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Sample records for mediate cell proliferation

  1. Regulation of cell proliferation and apoptosis by bioactive lipid mediators.

    PubMed

    Clària, Joan

    2006-11-01

    Bioactive lipid mediators are increasingly being recognized as important endogenous regulators of cell activation, signaling, apoptosis and proliferation. Most of these lipid mediators are originated from cleavage of constituents of cellular membranes under the activity of phospholipases and sphingomyelinases. One of the major cascades of bioactive lipid mediator production involves the release of arachidonic acid from membrane phospholipids followed by the formation of eicosanoids (i.e. prostaglandins, leukotrienes and lipoxins). These biologically active metabolites of arachidonic acid are emerging as key regulators of cell proliferation and neo-angiogenesis and agents that specifically target these lipid mediators are being investigated as potential anticancer drugs. On the other hand, the lysophospholipid family, which includes members of the sphingomyelin-ceramide-sphingosine-1-phosphate and lysophosphatidic acid subfamilies, has evolved as an important group of lipid signaling molecules implicated in cellular differentiation, cell growth and apoptosis. This article reviews the most recent patents in this field of research, covering the following strategies based on the modulation of bioactive lipid mediators: (1) prostaglandin H synthase-2 inhibitors, (2) lipoxin analogs and aspirin-triggered lipid mediators, and (3) lysophosphatidic acid and other lysophospholipids. PMID:18221047

  2. Role of STAT3 Phosphorylation in Ethanol-Mediated Proliferation of Breast Cancer Cells

    PubMed Central

    Narayanan, Poornima devi; Nandabalan, Sangeetha Kadapakkam

    2016-01-01

    Purpose In this study, we investigated the molecular mechanism involved in ethanol (EtOH)-mediated proliferation of breast cancer cells. Methods EtOH concentration was optimized by studying its effect on cell proliferation in MCF-7 and MDA MB-231 cells. We used flow cytometry and immunoblot analysis to evaluate the increased proliferation caused by the optimized concentrations of EtOH. The mechanism of EtOH-mediated proliferation was determined using reactive oxygen species (ROS) release assay, reverse transcription polymerase chain reaction, and immunoblot studies. Gene silencing followed by quantitative real-time polymerase chain reaction studies and inhibitor studies indicated the involvement of signal transducer and activator of transcription 3 (STAT3) in EtOH-mediated breast cancer proliferation. Results Exposure to EtOH caused an increase in cell proliferation and an accumulation of cells in S-phase in MCF-7 (347 µM EtOH) and MDA MB-231 (173 µM EtOH) cells. Additionally, increased release of ROS and the expression of pro-inflammatory cytokines, such as interleukin 6 and tumor necrosis factor α, confirmed that the proliferation was induced by the ROS-linked inflammatory response in breast cancer. The proinflammatory response was followed by phosphorylation of STAT3. The importance of STAT3 activation in EtOH-mediated proliferation was confirmed through the silencing of STAT3, followed by an investigation on the expression of cyclins and matrix metalloproteinases. Finally, studies using specific inhibitors indicated that the EtOH-mediated effect on STAT3 activation could be regulated by phosphoinositide-3-kinase and Janus kinase 2. Conclusion The study demonstrates the involvement of STAT3 signaling in EtOH-mediated breast cancer proliferation. PMID:27382387

  3. Gli1 mediates lung cancer cell proliferation and Sonic Hedgehog-dependent mesenchymal cell activation.

    PubMed

    Bermudez, Olga; Hennen, Elisabeth; Koch, Ina; Lindner, Michael; Eickelberg, Oliver

    2013-01-01

    Non-Small-Cell-Lung-Cancer (NSCLC) represents approximately 85% of all lung cancers and remains poorly understood. While signaling pathways operative during organ development, including Sonic Hedgehog (Shh) and associated Gli transcription factors (Gli1-3), have recently been found to be reactivated in NSCLC, their functional role remains unclear. Here, we hypothesized that Shh/Gli1-3 could mediate NSCLC autonomous proliferation and epithelial/stromal signaling in the tumoral tissue. In this context, we have investigated the activity of Shh/Gli1-3 signaling in NSCLC in both, cancer and stromal cells. We report here that inhibition of Shh signaling induces a significant decrease in the proliferation of NSCLC cells. This effect is mediated by Gli1 and Gli2, but not Gli3, through regulation of cyclin D1 and cyclin D2 expression. While exogenous Shh was unable to induce signaling in either A549 lung adenocarcinoma or H520 lung squamous carcinoma cells, both cells were found to secrete Shh ligand, which induced fibroblast proliferation, survival, migration, invasion, and collagen synthesis. Furthermore, Shh secreted by NSCLC mediates the production of proangiogenic and metastatic factors in lung fibroblasts. Our results thus provide evidence that Shh plays an important role in mediating epithelial/mesenchymal crosstalk in NSCLC. While autonomous Gli activity controls NSCLC proliferation, increased Shh expression by NSCLC is associated with fibroblast activation in tumor-associated stroma. Our study highlights the relevance of studying stromal-associated cells in the context of NSCLC regarding new prognosis and therapeutic options. PMID:23667589

  4. Gli1 Mediates Lung Cancer Cell Proliferation and Sonic Hedgehog-Dependent Mesenchymal Cell Activation

    PubMed Central

    Bermudez, Olga; Hennen, Elisabeth; Koch, Ina; Lindner, Michael; Eickelberg, Oliver

    2013-01-01

    Non-Small-Cell-Lung-Cancer (NSCLC) represents approximately 85% of all lung cancers and remains poorly understood. While signaling pathways operative during organ development, including Sonic Hedgehog (Shh) and associated Gli transcription factors (Gli1-3), have recently been found to be reactivated in NSCLC, their functional role remains unclear. Here, we hypothesized that Shh/Gli1-3 could mediate NSCLC autonomous proliferation and epithelial/stromal signaling in the tumoral tissue. In this context, we have investigated the activity of Shh/Gli1-3 signaling in NSCLC in both, cancer and stromal cells. We report here that inhibition of Shh signaling induces a significant decrease in the proliferation of NSCLC cells. This effect is mediated by Gli1 and Gli2, but not Gli3, through regulation of cyclin D1 and cyclin D2 expression. While exogenous Shh was unable to induce signaling in either A549 lung adenocarcinoma or H520 lung squamous carcinoma cells, both cells were found to secrete Shh ligand, which induced fibroblast proliferation, survival, migration, invasion, and collagen synthesis. Furthermore, Shh secreted by NSCLC mediates the production of proangiogenic and metastatic factors in lung fibroblasts. Our results thus provide evidence that Shh plays an important role in mediating epithelial/mesenchymal crosstalk in NSCLC. While autonomous Gli activity controls NSCLC proliferation, increased Shh expression by NSCLC is associated with fibroblast activation in tumor-associated stroma. Our study highlights the relevance of studying stromal-associated cells in the context of NSCLC regarding new prognosis and therapeutic options. PMID:23667589

  5. 20-HETE Mediates Proliferation of Renal Epithelial Cells in Polycystic Kidney Disease

    PubMed Central

    Park, Frank; Sweeney, William E.; Jia, Guangfu; Roman, Richard J.; Avner, Ellis D.

    2008-01-01

    Polycystic kidney diseases are characterized by abnormal proliferation of renal epithelial cells. In this study, the role of 20-hydroxyeicosatetraenoic acid (20-HETE), an endogenous cytochrome P450 metabolite of arachidonic acid with mitogenic properties, was evaluated in cystic renal disease. Daily administration of HET-0016, an inhibitor of 20-HETE synthesis, significantly reduced kidney size by half in the BPK mouse model of autosomal recessive polycystic kidney disease. In addition, compared with untreated BPK mice, this treatment significantly reduced collecting tubule cystic indices and approximately doubled survival. For evaluation of the role of 20-HETE as a mediator of epithelial cell proliferation, principal cells isolated from cystic BPK and noncystic Balb/c mice were genetically modified using lentiviral vectors. Noncystic Balb/c cells overproducing Cyp4a12 exhibited a four- to five-fold increase in cell proliferation compared with control Balb/c cells, and this increase was completely abolished when 20-HETE synthesis was inhibited; therefore, this study suggests that 20-HETE mediates proliferation of epithelial cells in the formation of renal cysts. PMID:18596124

  6. Wnt3a is critical for endothelial progenitor cell-mediated neural stem cell proliferation and differentiation.

    PubMed

    Du, Yibin; Zhang, Shuo; Yu, Tao; Du, Gongwen; Zhang, Hui; Yin, Zongsheng

    2016-09-01

    The present study aimed to determine whether co-culture with bone marrow‑derived endothelial progenitor cells (EPCs) affects the proliferation and differentiation of spinal cord-derived neural stem cells (NSCs), and to investigate the underlying mechanism. The proliferation and differentiation of the NSCs were evaluated by an MTT cell proliferation and cytotoxicity assay, and immunofluorescence, respectively. The number of neurospheres and the number of β‑tubulin III‑positive cells were detected by microscopy. The wingless‑type MMTV integration site family, member 3a (Wnt3a)/β-catenin signaling pathway was analyzed by western blot analysis and reverse transcription‑quantitative polymerase chain reaction to elucidate the possible mechanisms of EPC‑mediated NSC proliferation and differentiation. The results revealed that co‑culture with EPCs significantly induced NSC proliferation and differentiation. In addition, co‑culture with EPCs markedly induced the expression levels of Wnt3a and β‑catenin and inhibited the phosphorylation of glycogen synthase kinase 3β (GSK‑3β). By contrast, Wnt3a knockdown using a short hairpin RNA plasmid in the EPCs reduced EPC‑mediated NSC proliferation and differentiation, accompanied by inhibition of the EPC‑mediated expression of β‑catenin, and its phosphorylation and activation of GSK‑3β. Taken together, the findings of the present study demonstrated that Wnt3a was critical for EPC‑mediated NSC proliferation and differentiation. PMID:27484039

  7. Wnt3a is critical for endothelial progenitor cell-mediated neural stem cell proliferation and differentiation

    PubMed Central

    Du, Yibin; Zhang, Shuo; Yu, Tao; Du, Gongwen; Zhang, Hui; Yin, Zongsheng

    2016-01-01

    The present study aimed to determine whether co-culture with bone marrow-derived endothelial progenitor cells (EPCs) affects the proliferation and differentiation of spinal cord-derived neural stem cells (NSCs), and to investigate the underlying mechanism. The proliferation and differentiation of the NSCs were evaluated by an MTT cell proliferation and cytotoxicity assay, and immunofluorescence, respectively. The number of neurospheres and the number of β-tubulin III-positive cells were detected by microscopy. The wingless-type MMTV integration site family, member 3a (Wnt3a)/β-catenin signaling pathway was analyzed by western blot analysis and reverse transcription-quantitative polymerase chain reaction to elucidate the possible mechanisms of EPC-mediated NSC proliferation and differentiation. The results revealed that co-culture with EPCs significantly induced NSC proliferation and differentiation. In addition, co-culture with EPCs markedly induced the expression levels of Wnt3a and β-catenin and inhibited the phosphorylation of glycogen synthase kinase 3β (GSK-3β). By contrast, Wnt3a knockdown using a short hairpin RNA plasmid in the EPCs reduced EPC-mediated NSC proliferation and differentiation, accompanied by inhibition of the EPC-mediated expression of β-catenin, and its phosphorylation and activation of GSK-3β. Taken together, the findings of the present study demonstrated that Wnt3a was critical for EPC-mediated NSC proliferation and differentiation. PMID:27484039

  8. MCM2 mediates progesterone-induced endometrial stromal cell proliferation and differentiation in mice.

    PubMed

    Kong, Shuangbo; Han, Xue; Cui, Tongtong; Zhou, Chan; Jiang, Yufei; Zhang, Hangxiao; Wang, Bingyan; Wang, Haibin; Zhang, Shuang

    2016-08-01

    Uterine decidualization characterized by stromal cell proliferation and differentiation is critical to the establishment of pregnancy in many species. Progesterone is a key factor in regulating endometrial cell decidualization, however, the molecular basis involved in mediating the effects of progesterone during decidualization remains largely unknown. We report here that the DNA replication licensing factor MCM2, one of the conserved set of six-related proteins (MCM complex: MCM2-7) essential for eukaryotic DNA replication, is dynamically expressed in both proliferative and differentiated stromal cells during mouse periimplantation uterus. Applying PR-knockout mouse model and pharmacological strategy, we further found that the expression of Mcm2 is induced by progesterone action in the mouse uterine stroma. Employing a primary cell culture system, we further demonstrated that siRNA-mediated silencing of MCM2 arrests the cell cycle at G1-S transition during stromal cell proliferation. Moreover, the downregulation of Mcm2 could also compromise stromal cell differentiation. Collectively, our studies uncovered the role of a unique DNA replication licensing molecule MCM2 in mediating Progesterone-induced stromal cell decidualization in mouse uterus. PMID:26910396

  9. Polyphosphate induces matrix metalloproteinase-3-mediated proliferation of odontoblast-like cells derived from induced pluripotent stem cells

    SciTech Connect

    Ozeki, Nobuaki; Hase, Naoko; Yamaguchi, Hideyuki; Hiyama, Taiki; Kawai, Rie; Kondo, Ayami; Nakata, Kazuhiko; Mogi, Makio

    2015-05-01

    Inorganic polyphosphate [Poly(P)] may represent a physiological source of phosphate and has the ability to induce bone differentiation in osteoblasts. We previously reported that cytokine-induced matrix metalloproteinase (MMP)-3 accelerates the proliferation of purified odontoblast-like cells. In this study, MMP-3 small interfering RNA (siRNA) was transfected into odontoblast-like cells derived from induced pluripotent stem cells to investigate whether MMP-3 activity is induced by Poly(P) and/or is associated with cell proliferation and differentiation into odontoblast-like cells. Treatment with Poly(P) led to an increase in both cell proliferation and additional odontoblastic differentiation. Poly(P)-treated cells showed a small but significant increase in dentin sialophosphoprotein (DSPP) and dentin matrix protein-1 (DMP-1) mRNA expression, which are markers of mature odontoblasts. The cells also acquired additional odontoblast-specific properties including adoption of an odontoblastic phenotype typified by high alkaline phosphatase (ALP) activity and a calcification capacity. In addition, Poly(P) induced expression of MMP-3 mRNA and protein, and increased MMP-3 activity. MMP-3 siRNA-mediated disruption of the expression of these effectors potently suppressed the expression of odontoblastic biomarkers ALP, DSPP, and DMP-1, and blocked calcification. Interestingly, upon siRNA-mediated silencing of MMP-3, we noted a potent and significant decrease in cell proliferation. Using specific siRNAs, we revealed that a unique signaling cascade, Poly(P)→MMP-3→DSPP and/or DMP-1, was intimately involved in the proliferation of odontoblast-like cells. - Highlights: • Polyphosphate increases proliferation of iPS cell-derived odontoblast-like cells. • Polyphosphate-induced MMP-3 results in an increase of cell proliferation. • Induced cell proliferation involves MMP-3, DSPP, and/or DMP-1 sequentially. • Induced MMP-3 also results in an increase of odontoblastic

  10. Signaling pathways involved in megakaryocyte-mediated proliferation of osteoblast lineage cells.

    PubMed

    Cheng, Ying-Hua; Streicher, Drew A; Waning, David L; Chitteti, Brahmananda R; Gerard-O'Riley, Rita; Horowitz, Mark C; Bidwell, Joseph P; Pavalko, Fredrick M; Srour, Edward F; Mayo, Lindsey D; Kacena, Melissa A

    2015-03-01

    Recent studies suggest that megakaryocytes (MKs) may play a significant role in skeletal homeostasis, as evident by the occurrence of osteosclerosis in multiple MK related diseases (Lennert et al., 1975; Thiele et al., 1999; Chagraoui et al., 2006). We previously reported a novel interaction whereby MKs enhanced proliferation of osteoblast lineage/osteoprogenitor cells (OBs) by a mechanism requiring direct cell-cell contact. However, the signal transduction pathways and the downstream effector molecules involved in this process have not been characterized. Here we show that MKs contact with OBs, via beta1 integrin, activate the p38/MAPKAPK2/p90RSK kinase cascade in the bone cells, which causes Mdm2 to neutralizes p53/Rb-mediated check point and allows progression through the G1/S. Interestingly, activation of MAPK (ERK1/2) and AKT, collateral pathways that regulate the cell cycle, remained unchanged with MK stimulation of OBs. The MK-to-OB signaling ultimately results in significant increases in the expression of c-fos and cyclin A, necessary for sustaining the OB proliferation. Overall, our findings show that OBs respond to the presence of MKs, in part, via an integrin-mediated signaling mechanism, activating a novel response axis that de-represses cell cycle activity. Understanding the mechanisms by which MKs enhance OB proliferation will facilitate the development of novel anabolic therapies to treat bone loss associated with osteoporosis and other bone-related diseases. PMID:25160801

  11. Thyroid hormone suppresses cell proliferation through endoglin-mediated promotion of p21 stability.

    PubMed

    Lin, Y-H; Huang, Y-H; Wu, M-H; Wu, S-M; Chi, H-C; Liao, C-J; Chen, C-Y; Tseng, Y-H; Tsai, C-Y; Tsai, M-M; Lin, K-H

    2013-08-15

    Hypothyroidism has been associated with significantly elevated risk for hepatocellular carcinoma (HCC), although the precise underlying mechanisms remain unknown at present. Thyroid hormone (T3) and its receptor (TR) are involved in metabolism and growth. Endoglin is a T3/TR candidate target gene identified from our previous studies. Here, we demonstrated that T3 positively regulates endoglin mRNA and protein levels, both in vitro and in vivo. The thyroid hormone response elements of endoglin were identified at positions -2114/-2004 and -2032/-1973 of the promoter region using the electrophoretic mobility shift assay and chromatin immunoprecipitation assay. Endoglin was downregulated in the subgroups of HCC patients and significantly associated with histology grade (negative association, P=0.001), and this expression level was significantly associated with TRα1 in these HCC patients. Our results clearly indicate that p21 is involved in T3-mediated suppression of cell proliferation. Knock down of endoglin expression in HCC cells facilitated p21 polyubiquitination and promoted cell proliferation in the presence of T3. The data collectively suggest that T3/TR signaling suppresses cell proliferation by upregulating endoglin, in turn, affecting p21 stability. The results indicate that endoglin has a suppressor role to inhibit cell proliferation in HCC cell lines. PMID:23376845

  12. Phosphorylation Regulates Id2 Degradation and Mediates the Proliferation of Neural Precursor Cells.

    PubMed

    Sullivan, Jaclyn M; Havrda, Matthew C; Kettenbach, Arminja N; Paolella, Brenton R; Zhang, Zhonghua; Gerber, Scott A; Israel, Mark A

    2016-05-01

    Inhibitor of DNA binding proteins (Id1-Id4) function to inhibit differentiation and promote proliferation of many different cell types. Among the Id family members, Id2 has been most extensively studied in the central nervous system (CNS). Id2 contributes to cultured neural precursor cell (NPC) proliferation as well as to the proliferation of CNS tumors such as glioblastoma that are likely to arise from NPC-like cells. We identified three phosphorylation sites near the N-terminus of Id2 in NPCs. To interrogate the importance of Id2 phosphorylation, Id2(-/-) NPCs were modified to express wild type (WT) Id2 or an Id2 mutant protein that could not be phosphorylated at the identified sites. We observed that NPCs expressing this mutant lacking phosphorylation near the N-terminus had higher steady-state levels of Id2 when compared to NPCs expressing WT Id2. This elevated level was the result of a longer half-life and reduced proteasome-mediated degradation. Moreover, NPCs expressing constitutively de-phosphorylated Id2 proliferated more rapidly than NPCs expressing WT Id2, a finding consistent with the well-characterized function of Id2 in driving proliferation. Observing that phosphorylation of Id2 modulates the degradation of this important cell-cycle regulator, we sought to identify a phosphatase that would stabilize Id2 enhancing its activity in NPCs and extended our analysis to include human glioblastoma-derived stem cells (GSCs). We found that expression of the phosphatase PP2A altered Id2 levels. Our findings suggest that inhibition of PP2A may be a novel strategy to regulate the proliferation of normal NPCs and malignant GSCs by decreasing Id2 levels. Stem Cells 2016;34:1321-1331. PMID:26756672

  13. Integrins mediate adhesion of medulloblastoma cells to tenascin and activate pathways associated with survival and proliferation.

    PubMed

    Fiorilli, Paul; Partridge, Darren; Staniszewska, Izabela; Wang, Jin Y; Grabacka, Maja; So, Kelvin; Marcinkiewicz, Cezary; Reiss, Krzysztof; Khalili, Kamel; Croul, Sidney E

    2008-11-01

    Medulloblastoma spreads by leptomeningeal dissemination rather than by infiltration that characterizes other CNS tumors, eg, gliomas. This study represents an initial attempt to identify both the molecules that mediate medulloblastoma adhesion to leptomeninges and the pathways that are key to survival and proliferation of tumor following adhesion. As a first step in molecule identification, we produced adhesion of D283 medulloblastoma cells to the extracellular matrix (ECM) of H4 glioma cells in vitro. Within this context, D283 cells preferentially expressed the alpha9 and beta1 integrin subunits; antibody and disintegrin blockade of alpha9 and beta1 binding eliminated the adhesion. The H4 ECM was enriched in tenascin, a binding partner for the alpha9beta1 integrin heterodimer. Purified tenascin-C supported D283 cell adhesion. The adhesion was blocked by antibodies to alpha9 and beta1 integrin. In vivo data were similar; immunohistochemistry of primary human medulloblastomas with leptomeningeal extension demonstrated increased expression of alpha9 and beta1 integrins as well as tenascin at the interface of brain and leptomeningeal tumor. These data suggest that tumor-cell expressions of alpha9 and beta1 integrins in combination with extracellular tenascin are necessary for medulloblastoma adhesion to the leptomeninges. As a first step in the identification of pathways that mediate survival and proliferation of tumor following adhesion, we demonstrated that adhesion to H4 ECM was associated with survival and proliferation of D283 cells as well as activation of the MAPK pathway in a growth factor deficient environment. Antibody blockade of alpha9 and beta1 integrin binding that eliminated adhesion also eliminated the in vitro survival benefit. These data suggest that adhesion of medulloblastoma to the meninges is necessary for the survival and proliferation of these tumor cells at the secondary site. PMID:18794852

  14. Androgen receptor-mediated non-genomic regulation of prostate cancer cell proliferation

    PubMed Central

    Liao, Ross S.; Ma, Shihong; Miao, Lu; Li, Rui; Yin, Yi

    2013-01-01

    Androgen receptor (AR)-mediated signaling is necessary for prostate cancer cell proliferation and an important target for therapeutic drug development. Canonically, AR signals through a genomic or transcriptional pathway, involving the translocation of androgen-bound AR to the nucleus, its binding to cognate androgen response elements on promoter, with ensuing modulation of target gene expression, leading to cell proliferation. However, prostate cancer cells can show dose-dependent proliferation responses to androgen within minutes, without the need for genomic AR signaling. This proliferation response known as the non-genomic AR signaling is mediated by cytoplasmic AR, which facilitates the activation of kinase-signaling cascades, including the Ras-Raf-1, phosphatidyl-inositol 3-kinase (PI3K)/Akt and protein kinase C (PKC), which in turn converge on mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) activation, leading to cell proliferation. Further, since activated ERK may also phosphorylate AR and its coactivators, the non-genomic AR signaling may enhance AR genomic activity. Non-genomic AR signaling may occur in an ERK-independent manner, via activation of mammalian target of rapamycin (mTOR) pathway, or modulation of intracellular Ca2+ concentration through plasma membrane G protein-coupled receptors (GPCRs). These data suggest that therapeutic strategies aimed at preventing AR nuclear translocation and genomic AR signaling alone may not completely abrogate AR signaling. Thus, elucidation of mechanisms that underlie non-genomic AR signaling may identify potential mechanisms of resistance to current anti-androgens and help developing novel therapies that abolish all AR signaling in prostate cancer. PMID:26816736

  15. Phosphorylation Regulates Id2 Degradation and Mediates the Proliferation of Neural Precursor Cells

    PubMed Central

    Sullivan, Jaclyn M.; Havrda, Matthew C.; Kettenbach, Arminja N.; Paolella, Brenton R.; Zhang, Zhonghua; Gerber, Scott A.; Israel, Mark A.

    2016-01-01

    Inhibitor of DNA binding proteins (Id1-Id4) function to inhibit differentiation and promote proliferation of many different cell types. Among the Id family members, Id2 has been most extensively studied in the central nervous system (CNS). Id2 contributes to cultured neural precursor cell (NPC) proliferation as well as to the proliferation of CNS tumors such as glioblastoma that are likely to arise from NPC-like cells. We identified three phosphorylation sites near the N-terminus of Id2 in NPCs. To interrogate the importance of Id2 phosphorylation, Id2−/− NPCs were modified to express wild type (WT) Id2 or an Id2 mutant protein that could not be phosphorylated at the identified sites. We observed that NPCs expressing this mutant lacking phosphorylation near the N-terminus had higher steady-state levels of Id2 when compared to NPCs expressing WT Id2. This elevated level was the result of a longer half-life and reduced proteasome-mediated degradation. Moreover, NPCs expressing constitutively de-phosphorylated Id2 proliferated more rapidly than NPCs expressing WT Id2, a finding consistent with the well-characterized function of Id2 in driving proliferation. Observing that phosphorylation of Id2 modulates the degradation of this important cell-cycle regulator, we sought to identify a phosphatase that would stabilize Id2 enhancing its activity in NPCs and extended our analysis to include human glioblastoma-derived stem cells (GSCs). We found that expression of the phosphatase PP2A altered Id2 levels. Our findings suggest that inhibition of PP2A may be a novel strategy to regulate the proliferation of normal NPCs and malignant GSCs by decreasing Id2 levels. PMID:26756672

  16. Lentivirus-mediated silencing of SCIN inhibits proliferation of human lung carcinoma cells.

    PubMed

    Liu, Hongxu; Shi, Daiwang; Liu, Tieqin; Yu, Zhanwu; Zhou, Chuanjiang

    2015-01-01

    SCIN (scinderin) is a calcium-dependent actin severing and capping protein. Homologue in zebrafish has been found to be related with cell death. In the present study, we found that SCIN is highly expressed in human lung cancer specimens. However, the role of SCIN in lung cancer has not yet been determined. To investigate the function of SCIN in lung carcinoma cells, we took advantage of lentivirus-mediated RNA interference (RNAi) to knockdown SCIN expression in two lung carcinoma cell lines A549 and H1299. Silencing of SCIN significantly inhibited the proliferation and colony formation ability of both cell lines in vitro. Moreover, flow cytometry analysis showed that knockdown of SCIN led to G0/G1 phase cell cycle arrest as well as an excess accumulation of cells in the sub-G1 phase. Furthermore, depletion of SCIN resulted in a significant increase in Cyclin B1, p21 and PARP expression, and a little decrease in Cyclin D1 expression. These results suggest that SCIN plays an important role in lung carcinoma cell proliferation, and lentivirus-mediated silencing of SCIN might be a potential therapeutic approach for the treatment of lung cancer. PMID:25303873

  17. shRNA-mediated silencing of the RFC3 gene suppresses hepatocellular carcinoma cell proliferation.

    PubMed

    Yao, Zhicheng; Hu, Kunpeng; Huang, He; Xu, Shilei; Wang, Qingliang; Zhang, Peng; Yang, Peisheng; Liu, Bo

    2015-11-01

    Hepatocellular carcinoma (HCC) is one of the most common and lethal malignancies worldwide. Replication factor C (RFC) plays an important role in DNA replication and checkpoint control during the cell cycle. RFC is comprised of one large subunit [replication factor C, subunit 1 (RFC1)] and four small subunits [replication factor C, subunits 2-5 (RFC2‑5)]. The role of RFC3 in the development of HCC is, as of yet, not fully understood. In the present study, western blot analysis and reverse-transcription-quantitative PCR were used to measure the expression levels of replication factor C, subunit 3 (RFC3) in HCC tissues and HCC cells. Lentivirus-mediated RFC3-specific short hairpin RNA (shRNA) was used to knock down the expression of RFC3 in HCC cells in order to examine the effects of RFC3 on HCC cell proliferation and growth. Furthermore, the expression levels of cell cycle‑related proteins were also measured in the HCC cells in which RFC3 was knocked down. Our results revealed that the expression level of RFC3 was markedly upregulated in the HCC tissues and cells. In addition, MTS and cell growth assays were used to determine the viability and proliferation of the HCC cells in which RFC3 was knocked down, and the results revealed that both cell viability and proliferation were effectively suppressed. The downregulation of RFC3 expression led to HCC cell cycle arrest in the S phase, partly by regulating the epression of cell cycle-related proteins, such as p21, p53, p57 and cyclin A. The results of the present study suggest that RFC3 plays an important role in the development of HCC, and may thus be a potential biological target in the treatment of HCC. PMID:26397132

  18. The membrane estrogen receptor GPR30 mediates cadmium-induced proliferation of breast cancer cells

    SciTech Connect

    Yu Xinyuan; Filardo, Edward J.; Shaikh, Zahir A.

    2010-05-15

    Cadmium (Cd) is a nonessential metal that is dispersed throughout the environment. It is an endocrine-disrupting element which mimics estrogen, binds to estrogen receptor alpha (ERalpha), and promotes cell proliferation in breast cancer cells. We have previously published that Cd promotes activation of the extracellular regulated kinases, erk-1 and -2 in both ER-positive and ER-negative human breast cancer cells, suggesting that this estrogen-like effect of Cd is not associated with the ER. Here, we have investigated whether the newly appreciated transmembrane estrogen receptor, G-protein coupled receptor 30 (GPR30), may be involved in Cd-induced cell proliferation. Towards this end, we compared the effects of Cd in ER-negative human SKBR3 breast cancer cells in which endogenous GPR30 signaling was selectively inhibited using a GPR30 interfering mutant. We found that Cd concentrations from 50 to 500 nM induced a proliferative response in control vector-transfected SKBR3 cells but not in SKBR3 cells stably expressing interfering mutant. Similarly, intracellular cAMP levels increased about 2.4-fold in the vector transfectants but not in cells in which GPR30 was inactivated within 2.5 min after treatment with 500 nM Cd. Furthermore, Cd treatment rapidly activated (within 2.5 min) raf-1, mitogen-activated protein kinase kinase, mek-1, extracellular signal regulated kinases, erk-1/2, ribosomal S6 kinase, rsk, and E-26 like protein kinase, elk, about 4-fold in vector transfectants. In contrast, the activation of these signaling molecules in SKBR3 cells expressing the GPR30 mutant was only about 1.4-fold. These results demonstrate that Cd-induced breast cancer cell proliferation occurs through GPR30-mediated activation in a manner that is similar to that achieved by estrogen in these cells.

  19. Nicotine-mediated cell proliferation and angiogenesis: new twists to an old story.

    PubMed

    Dasgupta, Piyali; Chellappan, Srikumar P

    2006-10-01

    Tobacco smoking is one of the major etiologic factors associated with cancer. While there are many carcinogenic compounds present in tobacco smoke, its main addictive component, nicotine, is not carcinogenic by itself. The addictive properties of nicotine are achieved through the nicotinic acetylcholine receptors (nAChRs) that are widely distributed in the brain and neuromuscular junctions; at the same time, they were found to be expressed in a variety of non-neuronal tissues in the body including those of the lung. Recent studies show that these non-neuronal nAChRs can induce cell proliferation and angiogenesis. Analysis of the molecular mechanisms underlying nicotine-mediated cell proliferation showed the involvement of Src kinase and the scaffolding protein beta-arrestin-1. Further, nAChRs were found to activate the basic components of the cell cycle machinery similar to growth factor receptors. This involved increased binding of Raf-1 kinase to the Rb protein, activation of cyclins D and E as well as induction of proliferative promoters. This article describes pathway involved in nicotine-induced cell proliferation and angiogenesis and the potential steps that are amenable for developing novel anti-cancer therapies. PMID:17102610

  20. Regulation of MET-mediated proliferation of thyroid carcinoma cells by miR-449b.

    PubMed

    Chen, Lei; Xu, Lei; Wang, Gang

    2015-11-01

    Thyroid carcinoma (TC) is a lethal cancer worldwide, whereas its carcinogenesis is not fully understood. Although growing evidence has demonstrated that dysregulation of microRNAs (miRNAs) contributes to the development of various cancers, the role of miRNAs in the pathogenesis of TC is poorly characterized. Here, we analyzed the levels of miR-449b in TC tissues and detected significantly lower miR-449b levels in TC tissues. Moreover, the low miR-449b levels were associated with poor survival. We then overexpressed miR-449b by miRNA mimic transfection and inhibited miR-449b by miRNA antisense transfection. Cell growth was analyzed by CCK-8 assay and MTT assay, and apoptosis and cell proliferation were analyzed by flow cytometry. Overexpression of miR-449b significantly inhibited cell growth, while depletion of miR-449b increased cell growth. Moreover, the effects of miR-449b on cell growth were through modulation of cell proliferation rather than through modulation of cell apoptosis. Targeted genes were predicted by a bioinformatics algorithm and confirmed by a dual luciferase reporter assay, showing that miR-449b binds to the 3'-UTR of MET (hepatocyte growth factor receptor) mRNA, to inhibit its expression in TC cells. MET levels were regulated by miR-449b in TT cells. Together, we show that reduced miR-449b levels in TT tissues may promote TC growth, through MET-mediated cell proliferation. PMID:26044563

  1. IL1{beta}-mediated Stromal COX-2 signaling mediates proliferation and invasiveness of colonic epithelial cancer cells

    SciTech Connect

    Zhu, Yingting; Zhu, Min; Lance, Peter

    2012-11-15

    COX-2 is a major inflammatory mediator implicated in colorectal inflammation and cancer. However, the exact origin and role of COX-2 on colorectal inflammation and carcinogenesis are still not well defined. Recently, we reported that COX-2 and iNOS signalings interact in colonic CCD18Co fibroblasts. In this article, we investigated whether activation of COX-2 signaling by IL1{beta} in primary colonic fibroblasts obtained from normal and cancer patients play a critical role in regulation of proliferation and invasiveness of human colonic epithelial cancer cells. Our results demonstrated that COX-2 level was significantly higher in cancer associated fibroblasts than that in normal fibroblasts with or without stimulation of IL-1{beta}, a powerful stimulator of COX-2. Using in vitro assays for estimating proliferative and invasive potential, we discovered that the proliferation and invasiveness of the epithelial cancer cells were much greater when the cells were co-cultured with cancer associated fibroblasts than with normal fibroblasts, with or without stimulation of IL1{beta}. Further analysis indicated that the major COX-2 product, prostaglandin E{sub 2}, directly enhanced proliferation and invasiveness of the epithelial cancer cells in the absence of fibroblasts. Moreover, a selective COX-2 inhibitor, NS-398, blocked the proliferative and invasive effect of both normal and cancer associate fibroblasts on the epithelial cancer cells, with or without stimulation of IL-1{beta}. Those results indicate that activation of COX-2 signaling in the fibroblasts plays a major role in promoting proliferation and invasiveness of the epithelial cancer cells. In this process, PKC is involved in the activation of COX-2 signaling induced by IL-1{beta} in the fibroblasts.

  2. Lentivirus-Mediated knockdown of tectonic family member 1 inhibits medulloblastoma cell proliferation

    PubMed Central

    Jing, Junjie; Wang, Chengfeng; Liang, Qinchuan; Zhao, Yang; Zhao, Qingshuang; Wang, Shousen; Ma, Jie

    2015-01-01

    Tectonic family member 1 (TCTN1) encodes a member of the tectonic family which are evolutionarily conserved secreted and transmembrane proteins, involving in a diverse variety of developmental processes. It has been demonstrated that tectonics expressed in regions that participate in Hedgehog (Hh) signaling during mouse embryonic development and was imperative for Hh-mediated patterning of the ventral neural tube. However, the expression and regulation of tectonics in human tumor is still not clear. In this study, shRNA-expressing lentivirus was constructed to knockdown TCTN1 in medulloblastoma cell line Daoy. The results showed that knockdown of TCTN1 inhibited cell proliferation and colony formation in Daoy cell line, also caused cell cycle arrest at the G2/M boundary. Taken all together, our data suggest that TCTN1 might play an important role in the progression of medulloblastoma. PMID:26550235

  3. Role of the retinoblastoma protein in cell cycle arrest mediated by a novel cell surface proliferation inhibitor

    NASA Technical Reports Server (NTRS)

    Enebo, D. J.; Fattaey, H. K.; Moos, P. J.; Johnson, T. C.; Spooner, B. S. (Principal Investigator)

    1994-01-01

    A novel cell regulatory sialoglycopeptide (CeReS-18), purified from the cell surface of bovine cerebral cortex cells has been shown to be a potent and reversible inhibitor of proliferation of a wide array of fibroblasts as well as epithelial-like cells and nontransformed and transformed cells. To investigate the possible mechanisms by which CeReS-18 exerts its inhibitory action, the effect of the inhibitor on the posttranslational regulation of the retinoblastoma susceptibility gene product (RB), a tumor suppressor gene, has been examined. It is shown that CeReS-18 mediated cell cycle arrest of both human diploid fibroblasts (HSBP) and mouse fibroblasts (Swiss 3T3) results in the maintenance of the RB protein in the hypophosphorylated state, consistent with a late G1 arrest site. Although their normal nontransformed counterparts are sensitive to cell cycle arrest mediated by CeReS-18, cell lines lacking a functional RB protein, through either genetic mutation or DNA tumor virus oncoprotein interaction, are less sensitive. The refractory nature of these cells is shown to be independent of specific surface receptors for the inhibitor, and another tumor suppressor gene (p53) does not appear to be involved in the CeReS-18 inhibition of cell proliferation. The requirement for a functional RB protein product, in order for CeReS-18 to mediate cell cycle arrest, is discussed in light of regulatory events associated with density-dependent growth inhibition.

  4. Wnt5a promotes cancer cell invasion and proliferation by receptor-mediated endocytosis-dependent and -independent mechanisms, respectively

    PubMed Central

    Shojima, Kensaku; Sato, Akira; Hanaki, Hideaki; Tsujimoto, Ikuko; Nakamura, Masahiro; Hattori, Kazunari; Sato, Yuji; Dohi, Keiji; Hirata, Michinari; Yamamoto, Hideki; Kikuchi, Akira

    2015-01-01

    Wnt5a activates the Wnt/β-catenin-independent pathway and its overexpression is associated with tumor aggressiveness enhancing invasive activity. For this action, Wnt5a-induced receptor endocytosis with clathrin is required. Wnt5a expression was previously believed to be associated with cancer cell motility but not proliferation. Recently, it was reported that Wnt5a is also implicated in cancer cell proliferation, but the mechanism was not clear. In this study, we generated a neutralizing anti-Wnt5a monoclonal antibody (mAb5A16) to investigate the mechanism by which Wnt5a regulates cancer cell proliferation. Wnt5a stimulated both invasion and proliferation of certain types of cancer cells, including HeLaS3 cervical cancer cells and A549 lung cancer cells although Wnt5a promoted invasion but not proliferation in other cancer cells such as KKLS gastric cancer cells. mAb5A16 did not affect the binding of Wnt5a to its receptor, but it suppressed Wnt5a-induced receptor-mediated endocytosis. mAb5A16 inhibited invasion but not proliferation of HeLaS3 and A549 cells. Wnt5a activated Src family kinases (SFKs) and Wnt5a-dependent cancer cell proliferation was dependent on SFKs, yet blockade of receptor-mediated endocytosis did not affect cancer cell proliferation and SFK activity. These results suggest that Wnt5a promotes invasion and proliferation of certain types of cancer cells through receptor-mediated endocytosis-dependent and -independent mechanisms, respectively. PMID:25622531

  5. Aurora Kinase A is critical for the Nkx6.1 mediated β-cell proliferation pathway

    PubMed Central

    Hobson, Amanda; Draney, Carrie; Stratford, Andrew; Becker, Thomas C; Lu, Danhong; Arlotto, Michelle; Tessem, Jeffery S

    2015-01-01

    Type 1 and type 2 diabetes are ultimately characterized by depleted β-cell mass. Characterization of the molecular pathways that control β-cell proliferation could be harnessed to restore these cells. The homeobox β-cell transcription factor Nkx6.1 induces β-cell proliferation by activating the orphan nuclear receptors Nr4a1 and Nr4a3. Here, we demonstrate that Nkx6.1 localizes to the promoter of the mitotic kinase AURKA (Aurora Kinase A) and induces its expression. Adenovirus mediated overexpression of AURKA is sufficient to induce proliferation in primary rat islets while maintaining glucose stimulated insulin secretion. Furthermore, AURKA is necessary for Nkx6.1 mediated β-cell proliferation as demonstrated by shRNA mediated knock down and pharmacological inhibition of AURKA kinase activity. AURKA preferentially induces DNA replication in β-cells as measured by BrdU incorporation, and enhances the rate of histone H3 phosphorylation in primary β-cells, demonstrating that AURKA induces the replicative and mitotic cell cycle phases in rat β-cells. Finally, overexpression of AURKA results in phosphorylation of the cell cycle regulator p53, which targets p53 for degradation and permits cell cycle progression. These studies define a pathway by which AURKA upregulation by Nkx6.1 results in phosphorylation and degradation of p53, thus removing a key inhibitory factor and permitting engagement of the β-cell proliferation pathway. PMID:26030060

  6. Regulation of FOXO1-mediated transcription and cell proliferation by PARP-1

    SciTech Connect

    Sakamaki, Jun-ichi; Daitoku, Hiroaki; Yoshimochi, Kenji; Miwa, Masanao; Fukamizu, Akiyoshi

    2009-05-08

    Forkhead box O (FOXO) transcription factors play an important role in a wide range of biological processes, including cell cycle control, apoptosis, detoxification of reactive oxygen species, and gluconeogenesis through regulation of gene expression. In this study, we demonstrated that PARP-1 functions as a negative regulator of FOXO1. We showed that PARP-1 directly binds to and poly(ADP-ribosyl)ates FOXO1 protein. PARP-1 represses FOXO1-mediated expression of cell cycle inhibitor p27{sup Kip1} gene. Notably, poly(ADP-ribosyl)ation activity was not required for the repressive effect of PARP-1 on FOXO1 function. Furthermore, knockdown of PARP-1 led to a decrease in cell proliferation in a manner dependent on FOXO1 function. Chromatin immunoprecipitation experiments confirmed that PARP-1 is recruited to the p27{sup Kip1} gene promoter through a binding to FOXO1. These results suggest that PARP-1 acts as a corepressor for FOXO1, which could play an important role in proper cell proliferation by regulating p27{sup Kip1} gene expression.

  7. BRK Targets Dok1 for Ubiquitin-Mediated Proteasomal Degradation to Promote Cell Proliferation and Migration

    PubMed Central

    Miah, Sayem; Goel, Raghuveera Kumar; Dai, Chenlu; Kalra, Natasha; Beaton-Brown, Erika; Bagu, Edward T.; Bonham, Keith; Lukong, Kiven E.

    2014-01-01

    Breast tumor kinase (BRK), also known as protein tyrosine kinase 6 (PTK6), is a non-receptor tyrosine kinase overexpressed in more that 60% of human breast carcinomas. The overexpression of BRK has been shown to sensitize mammary epithelial cells to mitogenic signaling and to promote cell proliferation and tumor formation. The molecular mechanisms of BRK have been unveiled by the identification and characterization of BRK target proteins. Downstream of tyrosine kinases 1 or Dok1 is a scaffolding protein and a substrate of several tyrosine kinases. Herein we show that BRK interacts with and phosphorylates Dok1 specifically on Y362. We demonstrate that this phosphorylation by BRK significantly downregulates Dok1 in a ubiquitin-proteasome-mediated mechanism. Together, these results suggest a novel mechanism of action of BRK in the promotion of tumor formation, which involves the targeting of tumor suppressor Dok1 for degradation through the ubiquitin proteasomal pathway. PMID:24523872

  8. [Production of a dialysable transfer factor of cell mediated immunity by lymphoblastoid cells in continuous proliferation].

    PubMed

    Goust, J M; Viza, D; Moulias, R; Trejdosiewicz, L; Lesourd, B; Marescot, M R; Prévot, A

    1975-01-20

    Four lymphoblastoid cell lines tested in this work contain normally a dialysable moiety having by ultraviolet spectroscopy, column chromatography (Biogel P 10) and chemically the same properties than human dialysable Transfer Factor (TFd), but unable to transfer cell mediated immune response against common antigens. Two of them are able to do so after incubation with minimal amounts of TFd. Production of a molecule identical to human TFd is possible in some lymphoblastoid cell lines after induction with TFd. PMID:808340

  9. Leukotriene D4-induced Caco-2 cell proliferation is mediated by prostaglandin E2 synthesis

    PubMed Central

    Cabral, Marisol; Martín-Venegas, Raquel; Moreno, Juan J

    2015-01-01

    Leukotriene D4 (LTD4) is a pro-inflammatory mediator formed from arachidonic acid through the action of 5-lipoxygenase (5-LOX). Its biological effects are mediated by at least two G-coupled plasmatic cysteinyl LT receptors (CysLT1-2R). It has been reported an upregulation of the 5-LOX pathway in tumor tissue unlike in normal colon mucosa. Colon tumors generally have an increased expression of CysLT1R and colon cancer patients with high expression levels of CysLT1R have poor prognosis. We previously observed that the cyclooxygenase pathway is involved in the control of intestinal epithelial cancer cell growth through PGE2 production. The aim of this study was therefore to assess the effect of LTD4 binding with CysLT1R on Caco-2 cell growth. We note a number of key findings from this research. We observed that at a concentration similar to that found under inflammatory conditions, LTD4 was able to induce Caco-2 cell proliferation and DNA synthesis. Moreover, with the use of a specific receptor antagonist this study has demonstrated that the effect of LTD4 is a result of its interaction with CystLT1R. We also note the possible participation of the PLC-IP3-Ca2+/DAG-PKC signaling pathways in cytosolic PLA2 and [3H]AA release induced by LTD4-CystLT1R interaction. Finally, we found that the resulting activation of the AA cascade and the production of PGE2 eicosanoid could be related to the activation of cell signaling pathways such as ERK and CREB. These findings will help facilitate our understanding of how inflammatory mediators can affect the survival and dissemination of intestinal carcinoma cells. PMID:26216432

  10. RIP1 maintains DNA integrity and cell proliferation by regulating PGC-1α-mediated mitochondrial oxidative phosphorylation and glycolysis

    PubMed Central

    Chen, W; Wang, Q; Bai, L; Chen, W; Wang, X; Tellez, C S; Leng, S; Padilla, M T; Nyunoya, T; Belinsky, S A; Lin, Y

    2014-01-01

    Aerobic glycolysis or the Warburg effect contributes to cancer cell proliferation; however, how this glucose metabolism pathway is precisely regulated remains elusive. Here we show that receptor-interacting protein 1 (RIP1), a cell death and survival signaling factor, regulates mitochondrial oxidative phosphorylation and aerobic glycolysis. Loss of RIP1 in lung cancer cells suppressed peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) expression, impairing mitochondrial oxidative phosphorylation and accelerating glycolysis, resulting in spontaneous DNA damage and p53-mediated cell proliferation inhibition. Thus, although aerobic glycolysis within a certain range favors cancer cell proliferation, excessive glycolysis causes cytostasis. Our data suggest that maintenance of glycolysis by RIP1 is pivotal to cancer cell energy homeostasis and DNA integrity and may be exploited for use in anticancer therapy. PMID:24583643

  11. Wnt5a-mediated non-canonical Wnt signalling regulates human endothelial cell proliferation and migration

    SciTech Connect

    Cheng Chingwen Yeh Juching; Fan Taiping; Smith, Stephen K.; Charnock-Jones, D. Stephen

    2008-01-11

    Cell to cell interaction is one of the key processes effecting angiogenesis and endothelial cell function. Wnt signalling is mediated through cell-cell interaction and is involved in many developmental processes and cellular functions. In this study, we investigated the possible function of Wnt5a and the non-canonical Wnt pathway in human endothelial cells. We found that Wnt5a-mediated non-canonical Wnt signalling regulated endothelial cell proliferation. Blocking this pathway using antibody, siRNA or a down-stream inhibitor led to suppression of endothelial cell proliferation, migration, and monolayer wound closure. We also found that the mRNA level of Wnt5a is up-regulated when endothelial cells are treated with a cocktail of inflammatory cytokines. Our findings suggest non-canonical Wnt signalling plays a role in regulating endothelial cell growth and possibly in angiogenesis.

  12. EPAS-1 Mediates SP-1-Dependent FBI-1 Expression and Regulates Tumor Cell Survival and Proliferation

    PubMed Central

    Wang, Xiaogang; Cao, Peng; Li, Zhiqing; Wu, Dongyang; Wang, Xi; Liang, Guobiao

    2014-01-01

    Factor binding IST-1 (FBI-1) plays an important role in oncogenic transformation and tumorigenesis. As FBI-1 is over-expressed in multiple human cancers, the regulation of itself would provide new effective options for cancer intervention. In this work, we aimed to study the role that EPAS-1 plays in regulating FBI-1. We use the fact that specificity protein-1 (SP-1) is one of the crucial transcription factors of FBI-1, and that SP-1 can interact with the endothelial pas domain protein-1 (EPAS-1) for the induction of hypoxia related genes. The study showed that EPAS-1 plays an indispensible role in SP-1 transcription factor-mediated FBI-1 induction, and participated in tumor cell survival and proliferation. Thus, EPAS-1 could be a novel target for cancer therapeutics. PMID:25192290

  13. EPAS-1 mediates SP-1-dependent FBI-1 expression and regulates tumor cell survival and proliferation.

    PubMed

    Wang, Xiaogang; Cao, Peng; Li, Zhiqing; Wu, Dongyang; Wang, Xi; Liang, Guobiao

    2014-01-01

    Factor binding IST-1 (FBI-1) plays an important role in oncogenic transformation and tumorigenesis. As FBI-1 is over-expressed in multiple human cancers, the regulation of itself would provide new effective options for cancer intervention. In this work, we aimed to study the role that EPAS-1 plays in regulating FBI-1. We use the fact that specificity protein-1 (SP-1) is one of the crucial transcription factors of FBI-1, and that SP-1 can interact with the endothelial pas domain protein-1 (EPAS-1) for the induction of hypoxia related genes. The study showed that EPAS-1 plays an indispensible role in SP-1 transcription factor-mediated FBI-1 induction, and participated in tumor cell survival and proliferation. Thus, EPAS-1 could be a novel target for cancer therapeutics. PMID:25192290

  14. KSHV-Mediated Regulation of Par3 and SNAIL Contributes to B-Cell Proliferation.

    PubMed

    Jha, Hem C; Sun, Zhiguo; Upadhyay, Santosh K; El-Naccache, Darine W; Singh, Rajnish K; Sahu, Sushil K; Robertson, Erle S

    2016-07-01

    Studies have suggested that Epithelial-Mesenchymal Transition (EMT) and transformation is an important step in progression to cancer. Par3 (partitioning-defective protein) is a crucial factor in regulating epithelial cell polarity. However, the mechanism by which the latency associated nuclear antigen (LANA) encoded by Kaposi's Sarcoma associated herpesvirus (KSHV) regulates Par3 and EMTs markers (Epithelial-Mesenchymal Transition) during viral-mediated B-cell oncogenesis has not been fully explored. Moreover, several studies have demonstrated a crucial role for EMT markers during B-cell malignancies. In this study, we demonstrate that Par3 is significantly up-regulated in KSHV-infected primary B-cells. Further, Par3 interacted with LANA in KSHV positive and LANA expressing cells which led to translocation of Par3 from the cell periphery to a predominantly nuclear signal. Par3 knockdown led to reduced cell proliferation and increased apoptotic induction. Levels of SNAIL was elevated, and E-cadherin was reduced in the presence of LANA or Par3. Interestingly, KSHV infection in primary B-cells led to enhancement of SNAIL and down-regulation of E-cadherin in a temporal manner. Importantly, knockdown of SNAIL, a major EMT regulator, in KSHV cells resulted in reduced expression of LANA, Par3, and enhanced E-cadherin. Also, SNAIL bound to the promoter region of p21 and can regulate its activity. Further a SNAIL inhibitor diminished NF-kB signaling through upregulation of Caspase3 in KSHV positive cells in vitro. This was also supported by upregulation of SNAIL and Par3 in BC-3 transplanted NOD-SCID mice which has potential as a therapeutic target for KSHV-associated B-cell lymphomas. PMID:27463802

  15. KSHV-Mediated Regulation of Par3 and SNAIL Contributes to B-Cell Proliferation

    PubMed Central

    Jha, Hem C.; Sun, Zhiguo; Upadhyay, Santosh K.; El-Naccache, Darine W.; Singh, Rajnish K.; Sahu, Sushil K.; Robertson, Erle S.

