC1L1) protein, the present study is to investigate whether curcumin affects cholesterol uptake in the intestinal Caco-2 cells. Caco-2 cells were cultured to confluence. The micelles composed of bile salt, monoolein, and 14C-cholesterol were prepared. We first incubated the cells with the micelles in the presence and absence of ezetimibe, the specific inhibitor of NPC1L1, to see whether the uptake of the cholesterol in the cells was mediated by NPC1L1. We then pretreated the cells with curcumin at different concentrations for 24 h followed by examination of the changes of cholesterol uptake in these curcumin-treated cells. Finally we determined whether curcumin affects the expression of NPC1L1 by both Western blot analysis and qPCR quantification. We found that the uptake of radioactive cholesterol in Caco-2 cells was inhibited by ezetimibe in a dose-dependent manner. The results indicate that the uptake of cholesterol in this study was mediated by NPC1L1. We then pretreated the cells with 25-100 muM curcumin for 24 h and found that such a treatment dose-dependently inhibited cholesterol uptake with 40% inhibition obtained by 100 muM curcumin. In addition, we found that the curcumin-induced inhibition of cholesterol uptake was associated with significant decrease in the levels of NPC1L1 protein and NPC1L1 mRNA, as analyzed by Western blot and qPCR, respectively. Curcumin inhibits cholesterol uptake through suppression of NPC1L1

  4. Fluorescent Dansyl-Guanosine Conjugates that Bind c-MYC Promoter G-Quadruplex and Downregulate c-MYC Expression.

    Science.gov (United States)

    Pavan Kumar, Y; Saha, Puja; Saha, Dhurjhoti; Bessi, Irene; Schwalbe, Harald; Chowdhury, Shantanu; Dash, Jyotirmayee

    2016-03-02

    The four-stranded G-quadruplex present in the c-MYC P1 promoter has been shown to play a pivotal role in the regulation of c-MYC transcription. Small-molecule compounds capable of inhibiting the c-MYC promoter activity by stabilising the c-MYC G-quadruplex could potentially be used as anticancer agents. In this context, here we report the synthesis of dansyl-guanosine conjugates through one-pot modular click reactions. The dansyl-guanosine conjugates can selectively detect c-MYC G-quadruplex over other biologically relevant quadruplexes and duplex DNA and can be useful as staining reagents for selective visualisation of c-MYC G-quadruplex over duplex DNA by gel electrophoresis. NMR spectroscopic titrations revealed the preferential binding sites of these dansyl ligands to the c-MYC G-quadruplex. A dual luciferase assay and qRT-PCR revealed that a dansyl-bisguanosine ligand represses the c-MYC expression, possibly by stabilising the c-MYC G-quadruplex. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  5. Transforming growth factor-β1 promotes breast cancer metastasis by downregulating miR-196a-3p expression.

    Science.gov (United States)

    Chen, Yan; Huang, Shai; Wu, Bo; Fang, Jiankai; Zhu, Minsheng; Sun, Li; Zhang, Lifeng; Zhang, Yongsheng; Sun, Maomin; Guo, Lingling; Wang, Shouli

    2017-07-25

    Transforming growth factor-β1 is considered a key contributor to the progression of breast cancer. MicroRNAs are important factors in the development and progression of many malignancies. In the present study, upon studies of breast cancer cell lines and tissues, we showed that microRNA -196a-3p is decreased by transforming growth factor-β1 in breast cancer cells and associated with breast cancer progression. We identified neuropilin-2 as a target gene of microRNA -196a-3p and showed that it is regulated by transforming growth factor-β1. Moreover, transforming growth factor-β1-mediated inhibition of microRNA -196a-3p and activation of neuropilin-2were required for transforming growth factor-β1-induced migration and invasion of breast cancer cells. In addition, neuropilin-2 expression was suppressed in breast tumors, particularly in triple-negative breast cancers. Collectively, our findings strongly indicate that microRNA -196a-3p is a predictive biomarker of breast cancer metastasis and patient survival and a potential therapeutic target in metastatic breast cancer.

  6. Zearalenone exposure impairs ovarian primordial follicle formation via down-regulation of Lhx8 expression in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Guo-Liang [College of Animal Science and Technology, Northwest A& F University, Yangling, Shaanxi 712100 (China); Sun, Xiao-Feng [Institute of Reproductive Sciences, College of Animal Science and Technology, Qingdao Agricultural University, Qingdao, Shandong 266109 (China); Feng, Yan-Zhong [Institute of Animal Sciences, Heilongjiang Academy of Agricultural Sciences, Harbin, Heilongjiang 150086 (China); Li, Bo [Chengguo Station of Animal Husbandry and Veterinary, Laizhou 261437 (China); Li, Ya-Peng; Yang, Fan [Institute of Reproductive Sciences, College of Animal Science and Technology, Qingdao Agricultural University, Qingdao, Shandong 266109 (China); Nyachoti, Charles Martin [Department of Animal Science, University of Manitoba, Winnipeg, MB, R3T 2N2 (Canada); Shen, Wei [Institute of Reproductive Sciences, College of Animal Science and Technology, Qingdao Agricultural University, Qingdao, Shandong 266109 (China); Sun, Shi-Duo, E-mail: ssdsm@tom.com [College of Animal Science and Technology, Northwest A& F University, Yangling, Shaanxi 712100 (China); Li, Lan, E-mail: lilan9600@126.com [Institute of Reproductive Sciences, College of Animal Science and Technology, Qingdao Agricultural University, Qingdao, Shandong 266109 (China)

    2017-02-15

    Zearalenone (ZEA) is an estrogenic mycotoxin mainly produced as a secondary metabolite by numerous species of Fusarium. Previous work showed that ZEA had a negative impact on domestic animals with regard to reproduction. The adverse effects and the mechanisms of ZEA on mammalian ovarian folliculogenesis remain largely unknown, particularly its effect on primordial follicle formation. Thus, we investigated the biological effects of ZEA exposure on murine ovarian germ cell cyst breakdown and primordial follicle assembly. Our results demonstrated that newborn mouse ovaries exposed to 10 or 30 μM ZEA in vitro had significantly less germ cell numbers compared to the control group. Moreover, the presence of ZEA in vitro increased the numbers of TUNEL and γH2AX positive cells within mouse ovaries and the ratio of mRNA levels of the apoptotic genes Bax/Bcl-2. Furthermore, ZEA exposure reduced the mRNA of oocyte specific genes such as LIM homeobox 8 (Lhx8), newborn ovary homeobox (Nobox), spermatogenesis and oogenesis helix-loop-helix (Sohlh2), and factor in the germline alpha (Figlα) in a dose dependent manner. Exposure to ZEA led to remarkable changes in the Lhx8 3′-UTR DNA methylation dynamics in oocytes and severely impaired folliculogenesis in ovaries after transplantation under the kidney capsules of immunodeficient mice. In conclusion, ZEA exposure impairs mouse primordial follicle formation in vitro. - Highlights: • First time to evaluate the impact of ZEA on primordial follicle formation • ZEA exposure increases oocyte apoptosis and delays germ cell cyst breakdown. • ZEA exposure impairs the expression of LHX8 by affecting its DNA methylation.

  7. Antihypoxic effect of miR-24 in SH-SY5Y cells under hypoxia via downregulating expression of neurocan

    Energy Technology Data Exchange (ETDEWEB)

    Sun, Xingyuan, E-mail: sunxingyuan@sina.com; Ren, Zhanjun; Pan, Yunzhi; Zhang, Chenxin

    2016-09-02

    Hypoxia-induced apoptosis-related mechanisms involved in the brain damage following cerebral ischemia injury. A subset of the small noncoding microRNA (miRNAs) is regulated by tissue oxygen levels, and miR-24 was found to be activated by hypoxic conditions. However, the roles of miR-24 and its target gene in neuron are not well understood. Here, we validated miRNA-24 is down-regulated in patients with cerebral infarction. Hypoxia suppressed the expression of miR-24, but increased the expression of neurocan in both mRNA and protein levels in SH-SY5Y cells. MiR-24 mimics reduced the expression of neurocan, suppressed cell apoptosis, induced cell cycle progression and cell proliferation in SH-SY5Y cells under hypoxia. By luciferase reporter assay, neurocan is validated a direct target gene of miR-24. Furthermore, knockdown of neurocan suppressed cell apoptosis, induced cell cycle progression and cell proliferation in SH-SY5Y cells under hypoxia. Taken together, miR-24 overexpression or silencing of neurocan shows an antihypoxic effect in SH-SY5Y cells. Therefore, miR-24 and neurocan play critical roles in neuron cell apoptosis and are potential therapeutic targets for ischemic brain disease. - Highlights: • miR-24 and neurocan play critical roles in neuron cell apoptosis. • miR-24 and neurocan are potential therapeutic targets for ischemic brain disease. • Antihypoxic effect of miR-24 and neurocan in SH-SY5Y cells.

  8. p75 neurotrophin receptor is involved in proliferation of undifferentiated mouse embryonic stem cells

    International Nuclear Information System (INIS)

    Moscatelli, Ilana; Pierantozzi, Enrico; Camaioni, Antonella; Siracusa, Gregorio; Campagnolo, Luisa

    2009-01-01

    Neurotrophins and their receptors are known to play a role in the proliferation and survival of many different cell types of neuronal and non-neuronal lineages. In addition, there is much evidence in the literature showing that the p75 neurotrophin receptor (p75 NTR ), alone or in association with members of the family of Trk receptors, is expressed in a wide variety of stem cells, although its role in such cells has not been completely elucidated. In the present work we have investigated the expression of p75 NTR and Trks in totipotent and pluripotent cells, the mouse pre-implantation embryo and embryonic stem and germ cells (ES and EG cells). p75 NTR and TrkA can be first detected in the blastocyst from which ES cell lines are derived. Mouse ES cells retain p75 NTR /TrkA expression. Nerve growth factor is the only neurotrophin able to stimulate ES cell growth in culture, without affecting the expression of stem cell markers, alkaline phosphatase, Oct4 and Nanog. Such proliferation effect was blocked by antagonizing either p75 NTR or TrkA. Interestingly, immunoreactivity to anti-p75 NTR antibodies is lost upon ES cell differentiation. The expression pattern of neurotrophin receptors in murine ES cells differs from human ES cells, that only express TrkB and C, and do not respond to NGF. In this paper we also show that, while primordial germ cells (PGC) do not express p75 NTR , when they are made to revert to an ES-like phenotype, becoming EG cells, expression of p75 NTR is turned on.

  9. Exposure to nerve growth factor worsens nephrotoxic effect induced by Cyclosporine A in HK-2 cells.

    Directory of Open Access Journals (Sweden)

    Donatella Vizza

    Full Text Available Nerve growth factor is a neurotrophin that promotes cell growth, differentiation, survival and death through two different receptors: TrkA(NTR and p75(NTR. Nerve growth factor serum concentrations increase during many inflammatory and autoimmune diseases, glomerulonephritis, chronic kidney disease, end-stage renal disease and, particularly, in renal transplant. Considering that nerve growth factor exerts beneficial effects in the treatment of major central and peripheral neurodegenerative diseases, skin and corneal ulcers, we asked whether nerve growth factor could also exert a role in Cyclosporine A-induced graft nephrotoxicity. Our hypothesis was raised from basic evidence indicating that Cyclosporine A-inhibition of calcineurin-NFAT pathway increases nerve growth factor expression levels. Therefore, we investigated the involvement of nerve growth factor and its receptors in the damage exerted by Cyclosporine A in tubular renal cells, HK-2. Our results showed that in HK-2 cells combined treatment with Cyclosporine A + nerve growth factor induced a significant reduction in cell vitality concomitant with a down-regulation of Cyclin D1 and up-regulation of p21 levels respect to cells treated with Cyclosporine A alone. Moreover functional experiments showed that the co-treatment significantly up-regulated human p21promoter activity by involvement of the Sp1 transcription factor, whose nuclear content was negatively regulated by activated NFATc1. In addition we observed that the combined exposure to Cyclosporine A + nerve growth factor promoted an up-regulation of p75 (NTR and its target genes, p53 and BAD leading to the activation of intrinsic apoptosis. Finally, the chemical inhibition of p75(NTR down-regulated the intrinsic apoptotic signal. We describe two new mechanisms by which nerve growth factor promotes growth arrest and apoptosis in tubular renal cells exposed to Cyclosporine A.

  10. Propofol effectively inhibits lithium-pilocarpine- induced status epilepticus in rats via downregulation of N-methyl-D-aspartate receptor 2B subunit expression

    Science.gov (United States)

    Wang, Henglin; Wang, Zhuoqiang; Mi, Weidong; Zhao, Cong; Liu, Yanqin; Wang, Yongan; Sun, Haipeng

    2012-01-01

    Status epilepticus was induced via intraperitoneal injection of lithium-pilocarpine. The inhibitory effects of propofol on status epilepticus in rats were judged based on observation of behavior, electroencephalography and 24-hour survival rate. Propofol (12.5–100 mg/kg) improved status epilepticus in a dose-dependent manner, and significantly reduced the number of deaths within 24 hours of lithium-pilocarpine injection. Western blot results showed that, 24 hours after induction of status epilepticus, the levels of N-methyl-D-aspartate receptor 2A and 2B subunits were significantly increased in rat cerebral cortex and hippocampus. Propofol at 50 mg/kg significantly suppressed the increase in N-methyl-D-aspartate receptor 2B subunit levels, but not the increase in N-methyl-D-aspartate receptor 2A subunit levels. The results suggest that propofol can effectively inhibit status epilepticus induced by lithium-pilocarpine. This effect may be associated with downregulation of N-methyl-D-aspartate receptor 2B subunit expression after seizures. PMID:25737709

  11. Transgenic expression of B-50/GAP-43 in mature olfactory neurons triggers downregulation of native B-50/GAP-43 expression in immature olfactory neurons

    NARCIS (Netherlands)

    Holtmaat, Anthony J D G; Huizinga, C T; Margolis, F L; Gispen, Willem Hendrik; Verhaagen, J

    1999-01-01

    The adult mammalian olfactory neuroepithelium is an unusual neural tissue, since it maintains its capacity to form new neurons throughout life. Newly formed neurons differentiate in the basal layers of the olfactory neuroepithelium and express B-50/GAP-43, a protein implicated in neurite outgrowth.

  12. Down-regulation of human topoisomerase IIα expression correlates with relative amounts of specificity factors Sp1 and Sp3 bound at proximal and distal promoter regions

    Directory of Open Access Journals (Sweden)

    Isaacs Richard J

    2007-05-01

    Full Text Available Abstract Background Topoisomerase IIα has been shown to be down-regulated in doxorubicin-resistant cell lines. The specificity proteins Sp1 and Sp3 have been implicated in regulation of topoisomerase IIα transcription, although the mechanism by which they regulate expression is not fully understood. Sp1 has been shown to bind specifically to both proximal and distal GC elements of the human topoisomerase IIα promoter in vitro, while Sp3 binds only to the distal GC element unless additional flanking sequences are included. While Sp1 is thought to be an activator of human topoisomerase IIα, the functional significance of Sp3 binding is not known. Therefore, we sought to determine the functional relationship between Sp1 and Sp3 binding to the topoisomerase IIα promoter in vivo. We investigated endogenous levels of Sp1, Sp3 and topoisomerase IIα as well as binding of both Sp1 and Sp3 to the GC boxes of the topoisomerase IIα promoter in breast cancer cell lines in vivo after short term doxorubicin exposure. Results Functional effects of Sp1 and Sp3 were studied using transient cotransfection assays using a topoisomerase IIα promoter reporter construct. The in vivo interactions of Sp1 and Sp3 with the GC elements of the topoisomerase IIα promoter were studied in doxorubicin-treated breast cancer cell lines using chromatin immunoprecipitation assays. Relative amounts of endogenous proteins were measured using immunoblotting. In vivo DNA looping mediated by proteins bound at the GC1 and GC2 elements was studied using the chromatin conformation capture assay. Both Sp1 and Sp3 bound to the GC1 and GC2 regions. Sp1 and Sp3 were transcriptional activators and repressors respectively, with Sp3 repression being dominant over Sp1-mediated activation. The GC1 and GC2 elements are linked in vivo to form a loop, thus bringing distal regulatory elements and their cognate transcription factors into close proximity with the transcription start site

  13. Culture supernatants of oral cancer cells induce impaired IFN-α production of pDCs partly through the down-regulation of TLR-9 expression.

    Science.gov (United States)

    Han, Nannan; Zhang, Zun; Jv, Houyu; Hu, Jingzhou; Ruan, Min; Zhang, Chenping

    2018-06-05

    The aim of the present study was to investigate whether tumor-derived supernatants down-regulate the immune function of plasmacytoid dendritic cells (pDCs) in oral cancer and the potential molecular mechanisms of this effect. Immunohistochemistry (IHC) and flow cytometry were used to detect tumor-infiltrating and peripheral blood pDCs. MTS and flow cytometry were employed to evaluate the immune response of CD4 + T cells. Real-time PCR and ELISA assays were used to identify TLR-7 and TLR-9 expression, IFN-α production and tumor-secreted soluble cytokines. The proportion of pDCs (0.121%±0.043%) was significantly higher in Oral squamous cell carcinoma (OSCC) samples than in normal tissue (0.023%±0.016%) (P = 0.021). TLR9 mRNA was significantly lower in tumor-infiltrating pDCs and positively correlated to low IFN-α production (r = 0.956; Poral cancer cells negatively regulated TLR9 mRNA expression and the subsequent IFN-α production of pDCs, which inhibited the immune response of CD4 + T cells. The neutralizing antibodies blocking assay showed that the specific inhibitory effect of pDC functionality was associated with the soluble fraction of the oral cancer environment, which is mainly mediated by IL-10 and TGF-β cooperation. Tumor-derived supernatants may impair the function of tumor-infiltrating pDCs, which subsequently decreases the immune response of CD4 + T cells in human oral cancer through TGF-β- and IL-10- dependent mechanisms. Careful manipulation of these impaired pDCs may help develop an important alternative immunotherapy for the treatment of oral cancer. Copyright © 2018 Elsevier Ltd. All rights reserved.

  14. The Natural Compound Dansameum Reduces foam Cell Formation by Downregulating CD36 and Peroxisome Proliferator-activated Receptor-gamma; Expression.

    Science.gov (United States)

    Park, Kang-Seo; Ahn, Sang Hyun; Lee, Kang Pa; Park, Sun-Young; Cheon, Jin Hong; Choi, Jun-Yong; Kim, Kibong

    2018-01-01

    Atherosclerosis-induced vascular disorders are major causes of death in most western countries. During the development of atherosclerotic lesions, foam cell formation is essential and formed through the expression of CD36 and the peroxisome proliferator-activated receptor gamma (PPAR-γ). To investigate whether dansameum extract (DSE) could show anti-atherosclerotic effect through down-regulating cellular redox state including CD36 and PARP-γ expression in oxidative low-density lipoprotein (oxLDL)-treated RAW264.7 cells and on differentiated foam cells in ApoE Knockout (ApoE-/-) mice. The Korean polyherbal medicine DSE was prepared from three plants in the following proportions: 40 g of Salvia miltiorrhiza root, 4 g of Amomumxanthioides fruit, and 4 g of Santalum album lignum. The immunohistochemistry and reverse transcription-polymerase chain reaction was used for analysis of protein and mRNA involved in foam cell formation. We first showed that effects of DSE on foam cell formation in both oxLDL-induced RAW264.7 cells and in blood vessels from apolipoprotein E deficientApoE-/- mice with high fat diet-fed. DSE treatment significantly reduced the expression of CD36 and PPAR-γ in oxLDL-stimulated RAW264.7 cells and ApoE-/-mice, in the latter case by regulating heme oxygenase-1. Furthermore, DSE treatment also reduced cellular lipid content in vitro and in vivo experiments. Our data suggest that DSE may have anti-atherosclerotic properties through regulating foam cell formation. Dansameum extract (DSE) Regulates the expression of CD36 and peroxisome proliferator-activated receptor gamma in oxidative low-density lipoprotein-stimulated RAW264.7 Cells and ApoE Knockout (ApoE Knockout [ApoE-/-]) miceDSE Regulates Cholesterol Levels in the Serum of ApoE-deficient (ApoE-/-) miceDSE Reduced the Formation of Foam Cells by Regulating heme oxygenase-1 in ApoE-/- mice with high fat diet-fed. Abbreviations used: DSE: Dansameum extract, PPAR-γ: Peroxisome proliferator

  15. 17 beta-estradiol modifies nitric oxide-sensitive guanylyl cyclase expression and down-regulates its activity in rat anterior pituitary gland.

    Science.gov (United States)

    Cabilla, Jimena P; Díaz, María del Carmen; Machiavelli, Leticia I; Poliandri, Ariel H; Quinteros, Fernanda A; Lasaga, Mercedes; Duvilanski, Beatriz H

    2006-09-01

    Previous studies showed that 17 beta-estradiol (17 beta-E2) regulates the nitric oxide (NO)/soluble guanylyl cyclase (sGC)/cGMP pathway in many tissues. Evidence from our laboratory indicates that 17 beta-E2 disrupts the inhibitory effect of NO on prolactin release, decreasing sGC activity and affecting the cGMP pathway in anterior pituitary gland of adult ovariectomized and estrogenized rats. To ascertain the mechanisms by which 17 beta-E2 affects sGC activity, we investigated the in vivo and in vitro effects of 17 beta-E2 on sGC protein and mRNA expression in anterior pituitary gland from immature female rats. In the present work, we showed that 17 beta-E2 acute treatment exerted opposite effects on the two sGC subunits, increasing alpha1 and decreasing beta1 subunit protein and mRNA expression. This action on sGC protein expression was maximal 6-9 h after 17 beta-E2 administration. 17beta-E2 also caused the same effect on mRNA expression at earlier times. Concomitantly, 17 beta-E2 dramatically decreased sGC activity 6 and 9 h after injection. These effects were specific of 17 beta-E2, because they were not observed with the administration of other steroids such as progesterone and 17 alpha-estradiol. This inhibitory action of 17beta-E2 on sGC also required the activation of estrogen receptor (ER), because treatment with the pure ER antagonist ICI 182,780 completely blocked 17 beta-E2 action. 17 beta-E2 acute treatment caused the same effects on pituitary cells in culture. These results suggest that 17 beta-E2 exerts an acute inhibitory effect on sGC in anterior pituitary gland by down-regulating sGC beta 1 subunit and sGC activity in a specific, ER-dependent manner.

  16. Hypoxia in Tumor Angiogenesis and Metastasis: Evaluation of VEGF and MMP Over-expression and Down-Regulation of HIF-1alpha with RNAi in Hypoxic Tumor Cells

    Science.gov (United States)

    Shah, Shruti

    Background: As tumor mass grows beyond a few millimeters in diameter, the angiogenic "switch" is turned on leading to recruitment of blood vessels from surrounding artery and veins. However, the tumor mass is poorly perfused and there are pockets of hypoxia or lower oxygen concentrations relative to normal tissue. Hypoxia-inducing factor-1a (HIF-1a), a transcription factor, is activated when the oxygen concentration is low. Upon activation of HIF-1a, a number of other genes also turn on that allows the tumor to become more aggressive and resistant to therapy. Purpose: The main objectives of this study were to evaluate the effect of hypoxia-induced HIF-1a followed by over-expression of angiogenic and metastatic markers in tumor cells and down-regulation of HIF-1a using nanoparticle-delivered RNA interference therapy. Methods: Human ovarian (SKOV3) and breast (MDA-MB-231) adenocarcinoma cells were incubated under normoxic and hypoxic conditions. Following hypoxia treatment of the cells, HIF-1α, vascular endothelial growth factor (VEGF), matrix metalloproteinase 2 (MMP-2), and MMP-9 expression was analyzed qualitatively and quantitatively. For intracellular delivery of HIF-1a gene silencing small interfering RNA (siRNA), type B gelatin nanoparticles were fabricated using the solvent displacement method and the surface was modified with poly(ethylene glycol) (PEG, Mol. wt. 2kDa). Cellular uptake and distribution of the nanoparticles was observed with Cy3-siRNA loaded, FITC-conjugated gelatin nanoparticles. Cytotoxicity of the nanoparticle formulations was evaluated in both the cell lines. siRNA was transfected in the gelatin nanoparticles under hypoxic conditions. Total cellular protein and RNA were extracted for analysis of HIF1a, VEGF, MMP-2 and MMP-9 expression. Results: MDA-MB-231 and SKOV3 cells show increased expression of HIF1a under hypoxic conditions compared to baseline levels at normoxic conditions. ELISA and western blots of VEGF, MMP-2 and MMP-9 appear to

  17. Comparative Analysis of RNAi-Based Methods to Down-Regulate Expression of Two Genes Expressed at Different Levels in Myzus persicae

    Directory of Open Access Journals (Sweden)

    Michaël Mulot

    2016-11-01

    Full Text Available With the increasing availability of aphid genomic data, it is necessary to develop robust functional validation methods to evaluate the role of specific aphid genes. This work represents the first study in which five different techniques, all based on RNA interference and on oral acquisition of double-stranded RNA (dsRNA, were developed to silence two genes, ALY and Eph, potentially involved in polerovirus transmission by aphids. Efficient silencing of only Eph transcripts, which are less abundant than those of ALY, could be achieved by feeding aphids on transgenic Arabidopsis thaliana expressing an RNA hairpin targeting Eph, on Nicotiana benthamiana infected with a Tobacco rattle virus (TRV-Eph recombinant virus, or on in vitro-synthesized Eph-targeting dsRNA. These experiments showed that the silencing efficiency may differ greatly between genes and that aphid gut cells seem to be preferentially affected by the silencing mechanism after oral acquisition of dsRNA. In addition, the use of plants infected with recombinant TRV proved to be a promising technique to silence aphid genes as it does not require plant transformation. This work highlights the need to pursue development of innovative strategies to reproducibly achieve reduction of expression of aphid genes.

  18. Hepatic expression of spermatogenic genes and their transiently remarkable downregulations in Wistar-Kyoto rats in response to lead-nitrate administration: strain-difference in the gene expression patterns.

