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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 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

  4. 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

  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 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

  12. 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

  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 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

  19. 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

  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. 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

  3. 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

  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 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

  2. 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

  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. 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

  14. 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

  15. 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

  16. 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

  17. 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

  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. 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

  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. 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

  7. 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.

  8. 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

  9. 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

  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. 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

  4. 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

  5. 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

  6. 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

  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. 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

  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. 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

  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. 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

  9. 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

  10. 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

  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

    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 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

  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.

    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

  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

    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.

  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.

  11. Germ Cells Need Folate to Proliferate.

    PubMed

    Walker, Amy K

    2016-07-11

    In this issue of Developmental Cell, Chaudhari and colleagues (2016) use a novel method to create an in vitro proliferative cell line from tumorous C. elegans germ cells, and in the process discover that bacterial folates act as signals for proliferation, independent of their roles as vitamins. PMID:27404353

  12. Blue light inhibits proliferation of melanoma cells

    NASA Astrophysics Data System (ADS)

    Becker, Anja; Distler, Elisabeth; Klapczynski, Anna; Arpino, Fabiola; Kuch, Natalia; Simon-Keller, Katja; Sticht, Carsten; van Abeelen, Frank A.; Gretz, Norbert; Oversluizen, Gerrit

    2016-03-01

    Photobiomodulation with blue light is used for several treatment paradigms such as neonatal jaundice, psoriasis and back pain. However, little is known about possible side effects concerning melanoma cells in the skin. The aim of this study was to assess the safety of blue LED irradiation with respect to proliferation of melanoma cells. For that purpose we used the human malignant melanoma cell line SK-MEL28. Cell proliferation was decreased in blue light irradiated cells where the effect size depended on light irradiation dosage. Furthermore, with a repeated irradiation of the melanoma cells on two consecutive days the effect could be intensified. Fluorescence-activated cell sorting with Annexin V and Propidium iodide labeling did not show a higher number of dead cells after blue light irradiation compared to non-irradiated cells. Gene expression analysis revealed down-regulated genes in pathways connected to anti-inflammatory response, like B cell signaling and phagosome. Most prominent pathways with up-regulation of genes were cytochrome P450, steroid hormone biosynthesis. Furthermore, even though cells showed a decrease in proliferation, genes connected to the cell cycle were up-regulated after 24h. This result is concordant with XTT test 48h after irradiation, where irradiated cells showed the same proliferation as the no light negative control. In summary, proliferation of melanoma cells can be decreased using blue light irradiation. Nevertheless, the gene expression analysis has to be further evaluated and more studies, such as in-vivo experiments, are warranted to further assess the safety of blue light treatment.

  13. Proliferating pancreatic beta-cells upregulate ALDH.

    PubMed

    Liu, Yinglan; Jiang, Xiaoxin; Zeng, Yong; Zhou, Hui; Yang, Jing; Cao, Renxian

    2014-12-01

    High levels of aldehyde dehydrogenase (ALDH) activity have been regarded as a specific feature of progenitor cells and stem cells. Hence, as an indicator of ALDH activity, aldefluor fluorescence has been widely used for the identification and isolation of stem and progenitor cells. ALDH activity was recently detected in embryonic mouse pancreas, and specifically and exclusively in adult centroacinar and terminal duct cells, suggesting that these duct cells may harbor cells of endocrine and exocrine differentiation potential in the adult pancreas. Here, we report the presence of aldefluor+ beta-cells in a beta-cell proliferation model, partial pancreatectomy. The aldefluor+ beta-cells are essentially all positive for Ki-67 and expressed high levels of cell-cycle activators such as CyclinD1, CyclinD2, and CDK4, suggesting that they are mitotic cells. Our data thus reveal a potential change in ALDH activity of proliferating beta-cells, which provides a novel method for the isolation and analysis of proliferating beta-cells. Moreover, our data also suggest that aldefluor lineage-tracing is not a proper method for analyzing progenitor or stem activity in the adult pancreas. PMID:25028343

  14. Lensless imaging system to quantify cell proliferation

    NASA Astrophysics Data System (ADS)

    Vinjimore Kesavan, S.; Allier, C. P.; Navarro, F.; Mittler, F.; Chalmond, B.; Dinten, J.-M.