    2016-01-01

    Studies have suggested that Epithelial–Mesenchymal Transition (EMT) and transformation is an important step in progression to cancer. Par3 (partitioning-defective protein) is a crucial factor in regulating epithelial cell polarity. However, the mechanism by which the latency associated nuclear antigen (LANA) encoded by Kaposi's Sarcoma associated herpesvirus (KSHV) regulates Par3 and EMTs markers (Epithelial-Mesenchymal Transition) during viral-mediated B-cell oncogenesis has not been fully explored. Moreover, several studies have demonstrated a crucial role for EMT markers during B-cell malignancies. In this study, we demonstrate that Par3 is significantly up-regulated in KSHV-infected primary B-cells. Further, Par3 interacted with LANA in KSHV positive and LANA expressing cells which led to translocation of Par3 from the cell periphery to a predominantly nuclear signal. Par3 knockdown led to reduced cell proliferation and increased apoptotic induction. Levels of SNAIL was elevated, and E-cadherin was reduced in the presence of LANA or Par3. Interestingly, KSHV infection in primary B-cells led to enhancement of SNAIL and down-regulation of E-cadherin in a temporal manner. Importantly, knockdown of SNAIL, a major EMT regulator, in KSHV cells resulted in reduced expression of LANA, Par3, and enhanced E-cadherin. Also, SNAIL bound to the promoter region of p21 and can regulate its activity. Further a SNAIL inhibitor diminished NF-kB signaling through upregulation of Caspase3 in KSHV positive cells in vitro. This was also supported by upregulation of SNAIL and Par3 in BC-3 transplanted NOD-SCID mice which has potential as a therapeutic target for KSHV-associated B-cell lymphomas. PMID:27463802

  16. Identification of mediators stimulating proliferation and matrix synthesis of rat pancreatic stellate cells.

    PubMed

    Schneider, E; Schmid-Kotsas, A; Zhao, J; Weidenbach, H; Schmid, R M; Menke, A; Adler, G; Waltenberger, J; Grünert, A; Bachem, M G

    2001-08-01

    The aim of this study was to identify fibrogenic mediators stimulating activation, proliferation, and/or matrix synthesis of rat pancreatic stellate cells (PSC). PSC were isolated from the pancreas of normal Wistar rats and from rats with cerulein pancreatitis. Cell activation was demonstrated by immunofluorescence microscopy of smooth muscle alpha-actin (SMA) and real-time quantitative RT-PCR of SMA, fibronectin, and transforming growth factor (TGF)-beta(1). Proliferation was measured by bromodeoxyuridine incorporation. Matrix synthesis was demonstrated on the protein and mRNA level. Within a few days in primary culture, PSC changed their phenotype from fat-storing to SMA-positive myofibroblast-like cells expressing platelet-derived growth factor (PDGF) alpha- and PDGF beta-receptors. TGF-beta(1) and tumor necrosis factor (TNF)-alpha accelerated the change in the cells' phenotype. Addition of 50 ng/ml PDGF and 5 ng/ml basic fibroblast growth factor (bFGF) to cultured PSC significantly stimulated cell proliferation (4.37 +/- 0.49- and 2.96 +/- 0.39-fold of control). Fibronectin synthesis calculated on the basis of DNA was stimulated by 5 ng/ml bFGF (3.44 +/- 1.13-fold), 5 ng/ml TGF-beta(1) (2.46 +/- 0.89-fold), 20 ng/ml PDGF (2.27 +/- 0.68-fold), and 50 ng/ml TGF-alpha (1.87 +/- 0.19-fold). As shown by RT-PCR, PSC express predominantly the splice variant EIII-A of fibronectin. Immunofluorescence microscopy and Northern blot confirmed that in particular bFGF and TGF-beta(1) stimulated the synthesis of fibronectin and collagens type I and III. In conclusion, our data demonstrate that 1) TGF-beta(1) and TNF-alpha accelerate the change in the cell phenotype, 2) PDGF represents the most effective mitogen, and 3) bFGF, TGF-beta(1), PDGF, and, to a lesser extent, TGF-alpha stimulate extracellular matrix synthesis of cultured rat PSC. PMID:11443052

  17. SETDB1 mediated FosB expression increases the cell proliferation rate during anticancer drug therapy

    PubMed Central

    Na, Han-Heom; Noh, Hee-Jung; Cheong, Hyang-Min; Kang, Yoonsung; Kim, Keun-Cheol

    2016-01-01

    The efficacy of anticancer drugs depends on a variety of signaling pathways, which can be positively or negatively regulated. In this study, we show that SETDB1 HMTase is down-regulated at the transcriptional level by several anticancer drugs, due to its inherent instability. Using RNA sequence analysis, we identified FosB as being regulated by SETDB1 during anticancer drug therapy. FosB expression was increased by treatment with doxorubicin, taxol and siSETDB1. Moreover, FosB was associated with an increased rate of proliferation. Combinatory transfection of siFosB and siSETDB1 was slightly increased compared to transfection of siFosB. Furthermore, FosB was regulated by multiple kinase pathways. ChIP analysis showed that SETDB1 and H3K9me3 interact with a specific region of the FosB promoter. These results suggest that SETDB1-mediated FosB expression is a common molecular phenomenon, and might be a novel pathway responsible for the increase in cell proliferation that frequently occurs during anticancer drug therapy. [BMB Reports 2016; 49(4): 238-243] PMID:26949019

  18. Downregulation of TRAF2 Mediates NIK-Induced Pancreatic Cancer Cell Proliferation and Tumorigenicity

    PubMed Central

    Döppler, Heike; Liou, Geou-Yarh; Storz, Peter

    2013-01-01

    Background Increased levels of NF-κB are hallmarks of pancreatic ductal adenocarcinoma (PDAC) and both classical and alternative NF-κB activation pathways have been implicated. Methodology/Principal Findings Here we show that activation of the alternative pathway is a source for the high basal NF-κB activity in PDAC cell lines. Increased activity of the p52/RelB NF-κB complex is mediated through stabilization and activation of NF-κB-inducing kinase (NIK). We identify proteasomal downregulation of TNF receptor-associated factor 2 (TRAF2) as a mechanism by which levels of active NIK are increased in PDAC cell lines. Such upregulation of NIK expression and activity levels relays to increased proliferation and anchorage-independent growth, but not migration or survival of PDAC cells. Conclusions/Significance Rapid growth is one characteristic of pancreatic cancer. Our data indicates that the TRAF2/NIK/NF-κB2 pathway regulates PDAC cell tumorigenicity and could be a valuable target for therapy of this cancer. PMID:23301098

  19. PKB/Akt: a key mediator of cell proliferation, survival and insulin responses?

    PubMed

    Lawlor, M A; Alessi, D R

    2001-08-01

    The serine/threonine protein kinase PKB (also known as Akt) is thought to be a key mediator of signal transduction processes. The identification of PKB substrates and the role PKB phosphorylation plays in regulating these molecules have been a major focus of research in recent years. A recently developed motif-profile scoring algorithm that can be used to scan the genome for potential PKB substrates is therefore a useful tool, although additional considerations, such as the evolutionary conservation of the phosphorylation site, must also be taken into account. Recent evidence indicates that PKB plays a key role in cancer progression by stimulating cell proliferation and inhibiting apoptosis and is also probably a key mediator of insulin signalling. These findings indicate that PKB is likely to be a hot drug target for the treatment of cancer, diabetes and stroke. There are, however, a number of pitfalls of methodologies currently employed to study PKB function, and therefore caution should be used in interpretation of such experiments. PMID:11686294

  20. Stromal inhibition of prostatic epithelial cell proliferation not mediated by transforming growth factor beta.

    PubMed Central

    Kooistra, A.; van den Eijnden-van Raaij, A. J.; Klaij, I. A.; Romijn, J. C.; Schröder, F. H.

    1995-01-01

    The paracrine influence of prostatic stroma on the proliferation of prostatic epithelial cells was investigated. Stromal cells from the human prostate have previously been shown to inhibit anchorage-dependent as well as anchorage-independent growth of the prostatic tumour epithelial cell lines PC-3 and LNCaP. Antiproliferative activity, mediated by a diffusible factor in the stromal cell conditioned medium, was found to be produced specifically by prostatic stromal cells. In the present study the characteristics of this factor were examined. It is demonstrated that prostate stroma-derived inhibiting factor is an acid- and heat-labile, dithiothreitol-sensitive protein. Although some similarities with type beta transforming growth factor (TGF-beta)-like inhibitors are apparent, evidence is presented that the factor is not identical to TGF-beta or to the TGF-beta-like factors activin and inhibin. Absence of TGF-beta activity was shown by the lack of inhibitory response of the TGF-beta-sensitive mink lung cell line CCL-64 to prostate stromal cell conditioned medium and to concentrated, partially purified preparations of the inhibitor. Furthermore, neutralising antibodies against TGF-beta 1 or TGF-beta 2 did not cause a decline in the level of PC-3 growth inhibition caused by partially purified inhibitor. Using Northern blot analyses, we excluded the involvement of inhibin or activin. It is concluded that the prostate stroma-derived factor may be a novel growth inhibitor different from any of the currently described inhibiting factors. Images Figure 5 PMID:7543773

  1. Lentivirus-Mediated Knockdown of Myosin VI Inhibits Cell Proliferation of Breast Cancer Cell.

    PubMed

    Wang, Hong; Wang, Biyun; Zhu, Wei; Yang, Ziang

    2015-10-01

    Myosin VI (MYO6) is a unique member of the myosin superfamily, and almost no experimental studies link MYO6 to tumorigenesis of breast cancer. However, previous microarray data demonstrated that MYO6 was frequently overexpressed in breast cancer tissues. In this study, to further develop its role in breast cancer, endogenous expression of MYO6 was significantly inhibited in breast cancer ZR-75-30 and MDA-MB-231 cells using lentivirus-mediated RNA interference. Quantitative polymerase chain reaction and western blot were applied to detect the expression level of MYO6. Cell viability of both cell lines was measured by methylthiazol tetrazolium and colony formation assays. Besides, cell cycle assay was utilized to acquire the distribution information of cell phase. The results demonstrated that knockdown of MYO6 markedly reduced cell viability and colony formation, as well as suppressed cell cycle progression in breast cancer cells. The results suggested that MYO6 played a vital role in breast cancer cells and might provide useful information for diagnosis and therapy of human breast cancer in future. PMID:26407123

  2. Oxidative stress-mediated inhibition of intestinal epithelial cell proliferation by silver nanoparticles.

    PubMed

    McCracken, Christie; Zane, Andrew; Knight, Deborah A; Hommel, Elizabeth; Dutta, Prabir K; Waldman, W James

    2015-10-01

    Given the increasing use of silver nanoparticles (Ag NP) by the food and food packaging industries, this study investigated potential consequences of Ag NP ingestion in intestinal epithelial C2BBe1 cells. Treatment of proliferating cells (<10,000 cells/cm(2)) with 0.25 μg/cm(2) (1.25 μg/mL) of 23 nm Ag NP for 24 h induced 15% necrotic cell death and an 80% reduction in metabolic activity and decreased the GSH/GSSG ratio, indicating oxidative stress. G2/M phase cell cycle arrest and complete inhibition of cell proliferation was also induced by Ag NP treatment. Simulated in vitro digestion of Ag NP prior to cell exposure required the use of slightly higher doses to induce the same toxicity, likely due to slower Ag dissolution. Treatment of cells with silica, titania, and ZnO NP partially inhibited cell proliferation, but inhibition at low doses was unique to Ag NP. These data suggest that Ag NP induces oxidative stress, cell cycle arrest, and the inhibition of cell proliferation. However, toxicity and induction of oxidative stress were not observed in confluent cells (>100,000 cells/cm(2)) treated with 10 μg/cm(2) (40-50 μg/mL) Ag NP, indicating that these cells are less sensitive to Ag NP. PMID:26196530

  3. The histone demethylase JMJD1A regulates adrenomedullin-mediated cell proliferation in hepatocellular carcinoma under hypoxia

    SciTech Connect

    Park, Seong-Joon; Kim, Joong-Gook; Son, Tae Gen; Yi, Joo Mi; Kim, Nam Deuk; Yang, Kwangmo; Heo, Kyu

    2013-05-17

    Highlights: •Hypoxia stimulates HepG2 and Hep3B cell proliferation. •The JMJD1A and ADM expressions are enhanced by hypoxia in HCCs. •Increased JMJD1A expression reduces a H3K9 di-methylation in the ADM promoter region. •Knock-down of JMJD1A abrogates the hypoxia-induced HepG2 and Hep3B cell growth. -- Abstract: We studied the roles of JMJD1A and its target gene ADM in the growth of hepatocellular carcinomas (HCCs) and breast cancer cells under hypoxic conditions. Hypoxia stimulated HepG2 and Hep3B cell proliferation but had no effect on MDA-MB-231 cell proliferation. Interestingly, the JMJD1A and ADM expressions were enhanced by hypoxia only in HepG2 and Hep3B cells. Our ChIP results showed that hypoxia-induced HepG2 and Hep3B cell proliferation is mediated by JMJD1A upregulation and subsequent decrease in methylation in the ADM promoter region. Furthermore, JMJD1A gene silencing abrogated the hypoxia-induced ADM expression and inhibited HepG2 and Hep3B cell growth. These data suggest that JMJD1A might function as a proliferation regulator in some cancer cell types.

  4. The Hippo pathway mediates inhibition of vascular smooth muscle cell proliferation by cAMP

    PubMed Central

    Kimura, Tomomi E.; Duggirala, Aparna; Smith, Madeleine C.; White, Stephen; Sala-Newby, Graciela B.; Newby, Andrew C.; Bond, Mark

    2016-01-01

    Aims Inhibition of vascular smooth muscle cell (VSMC) proliferation by intracellular cAMP prevents excessive neointima formation and hence angioplasty restenosis and vein-graft failure. These protective effects are mediated via actin-cytoskeleton remodelling and subsequent regulation of gene expression by mechanisms that are incompletely understood. Here we investigated the role of components of the growth-regulatory Hippo pathway, specifically the transcription factor TEAD and its co-factors YAP and TAZ in VSMC. Methods and results Elevation of cAMP using forskolin, dibutyryl-cAMP or the physiological agonists, Cicaprost or adenosine, significantly increased phosphorylation and nuclear export YAP and TAZ and inhibited TEAD-luciferase report gene activity. Similar effects were obtained by inhibiting RhoA activity with C3-transferase, its downstream kinase, ROCK, with Y27632, or actin-polymerisation with Latrunculin-B. Conversely, expression of constitutively-active RhoA reversed the inhibitory effects of forskolin on TEAD-luciferase. Forskolin significantly inhibited the mRNA expression of the pro-mitogenic genes, CCN1, CTGF, c-MYC and TGFB2 and this was reversed by expression of constitutively-active YAP or TAZ phospho-mutants. Inhibition of YAP and TAZ function with RNAi or Verteporfin significantly reduced VSMC proliferation. Furthermore, the anti-mitogenic effects of forskolin were reversed by overexpression of constitutively-active YAP or TAZ. Conclusion Taken together, these data demonstrate that cAMP-induced actin-cytoskeleton remodelling inhibits YAP/TAZ–TEAD dependent expression of pro-mitogenic genes in VSMC. This mechanism contributes novel insight into the anti-mitogenic effects of cAMP in VSMC and suggests a new target for intervention. PMID:26625714

  5. During Drosophila disc regeneration, JAK/STAT coordinates cell proliferation with Dilp8-mediated developmental delay.

    PubMed

    Katsuyama, Tomonori; Comoglio, Federico; Seimiya, Makiko; Cabuy, Erik; Paro, Renato

    2015-05-01

    Regeneration of fragmented Drosophila imaginal discs occurs in an epimorphic manner involving local cell proliferation at the wound site. After disc fragmentation, cells at the wound site activate a restoration program through wound healing, regenerative cell proliferation, and repatterning of the tissue. However, the interplay of signaling cascades driving these early reprogramming steps is not well-understood. Here, we profiled the transcriptome of regenerating cells in the early phase within 24 h after wounding. We found that JAK/STAT signaling becomes activated at the wound site and promotes regenerative cell proliferation in cooperation with Wingless (Wg) signaling. In addition, we showed that the expression of Drosophila insulin-like peptide 8 (dilp8), which encodes a paracrine peptide to delay the onset of pupariation, is controlled by JAK/STAT signaling in early regenerating discs. Our findings suggest that JAK/STAT signaling plays a pivotal role in coordinating regenerative disc growth with organismal developmental timing. PMID:25902518

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

    PubMed

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

    2016-08-01

    Cadmium (Cd) is a heavy metal widely found in a number of environmental matrices, and the exposure to Cd is increasing nowadays. In this study, the role of high mobility group A2 (HMGA2) in Cd-induced proliferation was investigated in MRC-5 cells. Exposure to Cd (2μM) for 48h significantly enhanced the growth of MRC-5 cells, increased reactive oxygen species (ROS) production, and induced both mRNA and protein expression of HMGA2. Evidence for Cd-induced reduction of the number of G0/G1 phase cells and an increase in the number of cells in S phase and G2/M phase was sought by flow cytometric analysis. Western blot analysis showed that cyclin D1, cyclin B1, and cyclin E were upregulated in Cd-treated cells. Further study revealed that N-acetyl cysteine (NAC) markedly prevented Cd-induced proliferation of MRC-5 cells, ROS generation, and the increasing protein level of HMGA2. Silencing of HMGA2 gene by siRNA blocked Cd-induced cyclin D1, cyclin B1, and cyclin E expression and reduction of the number of G0/G1 phase cells. Combining, our data showed that Cd-induced ROS formation provoked HMGA2 upregulation, caused cell cycle changes, and led to cell proliferation. This suggests that HMGA2 might be an important biomarker in Cd-induced cell proliferation. PMID:27071802

  7. The inhibition of human T cell proliferation by the caspase inhibitor z-VAD-FMK is mediated through oxidative stress

    SciTech Connect

    Rajah, T.; Chow, S.C.

    2014-07-15

    The caspase inhibitor benzyloxycarbony (Cbz)-L-Val-Ala-Asp (OMe)-fluoromethylketone (z-VAD-FMK) has recently been shown to inhibit T cell proliferation without blocking caspase-8 and caspase-3 activation in primary T cells. We showed in this study that z-VAD-FMK treatment leads to a decrease in intracellular glutathione (GSH) with a concomitant increase in reactive oxygen species (ROS) levels in activated T cells. The inhibition of anti-CD3-mediated T cell proliferation induced by z-VAD-FMK was abolished by the presence of low molecular weight thiols such as GSH, N-acetylcysteine (NAC) and L-cysteine, whereas D-cysteine which cannot be metabolised to GSH has no effect. These results suggest that the depletion of intracellular GSH is the underlying cause of z-VAD-FMK-mediated inhibition of T cell activation and proliferation. The presence of exogenous GSH also attenuated the inhibition of anti-CD3-induced CD25 and CD69 expression mediated by z-VAD-FMK. However, none of the low molecular weight thiols were able to restore the caspase-inhibitory properties of z-VAD-FMK in activated T cells where caspase-8 and caspase-3 remain activated and processed into their respective subunits in the presence of the caspase inhibitor. This suggests that the inhibition of T cell proliferation can be uncoupled from the caspase-inhibitory properties of z-VAD-FMK. Taken together, the immunosuppressive effects in primary T cells mediated by z-VAD-FMK are due to oxidative stress via the depletion of GSH.

  8. G-Protein-Coupled Receptor 35 Mediates Human Saphenous Vein Vascular Smooth Muscle Cell Migration and Endothelial Cell Proliferation

    PubMed Central

    McCallum, Jennifer E.; Mackenzie, Amanda E.; Divorty, Nina; Clarke, Carolyn; Delles, Christian; Milligan, Graeme; Nicklin, Stuart A.

    2016-01-01

    Vascular smooth muscle cell (VSMC) migration and proliferation is central to neointima formation in vein graft failure following coronary artery bypass. However, there are currently no pharmacological interventions that prevent vein graft failure through intimal occlusion. It is hence a therapeutic target. Here, we investigated the contribution of GPR35 to human VSMC and endothelial cell (EC) migration, using a scratch-wound assay, and also the contribution to proliferation, using MTS and BrdU assays, in in vitro models using recently characterized human GPR35 ortholog-selective small-molecule agonists and antagonists. Real-time PCR studies showed GPR35 to be robustly expressed in human VSMCs and ECs. Stimulation of GPR35, with either the human-selective agonist pamoic acid or the reference agonist zaprinast, promoted VSMC migration in the scratch-wound assay. These effects were blocked by coincubation with either of the human GPR35-specific antagonists, CID-2745687 or ML-145. These GPR35-mediated effects were produced by inducing alterations in the actin cytoskeleton via the Rho A/Rho kinase signaling axis. Additionally, the agonist ligands stimulated a proliferative response in ECs. These studies highlight the potential that small molecules that stimulate or block GPR35 activity can modulate vascular proliferation and migration. These data propose GPR35 as a translational therapeutic target in vascular remodeling. PMID:27064272

  9. Stimulation of proximal tubular cell apoptosis by albumin-bound fatty acids mediated by peroxisome proliferator activated receptor-gamma.

    PubMed

    Arici, Mustafa; Chana, Ravinder; Lewington, Andrew; Brown, Jez; Brunskill, Nigel John

    2003-01-01

    In nephrotic syndrome, large quantities of albumin enter the kidney tubule. This albumin carries with it a heavy load of fatty acids to which the proximal tubule cells are exposed at high concentration. It is postulated that exposure to fatty acids in this way is injurious to proximal tubule cells. This study has examined the ability of fatty acids to interact with peroxisome proliferator-activated receptors (PPAR) in primary cultures of human proximal tubule cells. Luciferase reporter assays in transiently transfected human proximal tubule cells were used to show that albumin bound fatty acids and other agonists activate PPARgamma in a dose-dependent manner. One of the consequences of this activation is apoptosis of the cells as determined by changes in cell morphology, evidence of PARP cleavage, and appearance of DNA laddering. Overexpression of PPARgamma in these cells also results in enhanced apoptosis. Both fatty acid-induced PPAR activation and apoptosis in these cells can be blocked by PPAR response element decoy oligonucleotides. Activation of PPARgamma by the specific agonist PGJ(2) is associated with inhibition of cell proliferation, whereas activation by albumin bound fatty acids is accompanied by increased proliferation. However, the net balance of apoptosis/proliferation favors deletion of cells. These results implicate albumin-bound fatty acids as important mediators of tubular injury in nephrosis and provide fresh impetus for pursuit of lipid-lowering strategies in proteinuric renal disease. PMID:12506134

  10. CDO, an Hh-Coreceptor, Mediates Lung Cancer Cell Proliferation and Tumorigenicity through Hedgehog Signaling

    PubMed Central

    Leem, Young-Eun; Ha, Hye-Lim; Bae, Ju-Hyeon; Baek, Kwan-Hyuck; Kang, Jong-Sun

    2014-01-01

    Hedgehog (Hh) signaling plays essential roles in various developmental processes, and its aberrant regulation results in genetic disorders or malignancies in various tissues. Hyperactivation of Hh signaling is associated with lung cancer development, and there have been extensive efforts to investigate how to control Hh signaling pathway and regulate cancer cell proliferation. In this study we investigated a role of CDO, an Hh co-receptor, in non-small cell lung cancer (NSCLC). Inhibition of Hh signaling by SANT-1 or siCDO in lung cancer cells reduced proliferation and tumorigenicity, along with the decrease in the expression of the Hh components. Histological analysis with NSCLC mouse tissue demonstrated that CDO was expressed in advanced grade of the cancer, and precisely co-localized with GLI1. These data suggest that CDO is required for proliferation and survival of lung cancer cells via Hh signaling. PMID:25369201

  11. Lentivirus-mediated gene silencing of NOB1 suppresses non-small cell lung cancer cell proliferation.

    PubMed

    Huang, Weiyi; Zhong, Weiqing; Xu, Jun; Su, Benhua; Huang, Guanghui; Du, Jiajun; Liu, Qi

    2015-09-01

    NIN/RPN12 binding protein 1 (NOB1p) encoded by NOB1 has been found to be an essential factor in 26S proteasome biogenesis which participates in protein degradation. However, the functions of NOB1 in non-small cell lung cancer cells are largely unknown. In the present study, lentivirus-mediated NOB1 shRNA transfection in two non-small cell lung cancer cell lines (A549 and H1299) was accomplished, as determined by fluorescence imaging. Downregulation of NOB1 expression was confirmed by real-time PCR and western blotting. NOB1 silencing resulted in a significant decline in the proliferation and colony formation capability of non-small cell lung cancer cells. Moreover, flow cytometry showed that A549 cells were arrested in the G0/G1 phase of the cell cycle after NOB1 suppression. Furthermore, depletion of NOB1 resulted in a significant decrease in CDK4 and cyclin D1 expression. These results suggest that NOB1 may act as an important regulator in non-small cell lung cancer growth and could be a therapeutic target of non‑small cell lung cancer. PMID:26178254

  12. Tumor suppressor KAI1 affects integrin {alpha}v{beta}3-mediated ovarian cancer cell adhesion, motility, and proliferation

    SciTech Connect

    Ruseva, Zlatna; Geiger, Pamina Xenia Charlotte; Hutzler, Peter; Kotzsch, Matthias; Luber, Birgit; Schmitt, Manfred; Gross, Eva; Reuning, Ute

    2009-06-10

    The tetraspanin KAI1 had been described as a metastasis suppressor in many different cancer types, a function for which associations of KAI1 with adhesion and signaling receptors of the integrin superfamily likely play a role. In ovarian cancer, integrin {alpha}v{beta}3 correlates with tumor progression and its elevation in vitro provoked enhanced cell adhesion accompanied by significant increases in cell motility and proliferation in the presence of its major ligand vitronectin. In the present study, we characterized integrin {alpha}v{beta}3-mediated tumor biological effects as a function of cellular KAI1 restoration and proved for the first time that KAI1, besides its already known physical crosstalk with {beta}1-integrins, also colocalizes with integrin {alpha}v{beta}3. Functionally, elevated KAI1 levels drastically increased integrin {alpha}v{beta}3/vitronectin-dependent ovarian cancer cell adhesion. Since an intermediate level of cell adhesive strength is required for optimal cell migration, we next studied ovarian cancer cell motility as a function of KAI1 restoration. By time lapse video microscopy, we found impaired integrin {alpha}v{beta}3/vitronectin-mediated cell migration most probably due to strongly enhanced cellular immobilization onto the adhesion-supporting matrix. Moreover, KAI1 reexpression significantly diminished cell proliferation. These data strongly indicate that KAI1 may suppress ovarian cancer progression by inhibiting integrin {alpha}v{beta}3/vitronectin-provoked tumor cell motility and proliferation as important hallmarks of the oncogenic process.

  13. Cytosolic Phospholipase A2-α Mediates Endothelial Cell Proliferation and Is Inactivated by Association with the Golgi Apparatus

    PubMed Central

    Herbert, S. P.; Ponnambalam, S.; Walker, J. H.

    2005-01-01

    Arachidonic acid and its metabolites are implicated in regulating endothelial cell proliferation. Cytosolic phospholipase A2-α (cPLA2α) is responsible for receptor-mediated arachidonic acid evolution. We tested the hypothesis that cPLA2α activity is linked to endothelial cell proliferation. The specific cPLA2α inhibitor, pyrrolidine-1, inhibited umbilical vein endothelial cell (HUVEC) proliferation in a dose-dependent manner. Exogenous arachidonic acid addition reversed this inhibitory effect. Inhibition of sPLA2 did not affect HUVEC proliferation. The levels of cPLA2α did not differ between subconfluent and confluent cultures of cells. However, using fluorescence microscopy we observed a novel, confluence-dependent redistribution of cPLA2α to the distal Golgi apparatus in HUVECs. Association of cPLA2α with the Golgi was linked to the proliferative status of HUVECs. When associated with the Golgi apparatus, cPLA2α activity was seen to be 87% inhibited. Relocation of cPLA2α to the cytoplasm and nucleus, and cPLA2α enzyme activity were required for cell cycle entry upon mechanical wounding of confluent monolayers. Thus, cPLA2α activity and function in controlling endothelial cell proliferation is regulated by reversible association with the Golgi apparatus. PMID:15930125

  14. At Least Three Transporters Likely Mediate Threonine Uptake Needed for Mouse Embryonic Stem Cell Proliferation

    PubMed Central

    Formisano, Tara M.; Van Winkle, Lon J.

    2016-01-01

    Stem cells are at the forefront of current regenerative and biomedical research. Thus, there exists an imperative and urgent need to understand the mechanisms that drive stem cell function in order to exploit their use as a therapeutic tool. Amino acids are potent inducers of signaling cascades that drive stem cell proliferation and differentiation. With a focus on mouse embryonic stem (mES) cells, Threonine (Thr) is the only amino acid required in culture media for mES cell proliferation. Current research associates this need for Thr with threonine dehydrogenase (TDH), which catabolizes Thr to glycine and acetyl-CoA in mES cells. This theory depends, in part, on the ability of 3- hydroxynorvaline (3-HNV) to inhibit both TDH and mES cell proliferation. However, the concentration of 3-HNV needed to inhibit mES cell proliferation is more than an order of magnitude less than its apparent Ki for TDH inhibition. Additionally, 3-HNV inhibits human embryonic stem (hES) cell proliferation, but hES cells do not express a functional tdh gene. Such findings indicate another mechanism for Thr stimulated mES and hES cell proliferation. Since amino acid transporters may be inducers of signaling cascades, we characterized the Thr transport systems in mES cells. We found that there is a Na+-dependent and a Na+-independent component of substrate-saturable transport, with the Na+-dependent component predominating. We also found that of 20 amino acids tested, the amino acids that were the strongest inhibitors of the Na+-dependent component of radiolabeled Thr transport were Ser, Cys, 4-OH-Pro, Asn, Met, and non-radiolabeled Thr itself. Such findings are consistent with characteristics of the ASC transport system, suggesting that this ASC system is responsible for the majority of Thr transport in mES cells. We confirmed expression of mRNA encoding the ASC system transporters, ASCT1 and ASCT2, in mES cells using RT-PCR. In conclusion, mES cells likely express at least three

  15. Domain of Dentine Sialoprotein Mediates Proliferation and Differentiation of Human Periodontal Ligament Stem Cells

    PubMed Central

    Yang, Guobin; Wang, Feng; Li, Wentong; Yang, Yuan; Guo, Feng; Gao, Qingping; Shoff, Lisa; Chen, Zhi; Gay, Isabel C.; Donly, Kevin J.; MacDougall, Mary; Chen, Shuo

    2013-01-01

    Classic embryological studies have documented the inductive role of root dentin on adjacent periodontal ligament differentiation.  The biochemical composition of root dentin includes collagens and cleavage products of dentin sialophosphoprotein (DSPP), such as dentin sialoprotein (DSP).  The high abundance of DSP in root dentin prompted us to ask the question whether DSP or peptides derived thereof would serve as potent biological matrix components to induce periodontal progenitors to further differentiate into periodontal ligament cells. Here, we test the hypothesis that domain of DSP influences cell fate. In situ hybridization and immunohistochemical analyses showed that the COOH-terminal DSP domain is expressed in mouse periodontium at various stages of root development. The recombinant COOH-terminal DSP fragment (rC-DSP) enhanced attachment and migration of human periodontal ligament stem cells (PDLSC), human primary PDL cells without cell toxicity. rC-DSP induced PDLSC cell proliferation as well as differentiation and mineralization of PDLSC and PDL cells by formation of mineralized tissue and ALPase activity. Effect of rC-DSP on cell proliferation and differentiation was to promote gene expression of tooth/bone-relate markers, transcription factors and growth factors. The results for the first time showed that rC-DSP may be one of the components of cell niche for stimulating stem/progenitor cell proliferation and differentiation and a natural scaffold for periodontal regeneration application. PMID:24400037

  16. MiR-15b mediates liver cancer cells proliferation through targeting BCL-2

    PubMed Central

    Zhang, Yuping; Huang, Feizhou; Wang, Jian; Peng, Lin; Luo, Hongwu

    2015-01-01

    The incidence and mortality of liver cancer increased year by year. Our country presents high incidence of liver cancer. MicroRNAs have tissue sensitivity as tumor biomarkers that play a role by promoting tumor growth as oncogenes or inhibit malignant cell growth as tumor suppressor genes. Studies showed that miR-15b abnormal expression in the tumor and can be treated as one of the tumor molecular markers. However, miR-15b expression and role in the liver cancer cells have not been elucidated. This study intended to explore the mechanism of miR-15b effect on liver cancer occurrence and development. Liver cancer cell line HepG2 was transfected with miR-15b mimic or inhibitor. Real-time PCR was applied to detect miR-15b expression. MTT was used to test cell proliferation. Transwell assay was performed to determine cell invasive ability. Real-time PCR and Western blot were used to detect BCL2 expression. MiR-15b mimic transfection promoted miR-15b overexpression and inhibited HepG2 cell proliferation significantly (P < 0.05). MiR-15b overexpression downregulated BCL2 mRNA and protein expression obviously (P < 0.05). On the contrary, miR-15b inhibitor transfection markedly reduced miR-15b expression in liver cancer cells (P < 0.05), promoted tumor cell proliferation, and increased BCL2 mRNA and protein expression. MiR-15b expression changes did not affect cell invasion (P > 0.05). MiR-15b can inhibit HepG2 cell proliferation and down-regulate BCL2 mRNA and protein expression. PMID:26884837

  17. microRNA 21-mediated suppression of Sprouty1 by Pokemon affects liver cancer cell growth and proliferation.

    PubMed

    Jin, Xiu-Li; Sun, Qin-Sheng; Liu, Feng; Yang, Hong-Wei; Liu, Min; Liu, Hong-Xia; Xu, Wei; Jiang, Yu-Yang

    2013-07-01

    Transcriptional repressor Pokemon is a critical factor in embryogenesis, development, cell proliferation, differentiation, and oncogenesis, thus behaving as an oncogene. Oncomine database suggests a potential correlation between the expressions of Pokemon and Sprouty1. This study investigated the regulatory role of Pokemon in Sprouty1 expression and the effect on liver cancer cell growth and proliferation, revealing a novel miR-21-mediated regulatory circuit. In normal (HL-7702) and cancer (QGY-7703) liver cell lines, Sprouty1 expression is inversely correlated with Pokemon levels. Targeted expression or siRNA-mediated silencing showed that Pokemon is a repressor of Sprouty1 expression at both mRNA and protein levels, but Pokemon cannot affect the promoter activity of Sprouty1. Sprouty1 is a target of miR-21 and interestingly, we found that miR-21 is up-regulated by Pokemon in liver cancer cells. Luciferase reporter assays showed that Pokemon up-regulated miR-21 transcription in a dose-dependent manner, and ChIP assay exhibited a direct binding of Pokemon to the miR-21 promoter at -747 to -399 bp. Site-directed mutagenesis of the GC boxes at -684 to -679 bp and -652 to -647 bp of miR-21 promoter abolished the regulatory activity by Pokemon. Furthermore, we found that the modulation of Pokemon and miR-21 expression affected the growth and proliferation of liver cancer cells QGY-7703. In summary, our findings demonstrate that Pokemon suppresses Sprouty1 expression through a miR-21-mediated mechanism, affecting the growth and proliferation of liver cancer cells. This study recognized miR-21 and Sprouty1 as novel targets of the Pokemon regulatory network. PMID:23355454

  18. Inhibitory effect of mimosine on proliferation of human lung cancer cells is mediated by multiple mechanisms.

    PubMed

    Chang, H C; Lee, T H; Chuang, L Y; Yen, M H; Hung, W C

    1999-10-18

    The plant amino acid mimosine has been reported to block cell cycle progression in the late G1 phase. A recent study showed that mimosine might induce growth arrest by activating the expression of p21CIP1, a cyclin-dependent kinase inhibitor (CDKI), and by inhibiting the activity of cyclin E-associated kinases in human breast cancer cells. However, mimosine at higher concentrations also blocked proliferation of p21-/- cells by unknown mechanisms. In this study, we investigated the effect of mimosine on the expression of cyclins and CDKIs in human lung cancer cells. We found that mimosine specifically inhibited cyclin D1 expression in H226 cells. The expression of another G1 cyclin, cyclin E, was not regulated by mimosine in all lung cancer cell lines examined. Moreover, mimosine induced p21CIP1 expression in H226 and H358 cells, while it activated p27KIP1 expression in H322 cells. However, mimosine does not affect transcription of these genes directly because significant changes in cyclin D1 or CDKI expression were observed at 12-24 h after drug addition. Our results indicate that mimosine may block cell proliferation by multiple mechanisms and this amino acid is a useful agent for the study of cell cycle control. PMID:10530763

  19. Annexin A6 regulates interleukin-2-mediated T-cell proliferation.

    PubMed

    Cornely, Rhea; Pollock, Abigail H; Rentero, Carles; Norris, Sarah E; Alvarez-Guaita, Anna; Grewal, Thomas; Mitchell, Todd; Enrich, Carlos; Moss, Stephen E; Parton, Robert G; Rossy, Jérémie; Gaus, Katharina

    2016-07-01

    Annexin A6 (AnxA6) has been implicated in cell signalling by contributing to the organisation of the plasma membrane. Here we examined whether AnxA6 regulates signalling and proliferation in T cells. We used a contact hypersensitivity model to immune challenge wild-type (WT) and AnxA6(-/-) mice and found that the in vivo proliferation of CD4(+) T cells, but not CD8(+) T cells, was impaired in AnxA6(-/-) relative to WT mice. However, T-cell migration and signalling through the T-cell receptor ex vivo was similar between T cells isolated from AnxA6(-/-) and WT mice. In contrast, interleukin-2 (IL-2) signalling was reduced in AnxA6(-/-) compared with WT T cells. Further, AnxA6-deficient T cells had reduced membrane order and cholesterol levels. Taken together, our data suggest that AnxA6 regulates IL-2 homeostasis and sensitivity in T cells by sustaining a lipid raft-like membrane environment. PMID:26853809

  20. IL-1β-induced, matrix metalloproteinase-3-regulated proliferation of embryonic stem cell-derived odontoblastic cells is mediated by the Wnt5 signaling pathway

    SciTech Connect

    Ozeki, Nobuaki; Hase, Naoko; Hiyama, Taiki; Yamaguchi, Hideyuki; Kawai, Rie; Kondo, Ayami; Nakata, Kazuhiko; Mogi, Makio

    2014-10-15

    We previously established a method for differentiating induced pluripotent stem cells and embryonic stem (ES) cells into α2 integrin-positive odontoblast-like cells. We also reported that interleukin (IL)-1β induces matrix metalloproteinase (MMP)-3-regulated cell proliferation and suppresses apoptosis in these cells, suggesting that MMP-3 plays a potentially unique physiological role in the regeneration of odontoblast-like cells. Here, we examined whether up-regulation of MMP-3 activity by IL-1β was mediated by Wnt signaling and led to increased proliferation of odontoblast-like cells. IL-1β increased mRNA and protein levels of Wnt5a, Wnt5b and the Wnt receptor Lrp5. Exogenous Wnt5a and Wnt5b were found to increase MMP-3 mRNA, protein and activity, and interestingly the rate of proliferation in these cells. Treatment with siRNAs against Wnt5a, Wnt5b and Lrp5 suppressed the IL-1β-induced increase in MMP-3 expression and suppressed cell proliferation, an effect rescued by application of exogenous Wnt5. These results demonstrate the sequential involvement of Wnt5, Lrp5 and MMP-3 in effecting IL-1β-induced proliferation of ES cell-derived odontoblast-like cells. - Highlights: • IL-1β induces Wnt5, Lrp5/Fzd9 and MMP-3 in ES cell-derived odontoblast-like cells. • IL-1β-induced Wnt5 expression results in increased cell proliferation. • Exogenous Wnt5 increases MMP-3 activity and cell proliferation. • Exogenous Wnt5 rescues IL-1β-driven proliferation with anti-Wnt5 siRNA suppression. • IL-1β-induced cell proliferation involves Wnt5, Lrp5, and MMP-3 sequentially.

  1. Inhibition of AGS Cancer Cell Proliferation following siRNA-Mediated Downregulation of VEGFR2

    PubMed Central

    Zarei Mahmudabadi, Ali; Masoomi Karimi, Masoomeh; Bahabadi, Majid; Bagheri Hoseinabadi, Zahra; JafariSani, Moslem; Ahmadi, Reza

    2016-01-01

    Objective Vascular endothelial growth factor (VEGF) and VEGF receptors (VEGFRs) play important roles in angiogenesis of different developmental mechanisms such as wound healing, embryogenesis and diseases, including different types of cancer. VEGFR2 is associated with cell proliferation, migration, and vascular permeability of endothelial cells. Blocking VEGF and its receptors is suggested as a therapeutic approach to prevent tumor growth. In this study, we aim to block VEGF signaling via small interfering RNA (siRNA) inhibition of VEGFR2. Materials and Methods In this experimental study, we used the RNA interference (RNAi) mechanism to suppress expression of the VEGFR2 gene. We conducted the 3-(4,5-di- methylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay, real-time polymerase chain reaction (PCR), Western blot, and flow cytometry analyses of VEGFR2 expression. Results Real-time PCR and Western blot results showed that VEGFR2 expression significantly downregulated. This suppression was followed by inhibition of cell prolifera- tion, reduction of viability, and induction of apoptosis in the cancer cells. Conclusion These findings suggest that VEGFR2 has a role in cell proliferation and tumor growth. Accordingly, it is suggested that VEGFR2 can be a therapeutic target for controlling tumor growth and proliferation. PMID:27602320

  2. VEGF-mediated angiogenesis stimulates neural stem cell proliferation and differentiation in the premature brain

    SciTech Connect

    Sun, Jinqiao; Sha, Bin; Zhou, Wenhao; Yang, Yi

    2010-03-26

    This study investigated the effects of angiogenesis on the proliferation and differentiation of neural stem cells in the premature brain. We observed the changes in neurogenesis that followed the stimulation and inhibition of angiogenesis by altering vascular endothelial growth factor (VEGF) expression in a 3-day-old rat model. VEGF expression was overexpressed by adenovirus transfection and down-regulated by siRNA interference. Using immunofluorescence assays, Western blot analysis, and real-time PCR methods, we observed angiogenesis and the proliferation and differentiation of neural stem cells. Immunofluorescence assays showed that the number of vWF-positive areas peaked at day 7, and they were highest in the VEGF up-regulation group and lowest in the VEGF down-regulation group at every time point. The number of neural stem cells, neurons, astrocytes, and oligodendrocytes in the subventricular zone gradually increased over time in the VEGF up-regulation group. Among the three groups, the number of these cells was highest in the VEGF up-regulation group and lowest in the VEGF down-regulation group at the same time point. Western blot analysis and real-time PCR confirmed these results. These data suggest that angiogenesis may stimulate the proliferation of neural stem cells and differentiation into neurons, astrocytes, and oligodendrocytes in the premature brain.

  3. Lentivirus-mediated shRNA targeting Nanog inhibits cell proliferation and attenuates cancer stem cell activities in breast cancer.

    PubMed

    Hu, Chun; Xu, Liang; Liang, Shujing; Zhang, Zhiying; Zhang, Yanyun; Zhang, Fengchun

    2016-06-01

    Emerging evidences suggest that cancer stem cells (CSCs) are responsible for tumor growth, metastasis and treatment resistance. Nanog is one of the transcription factors that are essential for stem cellular physiology process. Previous studies reported that Nanog was detected in breast cancer and other solid tumors and indicated that it has oncogenic characteristics. However, expression feature of Nanog in breast cancer stem cells (BCSCs) enriched population and its biological function in BCSCs is poorly understood. In this study, CD44 + CD24- fraction sorting with Fluorescence Activated Cell Sorter and mammosphere culture were used for enriching BCSCs. We report here that Nanog was highly expressed in CSCs-enriched population from the breast cancer cells, as well as stemness-associated genes. In addition, we employed the lentivirus-mediated shRNA targeting Nanog to investigate function of Nanog in BCSCs. We found that targeted inhibition of Nanog could suppress proliferation and colony formation in breast cancer cells. Further studies showed that targeted inhibition of Nanog resulted in a decrease of BCSCs activities, including mammosphere formation, CD44 + CD24- proportion and expressions of stemness-associated genes. These data therefore suggest that Nanog possesses important function in BCSCs and targeted inhibition of Nanog may provide a novel means of targeting and eliminating BCSCs. PMID:26339994

  4. Everolimus inhibits anti-HLA I antibody-mediated endothelial cell signaling, migration and proliferation more potently than sirolimus.

    PubMed

    Jin, Y-P; Valenzuela, N M; Ziegler, M E; Rozengurt, E; Reed, E F

    2014-04-01

    Antibody (Ab) crosslinking of HLA I molecules on the surface of endothelial cells triggers proliferative and pro-survival intracellular signaling, which is implicated in the process of chronic allograft rejection, also known as transplant vasculopathy (TV). The purpose of this study was to investigate the role of mammalian target of rapamycin (mTOR) in HLA I Ab-induced signaling cascades. Everolimus provides a tool to establish how the mTOR signal network regulates HLA I-mediated migration, proliferation and survival. We found that everolimus inhibits mTOR complex 1 (mTORC1) by disassociating Raptor from mTOR, thereby preventing class I-induced phosphorylation of mTOR, p70S6K, S6RP and 4E-BP1, and resultant class I-stimulated cell migration and proliferation. Furthermore, we found that everolimus inhibits class I-mediated mTORC2 activation (1) by disassociating Rictor and Sin1 from mTOR; (2) by preventing class I-stimulated Akt phosphorylation and (3) by preventing class I-mediated ERK phosphorylation. These results suggest that everolimus is more effective than sirolimus at antagonizing both mTORC1 and mTORC2, the latter of which is critical in endothelial cell functional changes leading to TV in solid organ transplantation after HLA I crosslinking. Our findings point to a potential therapeutic effect of everolimus in prevention of chronic Ab-mediated rejection. PMID:24580843

  5. T-helper cell-mediated proliferation and cytokine responses against recombinant Merkel cell polyomavirus-like particles.