    Science.gov (United States)

    Nemoto, Kiyomitsu; Ito, Sei; Yoshida, Chiaki; Miyata, Misaki; Kojima, Misaki; Degawa, Masakuni

    2011-06-01

    Administration of lead ion (Pb) to rats and mice affects hepatic functions such as the induction of hepatic cell proliferation and upregulation of cholesterol biosynthesis. To identify the genes for which expression changes in response to Pb-administration, we analyzed hepatic gene expression patterns in stroke-prone spontaneously hypertensive rat (SHRSP), its normotensive control, Wistar-Kyoto rat (WKY), and Spraque-Dawley (SD) rat strains, 3, 6, and 12 hr later after single i.v. injection of lead nitrate (LN) at a dose of 100 µmol using a DNA microarray technique. The data analysis demonstrated that the expression of a great number of genes was transiently and remarkably downregulated 3 hr after LN-injection, and then recovered to control levels only in LN-injected WKY. These normal hepatic expression levels in WKY and SHRSP were much higher than those in SD rats. Furthermore, most of these genes were ones thought to be expressed specifically in the spermatids and/or testes; i.e. genes encoding protamin 1, transition protein 1, and transition protein 2. These findings suggest that the regulation system common to expression of all of these genes could be a target site of Pb-toxic action, at least, in the liver of WKY, and that this system might be similar to the system essential for spermatogenesis, especially spermiogenesis, in the testis. In addition, it appears that clarifying the cause of the difference between the systems of WKY and SHRSP might aid in identifying the pathologic genes in SHRSP. Finally, it will be an important to clarify how the products of the genes related to spermatogenesis, including spermiogenesis, are functional in the livers of WKY and SHRSP.

  19. Quinacrine induces apoptosis in human leukemia K562 cells via p38 MAPK-elicited BCL2 down-regulation and suppression of ERK/c-Jun-mediated BCL2L1 expression

    International Nuclear Information System (INIS)

    Changchien, Jung-Jung; Chen, Ying-Jung; Huang, Chia-Hui; Cheng, Tian-Lu; Lin, Shinne-Ren; Chang, Long-Sen

    2015-01-01

    Although previous studies have revealed the anti-cancer activity of quinacrine, its effect on leukemia is not clearly resolved. We sought to explore the cytotoxic effect and mechanism of quinacrine action in human leukemia K562 cells. Quinacrine induced K562 cell apoptosis accompanied with ROS generation, mitochondrial depolarization, and down-regulation of BCL2L1 and BCL2. Upon exposure to quinacrine, ROS-mediated p38 MAPK activation and ERK inactivation were observed in K562 cells. Quinacrine-induced cell death and mitochondrial depolarization were suppressed by the p38MAPK inhibitor SB202190 and constitutively active MEK1 over-expression. Activation of p38 MAPK was shown to promote BCL2 degradation. Further, ERK inactivation suppressed c-Jun-mediated transcriptional expression of BCL2L1. Over-expression of BCL2L1 and BCL2 attenuated quinacrine-evoked mitochondrial depolarization and rescued the viability of quinacrine-treated cells. Taken together, our data indicate that quinacrine-induced K562 cell apoptosis is mediated through mitochondrial alterations triggered by p38 MAPK-mediated BCL2 down-regulation and suppression of ERK/c-Jun-mediated BCL2L1 expression. - Highlights: • Quinacrine induces K562 cell apoptosis via down-regulation of BCL2 and BCL2L1. • Quinacrine induces p38 MAPK activation and ERK inactivation in K562 cells. • Quinacrine elicits p38 MAPK-mediated BCL2 down-regulation. • Quinacrine suppresses ERK/c-Jun-mediated BCL2L1 expression

  20. Comparative proteomic analyses reveal that FlbA down-regulates gliT expression and SOD activity in Aspergillus fumigatus.

    Science.gov (United States)

    Shin, Kwang-Soo; Park, Hee-Soo; Kim, Young-Hwan; Yu, Jae-Hyuk

    2013-07-11

    mutant and 8 proteins differentially secreted in wild type and the flbA mutant. Based on proteomic analyses, we further examined the role of FlbA and found that FlbA down-regulates gliT expression and SOD activity. Our results proposed that FlbA-mediated signaling control negatively affects cellular responses associated with detoxification of reactive oxygen species and exogenous gliotoxin in A. fumigatus. Copyright © 2013 Elsevier B.V. All rights reserved.

  1. Towards the PET radiotracer for p75 neurotrophin receptor: [(11)C]LM11A-24 shows biological activity in vitro, but unfavorable ex vivo and in vivo profile.

    Science.gov (United States)

    Gibon, Julien; Kang, Min Su; Aliaga, Arturo; Sharif, Behrang; Rosa-Neto, Pedro; Séguéla, Philippe; Barker, Philip A; Kostikov, Alexey

    2016-10-01

    Mature neurotrophins as well as their pro forms are critically involved in the regulation of neuronal functions. They are signaling through three distinct types of receptors: tropomyosin receptor kinase family (TrkA/B/C), p75 neurotrophin receptor (p75(NTR)) and sortilin. Aberrant expression of p75(NTR) in the CNS is implicated in a variety of neurodegenerative diseases, including Alzheimer's disease. The goal of this work was to evaluate one of the very few reported p75(NTR) small molecule ligands as a lead compound for development of novel PET radiotracers for in vivo p75(NTR) imaging. Here we report that previously described ligand LM11A-24 shows significant inhibition of carbachol-induced persistent firing (PF) of entorhinal cortex (EC) pyramidal neurons in wild-type mice via selective interaction with p75(NTR). Based on this electrophysiological assay, the compound has very high potency with an EC50<10nM. We optimized the radiosynthesis of [(11)C]LM11A-24 as the first attempt to develop PET radioligand for in vivo imaging of p75(NTR). Despite some weak interaction with CNS tissues, the radiolabeled compound showed unfavorable in vivo profile presumably due to high hydrophilicity. Copyright © 2016 Elsevier Ltd. All rights reserved.

  2. Subcutaneous administration of polymerized type I collagen downregulates interleukin (IL)-17A, IL-22 and transforming growth factor-β1 expression, and increases Foxp3-expressing cells in localized scleroderma.

    Science.gov (United States)

    Furuzawa-Carballeda, J; Ortíz-Ávalos, M; Lima, G; Jurado-Santa Cruz, F; Llorente, L

    2012-08-01

    Localized scleroderma (LS) is a disfiguring inflammatory autoimmune disease of the skin and underlying tissue. As in systemic sclerosis, a key feature is the presence of T cells in inflammatory lesions. To evaluate the effect of polymerized type I collagen vs. methylprednisolone (MP) in LS, and to determine the influence of this polymerized collagen (PC) on CD4+ peripheral T cells expressing interleukin (IL)-4, IL-17A, interferon-γ and Forkhead box protein (Foxp)3, and on cells expressing transforming growth factor (TGF)-β1, IL-17A, IL-22 and Foxp3 in the skin. In total, 16 patients with LS were treated for 3 months with monthly subcutaneous intralesional injections of 0.1 mL MP (giving a total dose of 20 mg/mL each month) and 15 patients were treated, with weekly subcutaneous intralesional injections of PC, ranging from 0.2 mL (equivalent to 1.66 mg collagen) for a lesion of 50 mm in size, up to a maximum of 1.0 mL (8.3 mg collagen) for a lesion > 100 mm in size, and followed up for a further 6 months. Skin biopsies were obtained from lesions at baseline (before treatment) and 9 months later (6 months after treatment end). Tissue sections were evaluated by histology and immunohistochemistry (IL-17A, IL-22, TGF-β1 and Foxp3). CD4+ T-cell subsets were determined in peripheral blood by flow cytometry. Abnormal tissue architecture was seen in the biopsies taken from patients treated with MP, whereas the PC treatment restored normal skin architecture. PC downregulated pro-inflammatory/profibrotic cytokine expression in peripheral cells, and upregulated the number of regulatory T cells (Tregs) in skin. PC was safe and well tolerated. PC is not only an antifibrotic/fibrolytic agent but also an immunomodulator biodrug that restores the balance between T helper (Th)1, Th2, Th17 and Tregs, downregulates production of pro-inflammatory or profibrogenic cytokines (IL-17A, IL-22 and TGF-β1), and renews skin architecture, without adverse effects. © The Author(s). CED

  3. A 4-Phenoxyphenol Derivative Exerts Inhibitory Effects on Human Hepatocellular Carcinoma Cells through Regulating Autophagy and Apoptosis Accompanied by Downregulating α-Tubulin Expression

    Directory of Open Access Journals (Sweden)

    Wen-Tsan Chang

    2017-05-01

    Full Text Available Hepatocellular carcinoma (HCC is a leading cancer worldwide. Advanced HCCs are usually resistant to anticancer drugs, causing unsatisfactory chemotherapy outcomes. In this study, we showed that a 4-phenoxyphenol derivative, 4-[4-(4-hydroxyphenoxyphenoxy]phenol (4-HPPP, exerts an inhibitory activity against two HCC cell lines, Huh7 and Ha22T. We further investigated the anti-HCC activities of 4-HPPP, including anti-proliferation and induction of apoptosis. Our results showed that higher dosage of 4-HPPP downregulates the expression of α-tubulin and causes nuclear enlargement in both the Huh-7 and Ha22T cell lines. Interestingly, the colony formation results showed a discrepancy in the inhibitory effect of 4-HPPP on HCC and rat liver epithelial Clone 9 cells, suggesting the selective cytotoxicity of 4-HPPP toward HCC cells. Furthermore, the cell proliferation and apoptosis assay results illustrated the differences between the two HCC cell lines. The results of cellular proliferation assays, including trypan blue exclusion and colony formation, revealed that 4-HPPP inhibits the growth of Huh7 cells, but exerts less cytotoxicity in Ha22T cells. Furthermore, the annexin V assay performed for detecting the apoptosis showed similar results. Western blotting results showed 4-HPPP caused the increase of pro-apoptotic factors including cleaved caspase-3, Bid and Bax in HCC cells, especially in Huh-7. Furthermore, an increase of autophagy-associated protein microtubule-associated protein-1 light chain-3B (LC3B-II and the decrease of Beclin-1 and p62/SQSTM1 were observed following 4-HPPP treatment. Additionally, the level of γH2A histone family, member X (γH2AX, an endogenous DNA damage biomarker, was dramatically increased in Huh7 cells after 4-HPPP treatment, suggesting the involvement of DNA damage pathway in 4-HPPP-induced apoptosis. On the contrary, the western blotting results showed that treatment up-regulates pro-survival proteins, including the

  4. Downregulation of eIF4G by microRNA-503 enhances drug sensitivity of MCF-7/ADR cells through suppressing the expression of ABC transport proteins.

    Science.gov (United States)

    Pan, Xia; Yang, Xiaoyan; Zang, Jinglei; Zhang, Si; Huang, Nan; Guan, Xinxin; Zhang, Jianhua; Wang, Zhihui; Li, Xi; Lei, Xiaoyong

    2017-06-01

    Overexpression of adenosine triphosphate-binding cassette (ABC) transport protein is emerging as a critical contributor to anticancer drug resistance. The eukaryotic translation initiation factor (eIF) 4F complex, the key modulator of mRNA translation, is regulated by the phosphoinositide 3-kinase-AKT-mammalian target of rapamycin pathway in anticancer drug-resistant tumors. The present study demonstrated the roles of ABC translation protein alterations in the acquisition of the Adriamycin (ADM)-resistant phenotype of MCF-7 human breast cells. Quantitative polymerase chain reaction and western blot analysis were applied to examine the differences in mRNA and protein levels, respectively. It was found that the expression of the ABC sub-family B member 1, ABC sub-family C member 1 and ABC sub-family G member 2 transport proteins were upregulated in MCF-7/ADR cells. An MTT assay was used to detect the cell viability, from the results MCF-7/ADR cells were less sensitive to ADM, tamoxifen (TAM) and taxol (TAX) treatment compared with MCF-7 cells. We predicted that the 3'-untranslated region of eukaryotic translation initiation factor 4-γ 1 (eIF4G) contains a potential miRNA binding site for microRNA (miR)-503 through using computational programs. These binding sites were confirmed by luciferase reporter assays. eIF4G mRNA degradation was accelerated in cells transfected with miR-503 mimics. Furthermore, it was demonstrated that eIF4G and ABC translation proteins were significantly downregulated in MCF-7/ADR cells after transfection with miR-503. It was found that miR-503 mimics could sensitize the cells to treatment with ADM, TAM and TAX. These findings demonstrated for the first time that eIF4G acted as a key factor in MCF-7/ADR cells, and may be an efficient agent for preventing and reversing multi-drug resistance in breast cancer.

  5. Loss of the polycomb protein Mel-18 enhances the epithelial-mesenchymal transition by ZEB1 and ZEB2 expression through the downregulation of miR-205 in breast cancer.

    Science.gov (United States)

    Lee, J-Y; Park, M K; Park, J-H; Lee, H J; Shin, D H; Kang, Y; Lee, C H; Kong, G

    2014-03-06

    The epithelial-mesenchymal transition (EMT) is the pivotal mechanism underlying the initiation of cancer invasion and metastasis. Although Mel-18 has been implicated in several biological processes in cancer, its function in the EMT of human cancers has not yet been studied. Here, we demonstrate that Mel-18 negatively regulates the EMT by epigenetically modulating miR-205. We identified miR-205 as a novel target of Mel-18 using a microRNA microarray analysis and found that Mel-18 increased miR-205 transcription by the inhibition of DNA methyltransferase-mediated DNA methylation of the miR-205 promoter, thereby downregulating its target genes, ZEB1 and ZEB2. Furthermore, the loss of Mel-18 promoted ZEB1- and ZEB2-mediated downregulation of E-cadherin transcription and also enhanced the expression of mesenchymal markers, leading to increased migration and invasion in MCF-7 cells. In MDA-MB-231 cells, Mel-18 overexpression restored E-cadherin expression, resulting in reduced migration and invasion. These effects were reversed by miR-205 overexpression or inhibition. A tumor xenograft with Mel-18 knockdown MCF-7 cells consistently showed increased ZEB1 and ZEB2 expression and decreased E-cadherin expression. Taken together, these results suggest that Mel-18 functions as a tumor suppressor by its novel negative control of the EMT, achieved through regulating the expression of miR-205 and its target genes, ZEB1 and ZEB2.

  6. TSA suppresses miR-106b-93-25 cluster expression through downregulation of MYC and inhibits proliferation and induces apoptosis in human EMC.

    Science.gov (United States)

    Zhao, Zhi-Ning; Bai, Jiu-Xu; Zhou, Qiang; Yan, Bo; Qin, Wei-Wei; Jia, Lin-Tao; Meng, Yan-Ling; Jin, Bo-Quan; Yao, Li-Bo; Wang, Tao; Yang, An-Gang

    2012-01-01

    Histone deacetylase (HDAC) inhibitors are emerging as a novel class of anti-tumor agents and have manifested the ability to decrease proliferation and increase apoptosis in different cancer cells. A significant number of genes have been identified as potential effectors responsible for the anti-tumor function of HDAC inhibitor. However, the molecular mechanisms of these HDAC inhibitors in this process remain largely undefined. In the current study, we searched for microRNAs (miRs) that were affected by HDAC inhibitor trichostatin (TSA) and investigated their effects in endometrial cancer (EMC) cells. Our data showed that TSA significantly inhibited the growth of EMC cells and induced their apoptosis. Among the miRNAs that altered in the presence of TSA, the miR-106b-93-25 cluster, together with its host gene MCM7, were obviously down-regulated in EMC cells. p21 and BIM, which were identified as target genes of miR-106b-93-25 cluster, increased in TSA treated tumor cells and were responsible for cell cycle arrest and apoptosis. We further identified MYC as a regulator of miR-106b-93-25 cluster and demonstrated its down-regulation in the presence of TSA resulted in the reduction of miR-106b-93-25 cluster and up-regulation of p21 and BIM. More important, we found miR-106b-93-25 cluster was up-regulated in clinical EMC samples in association with the overexpression of MCM7 and MYC and the down-regulation of p21 and BIM. Thus our studies strongly indicated TSA inhibited EMC cell growth and induced cell apoptosis and cell cycle arrest at least partially through the down-regulation of the miR-106b-93-25 cluster and up-regulation of it's target genes p21 and BIM via MYC.

  7. Lipopolysaccharide derived from the rumen down-regulates stearoyl-CoA desaturase 1 expression and alters fatty acid composition in the liver of dairy cows fed a high-concentrate diet.

    Science.gov (United States)

    Xu, Tianle; Tao, Hui; Chang, Guangjun; Zhang, Kai; Xu, Lei; Shen, Xiangzhen

    2015-03-07

    Dairy cows are often fed a high-concentrate diet to meet lactating demands, yet long-term concentrate feeding induces subacute ruminal acidosis (SARA) and leads to a decrease in milk fat. Stearoyl-CoA desaturase1 (SCD1) participates in fatty acid biosynthesis in the liver of lactating ruminants. Here, we conducted this study to investigate the impact of lipopolysaccharide derived from the rumen on SCD1 expression and on fatty acid composition in the liver of dairy cows fed a high-concentrate diet. Eight multiparous mid-lactating Holstein cows (455 ± 28 kg) were randomly assigned into two groups in the experiment and were fed a low-concentrate diet (LC) or high-concentrate diet (HC) for 18 weeks. The results showed that the total volatile fatty acids and lactic acid accumulated in the rumen, leading to a decreased rumen pH and elevated lipopolysaccharides (LPSs) in the HC group. The long chain fatty acid profile in the rumen and hepatic vein was remarkably altered in the animals fed the HC diet. The triglyceride (TG), non-esterified fatty acid (NEFA) and total cholesterol (TCH) content in the plasma was significantly decreased, whereas plasma glucose and insulin levels were increased. The expression of SCD1 in the liver was significantly down-regulated in the HC group. In regards to transcriptional regulators, the expression of sterol regulatory element binding transcription factors (SREBF1c, SREBF2) and SREBP cleavage activating protein (SCAP) was down-regulated, while peroxisome proliferator-activated receptor α (PPARα) was up-regulated. These data indicate that lipopolysaccharide derived from the rumen down-regulates stearoyl-CoA desaturase 1 expression and alters fatty acid composition in the liver of dairy cows fed a high-concentrate diet.

  8. Hath1 inhibits proliferation of colon cancer cells probably through up-regulating expression of Muc2 and p27 and down-regulating expression of cyclin D1.

    Science.gov (United States)

    Zhu, Dai-Hua; Niu, Bai-Lin; Du, Hui-Min; Ren, Ke; Sun, Jian-Ming; Gong, Jian-Ping

    2012-01-01

    Previous studies showed that Math1 homologous to human Hath1 can cause mouse goblet cells to differentiate. In this context it is important that the majority of colon cancers have few goblet cells. In the present study, the potential role of Hath1 in colon carcinogenesis was investigated. Sections of paraffin-embedded tissues were used to investigate the goblet cell population of normal colon mucosa, mucosa adjacent colon cancer and colon cancer samples from 48 patients. Hath1 and Muc2 expression in these samples were tested by immunohistochemistry, quantitative real-time reverse transcription -PCR and Western blotting. After the recombinant plasmid, pcDNA3.1(+)-Hath1 had been transfected into HT29 colon cancer cells, three clones were selected randomly to test the levels of Hath1 mRNA, Muc2 mRNA, Hath1, Muc2, cyclin D1 and p27 by quantitative real-time reverse transcription-PCR and Western blotting. Moreover, the proliferative ability of HT29 cells introduced with Hath1 was assessed by means of colony formation assay and xenografting. Expression of Hath1, Muc2, cyclin D1 and p27 in the xenograft tumors was also detected by Western blotting. No goblet cells were to be found in colon cancer and levels of Hath1 mRNA and Hath1, Muc2 mRNA and Muc2 were significantly down-regulated. Hath1 could decrease cyclin D1, increase p27 and Muc2 in HT29 cells and inhibit their proliferation. Hath1 may be an anti-oncogene in colon carcinogenesis.

  9. Cadmium(Cd)-induced oxidative stress down-regulates the gene expression of DNA mismatch recognition proteins MutS homolog 2 (MSH2) and MSH6 in zebrafish (Danio rerio) embryos

    Energy Technology Data Exchange (ETDEWEB)

    Hsu, Todd, E-mail: toddhsu@mail.ntou.edu.tw [Institute of Bioscience and Biotechnology and Center of Excellence for Marine Bioenvironment and Biotechnology, National Taiwan Ocean University, Keelung 20224, Taiwan (China); Huang, Kuan-Ming; Tsai, Huei-Ting; Sung, Shih-Tsung; Ho, Tsung-Nan [Institute of Bioscience and Biotechnology and Center of Excellence for Marine Bioenvironment and Biotechnology, National Taiwan Ocean University, Keelung 20224, Taiwan (China)

    2013-01-15

    DNA mismatch repair (MMR) of simple base mismatches and small insertion-deletion loops in eukaryotes is initiated by the binding of the MutS homolog 2 (MSH2)-MSH6 heterodimer to mismatched DNA. Cadmium (Cd) is a genotoxic heavy metal that has been recognized as a human carcinogen. Oxidant stress and inhibition of DNA repair have been proposed as major factors underlying Cd genotoxicity. Our previous studies indicated the ability of Cd to disturb the gene expression of MSH6 in zebrafish (Danio rerio) embryos. This study was undertaken to explore if Cd-induced oxidative stress down-regulated MSH gene activities. Following the exposure of zebrafish embryos at 1 h post fertilization (hpf) to sublethal concentrations of Cd at 3-5 {mu}M for 4 or 9 h, a parallel down-regulation of MSH2, MSH6 and Cu/Zn superoxide dismutase (Cu/Zn-SOD) gene expression was detected by real-time RT-PCR and the expression levels were 40-50% of control after a 9-h exposure. Cd exposure also induced oxidative stress, yet no inhibition of catalase gene activity was observed. Whole mount in situ hybridization revealed a wide distribution of msh6 mRNA in the head regions of 10 hpf embryos and pretreatment of embryos with antioxidants butylhydroxytoluene (BHT), D-mannitol or N-acetylcysteine (NAC) at 1-10 {mu}M restored Cd-suppressed msh6 expression. QPCR confirmed the protective effects of antioxidants on Cd-suppressed msh2/msh6 mRNA production. Down-regulated MSH gene activities reaching about 50% of control were also induced in embryos exposed to paraquat, a reactive oxygen species (ROS)-generating herbicide, or hydrogen peroxide at 200 {mu}M. Hence, Cd at sublethal levels down-regulates msh2/msh6 expression primarily via ROS as signaling molecules. The transcriptional activation of human msh6 is known to be fully dependent on the specificity factor 1 (Sp1). Cd failed to inhibit the DNA binding activity of zebrafish Sp1 unless at lethal concentrations based on band shift assay, therefore

  10. HA117 endows HL60 cells with a stem-like signature by inhibiting the degradation of DNMT1 via its ability to down-regulate expression of the GGL domain of RGS6.

    Directory of Open Access Journals (Sweden)

    Shuangshuang Li

    Full Text Available All-trans retinoic acid (ATRA induces complete remission in almost all patients with acute promyelocytic leukemia (APL via its ability to induce the in vivo differentiation of APL blasts. However, prolonged ATRA treatment can result in drug resistance. In previous studies, we generated a multi-drug-resistant HL60/ATRA cell line and found it to contain a new drug resistance-related gene segment, HA117. In this study, we demonstrate that ATRA induces multi-drug-resistant subpopulations of HL60 cells with a putative stem-like signature by up-regulating the expression of the new gene segment HA117. Western blot analysis and quantitative real-time PCR demonstrated that HA117 causes alternative splicing of regulator of G-protein signaling 6 (RGS6 and down-regulation of the expression of the GGL domain of RGS6, which plays an important role in DNA methyltransferase 1 (DNMT1 degradation. Moreover, DNMT1 expression was increased in multi-drug resistance HL60/ATRA cells. Knockdown of HA117 restored expression of the GGL domain and blocked DNMT1 expression. Moreover, resistant cells displayed a putative stem-like signature with increased expression of cancer steam cell markers CD133 and CD123. The stem cell marker, Nanog, was significantly up-regulated. In conclusion, our study shows that HA117 potentially promotes the stem-like signature of the HL60/ATRA cell line by inhibiting by the ubiquitination and degradation of DNMT1 and by down-regulating the expression of the GGL domain of RGS6. These results throw light on the cellular events associated with the ATRA-induced multi-drug resistance phenotype in acute leukemia.

  11. Gallic acid inhibits gastric cancer cells metastasis and invasive growth via increased expression of RhoB, downregulation of AKT/small GTPase signals and inhibition of NF-κB activity

    Energy Technology Data Exchange (ETDEWEB)

    Ho, Hsieh-Hsun [Institute of Biochemistry and Biotechnology, Chung Shan Medical University, Taichung 402, Taiwan (China); Chang, Chi-Sen [Department of Medicine, Chung Shan Medical University, Taichung 402, Taiwan (China); Division of Gastroenterology, Taichung Veterans General Hospital, Taichung 402, Taiwan (China); Ho, Wei-Chi [Division of Gastroenterology, Jen-Ai Hospital, Taichung 402, Taiwan (China); Liao, Sheng-You [Institute of Biochemistry and Biotechnology, Chung Shan Medical University, Taichung 402, Taiwan (China); Lin, Wea-Lung [Department of Pathology, School of Medicine, Chung Shan Medical University, Taichung 402, Taiwan (China); Department of Pathology, Chung Shan Medical University Hospital, Taichung 402, Taiwan (China); Wang, Chau-Jong, E-mail: wcj@csmu.edu.tw [Institute of Biochemistry and Biotechnology, Chung Shan Medical University, Taichung 402, Taiwan (China); Department of Medical Research, Chung Shan Medical University Hospital, Taichung 402, Taiwan (China)

    2013-01-01

    Our previous study demonstrated the therapeutic potential of gallic acid (GA) for controlling tumor metastasis through its inhibitory effect on the motility of AGS cells. A noteworthy finding in our previous experiment was increased RhoB expression in GA-treated cells. The aim of this study was to evaluate the role of RhoB expression on the inhibitory effects of GA on AGS cells. By applying the transfection of RhoB siRNA into AGS cells and an animal model, we tested the effect of GA on inhibition of tumor growth and RhoB expression. The results confirmed that RhoB-siRNA transfection induced GA to inhibit AGS cells’ invasive growth involving blocking the AKT/small GTPase signals pathway and inhibition of NF-κB activity. Finally, we evaluated the effect of GA on AGS cell metastasis by colonization of tumor cells in nude mice. It showed GA inhibited tumor cells growth via the expression of RhoB. These data support the inhibitory effect of GA which was shown to inhibit gastric cancer cell metastasis and invasive growth via increased expression of RhoB, downregulation of AKT/small GTPase signals and inhibition of NF-κB activity. Thus, GA might be a potential agent in treating gastric cancer. Highlights: ► GA could downregulate AKT signal via increased expression of RhoB. ► GA inhibits metastasis in vitro in gastric carcinoma. ► GA inhibits tumor growth in nude mice model.