    2013-02-01

    Owing to its simplicity, lensless imaging system is adept at continuous monitoring of adherent cells inside the incubator. The setup consists of a CMOS sensor with pixel pitch of 2.2 μm and field of view of 24 mm2, LED with a dominating wavelength of 525 nm, along with a pinhole of 150 μm as the source of illumination. The in-line hologram obtained from cells depends on the degree of cell-substrate adhesion. Drastic difference is observed between the holographic patterns of floating and adherent cells. In addition, the well-established fact of reduction of cell-substrate contact during cell division is observed with our system based on corresponding spontaneous transition in the holographic pattern. Here, we demonstrate that by recognizing this specific holographic pattern, number of cells undergoing mitosis in a cell culture with a population of approximately 5000 cells, can be estimated in real-time. The method is assessed on comparison with Edu-based proliferation assay. The approach is straightforward and it eliminates the use of markers to estimate the proliferation rate of a given cell culture. Unlike most proliferation assays, the cells are not harvested enabling continuous monitoring of cell culture.

  15. E-cadherin mediates contact inhibition of proliferation through Hippo signaling-pathway components

    PubMed Central

    Kim, Nam-Gyun; Koh, Eunjin; Chen, Xiao; Gumbiner, Barry M.

    2011-01-01

    Contact inhibition of cell growth is essential for embryonic development and maintenance of tissue architecture in adult organisms, and the growth of tumors is characterized by a loss of contact inhibition of proliferation. The recently identified Hippo signaling pathway has been implicated in contact inhibition of proliferation as well as organ size control. The modulation of the phosphorylation and nuclear localization of Yes-associated protein (YAP) by the highly conserved kinase cascade of the Hippo signaling pathway has been intensively studied. However, cell-surface receptors regulating the Hippo signaling pathway in mammals are not well understood. In this study, we show that Hippo signaling pathway components are required for E-cadherin–dependent contact inhibition of proliferation. Knockdown of the Hippo signaling components or overexpression of YAP inhibits the decrease in cell proliferation caused by E-cadherin homophilic binding at the cell surface, independent of other cell–cell interactions. We also demonstrate that the E-cadherin/catenin complex functions as an upstream regulator of the Hippo signaling pathway in mammalian cells. Expression of E-cadherin in MDA-MB-231 cells restores the density-dependent regulation of YAP nuclear exclusion. Knockdown of β-catenin in densely cultured MCF10A cells, which mainly depletes E-cadherin–bound β-catenin, induces a decrease in the phosphorylation of S127 residue of YAP and its nuclear accumulation. Moreover, E-cadherin homophilic binding independent of other cell interactions is sufficient to control the subcellular localization of YAP. Therefore, Our results indicate that, in addition to its role in cell–cell adhesion, E-cadherin-mediated cell–cell contact directly regulates the Hippo signaling pathway to control cell proliferation. PMID:21730131

  16. Exosomes Secreted by Toxoplasma gondii-Infected L6 Cells: Their Effects on Host Cell Proliferation and Cell Cycle Changes

    PubMed Central

    Kim, Min Jae; Jung, Bong-Kwang; Cho, Jaeeun; Song, Hyemi; Pyo, Kyung-Ho; Lee, Ji Min; Kim, Min-Kyung; Chai, Jong-Yil