    PubMed

    Kumar, Arun; Chen, Tingting; Pakkanen, Sari; Kantele, Anu; Söderlund-Venermo, Maria; Hedman, Klaus; Franssila, Rauli

    2011-01-01

    The newly discovered Merkel Cell Polyomavirus (MCPyV) resides in approximately 80% of Merkel cell carcinomas (MCC). Causal role of MCPyV for this rare and aggressive skin cancer is suggested by monoclonal integration and truncation of large T (LT) viral antigen in MCC cells. The mutated MCPyV has recently been found in highly purified leukemic cells from patients with chronic lymphocytic leukemia (CLL), suggesting a pathogenic role also in CLL. About 50-80% of adults display MCPyV-specific antibodies. The humoral immunity does not protect against the development of MCC, as neutralizing MCPyV antibodies occur in higher levels among MCC patients than healthy controls. Impaired T-cell immunity has been linked with aggressive MCC behavior. Therefore, cellular immunity appears to be important in MCPyV infection surveillance. In order to elucidate the role of MCPyV-specific Th-cell immunity, peripheral blood mononuclear cells (PBMC) of healthy adults were stimulated with MCPyV VP1 virus-like particles (VLPs), using human bocavirus (HBoV) VLPs and Candida albicans antigen as positive controls. Proliferation, IFN-γ, IL-13 and IL-10 responses were examined in 15 MCPyV-seropositive and 15 seronegative volunteers. With the MCPyV antigen, significantly stronger Th-cell responses were found in MCPyV-seropositive than MCPyV-seronegative subjects, whereas with the control antigens, the responses were statistically similar. The most readily detectable cytokine was IFN-γ. The MCPyV antigen tended to induce stronger IFN-γ responses than HBoV VLP antigen. Taken together, MCPyV-specific Th-cells elicit vigorous IFN-γ responses. IFN-γ being a cytokine with major antiviral and tumor suppressing functions, Th-cells are suggested to be important mediators of MCPyV-specific immune surveillance. PMID:21991346

  6. Fucosyltransferase 1 mediates angiogenesis, cell adhesion and rheumatoid arthritis synovial tissue fibroblast proliferation

    PubMed Central

    2014-01-01

    Introduction We previously reported that sialyl Lewisy, synthesized by fucosyltransferases, is involved in angiogenesis. Fucosyltransferase 1 (fut1) is an α(1,2)-fucosyltransferase responsible for synthesis of the H blood group and Lewisy antigens. However, the angiogenic involvement of fut 1 in the pathogenesis of rheumatoid arthritis synovial tissue (RA ST) has not been clearly defined. Methods Assay of α(1,2)-linked fucosylated proteins in RA was performed by enzyme-linked lectin assay. Fut1 expression was determined in RA ST samples by immunohistological staining. We performed angiogenic Matrigel assays using a co-culture system of human dermal microvascular endothelial cells (HMVECs) and fut1 small interfering RNA (siRNA) transfected RA synovial fibroblasts. To determine if fut1 played a role in leukocyte retention and cell proliferation in the RA synovium, myeloid THP-1 cell adhesion assays and fut1 siRNA transfected RA synovial fibroblast proliferation assays were performed. Results Total α(1,2)-linked fucosylated proteins in RA ST were significantly higher compared to normal (NL) ST. Fut1 expression on RA ST lining cells positively correlated with ST inflammation. HMVECs from a co-culture system with fut1 siRNA transfected RA synovial fibroblasts exhibited decreased endothelial cell tube formation compared to control siRNA transfected RA synovial fibroblasts. Fut1 siRNA also inhibited myeloid THP-1 adhesion to RA synovial fibroblasts and RA synovial fibroblast proliferation. Conclusions These data show that α(1,2)-linked fucosylated proteins are upregulated in RA ST compared to NL ST. We also show that fut1 in RA synovial fibroblasts is important in angiogenesis, leukocyte-synovial fibroblast adhesion, and synovial fibroblast proliferation, all key processes in the pathogenesis of RA. PMID:24467809

  7. Identification of chimeric antigen receptors that mediate constitutive or inducible proliferation of T cells.

    PubMed

    Frigault, Matthew J; Lee, Jihyun; Basil, Maria Ciocca; Carpenito, Carmine; Motohashi, Shinichiro; Scholler, John; Kawalekar, Omkar U; Guedan, Sonia; McGettigan, Shannon E; Posey, Avery D; Ang, Sonny; Cooper, Laurence J N; Platt, Jesse M; Johnson, F Brad; Paulos, Chrystal M; Zhao, Yangbing; Kalos, Michael; Milone, Michael C; June, Carl H

    2015-04-01

    This study compared second-generation chimeric antigen receptors (CAR) encoding signaling domains composed of CD28, ICOS, and 4-1BB (TNFRSF9). Here, we report that certain CARs endow T cells with the ability to undergo long-term autonomous proliferation. Transduction of primary human T cells with lentiviral vectors encoding some of the CARs resulted in sustained proliferation for up to 3 months following a single stimulation through the T-cell receptor (TCR). Sustained numeric expansion was independent of cognate antigen and did not require the addition of exogenous cytokines or feeder cells after a single stimulation of the TCR and CD28. Results from gene array and functional assays linked sustained cytokine secretion and expression of T-bet (TBX21), EOMES, and GATA-3 to the effect. Sustained expression of the endogenous IL2 locus has not been reported in primary T cells. Sustained proliferation was dependent on CAR structure and high expression, the latter of which was necessary but not sufficient. The mechanism involves constitutive signaling through NF-κB, AKT, ERK, and NFAT. The propagated CAR T cells retained a diverse TCR repertoire, and cellular transformation was not observed. The CARs with a constitutive growth phenotype displayed inferior antitumor effects and engraftment in vivo. Therefore, the design of CARs that have a nonconstitutive growth phenotype may be a strategy to improve efficacy and engraftment of CAR T cells. The identification of CARs that confer constitutive or nonconstitutive growth patterns may explain observations that CAR T cells have differential survival patterns in clinical trials. PMID:25600436

  8. VEGF induces proliferation of human hair follicle dermal papilla cells through VEGFR-2-mediated activation of ERK

    SciTech Connect

    Li, Wei; Man, Xiao-Yong; Li, Chun-Ming; Chen, Jia-Qi; Zhou, Jiong; Cai, Sui-Qing; Lu, Zhong-Fa; Zheng, Min

    2012-08-15

    Vascular endothelial growth factor (VEGF) is one of the strongest regulators of physiological and pathological angiogenesis. VEGF receptor 2 (VEGFR-2), the primary receptor for VEGF, is thought to mediate major functional effects of VEGF. Previously, we have localized both VEGF and VEGFR-2 in human hair follicles. In this study, we further defined the expression and roles of VEGFR-2 on human hair follicle dermal papilla (DP) cells. The expression of VEGFR-2 on DP cells was examined by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analysis separately, and localization of VEGFR-2 was defined by immunofluorescence. The effect of VEGF on DP cells was analyzed by MTT assays and specific inhibitors. Finally, the role of VEGF involved in the signaling pathways was investigated by Western blot. RT-PCR and Western blot analysis demonstrated the expression of VEGFR-2 on DP cells. Immunostaining for VEGFR-2 showed strong signal on cultured human DP cells in vitro. Exogenous VEGF{sub 165} stimulated proliferation of DP cells in a dose-dependent manner. Furthermore, this stimulation was blocked by a VEGFR-2 neutralizing antibody (MAB3571) and an ERK inhibitor (PD98059). VEGF{sub 165}-induced phosphorylation of ERK1/2 was abolished by MAB3571 and PD98059, while the phosphorylation of p38, JNK and AKT were not changed by VEGF{sub 165}. Taken together, VEGFR-2 is expressed on primary human hair follicle DP cells and VEGF induces proliferation of DP cells through VEGFR-2/ERK pathway, but not p38, JNK or AKT signaling. -- Highlights: Black-Right-Pointing-Pointer We examine the expression of VEGFR-2 on cultured human dermal papilla (DP) cells. Black-Right-Pointing-Pointer VEGF{sub 165} stimulated proliferation of human DP cells in a dose-dependent manner. Black-Right-Pointing-Pointer This stimulation was through VEGFR-2-mediated activation of ERK.

  9. VEGF-Mediated Proliferation of Human Adipose Tissue-Derived Stem Cells

    PubMed Central

    Sun, Chen; Li, Min; Zhou, Qing; Zhang, Chen; Huang, Jun; Qiu, Yu; Wen, Xiangyi; Zhang, Yan; Zhang, Yushan; Yang, Shuzhang; Lu, Lixia; Zhang, Jieping; Yuan, Qionglan; Lu, Jianwei; Xu, Guotong; Xue, Yunyun; Jin, Zibing; Jiang, Cizhong; Ying, Ming; Liu, Xiaoqing

    2013-01-01

    Human adipose tissue-derived stem cells (ADSCs) are an attractive multipotent stem cell source with therapeutic applicability across diverse fields for the repair and regeneration of acute and chronically damaged tissues. In recent years, there has been increasing interest in ADSC for tissue engineering applications. However, the mechanisms underlying the regulation of ADSC proliferation are not fully understood. Here we show that 47 transcripts are up-regulated while 23 are down-regulated in ADSC compared to terminally differentiated cells based on global mRNA profiling and microRNA profiling. Among the up-regulated genes, the expression of vascular endothelial growth factor (VEGF) is fine-tuned by miR-199a-5p. Further investigation indicates that VEGF accelerates ADSC proliferation whereas the multipotency of ADSC remains stable in terms of adipogenic, chondrogenic and osteogenic potentials after VEGF treatment, suggesting that VEGF may serve as an excellent supplement for accelerating ADSC proliferation during in vitro expansion. PMID:24098328

  10. CO-029 is overexpressed in gastric cancer and mediates the effects of EGF on gastric cancer cell proliferation and invasion.

    PubMed

    Zhu, Hongyu; Wu, Yulian; Zheng, Wen; Lu, Shiliu

    2015-03-01

    Tetraspanins are cell-surface glycoproteins and have received attention recently as both suppressors and promoters of metastasis. CO-029 is a member of the tetraspanin family and is implicated to be a metastasis-promoting tetraspanin in some cancers. However, the role of CO-029 in gastric cancer remains unexplored. The present study aimed to investigate the expression of CO-029 in gastric cancer tissues and to determine whether CO-029 is involved in the effects of epidermal growth factor (EGF) on gastric cancer cell proliferation and invasion. We collected clinical samples and found that the expression of CO-029 was increased both at the mRNA level and protein level in gastric cancer tissues in comparison to normal and tumor-adjacent tissues, as demonstrated by RT-qPCR and western blot analysis, respectively. Furthermore, we performed an in vitro experiment using AGS cells and observed that EGF promoted AGS cell proliferation and enhanced the invasion ability of the AGS cells, as shown by MTT assay and cell invasion assay, respectively. To the best of our knowledge, our results reveal for the first time, that CO-029 expression was affected by EGF in a concentration- time-dependent manner. The knockdown of CO-029 attenuated the effects of EGF on gastric cancer cell proliferation and invasion. These findings suggest that CO-029 is an oncogene in human gastric cancer and that CO-029 at least partially mediates the effects of EGF on gastric cancer cell proliferation and invasion. Our data may provide a novel target for therapeutic intervention in human gastric cancer. PMID:25592989

  11. Normal stroma suppresses cancer cell proliferation via mechanosensitive regulation of JMJD1a-mediated transcription.

    PubMed

    Kaukonen, Riina; Mai, Anja; Georgiadou, Maria; Saari, Markku; De Franceschi, Nicola; Betz, Timo; Sihto, Harri; Ventelä, Sami; Elo, Laura; Jokitalo, Eija; Westermarck, Jukka; Kellokumpu-Lehtinen, Pirkko-Liisa; Joensuu, Heikki; Grenman, Reidar; Ivaska, Johanna

    2016-01-01

    Tissue homeostasis is dependent on the controlled localization of specific cell types and the correct composition of the extracellular stroma. While the role of the cancer stroma in tumour progression has been well characterized, the specific contribution of the matrix itself is unknown. Furthermore, the mechanisms enabling normal-not cancer-stroma to provide tumour-suppressive signals and act as an antitumorigenic barrier are poorly understood. Here we show that extracellular matrix (ECM) generated by normal fibroblasts (NFs) is softer than the CAF matrix, and its physical and structural features regulate cancer cell proliferation. We find that normal ECM triggers downregulation and nuclear exit of the histone demethylase JMJD1a resulting in the epigenetic growth restriction of carcinoma cells. Interestingly, JMJD1a positively regulates transcription of many target genes, including YAP/TAZ (WWTR1), and therefore gene expression in a stiffness-dependent manner. Thus, normal stromal restricts cancer cell proliferation through JMJD1a-dependent modulation of gene expression. PMID:27488962

  12. Normal stroma suppresses cancer cell proliferation via mechanosensitive regulation of JMJD1a-mediated transcription

    PubMed Central

    Kaukonen, Riina; Mai, Anja; Georgiadou, Maria; Saari, Markku; De Franceschi, Nicola; Betz, Timo; Sihto, Harri; Ventelä, Sami; Elo, Laura; Jokitalo, Eija; Westermarck, Jukka; Kellokumpu-Lehtinen, Pirkko-Liisa; Joensuu, Heikki; Grenman, Reidar; Ivaska, Johanna

    2016-01-01

    Tissue homeostasis is dependent on the controlled localization of specific cell types and the correct composition of the extracellular stroma. While the role of the cancer stroma in tumour progression has been well characterized, the specific contribution of the matrix itself is unknown. Furthermore, the mechanisms enabling normal—not cancer—stroma to provide tumour-suppressive signals and act as an antitumorigenic barrier are poorly understood. Here we show that extracellular matrix (ECM) generated by normal fibroblasts (NFs) is softer than the CAF matrix, and its physical and structural features regulate cancer cell proliferation. We find that normal ECM triggers downregulation and nuclear exit of the histone demethylase JMJD1a resulting in the epigenetic growth restriction of carcinoma cells. Interestingly, JMJD1a positively regulates transcription of many target genes, including YAP/TAZ (WWTR1), and therefore gene expression in a stiffness-dependent manner. Thus, normal stromal restricts cancer cell proliferation through JMJD1a-dependent modulation of gene expression. PMID:27488962

  13. MMP13 mediates cell cycle progression in melanocytes and melanoma cells: in vitro studies of migration and proliferation

    PubMed Central

    2010-01-01

    Background Melanoma cells are usually characterized by a strong proliferative potential and efficient invasive migration. Among the multiple molecular changes that are recorded during progression of this disease, aberrant activation of receptor tyrosine kinases (RTK) is often observed. Activation of matrix metalloproteases goes along with RTK activation and usually enhances RTK-driven migration. The purpose of this study was to examine RTK-driven three-dimensional migration of melanocytes and the pro-tumorigenic role of matrix metalloproteases for melanocytes and melanoma cells. Results Using experimental melanocyte dedifferentiation as a model for early melanomagenesis we show that an activated EGF receptor variant potentiates migration through three-dimensional fibrillar collagen. EGFR stimulation also resulted in a strong induction of matrix metalloproteases in a MAPK-dependent manner. However, neither MAPK nor MMP activity were required for migration, as the cells migrated in an entirely amoeboid mode. Instead, MMPs fulfilled a function in cell cycle regulation, as their inhibition resulted in strong growth inhibition of melanocytes. The same effect was observed in the human melanoma cell line A375 after stimulation with FCS. Using sh- and siRNA techniques, we could show that MMP13 is the protease responsible for this effect. Along with decreased proliferation, knockdown of MMP13 strongly enhanced pigmentation of melanocytes. Conclusions Our data show for the first time that growth stimuli are mediated via MMP13 in melanocytes and melanoma, suggesting an autocrine MMP13-driven loop. Given that MMP13-specific inhibitors are already developed, these results support the evaluation of these inhibitors in the treatment of melanoma. PMID:20667128

  14. TRIM45 negatively regulates NF-{kappa}B-mediated transcription and suppresses cell proliferation

    SciTech Connect

    Shibata, Mio; Sato, Tomonobu; Nukiwa, Ryota; Ariga, Tadashi; Hatakeyama, Shigetsugu

    2012-06-22

    Highlights: Black-Right-Pointing-Pointer NF-{kappa}B plays an important role in cell survival and carcinogenesis. Black-Right-Pointing-Pointer TRIM45 negatively regulates TNF{alpha}-induced NF-{kappa}B-mediated transcription. Black-Right-Pointing-Pointer TRIM45 overexpression suppresses cell growth. Black-Right-Pointing-Pointer TRIM45 acts as a repressor for the NF-{kappa}B signal and regulates cell growth. -- Abstract: The NF-{kappa}B signaling pathway plays an important role in cell survival, immunity, inflammation, carcinogenesis, and organogenesis. Activation of NF-{kappa}B is regulated by several posttranslational modifications including phosphorylation, neddylation and ubiquitination. The NF-{kappa}B signaling pathway is activated by two distinct signaling mechanisms and is strictly modulated by the ubiquitin-proteasome system. It has been reported that overexpression of TRIM45, one of the TRIM family ubiquitin ligases, suppresses transcriptional activities of Elk-1 and AP-1, which are targets of the MAPK signaling pathway. In this study, we showed that TRIM45 also negatively regulates TNF{alpha}-induced NF-{kappa}B-mediated transcription by a luciferase reporter assay and that TRIM45 lacking a RING domain also has an activity to inhibit the NF-{kappa}B signal. Moreover, we found that TRIM45 overexpression suppresses cell growth. These findings suggest that TRIM45 acts as a repressor for the NF-{kappa}B signal and regulates cell growth.

  15. EGFR tyrosine kinase inhibitory peptide attenuates Helicobacter pylori-mediated hyper-proliferation in AGS enteric epithelial cells

    SciTech Connect

    Himaya, S.W.A.; Dewapriya, Pradeep; Kim, Se-Kwon

    2013-06-15

    Helicobacter pylori infection is one of the most critical causes of stomach cancer. The current study was conducted to explore the protective effects of an isolated active peptide H-P-6 (Pro-Gln-Pro-Lys-Val-Leu-Asp-Ser) from microbial hydrolysates of Chlamydomonas sp. against H. pylori-induced carcinogenesis. The peptide H-P-6 has effectively suppressed H. pylori-induced hyper-proliferation and migration of gastric epithelial cells (AGS). However, the peptide did not inhibit the viability of the bacteria or invasion into AGS cells. Therefore, the effect of the peptide on regulating H. pylori-induced molecular signaling was investigated. The results indicated that H. pylori activates the EGFR tyrosine kinase signaling and nuclear translocation of the β-catenin. The EGFR activation has led to the up-regulation of PI3K/Akt signaling pathway. Moreover, the nuclear translocation levels of β-catenin were significantly increased as a result of Akt mediated down-regulation of GSK3/β protein levels in the cytoplasm. Both of these consequences have resulted in increased expression of cell survival and migration related genes such as c-Myc, cyclin-D, MMP-2 and matrilysin. Interestingly, the isolated peptide potently inhibited H. pylori-mediated EGFR activation and thereby down-regulated the subsequent P13K/Akt signaling leading to β-catenin nuclear translocation. The effect of the peptide was confirmed with the use of EGFR tyrosine kinase inhibitor AG1487 and molecular docking studies. Collectively this study identifies a potent peptide which regulates the H. pylori-induced hyper-proliferation and migration of AGS cells at molecular level. - Highlights: • Chlamydomonas sp. derived peptide H-P-6 inhibits H. pylori-induced pathogenesis. • H-P-6 suppresses H. pylori-induced hyper-proliferation and migration of AGS cells. • The peptide inhibits H. pylori-induced EGFR activation.

  16. Herpesvirus ateles and herpesvirus saimiri transform marmoset T cells into continuously proliferating cell lines that can mediate natural killer cell-like cytotoxicity.

    PubMed Central

    Johnson, D R; Jondal, M

    1981-01-01

    Herpesvirus ateles (HVA) and herpesvirus saimiri (HVS) have the capacity to transform cotton-topped marmoset T lymphocytes into continuously proliferating cell lines that retain some functions associated with cell-mediated immunity. In the present paper, we demonstrate that HVA/HVS-transformed T cell lines are cytotoxic in a short-term 51Cr release assay and that this killing resembles killing by marmoset natural killer (NK) cells. The relationship between NK cells and HVA/HVS-transformed killer cell lines is discussed in view of present knowledge of the origin and function of the NK system. It is suggested that the described cytotoxic cell lines may be useful for further defining cellular cytotoxicity with regard to cell surface recognition, regulatory events, and lytic mechanisms. PMID:6273869

  17. ENO1 promotes tumor proliferation and cell adhesion mediated drug resistance (CAM-DR) in Non-Hodgkin's Lymphomas

    SciTech Connect

    Zhu, Xinghua; Miao, Xiaobing; Wu, Yaxun; Li, Chunsun; Guo, Yan; Liu, Yushan; Chen, Yali; Lu, Xiaoyun; Wang, Yuchan; He, Song

    2015-07-15

    Enolases are glycolytic enzymes responsible for the ATP-generated conversion of 2-phosphoglycerate to phosphoenolpyruvate. In addition to the glycolytic function, Enolase 1 (ENO1) has been reported up-regulation in several tumor tissues. In this study, we investigated the expression and biologic function of ENO1 in Non-Hodgkin's Lymphomas (NHLs). Clinically, by western blot analysis we observed that ENO1 expression was apparently higher in diffuse large B-cell lymphoma than in the reactive lymphoid tissues. Subsequently, immunohistochemical staining of 144 NHLs suggested that the expression of ENO1 was significantly lower in the indolent lymphomas compared with the progressive lymphomas. Further, we identified ENO1 as an independent prognostic factor, and it was significantly correlated with overall survival of NHL patients. In addition, we found that ENO1 could promote cell proliferation, regulate cell cycle associated gene and PI3K/AKT signaling pathway in NHLs. Finally, we verified that ENO1 participated in the process of lymphoma cell adhesion mediated drug resistance (CAM-DR). Adhesion to FN or HS5 cells significantly protected OCI-Ly8 and Daudi cells from cytotoxicity compared with those cultured in suspension, and these effects were attenuated when transfected with ENO1-siRNA. Based on the study, we propose that inhibition of ENO1 expression may be a novel strategy for therapy for NHLs patients, and it may be a target for drug resistance. - Highlights: • ENO1 expression is reversely correlated with clinical outcomes of patients with NHLs. • ENO1 promotes the proliferation of NHL cells. • ENO1 regulates cell adhesion mediated drug resistance.

  18. Down-Regulation of NDUFB9 Promotes Breast Cancer Cell Proliferation, Metastasis by Mediating Mitochondrial Metabolism.

    PubMed

    Li, Liang-Dong; Sun, He-Fen; Liu, Xue-Xiao; Gao, Shui-Ping; Jiang, Hong-Lin; Hu, Xin; Jin, Wei

    2015-01-01

    Despite advances in basic and clinical research, metastasis remains the leading cause of death in breast cancer patients. Genetic abnormalities in mitochondria, including mutations affecting complex I and oxidative phosphorylation, are found in breast cancers and might facilitate metastasis. Genes encoding complex I components have significant breast cancer prognostic value. In this study, we used quantitative proteomic analyses to compare a highly metastatic cancer cell line and a parental breast cancer cell line; and observed that NDUFB9, an accessory subunit of the mitochondrial membrane respiratory chain NADH dehydrogenase (complex I), was down-regulated in highly metastatic breast cancer cells. Furthermore, we demonstrated that loss of NDUFB9 promotes MDA-MB-231 cells proliferation, migration, and invasion because of elevated levels of mtROS, disturbance of the NAD+/NADH balance, and depletion of mtDNA. We also showed that, the Akt/mTOR/p70S6K signaling pathway and EMT might be involved in this mechanism. Thus, our findings contribute novel data to support the hypothesis that misregulation of mitochondrial complex I NADH dehydrogenase activity can profoundly enhance the aggressiveness of human breast cancer cells, suggesting that complex I deficiency is a potential and important biomarker for further basic research or clinical application. PMID:26641458

  19. Down-Regulation of NDUFB9 Promotes Breast Cancer Cell Proliferation, Metastasis by Mediating Mitochondrial Metabolism

    PubMed Central

    Gao, Shui-Ping; Jiang, Hong-Lin; Hu, Xin; Jin, Wei

    2015-01-01

    Despite advances in basic and clinical research, metastasis remains the leading cause of death in breast cancer patients. Genetic abnormalities in mitochondria, including mutations affecting complex I and oxidative phosphorylation, are found in breast cancers and might facilitate metastasis. Genes encoding complex I components have significant breast cancer prognostic value. In this study, we used quantitative proteomic analyses to compare a highly metastatic cancer cell line and a parental breast cancer cell line; and observed that NDUFB9, an accessory subunit of the mitochondrial membrane respiratory chain NADH dehydrogenase (complex I), was down-regulated in highly metastatic breast cancer cells. Furthermore, we demonstrated that loss of NDUFB9 promotes MDA-MB-231 cells proliferation, migration, and invasion because of elevated levels of mtROS, disturbance of the NAD+/NADH balance, and depletion of mtDNA. We also showed that, the Akt/mTOR/p70S6K signaling pathway and EMT might be involved in this mechanism. Thus, our findings contribute novel data to support the hypothesis that misregulation of mitochondrial complex I NADH dehydrogenase activity can profoundly enhance the aggressiveness of human breast cancer cells, suggesting that complex I deficiency is a potential and important biomarker for further basic research or clinical application. PMID:26641458

  20. mir-1-mediated paracrine effect of cancer-associated fibroblasts on lung cancer cell proliferation and chemoresistance.

    PubMed

    Li, Jianmin; Guan, Jing; Long, Xiaoping; Wang, Yang; Xiang, Xudong

    2016-06-01

    Lung cancer is the leading cause of cancer-related mortality in humans worldwide. Moreover, the overall 5-year survival rate is only 15%. Pathologically almost 80% of all lung cancer cases are non-small cell lung cancer (NSCLC). Cancer-associated fibroblasts (CAFs) have been found to exist in a large number of NSCLCs. CAFs have been proven to promote tumor progression, metastasis and resistance to therapy through paracrine effects in most solid tumors. In the present study, firstly we isolated CAFs from patient tissues and demonstrated that they promoted cell proliferation and chemoresistance to cisplatin in the lung cancer cell lines A549 and 95D in a paracrine manner. Secondly, using ELISA and quantative PCR, we found that a higher amount of stromal cell-derived factor 1 (SDF-1) existed in the CAFs rather than that observed in the normal fibroblasts (NFs). Thirdly, we detected that SDF-1 facilitated lung cancer cell proliferation and drug resistance via the CXCR4-mediated signaling pathway which involved NF-κB and Bcl-xL. Moreover, we also confirmed that the expression level of SDF-1 in the CAFs was negatively regulated by microRNA mir-1 through microRNA overexpression and quantitative PCR. Overall, our data provide one explanation for the effects of CAFs on lung cancer cells. Meanwhile, our results also suggest CAFs as a potential therapeutic target in tumor treatment. PMID:27035564

  1. TRPV1 mediates cellular uptake of anandamide and thus promotes endothelial cell proliferation and network-formation

    PubMed Central

    Hofmann, Nicole A.; Barth, Sonja; Waldeck-Weiermair, Markus; Klec, Christiane; Strunk, Dirk; Malli, Roland; Graier, Wolfgang F.

    2014-01-01

    ABSTRACT Anandamide (N-arachidonyl ethanolamide, AEA) is an endogenous cannabinoid that is involved in various pathological conditions, including cardiovascular diseases and tumor-angiogenesis. Herein, we tested the involvement of classical cannabinoid receptors (CBRs) and the Ca2+-channel transient receptor potential vanilloid 1 (TRPV1) on cellular AEA uptake and its effect on endothelial cell proliferation and network-formation. Uptake of the fluorescence-labeled anandamide (SKM4-45-1) was monitored in human endothelial colony-forming cells (ECFCs) and a human endothelial-vein cell line (EA.hy926). Involvement of the receptors during AEA translocation was determined by selective pharmacological inhibition (AM251, SR144528, CID16020046, SB366791) and molecular interference by TRPV1-selective siRNA-mediated knock-down and TRPV1 overexpression. We show that exclusively TRPV1 contributes essentially to AEA transport into endothelial cells in a Ca2+-independent manner. This TRPV1 function is a prerequisite for AEA-induced endothelial cell proliferation and network-formation. Our findings point to a so far unknown moonlighting function of TRPV1 as Ca2+-independent contributor/regulator of AEA uptake. We propose TRPV1 as representing a promising target for development of pharmacological therapies against AEA-triggered endothelial cell functions, including their stimulatory effect on tumor-angiogenesis. PMID:25395667

  2. IFNγ-mediated inhibition of cell proliferation through increased PKCδ-induced overexpression of EC-SOD

    PubMed Central

    Jeon, Yoon-Jae; Yoo, Hyun; Kim, Byung Hak; Lee, Yun Sang; Jeon, Byeongwook; Kim, Sung-Sub; Kim, Tae-Yoon

    2012-01-01

    Extracellular superoxide dismutase (EC-SOD) overexpression modulates cellular responses such as tumor cell suppression and is induced by IFNγ. Therefore, we examined the role of EC-SOD in IFNγ-mediated tumor cell suppression. We observed that the dominant-negative protein kinase C delta (PKCδ) suppresses IFNγ-induced EC-SOD expression in both keratinocytes and melanoma cells. Our results also showed that PKCδ-induced ECSOD expression was reduced by pretreatment with a PKCspecific inhibitor or a siRNA against PKCδ. PKCδ-induced ECSOD expression suppressed cell proliferations by the up-regulation of p21 and Rb, and the downregulation of cyclin A and D. Finally, we demonstrated that increased expression of EC-SOD drastically suppressed lung melanoma proliferation in an EC-SOD transgenic mouse via p21 expression. In summary, our findings suggest that IFNγ-induced EC-SOD expression occurs via activation of PKCδ. Therefore, the upregulation of EC-SOD may be effective for prevention of various cancers, including melanoma, via cell cycle arrest. [BMB Reports 2012; 45(11): 659-664] PMID:23187006

  3. The role of IGFBP-5 in mediating the anti-proliferation effect of tetrandrine in human colon cancer cells.

    PubMed

    Wu, Ke; Zhou, Mi; Wu, Qiu-Xiang; Yuan, Shuang-Xu; Wang, Dong-Xu; Jin, Jie-Li; Huang, Jun; Yang, Jun-Qin; Sun, Wen-Juan; Wan, Li-Hua; He, Bai-Cheng

    2015-03-01

    Colon cancer is one of the most common malignancies, causes considerable morbidity and mortality. The current treatment for colon cancer is more modest than had been hoped. There is an urgent clinical need to explore new agents or adjuvants for colon cancer treatment. Natural products and their derivates act as one of the major source for anticancer agent. In the present study, we investigated the anti-proliferation and chemoprevention effects of tetrandrine (Tet) on colon cancer cells to uncover the possible molecular basis of this effect. We found that Tet can inhibit proliferation and induce apoptosis in LoVo cells. With dimethylhydrazine (DMH) and dextran sodium sulfate (DSS) induced colon cancer model, we found that Tet can prevent or inhibit DMH plus DSS induced aberrant crypt foci (ACF) and colon cancer formation, as well as suppress tumor growth in the xenograft colon cancer model. Tet can downregulate the expression of IGFBP-5 in LoVo cells. Exogenous expression of IGFBP-5 can attenuate the anti-cancer activity of Tet, while IGFBP-5 knockdown potentiates this effect of Tet on LoVo cells. Tet can inhibit Wnt/β-catenin signaling transduction, which can be partly reversed by exogenous expression of IGFBP-5, but is enhanced by IGFBP-5 knockdown. Our results demonstrated that the anticancer activity of Tet in colon cancer cells may be mediated partly by downregulating the expression of IGFBP-5, thus inactivating Wnt/β-catenin signaling transduction. PMID:25524807

  4. Estrogen receptor beta (ERβ) mediates expression of β-catenin and proliferation in prostate cancer cell line PC-3.

    PubMed

    Lombardi, Ana Paola G; Pisolato, Raisa; Vicente, Carolina M; Lazari, Maria Fatima M; Lucas, Thaís F G; Porto, Catarina S

    2016-07-15

    The aim of the present study was to characterize the mechanism underlying estrogen effects on the androgen-independent prostate cancer cell line PC-3. 17β-estradiol and the ERβ-selective agonist DPN, but not the ERα-selective agonist PPT, increased the incorporation of [methyl-(3)H]thymidine and the expression of Cyclin D2, suggesting that ERβ mediates the proliferative effect of estrogen on PC-3 cells. In addition, upregulation of Cyclin D2 and incorporation of [methyl-(3)H]thymidine induced by 17β-estradiol and DPN were blocked by the ERβ-selective antagonist PHTPP in PC-3 cells. Upregulation of Cyclin D2 and incorporation of [methyl-(3)H]thymidine induced by DPN were also blocked by PKF118-310, a compound that disrupts β-catenin-TCF (T-cell-specific transcription factor) complex, suggesting the involvement of β-catenin in the estradiol effects in PC-3 cells. A diffuse immunostaining for non-phosphorylated β-catenin was detected in the cytoplasm of PC-3 cells. Low levels of non-phosphorylated β-catenin immunostaining were also detected near the plasma membrane and in nuclei. Treatment of PC-3 cells with 17β-estradiol or DPN markedly increased non-phosphorylated β-catenin expression. These effects were blocked by pretreatment with the ERβ-selective antagonist PHTPP, PI3K inhibitor Wortmannin or AKT inhibitor MK-2206, indicating that ERβ-PI3K/AKT mediates non-phosphorylated β-catenin expression. Cycloheximide blocked the DPN-induced upregulation of non-phosphorylated β-catenin, suggesting de novo synthesis of this protein. In conclusion, these results suggest that estrogen may play a role in androgen-independent prostate cancer cell proliferation through a novel pathway, involving ERβ-mediated activation of β-catenin. PMID:27107935

  5. NFAT5-mediated expression of S100A4 contributes to proliferation and migration of renal carcinoma cells.

    PubMed

    Küper, Christoph; Beck, Franz-Xaver; Neuhofer, Wolfgang

    2014-01-01

    The osmosensitive transcription factor nuclear factor of activated T-cells (NFAT) 5, also known as tonicity enhancer binding protein (TonEBP), has been associated with the development of a variety of tumor entities, among them breast cancer, colon carcinoma, and melanoma. The aim of the present study was to determine whether NFAT5 is also involved in the development of renal cell carcinoma (RCC). The most common type of RCC, the clear cell RCC, originates from the proximal convoluted tubule. We tested our hypothesis in the clear cell RCC cell line CaKi-1 and the non-cancerous proximal tubule cell line HK-2, as control. Basal expression of NFAT5 and NFAT5 activity in CaKi-1 cells was several times higher than in HK-2 cells. Osmotic stress induced an increased NFAT5 activity in both CaKi-1 and HK-2 cells, again with significantly higher activities in CaKi-1 cells. Analysis of NFAT5-regulating signaling pathways in CaKi-1 cells revealed that inhibition of the MAP kinases p38, c-Jun-terminal kinase (JNK) and extracellular regulated kinase (ERK) and of the focal adhesion kinase (FAK) partially blunted NFAT5 activity. FAK and ERK were both constitutively active, even under isotonic conditions, which may contribute to the high basal expression and activity of NFAT5 in CaKi-1 cells. In contrast, the MAP kinases p38 and JNK were inactive under isotonic conditions and became activated under osmotic stress conditions, indicating that p38 and JNK mediate upregulation of NFAT5 activity under these conditions. siRNA-mediated knockdown of NFAT5 in CaKi-1 cells reduced the expression of S100A4, a member of the S100 family of proteins, which promotes metastasis. Knockdown of NFAT5 was accompanied by a significant decrease in proliferation and migration activity. Taken together, our results indicate that NFAT5 induces S100A4 expression in CaKi-1 cells, thereby playing an important role in RCC proliferation and migration. PMID:25152734

  6. NFAT5-mediated expression of S100A4 contributes to proliferation and migration of renal carcinoma cells

    PubMed Central

    Küper, Christoph; Beck, Franz-Xaver; Neuhofer, Wolfgang

    2014-01-01

    The osmosensitive transcription factor nuclear factor of activated T-cells (NFAT) 5, also known as tonicity enhancer binding protein (TonEBP), has been associated with the development of a variety of tumor entities, among them breast cancer, colon carcinoma, and melanoma. The aim of the present study was to determine whether NFAT5 is also involved in the development of renal cell carcinoma (RCC). The most common type of RCC, the clear cell RCC, originates from the proximal convoluted tubule. We tested our hypothesis in the clear cell RCC cell line CaKi-1 and the non-cancerous proximal tubule cell line HK-2, as control. Basal expression of NFAT5 and NFAT5 activity in CaKi-1 cells was several times higher than in HK-2 cells. Osmotic stress induced an increased NFAT5 activity in both CaKi-1 and HK-2 cells, again with significantly higher activities in CaKi-1 cells. Analysis of NFAT5-regulating signaling pathways in CaKi-1 cells revealed that inhibition of the MAP kinases p38, c-Jun-terminal kinase (JNK) and extracellular regulated kinase (ERK) and of the focal adhesion kinase (FAK) partially blunted NFAT5 activity. FAK and ERK were both constitutively active, even under isotonic conditions, which may contribute to the high basal expression and activity of NFAT5 in CaKi-1 cells. In contrast, the MAP kinases p38 and JNK were inactive under isotonic conditions and became activated under osmotic stress conditions, indicating that p38 and JNK mediate upregulation of NFAT5 activity under these conditions. siRNA-mediated knockdown of NFAT5 in CaKi-1 cells reduced the expression of S100A4, a member of the S100 family of proteins, which promotes metastasis. Knockdown of NFAT5 was accompanied by a significant decrease in proliferation and migration activity. Taken together, our results indicate that NFAT5 induces S100A4 expression in CaKi-1 cells, thereby playing an important role in RCC proliferation and migration. PMID:25152734

  7. SCFβ-TRCP targets MTSS1 for ubiquitination-mediated destruction to regulate cancer cell proliferation and migration

    PubMed Central

    Tron, Adriana E.; Wang, Zhiwei; Sun, Liankun; Inuzuka, Hiroyuki; Wei, Wenyi

    2013-01-01

    Metastasis suppressor 1 (MTSS1) is an important tumor suppressor protein, and loss of MTSS1 expression has been observed in several types of human cancers. Importantly, decreased MTSS1 expression is associated with more aggressive forms of breast and prostate cancers, and with poor survival rate. Currently, it remains unclear how MTSS1 is regulated in cancer cells, and whether reduced MTSS1 expression contributes to elevated cancer cell proliferation and migration. Here we report that the SCFβ-TRCP regulates MTSS1 protein stability by targeting it for ubiquitination and subsequent destruction via the 26S proteasome. Notably, depletion of either Cullin 1 or β-TRCP1 led to increased levels of MTSS1. We further demonstrated a crucial role for Ser322 in the DSGXXS degron of MTSS1 in governing SCFβ-TRCP-mediated MTSS1 degradation. Mechanistically, we defined that Casein Kinase Iδ (CKIδ) phosphorylates Ser322 to trigger MTSS1's interaction with β-TRCP for subsequent ubiquitination and degradation. Importantly, introducing wild-type MTSS1 or a non-degradable MTSS1 (S322A) into breast or prostate cancer cells with low MTSS1 expression significantly inhibited cellular proliferation and migration. Moreover, S322A-MTSS1 exhibited stronger effects in inhibiting cell proliferation and migration when compared to ectopic expression of wild-type MTSS1. Therefore, our study provides a novel molecular mechanism for the negative regulation of MTSS1 by β-TRCP in cancer cells. It further suggests that preventing MTSS1 degradation could be a possible novel strategy for clinical treatment of more aggressive breast and prostate cancers. PMID:24318128

  8. The EIIIA domain from astrocyte‐derived fibronectin mediates proliferation of oligodendrocyte progenitor cells following CNS demyelination

    PubMed Central

    Stoffels, Josephine M. J.; Hoekstra, Dick; Franklin, Robin J. M.

    2014-01-01

    Central nervous system remyelination by oligodendrocyte progenitor cells (OPCs) ultimately fails in the majority of multiple sclerosis (MS) lesions. Remyelination benefits from transient expression of factors that promote migration and proliferation of OPCs, which may include fibronectin (Fn). Fn is present in demyelinated lesions in two major forms; plasma Fn (pFn), deposited following blood‐brain barrier disruption, and cellular Fn, synthesized by resident glial cells and containing alternatively spliced domains EIIIA and EIIIB. Here, we investigated the distinctive roles that astrocyte‐derived Fn (aFn) and pFn play in remyelination. We used an inducible Cre‐lox recombination strategy to selectively remove pFn, aFn or both from mice, and examined the impact on remyelination of toxin‐induced demyelinated lesions of spinal cord white matter. This approach revealed that astrocytes are a major source of Fn in demyelinated lesions. Furthermore, following aFn conditional knockout, the number of OPCs recruited to the demyelinated lesion decreased significantly, whereas OPC numbers were unaltered following pFn conditional knockout. However, remyelination completed normally following conditional knockout of aFn and pFn. Both the EIIIA and EIIIB domains of aFn were expressed following demyelination, and in vitro assays demonstrated that the EIIIA domain of aFn mediates proliferation of OPCs, but not migration. Therefore, although the EIIIA domain from aFn mediates OPC proliferation, aFn is not essential for successful remyelination. Since previous findings indicated that astrocyte‐derived Fn aggregates in chronic MS lesions inhibit remyelination, aFn removal may benefit therapeutic strategies to promote remyelination in MS. GLIA 2015;63:242–256 PMID:25156142

  9. Effects of RNA interference-mediated NRP-1 silencing on the proliferation and apoptosis of breast cancer cells.

    PubMed

    Han, Zhengxiang; Jiang, Guan; Zhang, Yingying; Xu, Jie; Chen, Chong; Zhang, Lansheng; Xu, Zhenyuan; Du, Xiuping

    2015-07-01

    Lentiviral expression vectors carrying human NRP-1 short hairpin RNA (shRNA) were constructed and selected to present highly efficient NRP-1/shRNA interference sequences, in order to investigate the effects of RNA interference (RNAi)-mediated NRP-1 silencing on the biological activities of breast cancer cells. Three pairs of human NRP-1 targeted specific interference sequences and one pair of non-specific control sequences were designed, synthesized and subcloned into pLB lentiviral vectors, which were further identified by polymerase chain reaction (PCR) and sequencing. Recombinant and lentiviral packaging plasmids were co-transfected into 293FT cell lines in order to produce lentiviral particles and to infect breast cancer cells with high NRP-1 expression. Flow cytometry was used to sort green fluorescent protein-positive cells. Fluorescence quantitative-reverse transcription-PCR and western blot analysis were employed to identify the interference silencing sequence with the most efficient silencing profile. A cell counting kit-8 assay and an Annexin V-propidium iodide method in combination with flow cytometry were used to examine the effects of RNA interference-mediated NRP-1 gene silencing on cell proliferation, apoptosis and sensitivity to chemotherapy. The recombinant lentiviral plasmid pLB-NRP-1/shRNA was constructed successfully, as confirmed by PCR and sequencing. After the infection of recombinant lentiviral plasmids, the expression profiles of NRP-1 mRNA, and proteins of MCF-7 and SK-BR-3 cell-specific interference group (pLB-NRP-1/shRNA3) were significantly lower than that of the control group (P<0.05). Compared with the control group, the MCF-7 and SK-BR-3 cell-specific interference group (pLB-NRP-1/shRNA3) showed lower optical density values and higher apoptotic rates at 48, 72 and 96 h; these differences were statistically significant (P<0.05). EPI administration resulted in increased apoptosis in the MCF-7 and SK-BR-3 cell-specific interference

  10. Claudin-18 inhibits cell proliferation and motility mediated by inhibition of phosphorylation of PDK1 and Akt in human lung adenocarcinoma A549 cells.

    PubMed

    Shimobaba, Shun; Taga, Saeko; Akizuki, Risa; Hichino, Asami; Endo, Satoshi; Matsunaga, Toshiyuki; Watanabe, Ryo; Yamaguchi, Masahiko; Yamazaki, Yasuhiro; Sugatani, Junko; Ikari, Akira

    2016-06-01

    Abnormal expression of claudin subtypes has been reported in various cancers. However, the pathological role of each claudin has not been clarified in detail. Claudin-18 was absent in human non-small cell and small cell lung cancers, although it is expressed in normal lung tissues. Here, we examined the effect of claudin-18 expression on the expression of junctional proteins, cell proliferation, and cell motility using human lung adenocarcinoma A549 cells. Real-time PCR and western blotting showed that exogenous expression of claudin-18 had no effect on the expression of junctional proteins including claudin-1, zonula occludens-1 (ZO-1), occludin, and E-cadherin. Claudin-18 was mainly distributed in cell-cell contact areas concomitant with ZO-1. Cell proliferation was significantly decreased at 48 and 72h after seeding of claudin 18-expressing cells. Claudin-18 suppressed cell motility, whereas it increased cell death in anoikis. Claudin-18 decreased phosphorylated (p)-3-phosphoinositide-dependent protein kinase-1 (PDK1) and p-Akt levels without affecting p-epidermal growth factor receptor and p-phosphatidylinositol-3 kinase (PI3K) levels. Furthermore, claudin-18 was bound with PDK1 and suppressed the nuclear localization of PDK1. We suggest that claudin-18 suppresses the abnormal proliferation and motility of lung epithelial cells mediated by inhibition of the PI3K/PDK1/Akt signaling pathway. PMID:26919807

  11. Phenylboronic acid-functionalized polyamidoamine-mediated Bcl-2 siRNA delivery for inhibiting the cell proliferation.

    PubMed

    Wu, Di; Yang, Jiebing; Xing, Zhen; Han, Haobo; Wang, Tingting; Zhang, Aijun; Yang, Yan; Li, Quanshun

    2016-10-01

    In this study, the conjugation of phenylboronic acid (PBA) to amine-terminated polyamidoamine (PAMAM) was successfully conducted to prepare a tumor-targeted gene carrier PBA-functionalized PAMAM (PPP) for Bcl-2 siRNA delivery, using a heterobifunctional crosslinker NHS-PEG5k-Mal. The carrier possessed favorable capacity for siRNA condensation and could protect siRNA from the degradation against RNase and serum. The introduction of PBA could facilitate the cellular uptake and further transfection of Bcl-2 siRNA demonstrated by confocal laser scanning microscopy and flow cytometry. Meanwhile, PPP-mediated transfection of Bcl-2 siRNA could significantly inhibit the expression of Bcl-2 gene at both mRNA and protein levels. Furthermore, owing to the knock-down of Bcl-2, PPP/siRNA could significantly inhibit the cell proliferation by inducing the cell apoptosis, and also enhance the antitumor efficiency of doxorubicin by suppressing the resistance of tumor cells to chemotherapeutics. In conclusion, the PPP-mediated Bcl-2 siRNA delivery could potentially be an effective platform for solving the drug resistance and further achieving the combined chemotherapy and gene therapy in tumor treatment. PMID:27371891

  12. Permeability Transition Pore-Mediated Mitochondrial Superoxide Flashes Mediate an Early Inhibitory Effect of Aβ1–42 on Neural Progenitor Cell Proliferation

    PubMed Central

    Hou, Yan; Ghosh, Paritosh; Wan, Ruiqian; Ouyang, Xin; Cheng, Heping; Mattson, Mark P.; Cheng, Aiwu

    2013-01-01

    Cellular damage by reactive oxygen species (ROS) and altered neurogenesis are implicated in the etiology of AD and the pathogenic actions of amyloid β-peptide (Aβ); the underlying mechanisms and the early oxidative intracellular events triggered by Aβ are not established. In the present study, we found that mouse embryonic cortical neural progenitor cells exhibit intermittent spontaneous mitochondrial superoxide (SO) flashes that require transient opening of mitochondrial permeability transition pores (mPTPs). The incidence of mitochondria SO flash activity in NPCs increased during the first 6 – 24 hours of exposure to aggregating amyloid β-peptide (Aβ1-42), indicating an increase in transient mPTP opening. Subsequently, the SO flash frequency progressively decreased and ceased between 48 and 72 hours of exposure to Aβ1-42, during which time global cellular ROS increased, mitochondrial membrane potential decreased, cytochrome C was released from mitochondria and the cells degenerated. Inhibition of mPTPs and selective reduction in mitochondrial SO flashes significantly ameliorated the negative effects of Aβ1-42 on NPC proliferation and survival. Our findings suggest that mPTP-mediated bursts of mitochondrial SO production is a relatively early and pivotal event in the adverse effects of Aβ1-42 on NPCs. If Aβ inhibits NPC proliferation in the brains of AD patients by a similar mechanism, then interventions that inhibit mPTP-mediated superoxide flashes would be expected to protect NPCs against the adverse effects of Aβ. PMID:24325797

  13. SPRY1 promotes the degradation of uPAR and inhibits uPAR-mediated cell adhesion and proliferation

    PubMed Central

    Liu, Xiufeng; Lan, Yan; Zhang, Di; Wang, Kai; Wang, Yao; Hua, Zi-Chun

    2014-01-01

    Urokinase plasminogen activator receptor (uPAR) is a GPI anchored cell surface protein that is closely associated with invasion, migration, and metastasis of cancer cells. Many functional extracellular proteins and transmembrane receptors interact with uPAR. However, few studies have examined the association of uPAR with cytoplasm proteins. We previously used yeast two-hybrid screening to isolate several novel uPAR-interacting cytoplasmic proteins, including Sprouty1 (SPRY1), an inhibitor of the (Ras-mitogen-activated protein kinase) MAPK pathway. In this study, we show that SPRY1 interacts with uPAR and directs it toward lysosomal-mediated degradation. Overexpression of SPRY1 decreased the cell surface and cytoplasmic uPAR protein level. Moreover, SPRY1 overexpression augmented uPAR-induced cell adhesion to vitronectin as well as proliferation of cancer cells. Our results also further support the critical role of SPRY1 contribution to tumor growth. In a subcutaneous tumor model, overexpression of SPRY1 in HCT116 or A549 xenograft in athymic nude mice led to great suppression of tumor growth. These results show that SPRY1 may affect tumor cell function through direct interaction with uPAR and promote its lysosomal degradation. PMID:25520860

  14. Nicotine promotes cell proliferation via {alpha}7-nicotinic acetylcholine receptor and catecholamine-synthesizing enzymes-mediated pathway in human colon adenocarcinoma HT-29 cells

    SciTech Connect

    Wong, Helen Pui Shan; Yu Le; Lam, Emily Kai Yee; Tai, Emily Kin Ki; Wu, William Ka Kei; Cho, Chi Hin . E-mail: chcho@cuhk.edu.hk

    2007-06-15

    Cigarette smoking has been implicated in colon cancer. Nicotine is a major alkaloid in cigarette smoke. In the present study, we showed that nicotine stimulated HT-29 cell proliferation and adrenaline production in a dose-dependent manner. The stimulatory action of nicotine was reversed by atenolol and ICI 118,551, a {beta}{sub 1}- and {beta}{sub 2}-selective antagonist, respectively, suggesting the role of {beta}-adrenoceptors in mediating the action. Nicotine also significantly upregulated the expression of the catecholamine-synthesizing enzymes [tyrosine hydroxylase (TH), dopamine-{beta}-hydroxylase (D{beta}H) and phenylethanolamine N-methyltransferase]. Inhibitor of TH, a rate-limiting enzyme in the catecholamine-biosynthesis pathway, reduced the actions of nicotine on cell proliferation and adrenaline production. Expression of {alpha}7-nicotinic acetylcholine receptor ({alpha}7-nAChR) was demonstrated in HT-29 cells. Methyllycaconitine, an {alpha}7-nAChR antagonist, reversed the stimulatory actions of nicotine on cell proliferation, TH and D{beta}H expression as well as adrenaline production. Taken together, through the action on {alpha}7-nAChR nicotine stimulates HT-29 cell proliferation via the upregulation of the catecholamine-synthesis pathway and ultimately adrenaline production and {beta}-adrenergic activation. These data reveal the contributory role {alpha}7-nAChR and {beta}-adrenoceptors in the tumorigenesis of colon cancer cells and partly elucidate the carcinogenic action of cigarette smoke on colon cancer.

  15. GPER mediates estrogen-induced signaling and proliferation in human breast epithelial cells and normal and malignant breast.

    PubMed

    Scaling, Allison L; Prossnitz, Eric R; Hathaway, Helen J

    2014-06-01

    17β-Estradiol (estrogen), through receptor binding and activation, is required for mammary gland development. Estrogen stimulates epithelial proliferation in the mammary gland, promoting ductal elongation and morphogenesis. In addition to a developmental role, estrogen promotes proliferation in tumorigenic settings, particularly breast cancer. The proliferative effects of estrogen in the normal breast and breast tumors are attributed to estrogen receptor α. Although in vitro studies have demonstrated that the G protein-coupled estrogen receptor (GPER, previously called GPR30) can modulate proliferation in breast cancer cells both positively and negatively depending on cellular context, its role in proliferation in the intact normal or malignant breast remains unclear. Estrogen-induced GPER-dependent proliferation was assessed in the immortalized nontumorigenic human breast epithelial cell line, MCF10A, and an ex vivo organ culture model employing human breast tissue from reduction mammoplasty or tumor resections. Stimulation by estrogen and the GPER-selective agonist G-1 increased the mitotic index in MCF10A cells and proportion of cells in the cell cycle in human breast and breast cancer explants, suggesting increased proliferation. Inhibition of candidate signaling pathways that may link GPER activation to proliferation revealed a dependence on Src, epidermal growth factor receptor transactivation by heparin-bound EGF and subsequent ERK phosphorylation. Proliferation was not dependent on matrix metalloproteinase cleavage of membrane-bound pro-HB-EGF. The contribution of GPER to estrogen-induced proliferation in MCF10A cells and breast tissue was confirmed by the ability of GPER-selective antagonist G36 to abrogate estrogen- and G-1-induced proliferation, and the ability of siRNA knockdown of GPER to reduce estrogen- and G-1-induced proliferation in MCF10A cells. This is the first study to demonstrate GPER-dependent proliferation in primary normal and malignant

  16. GPER mediates estrogen-induced signaling and proliferations in human breast epithelial cells, and normal and malignant breast

    PubMed Central

    Scaling, Allison L.