  12. Hyperbaric environment up-regulates CD15s expression on leukocytes, down-regulates CD77 expression on renal cells and up-regulates CD34 expression on pulmonary and cardiac cells in rat

    Directory of Open Access Journals (Sweden)

    Danka Đevenica

    2016-08-01

    Full Text Available Objective(s: The aim of this study was to estimate effects of hyperbaric (HB treatment by determination of CD15s and CD11b leukocyte proinflammatory markers expression.  In addition, this study describes changes in CD77 and CD34 expression on rat endothelial cells in renal, pulmonary and cardiac tissue following exposure to hyperbaric pressure. Materials and Methods:Expression of CD11b and CD15s on leukocytes, as well as CD77 and CD34 expression on endothelial cells in cell suspensions of renal, pulmonary and cardiac tissue in rats after hyperbaric treatment and in control rats were determined by flow cytometry. Results: Hyperbaric treatment significantly increased percentage of leukocytes expressing CD15s+CD11b- (from 1.71±1.11 to 23.42±2.85, P

  13. TIMP1 overexpression mediates resistance of MCF-7 human breast cancer cells to fulvestrant and down-regulates progesterone receptor expression

    DEFF Research Database (Denmark)

    Bjerre, Christina; Vinther, Lena; Belling, Kirstine C.

    2013-01-01

    is associated with endocrine sensitivity. We established a panel of 11 MCF-7 subclones with a wide range of TIMP1 mRNA and protein expression levels. Cells with high expression of TIMP1 versus low TIMP1 displayed significantly reduced sensitivity to the antiestrogen fulvestrant (ICI 182,780, Faslodex®), while......, the effects of fulvestrant, 4-hydroxytamoxifen, or estrogen on estrogen receptor expression were not associated with TIMP1 levels. Gene expression analyses revealed associations between expression of TIMP1 and genes involved in metabolic pathways, epidermal growth factor receptor 1/cancer signaling pathways......, and cell cycle. Gene and protein expression analyses showed no general defects in estrogen receptor signaling except from lack of progesterone receptor expression and estrogen inducibility in clones with high TIMP1. The present study suggests a relation between high expression level of TIMP1 and loss...

  14. Circadian control of p75 neurotrophin receptor leads to alternate activation of Nrf2 and c-Rel to reset energy metabolism in astrocytes via brain-derived neurotrophic factor.

    Science.gov (United States)

    Ishii, Tetsuro; Warabi, Eiji; Mann, Giovanni E

    2018-05-01

    Circadian clock genes regulate energy metabolism partly through neurotrophins in the body. The low affinity neurotrophin receptor p75 NTR is a clock component directly regulated by the transcriptional factor Clock:Bmal1 complex. Brain-derived neurotrophic factor (BDNF) is expressed in the brain and plays a key role in coordinating metabolic interactions between neurons and astrocytes. BDNF transduces signals through TrkB and p75 NTR receptors. This review highlights a novel molecular mechanism by which BDNF via circadian control of p75 NTR leads to daily resetting of glucose and glycogen metabolism in brain astrocytes to accommodate their functional interaction with neurons. Astrocytes store glycogen as an energy reservoir to provide active neurons with the glycolytic metabolite lactate. Astrocytes predominantly express the truncated receptor TrkB.T1 which lacks an intracellular receptor tyrosine kinase domain. TrkB.T1 retains the capacity to regulate cell morphology through regulation of Rho GTPases. In contrast, p75 NTR mediates generation of the bioactive lipid ceramide upon stimulation with BDNF and inhibits PKA activation. As ceramide directly activates PKCζ, we discuss the importance of the TrkB.T1-p75 NTR -ceramide-PKCζ signaling axis in the stimulation of glycogen and lipid synthesis and activation of RhoA. Ceramide-PKCζ-casein kinase 2 signaling activates Nrf2 to support oxidative phosphorylation via upregulation of antioxidant enzymes. In the absence of p75 NTR , TrkB.T1 functionally interacts with adenosine A 2A R and dopamine D1R receptors to enhance cAMP-PKA signaling and activate Rac1 and NF-κB c-Rel, favoring glycogen hydrolysis, gluconeogenesis and aerobic glycolysis. Thus, diurnal changes in p75 NTR levels in astrocytes resets energy metabolism via BDNF to accommodate their metabolic interaction with neurons. Copyright © 2018 Elsevier Inc. All rights reserved.

  15. Gene expression profile of zeitlupe/lov kelch protein1 T-DNA insertion mutants in Arabidopsis thaliana: Downregulation of auxin-inducible genes in hypocotyls.

    Science.gov (United States)

    Saitoh, Aya; Takase, Tomoyuki; Kitaki, Hiroyuki; Miyazaki, Yuji; Kiyosue, Tomohiro

    2015-01-01

    Elongation of hypocotyl cells has been studied as a model for elucidating the contribution of cellular expansion to plant organ growth. ZEITLUPE (ZTL) or LOV KELCH PROTEIN1 (LKP1) is a positive regulator of warmth-induced hypocotyl elongation under white light in Arabidopsis, although the molecular mechanisms by which it promotes hypocotyl cell elongation remain unknown. Microarray analysis showed that 134 genes were upregulated and 204 genes including 15 auxin-inducible genes were downregulated in the seedlings of 2 ztl T-DNA insertion mutants grown under warm conditions with continuous white light. Application of a polar auxin transport inhibitor, an auxin antagonist or an auxin biosynthesis inhibitor inhibited hypocotyl elongation of control seedlings to the level observed with the ztl mutant. Our data suggest the involvement of auxin and auxin-inducible genes in ZTL-mediated hypocotyl elongation.

  16. Role of protein kinase A and class II phosphatidylinositol 3-kinase C2β in the downregulation of KCa3.1 channel synthesis and membrane surface expression by lyso-globotriaosylceramide

    Energy Technology Data Exchange (ETDEWEB)

    Choi, Ju Yeon; Park, Seonghee, E-mail: sp@ewha.ac.kr

    2016-02-19

    The intermediate conductance calcium-activated potassium channel (KCa3.1) mediates proliferation of many cell types including fibroblasts, and is a molecular target for intervention in various cell proliferative diseases. Our previous study showed that reduction of KCa3.1 channel expression by lyso-globotriaosylceramide (lyso-Gb3) inhibits differentiation into myofibroblasts and collagen synthesis, which might lead to development of ascending thoracic aortic aneurysm secondary to Fabry disease. However, how lyso-Gb3 downregulates KCa3.1 channel expression is unknown. Therefore, we aimed to investigate the underlying mechanisms of lyso-Gb3-mediated KCa3.1 channel downregulation, focusing on the cAMP signaling pathway. We found that lyso-Gb3 increased the intracellular cAMP concentration by upregulation of adenylyl cyclase 6 and inhibited ERK 1/2 phosphorylation through the protein kinase A (PKA) pathway, leading to the inhibition of KCa3.1 channel synthesis, not the exchange protein directly activated by cAMP (Epac) pathway. Moreover, lyso-Gb3 suppressed expression of class II phosphatidylinositol 3-kinase C2β (PI3KC2β) by PKA activation, which reduces the production of phosphatidylinositol 3-phosphate [PI(3)P], and the reduced membrane surface expression of KCa3.1 channel was recovered by increasing the intracellular levels of PI(3)P. Consequently, our findings that lyso-Gb3 inhibited both KCa3.1 channel synthesis and surface expression by increasing intracellular cAMP, and controlled surface expression through changes in PI3KC2β-mediated PI(3)P production, suggest that modulation of PKA and PI3KC2β activity to control of KCa3.1 channel expression can be an alternative important target to attenuate ascending thoracic aortic aneurysms in Fabry disease. - Highlights: • Lyso-Gb3 causes elevation of intracellular cAMP. • Lyso-Gb3 inhibits the ERK 1/2 phosphorylation through PKA, thereby reducing KCa3.1 channel synthesis. • Lyso-Gb3 reduces PI3KC2

  17. Expression of background potassium channels in rat DRG is cell-specific and down-regulated in a neuropathic pain model.

    Science.gov (United States)

    Pollema-Mays, Sarah L; Centeno, Maria Virginia; Ashford, Crystle J; Apkarian, A Vania; Martina, Marco

    2013-11-01

    Neuropathic pain is associated with hyperexcitability of DRG neurons. Despite the importance of leakage potassium channels for neuronal excitability, little is known about their cell-specific expression in DRGs and possible modulation in neuropathic pain. Multiple leakage channels are expressed in DRG neurons, including TASK1, TASK3, TRESK, TRAAK, TWIK1, TREK1 and TREK2 but little is known about their distribution among different cell types. Our immunohistochemical studies show robust TWIK1 expression in large and medium size neurons, without overlap with TRPV1 or IB4 staining. TASK1 and TASK3, on the contrary, are selectively expressed in small cells; TASK1 expression closely overlaps TRPV1-positive cells, while TASK3 is expressed in TRPV1- and IB4-negative cells. We also studied mRNA expression of these channels in L4-L5 DRGs in control conditions and up to 4 weeks after spared nerve injury lesion. We found that TWIK1 expression is much higher than TASK1 and TASK3 and is strongly decreased 1, 2 and 4 weeks after neuropathic injury. TASK3 expression, on the other hand, decreases 1 week after surgery but reverts to baseline by 2weeks; TASK1 shows no significant change at any time point. These data suggest an involvement of TWIK1 in the maintenance of the pain condition. © 2013.

  18. A viral long terminal repeat expressed in CD4+CD8+ precursors is downregulated in mature peripheral CD4-CD8+ or CD4+CD8- T cells.

    OpenAIRE

    Paquette, Y; Doyon, L; Laperrière, A; Hanna, Z; Ball, J; Sekaly, R P; Jolicoeur, P

    1992-01-01

    The long terminal repeat from a thymotropic mouse mammary tumor virus variant, DMBA-LV, was used to drive the expression of two reporter genes, murine c-myc and human CD4, in transgenic mice. Expression was observed specifically in thymic immature cells. Expression of c-myc in these cells induced oligoclonal CD4+ CD8+ T-cell thymomas. Expression of human CD4 was restricted to thymic progenitor CD4- CD8- and CD4+ CD8+ T cells and was shut off in mature CD4+ CD8- and CD4- CD8+ T cells, known to...

  19. Interferon-β induced microRNA-129-5p down-regulates HPV-18 E6 and E7 viral gene expression by targeting SP1 in cervical cancer cells.

    Directory of Open Access Journals (Sweden)

    Jiarong Zhang

    Full Text Available Infection by human papillomavirus (HPV can cause cervical intraepithelial neoplasia (CIN and cancer. Down-regulation of E6 and E7 expression may be responsible for the positive clinical outcomes observed with IFN treatment, but the molecular basis has not been well determined. As miRNAs play an important role in HPV induced cervical carcinogenesis, we hypothesize that IFN-β can regulate the expressions of specific miRNAs in cervical cancer cells, and that these miRNAs can mediate E6 and E7 expression, thus modulate their oncogenic potential. In this study, we found that miR-129-5p to be a candidate IFN-β inducible miRNA. MiR-129-5p levels gradually decrease with the development of cervical intraepithelial lesions. Manipulation of miR-129-5p expression in Hela cells modulates HPV-18 E6 and E7 viral gene expression. Exogenous miR-129-5p inhibits cell proliferation in Hela cells, promotes apoptosis and blocks cell cycle progression in Hela cells. SP1 is a direct target of miR-129-5p in Hela cells. This study is the first report of a cellular miRNA with anti-HPV activity and provides new insights into regulatory mechanisms between the HPV and the IFN system in host cells at the miRNA level.

  20. Low intensity ultrasound promotes the sensitivity of rat brain glioma to Doxorubicin by down-regulating the expressions of p-glucoprotein and multidrug resistance protein 1 in vitro and in vivo.

    Directory of Open Access Journals (Sweden)

    Zhen Zhang

    Full Text Available The overall prognosis for malignant glioma is extremely poor, and treatment options are limited in part because of multidrug resistant proteins. Our previous findings suggest low intensity ultrasound (LIUS can induce apoptosis of glioma cells. Given this finding, we were interested in determining if LIUS could help treat glioma by inhibiting multidrug resistant proteins, and if so, which pathways are involved. In this study, the toxicity sensitivity and multidrug resistance proteins of glioma induced by LIUS were investigated using CCK-8, immunohistochemistry, immunofluorency, and RT-PCR in tissue samples and cultured cells. LIUS inhibited increase of C6 cells in an intensity- and time-dependent manner. The toxicity sensitivity of C6 cells increased significantly after LIUS sonication (intensity of 142.0 mW/cm(2 or Doxorubicin (DOX at different concentration, particularly by the combination of LIUS sonication and DOX. The expressions of P-gp and MRP1 decreased significantly post-sonication at intensity of 142.0 mW/cm(2 both in vitro and in vivo. The expressions of p110 delta (PI3K, NF-κB-p65, Akt/PKB, and p-Akt/PKB were downregulated by LIUS sonication and DOX treatment separately or in combination at the same parameters in rat glioma. These results indicate that LIUS could increase the toxicity sensitivity of glioma by down-regulating the expressions of P-gp and MRP1, which might be mediated by the PI3K/Akt/NF-κB pathway.

  1. Training induced decrease in oxygen cost of cycling is accompanied by down-regulation of SERCA expression in human vastus lateralis muscle.

    Science.gov (United States)

    Majerczak, J; Karasinski, J; Zoladz, J A

    2008-09-01

    We have examined the effect of 5 week cycling endurance training program on the sarco(endo)plasmic reticulum Ca2+ ATPase isoforms (SERCA1 and 2) and myosin heavy chain (MyHC) in the vastus lateralis muscle as well as on the oxygen uptake to power output ratio (VO2/PO) during incremental cycling. Fifteen untrained men performed an incremental cycling exercise until exhaustion before and after moderate intensity training. Muscle biopsies were taken from vastus lateralis before and after training program. Training resulted in higher (P = 0.048) maximal oxygen uptake (VO(2max)) as well as in higher power output reached at VO(2max) (P = 0.0001). Moreover, lower (P = 0.02) VO2/PO ratio determined during incremental moderate intensity cycling (i.e. 30-120 W) as well as lower (P = 0.003) VO2/PO ratio reached at VO(2max) were observed after the training. A significant down regulation of SERCA2 protein (P = 0.03) and tendency (P = 0.055) to lower SERCA1 content accompanied by lower (P<10(-4)) plasma thyroid hormone concentration, with no changes (P = 0.67) in MyHC composition in vastus lateralis muscle were found after training. We have concluded that the increase in mechanical efficiency of cycling occurring during first weeks of endurance training is not related to changes in MyHC composition but it may be due to down-regulation of SERCA pumps.

  2. TGF-β1 downregulates COX-2 expression leading to decrease of PGE2 production in human lung cancer A549 cells, which is involved in fibrotic response to TGF-β1.

    Directory of Open Access Journals (Sweden)

    Erina Takai

    Full Text Available Transforming growth factor-ß1 (TGF-β1 is a multifunctional cytokine that is involved in various pathophysiological processes, including cancer progression and fibrotic disorders. Here, we show that treatment with TGF-β1 (5 ng/mL induced downregulation of cyclooxygenase-2 (COX-2, leading to reduced synthesis of prostaglandin E2 (PGE2, in human lung cancer A549 cells. Treatment of cells with specific inhibitors of COX-2 or PGE2 receptor resulted in growth inhibition, indicating that the COX-2/PGE2 pathway contributes to proliferation in an autocrine manner. TGF-β1 treatment induced growth inhibition, which was attenuated by exogenous PGE2. TGF-β1 is also a potent inducer of epithelial mesenchymal transition (EMT, a phenotype change in which epithelial cells differentiate into fibroblastoid cells. Supplementation with PGE2 or PGE2 receptor EP4 agonist PGE1-alcohol, as compared with EP1/3 agonist sulprostone, inhibited TGF-β1-induced expression of fibronectin and collagen I (extracellular matrix components. Exogenous PGE2 or PGE2 receptor agonists also suppressed actin remodeling induced by TGF-β1. These results suggest that PGE2 has an anti-fibrotic effect. We conclude that TGF-β1-induced downregulation of COX-2/PGE2 signaling is involved in facilitation of fibrotic EMT response in A549 cells.

  3. Downregulation of survivin expression and concomitant induction of apoptosis by celecoxib and its non-cyclooxygenase-2-inhibitory analog, dimethyl-celecoxib (DMC, in tumor cells in vitro and in vivo

    Directory of Open Access Journals (Sweden)

    Hofman Florence M

    2006-05-01

    Full Text Available Abstract Background 2,5-Dimethyl-celecoxib (DMC is a close structural analog of the selective cyclooxygenase-2 (COX-2 inhibitor celecoxib (Celebrex® that lacks COX-2-inhibitory function. However, despite its inability to block COX-2 activity, DMC is able to potently mimic the anti-tumor effects of celecoxib in vitro and in vivo, indicating that both of these drugs are able to involve targets other than COX-2 to exert their recognized cytotoxic effects. However, the molecular components that are involved in mediating these drugs' apoptosis-stimulatory consequences are incompletely understood. Results We present evidence that celecoxib and DMC are able to down-regulate the expression of survivin, an anti-apoptotic protein that is highly expressed in tumor cells and known to confer resistance of such cells to anti-cancer treatments. Suppression of survivin is specific to these two drugs, as other coxibs (valdecoxib, rofecoxib or traditional NSAIDs (flurbiprofen, indomethacin, sulindac do not affect survivin expression at similar concentrations. The extent of survivin down-regulation by celecoxib and DMC in different tumor cell lines is somewhat variable, but closely correlates with the degree of drug-induced growth inhibition and apoptosis. When combined with irinotecan, a widely used anticancer drug, celecoxib and DMC greatly enhance the cytotoxic effects of this drug, in keeping with a model that suppression of survivin may be beneficial to sensitize cancer cells to chemotherapy. Remarkably, these effects are not restricted to in vitro conditions, but also take place in tumors from drug-treated animals, where both drugs similarly repress survivin, induce apoptosis, and inhibit tumor growth in vivo. Conclusion In consideration of survivin's recognized role as a custodian of tumor cell survival, our results suggest that celecoxib and DMC might exert their cytotoxic anti-tumor effects at least in part via the down-regulation of survivin – in a

  4. Fibrates suppress bile acid synthesis via peroxisome proliferator-activated receptor-α-mediated downregulation of cholesterol 7α-hydroxylase and sterol 27-hydroxylase expression

    NARCIS (Netherlands)

    Post, S.M.; Duez, H.; Gervois, P.P.; Staels, B.; Kuipers, F.; Princen, H.M.G.

    2001-01-01

    Fibrates are hypolipidemic drugs that affect the expression of genes involved in lipid metabolism by activating peroxisome proliferator-activated receptors (PPARs). Fibrate treatment causes adverse changes in biliary lipid composition and decreases bile acid excretion, leading to an increased

  5. Fibrates suppress bile acid synthesis via peroxisome proliferator-activated receptor-alpha-mediated downregulation of cholesterol 7 alpha-hydroxylase and sterol 27-hydroxylase expression

    NARCIS (Netherlands)

    Post, SM; Duez, H; Gervois, PP; Staels, B; Kuipers, F; Princen, HMG

    2001-01-01

    Fibrates are hypolipidemic drugs that affect the expression of genes involved in lipid metabolism by activating peroxisome proliferator-activated receptors (PPARs). Fibrate treatment causes adverse changes in biliary lipid composition and decreases bile acid excretion, leading to an increased

  6. Benfotiamine increases glucose oxidation and downregulates NADPH oxidase 4 expression in cultured human myotubes exposed to both normal and high glucose concentrations

    OpenAIRE

    Fraser, D. A.; Hessvik, N. P.; Nikolić, N.; Aas, V.; Hanssen, K. F.; Bøhn, S. K.; Thoresen, G. H.; Rustan, A. C.

    2011-01-01

    The aim of the present work was to study the effects of benfotiamine (S-benzoylthiamine O-monophosphate) on glucose and lipid metabolism and gene expression in differentiated human skeletal muscle cells (myotubes) incubated for 4 days under normal (5.5 mM glucose) and hyperglycemic (20 mM glucose) conditions. Myotubes established from lean, healthy volunteers were treated with benfotiamine for 4 days. Glucose and lipid metabolism were studied with labeled precursors. Gene expression was measu...

  7. Downregulation of CD147 expression by RNA interference inhibits HT29 cell proliferation, invasion and tumorigenicity in vitro and in vivo.

    Science.gov (United States)

    Li, Rui; Pan, Yuqin; He, Bangshun; Xu, Yeqiong; Gao, Tianyi; Song, Guoqi; Sun, Huiling; Deng, Qiwen; Wang, Shukui

    2013-12-01

    We investigated the effect of CD147 silencing on HT29 cell proliferation and invasion. We constructed a novel short hairpin RNA (shRNA) expression vector pYr-mir30-shRNA. The plasmid was transferred to HT29 cells. The expression of CD147, MCT1 (lactate transporters monocarboxylate transporter 1) and MCT4 (lactate transporters monocarboxylate transporter 4) were monitored by quantitative PCR and western blotting, respectively. The MMP-2 (matrix metalloproteinase-2) and MMP-9 (matrix metalloproteinase-9) activities were determined by gelatin zymography assay, while the intracellular lactate concentration was determined by the lactic acid assay kit. WST-8 assay was used to determine the HT29 cell proliferation and the chemosensitivity. Invasion assay was used to determine the invasion of HT29 cells. In addition, we established a colorectal cancer model, and detected CD147 expression in vivo. The results showed that the expression of CD147 and MCT1 was significantly reduced at both mRNA and protein levels, and also the activity of MMP-2 and MMP-9 was reduced. The proliferation and invasion were decreased, but chemosensitivity to cisplatin was increased. In vivo, the CD147 expression was also significantly decreased, and reduced the tumor growth after CD147 gene silencing. The results demonstrated that silencing of CD147 expression inhibited the proliferation and invasion, suggesting CD147 silencing might be an adjuvant gene therapy strategy to chemotherapy.

  8. Degradation of the HilC and HilD regulator proteins by ATP-dependent Lon protease leads to downregulation of Salmonella pathogenicity island 1 gene expression.

    Science.gov (United States)

    Takaya, Akiko; Kubota, Yohsuke; Isogai, Emiko; Yamamoto, Tomoko

    2005-02-01

    Salmonella pathogenicity island 1 (SPI1) enables infecting Salmonella to cross the small intestinal barrier and to escape phagocytosis by inducing apoptosis. Several environmental signals and transcriptional regulators modulate the expression of hilA, which encodes a protein playing a central role in the regulatory hierarchy of SPI1 gene expression. We have previously shown that Lon, a stress-induced ATP-dependent protease, is a negative regulator of hilA, suggesting that it targets factors required for activating hilA expression. To elucidate the mechanisms by which Lon protease negatively regulates SPI1 transcription, we looked for its substrate proteins. We found that HilC and HilD, which are positive regulators of hilA expression, accumulate in Lon-depleted cells, and that the enhancement of SPI1 expression that occurs in a lon-disrupted mutant is not observed in the lon hilC hilD triple null mutant. Furthermore, we demonstrated that the half-lives of HilC and HilD are, respectively, about 12 times and three times longer in the Lon-depleted mutant, than in the Lon+ cells, suggesting that Lon targets both of HilC and HilD. In view of these findings, we suggest that the regulation of SPI1 expression is negatively controlled through degradation of the HilC and HilD transcriptional regulators by Lon.

  9. IL-10-produced by human transitional B-cells down-regulates CD86 expression on B-cells leading to inhibition of CD4+T-cell responses.

    Science.gov (United States)

    Nova-Lamperti, Estefania; Fanelli, Giorgia; Becker, Pablo D; Chana, Prabhjoat; Elgueta, Raul; Dodd, Philippa C; Lord, Graham M; Lombardi, Giovanna; Hernandez-Fuentes, Maria P

    2016-01-22

    A novel subset of human regulatory B-cells has recently been described. They arise from within the transitional B-cell subpopulation and are characterised by the production of IL-10. They appear to be of significant importance in regulating T-cell immunity in vivo. Despite this important function, the molecular mechanisms by which they control T-cell activation are incompletely defined. Here we show that transitional B-cells produced more IL-10 and expressed higher levels of IL-10 receptor after CD40 engagement compared to other B-cell subsets. Furthermore, under this stimulatory condition, CD86 expressed by transitional B-cells was down regulated and T-cell proliferation was reduced. We provide evidence to demonstrate that the down-regulation of CD86 expression by transitional B-cells was due to the autocrine effect of IL-10, which in turn leads to decreased T-cell proliferation and TNF-α production. This analysis was further extended to peripheral B-cells in kidney transplant recipients. We observed that B-cells from patients tolerant to the graft maintained higher IL-10 production after CD40 ligation, which correlates with lower CD86 expression compared to patients with chronic rejection. Hence, the results obtained in this study shed light on a new alternative mechanism by which transitional B-cells inhibit T-cell proliferation and cytokine production.

  10. IL-10-produced by human transitional B-cells down-regulates CD86 expression on B-cells leading to inhibition of CD4+T-cell responses

    Science.gov (United States)

    Nova-Lamperti, Estefania; Fanelli, Giorgia; Becker, Pablo D.; Chana, Prabhjoat; Elgueta, Raul; Dodd, Philippa C.; Lord, Graham M.; Lombardi, Giovanna; Hernandez-Fuentes, Maria P.

    2016-01-01

    A novel subset of human regulatory B-cells has recently been described. They arise from within the transitional B-cell subpopulation and are characterised by the production of IL-10. They appear to be of significant importance in regulating T-cell immunity in vivo. Despite this important function, the molecular mechanisms by which they control T-cell activation are incompletely defined. Here we show that transitional B-cells produced more IL-10 and expressed higher levels of IL-10 receptor after CD40 engagement compared to other B-cell subsets. Furthermore, under this stimulatory condition, CD86 expressed by transitional B-cells was down regulated and T-cell proliferation was reduced. We provide evidence to demonstrate that the down-regulation of CD86 expression by transitional B-cells was due to the autocrine effect of IL-10, which in turn leads to decreased T-cell proliferation and TNF-α production. This analysis was further extended to peripheral B-cells in kidney transplant recipients. We observed that B-cells from patients tolerant to the graft maintained higher IL-10 production after CD40 ligation, which correlates with lower CD86 expression compared to patients with chronic rejection. Hence, the results obtained in this study shed light on a new alternative mechanism by which transitional B-cells inhibit T-cell proliferation and cytokine production. PMID:26795594

  11. Elabela/Toddler Is an Endogenous Agonist of the Apelin APJ Receptor in the Adult Cardiovascular System, and Exogenous Administration of the Peptide Compensates for the Downregulation of Its Expression in Pulmonary Arterial Hypertension.