    2016-01-01

    Toxoplasma gondii infection induces alteration of the host cell cycle and cell proliferation. These changes are not only seen in directly invaded host cells but also in neighboring cells. We tried to identify whether this alteration can be mediated by exosomes secreted by T. gondii-infected host cells. L6 cells, a rat myoblast cell line, and RH strain of T. gondii were selected for this study. L6 cells were infected with or without T. gondii to isolate exosomes. The cellular growth patterns were identified by cell counting with trypan blue under confocal microscopy, and cell cycle changes were investigated by flow cytometry. L6 cells infected with T. gondii showed decreased proliferation compared to uninfected L6 cells and revealed a tendency to stay at S or G2/M cell phase. The treatment of exosomes isolated from T. gondii-infected cells showed attenuation of cell proliferation and slight enhancement of S phase in L6 cells. The cell cycle alteration was not as obvious as reduction of the cell proliferation by the exosome treatment. These changes were transient and disappeared at 48 hr after the exosome treatment. Microarray analysis and web-based tools indicated that various exosomal miRNAs were crucial for the regulation of target genes related to cell proliferation. Collectively, our study demonstrated that the exosomes originating from T. gondii could change the host cell proliferation and alter the host cell cycle. PMID:27180572

  17. TWEAK induces liver progenitor cell proliferation.

    PubMed

    Jakubowski, Aniela; Ambrose, Christine; Parr, Michael; Lincecum, John M; Wang, Monica Z; Zheng, Timothy S; Browning, Beth; Michaelson, Jennifer S; Baetscher, Manfred; Baestcher, Manfred; Wang, Bruce; Bissell, D Montgomery; Burkly, Linda C

    2005-09-01

    Progenitor ("oval") cell expansion accompanies many forms of liver injury, including alcohol toxicity and submassive parenchymal necrosis as well as experimental injury models featuring blocked hepatocyte replication. Oval cells can potentially become either hepatocytes or biliary epithelial cells and may be critical to liver regeneration, particularly when hepatocyte replication is impaired. The regulation of oval cell proliferation is incompletely understood. Herein we present evidence that a TNF family member called TWEAK (TNF-like weak inducer of apoptosis) stimulates oval cell proliferation in mouse liver through its receptor Fn14. TWEAK has no effect on mature hepatocytes and thus appears to be selective for oval cells. Transgenic mice overexpressing TWEAK in hepatocytes exhibit periportal oval cell hyperplasia. A similar phenotype was obtained in adult wild-type mice, but not Fn14-null mice, by administering TWEAK-expressing adenovirus. Oval cell expansion induced by 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) was significantly reduced in Fn14-null mice as well as in adult wild-type mice with a blocking anti-TWEAK mAb. Importantly, TWEAK stimulated the proliferation of an oval cell culture model. Finally, we show increased Fn14 expression in chronic hepatitis C and other human liver diseases relative to its expression in normal liver, which suggests a role for the TWEAK/Fn14 pathway in human liver injury. We conclude that TWEAK has a selective mitogenic effect for liver oval cells that distinguishes it from other previously described growth factors. PMID:16110324

  18. Peroxisome Proliferator-activated Receptor α Positively Regulates Complement C3 Expression but Inhibits Tumor Necrosis Factor α-mediated Activation of C3 Gene in Mammalian Hepatic-derived Cells*

    PubMed Central

    Mogilenko, Denis A.; Kudriavtsev, Igor V.; Shavva, Vladimir S.; Dizhe, Ella B.; Vilenskaya, Ekaterina G.; Efremov, Alexander M.; Perevozchikov, Andrej P.; Orlov, Sergey V.