    2014-01-01

    17β-estradiol (estrogen), through receptor binding and activation, is required for mammary gland development. Estrogen stimulates epithelial proliferation in the mammary gland, promoting ductal elongation and morphogenesis. In addition to a developmental role, estrogen promotes proliferation in tumorigenic settings, particularly breast cancer. The proliferative effects of estrogen in the normal breast and breast tumors are attributed to estrogen receptor α. Although in vitro studies have demonstrated that the G protein-coupled estrogen receptor (GPER, previously called GPR30) can modulate proliferation in breast cancer cells both positively and negatively depending on cellular context, its role in proliferation in the intact normal or malignant breast remains unclear. Estrogen-induced GPER-dependent proliferation was assessed in the immortalized non-tumorigenic human breast epithelial cell line, MCF10A, and an ex vivo organ culture model employing human breast tissue from reduction mammoplasty or tumor resections. Stimulation by estrogen and the GPER-selective agonist G-1 increased the mitotic index in MCF10A cells and proportion of cells in the cell cycle in human breast and breast cancer explants, suggesting increased proliferation. Inhibition of candidate signaling pathways that may link GPER activation to proliferation revealed a dependence on Src, epidermal growth factor receptor transactivation by heparin-bound EGF and subsequent ERK phosphorylation. Proliferation was not dependent on matrix metalloproteinase cleavage of membrane bound pro-HB-EGF. The contribution of GPER to estrogen-induced proliferation in MCF10A cells and breast tissue was confirmed by the ability of GPER-selective antagonist G36 to abrogate estrogen- and G-1-induced proliferation, and the ability of siRNA knockdown of GPER to reduce estrogen- and G-1-induced proliferation in MCF10A cells. This is the first study to demonstrate GPER-dependent proliferation in primary normal and malignant

  17. Suppression of Human T Cell Proliferation Mediated by the Cathepsin B Inhibitor, z-FA-FMK Is Due to Oxidative Stress

    PubMed Central

    Rajah, Tanuja; Chow, Sek Chuen

    2015-01-01

    The cathepsin B inhibitor, benzyloxycarbonyl-phenylalanine-alanine-fluoromethyl ketone (z-FA-FMK) readily inhibits anti-CD3-induced human T cell proliferation, whereas the analogue benzyloxycarbonyl-phenylalanine-alanine-diazomethyl ketone (z-FA-DMK) had no effect. In contrast, benzyloxycarbonyl-phenylalanine-alanine-chloromethyl ketone (z-FA-CMK) was toxic. The inhibition of T cell proliferation mediated by z-FA-FMK requires not only the FMK moiety, but also the benzyloxycarbonyl group at the N-terminal, suggesting some degree of specificity in z-FA-FMK-induced inhibition of primary T cell proliferation. We showed that z-FA-FMK treatment leads to a decrease in intracellular glutathione (GSH) with a concomitant increase in reactive oxygen species (ROS) levels in activated T cells. The inhibition of anti-CD3-induced T cell proliferation mediated by z-FA-FMK was abolished by the presence of low molecular weight thiols such as GSH, N-acetylcysteine (NAC) and L-cysteine, whereas D-cysteine which cannot be metabolised to GSH has no effect. The inhibition of anti-CD3-induced up-regulation of CD25 and CD69 expression mediated by z-FA-FMK was also attenuated in the presence of exogenous GSH. Similar to cell proliferation, GSH, NAC and L-cysteine but not D-cysteine, completely restored the processing of caspase-8 and caspase-3 to their respective subunits in z-FA-FMK-treated activated T cells. Our collective results demonstrated that the inhibition of T cell activation and proliferation mediated by z-FA-FMK is due to oxidative stress via the depletion of GSH. PMID:25915766

  18. Dendritic Cell-Mediated T Cell Proliferation -A Functional Bioindicator of Inflammatory Source-Specific Particulate Matter

    EPA Science Inventory

    Previously we found that dendritic cells (DC) were sensitive functional bioindicators of ambient PM (APM) exposure mediating Th2-allergic inflammation in the draining lymph nodes. Here, the ability of bone-marrow-derived DC (DC) and putative BM-derived basophils (Ba) to present a...

  19. Lentivirus-mediated knockdown of eukaryotic translation initiation factor 3 subunit D inhibits proliferation of HCT116 colon cancer cells.

    PubMed

    Yu, Xiaojun; Zheng, Bo'an; Chai, Rui

    2014-01-01

    Dysregulation of protein synthesis is emerging as a major contributory factor in cancer development. eIF3D (eukaryotic translation initiation factor 3 subunit D) is one member of the eIF3 (eukaryotic translation initiation factor 3) family, which is essential for initiation of protein synthesis in eukaryotic cells. Acquaintance with eIF3D is little since it has been identified as a dispensable subunit of eIF3 complex. Recently, eIF3D was found to embed somatic mutations in human colorectal cancers, indicating its importance for tumour progression. To further probe into its action in colon cancer, we utilized lentivirus-mediated RNA interference to knock down eIF3D expression in one colon cancer cell line HCT116. Knockdown of eIF3D in HCT116 cells significantly inhibited cell proliferation and colony formation in vitro. Flow cytometry analysis indicated that depletion of eIF3D led to cell-cycle arrest in the G2/M phase, and induced an excess accumulation of HCT116 cells in the sub-G1 phase representing apoptotic cells. Signalling pathways responsible for cell growth and apoptosis have also been found altered after eIF3D silencing, such as AMPKα (AMP-activated protein kinase alpha), Bad, PRAS40 [proline-rich Akt (PKB) substrate of 40 kDa], SAPK (stress-activated protein kinase)/JNK (c-Jun N-terminal kinase), GSK3β and PARP [poly(ADP-ribose) polymerase]. Taken together, these findings suggest that eIF3D might play an important role in colon cancer progression. PMID:25370813

  20. Increased levels of palmitoylethanolamide and other bioactive lipid mediators and enhanced local mast cell proliferation in canine atopic dermatitis

    PubMed Central

    2014-01-01

    Background Despite the precise pathogenesis of atopic dermatitis (AD) is unknown, an immune dysregulation that causes Th2-predominant inflammation and an intrinsic defect in skin barrier function are currently the two major hypotheses, according to the so-called outside-inside-outside model. Mast cells (MCs) are involved in AD both by releasing Th2 polarizing cytokines and generating pruritus symptoms through release of histamine and tryptase. A link between MCs and skin barrier defects was recently uncovered, with histamine being found to profoundly contribute to the skin barrier defects. Palmitoylethanolamide and related lipid mediators are endogenous bioactive compounds, considered to play a protective homeostatic role in many tissues: evidence collected so far shows that the anti-inflammatory effect of palmitoylethanolamide depends on the down-modulation of MC degranulation. Based on this background, the purpose of the present study was twofold: (a) to determine if the endogenous levels of palmitoylethanolamide and other bioactive lipid mediators are changed in the skin of AD dogs compared to healthy animals; (b) to examine if MC number is increased in the skin of AD dogs and, if so, whether it depends on MC in-situ proliferation. Results The amount of lipid extract expressed as percent of biopsy tissue weight was significantly reduced in AD skin while the levels of all analyzed bioactive lipid mediators were significantly elevated, with palmitoylethanolamide showing the highest increase. In dogs with AD, the number of MCs was significantly increased in both the subepidermal and the perifollicular compartments and their granule content was significantly decreased in the latter. Also, in situ proliferation of MCs was documented. Conclusions The levels of palmitoylethanolamide and other bioactive lipid mediators were shown to increase in AD skin compared to healthy samples, leading to the hypothesis that they may be part of the body’s innate mechanisms to

  1. Toll-like receptors 2 and 4 mediate the capacity of mesenchymal stromal cells to support the proliferation and differentiation of CD34{sup +} cells

    SciTech Connect

    Wang, Xingbing; Cheng, Qiansong; Li, Lailing; Wang, Jian; Xia, Liang; Xu, Xiucai; Sun, Zimin

    2012-02-01

    Bone marrow derived-mesenchymal stromal cells (BM-MSCs) are multipotent, nonhematopoietic progenitors in a hematopoietic microenvironment and indispensable for regulating hematopoiesis. Several studies have reported that toll-like receptors (TLRs) are expressed in mesenchymal stromal cells (MSCs) to modulate their biological functions. In this study, we investigated the possible role(s) of TLRs in mediating the hematopoiesis-supporting role of human BM-MSCs. Human BM-MSCs were analyzed for mRNA expression of TLR1-10 by reverse transcription-polymerase chain reaction. TLR1-6, but not TLR7-10 were expressed by BM-MSCs. The protein expression of TLR2 and TLR4 was also confirmed by flow cytometry. We further explored the role of TLR2 and TLR4 in mediating the capacity of BM-MSCs to support the proliferation and differentiation of CD34{sup +} hematopoietic stem/progenitor cells obtained from cord blood. BM-MSCs increased proliferation of CD34{sup +} cells and promoted the differentiation towards the myeloid lineage 7 or 14 days after co-culture, as well as colony formation by those cells and the production of interleukin 1 (IL-1), IL-8, IL-11, stem cell factor (SCF), granulocyte colony-stimulating factor (CSF), macrophage CSF and granulocyte-macrophage CSF, if MSCs had been stimulated with TLR2 agonist (PAM{sub 3}CSK{sub 4}) or TLR4 agonist (LPS). Interestingly, although these effects were elevated in a different degree, a synergistic effect was not observed in BM-MSCs co-stimulated with PAM{sub 3}CSK{sub 4} and LPS. Together, our findings suggest that TLR2 and TLR4 signaling may indirectly regulate hematopoiesis by modulating BM-MSCs' functions. The increased hematopoietic proliferation and differentiation could be mediated, at least in part, by augmented hematopoiesis-related cytokine production of BM-MSCs.

  2. Anti-inflammatory activity of chloroquine and amodiaquine through p21-mediated suppression of T cell proliferation and Th1 cell differentiation.

    PubMed

    Oh, Sera; Shin, Ji Hyun; Jang, Eun Jung; Won, Hee Yeon; Kim, Hyo Kyeong; Jeong, Mi-Gyeong; Kim, Kwang Soo; Hwang, Eun Sook

    2016-05-27

    Chloroquine (CQ) and amodiaquine (AQ) have been used for treating or preventing malaria for decades, and their application has expanded into treating inflammatory disease in humans. CQ and AQ are applicable for controlling rheumatoid arthritis, but their molecular mechanisms of anti-inflammatory activity remain to be elucidated. In this study, we examined the effects of CQ and AQ on T cell activation and T cell-mediated immune response. CQ had no significant effect on T cell numbers, but decreased the population of T cells with a high division rate. However, AQ treatment significantly increased the number of cells with low division rates and eliminated cells with high division rates, resulting in the inhibition of T cell proliferation triggered by T cell receptor stimulation, of which inhibition occurred in developing effector T helper and regulatory T cells, regardless of the different exogenous cytokines. Interestingly, the cyclin-dependent kinase inhibitor p21 was significantly and dose-dependently increased by CQ, and more potently by AQ, while other cell cycle regulators were unchanged. Both CQ and AQ elevated the transcription level of p21 though the activation of p53, but also blocked p21 protein degradation in the presence of cycloheximide, causing p21 protein accumulation mainly in the nucleus. Sustained treatment of developing T cells with either CQ or AQ suppressed IFN-γ production in a dose dependent manner and potently inhibited the differentiation of IFN-γ-producing Th1 cells. These results demonstrate that CQ and AQ increase the expression level of p21 and inhibit T cell proliferation and the development of IFN-γ-producing Th1 cells, thereby revealing beneficial roles in treating a wide range of chronic inflammatory diseases mediated by inflammatory T cells. PMID:27109480

  3. Protein kinase A-mediated cell proliferation in brown preadipocytes is independent of Erk1/2, PI{sub 3}K and mTOR

    SciTech Connect

    Wang, Yanling; Sato, Masaaki; Guo, Yuan; Bengtsson, Tore; Nedergaard, Jan

    2014-10-15

    The physiological agonist norepinephrine promotes cell proliferation of brown preadipocytes during the process of tissue recruitment. In a primary culture system, cAMP mediates these adrenergic effects. In the present study, we demonstrated that, in contrast to other systems where the mitogenic effect of cAMP requires the synergistic action of (serum) growth factors, especially insulin/IGF, the cAMP effect in brown preadipocytes was independent of serum and insulin. Protein kinase A, rather than Epac, mediated the cAMP mitogenic effect. The Erk 1/2 family of MAPK, the PI{sub 3}K system and the mTOR complexes were all activated by cAMP, but these activations were not necessary for cAMP-induced cell proliferation; a protein kinase C isoform may be involved in mediating cAMP-activated cell proliferation. We conclude that the generally acknowledged cellular mediators for induction of cell proliferation are not involved in this process in the brown preadipocyte system; this conclusion may be of relevance both for examination of mechanisms for induction of brown adipose tissue recruitment but also for understanding the mechanism behind e.g. certain endocrine neoplasias. - Highlights: • cAMP can mimick norepinephrine-induced proliferation of brown preadipocytes. • The cAMP-induced proliferation can occur in the absence of serum, of any other growth factors, and of insulin. • Erk1/2, PI{sub 3}K and mTOR are cAMP activated but not involved in induction of proliferation. • A Protein Kinase C member may be in the signalling cascade. • This pathway analysis may also be of importance for certain endocrine hyper- and neoplasias.

  4. NKX3.1 Activates Expression of IGFBP-3 to Mediate IGF-I Signaling and Cell Proliferation

    PubMed Central

    Muhlbradt, Erin; Asatiani, Ekaterina; Ortner, Elizabeth; Wang, Antai; Gelmann, Edward P.

    2013-01-01

    NKX3.1 is a homeobox gene that codes for a haploinsufficient prostate cancer tumor suppressor. NKX3.1 protein levels are down regulated in the majority of primary prostate cancer tissues. NKX3.1 expression in PC-3 cells increased IGFBP-3 mRNA expression 10-fold as determined by expression microarray analysis. In both stably and transiently transfected PC-3 cells and in LNCaP cells NKX3.1 expression increased IGFBP-3 mRNA and protein expression. In prostates of Nkx3.1 gene-targeted mice Igfbp-3 mRNA levels correlated with Nkx3.1 copy number. NKX3.1 expression in PC-3 cells attenuated the ability of IGF-I to induce phosphorylation of IGF-IR, IRS-1, PI3-kinase, and AKT. The effect of NKX3.1 on IGF-I signaling was not seen when cells were exposed to long-R3-IGF-I, an IGF-1 variant peptide that does not bind to IGFBP-3. Additionally, siRNA-induced knock down of IGFBP-3 expression partially reversed the attenuation of IGF-1R signaling by NKX3.1 and abrogated NKX3.1 suppression of PC-3 cell proliferation. Thus there is a close relationship in vitro and in vivo between NKX3.1 and IGFBP-3. The growth suppressive effects of NKX3.1 in prostate cells are mediated, in part, by activation of IGFBP-3 expression. PMID:19258508

  5. Myocardin inhibits estrogen receptor alpha-mediated proliferation of human breast cancer MCF-7 cells via regulating MicroRNA expression.

    PubMed

    Xiang, Yuan; Lu, Da-Lin; Li, Jia-Peng; Yu, Cheng-Xi; Zheng, De-Liang; Huang, Xuan; Wang, Zhen-Yu; Hu, Peng; Liao, Xing-Hua; Zhang, Tong-Cun

    2016-06-01

    Myocardin is frequently repressed during human malignant transformation, and restoration of myocardin expression in sarcoma cells contributes to the inhibition of malignant growth. However, its role in breast carcinoma has barely been addressed. Here, we reported that myocardin could inhibit the proliferation of MCF-7 cells. Notably, we show that myocardin inhibited ERα-mediated proliferation of breast cancer MCF-7 via impairing ER-dependent transcriptional activation, mainly through the inhibition of the activity of ERα. Importantly, the molecular mechanism for the inhibition of the ERα-mediated proliferation is that myocardin inhibited the transcription and expression of ERα-induced PCNA, Ki-67, and E2F1 to impair ERα-mediated proliferation of breast cancer MCF-7. Interestingly, myocardin significantly enhanced the transcription and expression of miR-885 depending on the CArG box in miR-885 promoter, and miR-885 targeted the 3' untranslated regions (UTR) of E2F1 to silence the expression of E2F1. Thus, our data provided important and novel insights into how myocardin may deeply influence ERα-mediated breast cancer proliferation. In conclusion, myocardin could be seen as a breast cancer tumor suppressor so that it will provide new ideas for the treatment of breast cancer. © 2016 IUBMB Life, 68(6):477-487, 2016. PMID:27156566

  6. High-mobility group boxes mediate cell proliferation and radiosensitivity via retinoblastoma-interaction-dependent and -independent mechanisms.

    PubMed

    Wang, Li-Li; Meng, Qing-Hui; Jiao, Yang; Xu, Jia-Ying; Ge, Chun-Min; Zhou, Ju-Ying; Rosen, Eliot M; Wang, Hai-Chao; Fan, Sai-Jun

    2012-06-01

    Our previous studies have shown that high-mobility group box 1 (HMGB1) could physically associate with the retinoblastoma (RB) protein via an LXCXE (leucine-X-cysteine-X-glutamic; X=any amino acid) motif. An identical LXCXE motif is present in the HMGB1-3 protein sequences, whereas a near-consensus LXCXD (leucine-X-cysteine-X-asparagine; X=any amino acid) motif is found in the HMGB4 protein. In this study, we have demonstrated that like HMGB1, HMGB2-3 also associated with the RB in vitro and in vivo, as evidenced by glutathione-s-transferase capture and immunoprecipitation-Western blot assays. A point mutation of the LXCXE or LXCXD motif led to disruption of RB:HMGB1-4 interactions. Enforced expression of HMGB1-3 or HMGB4 by adenoviral-vector-mediated gene transfer resulted in significant inhibition of breast cancer cell proliferation through an LXCXE- or LXCXD-dependent mechanism and an increased radiosensitivity through an LXCXE- or LXCXD-independent mechanism. These results suggest an important role of the LXCXE/D motif in RB:HMGB1-4 association and modulation of cancer cell growth, but not radiosensitivity. PMID:22655796

  7. Down-regulation of tumor endothelial marker 8 suppresses cell proliferation mediated by ERK1/2 activity

    PubMed Central

    Cao, Chuangjie; Wang, Zhuo; Huang, Leilei; Bai, Lihong; Wang, Yuefeng; Liang, Yingjie; Dou, Chengyun; Wang, Liantang

    2016-01-01

    Tumor endothelial marker 8 (TEM8) was recently suggested as a putative anti-tumor target in several types of human cancer based on its selective overexpression in tumor versus normal endothelial cells. The objective of this study was to detect the potential functions of TEM8 in osteosarcoma. Overall, TEM8 was mainly located in cytoplasm and was up-regulated in osteosarcoma compared to benign bone lesions and adjacent non tumor tissue (ANT). High TEM8 expression group had a significant lower overall survival rate than that in the low TEM8 expression group. TEM8 knock-down by siRNA or shRNA results in significant reduction of osteosarcoma cell growth and proliferation both in vitro and in vivo. Ablation of TEM8 led to increasing of p21 and p27 and suppression of cyclin D1 mediated by Erk1/2 activity. These findings suggest that down-regulation of TEM8 play an important role in the inhibition of tumorigenesis and development of osteosarcoma. PMID:26996335

  8. Stim1 and Orai1 Mediate CRAC Currents and Store-Operated Calcium Entry Important for Endothelial Cell Proliferation

    PubMed Central

    Abdullaev, Iskandar F.; Bisaillon, Jonathan M.; Potier, Marie; Gonzalez, Jose C.; Motiani, Rajender K.; Trebak, Mohamed

    2009-01-01

    Recent breakthroughs in the store-operated Calcium (Ca2+) entry (SOCE) pathway have identified Stim1 as the endoplasmic reticulum (ER) Ca2+ sensor and Orai1 as the pore forming subunit of the highly Ca2+ selective CRAC channel expressed in hematopoietic cells. Previous studies however, have suggested that endothelial cell (EC) SOCE is mediated by the non-selective Canonical Transient Receptor Potential (TRPC) channel family, TRPC1 or TRPC4. Here we show that passive store depletion by thapsigargin or receptor activation by either thrombin or the vascular endothelial growth factor (VEGF) activates the same pathway in primary EC with classical SOCE pharmacological features. EC possess the archetypical Ca2+ release-activated Ca2+ current (ICRAC), albeit of a very small amplitude. Using a maneuver that amplifies currents in divalent free bath solutions, we show that EC CRAC has similar characteristics to that recorded from RBL cells, namely a similar time course of activation, sensitivity to 2-Aminoethoxydiphenyl borate (2-APB) and low concentrations of lanthanides, and large Na+ currents displaying the typical depotentiation. RNA silencing of either Stim1 or Orai1 essentially abolished SOCE and ICRAC in EC which were rescued by ectopic expression of either Stim1 or Orai1, respectively. Surprisingly, knockdown of either TRPC1 or TRPC4 proteins had no effect on SOCE and ICRAC. Ectopic expression of Stim1 in EC increased their ICRAC to a size comparable to that in RBL cells. Knockdown of Stim1, Stim2 or Orai1 inhibited EC proliferation and caused cell cycle arrest at S and G2/M phase, although Orai1 knockdown was more efficient than that of Stim proteins. These results are first to establish the requirement of Stim1/Orai1 in the endothelial SOCE pathway. PMID:18845811

  9. Cell Proliferation in Neuroblastoma.

    PubMed

    Stafman, Laura L; Beierle, Elizabeth A

    2016-01-01

    Neuroblastoma, the most common extracranial solid tumor of childhood, continues to carry a dismal prognosis for children diagnosed with advanced stage or relapsed disease. This review focuses upon factors responsible for cell proliferation in neuroblastoma including transcription factors, kinases, and regulators of the cell cycle. Novel therapeutic strategies directed toward these targets in neuroblastoma are discussed. PMID:26771642

  10. Cell Proliferation in Neuroblastoma

    PubMed Central

    Stafman, Laura L.; Beierle, Elizabeth A.

    2016-01-01

    Neuroblastoma, the most common extracranial solid tumor of childhood, continues to carry a dismal prognosis for children diagnosed with advanced stage or relapsed disease. This review focuses upon factors responsible for cell proliferation in neuroblastoma including transcription factors, kinases, and regulators of the cell cycle. Novel therapeutic strategies directed toward these targets in neuroblastoma are discussed. PMID:26771642

  11. Glycogen synthase kinase 3 regulates PAX3-FKHR-mediated cell proliferation in human alveolar rhabdomyosarcoma cells

    SciTech Connect

    Zeng, Fu-Yue; Dong, Hanqing; Cui, Jimmy; Liu, Lingling; Chen, Taosheng

    2010-01-01

    Patients with alveolar rhabdomyosarcoma (ARMS) have poorer response to conventional chemotherapy and lower survival rates than those with embryonal RMS (ERMS). To identify compounds that preferentially block the growth of ARMS, we conducted a small-scale screen of 160 kinase inhibitors against the ARMS cell line Rh30 and ERMS cell line RD and identified inhibitors of glycogen synthase kinase 3 (GSK3), including TWS119 as ARMS-selective inhibitors. GSK3 inhibitors inhibited cell proliferation and induced apoptosis more effectively in Rh30 than RD cells. Ectopic expression of fusion protein PAX3-FKHR in RD cells significantly increased their sensitivity to TWS119. Down-regulation of GSK3 by GSK3 inhibitors or siRNA significantly reduced the transcriptional activity of PAX3-FKHR. These results suggest that GSK3 is directly involved in regulating the transcriptional activity of PAX3-FKHR. Also, GSK3 phosphorylated PAX3-FKHR in vitro, suggesting that GSK3 might regulate PAX3-FKHR activity via phosphorylation. These findings support a novel mechanism of PAX3-FKHR regulation by GSK3 and provide a novel strategy to develop GSK inhibitors as anti-ARMS therapies.

  12. Glycogen synthase kinase 3 regulates PAX3-FKHR-mediated cell proliferation in human alveolar rhabdomyosarcoma cells.

    PubMed

    Zeng, Fu-Yue; Dong, Hanqing; Cui, Jimmy; Liu, Lingling; Chen, Taosheng

    2010-01-01

    Patients with alveolar rhabdomyosarcoma (ARMS) have poorer response to conventional chemotherapy and lower survival rates than those with embryonal RMS (ERMS). To identify compounds that preferentially block the growth of ARMS, we conducted a small-scale screen of 160 kinase inhibitors against the ARMS cell line Rh30 and ERMS cell line RD and identified inhibitors of glycogen synthase kinase 3 (GSK3), including TWS119 as ARMS-selective inhibitors. GSK3 inhibitors inhibited cell proliferation and induced apoptosis more effectively in Rh30 than RD cells. Ectopic expression of fusion protein PAX3-FKHR in RD cells significantly increased their sensitivity to TWS119. Down-regulation of GSK3 by GSK3 inhibitors or siRNA significantly reduced the transcriptional activity of PAX3-FKHR. These results suggest that GSK3 is directly involved in regulating the transcriptional activity of PAX3-FKHR. Also, GSK3 phosphorylated PAX3-FKHR in vitro, suggesting that GSK3 might regulate PAX3-FKHR activity via phosphorylation. These findings support a novel mechanism of PAX3-FKHR regulation by GSK3 and provide a novel strategy to develop GSK inhibitors as anti-ARMS therapies. PMID:19995556

  13. UAP56 is an important mediator of Angiotensin II/platelet derived growth factor induced vascular smooth muscle cell DNA synthesis and proliferation

    SciTech Connect

    Sahni, Abha; Wang, Nadan; Alexis, Jeffrey

    2013-02-15

    Highlights: ► Knockdown of UAP56 inhibits Angiotensin II/PDGF induced vascular smooth muscle cell proliferation. ► UAP56 is a positive regulator of E2F transcriptional activation. ► UAP56 is present in the vessel wall of low flow carotid arteries. -- Abstract: Angiotensin (Ang) II and platelet-derived growth factor (PDGF) are important mediators of pathologic vascular smooth muscle cell (VSMC) proliferation. Identifying downstream mediators of Ang II and PDGF signaling may provide insights for therapies to improve vascular proliferative diseases. We have previously demonstrated that breakpoint cluster region (Bcr) is an important mediator of Ang II/PDGF signaling in VSMC. We have recently reported that the DExD/H box protein UAP56 is an interacting partner of Bcr in regulating VSMC DNA synthesis. We hypothesized that UAP56 itself is an important regulator of VSMC proliferation. In this report we demonstrate that knockdown of UAP56 inhibits Ang II/PDGF induced VSMC DNA synthesis and proliferation, and inhibits E2F transcriptional activity. In addition, we demonstrate that UAP56 is present in the vessel wall of low-flow carotid arteries. These findings suggest that UAP56 is a regulator of VSMC proliferation and identify UAP56 as a target for preventing vascular proliferative disease.

  14. Evidence for a role of Collapsin response mediator protein-2 in signaling pathways that regulate the proliferation of non-neuronal cells

    SciTech Connect

    Tahimic, Candice Ginn T.; Tomimatsu, Nozomi; Nishigaki, Ryuichi |; Fukuhara, Akiko; Toda, Tosifusa; Kaibuchi, Kozo; Shiota, Goshi; Oshimura, Mitsuo |; Kurimasa, Akihiro |. E-mail: kurimasa@grape.med.tottori-u.ac.jp

    2006-02-24

    Collapsin response mediator protein-2 or Crmp-2 plays a critical role in the establishment of neuronal polarity. In this study, we present evidence that apart from its functions in neurodevelopment, Crmp-2 is also involved in pathways that regulate the proliferation of non-neuronal cells through its phosphorylation by regulatory proteins. We show that Crmp-2 undergoes dynamic phosphorylation changes in response to contact inhibition-induced quiescence and that hyperphosphorylation of Crmp-2 occurs in a tumor. We further suggest that de-regulation of Crmp-2 phosphorylation levels at certain amino acid residues may lead to aberrant cell proliferation and consequently, tumorigenesis.

  15. CDC2 mediates progestin initiated endometrial stromal cell proliferation: a PR signaling to gene expression independently of its binding to chromatin.

    PubMed

    Vallejo, Griselda; La Greca, Alejandro D; Tarifa-Reischle, Inti C; Mestre-Citrinovitz, Ana C; Ballaré, Cecilia; Beato, Miguel; Saragüeta, Patricia

    2014-01-01

    Although non-genomic steroid receptor pathways have been studied over the past decade, little is known about the direct gene expression changes that take place as a consequence of their activation. Progesterone controls proliferation of rat endometrial stromal cells during the peri-implantation phase of pregnancy. We showed that picomolar concentration of progestin R5020 mimics this control in UIII endometrial stromal cells via ERK1-2 and AKT activation mediated by interaction of Progesterone Receptor (PR) with Estrogen Receptor beta (ERb) and without transcriptional activity of endogenous PR and ER. Here we identify early downstream targets of cytoplasmic PR signaling and their possible role in endometrial stromal cell proliferation. Microarray analysis of global gene expression changes in UIII cells treated for 45 min with progestin identified 97 up- and 341 down-regulated genes. The most over-represented molecular functions were transcription factors and regulatory factors associated with cell proliferation and cell cycle, a large fraction of which were repressors down-regulated by hormone. Further analysis verified that progestins regulate Ccnd1, JunD, Usf1, Gfi1, Cyr61, and Cdkn1b through PR-mediated activation of ligand-free ER, ERK1-2 or AKT, in the absence of genomic PR binding. ChIP experiments show that progestin promoted the interaction of USF1 with the proximal promoter of the Cdc2 gene. Usf1 knockdown abolished Cdc2 progestin-dependent transcriptional regulation and cell proliferation, which also blocked Cdc2 knockdown. We conclude that progestin-induced proliferation of endometrial stromal cells is mediated by ERK1-2 and AKT dependent early regulation of USF1, which directly induces Cdc2. To our knowledge, this is the first description of early target genes of progestin-activated classical PR via crosstalk with protein kinases and independently of hormone receptor binding to the genomic targets. PMID:24859236

  16. Gold(III) compounds-mediated inhibition of lung cancer cell proliferation.

    PubMed

    Bostancioğlu, Rakibe B; Kaya, Murat; Koparal, Ayşe T; Benkli, Kadriye

    2016-03-01

    Research on chemotherapeutics for lung cancer is crucial for designing a new therapeutic strategy against malignant lung tumors. Although radiotherapy and chemotherapy, which are not selective for cancer cells and exert toxic effects on healthy cells, have a limited advantage, they are the primary treatment modalities for non-small lung cancer. In addition to cytotoxicity, resistance of chemotherapeutics results in failure of treatment. This is why it is of utmost importance to focus on the creation of new chemotherapeutics without toxicity for the successful treatment and improved survival of cancer patients. New gold(III) and Pt(II) compounds were synthesized with a heterocyclic ligand using 2-phenylimidazo[4,5-f][1,10]phenanthroline as a ligand and bis-1,4-di[([1,10] phenanthroline-5-il)amino]-2-buten as a bridge molecule. The characterization of the compounds was carried out using a variety of spectroscopic methods (H NMR, IR, MS, and elemental analysis). Their antiproliferative, antitumoral, and apoptotic activities were determined. IR spectra and NMR results confirmed the formation of dinuclear heterocyclic complexes for two metal complexes. Cytotoxicity studies on lung cancer cells (A549) and healthy cells (CHL) showed a marked increase in cytotoxicity with the use of gold(III) complexes, and especially [Au(L)B](PF6)2 showed higher cytotoxic and apoptotic features than cisplatin at lower concentrations in cancer cells. These findings have been supported by results from DAPI staining and colorimetric measurement of the caspase-3 enzyme in both cell lines. Compounds showed selective toxicity on the cancer cells. In the light of the high efficacy of our newly synthesized gold complexes, they might be good and promising anticancer agents compared with cisplatin. PMID:26825752

  17. Negative regulators of cell proliferation

    NASA Technical Reports Server (NTRS)

    Johnson, T. C.; Spooner, B. S. (Principal Investigator)

    1994-01-01

    Cell proliferation is governed by the influence of both mitogens and inhibitors. Although cell contact has long been thought to play a fundamental role in cell cycling regulation, and negative regulators have long been suspected to exist, their isolation and purification has been complicated by a variety of technical difficulties. Nevertheless, over recent years an ever-expanding list of putative negative regulators have emerged. In many cases, their biological inhibitory activities are consistent with density-dependent growth inhibition. Most likely their interactions with mitogenic agents, at an intracellular level, are responsible for either mitotic arrest or continued cell cycling. A review of naturally occurring cell growth inhibitors is presented with an emphasis on those factors shown to be residents of the cell surface membrane. Particular attention is focused on a cell surface sialoglycopeptide, isolated from intact bovine cerebral cortex cells, which has been shown to inhibit the proliferation of an unusually wide range of target cells. The glycopeptide arrest cells obtained from diverse species, both fibroblasts and epithelial cells, and a broad variety of transformed cells. Signal transduction events and a limited spectrum of cells that are refractory to the sialoglycopeptide have provided insight into the molecular events mediated by this cell surface inhibitor.

  18. Contribution of phospholipase D in endothelin-1-mediated extracellular signal-regulated kinase activation and proliferation in rat uterine leiomyoma cells.

    PubMed

    Robin, Philippe; Chouayekh, Sondes; Bole-Feysot, Christine; Leiber, Denis; Tanfin, Zahra

    2005-01-01

    Endothelin (ET)-1 is a mitogenic factor in numerous cell types, including rat myometrial cells. In the present study, we investigated the potential role of ET-1 in the proliferation of tumoral uterine smooth muscle cells (ELT-3 cells). We found that ET-1 exerted a more potent mitogenic effect in ELT-3 cells than in normal myometrial cells, as indicated by the increase in [3H]thymidine incorporation, cell number, and bromodeoxyuridine incorporation. The ET-1 was more efficient than platelet-derived growth factor and epidermal growth factor to stimulate proliferation. The ET-1-mediated cell proliferation was inhibited in the presence of U0126, a specific inhibitor of (mitogen-activated protein kinase ERK kinase), indicating that extracellular signal-regulated kinase (ERK) activation is involved. Additionally, ET-1 induced the activation of phospholipase (PL) D, leading to the synthesis of phosphatidic acid (PA). The ET-1-induced activation of PLD was twofold higher in ELT-3 cells compared to that in normal cells. The two cell types expressed mRNA for PLD1a and PLD2, whereas PLD1b was expressed only in ELT-3 cells. The exposure of cells to butan-1-ol reduced ET-1-mediated production of PA by PLD and partially inhibited ERK activation and DNA synthesis. Addition of exogenous PLD or PA in the medium reproduced the effect of ET-1 on ERK activation and cell proliferation. Collectively, these data indicate that ET-1 is a potent mitogenic factor in ELT-3 cells via a signaling pathway involving a PLD-dependent activation of ERK. This highlights the potential role of ET-1 in the development of uterine leiomyoma, and it reinforces the role of PLD in tumor growth. PMID:15355882

  19. Cytoskeleton reorganization mediates alpha6beta1 integrin-associated actions of laminin on proliferation and survival, but not on steroidogenesis of ovine granulosa cells

    PubMed Central

    Bellego, Frédérique Le; Fabre, Stéphane; Pisselet, Claudine; Monniaux, Danielle

    2005-01-01

    Background Laminin (LN) is one of the most abundant extracellular matrix components of the basal lamina and granulosa cell layers of ovarian follicles. Culture of ovine granulosa cells (GC) on LN substratum induces cell spreading, enhances cell survival and proliferation, and promotes luteinization. Previous investigations have shown that these effects are mostly mediated by the alpha6beta1 integrin, but its signalization pathways have not been investigated. This study aimed to assess the importance of the cytoskeleton in the alpha6beta1 integrin-mediated actions of laminin on survival, proliferation and steroidogenesis of ovine GC. Methods The relationships between morphology and functions of ovine GC cultured on substrata containing LN or/and RGD peptides were investigated. The effects of (1) cytochalasin D, an actin cytoskeleton-disrupting drug, (2) a specific function-blocking antibody raised against alpha6 integrin subunit (anti-alpha6 IgG), and (3) an inhibitor of the ERK1/2 signalization pathway (PD98059) were assessed for GC shape, pyknosis and proliferation rates, oestradiol and progesterone secretions. Results Cytoskeleton disruption by cytochalasin D induced cell rounding, inhibited proliferation, promoted pyknosis, inhibited progesterone secretion and enhanced oestradiol secretion by GC cultured on LN. When GC were cultured on various substrata containing LN and/or RGD peptides in the presence or absence of anti-alpha6 IgG, both the existence of close correlations between the percentage of round cells, and the GC proliferation rate (r = -0.87) and pyknotic rate (r = 0.76) were established, but no relationship was found between cell shape and steroidogenesis. Inhibition of the ERK1/2 signalization pathway by PD98059 had no effect on GC shape, proliferation or pyknotic rates. However, it dramatically reduced progesterone secretion, expression of cytochrome P450 cholesterol side-chain cleavage and 3beta-hydroxysteroid deshydrogenase enzymes, and enhanced

  20. Tumor endothelial marker 5 expression in endothelial cells during capillary morphogenesis is induced by the small GTPase Rac and mediates contact inhibition of cell proliferation

    SciTech Connect

    Vallon, Mario; Rohde, Franziska; Janssen, Klaus-Peter; Essler, Markus

    2010-02-01

    Tumor endothelial marker (TEM) 5 is an adhesion G-protein-coupled receptor upregulated in endothelial cells during tumor and physiologic angiogenesis. So far, the mechanisms leading to upregulation of TEM5 and its function during angiogenesis have not been identified. Here, we report that TEM5 expression in endothelial cells is induced during capillary-like network formation on Matrigel, during capillary morphogenesis in a three-dimensional collagen I matrix, and upon confluence on a two-dimensional matrix. TEM5 expression was not induced by a variety of soluble angiogenic factors, including VEGF and bFGF, in subconfluent endothelial cells. TEM5 upregulation was blocked by toxin B from Clostridium difficile, an inhibitor of the small GTPases Rho, Rac, and Cdc42. The Rho inhibitor C3 transferase from Clostridium botulinum did not affect TEM5 expression, whereas the Rac inhibitor NSC23766 suppressed TEM5 upregulation. An excess of the soluble TEM5 extracellular domain or an inhibitory monoclonal TEM5 antibody blocked contact inhibition of endothelial cell proliferation resulting in multilayered islands within the endothelial monolayer and increased vessel density during capillary formation. Based on our results we conclude that TEM5 expression during capillary morphogenesis is induced by the small GTPase Rac and mediates contact inhibition of proliferation in endothelial cells.

  1. Citrus limon-derived nanovesicles inhibit cancer cell proliferation and suppress CML xenograft growth by inducing TRAIL-mediated cell death.

    PubMed

    Raimondo, Stefania; Naselli, Flores; Fontana, Simona; Monteleone, Francesca; Lo Dico, Alessia; Saieva, Laura; Zito, Giovanni; Flugy, Anna; Manno, Mauro; Di Bella, Maria Antonietta; De Leo, Giacomo; Alessandro, Riccardo

    2015-08-14

    Nanosized vesicles are considered key players in cell to cell communication, thus influencing physiological and pathological processes, including cancer. Nanovesicles have also been found in edible-plants and have shown therapeutic activity in inflammatory bowel diseases; however information on their role in affecting cancer progression is missing.Our study identify for the first time a fraction of vesicles from lemon juice (Citrus limon L.), obtained as a result of different ultracentrifugation, with density ranging from 1,15 to 1,19 g/ml and specific proteomic profile. By using an in vitro approach, we show that isolated nanovesicles inhibit cancer cell proliferation in different tumor cell lines, by activating a TRAIL-mediated apoptotic cell death. Furthermore, we demonstrate that lemon nanovesicles suppress CML tumor growth in vivo by specifically reaching tumor site and by activating TRAIL-mediated apoptotic cell processes. Overall, this study suggests the possible use of plant-edible nanovesicles as a feasible approach in cancer treatment. PMID:26098775

  2. Citrus limon-derived nanovesicles inhibit cancer cell proliferation and suppress CML xenograft growth by inducing TRAIL-mediated cell death

    PubMed Central

    Raimondo, Stefania; Naselli, Flores; Fontana, Simona; Monteleone, Francesca; Lo Dico, Alessia; Saieva, Laura; Zito, Giovanni; Flugy, Anna; Manno, Mauro; Di Bella, Maria Antonietta; De Leo, Giacomo; Alessandro, Riccardo

    2015-01-01

    Nanosized vesicles are considered key players in cell to cell communication, thus influencing physiological and pathological processes, including cancer. Nanovesicles have also been found in edible-plants and have shown therapeutic activity in inflammatory bowel diseases; however information on their role in affecting cancer progression is missing. Our study identify for the first time a fraction of vesicles from lemon juice (Citrus limon L.), obtained as a result of different ultracentrifugation, with density ranging from 1,15 to 1,19 g/ml and specific proteomic profile. By using an in vitro approach, we show that isolated nanovesicles inhibit cancer cell proliferation in different tumor cell lines, by activating a TRAIL-mediated apoptotic cell death. Furthermore, we demonstrate that lemon nanovesicles suppress CML tumor growth in vivo by specifically reaching tumor site and by activating TRAIL-mediated apoptotic cell processes. Overall, this study suggests the possible use of plant-edible nanovesicles as a feasible approach in cancer treatment. PMID:26098775

  3. CYP2S1 depletion enhances colorectal cell proliferation is associated with PGE2-mediated activation of β-catenin signaling

    SciTech Connect

    Yang, Chao; Li, Changyuan; Li, Minle; Tong, Xuemei; Hu, Xiaowen; Yang, Xuhan; Yan, Xiaomei; He, Lin; Wan, Chunling

    2015-02-15

    Colorectal epithelial cancer is one of the most common cancers in the world and its 5-year survival rate is still relatively low. Cytochrome P450 (CYP) enzymes in epithelial cells lining the alimentary tract play an important role in the oxidative metabolism of a wide range of xenobiotics, including (pro-)carcinogens and endogenous compounds. Although CYP2S1, a member of CYP family, strongly expressed in many extrahepatic tissues, the role of CYP2S1 in cancer remains unclear. To investigate whether CYP2S1 involves in colorectal carcinogenesis, cell proliferation was analyzed in HCT116 cells depleted of CYP2S1 using small hairpin interfering RNA. Our data show that CYP2S1 knockdown promotes cell proliferation through increasing the level of endogenous prostaglandin E2(PGE2). PGE2, in turn, reduces phosphorylation of β-catenin and activates β-catenin signaling, which contributes to the cell proliferation. Furthermore, CYP2S1 knockdown increase tumor growth in xenograft mouse model. In brief, these results demonstrate that CYP2S1 regulates colorectal cancer growth through associated with PGE2-mediated activation of β-catenin signaling. - Highlights: • Knockdown of CYP2S1 expression improve HCT116 cell proliferation in vitro and in vivo. • Elevate PGE2 production in CYP2S1 knockdown cell is associated with its proliferation. • Elevate PGE2 level in CYP2S1 knockdown cells enhance β-catenin accumulation. • β-catenin activate TCF/LEF and target gene expression thus promote cell proliferation.

  4. (E)-1-(3,4-dihydroxyphenethyl)-3-styrylurea inhibits proliferation of MCF-7 cells through G1 cell cycle arrest and mitochondria-mediated apoptosis.

    PubMed

    Yu, Ji-Yeon; Kim, Ji-Hae; Kim, Tae-Geum; Kim, Beom-Tae; Jang, Yong-Suk; Lee, Jeong-Chae

    2010-10-01

    Growing interest in the beneficial effects of antioxidants has inspired the synthesis of new phenolic acid phenethyl ureas (PAPUs) with enhanced antioxidant potential. We have previously shown the capacity of one PAPU compound, (E)-1-(3,4-dihydroxyphenethyl)-3-styrylurea (PAPU1), to induce caspase-dependent apoptosis in melanoma cells. In the present study, we examined the anti-proliferative effects of PAPU compounds on MCF-7 human breast cancer cells and determined the molecular mechanisms involved. Treatment with PAPU compounds inhibited predominantly proliferation in these cells, where the PAPU1 was the most efficient form. Flow cytometric analysis showed that PAPU1 blocked cell cycle progression in the G(0)/G(1) phase, and reduced the proportion of cells in G(2)/M phase. This was related to the inhibition of cell cycle regulatory factors, including cyclin D/E and cyclin-dependent kinase (CDK) 2/4, through induction of p21(Cip1). PAPU1 also induced the mitochondrial-mediated and caspase-dependent apoptosis in MCF-7 cells. This was evidenced by cellular changes in the levels of Bcl-2 and Bax, loss of the mitochondrial membrane potential, release of cytochrome c into the cytosol, and caspase-9 activation. Collectively, our results suggest that G(1) cell cycle regulatory proteins and mitochondrial pathways are the crucial targets of PAPU1 in the chemoprevention of breast cancer cells. PMID:20811815

  5. Effect of m-calpain in PKCalpha-mediated proliferation of pulmonary artery smooth muscle cells by low dose of ouabain.

    PubMed

    Shaikh, Soni; Sarkar, Jaganmay; Pramanik, Asmita; Karmakar, Kanchan; Chakraborti, Sajal

    2013-10-01

    There is growing evidence that ouabain, a cardiotonic steroid may promote growth of cardiac and vascular myocytes, indicating its novel role in cell growth and proliferation, without appreciable inhibition of the sodium pump. The mechanism(s) by which low dose of ouabain produces pulmonary artery smooth muscle cell proliferation, a prerequisite for right ventricular hypertrophy, is currently unknown. Here, we analyzed the effects of low dose of ouabain (10 nM) on increase in [Ca2+]i, m-calpain and protein kinase C (PKC) activities on pulmonary artery smooth muscle cell proliferation and determined their sequential involvement in this scenario. We treated bovine pulmonary artery smooth muscle cells with a low dose of ouabain (10 nM) and determined [Ca2+]i in the cells by fluorometric assay using fura2-AM, m-calpain activity by fluorometric assay using SLLVY-AMC as the substrate. PKC activity using an assay kit and assay of Na+/K+ ATPase activity spectrophotometrically. We purified m-calpain and PKCalpha by standard chromatographic procedure by HPLC and then studied cleavage of the purified PKCalpha by m-calpain using Western immunoblot method. Subsequently, we performed cell proliferation assay utilizing the redox dye resazunin. We used selective inhibitors of [Ca2+]i (BAPTA-AM), m-calpain (MDL28170), PKCalpha (Go6976) and determined their involvement in ouabain (10 nM)-mediated smooth muscle cell proliferation. Our results suggested that treatment of bovine pulmonary artery smooth muscle cells with a low dose of ouabain (10 nM) increased [Ca2+]i and subsequently stimulated m-calpain activity and proteolytically activated PKCalpha in caveolae (signaling microdomain also known as signalosomes) of the cells. Upon activation, PKCalpha increased the smooth muscle cell proliferation via Go/G1 to S/G2-M phase transition. Thus, [Ca2]i-mCalpain-PKCalpha signaling axis plays a crucial role during low dose of ouabain-mediated pulmonary artery smooth muscle cell proliferation

  6. Akt- and CREB-Mediated Prostate Cancer Cell Proliferation Inhibition by Nexrutine, a Phellodendron amurense Extract1

    PubMed Central

    Garcia, Gretchen E; Nicole, Arevalo; Bhaskaran, Shylesh; Gupta, Ashima; Kyprianou, Natasha; Kumar, Addanki P

    2006-01-01

    Abstract Evidence from epidemiological studies suggests that plant-based diets can reduce the risk of prostate cancer. However, very little information is available concerning the use of botanicals in preventing prostate cancer. As a first step toward developing botanicals as prostate cancer preventives, we examined the effect of Nexrutine on human prostate cancer cells. Nexrutine is a herbal extract developed from Phellodendron amurense. Phellodendron extracts have been used traditionally in Chinese medicine for hundreds of years as an anti-diarrheal, astringent, and anti-inflammatory agent. The present study investigated its potential antitumor effect on human prostate cancer cells. Our results suggest that it inhibits tumor cell proliferation through apoptosis induction and inhibition of cell survival signaling. The results of the present study indicate that Nexrutine treatment 1) inhibits the proliferation of both androgen-responsive and androgen-independent human prostate cancer cells through induction of apoptosis; 2) reduces levels of pAkt, phosphorylated cAMP response-binding protein (pCREB), and CREB DNA-binding activity; and 3) induces apoptosis in prostate cancer cells stably overexpressing Bcl-2. Further Akt kinase activity was reduced in cells treated with Nexrutine, and ectopic expression of myristoylated Akt protected from Nexrutine induced inhibition of proliferation, implicating a role for Akt signaling. PMID:16820098

  7. Inhibition of Cancer Cell Proliferation by PPARγ is Mediated by a Metabolic Switch that Increases Reactive Oxygen Species Levels

    PubMed Central

    Srivastava, Nishi; Kollipara, Rahul K.; Singh, Dinesh K.; Sudderth, Jessica; Hu, Zeping; Nguyen, Hien; Wang, Shan; Humphries, Caroline G.; Carstens, Ryan; Huffman, Kenneth E.; DeBerardinis, Ralph J.; Kittler, Ralf

    2014-01-01

    SUMMARY The nuclear receptor peroxisome-proliferation activated receptor gamma (PPARγ), a transcriptional master regulator of glucose and lipid metabolism, inhibits the growth of several common cancers including lung cancer. In this study, we show that the mechanism by which activation of PPARγ inhibits proliferation of lung cancer cells is based on metabolic changes. We found that treatment with the PPARγ agonist pioglitazone triggers a metabolic switch that inhibits pyruvate oxidation and reduces glutathione levels. These PPARγ-induced metabolic changes result in a marked increase of reactive oxygen species (ROS) levels that lead to rapid hypophosphorylation of retinoblastoma protein (RB) and cell cycle arrest. The antiproliferative effect of PPARγ activation can be prevented by suppressing pyruvate dehydrogenase kinase 4 (PDK4) or β-oxidation of fatty acids in vitro and in vivo. Our proposed mechanism also suggests that metabolic changes can rapidly and directly inhibit cell cycle progression of cancer cells by altering ROS levels. PMID:25264247

  8. Inhibition of cancer cell proliferation by PPARγ is mediated by a metabolic switch that increases reactive oxygen species levels.