    Science.gov (United States)

    Yang, Peiran; Read, Cai; Kuc, Rhoda E; Buonincontri, Guido; Southwood, Mark; Torella, Rubben; Upton, Paul D; Crosby, Alexi; Sawiak, Stephen J; Carpenter, T Adrian; Glen, Robert C; Morrell, Nicholas W; Maguire, Janet J; Davenport, Anthony P

    2017-03-21

    Elabela/toddler (ELA) is a critical cardiac developmental peptide that acts through the G-protein-coupled apelin receptor, despite lack of sequence similarity to the established ligand apelin. Our aim was to investigate the receptor pharmacology, expression pattern, and in vivo function of ELA peptides in the adult cardiovascular system, to seek evidence for alteration in pulmonary arterial hypertension (PAH) in which apelin signaling is downregulated, and to demonstrate attenuation of PAH severity with exogenous administration of ELA in a rat model. In silico docking analysis, competition binding experiments, and downstream assays were used to characterize ELA receptor binding in human heart and signaling in cells expressing the apelin receptor. ELA expression in human cardiovascular tissues and plasma was determined using real-time quantitative polymerase chain reaction, dual-labeling immunofluorescent staining, and immunoassays. Acute cardiac effects of ELA-32 and [Pyr 1 ]apelin-13 were assessed by MRI and cardiac catheterization in anesthetized rats. Cardiopulmonary human and rat tissues from PAH patients and monocrotaline- and Sugen/hypoxia-exposed rats were used to show changes in ELA expression in PAH. The effect of ELA treatment on cardiopulmonary remodeling in PAH was investigated in the monocrotaline rat model. ELA competed for binding of apelin in human heart with overlap for the 2 peptides indicated by in silico modeling. ELA activated G-protein- and β-arrestin-dependent pathways. We detected ELA expression in human vascular endothelium and plasma. Comparable to apelin, ELA increased cardiac contractility, ejection fraction, and cardiac output and elicited vasodilatation in rat in vivo. ELA expression was reduced in cardiopulmonary tissues from PAH patients and PAH rat models, respectively. ELA treatment significantly attenuated elevation of right ventricular systolic pressure and right ventricular hypertrophy and pulmonary vascular remodeling in

  12. Curcumin enhances the mitomycin C-induced cytotoxicity via downregulation of MKK1/2-ERK1/2-mediated Rad51 expression in non-small cell lung cancer cells

    International Nuclear Information System (INIS)

    Ko, Jen-Chung; Tsai, Min-Shao; Weng, Shao-Hsing; Kuo, Ya-Hsun; Chiu, Yu-Fan; Lin, Yun-Wei

    2011-01-01

    Curcumin (diferuloylmethane), a major active component of turmeric (Curcuma longa), has been reported to suppress the proliferation of a wide variety of tumor cells. Rad51 is a key protein in the homologous recombination (HR) pathway of DNA double-strand break repair, and HR represents a novel target for cancer therapy. A high expression of Rad51 has been reported in chemo- or radio-resistant carcinomas. Therefore, in the current study, we will examine whether curcumin could enhance the effects of mitomycin C (MMC), a DNA interstrand cross-linking agent, to induce cytotoxicity by decreasing Rad51 expression. Exposure of two human non-small lung cancer (NSCLC) cell lines (A549 and H1975) to curcumin could suppress MMC-induced MKK1/2-ERK1/2 signal activation and Rad51 protein expression. Enhancement of ERK1/2 activation by constitutively active MKK1/2 (MKK1/2-CA) increased Rad51 protein levels in curcumin and MMC co-treated human lung cells. Moreover, the synergistic cytotoxic effect induced by curcumin combined with MMC was decreased by MKK1-CA-mediated enhancement of ERK1/2 activation by a significant degree. In contrast, MKK1/2 inhibitor, U0126 was shown to augment the cytotoxicity of curcumin and MMC through downregulation of ERK1/2 activation and Rad51 expression. Depletion of endogenous Rad51 expression by siRad51 RNA transfection significantly enhanced MMC and/or curcumin induced cell death and cell growth inhibition. In contrast, an overexpression of Rad51 protected lung cancer cells from synergistic cytotoxic effects induced by curcumin and MMC. We concluded that Rad51 inhibition may be an additional action mechanism for enhancing the chemosensitization of MMC by curcumin in NSCLC. - Highlights: → Curcumin downregulates MKK-ERK-mediated Rad51 expression. → Curcumin enhances mitomycin C-induced cytotoxicity. → Rad51 protects cells from cytotoxic effects induced by curcumin and mitomycin C. → Rad51 inhibition enhances the chemosensitization of

  13. Astaxanthin down-regulates Rad51 expression via inactivation of AKT kinase to enhance mitomycin C-induced cytotoxicity in human non-small cell lung cancer cells.

    Science.gov (United States)

    Ko, Jen-Chung; Chen, Jyh-Cheng; Wang, Tai-Jing; Zheng, Hao-Yu; Chen, Wen-Ching; Chang, Po-Yuan; Lin, Yun-Wei

    2016-04-01

    Astaxanthin has been demonstrated to exhibit a wide range of beneficial effects, including anti-inflammatory and anti-cancer properties. However, the molecular mechanism of astaxanthin-induced cytotoxicity in non-small cell lung cancer (NSCLC) cells has not been identified. Rad51 plays a central role in homologous recombination, and studies show that chemo-resistant carcinomas exhibit high levels of Rad51 expression. In this study, astaxanthin treatment inhibited cell viability and proliferation of two NSCLC cells, A549 and H1703. Astaxanthin treatment (2.5-20 μM) decreased Rad51 expression and phospho-AKT(Ser473) protein level in a time and dose-dependent manner. Furthermore, expression of constitutively active AKT (AKT-CA) vector rescued the decreased Rad51 mRNA and protein levels in astaxanthin-treated NSCLC cells. Combined treatment with phosphatidylinositol 3-kinase (PI3K) inhibitors (LY294002 or wortmannin) further decreased the Rad51 expression in astaxanthin-exposed A549 and H1703 cells. Knockdown of Rad51 expression by transfection with si-Rad51 RNA or cotreatment with LY294002 further enhanced the cytotoxicity and cell growth inhibition of astaxanthin. Additionally, mitomycin C (MMC) as an anti-tumor antibiotic is widely used in clinical NSCLC chemotherapy. Combination of MMC and astaxanthin synergistically resulted in cytotoxicity and cell growth inhibition in NSCLC cells, accompanied with reduced phospho-AKT(Ser473) level and Rad51 expression. Overexpression of AKT-CA or Flag-tagged Rad51 reversed the astaxanthin and MMC-induced synergistic cytotoxicity. In contrast, pretreatment with LY294002 further decreased the cell viability in astaxanthin and MMC co-treated cells. In conclusion, astaxanthin enhances MMC-induced cytotoxicity by decreasing Rad51 expression and AKT activation. These findings may provide rationale to combine astaxanthin with MMC for the treatment of NSCLC. Copyright © 2016 Elsevier Inc. All rights reserved.

  14. Inhibition of hypoxia inducible factor-1α downregulates the expression of epithelial to mesenchymal transition early marker proteins without undermining cell survival in hypoxic lens epithelial cells.

    Science.gov (United States)

    Cammarata, Patrick R; Neelam, Sudha; Brooks, Morgan M

    2015-01-01

    The purpose of this study was to identify potential therapeutic strategies to slow down or prevent the expression of early-onset epithelial to mesenchymal transition (EMT) marker proteins (fibronectin and alpha smooth muscle actin, α-SMA) without sacrificing the synthesis and accumulation of the prosurvival protein vascular endothelial growth factor (VEGF) in cultured virally transformed human lens epithelial (HLE) cells. HLE-B3 cells, maintained in a continuous hypoxic environment (1% oxygen), were treated with SB216763, a specific inhibitor of glycogen synthase kinase-3β (GSK-3β) catalytic activity. Western blot analysis was employed to detect the cytoplasmic and nuclear levels of β-catenin, as well as the total lysate content of fibronectin and α-SMA. Enzyme-linked immunosorbent assay (ELISA) was used to measure the levels of VEGF in cell culture medium. A hypoxia-inducible factor-1α (HIF-1α) translation inhibitor and an HIF-2α translation inhibitor were independently employed to evaluate the effect of hypoxia inducible factor inhibition on EMT marker protein and VEGF expression. XAV932 was used to assess the suppression of nuclear β-catenin and its downstream effect on EMT marker proteins and VEGF expression. SB216763-treated HLE-B3 cells caused marked inhibition of GSK-3β activity prompting a significant increase in the translocation of cytoplasmic β-catenin to the nucleus. The enhancement of nuclear β-catenin looked as if it positively correlated with a significant increase in the basal expression of VEGF as well as increased expression of fibronectin and α-SMA. In conjunction with SB216763, coadministration of an HIF-1α translation inhibitor, but not an HIF-2α translation inhibitor, markedly suppressed the expression of fibronectin and α-SMA without affecting VEGF levels. Treatment with XAV932 significantly reduced the level of nuclear β-catenin, but the levels of neither the EMT marker proteins nor VEGF were changed. Recently, we reported

  15. Mining microarray datasets in nutrition: expression of the GPR120 (n-3 fatty acid receptor/sensor) gene is down-regulated in human adipocytes by macrophage secretions.

    Science.gov (United States)

    Trayhurn, Paul; Denyer, Gareth

    2012-01-01

    Microarray datasets are a rich source of information in nutritional investigation. Targeted mining of microarray data following initial, non-biased bioinformatic analysis can provide key insight into specific genes and metabolic processes of interest. Microarrays from human adipocytes were examined to explore the effects of macrophage secretions on the expression of the G-protein-coupled receptor (GPR) genes that encode fatty acid receptors/sensors. Exposure of the adipocytes to macrophage-conditioned medium for 4 or 24 h had no effect on GPR40 and GPR43 expression, but there was a marked stimulation of GPR84 expression (receptor for medium-chain fatty acids), the mRNA level increasing 13·5-fold at 24 h relative to unconditioned medium. Importantly, expression of GPR120, which encodes an n-3 PUFA receptor/sensor, was strongly inhibited by the conditioned medium (15-fold decrease in mRNA at 24 h). Macrophage secretions have major effects on the expression of fatty acid receptor/sensor genes in human adipocytes, which may lead to an augmentation of the inflammatory response in adipose tissue in obesity.

  16. Saikosaponin A Alleviates Symptoms of Attention Deficit Hyperactivity Disorder through Downregulation of DAT and Enhancing BDNF Expression in Spontaneous Hypertensive Rats

    Directory of Open Access Journals (Sweden)

    Sun Jichao

    2017-01-01

    Full Text Available The disturbed dopamine availability and brain-derived neurotrophic factor (BDNF expression are due in part to be associated with attention deficit hyperactivity disorder (ADHD. In this study, we investigated the therapeutical effect of saikosaponin a (SSa isolated from Bupleurum Chinese DC, against spontaneously hypertensive rat (SHR model of ADHD. Methylphenidate and SSa were orally administered for 3 weeks. Activity was assessed by open-field test and Morris water maze test. Dopamine (DA and BDNF were determined in specific brain regions. The mRNA or protein expression of tyrosine hydroxylase (TH, dopamine transporter (DAT, and vesicles monoamine transporter (VMAT was also studied. Both MPH and SSa reduced hyperactivity and improved the spatial learning memory deficit in SHRs. An increased DA concentration in the prefrontal cortex (PFC and striatum was also observed after treating with the SSa. The increased DA concentration may partially be attributed to the decreased mRNA and protein expression of DAT in PFC while SSa exhibited no significant effects on the mRNA expression of TH and VMAT in PFC of SHRs. In addition, BDNF expression in SHRs was also increased after treating with SSa or MPH. The obtained result suggested that SSa may be a potential drug for treating ADHD.

  17. Healthy Nordic diet downregulates the expression of genes involved in inflammation in subcutaneous adipose tissue in individuals with features of the metabolic syndrome

    DEFF Research Database (Denmark)

    Kolehmainen, Marjukka; Ulven, Stine M; Paananen, Jussi

    2015-01-01

    BACKGROUND: Previously, a healthy Nordic diet (ND) has been shown to have beneficial health effects close to those of Mediterranean diets. OBJECTIVE: The objective was to explore whether the ND has an impact on gene expression in abdominal subcutaneous adipose tissue (SAT) and whether changes...... in gene expression are associated with clinical and biochemical effects. DESIGN: Obese adults with features of the metabolic syndrome underwent an 18- to 24-wk randomized intervention study comparing the ND with the control diet (CD) (the SYSDIET study, carried out within Nordic Centre of Excellence...... sites for the nuclear transcription factor κB. CONCLUSION: A healthy Nordic diet reduces inflammatory gene expression in SAT compared with a control diet independently of body weight change in individuals with features of the metabolic syndrome. The study was registered at clinicaltrials.gov as NCT...

  18. Collagen V-induced nasal tolerance downregulates pulmonary collagen mRNA gene and TGF-beta expression in experimental systemic sclerosis

    Directory of Open Access Journals (Sweden)

    Parra Edwin R

    2010-01-01

    Full Text Available Abstract Background The purpose of this study was to evaluate collagen deposition, mRNA collagen synthesis and TGF-beta expression in the lung tissue in an experimental model of scleroderma after collagen V-induced nasal tolerance. Methods Female New Zealand rabbits (N = 12 were immunized with 1 mg/ml of collagen V in Freund's adjuvant (IM. After 150 days, six immunized animals were tolerated by nasal administration of collagen V (25 μg/day (IM-TOL daily for 60 days. The collagen content was determined by morphometry, and mRNA expressions of types I, III and V collagen were determined by Real-time PCR. The TGF-beta expression was evaluated by immunostaining and quantified by point counting methods. To statistic analysis ANOVA with Bonferroni test were employed for multiple comparison when appropriate and the level of significance was determined to be p Results IM-TOL, when compared to IM, showed significant reduction in total collagen content around the vessels (0.371 ± 0.118 vs. 0.874 ± 0.282, p p p = 0.026. The lung tissue of IM-TOL, when compared to IM, showed decreased immunostaining of types I, III and V collagen, reduced mRNA expression of types I (0.10 ± 0.07 vs. 1.0 ± 0.528, p = 0.002 and V (1.12 ± 0.42 vs. 4.74 ± 2.25, p = 0.009 collagen, in addition to decreased TGF-beta expression (p Conclusions Collagen V-induced nasal tolerance in the experimental model of SSc regulated the pulmonary remodeling process, inhibiting collagen deposition and collagen I and V mRNA synthesis. Additionally, it decreased TGF-beta expression, suggesting a promising therapeutic option for scleroderma treatment.

  19. Epithelial Na, K-ATPase expression is down-regulated in canine prostate cancer; a possible consequence of metabolic transformation in the process of prostate malignancy

    Directory of Open Access Journals (Sweden)

    Martín-Vasallo Pablo

    2003-06-01

    Full Text Available Abstract Background An important physiological function of the normal prostate gland is the synthesis and secretion of a citrate rich prostatic fluid. In prostate cancer, citrate production levels are reduced as a result of altered cellular metabolism and bioenergetics. Na, K-ATPase is essential for citrate production since the inward Na+ gradients it generates are utilized for the Na+ dependent uptake of aspartate, a major substrate for citrate synthesis. The objective of this study was to compare the expression of previously identified Na, K-ATPase isoforms in normal canine prostate, benign prostatic hyperplasia (BPH and prostatic adenocarcinoma (PCa using immunohistochemistry in order to determine whether reduced citrate levels in PCa are also accompanied by changes in Na, K-ATPase expression. Results Expression of Na, K-ATPase α1 and β1 isoforms was observed in the lateral and basolateral plasma membrane domains of prostatic epithelial cells in normal and BPH prostates. Canine kidney was used as positive control for expression of Na, K-ATPase α1 and γ isoforms. The α1 isoform was detected in abundance in prostatic epithelial cells but there was no evidence of α2, α3 or γ subunit expression. In advanced PCa, Na, K-ATPase α1 isoform expression was significantly lower compared to normal and BPH glands. The abundant basolateral immunostaining observed in normal and BPH tissue was significantly attenuated in PCa. Conclusion The loss of epithelial structure and function and the transformation of normal epithelial cells to malignant cells in the canine prostate have important implications for cellular metabolism and are accompanied by a down regulation of Na, K-ATPase.

  20. Nerve Growth Factor in Cancer Cell Death and Survival

    International Nuclear Information System (INIS)

    Molloy, Niamh H.; Read, Danielle E.; Gorman, Adrienne M.

    2011-01-01

    One of the major challenges for cancer therapeutics is the resistance of many tumor cells to induction of cell death due to pro-survival signaling in the cancer cells. Here we review the growing literature which shows that neurotrophins contribute to pro-survival signaling in many different types of cancer. In particular, nerve growth factor, the archetypal neurotrophin, has been shown to play a role in tumorigenesis over the past decade. Nerve growth factor mediates its effects through its two cognate receptors, TrkA, a receptor tyrosine kinase and p75 NTR , a member of the death receptor superfamily. Depending on the tumor origin, pro-survival signaling can be mediated by TrkA receptors or by p75 NTR . For example, in breast cancer the aberrant expression of nerve growth factor stimulates proliferative signaling through TrkA and pro-survival signaling through p75 NTR . This latter signaling through p75 NTR promotes increased resistance to the induction of cell death by chemotherapeutic treatments. In contrast, in prostate cells the p75 NTR mediates cell death and prevents metastasis. In prostate cancer, expression of this receptor is lost, which contributes to tumor progression by allowing cells to survive, proliferate and metastasize. This review focuses on our current knowledge of neurotrophin signaling in cancer, with a particular emphasis on nerve growth factor regulation of cell death and survival in cancer

  1. DHRS7c, a novel cardiomyocyte-expressed gene that is down-regulated by adrenergic stimulation and in heart failure

    NARCIS (Netherlands)

    Lu, Bo; Tigchelaar, Wardit; Ruifrok, Willem P. T.; van Gilst, Wiek H.; de Boer, Rudolf A.; Sillje, Herman H. W.

    Aims Although cardiac diseases account for the highest mortality and morbidity rates in Western society, there is still a considerable gap in our knowledge of genes that contribute to cardiac (dys) function. Here we screened for gene expression profiles correlated to heart failure. Methods and

  2. Fenofibrate down-regulates the expressions of androgen receptor (AR) and AR target genes and induces oxidative stress in the prostate cancer cell line LNCaP

    Energy Technology Data Exchange (ETDEWEB)

    Zhao, Hu; Zhu, Chen; Qin, Chao [State Key Laboratory of Reproductive Medicine, Department of Urology, First Affiliated Hospital of Nanjing Medical University, Nanjing (China); Tao, Tao [Department of Neurosurgery, First Affiliated Hospital of Nanjing Medical University, Nanjing (China); Li, Jie; Cheng, Gong; Li, Pu; Cao, Qiang; Meng, Xiaoxin; Ju, Xiaobing; Shao, Pengfei; Hua, Lixin [State Key Laboratory of Reproductive Medicine, Department of Urology, First Affiliated Hospital of Nanjing Medical University, Nanjing (China); Gu, Min, E-mail: medzhao1980@163.com [State Key Laboratory of Reproductive Medicine, Department of Urology, First Affiliated Hospital of Nanjing Medical University, Nanjing (China); Yin, Changjun, E-mail: drcjyin@gmail.com [State Key Laboratory of Reproductive Medicine, Department of Urology, First Affiliated Hospital of Nanjing Medical University, Nanjing (China)

    2013-03-08

    Highlights: ► Fenofibrate induces cell cycle arrest in G1 phase and apoptosis in LNCaP cells. ► Fenofibrate reduces the expressions of androgen receptor in LNCaP cells. ► Fenofibrate induces oxidative stress in the prostate cancer cell line LNCaP. -- Abstract: Fenofibrate, a peroxisome proliferator-androgen receptor-alpha agonist, is widely used in treating different forms of hyperlipidemia and hypercholesterolemia. Recent reports have indicated that fenofibrate exerts anti-proliferative and pro-apoptotic properties. This study aims to investigate the effects of fenofibrate on the prostate cancer (PCa) cell line LNCaP. The effects of fenofibrate on LNCaP cells were evaluated by flow cytometry, reverse transcription-polymerase chain reaction, enzyme-linked immunosorbent assays, Western blot analysis, and dual-luciferase reporter assay. Fenofibrate induces cell cycle arrest in G1 phase and apoptosis in LNCaP cells, reduces the expressions of androgen receptor (AR) and AR target genes (prostate-specific antigen and TMPRSS2), and inhibits Akt phosphorylation. Fenofibrate can induce the accumulation of intracellular reactive oxygen species and malondialdehyde, and decrease the activities of total anti-oxidant and superoxide dismutase in LNCaP cells. Fenofibrate exerts an anti-proliferative property by inhibiting the expression of AR and induces apoptosis by causing oxidative stress. Therefore, our data suggest fenofibrate may have beneficial effects in fenofibrate users by preventing prostate cancer growth through inhibition of androgen activation and expression.

  3. Down-regulation of hepatic and intestinal Abcg5 and Abcg8 expression associated with altered sterol fluxes in rats with streptozotocin-induced diabetes

    NARCIS (Netherlands)

    Bloks, VW; Bakker-van Waarde, WM; Verkade, HJ; Kema, IP; Wolters, H; Vink, E; Groen, AK; Kuipers, F

    Aim/hypothesis., Type I diabetes is associated with altered hepatic bile formation and increased intestinal cholesterol absorption. The aim of this study was to evaluate whether altered expression of the ATP-Binding Cassette half-transporters Abcg5 and Abcg8, recently implicated in control of both

  4. The importance of 1,2-dithiolane structure in α-lipoic acid for the downregulation of cell surface β1-integrin expression of human bladder cancer cells.

    Science.gov (United States)

    Yamasaki, Masao; Soda, Shozen; Sakakibara, Yoichi; Suiko, Masahito; Nishiyama, Kazuo

    2014-01-01

    Here, we show that cell surface β1-integrin expression, cell adhesion to fibronectin, migration, and invasion were all significantly inhibited by α-lipoic acid. These effects were not observed when cells were treated with dihydrolipoic acid or caprylic acid. These data reveal that the 1,2-dithiolane structure plays an important role in the action of α-lipoic acid.

  5. Fibrates down-regulate IL-1-stimulated C-reactive protein gene expression in hepatocytes by reducing nuclear p50-NFκB-C/EBP-β complex formation

    NARCIS (Netherlands)

    Kleemann, R.; Gervois, P.P.; Verschuren, L.; Staels, B.; Princen, H.M.G.; Kooistra, T.

    2003-01-01

    C-reactive protein (CRP) is a major acute-phase protein in humans. Elevated plasma CRP levels are a risk factor for cardiovascular disease. CRP is predominantly expressed in hepatocytes and is induced by interleukin-1 (IL-1) and IL-6 under inflammatory situations, such as the acute phase. Fibrates

  6. Inhibitory effects of kaempferol on the invasion of human breast carcinoma cells by downregulating the expression and activity of matrix metalloproteinase-9.

    Science.gov (United States)

    Li, Chenglin; Zhao, Yuanwei; Yang, Dan; Yu, Yanyan; Guo, Hao; Zhao, Ziming; Zhang, Bei; Yin, Xiaoxing

    2015-02-01

    Matrix metalloproteinases (MMPs) have been regarded as major critical molecules assisting tumor cells during metastasis, for excessive ECM (ECM) degradation, and cancer cell invasion. In the present study, in vitro and in vivo assays were employed to examine the inhibitory effects of kaempferol, a natural polyphenol of flavonoid family, on tumor metastasis. Data showed that kaempferol could inhibit adhesion, migration, and invasion of MDA-MB-231 human breast carcinoma cells. Moreover, kaempferol led to the reduced activity and expression of MMP-2 and MMP-9, which were detected by gelatin zymography, real-time PCR, and western blot analysis, respectively. Further elucidation of the mechanism revealed that kaempferol treatment inhibited the activation of transcription factor activator protein-1 (AP-1) and MAPK signaling pathway. Moreover, kaempferol repressed phorbol-12-myristate-13-acetate (PMA)-induced MMP-9 expression and activity through suppressing the translocation of protein kinase Cδ (PKCδ) and MAPK signaling pathway. Our results also indicated that kaempferol could block the lung metastasis of B16F10 murine melanoma cells as well as the expression of MMP-9 in vivo. Taken together, these results demonstrated that kaempferol could inhibit cancer cell invasion through blocking the PKCδ/MAPK/AP-1 cascade and subsequent MMP-9 expression and its activity. Therefore, kaempferol might act as a therapeutic potential candidate for cancer metastasis.

  7. Down-regulated E-cadherin expression is associated with poor five-year overall survival in bone and soft tissue sarcoma: results of a meta-analysis.

    Directory of Open Access Journals (Sweden)

    Ning Wang

    Full Text Available To conduct a meta-analysis to evaluate the prognostic role of E-cadherin expression in bone and soft tissue sarcomas.The PubMed, EMBASE, and Web of Science databases were searched using terms related to E-cadherin, sarcoma, and prognosis for all articles published in English before March 2014. Pooled effect was calculated from the available data to evaluate the association between negative E-cadherin expression and 5-year overall survival and tumor clinicopathological features in sarcoma patients. Pooled odds ratios (OR and risk ratios (RR with 95% confidence intervals (CI were calculated using a fixed-effects model.Eight studies met the selection criteria and reported on 812 subjects. A total of 496 subjects showed positive E-cadherin expression (59.9%. Negative E-cadherin expression in bone and soft tissue sarcomas was correlated with lower 5-year overall survival (OR = 3.831; 95% CI: 2.246-6.534, and was associated with higher clinical stage (RR = 1.446; 95% CI: 1.030-2.028 and with male sex (RR = 0.678; 95% CI: 0.493-0.933.In the E-cadherin negative group, 5-year overall survival was significantly worse than in the E-cadherin positive group. However, further studies are required to confirm these results.

  8. Minocycline enhances mitomycin C-induced cytotoxicity through down-regulating ERK1/2-mediated Rad51 expression in human non-small cell lung cancer cells.

    Science.gov (United States)

    Ko, Jen-Chung; Wang, Tai-Jing; Chang, Po-Yuan; Syu, Jhan-Jhang; Chen, Jyh-Cheng; Chen, Chien-Yu; Jian, Yun-Ting; Jian, Yi-Jun; Zheng, Hao-Yu; Chen, Wen-Ching; Lin, Yun-Wei

    2015-10-01

    Minocycline is a semisynthetic tetracycline derivative; it has anti-inflammatory and anti-cancer effects distinct from its antimicrobial function. However, the molecular mechanism of minocycline-induced cytotoxicity in non-small cell lung cancer (NSCLC) cells has not been identified. Rad51 plays a central role in homologous recombination and high levels of Rad51 expression are observed in chemo- or radioresistant carcinomas. Our previous studies have shown that the MKK1/2-ERK1/2 signal pathway maintains the expression of Rad51 in NSCLC cells. In this study, minocycline treatment inhibited cell viability and proliferation of two NSCLC cells, A549 and H1975. Treatment with minocycline decreased Rad51 mRNA and protein levels through MKK1/2-ERK1/2 inactivation. Furthermore, expression of constitutively active MKK1 (MKK1-CA) vectors significantly rescued the decreased Rad51 protein and mRNA levels in minocycline-treated NSCLC cells. However, combined treatment with MKK1/2 inhibitor U0126 and minocycline further decreased the Rad51 expression and cell viability of NSCLC cells. Knocking down Rad51 expression by transfection with small interfering RNA of Rad51 enhanced the cytotoxicity and cell growth inhibition of minocycline. Mitomycin C (MMC) is typically used as a first or second line regimen to treat NSCLC. Compared to a single agent alone, MMC combined with minocycline resulted in cytotoxicity and cell growth inhibition synergistically in NSCLC cells, accompanied with reduced activation of phospho-ERK1/2, and reduced Rad51 protein levels. Overexpression of MKK1-CA or Flag-tagged Rad51 could reverse the minocycline and MMC-induced synergistic cytotoxicity. These findings may have implications for the rational design of future drug regimens incorporating minocycline and MMC for the treatment of NSCLC. Copyright © 2015 Elsevier Inc. All rights reserved.