    PubMed

    Srivastava, Nishi; Kollipara, Rahul K; Singh, Dinesh K; Sudderth, Jessica; Hu, Zeping; Nguyen, Hien; Wang, Shan; Humphries, Caroline G; Carstens, Ryan; Huffman, Kenneth E; DeBerardinis, Ralph J; Kittler, Ralf

    2014-10-01

    The nuclear receptor peroxisome-proliferation-activated receptor gamma (PPARγ), a transcriptional master regulator of glucose and lipid metabolism, inhibits the growth of several common cancers, including lung cancer. In this study, we show that the mechanism by which activation of PPARγ inhibits proliferation of lung cancer cells is based on metabolic changes. We found that treatment with the PPARγ agonist pioglitazone triggers a metabolic switch that inhibits pyruvate oxidation and reduces glutathione levels. These PPARγ-induced metabolic changes result in a marked increase of reactive oxygen species (ROS) levels that lead to rapid hypophosphorylation of retinoblastoma protein (RB) and cell-cycle arrest. The antiproliferative effect of PPARγ activation can be prevented by suppressing pyruvate dehydrogenase kinase 4 (PDK4) or β-oxidation of fatty acids in vitro and in vivo. Our proposed mechanism also suggests that metabolic changes can rapidly and directly inhibit cell-cycle progression of cancer cells by altering ROS levels. PMID:25264247

  9. A combination of desmopressin and docetaxel inhibit cell proliferation and invasion mediated by urokinase-type plasminogen activator (uPA) in human prostate cancer cells

    SciTech Connect

    Sasaki, Hiroshi; Klotz, Laurence H.; Sugar, Linda M.; Kiss, Alexander; Venkateswaran, Vasundara

    2015-08-28

    Background: This study was designed to assess the effectiveness of a combination treatment using both desmopressin and docetaxel in prostate cancer treatment. Desmopressin is a well-known synthetic analogue of the antidiuretic hormone vasopressin. It has recently been demonstrated to inhibit tumor progression and metastasis in in vivo models. Docetaxel is widely used for the treatment of castration resistant prostate cancer (CRPC) patients. However, durable responses have been uncommon to date. In this study, we investigated the anti-tumor effect of desmopressin in combination with docetaxel in vitro and in vivo. Methods: Two prostate cancer cells (PC3, LNCaP) were treated with different concentrations of desmopressin alone, docetaxel alone, and a combination of desmopressin and docetaxel. Cell proliferation was determined by MTS assay. The anti-invasive and anti-migration potential of desmopressin and in combination with docetaxel were examined by wound healing assay, migration chamber assay, and matrigel invasion assay. Results: The combination of desmopressin and docetaxel resulted in a significant inhibition of PC3 and LNCaP cell proliferation (p < 0.01). Additionally, cell migration and invasion were also inhibited by the combination when compared to that of either treatment alone in PC3 cells (p < 0.01). The anti-tumor effect of this combination treatment was associated with down-regulation of both urokinase-type plasminogen activator (uPA) and matrix metalloproteinase (MMP-2 and MMP-9) in PC3 cells. Conclusions: We are the first to elucidate the anti-tumor and anti-metastatic potential of desmopressin in combination with docetaxel in a prostate cancer model via the uPA-MMP pathway. Our finding could potentially contribute to the therapeutic profile of desmopressin and enhance the efficacy of docetaxel based treatment for CRPC. - Highlights: • Desmopressin inhibits cell proliferation in prostate cancer cells. • The expression of cyclin A and CDK2

  10. CAP1 (Cyclase-Associated Protein 1) Exerts Distinct Functions in the Proliferation and Metastatic Potential of Breast Cancer Cells Mediated by ERK

    PubMed Central

    Zhang, Haitao; Zhou, Guo-Lei

    2016-01-01

    The actin-regulating protein CAP1 is implicated in the invasiveness of human cancers. However, the exact role remains elusive and controversial given lines of conflicting evidence. Moreover, a potential role in the proliferative transformation has largely been overlooked. Further establishing the role and dissecting underlying mechanisms are imperative before targeting CAP1 can become a possibility for cancer treatment. Here we report our findings that CAP1 exerts cell type-dependent functions in the invasiveness of breast cancer cells. Depletion of CAP1 in the metastatic MDA-MB-231 and BT-549 cancer cells stimulated the metastatic potential while it actually inhibited it in the non-metastatic MCF-7 cancer cells or in normal cells. Moreover, we demonstrate functions for CAP1 in cancer cell proliferation and anchorage-independent growth, again in a cell context-dependent manner. Importantly, we identify pivotal roles for the ERK-centered signaling in mediating both CAP1 functions. Phosphor mutants of CAP1 at the S307/S309 regulatory site had compromised rescue effects for both the invasiveness and proliferation in CAP1-knockdown cells, suggesting that CAP1 likely mediates upstream cell signals to control both functions. These novel mechanistic insights may ultimately open up avenues for strategies targeting CAP1 in the treatment of breast cancer, tailored for specific types of the highly diverse disease. PMID:27173014

  11. UV Damage-Induced Phosphorylation of HBO1 Triggers CRL4DDB2-Mediated Degradation To Regulate Cell Proliferation.

    PubMed

    Matsunuma, Ryoichi; Niida, Hiroyuki; Ohhata, Tatsuya; Kitagawa, Kyoko; Sakai, Satoshi; Uchida, Chiharu; Shiotani, Bunsyo; Matsumoto, Masaki; Nakayama, Keiichi I; Ogura, Hiroyuki; Shiiya, Norihiko; Kitagawa, Masatoshi

    2016-02-01

    Histone acetyltransferase binding to ORC-1 (HBO1) is a critically important histone acetyltransferase for forming the prereplicative complex (pre-RC) at the replication origin. Pre-RC formation is completed by loading of the MCM2-7 heterohexameric complex, which functions as a helicase in DNA replication. HBO1 recruited to the replication origin by CDT1 acetylates histone H4 to relax the chromatin conformation and facilitates loading of the MCM complex onto replication origins. However, the acetylation status and mechanism of regulation of histone H3 at replication origins remain elusive. HBO1 positively regulates cell proliferation under normal cell growth conditions. Whether HBO1 regulates proliferation in response to DNA damage is poorly understood. In this study, we demonstrated that HBO1 was degraded after DNA damage to suppress cell proliferation. Ser50 and Ser53 of HBO1 were phosphorylated in an ATM/ATR DNA damage sensor-dependent manner after UV treatment. ATM/ATR-dependently phosphorylated HBO1 preferentially interacted with DDB2 and was ubiquitylated by CRL4(DDB2). Replacement of endogenous HBO1 in Ser50/53Ala mutants maintained acetylation of histone H3K14 and impaired cell cycle regulation in response to UV irradiation. Our findings demonstrate that HBO1 is one of the targets in the DNA damage checkpoint. These results show that ubiquitin-dependent control of the HBO1 protein contributes to cell survival during UV irradiation. PMID:26572825

  12. Decorin-Mediated Inhibition of Human Trophoblast Cells Proliferation, Migration, and Invasion and Promotion of Apoptosis In Vitro.

    PubMed

    Zou, Yanfen; Yu, Xiang; Lu, Jing; Jiang, Ziyan; Zuo, Qing; Fan, Mingsong; Huang, Shiyun; Sun, Lizhou

    2015-01-01

    Preeclampsia (PE) is a unique complication of pregnancy, the pathogenesis of which has been generally accepted to be associated with the dysfunctions of extravillous trophoblast (EVT) including proliferation, apoptosis, and migration and invasion. Decorin (DCN) has been proved to be a decidua-derived TGF-binding proteoglycan, which negatively regulates proliferation, migration, and invasiveness of human extravillous trophoblast cells. In this study, we identified a higher expression level of decorin in severe PE placentas by both real-time reverse transcription-polymerase chain reaction (qRT-PCR) and immunohistochemistry (IHC). And an inhibitory effect of decorin on proliferation, migration, and invasion and an enhanced effect on apoptosis in trophoblast cells HTR-8/SVneo and JEG-3 were validated in vitro. Also the modulations of decorin on trophoblast cells' metastasis and invasion functions were detected through regulating the matrix metalloproteinases (MMP2 and MMP9). Thus, we suggested that the contribution of decorin to the modulation of trophoblast cells might have implications for the pathogenesis of preeclampsia. PMID:26357650

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

    PubMed Central

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

    2015-01-01

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

  14. Members of the GCN5 histone acetyltransferase complex regulate PLETHORA-mediated root stem cell niche maintenance and transit amplifying cell proliferation in Arabidopsis.

    PubMed

    Kornet, Noortje; Scheres, Ben

    2009-04-01

    The PLETHORA (PLT) stem cell transcription factors form a developmentally instructive protein gradient in Arabidopsis thaliana roots. Histone acetylation is known to facilitate gene transcription and plays an important role in developmental processes. Here, we show that histone acetyltransferase GCN5 (for general control nonderepressible 5) attenuates the PLT gradient. Based on genetic evidence, we establish that GCN5 is essential for root stem cell niche maintenance and acts in the PLT pathway. The GCN5-associated factor ADA2b (for alteration/deficiency in activation 2b) is also positioned in the PLT pathway and regulates PLT expression, similar to GCN5. Both GCN5 and ADA2b mediate proliferation of the transit amplifying cells, but ADA2b does not affect stem cell niche maintenance. Overexpression of PLT2 rescues the stem cell niche defect of gcn5 mutants, indicating that GCN5 regulation of PLT expression is essential for maintenance of the root stem cell niche. We conclude that histone acetylation complexes play an important role in shaping a developmentally instructive gradient in the root. PMID:19376933

  15. Members of the GCN5 Histone Acetyltransferase Complex Regulate PLETHORA-Mediated Root Stem Cell Niche Maintenance and Transit Amplifying Cell Proliferation in Arabidopsis[W

    PubMed Central

    Kornet, Noortje; Scheres, Ben

    2009-01-01

    The PLETHORA (PLT) stem cell transcription factors form a developmentally instructive protein gradient in Arabidopsis thaliana roots. Histone acetylation is known to facilitate gene transcription and plays an important role in developmental processes. Here, we show that histone acetyltransferase GCN5 (for general control nonderepressible 5) attenuates the PLT gradient. Based on genetic evidence, we establish that GCN5 is essential for root stem cell niche maintenance and acts in the PLT pathway. The GCN5-associated factor ADA2b (for alteration/deficiency in activation 2b) is also positioned in the PLT pathway and regulates PLT expression, similar to GCN5. Both GCN5 and ADA2b mediate proliferation of the transit amplifying cells, but ADA2b does not affect stem cell niche maintenance. Overexpression of PLT2 rescues the stem cell niche defect of gcn5 mutants, indicating that GCN5 regulation of PLT expression is essential for maintenance of the root stem cell niche. We conclude that histone acetylation complexes play an important role in shaping a developmentally instructive gradient in the root. PMID:19376933

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

    PubMed

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

    2015-01-01

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

  17. The effect of estrogen on the proliferation of endometrial cancer cells is mediated by ERRγ through AKT and ERK1/2.

    PubMed

    Sun, Yan; Wang, Cuicui; Yang, Hui; Ma, Xiaoxin

    2014-09-01

    The objective of this study was to explore the effects and underlying mechanism of estrogen-related receptor γ (ERRγ) on the proliferation of endometrial carcinoma cells. Ishikawa cells, human endometrial cancer cells, were treated with estrogen. Immunofluorescence was used to observe the expression of ERRγ. Stable transfection with the plasmid containing ERRγ shRNA was carried out to knock down the expression of ERRγ in Ishikawa cells. MTT assays were performed to analyze the proliferation of Ishikawa cells. The activation of extracellular signal-regulated protein kinase (ERK)1/2 and activated protein kinase B (AKT) signaling pathways was identified using specific phosphorylated antibodies against ERK1/2 and AKT. PD98059, a MEK inhibitor, and LY294002, a PI3K inhibitor, were used in the inhibition experiments. ERRγ could translocate from the cytoplasm to the nucleus in Ishikawa cells after exposure to estrogen. Knockdown of ERRγ inhibited estrogen-induced proliferation of Ishikawa cells. More interestingly, knockdown of ERRγ abolished the activation of ERK1/2 and AKT in the Ishikawa cells treated with estrogen. Inhibition of AKT in Ishikawa cells with LY294002 resulted in a significant reduction in the levels of phospho-ERK1/2, whereas inhibition of ERK1/2 with PD98059 exerted no effects on AKT activation. Our data showed that ERRγ mediated the effects of estrogen on the proliferation of endometrial cancer cells through the activation AKT and ERK1/2 signaling pathways, which indicated that ERRγ plays a key role in endometrial cancer. PMID:25068805

  18. Stress-mediated progression of solid tumors: effect of mechanical stress on tissue oxygenation, cancer cell proliferation and drug delivery

    PubMed Central

    Mpekris, Fotios; Angeli, Stelios; Pirentis, Athanassios P.; Stylianopoulos, Triantafyllos

    2015-01-01

    Oxygen supply plays a central role in cancer cell proliferation. While vascular density increases at the early stages of carcinogenesis, mechanical solid stresses developed during growth compress tumor blood vessels and, thus, drastically reduce the supply of oxygen, but also the delivery of drugs at inner tumor regions. Among other effects, hypoxia and reduced drug delivery compromise the efficacy of radiation and chemo/nano therapy, respectively. In the present study, we developed a mathematical model of tumor growth to investigate the interconnections among tumor oxygenation that supports cancer cell proliferation, the heterogeneous accumulation of mechanical stresses owing to tumor growth, the non-uniform compression of intratumoral blood vessels due to the mechanical stresses, and the insufficient delivery of oxygen and therapeutic agents because of vessel compression. We found that the high vascular density and increased cancer cell proliferation often observed in the periphery compared to the interior of a tumor can be attributed to heterogeneous solid stress accumulation. Highly vascularized peripheral regions are also associated with greater oxygenation compared with the compressed, less vascularized inner regions. We also modeled the delivery of drugs of two distinct sizes, namely chemotherapy and nanomedicine. Model predictions suggest that drug delivery is affected negatively by vessel compression independently of the size of the therapeutic agent. Finally, we demonstrated the applicability of our model to actual geometries, employing a breast tumor model derived from MR images. PMID:25968141

  19. Stress-mediated progression of solid tumors: effect of mechanical stress on tissue oxygenation, cancer cell proliferation, and drug delivery.

    PubMed

    Mpekris, Fotios; Angeli, Stelios; Pirentis, Athanassios P; Stylianopoulos, Triantafyllos

    2015-11-01

    Oxygen supply plays a central role in cancer cell proliferation. While vascular density increases at the early stages of carcinogenesis, mechanical solid stresses developed during growth compress tumor blood vessels and, thus, drastically reduce not only the supply of oxygen, but also the delivery of drugs at inner tumor regions. Among other effects, hypoxia and reduced drug delivery compromise the efficacy of radiation and chemo/nanotherapy, respectively. In the present study, we developed a mathematical model of tumor growth to investigate the interconnections among tumor oxygenation that supports cancer cell proliferation, the heterogeneous accumulation of mechanical stresses owing to tumor growth, the non-uniform compression of intratumoral blood vessels due to the mechanical stresses, and the insufficient delivery of oxygen and therapeutic agents because of vessel compression. We found that the high vascular density and increased cancer cell proliferation often observed in the periphery compared to the interior of a tumor can be attributed to heterogeneous solid stress accumulation. Highly vascularized peripheral regions are also associated with greater oxygenation compared with the compressed, less vascularized inner regions. We also modeled the delivery of drugs of two distinct sizes, namely chemotherapy and nanomedicine. Model predictions suggest that drug delivery is affected negatively by vessel compression independently of the size of the therapeutic agent. Finally, we demonstrated the applicability of our model to actual geometries, employing a breast tumor model derived from MR images. PMID:25968141

  20. PIWIL2 induces c-Myc expression by interacting with NME2 and regulates c-Myc-mediated tumor cell proliferation

    PubMed Central

    Zhang, Yu; Lu, Yilu; Chen, Jianhui; Zheng, Xulei; Tao, Dachang; Liu, Yunqiang; Ma, Yongxin

    2014-01-01

    c-Myc serves as a crucial regulator in multiple cellular events. Cumulative evidences demonstrate that anomalous c-Myc overexpression correlates with proliferation, invasion and metastasis in various human tumors. However, the transcriptionally activating mechanisms responsible for c-Myc overexpression are complex and continue to be intangible. Here we showed that Piwi-Like RNA-Mediated Gene Silencing 2 (PIWIL2) can upregulate c-Myc via binding with NME/NM23 nucleoside diphosphate kinase 2 (NME2). PIWIL2 promotes c-Myc transcription by interacting with and facilitating NME2 to bind to G4-motif region within c-Myc promoter. Interestingly, in a c-Myc-mediated manner, PIWIL2 upregulates RhoA, which in turn induces filamentary F-actin. Deficiency of PIWIL2 results in obstacle for c-Myc expression, cell cycle progress and cell proliferation. Taken together, our present work demonstrates that PIWIL2 modulates tumor cell proliferation and F-actin filaments via promoting c-Myc expression. PMID:25193865

  1. Vitronectin absorbed on nanoparticles mediate cell viability/proliferation and uptake by 3T3 Swiss albino mouse fibroblasts: in vitro study.

    PubMed

    Rosso, F; Marino, G; Grimaldi, A; Cafiero, G; Chiellini, E; Chiellini, F; Barbarisi, M; Barbarisi, A

    2013-01-01

    We study the interaction of 3T3 Swiss albino mouse fibroblasts with polymeric nanoparticles (NPs) and investigate cellular behaviour in terms of viability/cytotoxicity, cell cycle, NPs uptake, MAP kinase (ERK1/2), and focal adhesion kinase (FAK) activation. After incubation of NPs with cell culture media, western blot analysis showed that Vitronectin is retained by NPs, while Fibronectin is not detected. From cytotoxicity studies (MTT and BrdU methods) an LD50 of about 1.5 mg/mL results for NPs. However, NPs in the range 0.01-0.30 mg/mL are able to trigger a statistically significant increase in proliferation and cell cycle progression in dose and time depending manner. Also, biochemical evaluation of ERK1/2 and FAK clearly shows an increasing phosphorylation in a dose and time depending manner. Finally, we found by transmission electron microscopy that NPs are internalised by cells. Competitively blocking VN-integrin receptors with echistatin (1 μg/mL) results in a decrease of viability/proliferation, cell cycle progression, cellular uptake, and FAK/ERK activation showing the involvement of Vitronectin receptors in signal transduction. In conclusion, our results show that cell surface NPs interactions are mediated by absorbed plasma proteins (i.e., Vitronectin) that represent an external stimuli, switched to the nucleus by FAK enzyme, which in turn modulate fibroblasts viability/proliferation. PMID:23710450

  2. Magnetofection Based on Superparamagnetic Iron Oxide Nanoparticles Weakens Glioma Stem Cell Proliferation and Invasion by Mediating High Expression of MicroRNA-374a

    PubMed Central

    Pan, Zhiguang; Shi, Zhifeng; Wei, Hua; Sun, Fengyan; Song, Jianping; Huang, Yongyi; Liu, Te; Mao, Ying

    2016-01-01

    Glioma stem cells belong to a special subpopulation of glioma cells that are characterized by strong proliferation, invasion and drug resistance capabilities. Magnetic nanoparticles are nanoscale biological materials with magnetic properties. In this study, CD133+ primary glioma stem cells were isolated from patients and cultured. Then, magnetic nanoparticles were used to mediate the transfection and expression of a microRNA-374a overexpression plasmid in the glioma stem cells. Transmission electron microscopy detected the presence of significant magnetic nanoparticle substances within the CD133+ glioma stem cells after transfection. The qRT-PCR and Northern blot results showed that the magnetic nanoparticles could be used to achieve the transfection of the microRNA-374a overexpression plasmid into glioma stem cells and the efficient expression of mature microRNA-374a. The MTT and flow cytometry results showed that the proliferation inhibition rate was significantly higher in cells from the microRNA-374a transfection group than in cells from the microRNA-mut transfection group; additionally, the former cells presented significant cell cycle arrest. The Transwell experiments confirmed that the overexpression of microRNA-374a could significantly reduce the invasiveness of CD133+ glioma stem cells. Moreover, the high expression of microRNA-374a mediated by the magnetic nanoparticles effectively reduced the tumourigenicity of CD133+ glioma stem cells in nude mice. The luciferase assays revealed that mature microRNA-374a fragments could bind to the 3'UTR of Neuritin (NRN1), thereby interfering with Neuritin mRNA expression. The qRT-PCR and Western blotting results showed that the overexpression of microRNA-374a significantly reduced the expression of genes such as NRN1, CCND1, CDK4 and Ki67 in glioma stem cells. Thus, magnetic nanoparticles can efficiently mediate the transfection and expression of microRNA expression plasmids in mammalian cells. The overexpression of

  3. Cisplatin suppresses the growth and proliferation of breast and cervical cancer cell lines by inhibiting integrin β5-mediated glycolysis

    PubMed Central

    Wang, Shaojia; Xie, Jie; Li, Jiajia; Liu, Fei; Wu, Xiaohua; Wang, Ziliang

    2016-01-01

    Cancer cells harbor lower energy consumption after rounds of anticancer drugs, but the underlying mechanism remains unclear. In this study, we investigated metabolic alterations in cancer cells exposed to cisplatin. The present study exhibited cisplatin, known as a chemotherapeutic agent interacting with DNA, also acted as an anti-metabolic agent. We found that glycolysis levels of breast and cervical cancer cells were reduced after cisplatin treatment, resulting in cells growth and proliferation inhibition. We demonstrated that cisplatin suppressed glycolysis-related proteins expression, including glucose transporter 1 (GLUT1), glucose transporter 4 (GLUT4) and lactate dehydrogenase B (LDHB), through down-regulating integrin β5 (ITGB5)/focal adhesion kinase (FAK) signaling pathway. ITGB5 overexpression rescued cisplatin-induced inhibition of cancer cell glycolysis, growth and proliferation. Conclusively, we reveal a novel insight into cisplatin-induced anticancer mechanism, suggesting alternative strategies to the current therapeutic approaches of targeting ITGB5, as well as of a combination of cisplatin with glucose up-regulation chemotherapeutic agents to enhance anticancer effect. PMID:27294003

  4. Adenosine A{sub 2A} receptor-dependent proliferation of pulmonary endothelial cells is mediated through calcium mobilization, PI3-kinase and ERK1/2 pathways

    SciTech Connect

    Ahmad, Aftab; Schaack, Jerome B.; White, Carl W.; Ahmad, Shama

    2013-05-10

    Highlights: •A{sub 2A} receptor-induced pulmonary endothelial growth is mediated by PI3K and ERK1/2. •Cytosolic calcium mobilization is also critical for pulmonary endothelial growth. •Effectors of A{sub 2A} receptor, like tyrosine kinases and cAMP increase PI3K/Akt signaling. •Activation of A{sub 2A} receptor can contribute to vascular remodeling. -- Abstract: Hypoxia and HIF-2α-dependent A{sub 2A} receptor expression and activation increase proliferation of human lung microvascular endothelial cells (HLMVECs). This study was undertaken to investigate the signaling mechanisms that mediate the proliferative effects of A{sub 2A} receptor. A{sub 2A} receptor-mediated proliferation of HLMVECs was inhibited by intracellular calcium chelation, and by specific inhibitors of ERK1/2 and PI3-kinase (PI3K). The adenosine A{sub 2A} receptor agonist CGS21680 caused intracellular calcium mobilization in controls and, to a greater extent, in A{sub 2A} receptor-overexpressing HLMVECs. Adenoviral-mediated A{sub 2A} receptor overexpression as well as receptor activation by CGS21680 caused increased PI3K activity and Akt phosphorylation. Cells overexpressing A{sub 2A} receptor also manifested enhanced ERK1/2 phosphorylation upon CGS21680 treatment. A{sub 2A} receptor activation also caused enhanced cAMP production. Likewise, treatment with 8Br-cAMP increased PI3K activity. Hence A{sub 2A} receptor-mediated cAMP production and PI3K and Akt phosphorylation are potential mediators of the A{sub 2A}-mediated proliferative response of HLMVECs. Cytosolic calcium mobilization and ERK1/2 phosphorylation are other critical effectors of HLMVEC proliferation and growth. These studies underscore the importance of adenosine A{sub 2A} receptor in activation of survival and proliferative pathways in pulmonary endothelial cells that are mediated through PI3K/Akt and ERK1/2 pathways.

  5. CD137 ligand-mediated reverse signaling inhibits proliferation and induces apoptosis in non-small cell lung cancer.

    PubMed

    Qian, Yingying; Pei, Dong; Cheng, Ting; Wu, Changling; Pu, Xiaolin; Chen, Xiaofeng; Liu, Yiqian; Shen, Hong; Zhang, Weiwei; Shu, Yongqian

    2015-03-01

    CD137 ligand (CD137L), a member of the tumor necrosis factor superfamily, is expressed on antigen-presenting cells and also on various tumor cells. Crosslinking of CD137L transmits signals that evoke different cellular responses in a variety of tumor cells. This study was designed to investigate signaling pathways activated by CD137L and its physiologic role in the progression of NSCLC. We investigated the expression of CD137L in tissues from 102 cases of human non-small cell lung cancer (NSCLC) using immunohistochemistry and analyzed the correlation with clinicopathological features using Fisher's exact test and overall survival using Kaplan-Meier curves and the log-rank test. The effect of CD137L reverse signaling induced by recombinant human CD137-Fc protein on NSCLC cell lines was assessed using proliferation and apoptosis assays, flow cytometry and Western blotting. Positive CD137L expression was observed in 53/102 (52.0%) of the NSCLC samples and correlated with early TNM stage (P = 0.046), well-differentiated tumors (P = 0.009) and better overall survival (P = 0.004). Moreover, induction of CD137L reverse signaling using CD137-Fc inhibited proliferation and induced apoptosis and cell cycle arrest in H1650 cells, which express high levels of CD137L; CD137L reverse signaling had no significant effects in PC9 cells, which express low levels of CD137L. In addition, CD137L reverse signaling-induced apoptosis occurred via activation of the intrinsic pathway and depended on phosphorylation of JNK. This study demonstrates a hitherto unrecognized role for CD137L reverse signaling in the development of NSCLC and indicates that CD137L has potential as a novel therapeutic target in NSCLC. PMID:25631633

  6. Novel roles for LIX1L in promoting cancer cell proliferation through ROS1-mediated LIX1L phosphorylation

    PubMed Central

    Nakamura, Satoki; Kahyo, Tomoaki; Tao, Hong; Shibata, Kiyoshi; Kurabe, Nobuya; Yamada, Hidetaka; Shinmura, Kazuya; Ohnishi, Kazunori; Sugimura, Haruhiko

    2015-01-01

    Herein, we report the characterization of Limb expression 1-like, (LIX1L), a putative RNA-binding protein (RBP) containing a double-stranded RNA binding motif, which is highly expressed in various cancer tissues. Analysis of MALDI-TOF/TOF mass spectrometry and RNA immunoprecipitation-sequencing of interacting proteins and the microRNAs (miRNAs) bound to LIX1L revealed that LIX1L interacts with proteins (RIOK1, nucleolin and PABPC4) and miRNAs (has-miRNA-520a-5p, −300, −216b, −326, −190a, −548b-3p, −7–5p and −1296) in HEK-293 cells. Moreover, the reduction of phosphorylated Tyr136 (pTyr136) in LIX1L through the homeodomain peptide, PY136, inhibited LIX1L-induced cell proliferation in vitro, and PY136 inhibited MKN45 cell proliferation in vivo. We also determined the miRNA-targeted genes and showed that was apoptosis induced through the reduction of pTyr136. Moreover, ROS1, HCK, ABL1, ABL2, JAK3, LCK and TYR03 were identified as candidate kinases responsible for the phosphorylation of Tyr136 of LIX1L. These data provide novel insights into the biological significance of LIX1L, suggesting that this protein might be an RBP, with implications for therapeutic approaches for targeting LIX1L in LIX1L-expressing cancer cells. PMID:26310847

  7. Novel roles for LIX1L in promoting cancer cell proliferation through ROS1-mediated LIX1L phosphorylation.

    PubMed

    Nakamura, Satoki; Kahyo, Tomoaki; Tao, Hong; Shibata, Kiyoshi; Kurabe, Nobuya; Yamada, Hidetaka; Shinmura, Kazuya; Ohnishi, Kazunori; Sugimura, Haruhiko

    2015-01-01

    Herein, we report the characterization of Limb expression 1-like, (LIX1L), a putative RNA-binding protein (RBP) containing a double-stranded RNA binding motif, which is highly expressed in various cancer tissues. Analysis of MALDI-TOF/TOF mass spectrometry and RNA immunoprecipitation-sequencing of interacting proteins and the microRNAs (miRNAs) bound to LIX1L revealed that LIX1L interacts with proteins (RIOK1, nucleolin and PABPC4) and miRNAs (has-miRNA-520a-5p, -300, -216b, -326, -190a, -548b-3p, -7-5p and -1296) in HEK-293 cells. Moreover, the reduction of phosphorylated Tyr(136) (pTyr(136)) in LIX1L through the homeodomain peptide, PY136, inhibited LIX1L-induced cell proliferation in vitro, and PY136 inhibited MKN45 cell proliferation in vivo. We also determined the miRNA-targeted genes and showed that was apoptosis induced through the reduction of pTyr(136). Moreover, ROS1, HCK, ABL1, ABL2, JAK3, LCK and TYR03 were identified as candidate kinases responsible for the phosphorylation of Tyr(136) of LIX1L. These data provide novel insights into the biological significance of LIX1L, suggesting that this protein might be an RBP, with implications for therapeutic approaches for targeting LIX1L in LIX1L-expressing cancer cells. PMID:26310847

  8. Overcrowding-mediated stress alters cell proliferation in key neuroendocrine areas during larval development in Rhinella arenarum.

    PubMed

    Distler, Mijal J; Jungblut, Lucas D; Ceballos, Nora R; Paz, Dante A; Pozzi, Andrea G

    2016-02-01

    Exposure to adverse environmental conditions can elicit a stress response, which results in an increase in endogenous corticosterone levels. In early life stages, it has been thoroughly demonstrated that amphibian larval growth and development is altered as a consequence of chronic stress by interfering with the metamorphic process, however, the underlying mechanisms involved have only been partially disentangled. We examined the effect of intraspecific competition on corticosterone levels during larval development of the toad Rhinella arenarum and its ultimate effects on cell proliferation in particular brain areas as well as the pituitary gland. While overcrowding altered the number of proliferating cells in the pituitary gland, hypothalamus, and third ventricle of the brain, no differences were observed in areas which are less associated with neuroendocrine processes, such as the first ventricle of the brain. Apoptosis was increased in hypothalamic regions but not in the pituitary. With regards to pituitary cell populations, thyrotrophs but not somatoatrophs and corticotrophs showed a decrease in the cell number in overcrowded larvae. Our study shows that alterations in growth and development, produced by stress, results from an imbalance in the neuroendocrine systems implicated in orchestrating the timing of metamorphosis. J. Exp. Zool. 9999A:XX-XX, 2016. © 2016 Wiley Periodicals, Inc. PMID:26817921

  9. Demonstration of. beta. -adrenergic receptors and catecholamine-mediated effects on cell proliferation in embryonic palatal tissue

    SciTech Connect

    Pisano, M.M.

    1986-01-01

    The ability of catecholamines to modulate cell proliferation, differentiation and morphogenesis in other systems, and modulate adenylate cyclase activity in the developing palate during the period of cellular differentiation, made it of interest to determine their involvement in palatal ontogenesis. Catecholamines exert their physiologic effects via interaction with distinct membrane-bound receptors, one class being the B-adrenergic receptors which are coupled to stimulation of adenylate cyclase and the generation of cAMP. A direct radioligand binding technique utilizing the B-adrenergic antagonist (/sup 3/H)-dihydroalprenolol ((/sup 3/H)-DHA) was employed in the identification of B-adrenergic receptors in the developing murine secondary palate. Specific binding of (/sup 3/H)-DHA in embryonic (day 13) palatal tissue homogenates was saturable and of high affinity. The functionality of B-adrenergic receptor binding sites was assessed from the ability of embryonic palate mesenchmyal cells in vitro to respond to catecholamines with elevations of cAMP. Embryonic palate mesenchymal cells responded to various B-adrenergic catecholamine agonists with significant, dose-dependent accumulations of intracellular cAMP. Embryonic (day 13) maxillary tissue homogenates were analyzed for the presence of catecholamines by high performance liquid chromatography and radioenzymatic assay. Since normal palatal and craniofacial morphogenesis depends on proper temporal and spatial patterns of growth, the effect of B-adrenergic catecholamines on embryonic palate mesenchymal cell proliferation was investigated.

  10. Metformin inhibits cell proliferation, migration and invasion by attenuating CSC function mediated by deregulating miRNAs in pancreatic cancer cells.

    PubMed

    Bao, Bin; Wang, Zhiwei; Ali, Shadan; Ahmad, Aamir; Azmi, Asfar S; Sarkar, Sanila H; Banerjee, Sanjeev; Kong, Dejuan; Li, Yiwei; Thakur, Shivam; Sarkar, Fazlul H

    2012-03-01

    Pancreatic cancer is the fourth leading cause of cancer-related deaths in the United States, which is, in part, due to intrinsic (de novo) and extrinsic (acquired) resistance to conventional therapeutics, suggesting that innovative treatment strategies are required for overcoming therapeutic resistance to improve overall survival of patients. Oral administration of metformin in patients with diabetes mellitus has been reported to be associated with reduced risk of pancreatic cancer and that metformin has been reported to kill cancer stem cells (CSC); however, the exact molecular mechanism(s) has not been fully elucidated. In the current study, we examined the effect of metformin on cell proliferation, cell migration and invasion, and self-renewal capacity of CSCs and further assessed the expression of CSC marker genes and microRNAs (miRNA) in human pancreatic cancer cells. We found that metformin significantly decreased cell survival, clonogenicity, wound-healing capacity, sphere-forming capacity (pancreatospheres), and increased disintegration of pancreatospheres in both gemcitabine-sensitive and gemcitabine-resistant pancreatic cancer cells. Metformin also decreased the expression of CSC markers,CD44, EpCAM,EZH2, Notch-1, Nanog and Oct4, and caused reexpression of miRNAs (let-7a,let-7b, miR-26a, miR-101, miR-200b, and miR-200c) that are typically lost in pancreatic cancer and especially in pancreatospheres. We also found that reexpression of miR-26a by transfection led to decreased expression of EZH2 and EpCAM in pancreatic cancer cells. These results clearly suggest that the biologic effects of metformin are mediated through reexpression of miRNAs and decreased expression of CSC-specific genes, suggesting that metformin could be useful for overcoming therapeutic resistance of pancreatic cancer cells. PMID:22086681

  11. Magnetofection based on superparamagnetic iron oxide nanoparticle-mediated low lncRNA HOTAIR expression decreases the proliferation and invasion of glioma stem cells.

    PubMed

    Fang, Kan; Liu, Peifeng; Dong, Suyan; Guo, Yanjie; Cui, Xinxin; Zhu, Xiaoying; Li, Xuan; Jiang, Lianghan; Liu, Te; Wu, Yuncheng

    2016-08-01

    Glioma stem cells (GSCs) are a special subpopulation of glioma cells that are key to the sensitivity of tumors to treatments and to the possibility of tumor recurrence. Identifying new strategies that inhibit the growth of GSCs are therefore important for developing novel therapies for glioblastoma multiforme (GBM). In this study, CD133+ human glioma stem cells were isolated and cultured. Magnetic nanoparticles were used to mediate the expression of siRNAs targeting the HOTAIR (si-HOTAIR) sequence in human gliomas. Effect of downregulation of HOTAIR expression on proliferation, invasion and in vivo tumorigenicity of human GSCs and underlying molecular mechanisms were further evaluated. The results of the MTT assay and flow cytometric analysis showed that downregulation of HOTAIR expression inhibited cell proliferation and induced cell cycle arrest. Transwell assays demonstrated that downregulation of HOTAIR expression resulted in a decrease in the invasive capability of GSCs. Moreover, magnetic nanoparticle-mediated low expression of HOTAIR effectively reduced the tumorigenic capacity of glioma stem cells in vivo. In addition, the results of qRT-PCR and western blot analysis demonstrated that downregulation of HOTAIR expression significantly increased the expression of PDCD4 in GSCs, in addition to reducing the expression of CCND1 and CDK4. An in-depth mechanistic analysis showed that downregulation of HOTAIR expression reduced the recruitment of downstream molecules, EZH2 and LSD1, thereby activating the expression of PDCD4 at the transcriptional level. In conclusion, downregulation of HOTAIR expression effectively promoted the expression of PDCD4, thereby inhibiting the proliferation, invasion and in vivo tumorigenicity of human GSCs. PMID:27277755

  12. Magnetofection based on superparamagnetic iron oxide nanoparticle-mediated low lncRNA HOTAIR expression decreases the proliferation and invasion of glioma stem cells

    PubMed Central

    Fang, Kan; Liu, Peifeng; Dong, Suyan; Guo, Yanjie; Cui, Xinxin; Zhu, Xiaoying; Li, Xuan; Jiang, Lianghan; Liu, Te; Wu, Yuncheng

    2016-01-01

    Glioma stem cells (GSCs) are a special subpopulation of glioma cells that are key to the sensitivity of tumors to treatments and to the possibility of tumor recurrence. Identifying new strategies that inhibit the growth of GSCs are therefore important for developing novel therapies for glioblastoma multiforme (GBM). In this study, CD133+ human glioma stem cells were isolated and cultured. Magnetic nanoparticles were used to mediate the expression of siRNAs targeting the HOTAIR (si-HOTAIR) sequence in human gliomas. Effect of downregulation of HOTAIR expression on proliferation, invasion and in vivo tumorigenicity of human GSCs and underlying molecular mechanisms were further evaluated. The results of the MTT assay and flow cytometric analysis showed that downregulation of HOTAIR expression inhibited cell proliferation and induced cell cycle arrest. Transwell assays demonstrated that downregulation of HOTAIR expression resulted in a decrease in the invasive capability of GSCs. Moreover, magnetic nanoparticle-mediated low expression of HOTAIR effectively reduced the tumorigenic capacity of glioma stem cells in vivo. In addition, the results of qRT-PCR and western blot analysis demonstrated that downregulation of HOTAIR expression significantly increased the expression of PDCD4 in GSCs, in addition to reducing the expression of CCND1 and CDK4. An in-depth mechanistic analysis showed that downregulation of HOTAIR expression reduced the recruitment of downstream molecules, EZH2 and LSD1, thereby activating the expression of PDCD4 at the transcriptional level. In conclusion, downregulation of HOTAIR expression effectively promoted the expression of PDCD4, thereby inhibiting the proliferation, invasion and in vivo tumorigenicity of human GSCs. PMID:27277755

  13. The GTPase ARF6 Controls ROS Production to Mediate Angiotensin II-Induced Vascular Smooth Muscle Cell Proliferation

    PubMed Central

    Bourmoum, Mohamed; Charles, Ricardo; Claing, Audrey

    2016-01-01

    High reactive oxygen species (ROS) levels and enhanced vascular smooth muscle cells (VSMC) proliferation are observed in numerous cardiovascular diseases. The mechanisms by which hormones such as angiotensin II (Ang II) acts to promote these cellular responses remain poorly understood. We have previously shown that the ADP-ribosylation factor 6 (ARF6), a molecular switch that coordinates intracellular signaling events can be activated by the Ang II receptor (AT1R). Whether this small GTP-binding protein controls the signaling events leading to ROS production and therefore Ang II-dependent VSMC proliferation, remains however unknown. Here, we demonstrate that in rat aortic VSMC, Ang II stimulation led to the subsequent activation of ARF6 and Rac1, a key regulator of NADPH oxidase activity. Using RNA interference, we showed that ARF6 is essential for ROS generation since in conditions where this GTPase was knocked down, Ang II could no longer promote superoxide anion production. In addition to regulating Rac1 activity, ARF6 also controlled expression of the NADPH oxidase 1 (Nox 1) as well as the ability of the EGFR to become transactivated. Finally, ARF6 also controlled MAPK (Erk1/2, p38 and Jnk) activation, a key pathway of VSMC proliferation. Altogether, our findings demonstrate that Ang II promotes activation of ARF6 to controls ROS production by regulating Rac1 activation and Nox1 expression. In turn, increased ROS acts to activate the MAPK pathway. These signaling events represent a new molecular mechanism by which Ang II can promote proliferation of VSMC. PMID:26824355

  14. Targeting histone deacetylase 6 mediates a dual anti-melanoma effect: Enhanced antitumor immunity and impaired cell proliferation

    PubMed Central

    Perez-Villaroel, P.; Lee, C.; Cheng, F.; Knox, T.; Woods, D.M.; Barrios, K.; Powers, J.; Sahakian, E.; Wang, H.W.; Canales, J.; Marante, D.; Smalley, K.S.M.; Bergman, J.; Seto, E.; Kozikowski, A.; Pinilla-Ibarz, J.; Sarnaik, A.; Celis, E.; Weber, J.; Sotomayor, E.M.; Villagra, A.

    2015-01-01

    The median survival for metastatic melanoma is in the realm of 8–16 months and there are few therapies that offer significant improvement in overall survival. One of the recent advances in cancer treatment focuses on epigenetic modifiers to alter the survivability and immunogenicity of cancer cells. Our group and others have previously demonstrated that pan-HDAC inhibitors induce apoptosis, cell cycle arrest and changes in the immunogenicity of melanoma cells. Here we interrogated specific HDACs which may be responsible for this effect. We found that both genetic abrogation and pharmacologic inhibition of HDAC6 decreases in vitro proliferation and induces G1 arrest of melanoma cell lines without inducing apoptosis. Moreover, targeting this molecule led to an important upregulation in the expression of tumor associated antigens and MHC class I, suggesting a potential improvement in the immunogenicity of these cells. Of note, this anti-melanoma activity was operative regardless of mutational status of the cells. These effects translated into a pronounced delay of in vivo melanoma tumor growth which was, at least in part, dependent on intact immunity as evidenced by the restoration of tumor growth after CD4+ and CD8+ depletion. Given our findings, we provide the initial rationale for the further development of selective HDAC6 inhibitors as potential therapeutic anti-melanoma agents. PMID:25957812

  15. RNAi-mediated knockdown of pituitary tumor-transforming gene-1 (PTTG1) suppresses the proliferation and invasive potential of PC3 human prostate cancer cells

    PubMed Central

    Huang, S.Q.; Liao, Q.J.; Wang, X.W.; Xin, D.Q.; Chen, S.X.; Wu, Q.J.; Ye, G.

    2012-01-01

    Pituitary tumor-transforming gene-1 (PTTG1) is a proto-oncogene that promotes tumorigenesis and metastasis in numerous cell types and is overexpressed in a variety of human tumors. We have demonstrated that PTTG1 expression was up-regulated in both human prostate cancer specimens and prostate cancer cell lines. For a more direct assessment of the function of PTTG1 in prostate tumorigenesis, RNAi-mediated knockdown was used to selectively decrease PTTG1 expression in PC3 human prostate tumor cells. After three weeks of selection, colonies stably transfected with PTTG1-targeted RNAi (the knockdown PC3 cell line) or empty vector (the control PC3 cell line) were selected and expanded to investigate the role of PTTG1 expression in PC3 cell growth and invasion. Cell proliferation rate was significantly slower (28%) in the PTTG1 knockdown line after 6 days of growth as indicated by an MTT cell viability assay (P < 0.05). Similarly, a soft agar colony formation assay revealed significantly fewer (66.7%) PTTG1 knockdown PC3 cell colonies than control colonies after three weeks of growth. In addition, PTTG1 knockdown resulted in cell cycle arrest at G1 as indicated by fluorescence-activated cell sorting. The PTTG1 knockdown PC3 cell line also exhibited significantly reduced migration through Matrigel in a transwell assay of invasive potential, and down-regulation of PTTG1 could lead to increased sensitivity of these prostate cancer cells to a commonly used anticancer drug, taxol. Thus, PTTG1 expression is crucial for PC3 cell proliferation and invasion, and could be a promising new target for prostate cancer therapy. PMID:22872288

  16. Inhibition of miR301 enhances Akt-mediated cell proliferation by accumulation of PTEN in nucleus and its effects on cell-cycle regulatory proteins

    PubMed Central

    Jain, Mayur V.; Shareef, Ahmad; Likus, Wirginia; Cieślar-Pobuda, Artur; Ghavami, Saeid; Łos, Marek J.

    2016-01-01

    Micro-RNAs (miRs) represent an innovative class of genes that act as regulators of gene expression. Recently, the aberrant expression of several miRs has been associated with different types of cancers. In this study, we show that miR301 inhibition influences PI3K-Akt pathway activity. Akt overexpression in MCF7 and MDAMB468 cells caused downregulation of miR301 expression. This effect was confirmed by co-transfection of miR301-modulators in the presence of Akt. Cells overexpressing miR301-inhibitor and Akt, exhibited increased migration and proliferation. Experimental results also confirmed PI3K, PTEN and FoxF2 as regulatory targets for miR301. Furthermore, Akt expression in conjunction with miR301-inhibitor increased nuclear accumulation of PTEN, thus preventing it from downregulating the PI3K-signalling. In summary, our data emphasize the importance of miR301 inhibition on PI3K-Akt pathway-mediated cellular functions. Hence, it opens new avenues for the development of new anti-cancer agents preferentially targeting PI3K-Akt pathway. PMID:26967567

  17. Dynamin-related Protein 1 Inhibition Mitigates Bisphenol A-mediated Alterations in Mitochondrial Dynamics and Neural Stem Cell Proliferation and Differentiation.

    PubMed

    Agarwal, Swati; Yadav, Anuradha; Tiwari, Shashi Kant; Seth, Brashket; Chauhan, Lalit Kumar Singh; Khare, Puneet; Ray, Ratan Singh; Chaturvedi, Rajnish Kumar

    2016-07-29

    The regulatory dynamics of mitochondria comprises well orchestrated distribution and mitochondrial turnover to maintain the mitochondrial circuitry and homeostasis inside the cells. Several pieces of evidence suggested impaired mitochondrial dynamics and its association with the pathogenesis of neurodegenerative disorders. We found that chronic exposure of synthetic xenoestrogen bisphenol A (BPA), a component of consumer plastic products, impaired autophagy-mediated mitochondrial turnover, leading to increased oxidative stress, mitochondrial fragmentation, and apoptosis in hippocampal neural stem cells (NSCs). It also inhibited hippocampal derived NSC proliferation and differentiation, as evident by the decreased number of BrdU- and β-III tubulin-positive cells. All these effects were reversed by the inhibition of oxidative stress using N-acetyl cysteine. BPA up-regulated the levels of Drp-1 (dynamin-related protein 1) and enhanced its mitochondrial translocation, with no effect on Fis-1, Mfn-1, Mfn-2, and Opa-1 in vitro and in the hippocampus. Moreover, transmission electron microscopy studies suggested increased mitochondrial fission and accumulation of fragmented mitochondria and decreased elongated mitochondria in the hippocampus of the rat brain. Impaired mitochondrial dynamics by BPA resulted in increased reactive oxygen species and malondialdehyde levels, disruption of mitochondrial membrane potential, and ATP decline. Pharmacological (Mdivi-1) and genetic (Drp-1siRNA) inhibition of Drp-1 reversed BPA-induced mitochondrial dysfunctions, fragmentation, and apoptosis. Interestingly, BPA-mediated inhibitory effects on NSC proliferation and neuronal differentiations were also mitigated by Drp-1 inhibition. On the other hand, Drp-1 inhibition blocked BPA-mediated Drp-1 translocation, leading to decreased apoptosis of NSC. Overall, our studies implicate Drp-1 as a potential therapeutic target against BPA-mediated impaired mitochondrial dynamics and

  18. nAChRs Mediate Human Embryonic Stem Cell-Derived Endothelial Cells: Proliferation, Apoptosis, and Angiogenesis

    PubMed Central

    Velotta, Jeffrey B.; Huang, Mei; Li, Zongjin; Lee, Andrew; Robbins, Robert C.; Cooke, John P.; Wu, Joseph C.

    2009-01-01

    Background Many patients with ischemic heart disease have cardiovascular risk factors such as cigarette smoking. We tested the effect of nicotine (a key component of cigarette smoking) on the therapeutic effects of human embryonic stem cell-derived endothelial cells (hESC-ECs). Methods and Results To induce endothelial cell differentiation, undifferentiated hESCs (H9 line) underwent 4-day floating EB formation and 8-day outgrowth differentiation in EGM-2 media. After 12 days, CD31+ cells (13.7±2.5%) were sorted by FACScan and maintained in EGM-2 media for further differentiation. After isolation, these hESC-ECs expressed endothelial specific markers such as vWF (96.3±1.4%), CD31 (97.2±2.5%), and VE-cadherin (93.7±2.8%), form vascular-like channels, and incorporated DiI-labeled acetylated low-density lipoprotein (DiI-Ac-LDL). Afterward, 5×106 hESC-ECs treated for 24 hours with nicotine (10−8 M) or PBS (as control) were injected into the hearts of mice undergoing LAD ligation followed by administration for two weeks of vehicle or nicotine (100 µg/ml) in the drinking water. Surprisingly, bioluminescence imaging (BLI) showed significant improvement in the survival of transplanted hESC-ECs in the nicotine treated group at 6 weeks. Postmortem analysis confirmed increased presence of small capillaries in the infarcted zones. Finally, in vitro mechanistic analysis suggests activation of the MAPK and Akt pathways following activation of nicotinic acetylcholine receptors (nAChRs). Conclusions This study shows for the first time that short-term systemic administrations of low dose nicotine can improve the survival of transplanted hESC-ECs, and enhance their angiogenic effects in vivo. Furthermore, activation of nAChRs has anti-apoptotic, angiogenic, and proliferative effects through MAPK and Akt signaling pathways. PMID:19753305

  19. Mild Glucose Starvation Induces KDM2A-Mediated H3K36me2 Demethylation through AMPK To Reduce rRNA Transcription and Cell Proliferation

    PubMed Central

    Tanaka, Yuji; Yano, Hirohisa; Ogasawara, Sachiko; Yoshioka, Sho-ichi; Imamura, Hiromi; Okamoto, Kengo

    2015-01-01

    Environmental conditions control rRNA transcription. Previously, we found that serum and glucose deprivation induces KDM2A-mediated H3K36me2 demethylation in the rRNA gene (rDNA) promoter and reduces rRNA transcription in the human breast cancer cell line MCF-7. However, the molecular mechanism and biological significance are still unclear. In the present study, we found that glucose starvation alone induced the KDM2A-dependent reduction of rRNA transcription. The treatment of cells with 2-deoxy-d-glucose, an inhibitor of glycolysis, reduced rRNA transcription and H3K36me2 in the rDNA promoter, both of which were completely dependent on KDM2A in low concentrations of 2-deoxy-d-glucose, that is, mild starvation conditions. The mild starvation induced these KDM2A activities through AMP-activated kinase (AMPK) but did not affect another AMPK effector of rRNA transcription, TIF-IA. In the triple-negative breast cancer cell line MDA-MB-231, the mild starvation also reduced rRNA transcription in a KDM2A-dependent manner. We detected KDM2A in breast cancer tissues irrespective of their estrogen receptor, progesterone receptor, and HER2 status, including triple-negative cancer tissues. In both MCF-7 and MDA-MB-231 cells, mild starvation reduced cell proliferation, and KDM2A knockdown suppressed the reduction of cell proliferation. These results suggest that under mild glucose starvation AMPK induces KDM2A-dependent reduction of rRNA transcription to control cell proliferation. PMID:26416883

  20. siRNA-mediated knockdown against CDCA1 and KNTC2, both frequently overexpressed in colorectal and gastric cancers, suppresses cell proliferation and induces apoptosis

    SciTech Connect

    Kaneko, Naoyuki; Miura, Koh; Gu, Zhaodi; Karasawa, Hideaki; Ohnuma, Shinobu; Sasaki, Hiroyuki; Tsukamoto, Nobukazu; Yokoyama, Satoru; Yamamura, Akihiro; Nagase, Hiroki; Shibata, Chikashi; Sasaki, Iwao; Horii, Akira

    2009-12-25

    Ndc80 has been shown to play an important role in stable microtubule-kinetochore attachment, chromosome alignment, and spindle checkpoint activation in mitosis. It is composed of two heterodimers, CDCA1-KNTC2 and SPC24-SPC25. Overexpression of CDCA1 and KNTC2 is reported to be associated with poor prognosis in non-small cell lung cancers (NSCLC), and siRNA-mediated knockdown against CDCA1 or KNTC2 has been found to inhibit cell proliferation and induction of apoptosis in NSCLC, ovarian cancer, cervical cancer and glioma. Therefore, CDCA1 and KNTC2 can be considered good candidates for molecular target therapy as well as diagnosis in some cancers. However, the role of the Ndc80 complex in colorectal and gastric cancers (CRC and GC) still remains unclear. In the present study, we used qRT-PCR to evaluate the expression levels of CDCA1, KNTC2, SPC24 and SPC25 in CRC and GC and employed siRNA-mediated knockdown to examine cell proliferation and apoptosis. mRNA overexpression of these four genes was observed in CRCs and GCs when compared with the corresponding normal mucosae. Additionally, the expression levels of tumor/normal ratios of CDCA1, KNTC2, SPC24 and SPC25 correlated with each other in CRCs. MTT assays revealed that cell growths after the siRNA-mediated knockdown of either CDCA1 or KNTC2 were significantly suppressed, and flow cytometry analyses revealed significant increases of the subG1 fractions after knockdown against both genes. Our present results suggest that expressional control of component molecules of Ndc80 can be utilized for molecular target therapy of patients with CRC and GC.

  1. miR-141 is involved in BRD7-mediated cell proliferation and tumor formation through suppression of the PTEN/AKT pathway in nasopharyngeal carcinoma

    PubMed Central

    Liu, Y; Zhao, R; Wang, H; Luo, Y; Wang, X; Niu, W; Zhou, Y; Wen, Q; Fan, S; Li, X; Xiong, W; Ma, J; Li, X; Tan, M; Li, G; Zhou, M

    2016-01-01

    Bromodomain containing 7 (BRD7) was identified as a nuclear transcriptional regulatory factor. BRD7 functions as a tumor suppressor in multiple cancers, including nasopharyngeal carcinoma (NPC). In this study, we reported a novel mechanism of BRD7 in NPC progression. We demonstrated that the expression of miR-141 was remarkably increased in NPC tissues and was negatively correlated with the expression of BRD7 and the survival rate of NPC patients. Decreased expression levels of miR-141, including the primary, the precursor and the mature forms of miR-141, were found in BRD7-overexpressing HEK293, 5-8F and HNE1 cells compared the control cells, while there was no obvious effect on the expression levels of the two critical enzymes Drosha and Dicer. BRD7 can negatively regulate the promoter activity of miR-141, while no obvious binding site of BRD7 was found in the potential promoter region of miR-141. Moreover, ectopic expression of miR-141 can significantly promote cell proliferation and inhibit apoptosis in NPC, and rescuing the expression of miR-141 in BRD7-overexpressing NPC cells could partially reverse the tumor suppressive effect of BRD7 on cell proliferation and tumor growth in vitro and in vivo. Furthermore, the activation of the PTEN/AKT pathway mediated by the overexpression of BRD7 could be inhibited by rescuing the expression of miR-141, which accordingly results in the partial restoration of cell proliferation and tumor growth. Our findings demonstrate that the BRD7/miR-141/PTEN/AKT axis has critical roles in the progression of NPC and provide some promising targets for the diagnosis and treatment of NPC. PMID:27010857

  2. DTX3L and ARTD9 inhibit IRF1 expression and mediate in cooperation with ARTD8 survival and proliferation of metastatic prostate cancer cells

    PubMed Central

    2014-01-01

    Background Prostate cancer (PCa) is one of the leading causes of cancer-related mortality and morbidity in the aging male population and represents the most frequently diagnosed malignancy in men around the world. The Deltex (DTX)-3-like E3 ubiquitin ligase (DTX3L), also known as B-lymphoma and BAL-associated protein (BBAP), was originally identified as a binding partner of the diphtheria-toxin-like macrodomain containing ADP-ribosyltransferase-9 (ARTD9), also known as BAL1 and PARP9. We have previously demonstrated that ARTD9 acts as a novel oncogenic survival factor in high-risk, chemo-resistant, diffuse large B cell lymphoma (DLBCL). The mono-ADP-ribosyltransferase ARTD8, also known as PARP14 functions as a STAT6-specific co-regulator of IL4-mediated proliferation and survival in B cells. Methods Co-expression of DTX3L, ARTD8, ARTD9 and STAT1 was analyzed in the metastatic PCa (mPCa) cell lines PC3, DU145, LNCaP and in the normal prostate luminal epithelial cell lines HPE and RWPE1. Effects on cell proliferation, survival and cell migration were determined in PC3, DU145 and/or LNCaP cells depleted of DTX3L, ARTD8, ARTD9, STAT1 and/or IRF1 compared to their proficient control cells, respectively. In further experiments, real-time RT-PCR, Western blot, immunofluorescence and co-immunoprecipitations were conducted to evaluate the physical and functional interactions between DTX3L, ARTD8 and ARTD9. Results Here we could identify DTX3L, ARTD9 and ARTD8 as novel oncogenic survival factors in mPCa cells. Our studies revealed that DTX3L forms a complex with ARTD8 and mediates together with ARTD8 and ARTD9 proliferation, chemo-resistance and survival of mPCa cells. In addition, DTX3L, ARTD8 and ARTD9 form complexes with each other. Our study provides first evidence that the enzymatic activity of ARTD8 is required for survival of mPCa cells. DTX3L and ARTD9 act together as repressors of the tumor suppressor IRF1 in mPCa cells. Furthermore, the present study shows that DTX

  3. Macrophage-Associated Osteoactivin/GPNMB Mediates Mesenchymal Stem Cell Survival, Proliferation, and Migration Via a CD44-Dependent Mechanism.

    PubMed

    Yu, Bing; Sondag, Gregory R; Malcuit, Christopher; Kim, Min-Ho; Safadi, Fayez F

    2016-07-01

    Although MSCs have been widely recognized to have therapeutic potential in the repair of injured or diseased tissues, it remains unclear how functional activities of mesenchymal stem cells (MSCs) are influenced by the surrounding inflammatory milieu at the site of tissue injury. Macrophages constitute an essential component of innate immunity and have been shown to exhibit a phenotypic plasticity in response to various stimuli, which play a central role in both acute inflammation and wound repair. Osteoactivin (OA)/Glycoprotein non-metastatic melanoma protein B (GPNMB), a transmembrane glycoprotein that plays a role in cell differentiation, survival, and angiogenesis. The objective of this study was to investigate the potential role of OA/GPNMB in macrophage-induced MSC function. We found that reparative M2 macrophages express significantly greater levels of OA/GPNMB than pro-inflammatory M1 macrophages. Furthermore, using loss of function and rescue studies, we demonstrated that M2 macrophages-secreted OA/GPNMB positively regulates the viability, proliferation, and migration of MSCs. More importantly, we demonstrated that OA/GPNMB acts through ERK and AKT signaling pathways in MSCs via CD44, to induce these effects. Taken together, our results provide pivotal insight into the mechanism by which OA/GPNMB contributes to the tissue reparative phenotype of M2 macrophages and positively regulates functional activities of MSCs. J. Cell. Biochem. 117: 1511-1521, 2016. © 2015 Wiley Periodicals, Inc. PMID:26442636

  4. YAP Mediates Tumorigenesis in Neurofibromatosis Type 2 by Promoting Cell Survival and Proliferation through a COX-2-EGFR Signaling Axis.

    PubMed

    Guerrant, William; Kota, Smitha; Troutman, Scott; Mandati, Vinay; Fallahi, Mohammad; Stemmer-Rachamimov, Anat; Kissil, Joseph L

    2016-06-15

    The Hippo-YAP pathway has emerged as a major driver of tumorigenesis in many human cancers. YAP is a transcriptional coactivator and while details of YAP regulation are quickly emerging, it remains unknown what downstream targets are critical for the oncogenic functions of YAP. To determine the mechanisms involved and to identify disease-relevant targets, we examined the role of YAP in neurofibromatosis type 2 (NF2) using cell and animal models. We found that YAP function is required for NF2-null Schwann cell survival, proliferation, and tumor growth in vivo Moreover, YAP promotes transcription of several targets including PTGS2, which codes for COX-2, a key enzyme in prostaglandin biosynthesis, and AREG, which codes for the EGFR ligand, amphiregulin. Both AREG and prostaglandin E2 converge to activate signaling through EGFR. Importantly, treatment with the COX-2 inhibitor celecoxib significantly inhibited the growth of NF2-null Schwann cells and tumor growth in a mouse model of NF2. Cancer Res; 76(12); 3507-19. ©2016 AACR. PMID:27216189

  5. Power Frequency Magnetic Fields Affect the p38 MAPK-Mediated Regulation of NB69 Cell Proliferation Implication of Free Radicals.

    PubMed

    Martínez, María Antonia; Úbeda, Alejandro; Moreno, Jorge; Trillo, María Ángeles

    2016-01-01

    The proliferative response of the neuroblastoma line NB69 to a 100 µT, 50 Hz magnetic field (MF) has been shown mediated by activation of the MAPK-ERK1/2 pathway. This work investigates the MF effect on the cell cycle of NB69, the participation of p38 and c-Jun N-terminal (JNK) kinases in the field-induced proliferative response and the potential involvement of reactive oxygen species (ROS) in the activation of the MAPK-ERK1/2 and -p38 signaling pathways. NB69 cultures were exposed to the 100 µT MF, either intermittently for 24, 42 or 63 h, or continuously for periods of 15 to 120 min, in the presence or absence of p38 or JNK inhibitors: SB203580 and SP600125, respectively. Antioxidant N-acetylcysteine (NAC) was used as ROS scavenger. Field exposure induced transient activation of p38, JNK and ERK1/2. The MF proliferative effect, which was mediated by changes in the cell cycle, was blocked by the p38 inhibitor, but not by the JNK inhibitor. NAC blocked the field effects on cell proliferation and p38 activation, but not those on ERK1/2 activation. The MF-induced proliferative effects are exerted through sequential upregulation of MAPK-p38 and -ERK1/2 activation, and they are likely mediated by a ROS-dependent activation of p38. PMID:27058530

  6. Power Frequency Magnetic Fields Affect the p38 MAPK-Mediated Regulation of NB69 Cell Proliferation Implication of Free Radicals

    PubMed Central

    Martínez, María Antonia; Úbeda, Alejandro; Moreno, Jorge; Trillo, María Ángeles

    2016-01-01

    The proliferative response of the neuroblastoma line NB69 to a 100 µT, 50 Hz magnetic field (MF) has been shown mediated by activation of the MAPK-ERK1/2 pathway. This work investigates the MF effect on the cell cycle of NB69, the participation of p38 and c-Jun N-terminal (JNK) kinases in the field-induced proliferative response and the potential involvement of reactive oxygen species (ROS) in the activation of the MAPK-ERK1/2 and -p38 signaling pathways. NB69 cultures were exposed to the 100 µT MF, either intermittently for 24, 42 or 63 h, or continuously for periods of 15 to 120 min, in the presence or absence of p38 or JNK inhibitors: SB203580 and SP600125, respectively. Antioxidant N-acetylcysteine (NAC) was used as ROS scavenger. Field exposure induced transient activation of p38, JNK and ERK1/2. The MF proliferative effect, which was mediated by changes in the cell cycle, was blocked by the p38 inhibitor, but not by the JNK inhibitor. NAC blocked the field effects on cell proliferation and p38 activation, but not those on ERK1/2 activation. The MF-induced proliferative effects are exerted through sequential upregulation of MAPK-p38 and -ERK1/2 activation, and they are likely mediated by a ROS-dependent activation of p38. PMID:27058530

  7. Stromal COX-2 signaling activated by deoxycholic acid mediates proliferation and invasiveness of colorectal epithelial cancer cells

    SciTech Connect

    Zhu, Yingting; Tissue Tech Inc., Miami, FL 33173 ; Zhu, Min; Lance, Peter

    2012-08-31

    Highlights: Black-Right-Pointing-Pointer Human colonic cancer associated fibroblasts are major sources of COX-2 and PGE{sub 2}. Black-Right-Pointing-Pointer The fibroblasts interact with human colonic epithelial cancer cells. Black-Right-Pointing-Pointer Activation of COX-2 signaling in the fibroblasts affects behavior of the epithelia. Black-Right-Pointing-Pointer Protein Kinase C controls the activation of COX-2 signaling. -- Abstract: COX-2 is a major regulator implicated in colonic cancer. However, how COX-2 signaling affects colonic carcinogenesis at cellular level is not clear. In this article, we investigated whether activation of COX-2 signaling by deoxycholic acid (DCA) in primary human normal and cancer associated fibroblasts play a significant role in regulation of proliferation and invasiveness of colonic epithelial cancer cells. Our results demonstrated while COX-2 signaling can be activated by DCA in both normal and cancer associated fibroblasts, the level of activation of COX-2 signaling is significantly greater in cancer associated fibroblasts than that in normal fibroblasts. In addition, we discovered that the proliferative and invasive potential of colonic epithelial cancer cells were much greater when the cells were co-cultured with cancer associated fibroblasts pre-treated with DCA than with normal fibroblasts pre-treated with DCA. Moreover, COX-2 siRNA attenuated the proliferative and invasive effect of both normal and cancer associate fibroblasts pre-treated with DCA on the colonic cancer cells. Further studies indicated that the activation of COX-2 signaling by DCA is through protein kinase C signaling. We speculate that activation of COX-2 signaling especially in cancer associated fibroblasts promotes progression of colonic cancer.

  8. PPARγ mediates the effects of WIN55,212-2, an synthetic cannabinoid, on the proliferation and apoptosis of the BEL-7402 hepatocarcinoma cells.

    PubMed

    Hong, Yuehui; Zhou, Yuting; Wang, Ying; Xiao, Shunhua; Liao, D Joshua; Zhao, Qing

    2013-11-01

    Cannabis sativa has long been used as a traditional medicine in China. Among its effective compounds are cannabinoids. This study determined the effect of WIN55,212-2 (WIN), a synthetic cannabinoid, on the BEL-7402 human hepatocellular carcinoma (HCC) cell line. The results showed that WIN could decrease the proliferation of BEL-7402 cells. Moreover, WIN could cause apoptosis of the cells via up-regulation of Bax expression, down-regulation of Bcl-2 expression, induction of the mitochondrial membrane potential, increase of caspase-3, -8 and -9 activities, and induction of the cleavage of caspase-3 and poly-ADP-ribose polymerase (PARP). The WIN-induced apoptosis was accompanied by the up-regulation of PPARγ expression, the activation of PPARγ DNA binding activity, and a down-regulation of PPARγ target oncogene c-myc. Conversely, the effects of WIN could be attenuated by PPARγ antagonist GW9662, and the WIN induced PPARγ expression was partially attenuated by AM630, a cannabinoid receptor-2 antagonist, whereas the WIN-induced reduction of c-myc expression was partially restored by GW9662. Collectively, our results suggest that WIN can decrease the proliferation and cause apoptosis of the BEL-7402 cells via a mitochondrial-caspase pathway and mediated by PPARγ. These results may provide a basis for the application of WIN in HCC treatment. PMID:24062073

  9. Carboxy terminus and pore-forming domain properties specific to Cx37 are necessary for Cx37-mediated suppression of insulinoma cell proliferation

    PubMed Central

    Nelson, Tasha K.; Sorgen, Paul L.

    2013-01-01

    Connexin 37 (Cx37) suppresses cell proliferation when expressed in rat insulinoma (Rin) cells, an effect also manifest in vivo during vascular development and in response to tissue injury. Mutant forms of Cx37 with nonfunctional channels but normally localized, wild-type carboxy termini are not growth suppressive. Here we determined whether the carboxy-terminal (CT) domain is required for Cx37-mediated growth suppression and whether the Cx37 pore-forming domain can be replaced with the Cx43 pore-forming domain and still retain growth-suppressive properties. We show that despite forming functional gap junction channels and hemichannels, Cx37 with residues subsequent to 273 replaced with a V5-epitope tag (Cx37–273tr*V5) had no effect on the proliferation of Rin cells, did not facilitate G1-cell cycle arrest with serum deprivation, and did not prolong cell cycle time comparably to the wild-type protein. The chimera Cx43*CT37, comprising the pore-forming domain of Cx43 and CT of Cx37, also did not suppress proliferation, despite forming functional gap junctions with a permselective profile similar to wild-type Cx37. Differences in channel behavior of both Cx37–273tr*V5 and Cx43*CT37 relative to their wild-type counterparts and failure of the Cx37-CT to interact as the Cx43-CT does with the Cx43 cytoplasmic loop suggest that the Cx37-CT and pore-forming domains are both essential to growth suppression by Cx37. PMID:24133065

  10. Apelin-13-induced proliferation and migration induced of rat vascular smooth muscle cells is mediated by the upregulation of Egr-1

    SciTech Connect

    Liu, Qi-Feng; Yu, Hong-Wei; You, Lu; Liu, Ming-Xin; Li, Ke-Yan; Tao, Gui-Zhou

    2013-09-20

    Highlights: •The mechanism underlying the effects of Apelin-13 on VSMC was investigated. •Apelin-13 induced VSMC migration, proliferation and Egr-1 and OPN upregulation. •These effects were inhibited by the Egr-1 specific deoxyribozyme, ED5. •The effects of Apelin-13 on VSMC are mediated via Egr-1 upregulation. •These data will help in attempts to prevent and treat vascular remodeling diseases. -- Abstract: Apelin-13 plays an important role in the migration and proliferation of vascular smooth muscle cells (VSMCs); however, the underlying mechanisms are still unclear. Egr-1 is a nuclear transcription factor, which is considered to be the critical initiating factor of the processes of VSMC proliferation and migration. Egr-1 is known to regulate the expression of osteopontin (OPN), which is a marker of the phenotypic modulation that is a necessary condition of VSMC proliferation and migration. We hypothesized that the role of Apelin-13 is mediated via upregulation of Egr-1. To test this hypothesis, we analyzed the effects of Apelin-13 treatment on Egr-1 mRNA and protein expression in A10 rat aortic VSMCs by RT-PCR and Western blotting, respectively. Results showed that, Apelin-13 upregulated the expression of Egr-1. Furthermore, treatment with the extracellular-regulated protein kinase (ERK) inhibitor, PD98059, inhibited the upregulation of Egr-1 by Apelin-13. In addition, this upregulation was inhibited by treatment of VSMCs with the Egr-1 specific deoxyribozyme ED5 (DNAenzyme/10-23 DRz). Furthermore, ED5 treatment was found to significantly inhibit Apelin-13-induced migration and proliferation of VSMCs using transwell and MTT assays, respectively. The evaluation of OPN mRNA and protein expression levels by RT-PCR and Western blot analyses revealed that ED5 treatment also inhibited Apelin-13-induced OPN upregulation. The results of this study indicated that Apelin-13 upregulates Egr-1 via ERK. Furthermore, Apelin-13 induced the proliferation and migration

  11. CD56brightCD16- NK Cells Produce Adenosine through a CD38-Mediated Pathway and Act as Regulatory Cells Inhibiting Autologous CD4+ T Cell Proliferation.

    PubMed

    Morandi, Fabio; Horenstein, Alberto L; Chillemi, Antonella; Quarona, Valeria; Chiesa, Sabrina; Imperatori, Andrea; Zanellato, Silvia; Mortara, Lorenzo; Gattorno, Marco; Pistoia, Vito; Malavasi, Fabio

    2015-08-01

    Recent studies suggested that human CD56(bright)CD16(-) NK cells may play a role in the regulation of the immune response. Since the mechanism(s) involved have not yet been elucidated, in the present study we have investigated the role of nucleotide-metabolizing enzymes that regulate the extracellular balance of nucleotides/nucleosides and produce the immunosuppressive molecule adenosine (ADO). Peripheral blood CD56(dim)CD16(+) and CD56(bright)CD16(-) NK cells expressed similar levels of CD38. CD39, CD73, and CD157 expression was higher in CD56(bright)CD16(-) than in CD56(dim)CD16(+) NK cells. CD57 was mostly expressed by CD56(dim)CD16(+) NK cells. CD203a/PC-1 expression was restricted to CD56(bright)CD16(-) NK cells. CD56(bright)CD16(-) NK cells produce ADO and inhibit autologous CD4(+) T cell proliferation. Such inhibition was 1) reverted pretreating CD56(bright)CD16(-) NK cells with a CD38 inhibitor and 2) increased pretreating CD56(bright)CD16(-) NK cells with a nucleoside transporter inhibitor, which increase extracellular ADO concentration. CD56(bright)CD16(-) NK cells isolated from the synovial fluid of juvenile idiopathic arthritis patients failed to inhibit autologous CD4(+) T cell proliferation. Such functional impairment could be related to 1) the observed reduced CD38/CD73 expression, 2) a peculiar ADO production kinetics, and 3) a different expression of ADO receptors. In contrast, CD56(bright)CD16(-) NK cells isolated from inflammatory pleural effusions display a potent regulatory activity. In conclusion, CD56(bright)CD16(-) NK cells act as "regulatory cells" through ADO produced by an ectoenzymes network, with a pivotal role of CD38. This function may be relevant for the modulation of the immune response in physiological and pathological conditions, and it could be impaired during autoimmune/inflammatory diseases. PMID:26091716

  12. RNAi-mediated knockdown of the CLN3 gene inhibits proliferation and promotes apoptosis in drug-resistant ovarian cancer cells

    PubMed Central

    MAO, DONGWEI; CHE, JIANHUA; HAN, SHIYU; ZHAO, HONGHUI; ZHU, YUMEI; ZHU, HONG

    2015-01-01

    CLN3 is a recently identified anti-apoptotic gene, which has been demonstrated to be highly expressed in a diverse range of cancer cell lines, including ovarian cancer. In the present study, RNA interference, mediated by a lentivirus expressing CLN3 short hairpin RNA (shRNA) was utilized to knockdown the expression of CLN3 in the A2780 human ovarian cancer cell line, and its cisplatin-resistant and carboplatin-resistant sublines, A2780/DDP and A2780/CBP cells. It was revealed that the mRNA and protein expression levels of CLN3 were significantly reduced in the CLN3-specific shRNA-transduced cells, compared with the untransduced and control shRNA-transduced cells. In addition, specific knockdown of CLN3 in these cells inhibited cell proliferation and led to cell cycle arrest at the G0/G1 phase, with eventual apoptosis. CLN3 knockdown caused increases in the levels of Bax, FAX, cleaved-caspase 3, cleaved-caspase 8 and cleaved-RARP, but decreased the level of Bcl-2. Finally, it was observed that CLN3 depletion markedly reduced the half maximum inhibitory concentration in the A2780/DDP and A2780/CBP cells. Taken together, these data suggested that CLN3 is involved in tumorigenesis and drug resistance in ovarian cancer, and may serve as a promising therapeutic target for its treatment. PMID:26299671

  13. Small-molecule inhibitors of ERK-mediated immediate early gene expression and proliferation of melanoma cells expressing mutated BRaf.

    PubMed

    Samadani, Ramin; Zhang, Jun; Brophy, Amanda; Oashi, Taiji; Priyakumar, U Deva; Raman, E Prabhu; St John, Franz J; Jung, Kwan-Young; Fletcher, Steven; Pozharski, Edwin; MacKerell, Alexander D; Shapiro, Paul

    2015-05-01

    Constitutive activation of the extracellular-signal-regulated kinases 1 and 2 (ERK1/2) are central to regulating the proliferation and survival of many cancer cells. The current inhibitors of ERK1/2 target ATP binding or the catalytic site and are therefore limited in their utility for elucidating the complex biological roles of ERK1/2 through its phosphorylation and regulation of over 100 substrate proteins. To overcome this limitation, a combination of computational and experimental methods was used to identify low-molecular-mass inhibitors that are intended to target ERK1/2 substrate-docking domains and selectively interfere with ERK1/2 regulation of substrate proteins. In the present study, we report the identification and characterization of compounds with a thienyl benzenesulfonate scaffold that were designed to inhibit ERK1/2 substrates containing an F-site or DEF (docking site for ERK, FXF) motif. Experimental evidence shows the compounds inhibit the expression of F-site containing immediate early genes (IEGs) of the Fos family, including c-Fos and Fra1, and transcriptional regulation of the activator protein-1 (AP-1) complex. Moreover, this class of compounds selectively induces apoptosis in melanoma cells containing mutated BRaf and constitutively active ERK1/2 signalling, including melanoma cells that are inherently resistant to clinically relevant kinase inhibitors. These findings represent the identification and initial characterization of a novel class of compounds that inhibit ERK1/2 signalling functions and their potential utility for elucidating ERK1/2 and other signalling events that control the growth and survival of cancer cells containing elevated ERK1/2 activity. PMID:25695333

  14. Small-molecule inhibitors of ERK-mediated immediate early gene expression and proliferation of melanoma cells expressing mutated BRaf

    PubMed Central

    Samadani, Ramin; Zhang, Jun; Brophy, Amanda; Oashi, Taiji; Priyakumar, U. Deva; Raman, E. Prabhu; St John, Franz J.; Jung, Kwan-Young; Fletcher, Steven; Pozharski, Edwin; MacKerell, Alexander D.; Shapiro, Paul

    2015-01-01

    Constitutive activation of the extracellular-signal-regulated kinases 1 and 2 (ERK1/2) are central to regulating the proliferation and survival of many cancer cells. The current inhibitors of ERK1/2 target ATP binding or the catalytic site and are therefore limited in their utility for elucidating the complex biological roles of ERK1/2 through its phosphorylation and regulation of over 100 substrate proteins. To overcome this limitation, a combination of computational and experimental methods was used to identify low-molecular-mass inhibitors that are intended to target ERK1/2 substrate-docking domains and selectively interfere with ERK1/2 regulation of substrate proteins. In the present study, we report the identification and characterization of compounds with a thienyl benzenesulfonate scaffold that were designed to inhibit ERK1/2 substrates containing an F-site or DEF (docking site for ERK, FXF) motif. Experimental evidence shows the compounds inhibit the expression of F-site containing immediate early genes (IEGs) of the Fos family, including c-Fos and Fra1, and transcriptional regulation of the activator protein-1 (AP-1) complex. Moreover, this class of compounds selectively induces apoptosis in melanoma cells containing mutated BRaf and constitutively active ERK1/2 signalling, including melanoma cells that are inherently resistant to clinically relevant kinase inhibitors. These findings represent the identification and initial characterization of a novel class of compounds that inhibit ERK1/2 signalling functions and their potential utility for elucidating ERK1/2 and other signalling events that control the growth and survival of cancer cells containing elevated ERK1/2 activity. PMID:25695333

  15. Ubiquitin carboxyl terminal hydrolase L1 negatively regulates TNF{alpha}-mediated vascular smooth muscle cell proliferation via suppressing ERK activation

    SciTech Connect

    Ichikawa, Tomonaga; Li, Jinqing; Dong, Xiaoyu; Potts, Jay D.; Tang, Dong-Qi; Li, Dong-Sheng; Cui, Taixing

    2010-01-01

    Deubiquitinating enzymes (DUBs) appear to be critical regulators of a multitude of processes such as proliferation, apoptosis, differentiation, and inflammation. We have recently demonstrated that a DUB of ubiquitin carboxyl terminal hydrolase L1 (UCH-L1) inhibits vascular lesion formation via suppressing inflammatory responses in vasculature. However, the precise underlying mechanism remains to be defined. Herein, we report that a posttranscriptional up-regulation of UCH-L1 provides a negative feedback to tumor necrosis factor alpha (TNF{alpha})-mediated activation of extracellular signal-regulated kinases (ERK) and proliferation in vascular smooth muscle cells (VSMCs). In rat adult VSMCs, adenoviral over-expression of UCH-L1 inhibited TNF{alpha}-induced activation of ERK and DNA synthesis. In contrast, over-expression of UCH-L1 did not affect platelet derived growth factor (PDGF)-induced VSMC proliferation and activation of growth stimulating cascades including ERK. TNF{alpha} hardly altered UCH-L1 mRNA expression and stability; however, up-regulated UCH-L1 protein expression via increasing UCH-L1 translation. These results uncover a novel mechanism by which UCH-L1 suppresses vascular inflammation.

  16. Thromboxane A2 Receptor Inhibition Suppresses Multiple Myeloma Cell Proliferation by Inducing p38/c-Jun N-terminal Kinase (JNK) Mitogen-activated Protein Kinase (MAPK)-mediated G2/M Progression Delay and Cell Apoptosis.

    PubMed

    Liu, Qian; Tao, Bo; Liu, Guizhu; Chen, Guilin; Zhu, Qian; Yu, Ying; Yu, Yu; Xiong, Hong

    2016-02-26

    Multiple myeloma (MM) is a plasma cell malignancy without effective therapeutics. Thromboxane A2 (TxA2)/TxA2 receptor (T prostanoid receptor (TP)) modulates the progression of some carcinomas; however, its effects on MM cell proliferation remain unclear. In this study, we evaluated cyclooxygenase (COX) enzymes and downstream prostaglandin profiles in human myeloma cell lines RPMI-8226 and U-266 and analyzed the effects of COX-1/-2 inhibitors SC-560 and NS-398 on MM cell proliferation. Our observations implicate COX-2 as being involved in modulating cell proliferation. We further incubated MM cells with prostaglandin receptor antagonists or agonists and found that only the TP antagonist, SQ29548, suppressed MM cell proliferation. TP silencing and the TP agonist, U46619, further confirmed this finding. Moreover, SQ29548 and TP silencing promoted MM cell G2/M phase delay accompanied by reducing cyclin B1/cyclin-dependent kinase-1 (CDK1) mRNA and protein expression. Notably, cyclin B1 overexpression rescued MM cells from G2/M arrest. We also found that the TP agonist activated JNK and p38 MAPK phosphorylation, and inhibitors of JNK and p38 MAPK depressed U46619-induced proliferation and cyclin B1/CDK1 protein expression. In addition, SQ29548 and TP silencing led to the MM cell apoptotic rate increasing with improving caspase 3 activity. The knockdown of caspase 3 reversed the apoptotic rate. Taken together, our results suggest that TxA2/TP promotes MM cell proliferation by reducing cell delay at G2/M phase via elevating p38 MAPK/JNK-mediated cyclin B1/CDK1 expression and hindering cell apoptosis. The TP inhibitor has potential as a novel agent to target kinase cascades for MM therapy. PMID:26724804

  17. Novel role of Zn(II)-curcumin in enhancing cell proliferation and adjusting proinflammatory cytokine-mediated oxidative damage of ethanol-induced acute gastric ulcers.

    PubMed

    Mei, Xueting; Xu, Donghui; Xu, Sika; Zheng, Yanping; Xu, Shibo

    2012-04-15

    Alcohol consumption can induce gastric ulcers and zinc deficiency. Zinc complexes were reported to have anti-ulcer activity as it acts as an anti-inflammatory and antioxidant. Zn(II)-curcumin complex and its solid dispersions (SDs) were synthesized and evaluated for its gastroprotective activity and mechanism against ethanol-induced ulcer. The Swiss murine fibroblast cell line (3T3) was used as an alternative in vitro model to evaluate the effects of Zn(II)-curcumin on cell proliferation. Zn(II)-curcumin were administered orally for seven consecutive days prior to induction of ulcers using ethanol. Gross and microscopic lesions, immunological and biochemical parameters were taken into consideration. The results showed that solid dispersions (SDs) of Zn(II)-curcumin (2.5-20 μM) enhanced the proliferation of 3T3 cells more significantly than curcumin at the same concentrations (P<0.01). Oral administration of Zn(II)-curcumin (12, 24 and 48 mg/kg) SDs dose-dependently prevented formation of ulcer lesions induced by ethanol. The levels of proinflammatory cytokines tumor necrosis factor-α (TNF-α), interleukin 6 (IL-6), and oxidative stress superoxide dismutase (SOD), glutathione peroxidase (GPX-Px), malonaldehyde (MDA) and H(+)-K(+)-ATPase were in the rats exposed to ethanol in ulceration have been altered. Zn(II)-curcumin prevented formation of ulcer lesions, significantly inhibited TNF-α and IL-6 mRNA expression, increased the activity of SOD and GSH-Px, reduced MDA levels and H(+)-K(+)-ATPase in mucosa of rats compared to controls (P<0.05). These findings suggest that the gastroprotective activity of Zn(II)-curcumin complex might contribute in stimulating cell proliferation and adjusting the proinflammatory cytokine-mediated oxidative damage to the gastric mucosa. PMID:22465177

  18. TNFα Signaling Regulates Cystic Epithelial Cell Proliferation through Akt/mTOR and ERK/MAPK/Cdk2 Mediated Id2 Signaling

    PubMed Central

    Zhou, Julie X.; Fan, Lucy X.; Li, Xiaoyan; Calvet, James P.; Li, Xiaogang

    2015-01-01

    Tumor necrosis factor alpha (TNFα) is present in cyst fluid and promotes cyst growth in autosomal dominant polycystic kidney disease (ADPKD). However, the cross-talk between TNFα and PKD associated signaling pathways remains elusive. In this study, we found that stimulation of renal epithelial cells with TNFα or RANKL (receptor activator of NF-κB ligand), a member of the TNFα cytokine family, activated either the PI3K pathway, leading to AKT and mTOR mediated the increase of Id2 protein, or MAPK and Cdk2 to induce Id2 nuclear translocation. The effects of TNFα/RANKL on increasing Id2 protein and its nuclear translocation caused significantly decreased mRNA and protein levels of the Cdk inhibitor p21, allowing increased cell proliferation. TNFα levels increase in cystic kidneys in response to macrophage infiltration and thus might contribute to cyst growth and enlargement during the progression of disease. As such, this study elucidates a novel mechanism for TNFα signaling in regulating cystic renal epithelial cell proliferation in ADPKD. PMID:26110849

  19. EZH2-mediated epigenetic suppression of long noncoding RNA SPRY4-IT1 promote s NSCLC cell proliferation and metastasis by affecting the epithelial–mesenchymal transition

    PubMed Central

    Sun, M; Liu, X-H; Lu, K-H; Nie, F-Q; Xia, R; Kong, R; Yang, J-S; Xu, T-P; Liu, Y-W; Zou, Y-F; Lu, B-B; Yin, R; Zhang, E-B; Xu, L; De, W; Wang, Z-X

    2014-01-01

    Recent evidence indicates that long noncoding RNAs (lncRNAs) have a critical role in the regulation of cellular processes such as differentiation, proliferation, and metastasis. These lncRNAs are dysregulated in a variety of cancers and many function as tumor suppressors; however, the regulatory factors involved in silencing lncRNA transcription are poorly understood. In this study, we showed that epigenetic silencing of lncRNA SPRY4 intronic transcript 1 (SPRY4-IT1) occurs in non-small-cell lung cancer (NSCLC) cells through direct transcriptional repression mediated by the Polycomb group protein enhancer of zeste homolog 2 (EZH2). SPRY4-IT1 is derived from an intron within SPRY4, and is upregulated in melanoma cells; knockdown of its expression leads to cell growth arrest, invasion inhibition, and elevated rates of apoptosis. Upon depletion of EZH2 by RNA interference, SPRY4-IT1 expression was restored, and transfection of SPRY4-IT1 into NSCLC cells resulted in a significant antitumoral effect, both in culture and in xenografted nude mice. Moreover, overexpression of SPRY4-IT1 was found to have a key role in the epithelial–mesenchymal transition through the regulation of E-cadherin and vimentin expression. In EZH2-knockdown cells, which characteristically showed impaired cell proliferation and metastasis, the induction of SPRY4-IT1 depletion partially rescued the oncogenic phenotype, suggesting that SPRY4-IT1 repression has an important role in EZH2 oncogenesis. Of most relevance, translation of these findings into human NSCLC tissue samples demonstrated that patients with low levels of SPRY4-IT1 expression had a shorter overall survival time, suggesting that SPRY4-IT1 could be a biomarker for poor prognosis of NSCLC. PMID:24967960

  20. Myogenic-specific ablation of Fgfr1 impairs FGF2-mediated proliferation of satellite cells at the myofiber niche but does not abolish the capacity for muscle regeneration

    PubMed Central

    Yablonka-Reuveni, Zipora; Danoviz, Maria E.; Phelps, Michael; Stuelsatz, Pascal

    2015-01-01

    Skeletal muscle satellite cells (SCs) are Pax7+ myogenic stem cells that reside between the basal lamina and the plasmalemma of the myofiber. In mature muscles, SCs are typically quiescent, but can be activated in response to muscle injury. Depending on the magnitude of tissue trauma, SCs may divide minimally to repair subtle damage within individual myofibers or produce a larger progeny pool that forms new myofibers in cases of overt muscle injury. SC transition through proliferation, differentiation and renewal is governed by the molecular blueprint of the cells as well as by the extracellular milieu at the SC niche. In particular, the role of the fibroblast growth factor (FGF) family in regulating SCs during growth and aging is well recognized. Of the several FGFs shown to affect SCs, FGF1, FGF2, and FGF6 proteins have been documented in adult skeletal muscle. These prototypic paracrine FGFs transmit their mitogenic effect through the FGFRs, which are transmembrane tyrosine kinase receptors. Using the mouse model, we show here that of the four Fgfr genes, only Fgfr1 and Fgfr4 are expressed at relatively high levels in quiescent SCs and their proliferating progeny. To further investigate the role of FGFR1 in adult myogenesis, we have employed a genetic (Cre/loxP) approach for myogenic-specific (MyoDCre-driven) ablation of Fgfr1. Neither muscle histology nor muscle regeneration following cardiotoxin-induced injury were overtly affected in Fgfr1-ablated mice. This suggests that FGFR1 is not obligatory for SC performance in this acute muscle trauma model, where compensatory growth factor/cytokine regulatory cascades may exist. However, the SC mitogenic response to FGF2 is drastically repressed in isolated myofibers prepared from Fgfr1-ablated mice. Collectively, our study indicates that FGFR1 is important for FGF-mediated proliferation of SCs and its mitogenic role is not compensated by FGFR4 that is also highly expressed in SCs. PMID:26074812

  1. The role of cyclooxygenase in n-6 and n-3 polyunsaturated fatty acid mediated effects on cell proliferation, PGE(2) synthesis and cytotoxicity in human colorectal carcinoma cell lines.

    PubMed

    Dommels, Yvonne E M; Haring, Merel M G; Keestra, Nynke G M; Alink, Gerrit M; van Bladeren, Peter J; van Ommen, Ben

    2003-03-01

    This study was conducted to investigate the role of the enzyme cyclooxygenase (COX) and its prostaglandin product PGE(2) in n-6 and n-3 polyunsaturated fatty acid (PUFA)-mediated effects on cellular proliferation of two human colorectal carcinoma cell lines. The long chain PUFAs eicosapentaenoic acid (EPA; 20:5n-3) and arachidonic acid (AA; 20:4n-6) both inhibited cell proliferation of Caco-2 cells compared with the long chain fatty acids alpha-linolenic acid (ALA; 18:3n-3) and linoleic acid (LA; 18:2n-6). Neither incubation with PGE(2) nor reduction in PGE(2) synthesis by EPA compared with AA led to differential effects on cell proliferation in Caco-2 cells. This suggests that n-6 and n-3 PUFA-mediated cell proliferation in Caco-2 cells is not regulated via PGE(2) levels. AA and EPA had no effect on growth of HT-29 colon cancer cells with a low COX activity. However, stimulation of COX-2 activity by IL-1 beta resulted in a decrease in cell proliferation and an induction of cytotoxicity by AA as well as by EPA. Both inhibition of the COX pathway by indomethacin as well as inhibition of direct lipid peroxidation by antioxidants such as vitamin E and C diminished the anti-proliferative effects of AA as well as EPA. Also, malondialdehyde, a product of lipid peroxidation and COX-activity was decreased by addition of vitamin E and partially decreased by indomethacin. These data support the hypothesis that growth inhibitory and cytotoxic effects of PUFAs with methylene-interrupted double bonds such as AA and EPA are due to peroxidation products that are generated during lipid peroxidation and COX activity. PMID:12663496

  2. Hypoxia inducible factor-1 mediates expression of miR-322: potential role in proliferation and migration of pulmonary arterial smooth muscle cells

    PubMed Central

    Zeng, Yan; Liu, Hongtao; Kang, Kang; Wang, Zhiwei; Hui, Gang; Zhang, Xiaoying; Zhong, Jiasheng; Peng, Wenda; Ramchandran, Ramaswamy; Usha Raj, J.; Gou, Deming

    2015-01-01

    There is growing evidence that microRNAs play important roles in cellular responses to hypoxia and in pulmonary hypertensive vascular remodeling, but the exact molecular mechanisms involved are not fully elucidated. In this study, we identified miR-322 as one of the microRNAs induced in lungs of chronically hypoxic mice and rats. The expression of miR-322 was also upregulated in primary cultured rat pulmonary arterial smooth muscle cells (PASMC) in response to hypoxia. We demonstrated that HIF-1α, but not HIF-2α, transcriptionally upregulates the expression of miR-322 in hypoxia. Furthermore, miR-322 facilitated the accumulation of HIF-1α in the nucleus and promoted hypoxia-induced cell proliferation and migration. Direct targeting BMPR1a and smad5 by miR-322 was demonstrated in PASMCs suggesting that downregulation of BMP-Smad signaling pathway may be mediating the hypoxia-induced PASMC proliferation and migration. Our study implicates miR-322 in the hypoxic proliferative response of PASMCs suggesting that it may be playing a role in pulmonary vascular remodeling associated with pulmonary hypertension. PMID:26166214

  3. MicroRNA-let-7a promotes E2F-mediated cell proliferation and NFκB activation in vitro

    PubMed Central

    Chafin, Cristen B; Regna, Nicole L; Caudell, David L; Reilly, Christopher M

    2014-01-01

    Epigenetic factors, including altered microRNA (miRNA) expression, may contribute to aberrant immune cell function in systemic lupus erythematosus (SLE). MiRNA-let-7a (let-7a) has been shown to directly alter cell cycle progression and proinflammatory cytokine production. Due to the crucial role of let-7a in cell division and inflammation, we investigated let-7a-mediated proliferation and NFκB translocation in J774A.1 macrophages and MES 13 mesangial cells in vitro. In immune-stimulated cells transfected with let-7a, cell proliferation was significantly increased over time. There was a significant increase in the number of immune-stimulated cells in S and G2 phases. Immune-stimulated cells overexpressing let-7a had increased nuclear translocation of NFκB. Bioinformatical analysis revealed that the E2F family, critical regulators of the G1–S transition, has potential binding sites for let-7a in their mRNA transcripts. Let-7a overexpression significantly increased the expression of the cell cycle activator E2F2 and increased retinoblastoma protein (Rb) phosphorylation in immune-stimulated cells. The cell cycle inhibitor E2F5 was significantly decreased in let-7a-transfected cells that were immune-stimulated. Bioinformatical analysis revealed E2F2 and NFκB are transcription factors predicted to regulate the let-7a promoter. We analyzed transcriptional regulation of let-7a by real-time RT-PCR using chromatin immunoprecipitation with E2F2 and NFκB antibodies. There was an increase in E2F2 and NFκB binding in DNA enriched for the let-7a promoter in immune-stimulated cells. Silencing E2F2 or NFκB significantly decreased let-7a expression and IL-6 production in immune-stimulated cells. Taken together, our results suggest that overexpression of let-7a may contribute to hyperplasia and the proinflammatory response in SLE. PMID:24240124

  4. c-Maf regulates pluripotency genes, proliferation/self-renewal, and lineage commitment in ROS-mediated senescence of human mesenchymal stem cells

    PubMed Central

    Li, Nan-Ting; Wu, Yao-Ming; Lin, Ming-Tsan; Hung, Shih-Chieh; Yen, Men-Luh

    2015-01-01

    Mesenchymal stem cells (MSCs) are therapeutically relevant multilineage and immunomodulatory progenitors. Ex vivo expansion of these rare cells is necessary for clinical application and can result in detrimental senescent effects, with mechanisms still largely unknown. We found that vigorous ex vivo expansion of human adipose tissue-derived MSCs (hAMSCs) results in proliferative decline, cell cycle arrest, and altered differentiation capacity. This senescent phenotype was associated with reactive oxygen species (ROS) accumulation, and with increased expression of G1 cell -cycle inhibitors— p15INK4b and p16INK4a — but decreased expression of pluripotency genes—Oct-4, Sox-2, Nanog, and c-Myc—as well as c-Maf a co-factor of MSC lineage-specific transcription factor and sensitive to oxidative stress. These global changes in the transcriptional and functional programs of proliferation, differentiation, and self-renewal were all mediated by ROS-induced suppression of c-Maf, as evidenced by binding of c-Maf to promoter regions of multiple relevant genes in hAMSCs which could be reduced by exogenous ROS. Our findings implicate the strong effects of ROS on multiple stem cell functions with a central role for c-Maf in stem cell senescence. PMID:26496036

  5. Toll-Like Receptor 2 Mediates Proliferation, Survival, NF-κB Translocation, and Cytokine mRNA Expression in LIF-Maintained Mouse Embryonic Stem Cells

    PubMed Central

    Taylor, Tammi; Kim, Young-June; Ou, Xuan; Derbigny, Wilbert

    2010-01-01

    Toll-like receptor (TLR) activation is important in immune responses and in differentiation of hematopoietic stem cells. We detected mRNA expression of TLRs 1, 2, 3, 5, and 6, but not TLRs 4, 7, 8, and 9 in murine (m)ESC line E14, and noted high cell surface protein expression of TLR2, but not TLR4, for mESC lines R1, CGR8, and E14. ESC lines were cultured in the presence of leukemia inhibitory factor (LIF). Pam3Cys enhanced proliferation and survival of the 3 ESC lines. In contrast, lipopolysaccharide (LPS) decreased proliferation and survival. Pam3Cys and LPS effects on proliferation and survival were blocked by antibody to TLR2, suggesting that effects of both Pam3Cys and LPS on these mESC lines were likely mediated through TLR2. E14 ESC line expressed MyD88. Pam3Cys stimulation of E14 ESCs was associated with induced NF-κB translocation, enhanced phosphorylation of IKK-α/β, and enhanced mRNA, but not protein, expression of tumor necrosis factor-α, interferon-γ, and IL-6. TLR2 activation by Pam3Cys or inhibition by LPS was not associated with changes in morphology or expression of alkaline phosphatase, Oct4, SSEA1, KLF4, or Sox2, markers of undifferentiated mESCs. Our studies identify TLR2 as present and functional in E14, R1, and CGR8 mESC lines. PMID:20132051

  6. DOT1L Activity Promotes Proliferation and Protects Cortical Neural Stem Cells from Activation of ATF4-DDIT3-Mediated ER Stress In Vitro.

    PubMed

    Roidl, Deborah; Hellbach, Nicole; Bovio, Patrick P; Villarreal, Alejandro; Heidrich, Stefanie; Nestel, Sigrun; Grüning, Björn A; Boenisch, Ulrike; Vogel, Tanja

    2016-01-01

    Growing evidence suggests that the lysine methyltransferase DOT1L/KMT4 has important roles in proliferation, survival, and differentiation of stem cells in development and in disease. We investigated the function of DOT1L in neural stem cells (NSCs) of the cerebral cortex. The pharmacological inhibition and shRNA-mediated knockdown of DOT1L impaired proliferation and survival of NSCs. DOT1L inhibition specifically induced genes that are activated during the unfolded protein response (UPR) in the endoplasmic reticulum (ER). Chromatin-immunoprecipitation analyses revealed that two genes encoding for central molecules involved in the ER stress response, Atf4 and Ddit3 (Chop), are marked with H3K79 methylation. Interference with DOT1L activity resulted in transcriptional activation of both genes accompanied by decreased levels of H3K79 dimethylation. Although downstream effectors of the UPR, such as Ppp1r15a/Gadd34, Atf3, and Tnfrsf10b/Dr5 were also transcriptionally activated, this most likely occurred in response to increased ATF4 expression rather than as a direct consequence of altered H3K79 methylation. While stem cells are particularly vulnerable to stress, the UPR and ER stress have not been extensively studied in these cells yet. Since activation of the ER stress program is also implicated in directing stem cells into differentiation or to maintain a proliferative status, the UPR must be tightly regulated. Our and published data suggest that histone modifications, including H3K4me3, H3K14ac, and H3K79me2, are implicated in the control of transcriptional activation of ER stress genes. In this context, the loss of H3K79me2 at the Atf4- and Ddit3-promoters appears to mark a point-of-no-return that activates the death program in NSCs. PMID:26299268

  7. Calcium signaling and cell proliferation.

    PubMed

    Pinto, Mauro Cunha Xavier; Kihara, Alexandre Hiroaki; Goulart, Vânia A M; Tonelli, Fernanda M P; Gomes, Katia N; Ulrich, Henning; Resende, Rodrigo R

    2015-11-01

    Cell proliferation is orchestrated through diverse proteins related to calcium (Ca(2+)) signaling inside the cell. Cellular Ca(2+) influx that occurs first by various mechanisms at the plasma membrane, is then followed by absorption of Ca(2+) ions by mitochondria and endoplasmic reticulum, and, finally, there is a connection of calcium stores to the nucleus. Experimental evidence indicates that the fluctuation of Ca(2+) from the endoplasmic reticulum provides a pivotal and physiological role for cell proliferation. Ca(2+) depletion in the endoplasmatic reticulum triggers Ca(2+) influx across the plasma membrane in an phenomenon called store-operated calcium entries (SOCEs). SOCE is activated through a complex interplay between a Ca(2+) sensor, denominated STIM, localized in the endoplasmic reticulum and a Ca(2+) channel at the cell membrane, denominated Orai. The interplay between STIM and Orai proteins with cell membrane receptors and their role in cell proliferation is discussed in this review. PMID:26275497

  8. Oridonin, a novel lysine acetyltransferases inhibitor, inhibits proliferation and induces apoptosis in gastric cancer cells through p53- and caspase-3-mediated mechanisms

    PubMed Central

    Zhang, Juan; Diao, Hua; Li, Guangming; Xu, Ling; Wang, Ting; Wei, Jue; Meng, Wenying; Ma, Jia-Li; Yu, Heguo; Wang, Yu-Gang

    2016-01-01

    Lysine acetylation has been reported to involve in the pathogenesis of multiple diseases including cancer. In our screening study to identify natural compounds with lysine acetyltransferase inhibitor (KATi) activity, oridonin was found to possess acetyltransferase-inhibitory effects on multiple acetyltransferases including P300, GCN5, Tip60, and pCAF. In gastric cancer cells, oridonin treatment inhibited cell proliferation in a concentration-dependent manner and down-regulated the expression of p53 downstream genes, whereas p53 inhibition by PFT-α reversed the antiproliferative effects of oridonin. Moreover, oridonin treatment induced cell apoptosis, increased the levels of activated caspase-3 and caspase-9, and decreased the mitochondrial membrane potential in gastric cancer cells in a concentration-dependent manner. Caspase-3 inhibition by Ac-DEVD-CHO reversed the proapoptosis effect of oridonin. In conclusion, our study identified oridonin as a novel KATi and demonstrated its tumor suppressive effects in gastric cancer cells at least partially through p53-and caspase-3-mediated mechanisms. PMID:26980707

  9. Arsenic Trioxide Induces Apoptosis and Incapacitates Proliferation and Invasive Properties of U87MG Glioblastoma Cells through a Possible NF-κB-Mediated Mechanism.