  9. Upregulation of inflammatory genes and downregulation of sclerostin gene expression are key elements in the early phase of fragility fracture healing.

    Directory of Open Access Journals (Sweden)

    Joana Caetano-Lopes

    Full Text Available BACKGROUND: Fracture healing is orchestrated by a specific set of events that culminates in the repair of bone and reachievement of its biomechanical properties. The aim of our work was to study the sequence of gene expression events involved in inflammation and bone remodeling occurring in the early phases of callus formation in osteoporotic patients. METHODOLOGY/PRINCIPAL FINDINGS: Fifty-six patients submitted to hip replacement surgery after a low-energy hip fracture were enrolled in this study. The patients were grouped according to the time interval between fracture and surgery: bone collected within 3 days after fracture (n = 13; between the 4(th and 7(th day (n = 33; and after one week from the fracture (n = 10. Inflammation- and bone metabolism-related genes were assessed at the fracture site. The expression of pro-inflammatory cytokines was increased in the first days after fracture. The genes responsible for bone formation and resorption were upregulated one week after fracture. The increase in RANKL expression occurred just before that, between the 4(th-7(th days after fracture. Sclerostin expression diminished during the first days after fracture. CONCLUSIONS: The expression of inflammation-related genes, especially IL-6, is highest at the very first days after fracture but from day 4 onwards there is a shift towards bone remodeling genes, suggesting that the inflammatory phase triggers bone healing. We propose that an initial inflammatory stimulus and a decrease in sclerostin-related effects are the key components in fracture healing. In osteoporotic patients, cellular machinery seems to adequately react to the inflammatory stimulus, therefore local promotion of these events might constitute a promising medical intervention to accelerate fracture healing.

  10. BDE-47 causes developmental retardation with down-regulated expression profiles of ecdysteroid signaling pathway-involved nuclear receptor (NR) genes in the copepod Tigriopus japonicus

    Energy Technology Data Exchange (ETDEWEB)

    Hwang, Dae-Sik; Han, Jeonghoon; Won, Eun-Ji; Kim, Duck-Hyun; Jeong, Chang-Bum [Department of Biological Science, College of Science, Sungkyunkwan University, Suwon 16419 (Korea, Republic of); Hwang, Un-Ki [Marine Ecological Risk Assessment Center, West Sea Fisheries Research Institute, National Fisheries Research & Development Institute, Incheon 46083 (Korea, Republic of); Zhou, Bingsheng [State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072 (China); Choe, Joonho [Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Daejeon 34141 (Korea, Republic of); Lee, Jae-Seong, E-mail: jslee2@skku.edu [Department of Biological Science, College of Science, Sungkyunkwan University, Suwon 16419 (Korea, Republic of)

    2016-08-15

    Highlights: • The developmental rate was significantly inhibited (P < 0.05) in response to BDE-47. • Expression profiles of nearly all NR genes were the highest at naupliar stages 5–6. • USP, HR96, and FTZ-F1 genes showed significant sex differences (P < 0.05) over different developmental stages. • NR gene expression patterns showed significant decreases (P<0.05) in response to BDE-47. • BDE-47 leads to molting and metamorphosis retardation and suppresses transcription of NR genes. - Abstract: 2,2′,4,4′-Tetrabromodiphenyl ether (BDE-47) is a persistent organic pollutant (POP) in marine environments. Despite its adverse effects (e.g. developmental retardation) in ecdysozoa, the effects of BDE-47 on transcription of ecdysteroid signaling pathway-involved-nuclear receptor (NR) genes and metamorphosis-related genes have not been examined in copepods. To examine the deleterious effect of BDE-47 on copepod molting and metamorphosis, BDE-47 was exposed to the harpacticoid copepod Tigriopus japonicus, followed by monitoring developmental retardation and transcriptional alteration of NR genes. The developmental rate was significantly inhibited (P < 0.05) in response to BDE-47 and the agricultural insecticide gamma-hexachlorocyclohexane. Conversely, the ecdysteroid agonist ponasterone A (PoA) led to decreased molting and metamorphosis time (P < 0.05) from the nauplius stage to the adult stage. In particular, expression profiles of all NR genes were the highest at naupliar stages 5–6 except for SVP, FTZ-F1, and HR96 genes. Nuclear receptor USP, HR96, and FTZ-F1 genes also showed significant sex differences (P < 0.05) in gene expression levels over different developmental stages, indicating that these genes may be involved in vitellogenesis. NR gene expression patterns showed significant decreases (P < 0.05) in response to BDE-47 exposure, implying that molting and metamorphosis retardation is likely associated with NR gene expression. In summary, BDE-47

  11. BDE-47 causes developmental retardation with down-regulated expression profiles of ecdysteroid signaling pathway-involved nuclear receptor (NR) genes in the copepod Tigriopus japonicus

    International Nuclear Information System (INIS)

    Hwang, Dae-Sik; Han, Jeonghoon; Won, Eun-Ji; Kim, Duck-Hyun; Jeong, Chang-Bum; Hwang, Un-Ki; Zhou, Bingsheng; Choe, Joonho; Lee, Jae-Seong

    2016-01-01

    Highlights: • The developmental rate was significantly inhibited (P < 0.05) in response to BDE-47. • Expression profiles of nearly all NR genes were the highest at naupliar stages 5–6. • USP, HR96, and FTZ-F1 genes showed significant sex differences (P < 0.05) over different developmental stages. • NR gene expression patterns showed significant decreases (P<0.05) in response to BDE-47. • BDE-47 leads to molting and metamorphosis retardation and suppresses transcription of NR genes. - Abstract: 2,2′,4,4′-Tetrabromodiphenyl ether (BDE-47) is a persistent organic pollutant (POP) in marine environments. Despite its adverse effects (e.g. developmental retardation) in ecdysozoa, the effects of BDE-47 on transcription of ecdysteroid signaling pathway-involved-nuclear receptor (NR) genes and metamorphosis-related genes have not been examined in copepods. To examine the deleterious effect of BDE-47 on copepod molting and metamorphosis, BDE-47 was exposed to the harpacticoid copepod Tigriopus japonicus, followed by monitoring developmental retardation and transcriptional alteration of NR genes. The developmental rate was significantly inhibited (P < 0.05) in response to BDE-47 and the agricultural insecticide gamma-hexachlorocyclohexane. Conversely, the ecdysteroid agonist ponasterone A (PoA) led to decreased molting and metamorphosis time (P < 0.05) from the nauplius stage to the adult stage. In particular, expression profiles of all NR genes were the highest at naupliar stages 5–6 except for SVP, FTZ-F1, and HR96 genes. Nuclear receptor USP, HR96, and FTZ-F1 genes also showed significant sex differences (P < 0.05) in gene expression levels over different developmental stages, indicating that these genes may be involved in vitellogenesis. NR gene expression patterns showed significant decreases (P < 0.05) in response to BDE-47 exposure, implying that molting and metamorphosis retardation is likely associated with NR gene expression. In summary, BDE-47

  12. Transcriptional down-regulation of thromboxane A(2) receptor expression via activation of MAPK ERK1/2, p38/NF-kappaB pathways

    DEFF Research Database (Denmark)

    Zhang, Wei; Zhang, Yaping; Edvinsson, Lars

    2009-01-01

    culture of the arteries, VSMC TP receptors were studied by using myography, real-time PCR and immunohistochemistry. We observed that organ culture for 24 and 48 h resulted in depressed TP receptor-mediated contraction in the VSMC, in parallel with decreased TP receptor mRNA and protein expressions....... Phosphorylation of extracellular signal-regulated kinase 1 and 2 (ERK1/2), p38 and nuclear factor-kappaB (NF-kappaB) was seen by Western blot within 1-3 h after organ culture. Inhibition of ERK1/2, p38 or NF-kappaB reversed depressed contraction as well as decreased receptor mRNA expression. Actinomycin D...

  13. Transcriptional Down-Regulation of Thromboxane A(2) Receptor Expression via Activation of MAPK ERK1/2, p38/NF-kappaB Pathways

    DEFF Research Database (Denmark)

    Zhang, Wei; Zhang, Yaping; Edvinsson, Lars

    2008-01-01

    culture of the arteries, VSMC TP receptors were studied by using myography, real-time PCR and immunohistochemistry. We observed that organ culture for 24 and 48 h resulted in depressed TP receptor-mediated contraction in the VSMC, in parallel with decreased TP receptor mRNA and protein expressions....... Phosphorylation of extracellular signal-regulated kinase 1 and 2 (ERK1/2), p38 and nuclear factor-kappaB (NF-kappaB) was seen by Western blot within 1-3 h after organ culture. Inhibition of ERK1/2, p38 or NF-kappaB reversed depressed contraction as well as decreased receptor mRNA expression. Actinomycin D...

  14. Pulsed Radiofrequency Applied to the Sciatic Nerve Improves Neuropathic Pain by Down-regulating The Expression of Calcitonin Gene-related Peptide in the Dorsal Root Ganglion

    Science.gov (United States)

    Ren, Hao; Jin, Hailong; Jia, Zipu; Ji, Nan; Luo, Fang

    2018-01-01

    Background: Clinical studies have shown that applying pulsed radiofrequency (PRF) to the neural stem could relieve neuropathic pain (NP), albeit through an unclear analgesic mechanism. And animal experiments have indicated that calcitonin gene-related peptide (CGRP) expressed in the dorsal root ganglion (DRG) is involved in generating and maintaining NP. In this case, it is uncertain whether PRF plays an analgesic role by affecting CGRP expression in DRG. Methods: Rats were randomly divided into four groups: Groups A, B, C, and D. In Groups C and D, the right sciatic nerve was ligated to establish the CCI model, while in Groups A and B, the sciatic nerve was isolated without ligation. After 14 days, the right sciatic nerve in Groups B and D re-exposed and was treated with PRF on the ligation site. Thermal withdrawal latency (TWL) and hindpaw withdrawal threshold (HWT) were measured before PRF treatment (Day 0) as well as after 2, 4, 8, and 14 days of treatment. At the same time points of the behavioral tests, the right L4-L6 DRG was sampled and analyzed for CGRP expression using RT-qPCR and an enzyme-linked immunosorbent assay (ELISA). Results: Fourteen days after sciatic nerve ligation, rats in Groups C and D had a shortened TWL (P 0.05). On the 8th and 14th days, the mRNA levels in Group D were restored to those of Groups A and B. Meanwhile, the CGRP content of Group D gradually dropped over time, from 76.4 pg/mg (Day 0) to 57.5 pg/mg (Day 14). Conclusions: In this study, we found that, after sciatic nerve ligation, rats exhibited apparent hyperalgesia and allodynia, and CGRP mRNA and CGRP contents in the L4-L6 DRG increased significantly. Through lowering CGRP expression in the DRG, PRF treatment might relieve the pain behaviors of NP. PMID:29333099

  15. DZNep, inhibitor of S-adenosylhomocysteine hydrolase, down-regulates expression of SETDB1 H3K9me3 HMTase in human lung cancer cells.

    Science.gov (United States)

    Lee, Ju-Kyung; Kim, Keun-Cheol

    2013-09-06

    3-Deazaneplanocin A (DZNep), an epigenetic anticancer drug, leads to the indirect suppression of S-adenosyl methionine-dependent cellular methylations by inhibiting S-adenosyl homocystein (AdoHcy) hydrolase. Although it is well known that DZNep targets the degradation of EZH2 protein, H3K27me3 HMTase, there are still uncertainties about the regulation of other types of HMTases during cell death. In this study, we describe that SETDB1 gene expression was regulated by DZNep treatment in human lung cancer cells. We confirm that DZNep induced growth inhibition and increased the dead cell population of lung cancer cells. DZNep treatment affected histone methylations, including H3K27me3 and H3K9me3, but not H3K4me3. Reduced levels of H3K27me3 and H3K9me3 were related with the decreased EZH2 and SETDB1 proteins. Real time PCR analysis showed that SETDB1 gene expression was decreased by DZNep treatment, but no effect was observed for EZH2 gene expression. We cloned the promoter region of SETDB1 and SUV39H1 genes, and performed luciferase assays. The promoter activity of SETDB1 gene was down regulated by DZNep treatment, whereas no effect on SUV39H1 promoter activity was observed. In conclusion, we suggest that DZNep regulates not only on H3K27me3 HMTase EZH2, but also H3K9 HMTase SETDB1 gene expression at the transcription level, implicating that the mechanism of action of DZNep targets multiple HMTases during the death of lung cancer cells. Copyright © 2013 Elsevier Inc. All rights reserved.

  16. BDE-47 causes developmental retardation with down-regulated expression profiles of ecdysteroid signaling pathway-involved nuclear receptor (NR) genes in the copepod Tigriopus japonicus.

    Science.gov (United States)

    Hwang, Dae-Sik; Han, Jeonghoon; Won, Eun-Ji; Kim, Duck-Hyun; Jeong, Chang-Bum; Hwang, Un-Ki; Zhou, Bingsheng; Choe, Joonho; Lee, Jae-Seong

    2016-08-01

    2,2',4,4'-Tetrabromodiphenyl ether (BDE-47) is a persistent organic pollutant (POP) in marine environments. Despite its adverse effects (e.g. developmental retardation) in ecdysozoa, the effects of BDE-47 on transcription of ecdysteroid signaling pathway-involved-nuclear receptor (NR) genes and metamorphosis-related genes have not been examined in copepods. To examine the deleterious effect of BDE-47 on copepod molting and metamorphosis, BDE-47 was exposed to the harpacticoid copepod Tigriopus japonicus, followed by monitoring developmental retardation and transcriptional alteration of NR genes. The developmental rate was significantly inhibited (P<0.05) in response to BDE-47 and the agricultural insecticide gamma-hexachlorocyclohexane. Conversely, the ecdysteroid agonist ponasterone A (PoA) led to decreased molting and metamorphosis time (P<0.05) from the nauplius stage to the adult stage. In particular, expression profiles of all NR genes were the highest at naupliar stages 5-6 except for SVP, FTZ-F1, and HR96 genes. Nuclear receptor USP, HR96, and FTZ-F1 genes also showed significant sex differences (P<0.05) in gene expression levels over different developmental stages, indicating that these genes may be involved in vitellogenesis. NR gene expression patterns showed significant decreases (P<0.05) in response to BDE-47 exposure, implying that molting and metamorphosis retardation is likely associated with NR gene expression. In summary, BDE-47 leads to molting and metamorphosis retardation and suppresses transcription of NR genes. This information will be helpful in understanding the molting and metamorphosis delay mechanism in response to BDE-47 exposure. Copyright © 2016 Elsevier B.V. All rights reserved.

  17. Aberrant KDM5B expression promotes aggressive breast cancer through MALAT1 overexpression and downregulation of hsa-miR-448

    International Nuclear Information System (INIS)

    Bamodu, Oluwaseun Adebayo; Huang, Wen-Chien; Lee, Wei-Hwa; Wu, Alexander; Wang, Liang Shun; Hsiao, Michael; Yeh, Chi-Tai; Chao, Tsu-Yi

    2016-01-01

    Triple negative breast cancers (TNBC) possess cell dedifferentiation characteristics, carry out activities connate to those of cancer stem cells (CSCs) and are associated with increased metastasis, as well as, poor clinical prognosis. The regulatory mechanism of this highly malignant phenotype is still poorly characterized. Accruing evidence support the role of non-coding RNAs (ncRNAs) as potent regulators of CSC and metastatic gene expression, with their dysregulation implicated in tumorigenesis and disease progression. In this study, we investigated TNBC metastasis, metastasis-associated genes and potential inhibitory mechanisms using bioinformatics, tissue microarray analyses, immunoblotting, polymerase chain reaction, loss and gain of gene function assays and comparative analyses of data obtained. Compared with other breast cancer types, the highly metastatic MDA-MB-231 cells concurrently exhibited increased expression levels of Lysine-specific demethylase 5B protein (KDM5B) and long non-coding RNA (lncRNA), MALAT1, suggesting their functional association. KDM5B-silencing in the TNBC cells correlated with the upregulation of hsa-miR-448 and led to suppression of MALAT1 expression with decreased migration, invasion and clonogenic capacity in vitro, as well as, poor survival in vivo. This projects MALAT1 as a mediator of KDM5B oncogenic potential and highlights the critical role of this microRNA, lncRNA and histone demethylase in cancer cell motility and metastatic colonization. Increased expression of KDM5B correlating with disease progression and poor clinical outcome in breast cancer was reversed by hsa-miR-448. Our findings demonstrate the critical role of KDM5B and its negative regulator hsa-miR-448 in TNBC metastasis and progression. Hsa-miR-448 disrupting KDM5B-MALAT1 signalling axis and associated activities in TNBC cells, projects it as a putative therapeutic factor for selective eradication of TNBC cells

  18. Healthy Nordic diet downregulates the expression of genes involved in inflammation in subcutaneous adipose tissue in individuals with features of the metabolic syndrome.

    Science.gov (United States)

    Kolehmainen, Marjukka; Ulven, Stine M; Paananen, Jussi; de Mello, Vanessa; Schwab, Ursula; Carlberg, Carsten; Myhrstad, Mari; Pihlajamäki, Jussi; Dungner, Elisabeth; Sjölin, Eva; Gunnarsdottir, Ingibjörg; Cloetens, Lieselotte; Landin-Olsson, Mona; Akesson, Björn; Rosqvist, Fredrik; Hukkanen, Janne; Herzig, Karl-Heinz; Dragsted, Lars O; Savolainen, Markku J; Brader, Lea; Hermansen, Kjeld; Risérus, Ulf; Thorsdottir, Inga; Poutanen, Kaisa S; Uusitupa, Matti; Arner, Peter; Dahlman, Ingrid

    2015-01-01

    Previously, a healthy Nordic diet (ND) has been shown to have beneficial health effects close to those of Mediterranean diets. The objective was to explore whether the ND has an impact on gene expression in abdominal subcutaneous adipose tissue (SAT) and whether changes in gene expression are associated with clinical and biochemical effects. Obese adults with features of the metabolic syndrome underwent an 18- to 24-wk randomized intervention study comparing the ND with the control diet (CD) (the SYSDIET study, carried out within Nordic Centre of Excellence of the Systems Biology in Controlled Dietary Interventions and Cohort Studies). The present study included participants from 3 Nordic SYSDIET centers [Kuopio (n = 20), Lund (n = 18), and Oulu (n = 18)] with a maximum weight change of ±4 kg, highly sensitive C-reactive protein concentration healthy Nordic diet reduces inflammatory gene expression in SAT compared with a control diet independently of body weight change in individuals with features of the metabolic syndrome. © 2015 American Society for Nutrition.

  19. Chromium downregulates the expression of Acetyl CoA Carboxylase 1 gene in lipogenic tissues of domestic goats: a potential strategy for meat quality improvement.

    Science.gov (United States)

    Najafpanah, Mohammad Javad; Sadeghi, Mostafa; Zali, Abolfazl; Moradi-Shahrebabak, Hossein; Mousapour, Hojatollah

    2014-06-15

    Acetyl CoA Carboxylase 1 (ACC1) is a biotin-dependent enzyme that catalyzes the carboxylation of Acetyl CoA to form Malonyl CoA, the key intermediate metabolite in fatty acid synthesis. In this study, the mRNA expression of the ACC1 gene was evaluated in four different tissues (liver, visceral fat, subcutaneous fat, and longissimus muscle) of the domestic goat (Capra hircus) kids feeding on four different levels of trivalent chromium (0, 0.5, 1, and 1.5mg/day) as food supplementation. RT-qPCR technique was used for expression analyses and heat shock protein 90 gene (HSP-90) was considered as reference gene for data normalization. Our results revealed that 1.5mg/day chromium significantly reduced the expression of the ACC1 gene in liver, visceral fat, and subcutaneous fat tissues, but not in longissimus muscles (Pmeat quality in domestic animals. Copyright © 2014 Elsevier B.V. All rights reserved.

  20. IL-4 and IL-13 mediated down-regulation of CD8 expression levels can dampen anti-viral CD8⁺ T cell avidity following HIV-1 recombinant pox viral vaccination.

    Science.gov (United States)

    Wijesundara, Danushka K; Jackson, Ronald J; Tscharke, David C; Ranasinghe, Charani

    2013-09-23

    We have shown that mucosal HIV-1 recombinant pox viral vaccination can induce high, avidity HIV-specific CD8(+) T cells with reduced interleukin (IL)-4 and IL-13 expression compared to, systemic vaccine delivery. In the current study how these cytokines act to regulate anti-viral CD8(+) T, cell avidity following HIV-1 recombinant pox viral prime-boost vaccination was investigated. Out of a panel of T cell avidity markers tested, only CD8 expression levels were found to be enhanced on, KdGag197-205 (HIV)-specific CD8(+) T cells obtained from IL-13(-/-), IL-4(-/-) and signal transducer and, activator of transcription of 6 (STAT6)(-/-) mice compared to wild-type (WT) controls following, vaccination. Elevated CD8 expression levels in this instance also correlated with polyfunctionality, (interferon (IFN)-γ, tumour necorsis factor (TNF)-α and IL-2 production) and the avidity of HIVspecific CD8(+) T cells. Furthermore, mucosal vaccination and vaccination with the novel adjuvanted IL-13 inhibitor (i.e. IL-13Rα2) vaccines significantly enhanced CD8 expression levels on HIV-specific CD8(+), T cells, which correlated with avidity. Using anti-CD8 antibodies that blocked CD8 availability on CD8(+), T cells, it was established that CD8 played an important role in increasing HIV-specific CD8(+) T cell avidity and polyfunctionality in IL-4(-/-), IL-13(-/-) and STAT6(-/-) mice compared to WT controls, following vaccination. Collectively, our data demonstrate that IL-4 and IL-13 dampen CD8 expression levels on anti-viral CD8(+) T cells, which can down-regulate anti-viral CD8(+) T cell avidity and, polyfunctionality following HIV-1 recombinant pox viral vaccination. These findings can be exploited to, design more efficacious vaccines not only against HIV-1, but many chronic infections where high, avidity CD8(+) T cells help protection. Copyright © 2013 Elsevier Ltd. All rights reserved.

  1. Tamoxifen enhances erlotinib-induced cytotoxicity through down-regulating AKT-mediated thymidine phosphorylase expression in human non-small-cell lung cancer cells.

    Science.gov (United States)

    Ko, Jen-Chung; Chiu, Hsien-Chun; Syu, Jhan-Jhang; Jian, Yi-Jun; Chen, Chien-Yu; Jian, Yun-Ting; Huang, Yi-Jhen; Wo, Ting-Yu; Lin, Yun-Wei

    2014-03-01

    Tamoxifen is a triphenylethylene nonsteroidal estrogen receptor (ER) antagonist used worldwide as an adjuvant hormone therapeutic agent in the treatment of breast cancer. However, the molecular mechanism of tamoxifen-induced cytotoxicity in non-small cell lung cancer (NSCLC) cells has not been identified. Thymidine phosphorylase (TP) is an enzyme of the pyrimidine salvage pathway which is upregulated in cancers. In this study, tamoxifen treatment inhibited cell survival in two NSCLC cells, H520 and H1975. Treatment with tamoxifen decreased TP mRNA and protein levels through AKT inactivation. Furthermore, expression of constitutively active AKT (AKT-CA) vectors significantly rescued the decreased TP protein and mRNA levels in tamoxifen-treated NSCLC cells. In contrast, combination treatment with PI3K inhibitors (LY294002 or wortmannin) and tamoxifen further decreased the TP expression and cell viability of NSCLC cells. Knocking down TP expression by transfection with small interfering RNA of TP enhanced the cytotoxicity and cell growth inhibition of tamoxifen. Erlotinib (Tarceva, OSI-774), an orally available small molecular inhibitor of epidermal growth factor receptor (EGFR) tyrosine kinase, is approved for clinical treatment of NSCLC. Compared to a single agent alone, tamoxifen combined with erlotinib resulted in cytotoxicity and cell growth inhibition synergistically in NSCLC cells, accompanied with reduced activation of phospho-AKT and phospho-ERK1/2, and reduced TP protein levels. These findings may have implications for the rational design of future drug regimens incorporating tamoxifen and erlotinib for the treatment of NSCLC. Copyright © 2014 Elsevier Inc. All rights reserved.

  2. Integration of HPV6 and downregulation of AKR1C3 expression mark malignant transformation in a patient with juvenile-onset laryngeal papillomatosis.

    Directory of Open Access Journals (Sweden)

    Christian Ulrich Huebbers

    Full Text Available Juvenile-onset recurrent respiratory papillomatosis (RRP is associated with low risk human papillomavirus (HPV types 6 and 11. Malignant transformation has been reported solely for HPV11-associated RRP in 2-4% of all RRP-cases, but not for HPV6. The molecular mechanisms in the carcinogenesis of low risk HPV-associated cancers are to date unknown. We report of a female patient, who presented with a laryngeal carcinoma at the age of 24 years. She had a history of juvenile-onset RRP with an onset at the age of three and subsequently several hundred surgical interventions due to multiple recurrences of RRP. Polymerase chain reaction (PCR or bead-based hybridization followed by direct sequencing identified HPV6 in tissue sections of previous papilloma and the carcinoma. P16(INK4A, p53 and pRb immunostainings were negative in all lesions. HPV6 specific fluorescence in situ hybridization (FISH revealed nuclear staining suggesting episomal virus in the papilloma and a single integration site in the carcinoma. Integration-specific amplification of papillomavirus oncogene transcripts PCR (APOT-PCR showed integration in the aldo-keto reductase 1C3 gene (AKR1C3 on chromosome 10p15.1. ArrayCGH detected loss of the other gene copy as part of a deletion at 10p14-p15.2. Western blot analysis and immunohistochemistry of the protein AKR1C3 showed a marked reduction of its expression in the carcinoma. In conclusion, we identified a novel molecular mechanism underlying a first case of HPV6-associated laryngeal carcinoma in juvenile-onset RRP, i.e. that HPV6 integration in the AKR1C3 gene resulted in loss of its expression. Alterations of AKR1C gene expression have previously been implicated in the tumorigenesis of other (HPV-related malignancies.