    PubMed

    Ghaffari, Seyed H; Yousefi, Meysam; Dizaji, Majid Zaki; Momeny, Majid; Bashash, Davood; Zekri, Ali; Alimoghaddam, Kamran; Ghavamzadeh, Ardeshir

    2016-01-01

    Identification of novel therapeutics in glioblastoma remains crucial due to the devastating and infiltrative capacity of this malignancy. The current study was aimed to appraise effect of arsenic trioxide (ATO) in U87MG cells. The results demonstrated that ATO induced apoptosis and impeded proliferation of U87MG cells in a dosedependent manner and also inhibited classical NF-κB signaling pathway. ATO further upregulated expression of Bax as an important proapoptotic target of NF-κB and also inhibited mRNA expression of survivin, c-Myc and hTERT and suppressed telomerase activity. Moreover, ATO significantly increased adhesion of U87MG cells and also diminished transcription of NF-κB down-stream targets involved in cell migration and invasion, including cathepsin B, uPA, MMP-2, MMP-9 and MMP-14 and suppressed proteolytic activity of cathepsin B, MMP-2 and MMP-9, demonstrating a possible mechanism of ATO effect on a well-known signaling in glioblastoma dissemination. Taken together, here we suggest that ATO inhibits survival and invasion of U87MG cells possibly through NF-κB-mediated inhibition of survivin and telomerase activity and NF-κB-dependent suppression of cathepsin B, MMP-2 and MMP-9. PMID:27039805

  10. Cardiac shockwave therapy improves myocardial function in patients with refractory coronary artery disease by promoting VEGF and IL-8 secretion to mediate the proliferation of endothelial progenitor cells

    PubMed Central

    CAI, HONG-YAN; LI, LIN; GUO, TAO; WANG, YU; MA, TIE-KUN; XIAO, JIAN-MING; ZHAO, LING; FANG, YIN; YANG, PING; ZHAO, HU

    2015-01-01

    Cardiac shockwave therapy (CSWT) is a potential and effective remedy to promote revascularization in the ischemic myocardium of patients with refractory coronary heart disease (CHD). The technique is both safe and non-invasive; however, the underlying molecular mechanism remains unclear. The aim of this study was to evaluate the efficacy of CSWT in treating CHD patients and investigate a potential mechanism. A total of 26 patients with CHD were enrolled in the study, and CSWT was performed over a 3-month period. The efficacy of CSWT was assessed using several clinical parameters. Peripheral blood (PB) was collected prior to and following treatment. The number of circulating endothelial progenitor cells (EPCs) in the PB was counted using a flow cytometer, and the levels of vascular endothelial growth factor (VEGF), interleukin-8 (IL-8), stromal cell-derived factor 1 and matrix metalloproteinase 9 in the PB were analyzed. Mononuclear cells were isolated from the PB and cultured in vitro. The EPCs and EPC-colony forming units (EPC-CFUs) in the PB mononuclear cell culture were counted using an inverted phase contrast microscope. Following CSWT, the tested clinical parameters were significantly improved. The levels of circulating EPCs, VEGF and IL-8 in the PB were significantly increased, as were the EPCs and EPC-CFUs from the PB mononuclear cell culture. We suggest that EPC proliferation, mediated by VEGF and IL-8 secretion, may be among the potential mechanisms associated with CSWT. PMID:26668649

  11. E2F1-Mediated Upregulation of p19INK4d Determines Its Periodic Expression during Cell Cycle and Regulates Cellular Proliferation

    PubMed Central

    Carcagno, Abel L.; Marazita, Mariela C.; Ogara, María F.; Ceruti, Julieta M.; Sonzogni, Silvina V.; Scassa, María E.; Giono, Luciana E.; Cánepa, Eduardo T.

    2011-01-01

    Background A central aspect of development and disease is the control of cell proliferation through regulation of the mitotic cycle. Cell cycle progression and directionality requires an appropriate balance of positive and negative regulators whose expression must fluctuate in a coordinated manner. p19INK4d, a member of the INK4 family of CDK inhibitors, has a unique feature that distinguishes it from the remaining INK4 and makes it a likely candidate for contributing to the directionality of the cell cycle. p19INK4d mRNA and protein levels accumulate periodically during the cell cycle under normal conditions, a feature reminiscent of cyclins. Methodology/Principal Findings In this paper, we demonstrate that p19INK4d is transcriptionally regulated by E2F1 through two response elements present in the p19INK4d promoter. Ablation of this regulation reduced p19 levels and restricted its expression during the cell cycle, reflecting the contribution of a transcriptional effect of E2F1 on p19 periodicity. The induction of p19INK4d is delayed during the cell cycle compared to that of cyclin E, temporally separating the induction of these proliferative and antiproliferative target genes. Specific inhibition of the E2F1-p19INK4d pathway using triplex-forming oligonucleotides that block E2F1 binding on p19 promoter, stimulated cell proliferation and increased the fraction of cells in S phase. Conclusions/Significance The results described here support a model of normal cell cycle progression in which, following phosphorylation of pRb, free E2F induces cyclin E, among other target genes. Once cyclinE/CDK2 takes over as the cell cycle driving kinase activity, the induction of p19 mediated by E2F1 leads to inhibition of the CDK4,6-containing complexes, bringing the G1 phase to an end. This regulatory mechanism constitutes a new negative feedback loop that terminates the G1 phase proliferative signal, contributing to the proper coordination of the cell cycle and provides an

  12. A novel miRNA-mediated STOP sign in lung cancer: miR-340 inhibits the proliferation of lung cancer cells through p27KIP1

    PubMed Central

    Fernandez, Serena; Risolino, Maurizio; Verde, Pasquale

    2015-01-01

    Oncosuppressor miRNAs inhibit cancer cell proliferation by targeting key components of the cell cycle machinery. In our recent report we showed that miR-340 is a novel tumor suppressor in non-small cell lung cancer. miR-340 inhibits neoplastic cell proliferation and induces p27KIP1 by targeting multiple translational and post-translational regulators of this cyclin-dependent kinase inhibitor. PMID:27308439

  13. Autocrine CSF1R signaling mediates switching between invasion and proliferation downstream of TGFβ in claudin-low breast tumor cells

    PubMed Central

    Patsialou, Antonia; Wang, Yarong; Pignatelli, Jeanine; Chen, Xiaoming; Entenberg, David; Oktay, Maja; Condeelis, John S.

    2014-01-01

    Patient data suggest that colony stimulating factor-1 (CSF1) and its receptor (CSF1R) play critical roles during breast cancer progression. We have previously shown that in human breast tumors expressing both CSF1 and CSF1R, invasion in vivo is dependent both on a paracrine interaction with tumor-associated macrophages and an autocrine regulation of CSF1R in the tumor cells themselves. Although the role of the paracrine interaction between tumor cells and macrophages has been extensively studied, very little is known about the mechanism by which the autocrine CSF1R signaling contributes to tumor progression. We show here that breast cancer patients of the claudin-low subtype have significantly increased expression of CSF1R. Using a panel of breast cancer cells lines, we confirm that CSF1R expression is elevated and regulated by TGFβ specifically in claudin-low cell lines. Abrogation of autocrine CSF1R signaling in MDA-MB-231 xenografts (a claudin-low cell line) leads to increased tumor size by enhanced proliferation, but significantly reduced invasion, dissemination and metastasis. Indeed, we show that proliferation and invasion are oppositely regulated by CSF1R downstream of TGFβ only in claudin-low cells lines. Intravital multiphoton imaging revealed that inhibition of CSF1R in the tumor cells leads to decreased in vivo motility and a more cohesive morphology. We show that, both in vitro and in vivo, CSF1R inhibition results in a reversal of claudin-low marker expression by significant upregulation of luminal keratins and tight junction proteins such as claudins. Finally, we show that artificial overexpression of claudins in MDA-MB-231 cells is sufficient to tip the cells from an invasive state to a proliferative state. Our results suggest that autocrine CSF1R signaling is essential in maintaining low claudin expression and that it mediates a switch between the proliferative and the invasive state in claudin-low tumor cells downstream of TGFβ. PMID:25088194

  14. Interference RNA (RNAi)-based silencing of endogenous thrombopoietin receptor (Mpl) in Dami cells resulted in decreased hNUDC-mediated megakaryocyte proliferation and differentiation

    SciTech Connect

    Pang, Shi-Feng; Li, Xiao-Kun; Zhang, Qiang; Yang, Fang; Xu, Peilin

    2009-12-10

    Recently our laboratory reported evidence showing that hNUDC acts as an additional cytokine for thrombopoietin receptor (Mpl). Previously known as the human homolog of a fungal nuclear migration protein, hNUDC plays a critical role in megakaryocyte differentiation and maturation. Here we sought to further clarify the hNUDC-Mpl ligand-receptor relationship by utilizing interference RNA (RNAi) to knockdown Mpl expression in a megakaryocyte cell line. We created U6 promoter driven constructs to express short hairpin RNAs (shRNA) with affinity for different sites on Mpl mRNA. By including Mpl-EGFP fusion protein in these constructs, we were able to effectively screen the shRNA that was most efficient in inhibiting Mpl mRNA expression. This shRNA was subsequently transferred into a lentivirus vector and transduced into Dami cells, a cell line which constitutively expresses endogenous Mpl. This lentiviral vector was also designed to simultaneously express EGFP to monitor transfection efficiency. Our results show that lentivirus can be used to effectively deliver shRNAs into Dami cells and cause specific inhibition of Mpl protein expression after transduction. Furthermore, we show the functional effects of shRNA-mediated Mpl silencing by demonstrating reduced hNUDC stimulated megakaryocyte proliferation and differentiation. Thus, the use of a RNAi knockdown strategy has allowed us to pinpoint the connection of hNUDC with Mpl in the regulation of megakaryocyte maturation.

  15. Contact-mediated cell-assisted cell proliferation in a model eukaryotic single-cell organism: An explanation for the lag phase in shaken cell culture

    NASA Astrophysics Data System (ADS)

    Franck, Carl; Ip, Wui; Bae, Albert; Franck, Nathan; Bogart, Elijah; Le, Thanhbinh Thi

    2008-04-01

    In cell culture, when cells are inoculated into fresh media, there can be a period of slow (or lag phase) growth followed by a transition to exponential growth. This period of slow growth is usually attributed to the cells’ adaptation to a new environment. However, we argue that, based on observations of shaken suspension culture of Dictyostelium discoideum, a model single-cell eukaryote, this transition is due to a density effect. Attempts to demonstrate the existence of implicit cell signaling via long-range diffusible messengers (i.e., soluble growth factors) through cell-medium separation and microfluidic flow perturbation experiments produced negative results. This, in turn, led to the development of a signaling model based on direct cell-to-cell contacts. Employing a scaling argument for the collision rate due to fluid shear, we reasonably estimate the crossover density for the transition into the exponential phase and fit the observed growth kinetics.

  16. NUP214-ABL1-mediated cell proliferation in T-cell acute lymphoblastic leukemia is dependent on the LCK kinase and various interacting proteins

    PubMed Central

    De Keersmaecker, Kim; Porcu, Michaël; Cox, Luk; Girardi, Tiziana; Vandepoel, Roel; de Beeck, Joyce Op; Gielen, Olga; Mentens, Nicole; Bennett, Keiryn L.; Hantschel, Oliver

    2014-01-01

    The NUP214-ABL1 fusion protein is a constitutively active protein tyrosine kinase that is found in 6% of patients with T-cell acute lymphoblastic leukemia and that promotes proliferation and survival of T-lymphoblasts. Although NUP214-ABL1 is sensitive to ABL1 kinase inhibitors, development of resistance to these compounds is a major clinical problem, underlining the need for additional drug targets in the sparsely studied NUP214-ABL1 signaling network. In this work, we identify and validate the SRC family kinase LCK as a protein whose activity is absolutely required for the proliferation and survival of T-cell acute lymphoblastic leukemia cells that depend on NUP214-ABL1 activity. These findings underscore the potential of SRC kinase inhibitors and of the dual ABL1/SRC kinase inhibitors dasatinib and bosutinib for the treatment of NUP214-ABL1-positive T-cell acute lymphoblastic leukemia. In addition, we used mass spectrometry to identify protein interaction partners of NUP214-ABL1. Our results strongly support that the signaling network of NUP214-ABL1 is distinct from that previously reported for BCR-ABL1. Moreover, we found that three NUP214-ABL1-interacting proteins, MAD2L1, NUP155, and SMC4, are strictly required for the proliferation and survival of NUP214-ABL1-positive T-cell acute lymphoblastic leukemia cells. In conclusion, this work identifies LCK, MAD2L1, NUP155 and SMC4 as four new potential drug targets in NUP214-ABL1-positive T-cell acute lymphoblastic leukemia. PMID:23872305

  17. NKX3.1 activates expression of insulin-like growth factor binding protein-3 to mediate insulin-like growth factor-I signaling and cell proliferation.

    PubMed

    Muhlbradt, Erin; Asatiani, Ekaterina; Ortner, Elizabeth; Wang, Antai; Gelmann, Edward P

    2009-03-15

    NKX3.1 is a homeobox gene that codes for a haploinsufficient prostate cancer tumor suppressor. NKX3.1 protein levels are down-regulated in the majority of primary prostate cancer tissues. NKX3.1 expression in PC-3 cells increased insulin-like growth factor binding protein-3 (IGFBP-3) mRNA expression 10-fold as determined by expression microarray analysis. In both stably and transiently transfected PC-3 cells and in LNCaP cells, NKX3.1 expression increased IGFBP-3 mRNA and protein expression. In prostates of Nkx3.1 gene-targeted mice Igfbp-3 mRNA levels correlated with Nkx3.1 copy number. NKX3.1 expression in PC-3 cells attenuated the ability of insulin-like growth factor-I (IGF-I) to induce phosphorylation of type I IGF receptor (IGF-IR), insulin receptor substrate 1, phosphatidylinositol 3-kinase, and AKT. The effect of NKX3.1 on IGF-I signaling was not seen when cells were exposed to long-R3-IGF-I, an IGF-I variant peptide that does not bind to IGFBP-3. Additionally, small interfering RNA-induced knockdown of IGFBP-3 expression partially reversed the attenuation of IGF-IR signaling by NKX3.1 and abrogated NKX3.1 suppression of PC-3 cell proliferation. Thus, there is a close relationship in vitro and in vivo between NKX3.1 and IGFBP-3. The growth-suppressive effects of NKX3.1 in prostate cells are mediated, in part, by activation of IGFBP-3 expression. PMID:19258508

  18. The phosphoinositide 3-kinase/Akt-signal pathway mediates proliferation and secretory function of hepatic sinusoidal endothelial cells in rats after partial hepatectomy

    SciTech Connect

    Chen Ping . E-mail: chenping@263.net; Zhang Lin; Ding Jiming; Zhu Jin; Li Ying; Duan Shigang; Yan Hongtao; Huan Yongwei; Dong Jiahong

    2006-04-14

    in the OG beyond 24 h. These findings suggest that the secretion of the cytokines by hepatic SECs is mediated by Akt signaling. Akt signaling pathway in relationship with proliferation of hepatic SECs and suppression of apoptosis. In OG group, the hepatic SECs in S and G2/M obviously increased. The proliferative index of hepatic SECs in OG group had significant differences with that in LY group at 6, 24, and 72 h, P < 0.05. Meanwhile, the cells of apoptosis in OG group were very low, and the cells in LY group gradually increased. Conclusions: These results suggest that AKT signaling pathway plays a crucial role in mediating proliferating and secreted signals in hepatic SECs. AKT has been suggested to play a pivotal role in early liver regeneration involved in the induction of secreted cytokines and proliferation of hepatic SECs.

  19. MicroRNA-H4-5p encoded by HSV-1 latency-associated transcript promotes cell proliferation, invasion and cell cycle progression via p16-mediated PI3K-Akt signaling pathway in SHSY5Y cells

    PubMed Central

    Zhao, Huiliang; Zhang, Chunying; Hou, Guangjun; Song, Jijun

    2015-01-01

    Herpes simplex virus 1 (HSV-1) microRNAs (miRNAs) mostly located in transcription-associated transcript (LAT) region have been identified that play critical roles in the intricate host-pathogen interaction networks. Increasing evidences throw new insight into the role of miRNA-mediated miRNA-mRNA cross-talk in HSV-1 latent or acute infection. In the present study, we found that hsv-1 miR-H4-5p (here termed as miR-H4b) can down-regulate the expression of cyclin-dependent kinase inhibitor 2A (CDKN2A, p16) in neuroblastoma (SHSY5Y) cell lines. Decreased expression of miR-H4b was directly related to attenuated cell proliferation and invasion as well as malfunction of cell cycle in recombinant SHSY5Y cells that stably expressing miR-H4b. Bioinformatics analysis and luciferase assays demonstrated miR-H4b can directly target p16 mRNA. MiR-H4b exerts its pro-proliferation function through inhibition of the p16-related PI3K-Akt pathways. Our findings provide, for the first time, significant clues regarding the role of herpesvirus-encoded miRNAs as a viral modulator to host cells. PMID:26221296

  20. Epigenetic involvement of Alien/ESET complex in thyroid hormone-mediated repression of E2F1 gene expression and cell proliferation

    SciTech Connect

    Hong, Wei; Li, Jinru; Wang, Bo; Chen, Linfeng; Niu, Wenyan; Yao, Zhi; Baniahmad, Aria

    2011-12-02

    Highlights: Black-Right-Pointing-Pointer Corepressor Alien interacts with histone methyltransferase ESET in vivo. Black-Right-Pointing-Pointer Alien/ESET complex is recruited to nTRE of T3-responsive gene by liganded TR{beta}1. Black-Right-Pointing-Pointer ESET-mediated H3K9 methylation is required for liganded TR{beta}1-repressed transcription. Black-Right-Pointing-Pointer ESET is involved in T3-repressed G1/S phase transition and proliferation. -- Abstract: The ligand-bound thyroid hormone receptor (TR) is known to repress via a negative TRE (nTRE) the expression of E2F1, a key transcription factor that controls the G1/S phase transition. Alien has been identified as a novel interacting factor of E2F1 and acts as a corepressor of E2F1. The detailed molecular mechanism by which Alien inhibits E2F1 gene expression remains unclear. Here, we report that the histone H3 lysine 9 (H3K9) methyltransferase (HMT) ESET is an integral component of the corepressor Alien complex and the Alien/ESET complex is recruited to both sites, the E2F1 and the nTRE site of the E2F1 gene while the recruitment to the negative thyroid hormone response element (nTRE) is induced by the ligand-bound TR{beta}1 within the E2F1 gene promoter. We show that, overexpression of ESET promotes, whereas knockdown of ESET releases, the inhibition of TR{beta}1-regulated gene transcription upon T3 stimulation; and H3K9 methylation is required for TR{beta}1-repressed transcription. Furthermore, depletion of ESET impairs thyroid hormone-repressed proliferation as well as the G1/S transition of the cell cycle. Taken together, our data indicate that ESET is involved in TR{beta}1-mediated transcription repression and provide a molecular basis of thyroid hormone-induced repression of proliferation.

  1. CD95/CD95L-mediated apoptosis of the hepatic stellate cell. A mechanism terminating uncontrolled hepatic stellate cell proliferation during hepatic tissue repair.

    PubMed Central

    Saile, B.; Knittel, T.; Matthes, N.; Schott, P.; Ramadori, G.

    1997-01-01

    During liver tissue repair, hepatic stellate cells (HSC), a pericyte-like mesenchymal liver cell population, transform from a "quiescent" status ("resting" HSC) into myofibroblast-like cells ("activated" HSC) with the latter representing the principle matrix synthesizing cell of the liver. Presently, the mechanisms that terminate HSC cell proliferation when tissue repair is concluded are poorly understood. Controlled cell death known as apoptosis could be a mechanism underlying this phenomenon. Therefore, apoptosis and its regulation were studied in HSC using an in vitro and in vivo approach. Spontaneous apoptosis became detectable in parallel with HSC activation because resting cells (2 days after isolation) displayed no sign of apoptosis, whereas apoptosis was present in 8% (+/- 5%) of "transitional" cells (day 4) and in 18% (+/- 8%) of fully activated cells (day 7). Both CD95 (APO-1/Fas) and CD95L (APO-1-/Fas-ligand) became increasingly expressed during the course of activation. Apoptosis could be fully blocked by CD95-blocking antibodies in normal cells and HSC already entering the apoptotic cycle. Using CD95-activating antibodies, transition of more than 95% cells into apoptosis was evident at each activation step. The apoptosis-regulating proteins Bcl-2 and p53 could not be detected in resting cells but were found in increasing amounts at days 4 and 7 of cultivation. Whereas p53 expression was induced by the CD95-activating antibody, no change was inducible in Bcl-2 expression. The Bcl-2-related protein bax could be found at days 2 and 4 in similar expression, was considerably up-regulated at day 7, but was not regulated by CD95-agonistic antibodies. In vivo, acute tissue damage was first accompanied by activation and proliferation of HSC displaying no sign of apoptosis. In the recovery phase, apoptotic HSC were detectable in parallel to a reduction in the total number of HSC present in the liver tissue. The data demonstrate that apoptosis becomes detectable

  2. Retinoic Acid-mediated Nuclear Receptor Activation and Hepatocyte Proliferation

    PubMed Central

    Bushue, Nathan; Wan, Yu-Jui Yvonne

    2016-01-01

    Due to their well-known differentiation and apoptosis-inducing abilities, retinoic acid (RA) and its analogs have strong anti-cancer efficacy in human cancers. However, in vivo RA is a liver mitogen. While speculation has persisted that RA-mediated signaling is likely involved in hepatocyte proliferation during liver regeneration, direct evidence is still required. Findings in support of this proposition include observations that a release of retinyl palmitate (the precursor of RA) occurs in liver stellate cells following liver injury. Nevertheless, the biological action of this released vitamin A is virtually unknown. More likely is that the released vitamin A is converted to RA, the biological form, and then bound to a specific receptor (retinoid x receptor; RXRα), which is most abundantly expressed in the liver. Considering the mitogenic effects of RA, the RA-activated RXRα would likely then influence hepatocyte proliferation and liver tissue repair. At present, the mechanism by which RA stimulates hepatocyte proliferation is largely unknown. This review summarizes the activation of nuclear receptors (peroxisome proliferator activated receptor-α, pregnane x receptor, constitutive androstane receptor, and farnesoid x receptor) in an RXRα dependent manner to induce hepatocyte proliferation, providing a link between RA and its proliferative role.

  3. Apo- and holo-lactoferrin are both internalized by lactoferrin receptor via clathrin-mediated endocytosis but differentially affect ERK-signaling and cell proliferation in Caco-2 cells

    PubMed Central

    Jiang, Rulan; Lopez, Veronica; Kelleher, Shannon L.; Lönnerdal, Bo

    2011-01-01

    Lactoferrin (Lf) is a major iron-binding and multi-functional protein in exocrine fluids such as breast milk and mucosal secretions. The functions of Lf appear dependent upon the iron-saturation of the Lf protein and are postulated to be mediated through Lf internalization by a Lf receptor (LfR). However, mechanisms by which LfR mediates Lf internalization in enterocytes are unknown. We now demonstrate that a LfR previously cloned from the small intestine mediates Lf endocytosis in a human enterocyte model (Caco-2 cells). LfR was detected at the plasma membrane by cell surface biotinylation; both apo-Lf and holo-Lf uptake were significantly inhibited in cells transfected with LfR siRNA. Treatments of hypertonic sucrose and clathrin siRNA and co-immunoprecipitation of LfR with clathrin adaptor AP2 indicate that LfR regulates Lf endocytosis via clathrin-mediated endocytosis. Although both iron-free Lf (apo-Lf) and iron-saturated Lf (holo-Lf) enter Caco-2 cells via a similar mechanism and no significant differences were observed in the binding and uptake of apo- and holo-Lf in Caco-2 cells, apo-Lf but not holo-Lf stimulates proliferation of Caco-2 cells. Interestingly, apo-Lf stimulated extracellular signal-regulated mitogen-activated protein kinase (ERK) cascade to a significantly greater extent than holo-Lf and the apo-Lf induced proliferation was significantly inhibited by an ERK cascade inhibitor (U0126) and clathrin siRNA. Taken together, our data suggest that LfR is a major pathway through which Lf is taken up by enterocytes, which occurs independently of iron saturation through clathrin-mediated endocytosis. The differential effects of apo- and holo-Lf are not due to differences in cellular internalization mechanisms. PMID:21935933

  4. Cavin-1/PTRF alters prostate cancer cell-derived extracellular vesicle content and internalization to attenuate extracellular vesicle-mediated osteoclastogenesis and osteoblast proliferation

    PubMed Central

    Inder, Kerry L.; Ruelcke, Jayde E.; Petelin, Lara; Moon, Hyeongsun; Choi, Eunju; Rae, James; Blumenthal, Antje; Hutmacher, Dietmar; Saunders, Nicholas A.; Stow, Jennifer L.; Parton, Robert G.; Hill, Michelle M.

    2014-01-01

    Background Tumour-derived extracellular vesicles (EVs) play a role in tumour progression; however, the spectrum of molecular mechanisms regulating EV secretion and cargo selection remain to be fully elucidated. We have reported that cavin-1 expression in prostate cancer PC3 cells reduced the abundance of a subset of EV proteins, concomitant with reduced xenograft tumour growth and metastasis. Methods We examined the functional outcomes and mechanisms of cavin-1 expression on PC3-derived EVs (PC3-EVs). Results PC3-EVs were internalized by osteoclast precursor RAW264.7 cells and primary human osteoblasts (hOBs) in vitro, stimulating osteoclastogenesis 37-fold and hOB proliferation 1.5-fold, respectively. Strikin gly, EVs derived from cavin-1-expressing PC3 cells (cavin-1-PC3-EVs) failed to induce multinucleate osteoblasts or hOB proliferation. Cavin-1 was not detected in EVs, indicating an indirect mechanism of action. EV morphology, size and quantity were also not affected by cavin-1 expression, suggesting that cavin-1 modulated EV cargo recruitment rather than release. While cavin-1-EVs had no osteoclastogenic function, they were internalized by RAW264.7 cells but at a reduced efficiency compared to control EVs. EV surface proteins are required for internalization of PC3-EVs by RAW264.7 cells, as proteinase K treatment abolished uptake of both control and cavin-1-PC3-EVs. Removal of sialic acid modifications by neuraminidase treatment increased the amount of control PC3-EVs internalized by RAW264.7 cells, without affecting cavin-1-PC3-EVs. This suggests that cavin-1 expression altered the glycosylation modifications on PC3-EV surface. Finally, cavin-1 expression did not affect EV in vivo tissue targeting as both control and cavin-1-PC3-EVs were predominantly retained in the lung and bone 24 hours after injection into mice. Discussion Taken together, our results reveal a novel pathway for EV cargo sorting, and highlight the potential of utilizing cavin-1-mediated

  5. Rac1 drives intestinal stem cell proliferation and regeneration

    PubMed Central

    Myant, Kevin B; Scopelliti, Alessandro; Haque, Sara; Vidal, Marcos; Sansom, Owen J; Cordero, Julia B

    2013-01-01

    Adult stem cells are responsible for maintaining the balance between cell proliferation and differentiation within self-renewing tissues. The molecular and cellular mechanisms mediating such balance are poorly understood. The production of reactive oxygen species (ROS) has emerged as an important mediator of stem cell homeostasis in various systems. Our recent work demonstrates that Rac1-dependent ROS production mediates intestinal stem cell (ISC) proliferation in mouse models of colorectal cancer (CRC). Here, we use the adult Drosophila midgut and the mouse small intestine to directly address the role of Rac1 in ISC proliferation and tissue regeneration in response to damage. Our results demonstrate that Rac1 is necessary and sufficient to drive ISC proliferation and regeneration in an ROS-dependent manner. Our data point to an evolutionarily conserved role of Rac1 in intestinal homeostasis and highlight the value of combining work in the mammalian and Drosophila intestine as paradigms to study stem cell biology. PMID:23974108

  6. The human HECA interacts with cyclins and CDKs to antagonize Wnt-mediated proliferation and chemoresistance of head and neck cancer cells

    SciTech Connect

    Dowejko, Albert; Bauer, Richard; Bauer, Karin; Mueller-Richter, Urs D.A.; Reichert, Torsten E.

    2012-03-10

    antagonizes Wnt-mediated cell proliferation through interaction with major cell cycle factors. Black-Right-Pointing-Pointer Modulating HECA level confers benefits for engaging tumor cells with cisplatin.

  7. miRNA-149* promotes cell proliferation and suppresses apoptosis by mediating JunB in T-cell acute lymphoblastic leukemia.

    PubMed

    Fan, Sheng-Jin; Li, Hui-Bo; Cui, Gang; Kong, Xiao-Lin; Sun, Li-Li; Zhao, Yan-Qiu; Li, Ying-Hua; Zhou, Jin

    2016-02-01

    MicroRNA-149* (miRNA-149*) functions as an oncogenic regulator in human melanoma. However, the effect of miRNA-149* on T-cell acute lymphoblastic leukemia (T-ALL) is unclear. Here we aimed to analyze the effects of miRNA-149* on in vitro T-ALL cells and to uncover the target for miRNA-149* in these cells. The miRNA-149* level was determined in multiple cell lines and bone marrow cells derived from patients with T-ALL, B acute lymphoblastic leukemia (B-ALL), acute myelocytic leukemia (AML), and healthy donors. We found that miRNA-149* was highly expressed in T-ALL cell lines and T-ALL patients' bone marrow samples. JunB was identified as a direct target of miR-149*. miRNA-149* mimics downregulated JunB levels in Molt-4 and Jurkat cells, while miRNA-149* inhibitors dramatically upregulated JunB expression in these cells. miRNA-149* mimics promoted proliferation, decreased the proportion of cells in G1 phase, and reduced cell apoptosis in T-ALL cells, while miRNA-149* inhibitors prevented these effects. miRNA-149* mimics downregulated p21 and upregulated cyclinD1, 4EBP1, and p70s6k in Molt-4 and Jurkat cells. Again, inhibitors prevented these effects. Our findings demonstrate that miRNA-149* may serve as an oncogenic regulator in T-ALL by negatively regulating JunB. PMID:26725775

  8. EBV Nuclear Antigen 3C Mediates Regulation of E2F6 to Inhibit E2F1 Transcription and Promote Cell Proliferation

    PubMed Central

    Sun, Zhiguo; Jha, Hem Chandra; Saha, Abhik; Robertson, Erle S.

    2016-01-01

    Epstein–Barr virus (EBV) is considered a ubiquitous herpesvirus with the ability to cause latent infection in humans worldwide. EBV-association is evidently linked to different types of human malignancies, mainly of epithelial and lymphoid origin. Of interest is the EBV nuclear antigen 3C (EBNA3C) which is critical for EBV-mediated immortalization. Recently, EBNA3C was shown to bind the E2F1 transcription regulator. The E2F transcription factors have crucial roles in various cellular functions, including cell cycle, DNA replication, DNA repair, cell mitosis, and cell fate. Specifically, E2F6, one of the unique E2F family members, is known to be a pRb-independent transcription repressor of E2F-target genes. In our current study, we explore the role of EBNA3C in regulating E2F6 activities. We observed that EBNA3C plays an important role in inducing E2F6 expression in LCLs. Our study also shows that EBNA3C physically interacts with E2F6 at its amino and carboxy terminal domains and they form a protein complex in human cells. In addition, EBNA3C stabilizes the E2F6 protein and is co-localized in the nucleus. We also demonstrated that both EBNA3C and E2F6 contribute to reduction in E2F1 transcriptional activity. Moreover, E2F1 forms a protein complex with EBNA3C and E2F6, and EBNA3C competes with E2F1 for E2F6 binding. E2F6 is also recruited by EBNA3C to the E2F1 promoter, which is critical for EBNA3C-mediated cell proliferation. These results demonstrate a critical role for E2F family members in EBV-induced malignancies, and provide new insights for targeting E2F transcription factors in EBV-associated cancers as potential therapeutic intervention strategies. PMID:27548379

  9. EBV Nuclear Antigen 3C Mediates Regulation of E2F6 to Inhibit E2F1 Transcription and Promote Cell Proliferation.

    PubMed

    Pei, Yonggang; Banerjee, Shuvomoy; Sun, Zhiguo; Jha, Hem Chandra; Saha, Abhik; Robertson, Erle S

    2016-08-01

    Epstein-Barr virus (EBV) is considered a ubiquitous herpesvirus with the ability to cause latent infection in humans worldwide. EBV-association is evidently linked to different types of human malignancies, mainly of epithelial and lymphoid origin. Of interest is the EBV nuclear antigen 3C (EBNA3C) which is critical for EBV-mediated immortalization. Recently, EBNA3C was shown to bind the E2F1 transcription regulator. The E2F transcription factors have crucial roles in various cellular functions, including cell cycle, DNA replication, DNA repair, cell mitosis, and cell fate. Specifically, E2F6, one of the unique E2F family members, is known to be a pRb-independent transcription repressor of E2F-target genes. In our current study, we explore the role of EBNA3C in regulating E2F6 activities. We observed that EBNA3C plays an important role in inducing E2F6 expression in LCLs. Our study also shows that EBNA3C physically interacts with E2F6 at its amino and carboxy terminal domains and they form a protein complex in human cells. In addition, EBNA3C stabilizes the E2F6 protein and is co-localized in the nucleus. We also demonstrated that both EBNA3C and E2F6 contribute to reduction in E2F1 transcriptional activity. Moreover, E2F1 forms a protein complex with EBNA3C and E2F6, and EBNA3C competes with E2F1 for E2F6 binding. E2F6 is also recruited by EBNA3C to the E2F1 promoter, which is critical for EBNA3C-mediated cell proliferation. These results demonstrate a critical role for E2F family members in EBV-induced malignancies, and provide new insights for targeting E2F transcription factors in EBV-associated cancers as potential therapeutic intervention strategies. PMID:27548379

  10. MiR-26b inhibits melanoma cell proliferation and enhances apoptosis by suppressing TRAF5-mediated MAPK activation.

    PubMed

    Li, Meng; Long, Chaoqin; Yang, Guilan; Luo, Yang; Du, Hua

    2016-03-11

    Alterations in microRNA-26b (miR-26b) expression have been shown to participate in various malignant tumor developments. However, the possible function of miR-26b in human melanoma cells remains unclarified. In this study, quantitative polymerase chain reaction was used to explore the expression profiles of miR-26b in melanoma cells. The effect of miR-26b on cell viability was determined by using MTT assays and colony formation assay. The apoptosis levels were evaluated by using Annexin V/fluorescein isothiocyanate (FITC) apoptosis detection kit and the apoptosis cells were confirmed by Transmission Electron Microscopy (TEM). Luciferase reporter plasmids were constructed to confirm direct targeting. Our study found that the expression of miR-26b was downregulated in human melanoma specimens. Overexpression of miR-26b significantly increased the anti-proliferative effects and apoptosis in A375 and B16F10 melanoma cells. In addition, luciferase gene reporter assays confirmed that TRAF5 was a direct target gene of miR-26b and the anti-tumor effect of miR-26b in melanoma cells was significantly counteracted by treatment with TRAF5 overexpression. Furthermore, the molecular mechanisms underlying the tumor suppressor of miR-26b in malignant melanomas may be due to the dephosphorylation of MAPK pathway caused by the decrease in TRAF5 expression when miR-26b is up-regulated in melanoma cells. These findings indicate that miR-26b might influence TRAF5-MAPK signaling pathways to facilitate the malignant progression of melanoma cells. PMID:26872428

  11. Activation of p38, p21, and NRF-2 Mediates Decreased Proliferation of Human Dental Pulp Stem Cells Cultured under 21% O2

    PubMed Central

    El Alami, Marya; Viña-Almunia, Jose; Gambini, Juan; Mas-Bargues, Cristina; Siow, Richard C.M.; Peñarrocha, Miguel; Mann, Giovanni E.; Borrás, Consuelo; Viña, Jose

    2014-01-01

    Summary High rates of stem cell proliferation are important in regenerative medicine and in stem cell banking for clinical use. Ambient oxygen tensions (21% O2) are normally used for in vitro culture, but physiological levels in vivo range between 3% and 6% O2. We compared proliferation of human dental pulp stem cells (hDPSCs) cultured under 21% versus 3% O2. The rate of hDPSC proliferation is significantly lower at 21% O2 compared to physiological oxygen levels due to enhanced oxidative stress. Under 21% O2, increased p38 phosphorylation led to activation of p21. Increased generation of reactive oxygen species and p21 led to activation of the NRF-2 signaling pathway. The upregulation of NRF-2 antioxidant defense genes under 21% O2 may interact with cell-cycle-related proteins involved in regulating cell proliferation. Activation of p38/p21/NRF-2 in hDPSCs cultured under ambient oxygen tension inhibits stem cell proliferation and upregulates NRF-2 antioxidant defenses. PMID:25358785

  12. Effects of thyroid hormones on human breast cancer cell proliferation.

    PubMed

    Hall, Linda C; Salazar, Eddie P; Kane, Staci R; Liu, Nan

    2008-03-01

    The involvement of estrogens in breast cancer development and growth has been well established. However, the effects of thyroid hormones and their combined effects with estrogens are not well studied. We investigated the response of human breast cancer cells to thyroid hormone, particularly the role of T3 in mediating cell proliferation and gene expression. We demonstrated that 17beta-estradiol (E2) or triiodothyronine (T3) promoted cell proliferation in a dose-dependent manner in both MCF-7 and T47-D cell lines. The E2- or T3-dependent cell proliferation was suppressed by co-administration of the ER antagonist ICI. We also demonstrated that T3 could enhance the effect of E2 on cell proliferation in T47-D cells. Using an estrogen response element (ERE)-mediated luciferase assay, we determined that T3 was able to induce the activation of ERE-mediated gene expression in MCF-7 cells, although the effects were much weaker than that induced by E2. These results suggest that T3 can promote breast cancer cell proliferation and increase the effect of E2 on cell proliferation in some breast cancer cell lines and thus that T3 may play a role in breast cancer development and progression. PMID:18328691

  13. eNOS signaling is essential in GLP-2-mediated stimulation of blood flow, but not cell proliferation in the mouse gut

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Through a G protein-coupled receptor, glucagon-like peptide-2 (GLP-2) stimulates intestinal crypt cell proliferation and mucosal blood flow. GLP-2 receptor is localized to enteric neurons, endocrine cells, and myofibroblasts, but not enterocytes. However, it is largely unknown how GLP-2 receptor-act...

  14. Molecular basis for the interplay of apoptosis and proliferation mediated by Bcl-xL:Bim interactions in pancreatic cancer cells

    SciTech Connect

    Abrol, Ravinder; Edderkaoui, Mouad; Goddard, William A.; Pandol, Stephen J.

    2012-06-15

    Highlights: Black-Right-Pointing-Pointer Direct role of Bcl-2 protein interactions in cell proliferation is not clear. Black-Right-Pointing-Pointer Designed Bcl-xL mutants show opposite effects on apoptosis and proliferation. Black-Right-Pointing-Pointer Disrupting Bcl-xL:Bim interaction increased apoptosis in pancreatic cancer. Black-Right-Pointing-Pointer Disrupting Bcl-xL:Bim interaction decreased proliferation in pancreatic cancer. Black-Right-Pointing-Pointer Bcl-xL:Bim interaction can control both apoptosis and proliferation. -- Abstract: A major mechanism through which cancer cells avoid apoptosis is by promoting the association of anti-apoptotic members of the pro-survival Bcl-2 protein family (like Bcl-2 and Bcl-xL) with BH{sub 3} domain-only proteins (like Bim and Bid). Apoptosis and cell proliferation have been shown to be linked for many cancers but the molecular basis for this link is far from understood. We have identified the Bcl-xL:Bim protein-protein interface as a direct regulator of proliferation and apoptosis in pancreatic cancer cells. We were able to predict and subsequently verify experimentally the effect of various Bcl-xL single-point mutants (at the position A142) on binding to Bim by structural analysis and computational modeling of the inter-residue interactions at the Bcl-xL:Bim protein-protein interface. The mutants A142N, A142Q, and A142Y decreased binding of Bim to Bcl-xL and A142S increased this binding. The Bcl-xL mutants, with decreased affinity for Bim, caused an increase in apoptosis and a corresponding decrease in cell proliferation. However, we could prevent these effects by introducing a small interfering RNA (siRNA) targeted at Bim. These results show a novel role played by the Bcl-xL:Bim interaction in regulating proliferation of pancreatic cancer cells at the expense of apoptosis. This study presents a physiologically relevant model of the Bcl-xL:Bim interface that can be used for rational therapeutic design for the

  15. Candidate Soluble Immune Mediators in Young Women with High-Risk Human Papillomavirus Infection: High Expression of Chemokines Promoting Angiogenesis and Cell Proliferation

    PubMed Central

    Zanotta, Nunzia; Tornesello, Maria Lina; Annunziata, Clorinda; Stellato, Giovanni; Buonaguro, Franco Maria; Comar, Manola

    2016-01-01

    Background The causal interpretation of cervical immune response to Human Papillomavirus (HPV) infection is complex and poorly characterized mainly due to the delicate balance that exists between viral infection, increase of inflammatory cytokines and host risk factors. This study aims to explore the significance of cervical immune mediators associated to cell survival, angiogenesis and interaction with immune response, in predicting the risk to develop HPV-related intraepithelial lesions. Methods A panel of 48 cytokines and growth factors were explored in a selected cohort of 168 immunocompetent women including 88 diagnosed with low (LSIL) or high (HSIL) squamous intraepithelial lesions of the cervix and 80 with normal cervical cytology (NIL). HPV genotyping was performed by Linear Array HPV test and the soluble concentration of 48 immune molecules was analyzed using the Bio-Plex platform. Results The prevalence of single HR-HPV infection was 30% in NIL and 100% in LSIL and HSIL women. The expression of 13 cytokines, including interleukins IL-6, IL-3, IL-12p40, IL-12p70, IL-16, IL-18, LIF, of chemokines CCL7 (MCP-3), CXCL9 (MIG), CXCL12 (SDF-1α) and of the tropic factors VEGF, G-CSF, M-CSF were significantly associated with the presence of infection, with levels being higher in women with precancerous lesions compared to NIL HPV negative women. Only the growth factor GM-CSF was positively associated with the cytological abnormalities. Conclusions The ability of HR-HPV to escape from innate immune recognition and to orchestrate the production of specific inflammatory and growth factors, involved in early inflammatory response and in the cell-proliferating phase of intraepithelial damage, was documented in women before the development of cervical lesions. PMID:26990868

  16. G protein-coupled receptor 30 mediates estrogen-induced proliferation of primordial germ cells via EGFR/Akt/β-catenin signaling pathway.

    PubMed

    Ge, Chutian; Yu, Minli; Zhang, Caiqiao

    2012-07-01

    In vertebrates, estrogens are required for the normal development and function of postnatal gonads. However, it remains unclear whether estrogens are able to modulate development of the fetal germ cells. Here, we show that, unexpectedly, chicken primordial germ cells (PGC) lacking estrogen receptor α/β still proliferate in response to 17β-estradiol (E(2)). This is due to the capacity of G protein-coupled receptor 30 (GPR30), existing on PGC, to directly bind E(2). Knockdown experiments suggest that GPR30 is required for E(2)-stimulated PGC proliferation. Furthermore, this estrogen-induced activation of GPR30 is revealed to occur through the Gβγ-subunit protein-dependent and through the matrix metalloproteinase-dependent transactivation of the epidermal growth factor receptor. Epidermal growth factor receptor activation results in a series of intracellular events, including activation of the phosphatidylinositol 3-kinase/serine-threonine kinase/β-catenin pathway, which are followed by the induction of c-fos, c-myc, cyclin D1/E, and B-cell lymphoma 2 expression, and the inhibition of B-cell lymphoma 2-associated X protein expression and caspase3/9 activity. This eventually leads to decreased apoptosis and increased PGC proliferation. Collectively, these findings offer novel insights into the dynamic mechanism of estrogen action on PGC proliferation and suggest that E(2)/GPR30 signaling might play an important role in regulating fetal germ cell development, particularly at the stage before sexual differentiation. PMID:22635679

  17. Biofilms’ Role in Planktonic Cell Proliferation

    PubMed Central

    Bester, Elanna; Wolfaardt, Gideon M.; Aznaveh, Nahid B.; Greener, Jesse

    2013-01-01

    The detachment of single cells from biofilms is an intrinsic part of this surface-associated mode of bacterial existence. Pseudomonas sp. strain CT07gfp biofilms, cultivated in microfluidic channels under continuous flow conditions, were subjected to a range of liquid shear stresses (9.42 mPa to 320 mPa). The number of detached planktonic cells was quantified from the effluent at 24-h intervals, while average biofilm thickness and biofilm surface area were determined by confocal laser scanning microscopy and image analysis. Biofilm accumulation proceeded at the highest applied shear stress, while similar rates of planktonic cell detachment was maintained for biofilms of the same age subjected to the range of average shear rates. The conventional view of liquid-mediated shear leading to the passive erosion of single cells from the biofilm surface, disregards the active contribution of attached cell metabolism and growth to the observed detachment rates. As a complement to the conventional conceptual biofilm models, the existence of a biofilm surface-associated zone of planktonic cell proliferation is proposed to highlight the need to expand the traditional perception of biofilms as promoting microbial survival, to include the potential of biofilms to contribute to microbial proliferation. PMID:24201127

  18. Activating transcription factor 2 expression mediates cell proliferation and is associated with poor prognosis in human non-small cell lung carcinoma

    PubMed Central

    YOU, ZHENYU; ZHOU, YONG; GUO, YULING; CHEN, WENYAN; CHEN, SHAOQING; WANG, XIAOLANG

    2016-01-01

    following knockdown of ATF2 expression using RNA interference, indicating that ATF2 may suppress cell proliferation. Taken together, the results of the present study demonstrated that ATF2 and p-ATF2 were significantly overexpressed in NSCLC tissues, and ATF2 and p-ATF2 overexpression predicted significantly worse outcomes for patients with NSCLC. PMID:26870280

  19. Cell proliferation in normal epidermis

    SciTech Connect

    Weinstein, G.D.; McCullough, J.L.; Ross, P.

    1984-06-01

    A detailed examination of cell proliferation kinetics in normal human epidermis is presented. Using tritiated thymidine with autoradiographic techniques, proliferative and differentiated cell kinetics are defined and interrelated. The proliferative compartment of normal epidermis has a cell cycle duration (Tc) of 311 h derived from 3 components: the germinative labeling index (LI), the duration of DNA synthesis (ts), and the growth fraction (GF). The germinative LI is 2.7% +/- 1.2 and ts is 14 h, the latter obtained from a composite fraction of labeled mitoses curve obtained from 11 normal subjects. The GF obtained from the literature and from human skin xenografts to nude mice is estimated to be 60%. Normal-appearing epidermis from patients with psoriasis appears to have a higher proliferation rate. The mean LI is 4.2% +/- 0.9, approximately 50% greater than in normal epidermis. Absolute cell kinetic values for this tissue, however, cannot yet be calculated for lack of other information on ts and GF. A kinetic model for epidermal cell renewal in normal epidermis is described that interrelates the rate of birth/entry, transit, and/or loss of keratinocytes in the 3 epidermal compartments: proliferative, viable differentiated (stratum malpighii), and stratum corneum. Expected kinetic homeostasis in the epidermis is confirmed by the very similar ''turnover'' rates in each of the compartments that are, respectively, 1246, 1417, and 1490 cells/day/mm2 surface area. The mean epidermal turnover time of the entire tissue is 39 days. The Tc of 311 h in normal cells in 8-fold longer than the psoriatic Tc of 36 h and is necessary for understanding the hyperproliferative pathophysiologic process in psoriasis.

  20. miR-493 mediated DKK1 down-regulation confers proliferation, invasion and chemo-resistance in gastric cancer cells

    PubMed Central

    Peng, Cong; Deng, Yingen; Wang, Jia; Deng, Min; Lu, Minying; Yin, Jiang; Zheng, Guopei; Liu, Haiying; He, Zhimin

    2016-01-01

    In the present study, we demonstrated that the levels of DKK1 were decreased in serums and tissues of GC. DKK1 levels inversely correlated with tumor class, TNM stage, distant metastasis and lymph node metastasis of GC. GC patients with low DKK1 levels had a poor overall survival. DKK1 inhibited the proliferation of GC cells in vitro and in vivo. DKK1 also inhibited invasion, but enhanced chemo-sensitivity of GC cells. Mechanically, miR-493 levels increased in GC and directly targeted and down-regulated DKK1 expression. In agreement, miR-493 promoted proliferation of GC cells in vitro and in vivo. MiR-493 also promoted invasion and chemo-resistance of GC cells. However, DKK1 overexpression reversed the effects of miR-493 on proliferation, invasion and chemo-sensitivity. Thus, our results provide new insight for the role of miR-493/DKK1 axis in GC. PMID:26799283

  1. miR-9-5p, miR-675-5p and miR-138-5p Damages the Strontium and LRP5-Mediated Skeletal Cell Proliferation, Differentiation, and Adhesion

    PubMed Central

    Sun, Tianhao; Leung, Frankie; Lu, William W.