  3. Down-regulation of viral replication by adenoviral-mediated expression of siRNA against cellular cofactors for hepatitis C virus

    International Nuclear Information System (INIS)

    Zhang Jing; Yamada, Osamu; Sakamoto, Takashi; Yoshida, Hiroshi; Iwai, Takahiro; Matsushita, Yoshihisa; Shimamura, Hideo; Araki, Hiromasa; Shimotohno, Kunitada

    2004-01-01

    Small interfering RNA (siRNA) is currently being evaluated not only as a powerful tool for functional genomics, but also as a potentially promising therapeutic agent for cancer and infectious diseases. Inhibitory effect of siRNA on viral replication has been demonstrated in multiple pathogenic viruses. However, because of the high sequence specificity of siRNA-mediated RNA degradation, antiviral efficacy of siRNA directed to viral genome will be largely limited by emergence of escape variants resistant to siRNA due to high mutation rates of virus, especially RNA viruses such as poliovirus and hepatitis C virus (HCV). To investigate the therapeutic feasibility of siRNAs specific for the putative cellular cofactors for HCV, we constructed adenovirus vectors expressing siRNAs against La, polypyrimidine tract-binding protein (PTB), subunit gamma of human eukaryotic initiation factors 2B (eIF2Bγ), and human VAMP-associated protein of 33 kDa (hVAP-33). Adenoviral-mediated expression of siRNAs markedly diminished expression of the endogenous genes, and silencing of La, PTB, and hVAP-33 by siRNAs substantially blocked HCV replication in Huh-7 cells. Thus, our studies demonstrate the feasibility and potential of adenoviral-delivered siRNAs specific for cellular cofactors in combating HCV infection, which can be used either alone or in combination with siRNA against viral genome to prevent the escape of mutant variants and provide additive or synergistic anti-HCV effects

  4. Synergism between Hedgehog-GLI and EGFR signaling in Hedgehog-responsive human medulloblastoma cells induces downregulation of canonical Hedgehog-target genes and stabilized expression of GLI1.

    Directory of Open Access Journals (Sweden)

    Frank Götschel

    Full Text Available Aberrant activation of Hedgehog (HH signaling has been identified as a key etiologic factor in many human malignancies. Signal strength, target gene specificity, and oncogenic activity of HH signaling depend profoundly on interactions with other pathways, such as epidermal growth factor receptor-mediated signaling, which has been shown to cooperate with HH/GLI in basal cell carcinoma and pancreatic cancer. Our experimental data demonstrated that the Daoy human medulloblastoma cell line possesses a fully inducible endogenous HH pathway. Treatment of Daoy cells with Sonic HH or Smoothened agonist induced expression of GLI1 protein and simultaneously prevented the processing of GLI3 to its repressor form. To study interactions between HH- and EGF-induced signaling in greater detail, time-resolved measurements were carried out and analyzed at the transcriptomic and proteomic levels. The Daoy cells responded to the HH/EGF co-treatment by downregulating GLI1, PTCH, and HHIP at the transcript level; this was also observed when Amphiregulin (AREG was used instead of EGF. We identified a novel crosstalk mechanism whereby EGFR signaling silences proteins acting as negative regulators of HH signaling, as AKT- and ERK-signaling independent process. EGFR/HH signaling maintained high GLI1 protein levels which contrasted the GLI1 downregulation on the transcript level. Conversely, a high-level synergism was also observed, due to a strong and significant upregulation of numerous canonical EGF-targets with putative tumor-promoting properties such as MMP7, VEGFA, and IL-8. In conclusion, synergistic effects between EGFR and HH signaling can selectively induce a switch from a canonical HH/GLI profile to a modulated specific target gene profile. This suggests that there are more wide-spread, yet context-dependent interactions, between HH/GLI and growth factor receptor signaling in human malignancies.

  5. The Curcumin Analogue 1,5-Bis(2-hydroxyphenyl-1,4-pentadiene-3-one Induces Apoptosis and Downregulates E6 and E7 Oncogene Expression in HPV16 and HPV18-Infected Cervical Cancer Cells

    Directory of Open Access Journals (Sweden)

    Felicia Paulraj

    2015-06-01

    Full Text Available In an effort to study curcumin analogues as an alternative to improve the therapeutic efficacy of curcumin, we screened the cytotoxic potential of four diarylpentanoids using the HeLa and CaSki cervical cancer cell lines. Determination of their EC50 values indicated relatively higher potency of 1,5-bis(2-hydroxyphenyl-1,4-pentadiene-3-one (MS17, 1.03 ± 0.5 μM; 2.6 ± 0.9 μM and 1,5-bis(4-hydroxy-3-methoxyphenyl-1,4-pentadiene-3-one (MS13, 2.8 ± 0.4; 6.7 ± 2.4 μM in CaSki and HeLa, respectively, with significantly greater growth inhibition at 48 and 72 h of treatment compared to the other analogues or curcumin. Based on cytotoxic and anti-proliferative activity, MS17 was selected for comprehensive apoptotic studies. At 24 h of treatment, fluorescence microscopy detected that MS17-exposed cells exhibited significant morphological changes consistent with apoptosis, corroborated by an increase in nucleosomal enrichment due to DNA fragmentation in HeLa and CaSki cells and activation of caspase-3 activity in CaSki cells. Quantitative real-time PCR also detected significant down-regulation of HPV18- and HPV16-associated E6 and E7 oncogene expression following treatment. The overall data suggests that MS17 treatment has cytotoxic, anti-proliferative and apoptosis-inducing potential in HPV-positive cervical cancer cells. Furthermore, its role in down-regulation of HPV-associated oncogenes responsible for cancer progression merits further investigation into its chemotherapeutic role for cervical cancer.

  6. Fluoxetine ameliorates cognitive impairments induced by chronic cerebral hypoperfusion via down-regulation of HCN2 surface expression in the hippocampal CA1 area in rats.

    Science.gov (United States)

    Luo, Pan; Zhang, Xiaoxue; Lu, Yun; Chen, Cheng; Li, Changjun; Zhou, Mei; Lu, Qing; Xu, Xulin; Shen, Guanxin; Guo, Lianjun

    2016-01-01

    Chronic cerebral hypoperfusion (CCH) causes cognitive impairments and increases the risk of Alzheimer's disease (AD) and vascular dementia (VD) through several biologically plausible pathways, yet the underlying neurobiological mechanisms are still poorly understood. In this study, we investigated whether fluoxetine, a selective serotonin reuptake inhibitor (SSRI), could play a neuroprotective role against chronic cerebral hypoperfusion injury and to clarify underlying mechanisms of its efficacy. Rats were subjected to permanent bilateral occlusion of the common carotid arteries (two-vessel occlusion, 2VO). Two weeks later, rats were treated with 30 mg/kg fluoxetine (intragastric injection, i.g.) for 6 weeks. Cognitive function was evaluated by Morris water maze (MWM) and novel objects recognition (NOR) test. Long-term potentiation (LTP) was used to address the underlying synaptic mechanisms. Western blotting was used to quantify the protein levels. Our results showed that fluoxetine treatment significantly improved the cognitive impairments caused by 2VO, accompanied with a reversion of 2VO-induced inhibitory of LTP. Furthermore, 2VO caused an up-regulation of hyperpolarization-activated cyclic nucleotide-gated channel 2 (HCN2) surface expressions in the hippocampal CA1 area and fluoxetine also effectively recovered the disorder of HCN2 surface expressions, which may be a possible mechanism that fluoxetine treatment ameliorates cognitive impairments in rats with CCH. Copyright © 2015 Elsevier Inc. All rights reserved.

  7. Constraint-induced movement therapy promotes motor function recovery and downregulates phosphorylated extracellular regulated protein kinase expression in ischemic brain tissue of rats

    Directory of Open Access Journals (Sweden)

    Bei Zhang

    2015-01-01

    Full Text Available Motor function impairment is a common outcome of stroke. Constraint-induced movement therapy (CIMT involving intensive use of the impaired limb while restraining the unaffected limb is widely used to overcome the effects of ′learned non-use′ and improve limb function after stroke. However, the underlying mechanism of CIMT remains unclear. In the present study, rats were randomly divided into a middle cerebral artery occlusion (model group, a CIMT + model (CIMT group, or a sham group. Restriction of the affected limb by plaster cast was performed in the CIMT and sham groups. Compared with the model group, CIMT significantly improved the forelimb functional performance in rats. By western blot assay, the expression of phosphorylated extracellular regulated protein kinase in the bilateral cortex and hippocampi of cerebral ischemic rats in the CIMT group was significantly lower than that in the model group, and was similar to sham group levels. These data suggest that functional recovery after CIMT may be related to decreased expression of phosphorylated extracellular regulated protein kinase in the bilateral cortex and hippocampi.

  8. miR-183 inhibits TGF-β1-induced apoptosis by downregulation of PDCD4 expression in human hepatocellular carcinoma cells

    International Nuclear Information System (INIS)

    Li, Jipeng; Fu, Hanjiang; Xu, Chengwang; Tie, Yi; Xing, Ruiyun; Zhu, Jie; Qin, Yide; Sun, Zhixian; Zheng, Xiaofei

    2010-01-01

    In recent years, some miRNAs have been reported to be connected closely with the development of human hepatocellular carcinoma. In our previous studies, a set of miRNAs were revealed to be dysregulated in HCC tissues. However, the functions of these miRNAs in HCC remain largely undefined. The expression profiles of miR-183 were compared between HCC tissues and adjacent normal liver tissues using qRT-PCR method. This method was used to screen the potential target genes of miR-183. A luciferase reporter assay was conducted to confirm target association. Finally, the functional effect of miR-183 in hepatoma cells was examined. Among the 25 HCC samples analyzed, microRNA-183 was significantly up-regulated (twofold to 367-fold) in 17 samples compared with the matching nontumoral liver tissues. Programmed cell death 4 (PDCD4) was identified as the target gene of miR-183. Moreover, PDCD4 is a proapoptotic molecule involved in TGF-β1-induced apoptosis in human HCC cells, we found that miR-183 transfectants were resistant to apoptosis induced by TGF-β1. We conclude that miR-183 can inhibit apoptosis in human HCC cells by repressing the PDCD4 expression, and miR-183 may play an important role in HCC development

  9. Lim Mineralization Protein 3 Induces the Osteogenic Differentiation of Human Amniotic Fluid Stromal Cells through Kruppel-Like Factor-4 Downregulation and Further Bone-Specific Gene Expression

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    Marta Barba

    2012-01-01

    Full Text Available Multipotent mesenchymal stem cells with extensive self-renewal properties can be easily isolated and rapidly expanded in culture from small volumes of amniotic fluid. These cells, namely, amniotic fluid-stromal cells (AFSCs, can be regarded as an attractive source for tissue engineering purposes, being phenotypically and genetically stable, plus overcoming all the safety and ethical issues related to the use of embryonic/fetal cells. LMP3 is a novel osteoinductive molecule acting upstream to the main osteogenic pathways. This study is aimed at delineating the basic molecular events underlying LMP3-induced osteogenesis, using AFSCs as a cellular model to focus on the molecular features underlying the multipotency/differentiation switch. For this purpose, AFSCs were isolated and characterized in vitro and transfected with a defective adenoviral vector expressing the human LMP3. LMP3 induced the successful osteogenic differentiation of AFSC by inducing the expression of osteogenic markers and osteospecific transcription factors. Moreover, LMP3 induced an early repression of the kruppel-like factor-4, implicated in MSC stemness maintenance. KLF4 repression was released upon LMP3 silencing, indicating that this event could be reasonably considered among the basic molecular events that govern the proliferation/differentiation switch during LMP3-induced osteogenic differentiation of AFSC.

  10. Downregulation of hepatic betaine:homocysteine methyltransferase (BHMT) expression in taurine-deficient mice is reversed by taurine supplementation in vivo.

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    Jurkowska, Halina; Niewiadomski, Julie; Hirschberger, Lawrence L; Roman, Heather B; Mazor, Kevin M; Liu, Xiaojing; Locasale, Jason W; Park, Eunkyue; Stipanuk, Martha H

    2016-03-01

    The cysteine dioxygenase (Cdo1)-null and the cysteine sulfinic acid decarboxylase (Csad)-null mouse are not able to synthesize hypotaurine/taurine by the cysteine/cysteine sulfinate pathway and have very low tissue taurine levels. These mice provide excellent models for studying the effects of taurine on biological processes. Using these mouse models, we identified betaine:homocysteine methyltransferase (BHMT) as a protein whose in vivo expression is robustly regulated by taurine. BHMT levels are low in liver of both Cdo1-null and Csad-null mice, but are restored to wild-type levels by dietary taurine supplementation. A lack of BHMT activity was indicated by an increase in the hepatic betaine level. In contrast to observations in liver of Cdo1-null and Csad-null mice, BHMT was not affected by taurine supplementation of primary hepatocytes from these mice. Likewise, CSAD abundance was not affected by taurine supplementation of primary hepatocytes, although it was robustly upregulated in liver of Cdo1-null and Csad-null mice and lowered to wild-type levels by dietary taurine supplementation. The mechanism by which taurine status affects hepatic CSAD and BHMT expression appears to be complex and to require factors outside of hepatocytes. Within the liver, mRNA abundance for both CSAD and BHMT was upregulated in parallel with protein levels, indicating regulation of BHMT and CSAD mRNA synthesis or degradation.

  11. Oligodeoxynucleotides Can Transiently Up- and Downregulate CHS Gene Expression in Flax by Changing DNA Methylation in a Sequence-Specific Manner

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    Magdalena Dzialo

    2017-05-01

    Full Text Available Chalcone synthase (CHS has been recognized as an essential enzyme in the phenylpropanoid biosynthesis pathway. Apart from the leading role in the production of phenolic compounds with many valuable biological activities beneficial to biomedicine, CHS is well appreciated in science. Genetic engineering greatly facilitates expanding knowledge on the function and genetics of CHS in plants. The CHS gene is one of the most intensively studied genes in flax. In our study, we investigated engineering of the CHS gene through genetic and epigenetic approaches. Considering the numerous restrictions concerning the application of genetically modified (GM crops, the main purpose of this research was optimization of the plant's modulation via epigenetics. In our study, plants modified through two methods were compared: a widely popular agrotransformation and a relatively recent oligodeoxynucleotide (ODN strategy. It was recently highlighted that the ODN technique can be a rapid and time-serving antecedent in quick analysis of gene function before taking vector-mediated transformation. In order to understand the molecular background of epigenetic variation in more detail and evaluate the use of ODNs as a tool for predictable and stable gene engineering, we concentrated on the integration of gene expression and gene-body methylation. The treatment of flax with a series of short oligonucleotides homologous to a different part of CHS gene isoforms revealed that those directed to regulatory gene regions (5′- and 3′-UTR activated gene expression, directed to non-coding region (introns caused gen activity reduction, while those homologous to a coding region may have a variable influence on its activity. Gene expression changes were accompanied by changes in its methylation status. However, only certain (CCGG motifs along the gene sequence were affected. The analyzed DNA motifs of the CHS flax gene are more accessible for methylation when located within a Cp

  12. In vitro and in vivo inhibition of proangiogenic retinal phenotype by an antisense oligonucleotide downregulating uPAR expression.

    Science.gov (United States)

    Lulli, Matteo; Cammalleri, Maurizio; Granucci, Irene; Witort, Ewa; Bono, Silvia; Di Gesualdo, Federico; Lupia, Antonella; Loffredo, Rosa; Casini, Giovanni; Dal Monte, Massimo; Capaccioli, Sergio

    2017-08-26

    Neoangiogenesis is the main pathogenic event involved in a variety of retinal diseases. It has been recently demonstrated that inhibiting the urokinase-type plasminogen activator receptor (uPAR) results in reduced angiogenesis in a mouse model of oxygen-induced retinopathy (OIR), establishing uPAR as a therapeutic target in proliferative retinopathies. Here, we evaluated in cultured human retinal endothelial cells (HRECs) and in OIR mice the potential of a specific antisense oligodeoxyribonucleotide (ASO) in blocking the synthesis of uPAR and in providing antiangiogenic effects. uPAR expression in HRECs was inhibited by lipofection with the phosphorotioated 5'-CGGCGGGTGACCCATGTG-3' ASO-uPAR, complementary to the initial translation site of uPAR mRNA. Inhibition of uPAR expression via ASO-uPAR was evaluated in HRECs by analyzing VEGF-induced tube formation and migration. In addition, the well-established and reproducible murine OIR model was used to induce retinal neovascularization in vivo. OIR mice were injected intraperitoneally with ASO-uPAR and retinopathy was evaluated considering the extent of the avascular area in the central retina and neovascular tuft formation. The ASO-uPAR specifically decreased uPAR mRNA and protein levels in HRECs and mitigated VEGF-induced tube formation and cell migration. Noteworthy, in OIR mice ASO-uPAR administration reduced both the avascular area and the formation of neovascular tufts. In conclusion, although the extrapolation of these experimental findings to the clinic is not straightforward, ASO-uPAR may be considered a potential therapeutic tool for treatment of proliferative retinal diseases. Copyright © 2017 Elsevier Inc. All rights reserved.

  13. A preliminary study of pamidronic acid downregulation of angiogenic factors IGF-1/PECAM-1 expression in circulating level in bone metastatic breast cancer patients

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    Wang Z

    2016-05-01

    Full Text Available Zeng Wang,1,2 Lei Lei,2,3 Xin-jun Cai,4 Ling Ya Chen,1,2 Meiqin Yuan,2,3 Guonong Yang,1,2 Ping Huang,1,2 Xiaojia Wang2,3 1Department of Pharmacy, 2Zhejiang Key Lab of Diagnosis & Treatment Technology on Thoracic Oncology, 3Department of Chemotherapy Center, Zhejiang Cancer Hospital, 4Department of Pharmacy, Integrated Chinese and Western Medicine Hospital of Zhejiang Province, Hangzhou, Zhejiang, People’s Republic of China Objective: To evaluate the expressions of circulating angiogenic factors affected by pamidronic acid (PA intravenous infusion in bone metastatic breast cancer patients and the impact on their prognosis.Methods: Peripheral blood of ten bone metastatic breast cancer patients was collected for serum insulin-like growth factor-1 (IGF-1 and platelet endothelial cell adhesion molecule-1 expression detection just before and 2 days after PA infusion.Results: Both IGF-1 and platelet endothelial cell adhesion molecule-1 concentrations decreased after PA treatment for 48 hours (P<0.05. Modification was defined as >20% decrease recorded 2 days after PA administration. The decrease of IGF-1 was more significant in breast cancer patients who had received previous hormonotherapy. Moreover, the progression-free survival of first-line chemotherapy treatment of IGF-1 modified patients was longer than that of IGF-1 unmodified patients (P=0.009.Conclusion: PA treatment could suppress circulating serum IGF-1 and platelet endothelial cell adhesion molecule-1 concentrations; moreover, the prognosis of patients in IGF-1 unmodified group was relatively poor. Keywords: pamidronic acid, insulin-like growth factor-1, platelet endothelial cell adhesion molecule-1, bone metastatic breast cancer, prognosis

  14. The Herb Medicine Formula “Chong Lou Fu Fang” Increases the Cytotoxicity of Chemotherapeutic Agents and Down-Regulates the Expression of Chemotherapeutic Agent Resistance-Related Genes in Human Gastric Cancer Cells In Vitro

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    Yongping Liu

    2011-01-01

    Full Text Available The herb medicine formula “Chong Lou Fu Fang” (CLFF has efficacy in inhibiting the proliferation of human gastric cancer in vitro and in vivo. To explore the potentially useful combination of CLFF with chemotherapeutic agents commonly used in gastric cancer therapy, we assess the interaction between CLFF and these chemotherapeutic agents in both SGC-7901 cell lines and BGC-823 cell lines using a median effect analysis and apoptosis analysis, and we also investigate the influence of CLFF on chemotherapeutic agent-associated gene expression. The synergistic analysis indicated that CLFF had a synergistic effect on the cytotoxicity of 5-fluorouracil (5-FU in a relative broad dose inhibition range (20–95% fraction affected in SGC-7901cell lines and 5–65% fraction affected in BGC-823 cell lines, while the synergistic interaction between CLFF and oxaliplatin or docetaxel only existed in a low dose inhibition range (≤50% fraction affected in both cell lines. Combination of CLFF and chemotherapeutic agents could also induce apoptosis in a synergistic manner. After 24 h, CLFF alone or CLFF combination with chemotherapeutic agents could significantly suppress the levels of expression of chemotherapeutic agent resistance related genes in gastric cancer cells. Our findings indicate that there are useful synergistic interactions between CLFF and chemotherapeutic agents in gastric cancer cells, and the possible mechanisms might be partially due to the down-regulation of chemotherapeutic agent resistance related genes and the synergistic apoptotic effect.

  15. Differentiation-Associated Downregulation of Poly(ADP-Ribose Polymerase-1 Expression in Myoblasts Serves to Increase Their Resistance to Oxidative Stress.

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    Gábor Oláh

    Full Text Available Poly(ADP-ribose polymerase 1 (PARP-1, the major isoform of the poly (ADP-ribose polymerase family, is a constitutive nuclear and mitochondrial protein with well-recognized roles in various essential cellular functions such as DNA repair, signal transduction, apoptosis, as well as in a variety of pathophysiological conditions including sepsis, diabetes and cancer. Activation of PARP-1 in response to oxidative stress catalyzes the covalent attachment of the poly (ADP-ribose (PAR groups on itself and other acceptor proteins, utilizing NAD+ as a substrate. Overactivation of PARP-1 depletes intracellular NAD+ influencing mitochondrial electron transport, cellular ATP generation and, if persistent, can result in necrotic cell death. Due to their high metabolic activity, skeletal muscle cells are particularly exposed to constant oxidative stress insults. In this study, we investigated the role of PARP-1 in a well-defined model of murine skeletal muscle differentiation (C2C12 and compare the responses to oxidative stress of undifferentiated myoblasts and differentiated myotubes. We observed a marked reduction of PARP-1 expression as myoblasts differentiated into myotubes. This alteration correlated with an increased resistance to oxidative stress of the myotubes, as measured by MTT and LDH assays. Mitochondrial function, assessed by measuring mitochondrial membrane potential, was preserved under oxidative stress in myotubes compared to myoblasts. Moreover, basal respiration, ATP synthesis, and the maximal respiratory capacity of mitochondria were higher in myotubes than in myoblasts. Inhibition of the catalytic activity of PARP-1 by PJ34 (a phenanthridinone PARP inhibitor exerted greater protective effects in undifferentiated myoblasts than in differentiated myotubes. The above observations in C2C12 cells were also confirmed in a rat-derived skeletal muscle cell line (L6. Forced overexpression of PARP1 in C2C12 myotubes sensitized the cells to oxidant

  16. Kaempferol Reduces Matrix Metalloproteinase-2 Expression by Down-Regulating ERK1/2 and the Activator Protein-1 Signaling Pathways in Oral Cancer Cells

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    Lin, Chiao-Wen; Chen, Pei-Ni; Chen, Mu-Kuan; Yang, Wei-En; Tang, Chih-Hsin; Yang, Shun-Fa; Hsieh, Yih-Shou

    2013-01-01

    Background Kaempferol has been proposed as a potential drug for cancer chemoprevention and treatment because it is a natural polyphenol contained in plant-based foods. Recent studies have demonstrated that kaempferol protects against cardiovascular disease and cancer. Based on this finding, we investigated the mechanisms by which kaempferol produces the anti-metastatic effect in human tongue squamous cell carcinoma SCC4 cells. Methodology/Principal Findings In this study, we provided molecular evidence associated with the anti-metastatic effect of kaempferol by demonstrating a substantial suppression of SCC4 cell migration and invasion. This effect was associated with reduced expressions of MMP-2 and TIMP-2 mRNA and protein levels. Analysis of the transcriptional regulation indicated that kaempferol inhibited MMP-2 transcription by suppressing c-Jun activity. Kaempferol also produced an inhibitory effect on the phosphorylation of ERK1/2. Conclusions These findings provide new insights into the molecular mechanisms involved in the anti-metastatic effect of kaempferol, and are valuable in the prevention of oral cancer metastasis. PMID:24278338

  17. Hydrogen sulfide inhibits opioid withdrawal-induced pain sensitization in rats by down-regulation of spinal calcitonin gene-related peptide expression in the spine.

    Science.gov (United States)

    Yang, Hai-Yu; Wu, Zhi-Yuan; Bian, Jin-Song

    2014-09-01

    Hyperalgesia often occurs in opioid-induced withdrawal syndrome. In the present study, we found that three hourly injections of DAMGO (a μ-opioid receptor agonist) followed by naloxone administration at the fourth hour significantly decreased rat paw nociceptive threshold, indicating the induction of withdrawal hyperalgesia. Application of NaHS (a hydrogen sulfide donor) together with each injection of DAMGO attenuated naloxone-precipitated withdrawal hyperalgesia. RT-PCR and Western blot analysis showed that NaHS significantly reversed the gene and protein expression of up-regulated spinal calcitonin gene-related peptide (CGRP) in naloxone-treated animals. NaHS also inhibited naloxone-induced cAMP rebound and cAMP response element-binding protein (CREB) phosphorylation in rat spinal cord. In SH-SY5Y neuronal cells, NaHS inhibited forskolin-stimulated cAMP production and adenylate cyclase (AC) activity. Moreover, NaHS pre-treatment suppressed naloxone-stimulated activation of protein kinase C (PKC) α, Raf-1, and extracellular signal-regulated kinase (ERK) 1/2 in rat spinal cord. Our data suggest that H2S prevents the development of opioid withdrawal-induced hyperalgesia via suppression of synthesis of CGRP in spine through inhibition of AC/cAMP and PKC/Raf-1/ERK pathways.

  18. Zuo-Gui and You-Gui pills, two traditional Chinese herbal formulas, downregulated the expression of NogoA, NgR, and RhoA in rats with experimental autoimmune encephalomyelitis.

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    Kou, Shuang; Zheng, Qi; Wang, Yizhou; Zhao, Hui; Zhang, Qiuxia; Li, Ming; Qi, Fang; Fang, Ling; Liu, Lei; Ouyang, Junyao; Zhao, Haiyu; Wang, Lei

    2014-12-02

    Zuo-Gui pills (ZGPs) and You-Gui pills (YGPs) are 2 traditional Chinese herbal formulas used for treating multiple sclerosis (MS) in the clinical setting and have been shown to have neuroprotective effects in experimental autoimmune encephalomyelitis (EAE), an animal model of MS. The aim of this study was to explore the mechanisms underlying the neuroprotective functions of ZGPs and YGPs. Female Lewis rats were randomly divided into normal control, EAE model, 2g/kg ZGP-treated EAE, 3g/kg YGP-treated EAE, and prednisone acetate-treated groups. EAE model was induced by subcutaneous injection of MBP68-86 antigen. The neurological function scores were estimated. Histological structures of the brains and spinal cords were observed, and myelinated and axons imaged. NogoA, Nogo receptor (NgR), and RhoA transcript and protein levels were measured by real-time quantitative RT-PCR and western blotting on postimmunization (PI) days 14 (acute stage) and 28 (remission stage). ZGPs and YGPs significantly reduced neurological functions scores and abrogated inflammatory infiltrates, demyelination, and axonal damage. Furthermore, treatment with ZGPs and YGPs inhibited NogoA, NgR, and RhoA mRNA and protein expression in rats at both the acute and remission stages. ZGPs exhibited stronger effects on NogoA and RhoA expressions, as well as neurological function, during the acute stage of EAE, while YGPs caused greater reductions in NogoA expression during the remission stage. Our findings suggested that ZGPs and YGPs exerted neuroprotective effects by downregulation of NogoA, NgR, and RhoA pathways, with differences in response times and targets observed between ZGPs and YGPs. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  19. Periplocin from Cortex periplocae inhibits cell growth and down-regulates survivin and c-myc expression in colon cancer in vitro and in vivo via beta-catenin/TCF signaling.

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    Zhao, Lianmei; Shan, Baoen; Du, Yanyan; Wang, Mingxia; Liu, Lihua; Ren, Feng-Zhi

    2010-08-01

    Cancer of the colon and rectum is the third most commonly diagnosed cancer and accounts for approximately 10% of all cancer-related deaths. Although surgical resection or radiotherapy are potentially curative for localized disease, advanced colon cancer is currently associated with poor prognosis. Therefore, the development of a new and effective chemotherapeutic agent is required to target critical pathways to induce responsiveness of colon cancer cells to death signals. Dysregulation of the beta-catenin/TCF pathway plays a central role in early activities of colorectal carcinogenesis. In this study, human colon cancer SW480 cells were used to investigate the effect of CPP (periplocin from Cortex periplocae) on the modulation of the beta-catenin/TCF signaling pathway. Our research results showed that CPP caused a dose- and time-dependent inhibition of cell growth as assessed by MTT assay and an induction in apoptosis as measured by flow cytometry and transmission electron microscopy. Furthermore, the CPP- treated cells were characterized by a decreased expression of beta-catenin protein in the total cell lysates and cytosolic and nuclear extracts. This expression alleviates the binding activity of T-cell factor (Tcf) complexes to its specific DNA-binding sites. Thus, the protein expression of the downstream elements survivin and c-myc was down-regulated. To determine the precise inhibitory mechanisms involved, further in-depth in vivo studies of CPP are warranted. In conclusion, our data suggest that CPP wields a multi-prong strategy to target the beta-catenin/Tcf signaling pathway, leading to the induction of apoptosis and inhibition of growth of colon cancer cells in vitro and in vivo. Therefore, CPP may become a potential agent against colon cancer.

  20. Ellagic acid derivatives from Terminalia chebula Retz. downregulate the expression of quorum sensing genes to attenuate Pseudomonas aeruginosa PAO1 virulence.

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    Sajal Sarabhai

    Full Text Available BACKGROUND: Burgeoning antibiotic resistance in Pseudomonas aeruginosa has necessitated the development of anti pathogenic agents that can quench acylhomoserine lactone (AHL mediated QS with least risk of resistance. This study explores the anti quorum sensing potential of T. chebula Retz. and identification of probable compounds(s showing anti QS activity and the mechanism of attenuation of P. aeruginosa PAO1 virulence factors. METHODS AND RESULTS: Methanol extract of T. chebula Retz. fruit showed anti QS activity using Agrobacterium tumefaciens A136. Bioactive fraction (F7, obtained by fractionation of methanol extract using Sephadex LH20, showed significant reduction (p<0.001 in QS regulated production of extracellular virulence factors in P. aeruginosa PAO1. Biofilm formation and alginate were significantly (p<0.05 reduced with enhanced (20% susceptibility to tobramycin. Real Time PCR of F7 treated P. aeruginosa showed down regulation of autoinducer synthase (lasI and rhlI and their cognate receptor (lasR and rhlR genes by 89, 90, 90 and 93%, respectively. Electrospray Ionization Mass Spectrometry also showed 90 and 64% reduction in the production of 3-oxo-C(12HSL and C(4HSL after treatment. Decrease in AHLs as one of the mechanisms of quorum quenching by F7 was supported by the reversal of inhibited swarming motility in F7-treated P. aeruginosa PAO1 on addition of C(4HSL. F7 also showed antagonistic activity against 3-oxo-C(12HSL-dependent QS in E. coli bioreporter. C. elegans fed on F7-treated P. aeruginosa showed enhanced survival with LT50 increasing from 24 to 72 h. LC-ESI-MS of F7 revealed the presence of ellagic acid derivatives responsible for anti QS activity in T. chebula extract. CONCLUSIONS: This is the first report on anti QS activity of T. chebula fruit linked to EADs which down regulate the expression of lasIR and rhlIR genes with concomitant decrease in AHLs in P. aeruginosa PAO1 causing attenuation of its virulence factors

  1. High Brain Ammonia Tolerance and Down-Regulation of Na+:K+:2Cl- Cotransporter 1b mRNA and Protein Expression in the Brain of the Swamp Eel, Monopterus albus, Exposed to Environmental Ammonia or Terrestrial Conditions

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    Ip, Yuen K.; Hou, Zhisheng; Chen, Xiu L.; Ong, Jasmine L. Y.; Chng, You R.; Ching, Biyun; Hiong, Kum C.; Chew, Shit F.

    2013-01-01

    Na+:K+:2Cl- cotransporter 1 (NKCC1) has been implicated in mediating ischemia-, trauma- or ammonia-induced astrocyte swelling/brain edema in mammals. This study aimed to determine the effects of ammonia or terrestrial exposure on ammonia concentrations in the plasma and brain, and the mRNA expression and protein abundance of nkcc/Nkcc in the brain, of the swamp eel Monopterus albus . Ammonia exposure led to a greater increase in the ammonia concentration in the brain of M. albus than terrestrial exposure. The brain ammonia concentration of M. albus reached 4.5 µmol g-1 and 2.7 µmol g-1 after 6 days of exposure to 50 mmol l-1 NH4Cl and terrestrial conditions, respectively. The full cDNA coding sequence of nkcc1b from M. albus brain comprised 3276 bp and coded for 1092 amino acids with an estimated molecular mass of 119.6 kDa. A molecular characterization indicated that it could be activated through phosphorylation and/or glycosylation by osmotic and/or oxidative stresses. Ammonia exposure for 1 day or 6 days led to significant decreases in the nkcc1b mRNA expression and Nkcc1b protein abundance in the brain of M. albus. In comparison, a significant decrease in nkcc1b mRNA expression was observed in the brain of M. albus only after 6 days of terrestrial exposure, but both 1 day and 6 days of terrestrial exposure resulted in significant decreases in the protein abundance of Nkcc1b. These results are novel because it has been established in mammals that ammonia up-regulates NKCC1 expression in astrocytes and NKCC1 plays an important role in ammonia-induced astrocyte swelling and brain edema. By contrast, our results indicate for the first time that M. albus is able to down-regulate the mRNA and protein expression of nkcc1b/Nkcc1b in the brain when confronted with ammonia toxicity, which could be one of the contributing factors to its extraordinarily high brain ammonia tolerance. PMID:24069137

  2. Fast Green FCF Alleviates Pain Hypersensitivity and Down-Regulates the Levels of Spinal P2X4 Expression and Pro-inflammatory Cytokines in a Rodent Inflammatory Pain Model

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    Fang Xu

    2018-05-01

    Full Text Available Fast Green FCF (FGF, a biocompatible dye, recently drew attention as a potential drug to treat amyloid-deposit diseases due to its effects against amyloid fibrillogenesis in vitro and a high degree of safety. However, its role in inflammatory pain is unknown. Our study aimed to investigate the effect of FGF in the inflammatory pain model induced by complete Freund’s adjuvant (CFA and to identify the associated mechanisms. We found that systemic administration of FGF reversed mechanical and thermal pain hypersensitivity evoked by CFA in a dose-dependent manner. FGF treatment decreased purinergic spinal P2X4 expression in the spinal cord of CFA-inflamed mice. FGF also down-regulated spinal and peripheral pro-inflammatory cytokines [tumor necrosis factor-α (TNF-α, interleukin-1β (IL-1β, and interleukin-6 (IL-6], but did not alter the spinal level of nerve growth factor (NGF or brain-derived neurotrophic factor (BDNF. In conclusion, our results suggest the potential of FGF for controlling the progress of inflammatory pain.

  3. The potential protective effect of Physalis peruviana L. against carbon tetrachloride-induced hepatotoxicity in rats is mediated by suppression of oxidative stress and downregulation of MMP-9 expression.

    Science.gov (United States)

    Al-Olayan, Ebtisam M; El-Khadragy, Manal F; Aref, Ahmed M; Othman, Mohamed S; Kassab, Rami B; Abdel Moneim, Ahmed E

    2014-01-01

    The active constituent profile in Cape gooseberry (Physalis peruviana L.) juice was determined by GC-MS. Quercetin and kaempferol were active components in the juice. In this study we have evaluated its potential protective effect on hepatic injury and fibrosis induced by carbon tetrachloride (CCl4). Twenty-eight rats divided into 4 groups: Group I served as control group, and Group II received weekly i.p. injection of 2 mL CCl4/kg bwt for 12 weeks. Group III were supplemented with Physalis juice via the drinking water. The animals of Group IV received Physalis juice as Group III and also were intraperitoneally injected weekly with 2 mL CCl4/kg bwt for 12 weeks. Hepatoprotective effect was evaluated by improvement in liver enzymes serum levels, reduction in collagen areas, downregulation in expression of the fibrotic marker MMP-9, reduction in the peroxidative marker malonaldehyde and the inflammatory marker nitric oxide, and restoration of the activity of antioxidant enzymatic and nonenzymatic systems, namely, glutathione content, superoxide dismutase, catalase, glutathione-S-transferase, glutathione peroxidase, and glutathione reductase activities. The results show that the potential hepatoprotective effects of Physalis peruviana may be due to physalis acts by promotion of processes that restore hepatolobular architecture and through the inhibition of oxidative stress pathway.

  4. Contribution of Schwann Cells to Remyelination in a Naturally Occurring Canine Model of CNS Neuroinflammation.

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    Kristel Kegler

    Full Text Available Gliogenesis under pathophysiological conditions is of particular clinical relevance since it may provide evidence for regeneration promoting cells recruitable for therapeutic purposes. There is evidence that neurotrophin receptor p75 (p75NTR-expressing cells emerge in the lesioned CNS. However, the phenotype and identity of these cells, and signals triggering their in situ generation under normal conditions and certain pathological situations has remained enigmatic. In the present study, we used a spontaneous, idiopathic and inflammatory CNS condition in dogs with prominent lympho-histiocytic infiltration as a model to study the phenotype of Schwann cells and their relation to Schwann cell remyelination within the CNS. Furthermore, the phenotype of p75NTR-expressing cells within the injured CNS was compared to their counter-part in control sciatic nerve and after peripheral nerve injury. In addition, organotypic slice cultures were used to further elucidate the origin of p75NTR-positive cells. In cerebral and cerebellar white and grey matter lesions as well as in the brain stem, p75NTR-positive cells co-expressed the transcription factor Sox2, but not GAP-43, GFAP, Egr2/Krox20, periaxin and PDGFR-α. Interestingly, and contrary to the findings in control sciatic nerves, p75NTR-expressing cells only co-localized with Sox2 in degenerative neuropathy, thus suggesting that such cells might represent dedifferentiated Schwann cells both in the injured CNS and PNS. Moreover, effective Schwann cell remyelination represented by periaxin- and P0-positive mature myelinating Schwann cells, was strikingly associated with the presence of p75NTR/Sox2-expressing Schwann cells. Intriguingly, the emergence of dedifferentiated Schwann cells was not affected by astrocytes, and a macrophage-dominated inflammatory response provided an adequate environment for Schwann cells plasticity within the injured CNS. Furthermore, axonal damage was reduced in brain stem areas

  5. Separate enrichment analysis of pathways for up- and downregulated genes.

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    Hong, Guini; Zhang, Wenjing; Li, Hongdong; Shen, Xiaopei; Guo, Zheng

    2014-03-06

    Two strategies are often adopted for enrichment analysis of pathways: the analysis of all differentially expressed (DE) genes together or the analysis of up- and downregulated genes separately. However, few studies have examined the rationales of these enrichment analysis strategies. Using both microarray and RNA-seq data, we show that gene pairs with functional links in pathways tended to have positively correlated expression levels, which could result in an imbalance between the up- and downregulated genes in particular pathways. We then show that the imbalance could greatly reduce the statistical power for finding disease-associated pathways through the analysis of all-DE genes. Further, using gene expression profiles from five types of tumours, we illustrate that the separate analysis of up- and downregulated genes could identify more pathways that are really pertinent to phenotypic difference. In conclusion, analysing up- and downregulated genes separately is more powerful than analysing all of the DE genes together.

  6. Natural indoles, indole-3-carbinol (I3C and 3,3'-diindolylmethane (DIM, attenuate staphylococcal enterotoxin B-mediated liver injury by downregulating miR-31 expression and promoting caspase-2-mediated apoptosis.

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    Philip B Busbee

    Full Text Available Staphylococcal enterotoxin B (SEB is a potent superantigen capable of inducing inflammation characterized by robust immune cell activation and proinflammatory cytokine release. Exposure to SEB can result in food poisoning as well as fatal conditions such as toxic shock syndrome. In the current study, we investigated the effect of natural indoles including indole-3-carbinol (I3C and 3,3'-diindolylmethane (DIM on SEB-mediated liver injury. Injection of SEB into D-galactosamine-sensitized female C57BL/6 mice resulted in liver injury as indicated by an increase in enzyme aspartate transaminase (AST levels, induction of inflammatory cytokines, and massive infiltration of immune cells into the liver. Administration of I3C and DIM (40 mg/kg, by intraperitonal injection, attenuated SEB-induced acute liver injury, as evidenced by decrease in AST levels, inflammatory cytokines and cellular infiltration in the liver. I3C and DIM triggered apoptosis in SEB-activated T cells primarily through activation of the intrinsic mitochondrial pathway. In addition, inhibitor studies involving caspases revealed that I3C and DIM-mediated apoptosis in these activated cells was dependent on caspase-2 but independent of caspase-8, 9 and 3. In addition, I3C and DIM caused a decrease in Bcl-2 expression. Both compounds also down-regulated miR-31, which directly targets caspase-2 and influences apoptosis in SEB-activated cells. Our data demonstrate for the first time that indoles can effectively suppress acute hepatic inflammation caused by SEB and that this may be mediated by decreased expression of miR-31 and consequent caspase-2-dependent apoptosis in T cells.

  7. Cosmetic applications of glucitol-core containing gallotannins from a proprietary phenolic-enriched red maple (Acer rubrum) leaves extract: inhibition of melanogenesis via down-regulation of tyrosinase and melanogenic gene expression in B16F10 melanoma cells.

    Science.gov (United States)

    Ma, Hang; Xu, Jialin; DaSilva, Nicholas A; Wang, Ling; Wei, Zhengxi; Guo, Liangran; Johnson, Shelby L; Lu, Wei; Xu, Jun; Gu, Qiong; Seeram, Navindra P

    2017-05-01

    The red maple (Acer rubrum) is a rich source of phenolic compounds which possess galloyl groups attached to different positions of a 1,5-anhydro-D-glucitol core. While these glucitol-core containing gallotannins (GCGs) have reported anti-oxidant and anti-glycative effects, they have not yet been evaluated for their cosmetic applications. Herein, the anti-tyrosinase and anti-melanogenic effects of a proprietary phenolic-enriched red maple leaves extract [Maplifa ™ ; contains ca. 45% ginnalin A (GA) along with other GCGs] were investigated using enzyme and cellular assays. The GCGs showed anti-tyrosinase activity with IC 50 values ranging from 101.4 to 1047.3 μM and their mechanism of tyrosinase inhibition (using GA as a representative GCG) was evaluated by chelating and computational/modeling studies. GA reduced melanin content in murine melanoma B16F10 cells by 79.1 and 56.7% (at non-toxic concentrations of 25 and 50 μM, respectively), and its mechanisms of anti-melanogenic effects were evaluated by using methods including fluorescent probe (DCF-DA), real-time PCR, and western blot experiments. These data indicated that GA was able to: (1) reduce the levels of reactive oxygen species, (2) down-regulate the expression of MITF, TYR, TRP-1, and TRP-2 gene levels in a time-dependent manner, and (3) significantly reduce protein expression of the TRP-2 gene. Therefore, the anti-melanogenic effects of red maple GCGs warrant further investigation of this proprietary natural product extract for potential cosmetic applications.

  8. Gibberellin Application at Pre-Bloom in Grapevines Down-Regulates the Expressions of VvIAA9 and VvARF7, Negative Regulators of Fruit Set Initiation, during Parthenocarpic Fruit Development

    Science.gov (United States)

    Jung, Chan Jin; Hur, Youn Young; Yu, Hee-Ju; Noh, Jung-Ho; Park, Kyo-Sun; Lee, Hee Jae

    2014-01-01

    Fruit set is initiated only after fertilization and is tightly regulated primarily by gibberellins (GAs) and auxins. The application of either of these hormones induces parthenocarpy, fruit set without fertilization, but the molecular mechanism underlying this induction is poorly understood. In the present study, we have shown that the parthenocarpic fruits induced by GA application at pre-bloom result from the interaction of GA with auxin signaling. The transcriptional levels of the putative negative regulators of fruit set initiation, including Vitis auxin/indole-3-acetic acid transcription factor 9 (VvIAA9), Vitis auxin response factor 7 (VvARF7), and VvARF8 were monitored during inflorescence development in seeded diploid ‘Tamnara’ grapevines with or without GA application. Without GA application, VvIAA9, VvARF7, and VvARF8 were expressed at a relatively high level before full bloom, but decreased thereafter following pollination. After GA application at 14 days before full bloom (DBF); however, the expression levels of VvIAA9 and VvARF7 declined at 5 DBF prior to pollination. The effects of GA application on auxin levels or auxin signaling were also analyzed by monitoring the expression patterns of auxin biosynthesis genes and auxin-responsive genes with or without GA application. Transcription levels of the auxin biosynthesis genes Vitis anthranilate synthase β subunit (VvASB1-like), Vitis YUCCA2 (VvYUC2), and VvYUC6 were not significantly changed by GA application. However, the expressions of Vitis Gretchen Hagen3.2 (VvGH3.2) and VvGH3.3, auxin-responsive genes, were up-regulated from 2 DBF to full bloom with GA application. Furthermore, the Vitis GA signaling gene, VvDELLA was up-regulated by GA application during 12 DBF to 7 DBF, prior to down-regulation of VvIAA9 and VvARF7. These results suggest that VvIAA9 and VvARF7 are negative regulators of fruit set initiation in grapevines, and GA signaling is integrated with auxin signaling via VvDELLA during

  9. Overexpression of a wheat (Triticum aestivum L.) bZIP transcription factor gene, TabZIP6, decreased the freezing tolerance of transgenic Arabidopsis seedlings by down-regulating the expression of CBFs.

    Science.gov (United States)

    Cai, Wangting; Yang, Yaling; Wang, Weiwei; Guo, Guangyan; Liu, Wei; Bi, Caili

    2018-03-01

    The basic leucine zipper (bZIP) proteins play important roles against abiotic stress in plants, including cold stress. However, most bZIPs involved in plant freezing tolerance are positive regulators. Only a few bZIPs function negatively in cold stress response. In this study, TabZIP6, a Group C bZIP transcription factor gene from common wheat (Triticum aestivum L.), was cloned and characterized. The transcript of TabZIP6 was strongly induced by cold treatment (4 °C). TabZIP6 is a nuclear-localized protein with transcriptional activation activity. Arabidopsis plants overexpressing TabZIP6 showed decreased tolerance to freezing stress. Microarray as well as quantitative real-time PCR (qRT-PCR) analysis showed that CBFs and some key COR genes, including COR47 and COR15B, were down-regulated by cold treatment in TabZIP6-overexpressing Arabidopsis lines. TabZIP6 was capable of binding to the G-box motif and the CBF1 and CBF3 promoters in yeast cells. A yeast two-hybrid assay revealed that TabZIP6, as well as the other two Group S bZIP proteins involved in cold stress tolerance in wheat, Wlip19 and TaOBF1, can form homodimers by themselves and heterodimers with each other. These results suggest that TabZIP6 may function negatively in the cold stress response by binding to the promoters of CBFs, and thereby decreasing the expression of downstream COR genes in TabZIP6-overexpressing Arabidopsis seedlings. Copyright © 2018. Published by Elsevier Masson SAS.

  10. Combination of intratumoral injections of vaccinia virus MVA expressing GM-CSF and immunization with DNA vaccine prolongs the survival of mice bearing HPV16 induced tumors with downregulated expression of MHC class I molecules

    Czech Academy of Sciences Publication Activity Database

    Němečková, Š.; Šmahel, M.; Hainz, P.; Macková, J.; Zurková, K.; Gabriel, P.; Indrová, Marie; Kutinová, L.

    2007-01-01

    Roč. 54, č. 4 (2007), s. 326-333 ISSN 0028-2685 R&D Projects: GA MZd NR8004 Institutional research plan: CEZ:AV0Z50520514 Keywords : vaccinia virus MVA expressing GM- CSF * DNA vaccine * HPV16 induced tumors Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 1.208, year: 2007

  11. Fraxinus rhynchophylla ethanol extract attenuates carbon tetrachloride-induced liver fibrosis in rats via down-regulating the expressions of uPA, MMP-2, MMP-9 and TIMP-1.

    Science.gov (United States)

    Peng, Wen-Huang; Tien, Yun-Chen; Huang, Chih-Yang; Huang, Tai-Hung; Liao, Jung-Chun; Kuo, Chao-Lin; Lin, Ying-Chih

    2010-02-17

    To investigate the effect of Fraxinus rhynchophylla ethanol extract (FR(EtOH)) on liver fibrosis induced by carbon tetrachloride (CCl(4)) in rats. Rat hepatic fibrosis was induced by oral administration of CCl(4). Sixty SD rats were divided randomly into 6 groups: control, CCl(4) group, silymarin group and three FR(EtOH)-treated groups. Except for the rats in control group, all rats were administered orally with CCl(4) (20%, 0.2 mL/100g body weight) twice a week for 8 weeks. Rats in FR(EtOH) groups were treated daily with FR(EtOH) (0.1, 0.5 and 1.0 g/kg, p.o.) throughout the whole experimental period. Liver function parameters (such as activities of serum GOT and GPT levels), activities of liver anti-oxidant enzymes (such as catalase, SOD, GPx) and expressions of uPA, tPA, MMP-2, MMP-9 and TIMP-1, -2, -3, -4 in the liver fibrosis pathway were detected. The results showed that FR(EtOH) (0.1, 0.5 and 1.0 g/kg BW) significantly reduced the elevated activities of sGOT and sGPT caused by CCl(4). FR(EtOH) (0.1 and 0.5 g/kg BW) and significantly increased the activities of GSH-Px. The histopathological study showed that FR(EtOH) (0.1 and 0.5 g/kg BW) reduced the incidence of liver lesions, including hepatic cells cloudy swelling, lymphocytes infiltration, cytoplasm vacuolization hepatic necrosis and fibrous connective tissue proliferated induced by CCl(4) in rats. In our study it was showed that CCl(4)-treated group significantly increased the protein levels of uPA, MMP-2, MMP-9 and TIMP-1. FR(EtOH) (0.1 and 0.5 g/kg BW) could inhibit the protein levels of uPA, MMP-2, MMP-9 and TIMP-1. Finally, the amount of esculetin in the FR(EtOH) was 33.54 mg/g extract. Oral administration of FR(EtOH) significantly reduces CCl(4)-induced hepatic fibrosis in rats, probably by exerting a protective effect against hepatocellular fibrosis by its free radical scavenging ability. FR(EtOH) down-regulated the expressions of uPA, MMP-2 and MMP-9 in CCl(4)-induced liver fibrosis in rats

  12. Chlamydia trachomatis Infection Is Associated with E-Cadherin Promoter Methylation, Downregulation of E-Cadherin Expression, and Increased Expression of Fibronectin and α-SMA—Implications for Epithelial-Mesenchymal Transition

    Directory of Open Access Journals (Sweden)

    Jovana Rajić

    2017-06-01

    Full Text Available Chlamydia trachomatis (Ct can induce scarring disease of the ocular mucosa, known as trachoma, the most common infectious cause of blindness worldwide. We hypothesized that epithelial-mesenchymal transition (EMT contributes to the fibrotic process in trachomatous scarring. Infection of human conjunctival epithelial cells (HCjE with Ct activated signaling pathways involved in EMT induction, which was correlated with decreased expression of E-cadherin, guardian of the epithelial phenotype. In addition, Ct infection was associated with increased expression of two mesenchymal cell markers: fibronectin and α-SMA. The DNA methylation statuses of selected regions of E-cadherin, fibronectin, and α-SMA genes revealed that Ct infection was accompanied with changes in DNA methylation of the E-cadherin promoter, while the expression of the two mesenchymal markers was not related with this epigenetic event. Our data suggest that Ct infection of conjunctival epithelial cells induces EMT-like changes that go along with modification of the methylation profile of the E-cadherin promoter and could, as one of the earliest events, contribute to processes triggering conjunctival scarring.

  13. The protein kinase SIK downregulates the polarity protein Par3

    DEFF Research Database (Denmark)

    Vanlandewijck, Michael; Dadras, Mahsa Shahidi; Lomnytska, Marta

    2018-01-01

    on Par3. Functionally, this mechanism impacts on tight junction downregulation. Furthermore, SIK contributes to the loss of epithelial polarity and examination of advanced and invasive human cancers of diverse origin displayed high levels of SIK expression and a corresponding low expression of Par3...

  14. Curcumin significantly enhances dual PI3K/Akt and mTOR inhibitor NVP-BEZ235-induced apoptosis in human renal carcinoma Caki cells through down-regulation of p53-dependent Bcl-2 expression and inhibition of Mcl-1 protein stability.