    2016-01-01

    This study was designed to evaluate the effects of strontium on the expression levels of microRNAs (miRNAs) and to explore their effects on skeletal cell proliferation, differentiation, adhesion, and apoptosis. The targets of these miRNAs were also studied. Molecular cloning, cell proliferation assay, cell apoptosis assay, quantitative real-time PCR, and luciferase reporter assay were used. Strontium altered the expression levels of miRNAs in vitro and in vivo. miR-9-5p, miR-675-5p, and miR-138-5p impaired skeletal cell proliferation, cell differentiation and cell adhesion. miR-9-5p and miR-675-5p induced MC3T3-E1 cell apoptosis more specifically than miR-138-5p. miR-9-5p, miR-675-5p, and miR-138-5p targeted glycogen synthase kinase 3 β (GSK3β), ATPase Aminophospholipid Transporter Class I Type 8A Member 2 (ATP8A2), and Eukaryotic Translation Initiation Factor 4E Binding Protein 1 (EIF4EBP1), respectively. Low-density lipoprotein receptor-related protein 5 (LRP5) played a positive role in skeletal development. miR-9-5p, miR-675-5p, and miR-138-5p damage strontium and LRP5-mediated skeletal cell proliferation, differentiation, and adhesion, and induce cell apoptosis by targeting GSK3β, ATP8A2, and EIF4EBP1, respectively. PMID:26891291

  2. MiR-9-5p, miR-675-5p and miR-138-5p Damages the Strontium and LRP5-Mediated Skeletal Cell Proliferation, Differentiation, and Adhesion.

    PubMed

    Sun, Tianhao; Leung, Frankie; Lu, William W

    2016-01-01

    This study was designed to evaluate the effects of strontium on the expression levels of microRNAs (miRNAs) and to explore their effects on skeletal cell proliferation, differentiation, adhesion, and apoptosis. The targets of these miRNAs were also studied. Molecular cloning, cell proliferation assay, cell apoptosis assay, quantitative real-time PCR, and luciferase reporter assay were used. Strontium altered the expression levels of miRNAs in vitro and in vivo. miR-9-5p, miR-675-5p, and miR-138-5p impaired skeletal cell proliferation, cell differentiation and cell adhesion. miR-9-5p and miR-675-5p induced MC3T3-E1 cell apoptosis more specifically than miR-138-5p. miR-9-5p, miR-675-5p, and miR-138-5p targeted glycogen synthase kinase 3 β (GSK3β), ATPase Aminophospholipid Transporter Class I Type 8A Member 2 (ATP8A2), and Eukaryotic Translation Initiation Factor 4E Binding Protein 1 (EIF4EBP1), respectively. Low-density lipoprotein receptor-related protein 5 (LRP5) played a positive role in skeletal development. miR-9-5p, miR-675-5p, and miR-138-5p damage strontium and LRP5-mediated skeletal cell proliferation, differentiation, and adhesion, and induce cell apoptosis by targeting GSK3β, ATP8A2, and EIF4EBP1, respectively. PMID:26891291

  3. Nifedipine inhibits advanced glycation end products (AGEs) and their receptor (RAGE) interaction-mediated proximal tubular cell injury via peroxisome proliferator-activated receptor-gamma activation

    SciTech Connect

    Matsui, Takanori; Yamagishi, Sho-ichi; Takeuchi, Masayoshi; Ueda, Seiji; Fukami, Kei; Okuda, Seiya

    2010-07-23

    Research highlights: {yields} Nifedipine inhibited the AGE-induced up-regulation of RAGE mRNA levels in tubular cells, which was prevented by GW9662, an inhibitor of peroxisome proliferator-activated receptor-{gamma}. {yields} GW9662 treatment alone increased RAGE mRNA levels in tubular cells. {yields} Nifedipine inhibited the AGE-induced reactive oxygen species generation, NF-{kappa}B activation and increases in intercellular adhesion molecule-1 and transforming growth factor-{beta} gene expression in tubular cells, all of which were blocked by GW9662. -- Abstract: There is a growing body of evidence that advanced glycation end products (AGEs) and their receptor (RAGE) interaction evokes oxidative stress generation and subsequently elicits inflammatory and fibrogenic reactions, thereby contributing to the development and progression of diabetic nephropathy. We have previously found that nifedipine, a calcium-channel blocker (CCB), inhibits the AGE-induced mesangial cell damage in vitro. However, effects of nifedipine on proximal tubular cell injury remain unknown. We examined here whether and how nifedipine blocked the AGE-induced tubular cell damage. Nifedipine, but not amlodipine, a control CCB, inhibited the AGE-induced up-regulation of RAGE mRNA levels in tubular cells, which was prevented by the simultaneous treatment of GW9662, an inhibitor of peroxisome proliferator-activated receptor-{gamma} (PPAR{gamma}). GW9662 treatment alone was found to increase RAGE mRNA levels in tubular cells. Further, nifedipine inhibited the AGE-induced reactive oxygen species generation, NF-{kappa}B activation and increases in intercellular adhesion molecule-1 and transforming growth factor-beta gene expression in tubular cells, all of which were blocked by GW9662. Our present study provides a unique beneficial aspect of nifedipine on diabetic nephropathy; it could work as an anti-oxidative and anti-inflammatory agent against AGEs in tubular cells by suppressing RAGE expression

  4. PPAR{gamma} activation abolishes LDL-induced proliferation of human aortic smooth muscle cells via SOD-mediated down-regulation of superoxide

    SciTech Connect

    Heo, Kyung-Sun; Kim, Dong-Uk; Ryoo, Sungwoo; Nam, Miyoung; Baek, Seung Tae; Kim, Lila; Park, Song-Kyu; Myung, Chang-Seon; Hoe, Kwang-Lae . E-mail: kwanghoe@kribb.re.kr

    2007-08-10

    Native LDL would be a mitogenic and chemotactic stimulus of VSMC proliferation and differentiation in the atherosclerotic lesion where endothelial disruption occurred. In previous studies, our group investigated the molecular mechanisms by which LDL induces IL-8 production and by which PPAR{alpha} activation abolishes LDL effects in human aortic SMCs (hAoSMCs). Herein is the first report of PPAR{gamma} activation by troglitazone (TG) exerting its inhibitory effects on LDL-induced cell proliferation via generation not of H{sub 2}O{sub 2}, but of O2?-, and the subsequent activation of Erk1/2 in hAoSMCs. Moreover, in this study TG abolished the LDL-accelerated G{sub 1}-S progression to control levels via down-regulation of active cyclinD1/CDK4 and cyclinE/CDK2 complexes and up-regulation of p21{sup Cip1} expression. TG exerted its anti-proliferative effects through the up-regulation of basal superoxide dismutase (SOD) expression. This data suggests that the regulation of O2?- is located at the crossroads between LDL signaling and cell proliferation.

  5. Capsaicin-mediated tNOX (ENOX2) up-regulation enhances cell proliferation and migration in vitro and in vivo.

    PubMed

    Liu, Nei-Chi; Hsieh, Pei-Fang; Hsieh, Ming-Kun; Zeng, Zih-Ming; Cheng, Hsiao-Ling; Liao, Jiunn-Wang; Chueh, Pin Ju

    2012-03-14

    Cancer chemoprevention is employed to block or reverse the progression of malignancies. To date, several thousands of agents have been found to possess chemopreventative activity, one of which is capsaicin, a component of chili peppers that exhibits antigrowth activity against various cancer cell lines. However, the role of capsaicin in tumorigenesis remains controversial because both cancer prevention and promotion have been proposed. Here, we made the unexpected discovery that treatment with low concentrations of capsaicin up-regulates tNOX (tumor-associated NADH oxidase) expression in HCT116 human colon carcinoma cells in association with enhanced cell proliferation and migration, as evidenced by down-regulation of epithelial markers and up-regulation of mesenchymal markers. Importantly, tNOX-knockdown in HCT116 cells by RNA interference reversed capsaicin-induced cell proliferation and migration in vitro and decreased tumor growth in vivo. Collectively, these findings provide a basis for explaining the tumor-promoting effect of capsaicin and might imply that caution should be taken when using capsaicin as a chemopreventive agent. PMID:22353011

  6. Heme-induced Trypanosoma cruzi proliferation is mediated by CaM kinase II

    SciTech Connect

    Souza, C.F.; Carneiro, A.B.; Silveira, A.B.; Laranja, G.A.T.; Silva-Neto, M.A.C.; Costa, S.C. Goncalves da; Paes, M.C.

    2009-12-18

    Trypanosoma cruzi, the etiologic agent of Chagas disease, is transmitted through triatomine vectors during their blood-meal on vertebrate hosts. These hematophagous insects usually ingest approximately 10 mM of heme bound to hemoglobin in a single meal. Blood forms of the parasite are transformed into epimastigotes in the crop which initiates a few hours after parasite ingestion. In a previous work, we investigated the role of heme in parasite cell proliferation and showed that the addition of heme significantly increased parasite proliferation in a dose-dependent manner . To investigate whether the heme effect is mediated by protein kinase signalling pathways, parasite proliferation was evaluated in the presence of several protein kinase (PK) inhibitors. We found that only KN-93, a classical inhibitor of calcium-calmodulin-dependent kinases (CaMKs), blocked heme-induced cell proliferation. KN-92, an inactive analogue of KN-93, was not able to block this effect. A T. cruzi CaMKII homologue is most likely the main enzyme involved in this process since parasite proliferation was also blocked when Myr-AIP, an inhibitory peptide for mammalian CaMKII, was included in the cell proliferation assay. Moreover, CaMK activity increased in parasite cells with the addition of heme as shown by immunological and biochemical assays. In conclusion, the present results are the first strong indications that CaMKII is involved in the heme-induced cell signalling pathway that mediates parasite proliferation.

  7. MiR-519d represses ovarian cancer cell proliferation and enhances cisplatin-mediated cytotoxicity in vitro by targeting XIAP

    PubMed Central

    Pang, Yingxin; Mao, Hongluan; Shen, Liang; Zhao, Zhe; Liu, Ruihan; Liu, Peishu

    2014-01-01

    Background MicroRNAs (miRNAs) are small, noncoding RNAs that are believed to play fundamental roles in tumorigenesis and tumor development at the posttranscriptional level, as negative regulators of gene expression. This study was designed to evaluate the expression and anticancer effect of miR-519d in ovarian cancer. Methods The expression levels of miR-519d in ovarian cancer cells and tissues were detected by TaqMan quantitative reverse transcriptase-polymerase chain reaction (TaqMan qRT-PCR; Life Technologies, Carlsbad, CA, USA). The effects of miR-519d on ovarian cancer cell proliferation and cisplatin chemosensitivity were analyzed by 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide, flow cytometry, and Western blotting assay. A luciferase reporter assay was performed to validate the miR-519d binding sites on the 3′ untranslated region of X-linked inhibitor of apoptosis protein (XIAP). The expression levels of XIAP mRNA and protein were examined by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting assay, respectively. Results miR-519d was significantly downregulated in human ovarian cancer cell lines and tissues. Overexpression of miR-519d in ovarian cancer cells decreased cell proliferation and sensitized ovarian cancer cells to cisplatin-induced cell death accompanied by increased activation of caspase 3 and cleavage of poly(adenosine diphosphate [ADP]-ribose) polymerase 1. Bioinformatics analysis indicated that XIAP was a putative target of miR-519d. Overexpression of miR-519d decreased XIAP expression at both the protein and mRNA levels. In contrast, inhibition of miR-519d increased XIAP expression. Luciferase reporter assay confirmed XIAP as a direct target of miR-519d. XIAP mRNA and protein expression levels were inversely correlated with miR-519d expression in ovarian cancer cell lines and tissues. Conclusion These findings indicate that miR-519d suppresses cell proliferation and sensitizes ovarian cancer

  8. Fullerene mediates proliferation and cardiomyogenic differentiation of adipose-derived stem cells via modulation of MAPK pathway and cardiac protein expression

    PubMed Central

    Hao, Tong; Zhou, Jin; Lü, Shuanghong; Yang, Boguang; Wang, Yan; Fang, Wancai; Jiang, Xiaoxia; Lin, Qiuxia; Li, Junjie; Wang, Changyong

    2016-01-01

    Zero-dimensional fullerenes can modulate the biological behavior of a variety of cell lines. However, the effects and molecular mechanisms of proliferation and cardiomyogenic differentiation in brown adipose-derived stem cells (BADSCs) are still unclear. In this study, we report the initial biological effects of fullerene-C60 on BADSCs at different concentrations. Results suggest that fullerene-C60 has no cytotoxic effects on BADSCs even at a concentration of 100 μg/mL. Fullerene-C60 improves the MAPK expression level and stem cell survival, proliferation, and cardiomyogenesis. Further, we found that the fullerene-C60 modulates cardiomyogenic differentiation. Fullerene-C60 improves the expression of cardiomyocyte-specific proteins (cTnT and α-sarcomeric actinin). At elevated concentration, fullerene-C60 reduces the incidence of diminished spontaneous cardiac differentiation of BADSCs with time. At the genetic level, fullerene-C60 (5 μg/mL) also improves the expression of cTnT. In addition, fullerene-C60 promotes the formation of gap junction among cells. These findings have important implications for clinical application of fullerenes in the treatment of myocardial infarction. PMID:26848263

  9. The Gαi AND Gαq Proteins Mediate the Effects of Melatonin on Steroid/Thyroid Hormone Receptor Transcriptional Activity and Breast Cancer Cell Proliferation

    PubMed Central

    Lai, Ling; Yuan, Lin; Chen, Qi; Dong, Chunmin; Mao, Lulu; Rowan, Brian; Frasch, Tripp; Hill, Steven M.

    2016-01-01

    Melatonin, via its MT1 receptor, but not the MT2 receptor, can modulate the transcriptional activity of various nuclear receptors (ERα and RARα, but not ERβ) in MCF-7, T47D and ZR-75-1 human breast cancer cell lines. The anti-proliferative and nuclear receptor modulatory actions of melatonin are mediated via the MT1 G protein-coupled receptor expressed in human breast cancer cells. However, the specific G proteins and associated pathways involved in nuclear receptor transcriptional regulation by melatonin are not yet clear. Upon activation, the MT1 receptor specifically couples to the Gαi2, Gαi3, Gαq and Gαll proteins, and via activation of Gαi2 proteins, melatonin suppresses forskolin-induced cyclic AMP (cAMP) production, while melatonin activation of Gαq, is able to inhibit phospholipid hydrolysis and ATP’s induction of inositol triphosphate (IP3) production in MCF-7 breast cancer cells. Employing dominant-negative (DN) and dominant-positive (DP) forms of these G proteins we demonstrate that Gαi2 proteins mediate the suppression of estrogen-induced ERα transcriptional activity by melatonin, while the Gq protein mediates the enhancement of retinoid-induced RARα transcriptional activity by melatonin. However, the growth-inhibitory actions of melatonin are mediated via both Gαi2 and Gαq proteins. PMID:18705646

  10. PP2A mediates diosmin p53 activation to block HA22T cell proliferation and tumor growth in xenografted nude mice through PI3K-Akt-MDM2 signaling suppression.

    PubMed

    Dung, Tran Duc; Day, Cecilia Hsuan; Binh, Truong Viet; Lin, Chih-Hsueh; Hsu, Hsi-Hsien; Su, Cheng-Chuan; Lin, Yueh-Min; Tsai, Fuu-Jen; Kuo, Wei-Wen; Chen, Li-Mien; Huang, Chih-Yang

    2012-05-01

    Hepatocellular carcinoma is a common type of cancer with poor prognosis. This study examines the in vitro and in vivo mechanisms of diosmin on human hepato-cellular carcinoma HA22T cell proliferation inhibition. HA22T cells were treated with different diosmin concentrations and analyzed with Western blot analysis, MTT assay, wound healing, flow cytometry, siRNA transfection assays and co-immuno-precipitation assay. The HA22T-implanted xeno-graft nude mice model was applied to confirm the cellular effects. Diosmin showed strong HA22T cell viability inhibition in a dose dependent manner and significantly reduced the cell proliferative proteins as well as inducing cell cycle arrest in the G2/M phase through p53 activation and PI3K-Akt-MDM2 signaling pathway inhibition. However, protein phosphatase 2A (PP2A) siRNA or PP2A inhibitor totally reversed the diosmin effects. The HA22T-implanted nude mice model further confirmed that diosmin inhibited HA22T tumor cell growth and down regulated the PI3K-Akt-MDM2 signaling and cell cycle regulating proteins, as well as activating PP2A and p53 proteins. Our findings indicate that HA22T cell proliferation inhibition and tumor growth suppression by diosmin are mediated through PP2A activation. PMID:22289577

  11. HDAC inhibitors suppress c-Jun/Fra-1-mediated proliferation through transcriptionally downregulating MKK7 and Raf1 in neuroblastoma cells

    PubMed Central

    Tang, Xiaomei; Xia, Yong; He, Guozhen; Min, Zhiqun; Li, Chun; Xiong, Shiqiu; Shi, Zhi; Lu, Yongjian; Yuan, Zhongmin

    2016-01-01

    Activator protein 1 (AP-1) is a transcriptional factor composed of the dimeric members of bZIP proteins, which are frequently deregulated in human cancer cells. In this study, we aimed to identify an oncogenic AP-1 dimer critical for the proliferation of neuroblastoma cells and to investigate whether histone deacetylase inhibitors (HDACIs), a new generation of anticancer agents, could target the AP-1 dimer. We report here that HDACIs including trichostatin A, suberoylanilidehydroxamic acid, valproic acid and M344 can transcriptionally suppress both c-Jun and Fra-1, preceding their inhibition of cell growth. c-Jun preferentially interacting with Fra-1 as a heterodimer is responsible for AP-1 activity and critical for cell growth. Mechanistically, HDACIs suppress Fra-1 expression through transcriptionally downregulating Raf1 and subsequently decreasing MEK1/2-ERK1/2 activity. Unexpectedly, HDACI treatment caused MKK7 downregulation at both the protein and mRNA levels. Deletion analysis of the 5′-flanking sequence of the MKK7 gene revealed that a major element responsible for the downregulation by HDACI is located at −149 to −3 relative to the transcriptional start site. Knockdown of MKK7 but not MKK4 remarkably decreased JNK/c-Jun activity and proliferation, whereas ectopic MKK7-JNK1 reversed HDACI-induced c-Jun suppression. Furthermore, suppression of both MKK-7/c-Jun and Raf-1/Fra-1 activities was involved in the tumor growth inhibitory effects induced by SAHA in SH-SY5Y xenograft mice. Collectively, these findings demonstrated that c-Jun/Fra-1 dimer is critical for neuroblastoma cell growth and that HDACIs act as effective suppressors of the two oncogenes through transcriptionally downregulating MKK7 and Raf1. PMID:26734995

  12. CXC195 suppresses proliferation and inflammatory response in LPS-induced human hepatocellular carcinoma cells via regulating TLR4-MyD88-TAK1-mediated NF-κB and MAPK pathway

    SciTech Connect

    Wang, Yiting; Tu, Qunfei; Yan, Wei; Xiao, Dan; Zeng, Zhimin; Ouyang, Yuming; Huang, Long; Cai, Jing; Zeng, Xiaoli; Chen, Ya-Jie; Liu, Anwen

    2015-01-02

    Highlights: • CXC195 exhibited significant anti-proliferative effect and induced cell cycle arrest in LPS-induced HepG2 cells. • CXC195 suppressed the release of pro-inflammatory mediators in LPS-induced HepG2 cells. • CXC195 regulated TLR4-MyD88-TAK1-mediated NF-κB and MAPK pathway in LPS-induced HepG2 cells. - Abstract: CXC195 showed strong protective effects in neuronal apoptosis by exerting its antioxidant activity. However, the anti-cancer effects of CXC195 is still with limited acquaintance. Here, we investigated the role of CXC195 in lipopolysaccharide (LPS)-induced human hepatocellular carcinoma (HCC) cells lines (HepG2) and the possible signaling pathways. CXC195 exhibited significant anti-proliferative effect and induced cell cycle arrest in LPS-induced HepG2 cells. In addition, CXC195 suppressed the release of pro-inflammatory mediators in LPS-induced HepG2 cells, including TNF-α, iNOS, IL-1β, IL-6, CC chemokine ligand (CCL)-2, CCL-22 and epidermal growth factor receptor (EGFR). Moreover, CXC195 inhibited the expressions and interactions of TLR4, MyD88 and TAK1, NF-κB translocation to nucleus and its DNA binding activity, phosphorylation of ERK1/2, p38 and JNK. Our results suggested that treatment with CXC195 could attenuate the TLR4-mediated proliferation and inflammatory response in LPS-induced HepG2 cells, thus might be beneficial for the treatment of HCC.

  13. Neuropeptide Y directly affects ovarian cell proliferation and apoptosis.

    PubMed

    Sirotkin, Alexander V; Kardošová, Diana; Alwasel, Saleh Hamad; Harrath, Abdel Halim

    2015-12-01

    The effects of neuropeptide Y (NPY; 0, 10, 100 and 1000 ng/mL) on the expression of PCNA, bax and p53 were examined by immunocytochemistry in porcine luteinized granulosa cells. NPY inhibited proliferation as well as promoted apoptosis and accumulation of p53 in the cells. This is the first report to demonstrate the direct action of NPY on ovarian cell proliferation and apoptosis. The results of the study suggest that the effect is mediated by transcription factor p53. PMID:26679167

  14. Lentiviral vector-mediated down-regulation of Notch1 in endometrial stem cells results in proliferation and migration in endometriosis.

    PubMed

    He, Hong; Liu, Rong; Xiong, Wei; Pu, Demin; Wang, Shixuan; Li, Tian

    2016-10-15

    The recent characterization of stem/progenitor cells in the endometrium has shed new light for pathogenesis of endometriosis. The present study was undertaken to investigate the role of Notch1, known as a cell fate regulator, in the mechanism of endometriosis. Influence of Notch1 on endometrial stem cells proliferation and migration was evaluated by knocking down Notch1 expression using shRNA. Furthermore, human endometrial stromal and epithelial stem cells with or without LV-Notch1-shRNA were injected into the peritoneal cavity of nude mice, to assess the in vivo effects of a specific antagonist of Notch1 on the progression of endometriosis. The results showed that LV-Notch1-shRNA led to a significant decline of clonogenicity and migration in human endometrial stem cells in vitro, as well as the size of endometriotic lesions in murine models. These data provide evidence that specific inhibition of Notch1 alters endometriotic tissue growth and progression, and may represent a promising potential therapeutic avenue. PMID:27389878

  15. Insulin-like growth factor binding protein (IGFBP)-3 and IGFBP-5 mediate TGF-{beta}- and myostatin-induced suppression of proliferation in porcine embryonic myogenic cell cultures

    SciTech Connect

    Kamanga-Sollo, E.; Pampusch, M.S.; White, M.E.; Hathaway, M.R.; Dayton, W.R. . E-mail: wdayton@umn.edu

    2005-11-15

    We have previously shown that cultured porcine embryonic myogenic cells (PEMC) produce both insulin-like growth factor binding protein (IGFBP)-3 and IGFBP-5 and secrete these proteins into their media. Exogenously added recombinant porcine (rp) IGFBP-3 and rpIGFBP-5 act via IGF-dependent and IGF-independent mechanisms to suppress proliferation of PEMC cultures. Furthermore, immunoneutralization of endogenous IGFBP-3 and IGFBP-5 in the PEMC culture medium results in increased DNA synthesis rate suggesting that endogenous IGFBP-3 and IGFBP-5 suppress PEMC proliferation. TGF-{beta} superfamily members myostatin and TGF-{beta}{sub 1} have also been shown to suppress proliferation of myogenic cells, and treatment of cultured PEMC with either TGF-{beta}{sub 1} or myostatin significantly (P < 0.01) increases levels of IGFBP-3 and -5 mRNA. We have previously shown that immunoneutralization of IGFBP-3 decreases the proliferation-suppressing activity of TGF-{beta}{sub 1} and myostatin. Here, we show that immunoneutralization of IGFBP-5 also significantly (P < 0.05) decreases the DNA synthesis-suppressing activity of these molecules. Simultaneous immunoneutralization of both IGFBP-3 and IGFBP-5 in TGF-{beta}{sub 1} or myostatin-treated PEMC cultures restores Long-R3-IGF-I-stimulated DNA synthesis rates to 90% of the levels observed in control cultures receiving no TGF-{beta}{sub 1} or myostatin treatment (P < 0.05). Even though immunoneutralization of IGFBP-3 and -5 increased DNA synthesis rates in TGF-{beta}{sub 1} or myostatin-treated PEMC cultures, phosphosmad2 levels in these cultures were not affected. These findings strongly suggest that IGFBP-3 and IGFBP-5 affect processes downstream from receptor-mediated Smad phosphorylation that facilitate the ability of TGF-{beta} and myostatin to suppress proliferation of PEMC.

  16. Insulin-like growth factor binding protein (IGFBP)-3 and IGFBP-5 mediate TGF-beta- and myostatin-induced suppression of proliferation in porcine embryonic myogenic cell cultures.

    PubMed

    Kamanga-Sollo, E; Pampusch, M S; White, M E; Hathaway, M R; Dayton, W R

    2005-11-15

    We have previously shown that cultured porcine embryonic myogenic cells (PEMC) produce both insulin-like growth factor binding protein (IGFBP)-3 and IGFBP-5 and secrete these proteins into their media. Exogenously added recombinant porcine (rp) IGFBP-3 and rpIGFBP-5 act via IGF-dependent and IGF-independent mechanisms to suppress proliferation of PEMC cultures. Furthermore, immunoneutralization of endogenous IGFBP-3 and IGFBP-5 in the PEMC culture medium results in increased DNA synthesis rate suggesting that endogenous IGFBP-3 and IGFBP-5 suppress PEMC proliferation. TGF-beta superfamily members myostatin and TGF-beta1 have also been shown to suppress proliferation of myogenic cells, and treatment of cultured PEMC with either TGF-beta1 or myostatin significantly (P < 0.01) increases levels of IGFBP-3 and -5 mRNA. We have previously shown that immunoneutralization of IGFBP-3 decreases the proliferation-suppressing activity of TGF-beta1 and myostatin. Here, we show that immunoneutralization of IGFBP-5 also significantly (P < 0.05) decreases the DNA synthesis-suppressing activity of these molecules. Simultaneous immunoneutralization of both IGFBP-3 and IGFBP-5 in TGF-beta1 or myostatin-treated PEMC cultures restores Long-R3-IGF-I-stimulated DNA synthesis rates to 90% of the levels observed in control cultures receiving no TGF-beta1 or myostatin treatment (P < 0.05). Even though immunoneutralization of IGFBP-3 and -5 increased DNA synthesis rates in TGF-beta1 or myostatin-treated PEMC cultures, phosphosmad2 levels in these cultures were not affected. These findings strongly suggest that IGFBP-3 and IGFBP-5 affect processes downstream from receptor-mediated Smad phosphorylation that facilitate the ability of TGF-beta and myostatin to suppress proliferation of PEMC. PMID:16214131

  17. NML-mediated rRNA base methylation links ribosomal subunit formation to cell proliferation in a p53-dependent manner.

    PubMed

    Waku, Tsuyoshi; Nakajima, Yuka; Yokoyama, Wataru; Nomura, Naoto; Kako, Koichiro; Kobayashi, Akira; Shimizu, Toshiyuki; Fukamizu, Akiyoshi

    2016-06-15

    Ribosomal RNAs (rRNAs) act as scaffolds and ribozymes in ribosomes, and these functions are modulated by post-transcriptional modifications. However, the biological role of base methylation, a well-conserved modification of rRNA, is poorly understood. Here, we demonstrate that a nucleolar factor, nucleomethylin (NML; also known as RRP8), is required for the N(1)-methyladenosine (m(1)A) modification in 28S rRNAs of human and mouse cells. NML also contributes to 60S ribosomal subunit formation. Intriguingly, NML depletion increases 60S ribosomal protein L11 (RPL11) levels in the ribosome-free fraction and protein levels of p53 through an RPL11-MDM2 complex, which activates the p53 pathway. Consequently, the growth of NML-depleted cells is suppressed in a p53-dependent manner. These observations reveal a new biological function of rRNA base methylation, which links ribosomal subunit formation to p53-dependent inhibition of cell proliferation in mammalian cells. PMID:27149924

  18. GL-1196 Suppresses the Proliferation and Invasion of Gastric Cancer Cells via Targeting PAK4 and Inhibiting PAK4-Mediated Signaling Pathways

    PubMed Central

    Zhang, Jian; Zhang, Hong-Yan; Wang, Jian; You, Liang-Hao; Zhou, Rui-Zhi; Zhao, Dong-Mei; Cheng, Mao-Sheng; Li, Feng

    2016-01-01

    Gastric cancer, which is the most common malignant gastrointestinal tumor, has jumped to the third leading cause of cancer-related mortality worldwide. It is of great importance to identify novel and potent drugs for gastric cancer treatment. P21-activated kinase 4 (PAK4) has emerged as an attractive target for the development of anticancer drugs in consideration of its vital functions in tumorigenesis and progression. In this paper, we reported that GL-1196, as a small molecular compound, effectively suppressed the proliferation of human gastric cancer cells through downregulation of PAK4/c-Src/EGFR/cyclinD1 pathway and CDK4/6 expression. Moreover, GL-1196 prominently inhibited the invasion of human gastric cancer cells in parallel with blockage of the PAK4/LIMK1/cofilin pathway. Interestingly, GL-1196 also inhibited the formation of filopodia and induced cell elongation in SGC7901 and BGC823 cells. Taken together, these results provided novel insights into the potential therapeutic strategy for gastric cancer. PMID:27077843

  19. Hexachlorobenzene induces cell proliferation, and aryl hydrocarbon receptor expression (AhR) in rat liver preneoplastic foci, and in the human hepatoma cell line HepG2. AhR is a mediator of ERK1/2 signaling, and cell cycle regulation in HCB-treated HepG2 cells.

    PubMed

    de Tomaso Portaz, Ana Clara; Caimi, Giselle Romero; Sánchez, Marcela; Chiappini, Florencia; Randi, Andrea S; Kleiman de Pisarev, Diana L; Alvarez, Laura

    2015-10-01

    are mediated by ERK1/2. Pretreatment with an AhR antagonist, prevented HCB-induced PCNA protein levels, ERK1/2 phosphorylation and alterations in cell cycle distribution. These results demonstrate that HCB-induced HepG2 proliferation and cell cycle progression depend on ERK1/2 phosphorylation which is mediated by the AhR. Our results provide a clue to the molecular events involved in the mechanism of action of HCB-induced hepatocarcinogenesis. PMID:26219504

  20. Effects of shRNA-Mediated SOX9 Inhibition on Cell Proliferation and Apoptosis in Human HCC Cell Line Hep3B Mediated by Ultrasound-Targeted Microbubble Destruction (UTMD).

    PubMed

    Xue, Yan; Yang, Guilun; Wang, Changmei; Li, Xiaodong; Du, Guijin

    2015-11-01

    Hepatocellular carcinoma (HCC) is one of the most aggressive tumors in humans. The survival rate of patients is still very poor as current therapies offer limited treatment efficacy. Therefore, it is necessary to explore novel and more effective strategies to treat HCC. Recently, Ultrasound-targeted microbubble destruction (UTMD) has been shown to be a better alternative to viral vectors in delivering plasmid DNA into cells. In this study, we thus first determined the effect of combining UTMD with effectene on the transfection efficiency in human Hep3B cells. Transfection rate of the [effectene + shRNA-SOX9 + UTMD] group was the highest among the five groups, and were significantly higher than that of the [effectene + shRNA-SOX9] or [shRNA-SOX9 + UTMD] groups, while there was no significant difference between [shRNA-SOX9 alone] and [shRNA-SOX9 + UTMD] groups. Expression of SOX9 mRNA and protein was the lowest in effectene + shRNA-SOX9 + UTMD group. Moreover, transfection of shRNA-SOX9 with UTMD and effectene in combination could markedly inhibit the proliferation and induced cell apoptosis of Hep3B cells. These results suggest that the efficiency of gene delivery is remarkably increased when UTMD is combined with other transfection strategies, such as effectene. In conclusion, our research demonstrates that combining conventional transfection methods with UTMD achieves better transfection efficiency and that this can provide an improved gene delivery system for gene therapy. PMID:27352352

  1. Reactive Oxygen Species (ROS) Mediate p300-dependent STAT1 Protein Interaction with Peroxisome Proliferator-activated Receptor (PPAR)-γ in CD36 Protein Expression and Foam Cell Formation.

    PubMed

    Kotla, Sivareddy; Rao, Gadiparthi N

    2015-12-18

    Previously, we have demonstrated that 15(S)-hydroxyeicosatetranoic acid (15(S)-HETE) induces CD36 expression involving STAT1. Many studies have shown that peroxisome proliferator-activated receptor (PPAR)-γ mediates CD36 expression. Therefore, we asked the question whether these transcriptional factors interact with each other in the regulation of CD36 expression by 15(S)-HETE. Here, we show that STAT1 interacts with PPARγ in the induction of CD36 expression and foam cell formation by 15(S)-HETE. In addition, using molecular biological approaches such as EMSA, supershift EMSA, ChIP, re-ChIP, and promoter-reporter gene assays, we demonstrate that the STAT1 and PPARγ complex binds to the STAT-binding site at -107 nucleotides in the CD36 promoter and enhances its activity. Furthermore, the interaction of STAT1 with PPARγ depends on STAT1 acetylation, which is mediated by p300. In addition, our findings show that reactive oxygen species-dependent Syk and Pyk2 stimulation is required for p300 tyrosine phosphorylation and activation. Together, these results demonstrate that an interaction between STAT1, p300, and peroxisome proliferator-activated receptor-γ is required for 15(S)-HETE-induced CD36 expression, oxidized low density lipoprotein uptake, and foam cell formation, critical events underlying the pathogenesis of atherosclerosis. PMID:26504087

  2. Laminin-111 stimulates proliferation of mouse embryonic stem cells through a reduction of gap junctional intercellular communication via RhoA-mediated Cx43 phosphorylation and dissociation of Cx43/ZO-1/drebrin complex.

    PubMed

    Suh, Han Na; Kim, Mi Ok; Han, Ho Jae

    2012-07-20

    Gap junctions within extracellular matrix (ECM)-defined boundaries ensure synchronous activity between cells destined to become functional mediators that regulate cell behavior. However, the role of ECM in connexin (Cx) function in mouse embryonic stem cells (mESCs) has not been elucidated. Therefore, we examined the role of laminin-111 in the control of Cx43 functions and related signal pathways in mESCs. ECM components (laminin-111, fibronectin, and collagen I) increased Cx43 phosphorylation and decreased Lucifer yellow (Ly) diffusion. In addition, laminin-111 increased the proliferation index through reduction of gap junctional intercellular communication (GJIC), which was confirmed by 18α-glycyrrhetinic acid (18α-GA). Laminin-111 increased phosphorylation of focal adhesion kinase (FAK)/Src and protein kinase C (PKC), which were inhibited by integrin β1 antibody (Ab) and laminin receptor-1 (LR-1) Ab, respectively. In addition, inhibition of both FAK/Src and PKC blocked Cx43 phosphorylation. Laminin-111 increased the Ras homolog gene family, member A (RhoA) activation, which was blocked by FAK/Src and PKC inhibitors, suggesting the existence of parallel pathways that merge at RhoA. Inhibition of RhoA reversed the laminin-111-induced increase of Cx43 phosphorylation and reduction of GJIC. Laminin-111 also stimulated the dissociation of Cx43/ZO-1 complex followed by disruption of Cx43/drebrin and Cx43/F-actin complexes, which were reversed by C3 (RhoA inhibitor). ZO-1 small interfering (si) RNA significantly decreased Ly diffusion. Moreover, laminin-111 decreased Cx43 labeling at the intercellular junction, whereas pretreatment with degradation inhibitors (lysosomal protease inhibitor, chloroquine; proteasome inhibitor, lactacystin) increased Cx43 expression, reversely. In conclusion, laminin-111 stimulated mESC proliferation through a reduction of GJIC via RhoA-mediated Cx43 phosphorylation and Cx43/ZO-1/drebrin complex instability-mediated Cx43 degradation

  3. Ethylene Inhibits Cell Proliferation of the Arabidopsis Root Meristem.

    PubMed

    Street, Ian H; Aman, Sitwat; Zubo, Yan; Ramzan, Aleena; Wang, Xiaomin; Shakeel, Samina N; Kieber, Joseph J; Schaller, G Eric

    2015-09-01

    The root system of plants plays a critical role in plant growth and survival, with root growth being dependent on both cell proliferation and cell elongation. Multiple phytohormones interact to control root growth, including ethylene, which is primarily known for its role in controlling root cell elongation. We find that ethylene also negatively regulates cell proliferation at the root meristem of Arabidopsis (Arabidopsis thaliana). Genetic analysis indicates that the inhibition of cell proliferation involves two pathways operating downstream of the ethylene receptors. The major pathway is the canonical ethylene signal transduction pathway that incorporates CONSTITUTIVE TRIPLE RESPONSE1, ETHYLENE INSENSITIVE2, and the ETHYLENE INSENSITIVE3 family of transcription factors. The secondary pathway is a phosphorelay based on genetic analysis of receptor histidine kinase activity and mutants involving the type B response regulators. Analysis of ethylene-dependent gene expression and genetic analysis supports SHORT HYPOCOTYL2, a repressor of auxin signaling, as one mediator of the ethylene response and furthermore, indicates that SHORT HYPOCOTYL2 is a point of convergence for both ethylene and cytokinin in negatively regulating cell proliferation. Additional analysis indicates that ethylene signaling contributes but is not required for cytokinin to inhibit activity of the root meristem. These results identify key elements, along with points of cross talk with cytokinin and auxin, by which ethylene negatively regulates cell proliferation at the root apical meristem. PMID:26149574

  4. FOXL2-induced follistatin attenuates activin A-stimulated cell proliferation in human granulosa cell tumors

    SciTech Connect

    Cheng, Jung-Chien; Chang, Hsun-Ming; Qiu, Xin; Fang, Lanlan; Leung, Peter C.K.

    2014-01-10

    Highlights: •Activin A stimulates cell proliferation in KGN human granulosa cell tumor-derived cell line. •Cyclin D2 mediates activin A-induced KGN cell proliferation. •FOXL2 induces follistatin expression in KGN cells. •FOXL2-induced follistatin attenuates activin A-stimulated KGN cell proliferation. -- Abstract: Human granulosa cell tumors (GCTs) are rare, and their etiology remains largely unknown. Recently, the FOXL2 402C > G (C134W) mutation was found to be specifically expressed in human adult-type GCTs; however, its function in the development of human GCTs is not fully understood. Activins are members of the transforming growth factor-beta superfamily, which has been shown to stimulate normal granulosa cell proliferation; however, little is known regarding the function of activins in human GCTs. In this study, we examined the effect of activin A on cell proliferation in the human GCT-derived cell line KGN. We show that activin A treatment stimulates KGN cell proliferation. Treatment with the activin type I receptor inhibitor SB431542 blocks activin A-stimulated cell proliferation. In addition, our results show that cyclin D2 is induced by treatment with activin A and is involved in activin A-stimulated cell proliferation. Moreover, the activation of Smad signaling is required for activin A-induced cyclin D2 expression. Finally, we show that the overexpression of the wild-type FOXL2 but not the C134W mutant FOXL2 induced follistatin production. Treatment with exogenous follistatin blocks activin A-stimulated cell proliferation, and the overexpression of wild-type FOXL2 attenuates activin A-stimulated cell proliferation. These results suggest that FOXL2 may act as a tumor suppressor in human adult-type GCTs by inducing follistatin expression, which subsequently inhibits activin-stimulated cell proliferation.

  5. Nesfatin-1 inhibits ovarian epithelial carcinoma cell proliferation in vitro

    SciTech Connect

    Xu, Yang; Pang, Xiaoyan; Dong, Mei; Wen, Fang Zhang, Yi

    2013-11-01

    Highlights: •Nesfatin-1 inhibits the proliferation and growth of HO-8910 cells by G1 phase arrest. •Nesfatin-1 enhances HO-8910 cell apoptosis. •Nesfatin-1 inhibits HO-8910 cell proliferation via mTOR and RhoA/ROCK signaling pathway. •The first report of nesfatin-1-mediated proliferation in ovarian epithelial carcinoma. -- Abstract: Nesfatin-1, an 82-amino-acid peptide derived from a 396-amino-acid precursor protein nucleobindin 2 (NUCB2), was originally identified in hypothalamic nuclei involved in the regulation of food intake. It was recently reported that nesfatin-1 is a novel depot specific adipokine preferentially produced by subcutaneous tissue, with obesity- and food deprivation-regulated expression. Although a relation between ovarian cancer mortality and obesity has been previously established, a role of nesfatin-1 in ovarian epithelial carcinoma remains unknown. The aim of the present study is to examine the effect of nesfatin-1 on ovary carcinoma cells proliferation. We found that nesfatin-1 inhibits the proliferation and growth of HO-8910 cells by G1 phase arrest, this inhibition could be abolished by nesfatin-1 neutralizing antibody. Nesfatin-1 enhances HO-8910 cell apoptosis, activation of mammalian target of rapamycin (mTOR) and RhoA/ROCK signaling pathway block the effects of nesfatin-1-induced apoptosis, therefore reverses the inhibition of HO-8910 cell proliferation by nesfatin-1. In conclusion, the present study demonstrated that nesfatin-1 can inhibit the proliferation in human ovarian epithelial carcinoma cell line HO-8910 cells through inducing apoptosis via mTOR and RhoA/ROCK signaling pathway. This study provides a novel regulatory signaling pathway of nesfatin-1-regulated ovarian epithelial carcinoma growth and may contribute to ovarian cancer prevention and therapy, especially in obese patients.

  6. COMP-angiopoietin 1 increases proliferation, differentiation, and migration of stem-like cells through Tie-2-mediated activation of p38 MAPK and PI3K/Akt signal transduction pathways

    SciTech Connect

    Kook, Sung-Ho; Lim, Shin-Saeng; Cho, Eui-Sic; Lee, Young-Hoon; Han, Seong-Kyu; Lee, Kyung-Yeol; Kwon, Jungkee; Hwang, Jae-Won; Bae, Cheol-Hyeon; Seo, Young-Kwon; Lee, Jeong-Chae

    2014-12-12

    Highlights: • COMP-Ang1 induces Tie-2 activation in BMMSCs, but not in primary osteoblasts. • Tie-2 knockdown inhibits COMP-Ang1-stimulated proliferation and osteoblastogenesis. • Tie-2 knockdown prevents COMP-Ang1-induced activation of PI3K/Akt and p38 MAPK. • COMP-Ang1 induces migration of cells via activation of PI3K/Akt and CXCR4 pathways. • COMP-Ang1 stimulates in vivo migration of PDLSCs into a calvarial defect site of rats. - Abstract: Recombinant COMP-Ang1, a chimera of angiopoietin-1 (Ang1) and a short coiled-coil domain of cartilage oligomeric matrix protein (COMP), is under consideration as a therapeutic agent capable of inducing the homing of cells with increased angiogenesis. However, the potentials of COMP-Ang1 to stimulate migration of mesenchymal stem cells (MSCs) and the associated mechanisms are not completely understood. We examined the potential of COMP-Ang1 on bone marrow (BM)-MSCs, human periodontal ligament stem cells (PDLSCs), and calvarial osteoblasts. COMP-Ang1 augmented Tie-2 induction at protein and mRNA levels and increased proliferation and expression of runt-related transcription factor 2 (Runx2), osterix, and CXCR4 in BMMSCs, but not in osteoblasts. The COMP-Ang1-mediated increases were inhibited by Tie-2 knockdown and by treating inhibitors of phosphoinositide 3-kinase (PI3K), LY294002, or p38 mitogen-activated protein kinase (MAPK), SB203580. Phosphorylation of p38 MAPK and Akt was prevented by siRNA-mediated silencing of Tie-2. COMP-Ang1 also induced in vitro migration of BMMSCs and PDLSCs. The induced migration was suppressed by Tie-2 knockdown and by CXCR4-specific peptide antagonist or LY294002, but not by SB203580. Furthermore, COMP-Ang1 stimulated the migration of PDLSCs into calvarial defect site of rats. Collectively, our results demonstrate that COMP-Ang1-stimulated proliferation, differentiation, and migration of progenitor cells may involve the Tie-2-mediated activation of p38 MAPK and PI3K/Akt pathways.

  7. MiR-126 regulates proliferation and invasion in the bladder cancer BLS cell line by targeting the PIK3R2-mediated PI3K/Akt signaling pathway

    PubMed Central

    Xiao, Jun; Lin, Huan-Yi; Zhu, Yuan-Yuan; Zhu, Yu-Ping; Chen, Ling-Wu

    2016-01-01

    Objective To assess whether microRNA-126 (miR-126) targets phosphatidylinositol 3-kinase regulatory subunit beta (PIK3R2) and to determine the potential roles of miR-126 in regulating proliferation and invasion via the PIK3R2-mediated phosphatidylinositol 3 kinase (PI3K)-protein kinase B (Akt) signaling pathway in the human bladder BLS cell line. Materials and methods A recombinant lentivirus (Lv) vector expressing miR-216 (Lv-miR-126) was successfully constructed, and Lv-miR-126 and Lv vector were transfected into the BLS cell line. A direct regulatory relationship between miR-126 and the PIK3R2 gene was demonstrated by luciferase reporter assays. To determine whether PIK3R2 directly participates in the miR-126-induced effects in BLS cells, anti-miR-126 and a PIK3R2 small interfering RNA (siRNA) were transfected into the BLS cells. Quantitative real-time polymerase chain reaction was used to measure miR-126 and PIK3R2 expressions. 5-Ethynyl-2′-deoxyuridine and colony formation assays to assess cell proliferation, flow cytometry for cell apoptosis and cell cycle analysis, Transwell assays for cell migration and invasion, and Western blots for PIK3R2, PI3K, phosphorylated PI3K (p-PI3K), Akt, and phosphorylated Akt (p-Akt) protein expressions were performed. Results Lv-miR-126 significantly enhanced the relative expression of miR-126 in the BLS cells after infection (P<0.0001). MiR-126 overexpression inhibited the proliferation, cloning, migration, and invasion of BLS cells, promoted cell apoptosis, and induced S phase arrest (all P<0.05). PIK3R2, p-PI3K, and p-Akt protein expressions were significantly decreased in the BLS cells infected with Lv-miR-126. Luciferase assays showed that miR-126 significantly inhibited the PIK3R2 3′ untranslated region (3′UTR) luciferase reporter activity (P<0.05). The anti-miR-126 + PIK3R2 siRNA group had significantly decreased PIK3R2, p-PI3K, and p-Akt expressions compared with those of anti-miR-126 alone, as well as

  8. Echinococcus multilocularis proliferation in mice and respective parasite 14-3-3 gene expression is mainly controlled by an αβ+ CD4+ T-cell-mediated immune response

    PubMed Central

    Dai, Wen Juan; Waldvogel, Andreas; Siles-Lucas, Mar; Gottstein, Bruno

    2004-01-01

    The role of specific B lymphocytes and T-cell populations in the control of experimental Echinococus multilocularis infection was studied in µMT, nude, T-cell receptor (TCR)-β–/–, major histocompatibility complex (MHC)-I–/– and MHC-II–/– mice. At 2 months postinfection, the parasite mass was more than 10 times higher in nude, TCR-β–/– and MHC-II–/– mice than in infected C57BL/6 wild-type (WT) mice, and these T-cell-deficient mice started to die of the high parasite load at this time-point. In contrast, MHC-I–/– and µMT mice exhibited parasite growth rates similar to those found in WT controls. These findings clearly point to the major role that CD4+ αβ+ T cells play in limiting the E. multilocularis proliferation, while CD8+ T and B cells appeared to play a minor role in the control of parasite growth. In the absence of T cells, especially CD4+ or αβ+ T cells, the cellular immune response to infection was impaired, as documented by the lack of hepatic granuloma formation around the parasite and by a decreased splenocyte responsiveness to concanavalin A (Con A) and parasite antigen stimulation. Surprisingly, in T-cell-deficient mice, the ex vivo expression of interferon-γ (IFN-γ) and other inflammatory cytokines (except for interleukin-6) were increased in association with a high parasite load. Thus, the relative protection mediated by CD4+ αβ+ T cells against E. multilocularis infection seemed not be IFN-γ dependent, but rather to rely on the effector's function of CD4+ αβ+ T cells. The local restriction of parasite germinal cell proliferation was reflected by a regulatory effect on the expression of 14-3-3 protein within the parasite tissue in T-cell-deficient mice. These results provide a strong indication that the CD4+ αβ+ T-cell-mediated immune response contributes to the control of the parasite growth and to the regulation of production of the parasite 14-3-3 protein in metacestode tissues. PMID:15196217

  9. Satellite cell proliferation in adult skeletal muscle

    NASA Technical Reports Server (NTRS)

    Booth, Frank W. (Inventor); Thomason, Donald B. (Inventor); Morrison, Paul R. (Inventor); Stancel, George M. (Inventor)

    1995-01-01

    Novel methods of retroviral-mediated gene transfer for the in vivo corporation and stable expression of eukaryotic or prokaryotic foreign genes in tissues of living animals is described. More specifically, methods of incorporating foreign genes into mitotically active cells are disclosed. The constitutive and stable expression of E. coli .beta.-galactosidase gene under the promoter control of the Moloney murine leukemia virus long terminal repeat is employed as a particularly preferred embodiment, by way of example, establishes the model upon which the incorporation of a foreign gene into a mitotically-active living eukaryotic tissue is based. Use of the described methods in therapeutic treatments for genetic diseases, such as those muscular degenerative diseases, is also presented. In muscle tissue, the described processes result in genetically-altered satellite cells which proliferate daughter myoblasts which preferentially fuse to form a single undamaged muscle fiber replacing damaged muscle tissue in a treated animal. The retroviral vector, by way of example, includes a dystrophin gene construct for use in treating muscular dystrophy. The present invention also comprises an experimental model utilizable in the study of the physiological regulation of skeletal muscle gene expression in intact animals.

  10. Cell proliferation and differentiation in chemical leukemogenesis

    NASA Technical Reports Server (NTRS)

    Irons, R. D.; Stillman, W. S.; Clarkson, T. W. (Principal Investigator)

    1993-01-01

    In tissues such as bone marrow with normally high rates of cell division, proliferation is tightly coordinated with cell differentiation. Survival, proliferation and differentiation of early hematopoietic progenitor cells depend on the growth factors, interleukin 3 (IL-3) and/or granulocyte-macrophage colony stimulating factor (GM-CSF) and their synergism with other cytokines. We provide evidence that a characteristic shared by a diverse group of compounds with demonstrated leukemogenic potential is the ability to act synergistically with GM-CSF. This results in an increase in recruitment of a resting population of hematopoietic progenitor cells normally unresponsive to the cytokine and a twofold increase in the size of the proliferating cell population normally regarded to be at risk of transformation in leukemogenesis. These findings support the possibility that transient alterations in hematopoietic progenitor cell differentiation may be an important factor in the early stages of development of leukemia secondary to chemical or drug exposure.