    Directory of Open Access Journals (Sweden)

    Bo Ram Seo

    Full Text Available The PI3K/Akt and mTOR signaling pathways are important for cell survival and growth, and they are highly activated in cancer cells compared with normal cells. Therefore, these signaling pathways are targets for inducing cancer cell death. The dual PI3K/Akt and mTOR inhibitor NVP-BEZ235 completely inhibited both signaling pathways. However, NVP-BEZ235 had no effect on cell death in human renal carcinoma Caki cells. We tested whether combined treatment with natural compounds and NVP-BEZ235 could induce cell death. Among several chemopreventive agents, curcumin, a natural biologically active compound that is extracted from the rhizomes of Curcuma species, markedly induced apoptosis in NVP-BEZ235-treated cells. Co-treatment with curcumin and NVP-BEZ235 led to the down-regulation of Mcl-1 protein expression but not mRNA expression. Ectopic expression of Mcl-1 completely inhibited curcumin plus NVP-NEZ235-induced apoptosis. Furthermore, the down-regulation of Bcl-2 was involved in curcumin plus NVP-BEZ235-induced apoptosis. Curcumin or NVP-BEZ235 alone did not change Bcl-2 mRNA or protein expression, but co-treatment reduced Bcl-2 mRNA and protein expression. Combined treatment with NVP-BEZ235 and curcumin reduced Bcl-2 expression in wild-type p53 HCT116 human colon carcinoma cells but not p53-null HCT116 cells. Moreover, Bcl-2 expression was completely reversed by treatment with pifithrin-α, a p53-specific inhibitor. Ectopic expression of Bcl-2 also inhibited apoptosis in NVP-BE235 plus curcumin-treated cells. In contrast, NVP-BEZ235 combined with curcumin did not have a synergistic effect on normal human skin fibroblasts and normal human mesangial cells. Taken together, combined treatment with NVP-BEZ235 and curcumin induces apoptosis through p53-dependent Bcl-2 mRNA down-regulation at the transcriptional level and Mcl-1 protein down-regulation at the post-transcriptional level.

  15. Beneficial effect of all-trans retinoic acid (ATRA) on glomerulosclerosis rats via the down-regulation of the expression of alpha-smooth muscle actin: a comparative study between ATRA and benazepril.

    Science.gov (United States)

    Hu, Peng; Qin, Yuan Han; Pei, Juan; Lei, Feng Ying; Hu, Bo; Lu, Ling

    2010-08-01

    Although ATRA is a potent renoprotective agent, relatively little is known regarding the mechanisms of its action. The present study was designed to further elucidate the mechanisms of ATRA's action to GS rats and compare that with the beneficial effect of benazepril. Male SD rats weighting 160 to 200g were used in this study. GS was induced by unilateral nephrectomy and intravenous injection of adriamycin (6mg/kg). They were divided randomly 20 ones per group into GS group, GS treated with ATRA (20mg/kg/day) group, and GS treated with benazepril (10mg/kg/day) group. The other 20 ones were taken as sham-operation group, injected normal saline into caudal vein. 12weeks later, all rats were subjected to sacrifice. As expected, the GS group exhibited significant lower serum TP and Alb, and higher BUN, Cr and proteinuria than those of the sham group. Administration of ATRA or benazepril did ameliorate these above disorders of biochemical parameters in GS rats. Extensive renal damage was observed in the GS group, such as mononuclear infiltration, mesangial proliferation, focal segment glomerular sclerosis, and tubulointerstitial fibrosis. The pathological changes in both ATRA and benazepril group were alleviated remarkably. Semiquantitative GSI was used to evaluate the degree of GS in all groups. GSI was significantly higher in the GS group than in sham group. GSI decreased from 21.9+/-6.7 in the GS group to 6.9+/-2.8 in the ATRA group and 7.0+/-2.7 in benazepril group respectively. However, no significant difference in GSI between rats treated with ATRA and rats treated with benazepril was found. RT-PCR analysis revealed the renal expression of alpha-SMA mRNA was induced substantially in GS group as compared to sham group, which could be offset completely by ATRA or benazepril administration. However, expression level of alpha-SMA mRNA in GS rats treated with ATRA was identical to that in GS rats treated with benazepril. We also examined immunohistochemical staining

  16. Polymerized-Type I Collagen Induces Upregulation of Foxp3-Expressing CD4 Regulatory T Cells and Downregulation of IL-17-Producing CD4+ T Cells (Th17 Cells in Collagen-Induced Arthritis

    Directory of Open Access Journals (Sweden)

    Janette Furuzawa-Carballeda

    2012-01-01

    Full Text Available Previous studies showed that polymerized-type I collagen (polymerized collagen exhibits potent immunoregulatory properties. This work evaluated the effect of intramuscular administration of polymerized collagen in early and established collagen-induced arthritis (CIA in mice and analyzed changes in Th subsets following therapy. Incidence of CIA was of 100% in mice challenged with type II collagen. Clinimorphometric analysis showed a downregulation of inflammation after administration of all treatments (P<0.05. Histological analysis showed that the CIA-mice group had extensive bone erosion, pannus and severe focal inflammatory infiltrates. In contrast, there was a remarkable reduction in the severity of arthritis in mice under polymerized collagen, methotrexate or methotrexate/polymerized collagen treatment. Polymerized Collagen but not methotrexate induced tissue joint regeneration. Polymerized Collagen and methotrexate/polymerized collagen but not methotrexate alone induces downregulation of CD4+/IL17A+ T cells and upregulation of Tregs and CD4+/IFN-γ+ T cells. Thus, Polymerized Collagen could be an effective therapeutic agent in early and established rheumatoid arthritis by exerting downregulation of autoimmune inflammation.

  17. Mechanisms Down-Regulating Sprouty1, a Growth Inhibitor in Prostate Cancer

    National Research Council Canada - National Science Library

    Kwabi-Addo, Bernard

    2006-01-01

    .... I have demonstrated that Sprouty1 is down-regulated in human prostate cancer (PCa). The purpose of the present study is to characterize the molecular mechanisms regulating Sprouty1 expression in the human PCa. Results...

  18. Human T cell leukemia virus type I prevents cell surface expression of the T cell receptor through down-regulation of the CD3-gamma, -delta, -epsilon, and -zeta genes

    NARCIS (Netherlands)

    de Waal Malefyt, R.; Yssel, H.; Spits, H.; de Vries, J. E.; Sancho, J.; Terhorst, C.; Alarcon, B.

    1990-01-01

    Infection and transformation by human T cell leukemia virus type I (HTLV-I) up-regulates expression of several inducible genes including those coding for cytokines involved in the proliferation of normal and leukemic T cells. We demonstrate that HTLV-I can also shut off expression of the CD3-gamma,

  19. Downregulation of rRNA transcription triggers cell differentiation.

    Directory of Open Access Journals (Sweden)

    Yuki Hayashi

    Full Text Available Responding to various stimuli is indispensable for the maintenance of homeostasis. The downregulation of ribosomal RNA (rRNA transcription is one of the mechanisms involved in the response to stimuli by various cellular processes, such as cell cycle arrest and apoptosis. Cell differentiation is caused by intra- and extracellular stimuli and is associated with the downregulation of rRNA transcription as well as reduced cell growth. The downregulation of rRNA transcription during differentiation is considered to contribute to reduced cell growth. However, the downregulation of rRNA transcription can induce various cellular processes; therefore, it may positively regulate cell differentiation. To test this possibility, we specifically downregulated rRNA transcription using actinomycin D or a siRNA for Pol I-specific transcription factor IA (TIF-IA in HL-60 and THP-1 cells, both of which have differentiation potential. The inhibition of rRNA transcription induced cell differentiation in both cell lines, which was demonstrated by the expression of the common differentiation marker CD11b. Furthermore, TIF-IA knockdown in an ex vivo culture of mouse hematopoietic stem cells increased the percentage of myeloid cells and reduced the percentage of immature cells. We also evaluated whether differentiation was induced via the inhibition of cell cycle progression because rRNA transcription is tightly coupled to cell growth. We found that cell cycle arrest without affecting rRNA transcription did not induce differentiation. To the best of our knowledge, our results demonstrate the first time that the downregulation of rRNA levels could be a trigger for the induction of differentiation in mammalian cells. Furthermore, this phenomenon was not simply a reflection of cell cycle arrest. Our results provide a novel insight into the relationship between rRNA transcription and cell differentiation.

  20. Synergistic Effect of Subtoxic-dose Cisplatin and TRAIL to Mediate Apoptosis by Down-regulating Decoy Receptor 2 and Up-regulating Caspase-8, Caspase-9 and Bax Expression on NCI-H460 and A549 Cells

    Directory of Open Access Journals (Sweden)

    Xiaoyan Zhang

    2013-05-01

    Full Text Available Objective(s: Although tumor necrosis factor-related apoptosis-inducing ligand (TRAIL can selectively induce apoptosis in tumor cells, more than half of tumors including non-small cell lung cancer (NSCLC exhibit TRAIL-resistance. The purpose of this study was to determine whether subtoxic-dose cisplatin and TRAIL could synergistically enhance apoptosis on NSCLC cells and investigate its underlying mechanisms. Materials and Methods:NCI-H460 and A549 cells were treated with TRAIL alone, cisplatin alone or combination treatment in this study. The cytotoxicity was evaluated according to Sulforhodamine B assay, and apoptosis was examined using Hoechst 33342 staining and flow cytometry. The mRNA and protein levels of TRAIL receptors and apoptotic proteins including caspase-8, caspase-9, Bcl-2 and Bax were determined by RT-PCR and Western blotting, respectively. Results:Our results showed that NCI-H460 cells were sensitive to TRAIL, whereas A549 cells were resistant. However, subtoxic-dose cisplatin could enhance the both cells to TRAIL-mediated cell proliferation inhibition and apoptosis. The underlying mechanisms might be associated with the down-regulation of DcR2 and up-regulation of Caspase-8, Caspase-9 and Bax. Conclusion:Subtoxic-dose cisplatin could enhance both TRAIL- sensitive and TRAIL- resistant NSCLC cells to TRAIL-mediated apoptosis. These findings motivated further studies to evaluate such a combinatory therapeutic strategy against NSCLC in the animal models.

  1. Transient up- and down-regulation of expression of myosin light chain 2 and myostatin mRNA mark the changes from stratified hyperplasia to muscle fiber hypertrophy in larvae of gilthead sea bream (Sparus aurata L.).

    Science.gov (United States)

    Georgiou, Stella; Alami-Durante, Hélène; Power, Deborah M; Sarropoulou, Elena; Mamuris, Zissis; Moutou, Katerina A

    2016-02-01

    Hyperplasia and hypertrophy are the two mechanisms by which muscle develops and grows. We study these two mechanisms, during the early development of white muscle in Sparus aurata, by means of histology and the expression of structural and regulatory genes. A clear stage of stratified hyperplasia was identified early in the development of gilthead sea bream but ceased by 35 dph when hypertrophy took over. Mosaic recruitment of new white fibers began as soon as 60 dph. The genes mlc2a and mlc2b were expressed at various levels during the main phases of hyperplasia and hypertrophy. The genes myog and mlc2a were significantly up-regulated during the intensive stratified formation of new fibers and their expression was significantly correlated. Expression of mstn1 and igf1 increased at 35 dph, appeared to regulate the hyperplasia-to-hypertrophy transition, and may have stimulated the expression of mlc2a, mlc2b and col1a1 at the onset of mosaic hyperplasia. The up-regulation of mstn1 at transitional phases in muscle development indicates a dual regulatory role of myostatin in fish larval muscle growth.

  2. Induction of cell scattering by expression of beta1 integrins in beta1-deficient epithelial cells requires activation of members of the rho family of GTPases and downregulation of cadherin and catenin function

    DEFF Research Database (Denmark)

    Gimond, C; van Der Flier, A; van Delft, S

    1999-01-01

    different beta1-null cell lines, epithelial GE11 and fibroblast-like GD25 cells. Expression of beta1A or the cytoplasmic splice variant beta1D, induced the disruption of intercellular adherens junctions and cell scattering in both GE11 and GD25 cells. In GE11 cells, the morphological change correlated...... for a complete morphological transition towards the spindle-shaped fibroblast-like phenotype. The expression of an interleukin-2 receptor (IL2R)-beta1A chimera and its incorporation into focal adhesions also induced the disruption of cadherin-based adhesions and the reorganization of ECM-cell contacts...

  3. The neurotoxicant PCB-95 by increasing the neuronal transcriptional repressor REST down-regulates caspase-8 and increases Ripk1, Ripk3 and MLKL expression determining necroptotic neuronal death.

    Science.gov (United States)

    Guida, Natascia; Laudati, Giusy; Serani, Angelo; Mascolo, Luigi; Molinaro, Pasquale; Montuori, Paolo; Di Renzo, Gianfranco; Canzoniero, Lorella M T; Formisano, Luigi

    2017-10-15

    Our previous study showed that the environmental neurotoxicant non-dioxin-like polychlorinated biphenyl (PCB)-95 increases RE1-silencing transcription factor (REST) expression, which is related to necrosis, but not apoptosis, of neurons. Meanwhile, necroptosis is a type of a programmed necrosis that is positively regulated by receptor interacting protein kinase 1 (RIPK1), RIPK3 and mixed lineage kinase domain-like (MLKL) and negatively regulated by caspase-8. Here we evaluated whether necroptosis contributes to PCB-95-induced neuronal death through REST up-regulation. Our results demonstrated that in cortical neurons PCB-95 increased RIPK1, RIPK3, and MLKL expression and decreased caspase-8 at the gene and protein level. Furthermore, the RIPK1 inhibitor necrostatin-1 or siRNA-mediated RIPK1, RIPK3 and MLKL expression knockdown significantly reduced PCB-95-induced neuronal death. Intriguingly, PCB-95-induced increases in RIPK1, RIPK3, MLKL expression and decreases in caspase-8 expression were reversed by knockdown of REST expression with a REST-specific siRNA (siREST). Notably, in silico analysis of the rat genome identified a REST consensus sequence in the caspase-8 gene promoter (Casp8-RE1), but not the RIPK1, RIPK3 and MLKL promoters. Interestingly, in PCB-95-treated neurons, REST binding to the Casp8-RE1 sequence increased in parallel with a reduction in its promoter activity, whereas under the same experimental conditions, transfection of siREST or mutation of the Casp8-RE1 sequence blocked PCB-95-induced caspase-8 reduction. Since RIPK1, RIPK3 and MLKL rat genes showed no putative REST binding site, we assessed whether the transcription factor cAMP Responsive Element Binding Protein (CREB), which has a consensus sequence in all three genes, affected neuronal death. In neurons treated with PCB-95, CREB protein expression decreased in parallel with a reduction in binding to the RIPK1, RIPK3 and MLKL gene promoter sequence. Furthermore, CREB overexpression was

  4. PGC-1beta is downregulated by training in human skeletal muscle: no effect of training twice every second day vs. once daily on expression of the PGC-1 family

    DEFF Research Database (Denmark)

    Mortensen, Ole Hartvig; Plomgaard, Peter; Fischer, Christian P

    2007-01-01

    We hypothesized that the peroxisome proliferator-activated receptor-gamma coactivator-1 (PGC-1) family of transcriptional coactivators (PGC-1alpha, PGC-1beta, and PRC) is differentially regulated by training once daily vs. training twice daily every second day and that this difference might...... be observed in the acute response to endurance exercise. Furthermore, we hypothesized that expression levels of the PGC-1 family differ with muscular fiber-type composition. Thus, before and after 10 wk of knee extensor endurance training, training one leg once daily and the other leg twice daily every second...... day, keeping the total amount of training for the legs equal, skeletal muscle mRNA expression levels of PGC-1alpha, PGC-1beta, and PRC were determined in young healthy men (n = 7) in response to 3 h of acute exercise. No significant difference was found between the two legs, suggesting that regulation...

  5. Down-regulation of activity and expression of three transport-related proteins in the gills of the euryhaline green crab, Carcinus maenas, in response to high salinity acclimation.

    Science.gov (United States)

    Jillette, Nathaniel; Cammack, Lauren; Lowenstein, Margaret; Henry, Raymond P

    2011-02-01

    The euryhaline green crab, Carcinus maenas, undergoes an annual cycle of salinity exposure, having to adapt to low salinity during its annual spring migration into estuaries, and then having to re-adapt to high salinity when it moves off-shore at the end of summer. Most studies have focused on low salinity acclimation, the activation of osmoregulatory mechanisms, and the induction of transport protein and transport-related enzyme activity and gene expression. In this study we followed the changes in hemolymph osmolality, carbonic anhydrase activity, and mRNA expression of three proteins through a complete cycle of low (15 ppt) and high (32 ppt) salinity acclimation. One week of low salinity acclimation resulted in hemolymph osmoregulation and a four-fold induction of branchial carbonic anhydrase activity. Relative mRNA expression increased for two CA isoforms (CAc 100-fold, and CAg 7-fold) and the α-subunit of the Na/K-ATPase (8-fold). Upon re-exposure to high salinity, hemolymph osmolality increased to 32 ppt acclimated levels by 6 h, and mRNA levels returned to high salinity, baseline levels within 1 week. However, CA activity remained unchanged in response to high salinity exposure for the first week and then gradually declined to baseline levels over 4 weeks. The relative timing of these changes suggests that while whole-organism physiological adaptations and regulation at the gene level can be very rapid, changes at the level of protein expression and turnover are much slower. It is possible that the high metabolic cost of protein synthesis and/or processing could be the underlying reason for long biological life spans of physiologically important proteins. Published by Elsevier Inc.

  6. Downregulated miR-23b-3p expression acts as a predictor of hepatocellular carcinoma progression: A study based on public data and RT-qPCR verification

    Science.gov (United States)

    Xiang, Xue-Lian; Yang, Xia; Liang, Hai-Wei; Qiu, Xiao-Hui; Yang, Li-Hua; Peng, Zhi-Gang; Chen, Gang

    2018-01-01

    Mounting evidence has shown that miR-23b-3p, which is associated with cell proliferation, invasion, and apoptosis, acts as a biomarker for diagnosis and outcomes in numerous cancers. However, the clinicopathological implication of miR-23b-3p in hepatocellular carcinoma (HCC) remains unclear. Our study evaluated the role of miR-23b-3p in HCC and investigated its potential application as a marker for preliminary diagnosis and therapy in HCC. High-throughput data from the NCBI Gene Expression Omnibus (GEO) and The Cancer Genome Atlas (TCGA) were collected and analyzed. One hundred and one tissue sections of HCC were paired with adjacent non-cancerous HCC as further supplements. miR-23b-3p expression was detected using quantitative real-time PCR. Additionally, the relationship between miR-23b-3p expression and HCC progression and Time-to-recurrence (months) was explored. Ten algorithms were applied to predict the prospective target genes of miR-23b-3p. Next, we conducted bioinformatics analysis for further study. miR-23b-3p expression was pronouncedly decreased in HCC tissues in contrast with their paired adjacent non-cancerous HCC (PAKT1, EGFR, CTNNB1, BCL2, SMAD3, PTEN and KDM6A, were located in the key nodes with high degree (>35). In conclusion, this study provides impressive illumination of the potential role of miR-23b-3p in HCC tumorigenesis and progression. Furthermore, miR-23b-3p may act as a predictor of HCC and could be a new treatment target. PMID:29484429

  7. 8-Hydroxyquinoline inhibits iNOS expression and nitric oxide production by down-regulating LPS-induced activity of NF-κB and C/EBPβ in Raw 264.7 cells

    International Nuclear Information System (INIS)

    Kim, Young-Ho; Woo, Kyung Jin; Lim, Jun Hee; Kim, Shin; Lee, Tae Jin; Jung, Eun Mi; Lee, Jin-Man; Park, Jong-Wook; Kwon, Taeg Kyu

    2005-01-01

    In activated macrophage, large amounts of nitric oxide (NO) are generated by inducible nitric oxide synthase (iNOS), resulting in acute or chronic inflammatory disorders. In Raw 264.7 cells stimulated with lipopolysaccharide (LPS) to mimic inflammation, 8-hydroxyquinoline (8HQ) inhibited the LPS-induced expression of both iNOS protein and mRNA in a parallel dose-dependent manner. 8HQ did not enhance the degradation of iNOS mRNA. To investigate the mechanism by which 8HQ inhibits iNOS gene expression, we examined the activation of MAP kinases in Raw 264.7 cells. We did not observe any significant change in the phosphorylation of MAPKs between LPS alone and LPS plus 8HQ-treated cells. Moreover, 8HQ significantly inhibited the DNA-binding activity of nuclear factor-κB (NF-κB) and CCAAT/enhancer-binding protein β (C/EBPβ), but not activator protein-1 and cAMP response element-binding protein. Taken together, these results suggest that 8HQ acts to inhibit inflammation through inhibition of NO production and iNOS expression through blockade of C/EBPβ DNA-binding activity and NF-κB activation

  8. Chronic restraint stress causes anxiety- and depression-like behaviors, downregulates glucocorticoid receptor expression, and attenuates glutamate release induced by brain-derived neurotrophic factor in the prefrontal cortex.

    Science.gov (United States)

    Chiba, Shuichi; Numakawa, Tadahiro; Ninomiya, Midori; Richards, Misty C; Wakabayashi, Chisato; Kunugi, Hiroshi

    2012-10-01

    Stress and the resulting increase in glucocorticoid levels have been implicated in the pathophysiology of depressive disorders. We investigated the effects of chronic restraint stress (CRS: 6 hours × 28 days) on anxiety- and depression-like behaviors in rats and on the possible changes in glucocorticoid receptor (GR) expression as well as brain-derived neurotrophic factor (BDNF)-dependent neural function in the prefrontal cortex (PFC). We observed significant reductions in body weight gain, food intake and sucrose preference from 1 week after the onset of CRS. In the 5th week of CRS, we conducted open-field (OFT), elevated plus-maze (EPM) and forced swim tests (FST). We observed a decrease in the number of entries into open arms during the EPM (anxiety-like behavior) and increased immobility during the FST (depression-like behavior). When the PFC was removed after CRS and subject to western blot analysis, the GR expression reduced compared with control, while the levels of BDNF and its receptors remained unchanged. Basal glutamate concentrations in PFC acute slice which were measured by high performance liquid chromatography were not influenced by CRS. However, BDNF-induced glutamate release was attenuated after CRS. These results suggest that reduced GR expression and altered BDNF function may be involved in chronic stress-induced anxiety--and depression-like behaviors. Copyright © 2012 Elsevier Inc. All rights reserved.

  9. Basal and β-Adrenergic Cardiomyocytes Contractility Dysfunction Induced by Dietary Protein Restriction is Associated with Downregulation of SERCA2a Expression and Disturbance of Endoplasmic Reticulum Ca2+ Regulation in Rats

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    Arlete R. Penitente

    2014-07-01

    Full Text Available Background: The mechanisms responsible for the cardiac dysfunction associated with dietary protein restriction (PR are poorly understood. Thus, this study was designed to evaluate the effects of PR on calcium kinetics, basal and β-adrenergic contractility in murine ventricular cardiomyocytes. Methods: After breastfeeding male Fisher rats were distributed into a control group (CG, n = 20 and a protein-restricted group (PRG, n = 20, receiving isocaloric diets for 35 days containing 15% and 6% protein, respectively. Biometric and hemodynamic variables were measured. After euthanasia left ventricles (LV were collected for histopathological evaluation, SERCA2a expression, cardiomyocytes contractility and Ca2+sparks analysis. Results: PRG animals showed reduced general growth, increased heart rate and arterial pressure. These animals presented extracellular matrix expansion and disorganization, cardiomyocytes hypotrophy, reduced amplitudes of shortening and maximum velocity of contraction and relaxation at baseline and after β-adrenergic stimulation. Reduced SERCA2a expression as well as higher frequency and lower amplitude of Ca2+sparks were observed in PRG cardiomyocytes. Conclusion: The observations reveal that protein restriction induces marked myocardial morphofunctional damage. The pathological changes of cardiomyocyte mechanics suggest the potential involvement of the β-adrenergic system, which is possibly associated with changes in SERCA2a expression and disturbances in Ca2+ intracellular kinetics.

  10. TGF-beta receptor 2 downregulation in tumour-associated stroma worsens prognosis and high-grade tumours show more tumour-associated macrophages and lower TGF-beta1 expression in colon carcinoma: a retrospective study

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    Bacman, David; Merkel, Susanne; Croner, Roland; Papadopoulos, Thomas; Brueckl, Wolfgang; Dimmler, Arno

    2007-01-01

    Histological phenotype and clinical behaviour of malignant tumours are not only dependent on alterations in the epithelial cell compartment, but are affected by their interaction with inflammatory cells and tumour-associated stroma. Studies in animal models have shown influence of tumour-associated macrophages (TAM) on histological grade of differentiation in colon carcinoma. Disruption of transforming growth factor beta (TGF-beta) signalling in tumour cells is related to more aggressive clinical behaviour. Expression data of components of this pathway in tumour-associated stroma is limited. Tissue micro arrays of 310 colon carcinomas from curatively resected patients in UICC stage II and III were established. In a first step we quantified amount of CD68 positive TAMs and expression of components of TGF-beta signalling (TGF-beta1, TGF-beta receptors type 1 and 2, Smad 3 and 4) in tumour and associated stroma. Further we analyzed correlation to histological and clinical parameters (histological grade of differentiation (low-grade (i.e. grade 1 and 2) vs. high-grade (i.e. grade 3 and 4)), lymph node metastasis, distant metastasis, 5 year cancer related survival) using Chi-square or Fisher's exact test, when appropriate, to compare frequencies, Kaplan-Meier method to calculate 5-year rates of distant metastases and cancer-related survival and log rank test to compare the rates of distant metastases and survival. To identify independent prognostic factors Cox regression analysis including lymph node status and grading was performed. High-grade tumours and those with lymph node metastases showed higher rates of TAMs and lower expression of TGF-beta1. Loss of nuclear Smad4 expression in tumor was associated with presence of lymph node metastasis, but no influence on prognosis could be demonstrated. Decrease of both TGF-beta receptors in tumour-associated stroma was associated with increased lymph node metastasis and shorter survival. Stromal TGF-beta receptor 2

  11. TGF-beta receptor 2 downregulation in tumour-associated stroma worsens prognosis and high-grade tumours show more tumour-associated macrophages and lower TGF-beta1 expression in colon carcinoma: a retrospective study

    Directory of Open Access Journals (Sweden)

    Papadopoulos Thomas

    2007-08-01

    Full Text Available Abstract Background Histological phenotype and clinical behaviour of malignant tumours are not only dependent on alterations in the epithelial cell compartment, but are affected by their interaction with inflammatory cells and tumour-associated stroma. Studies in animal models have shown influence of tumour-associated macrophages (TAM on histological grade of differentiation in colon carcinoma. Disruption of transforming growth factor beta (TGF-beta signalling in tumour cells is related to more aggressive clinical behaviour. Expression data of components of this pathway in tumour-associated stroma is limited. Methods Tissue micro arrays of 310 colon carcinomas from curatively resected patients in UICC stage II and III were established. In a first step we quantified amount of CD68 positive TAMs and expression of components of TGF-beta signalling (TGF-beta1, TGF-beta receptors type 1 and 2, Smad 3 and 4 in tumour and associated stroma. Further we analyzed correlation to histological and clinical parameters (histological grade of differentiation (low-grade (i.e. grade 1 and 2 vs. high-grade (i.e. grade 3 and 4, lymph node metastasis, distant metastasis, 5 year cancer related survival using Chi-square or Fisher's exact test, when appropriate, to compare freque