    2013-01-01

    Complement C3 is a pivotal component of three cascades of complement activation. The liver is the main source of C3 in circulation and expression and secretion of C3 by hepatocytes is increased during acute inflammation. However, the mechanism of the regulation of the C3 gene in hepatocytes is not well elucidated. We showed that the C3 gene is the direct target for peroxisome proliferator-activated receptor α (PPARα) in human hepatoma HepG2 cells and mouse liver. Using PPARα siRNA and synthetic PPARα agonist WY-14643 and antagonist MK886 we showed that activation of PPARα results in up-regulation of C3 gene expression and protein secretion by HepG2 cells. The PPAR response element (PPRE), which is able to bind PPARα in vitro and in vivo, was found in the human C3 promoter. PPRE is conserved between human and mouse, and WY-14643 stimulates mouse C3 expression in the liver. TNFα increases C3 gene via NF-κB and, to a lesser extent, MEK1/2 signaling pathways, whereas TNFα-mediated stimulation of C3 protein secretion depends on activation of MEK1/2, p38, and JNK in HepG2 cells. Activation of PPARα abolishes TNFα-mediated up-regulation of C3 gene expression and protein secretion due to interference with NF-κB via PPRE-dependent mechanism in HepG2 cells. TNFα decreases PPARα protein content via NF-κB and MEK1/2 signaling pathways and inhibits PPARα binding with the human C3 promoter in HepG2 cells. These results suggest novel mechanism controlling C3 expression in hepatocytes during acute phase inflammation and demonstrate a crosstalk between PPARα and TNFα in the regulation of complement system. PMID:23168409

  19. Predators inhibit brain cell proliferation in natural populations of electric fish, Brachyhypopomus occidentalis.

    PubMed

    Dunlap, Kent D; Tran, Alex; Ragazzi, Michael A; Krahe, Rüdiger; Salazar, Vielka L

    2016-02-10

    Compared with laboratory environments, complex natural environments promote brain cell proliferation and neurogenesis. Predators are one important feature of many natural environments, but, in the laboratory, predatory stimuli tend to inhibit brain cell proliferation. Often, laboratory predatory stimuli also elevate plasma glucocorticoids, which can then reduce brain cell proliferation. However, it is unknown how natural predators affect cell proliferation or whether glucocorticoids mediate the neurogenic response to natural predators. We examined brain cell proliferation in six populations of the electric fish, Brachyhypopomus occidentalis, exposed to three forms of predator stimuli: (i) natural variation in the density of predatory catfish; (ii) tail injury, presumably from predation attempts; and (iii) the acute stress of capture. Populations with higher predation pressure had lower density of proliferating (PCNA+) cells, and fish with injured tails had lower proliferating cell density than those with intact tails. However, plasma cortisol did not vary at the population level according to predation pressure or at the individual level according to tail injury. Capture stress significantly increased cortisol, but only marginally decreased cell proliferation. Thus, it appears that the presence of natural predators inhibits brain cell proliferation, but not via mechanisms that depend on changes in basal cortisol levels. This study is the first demonstration of predator-induced alteration of brain cell proliferation in a free-living vertebrate. PMID:26842566

  20. Spatial constraints control cell proliferation in tissues

    PubMed Central

    Streichan, Sebastian J.; Hoerner, Christian R.; Schneidt, Tatjana; Holzer, Daniela; Hufnagel, Lars

    2014-01-01

    Control of cell proliferation is a fundamental aspect of tissue formation in development and regeneration. Cells experience various spatial and mechanical constraints depending on their environmental context in the body, but we do not fully understand if and how such constraints influence cell cycle progression and thereby proliferation patterns in tissues. Here, we study the impact of mechanical manipulations on the cell cycle of individual cells within a mammalian model epithelium. By monitoring the response to experimentally applied forces, we find a checkpoint at the G1–S boundary that, in response to spatial constraints, controls cell cycle progression. This checkpoint prevents cells from entering S phase if the available space remains below a characteristic threshold because of crowding. Stretching the tissue results in fast cell cycle reactivation, whereas compression rapidly leads to cell cycle arrest. Our kinetic analysis of this response shows that cells have no memory of past constraints and allows us to formulate a biophysical model that predicts tissue growth in response to changes in spatial constraints in the environment. This characteristic biomechanical cell cycle response likely serves as a fundamental control mechanism to maintain tissue integrity and to ensure control of tissue growth during development and regeneration. PMID:24706777

  1. Microfluidic devices for cell cultivation and proliferation

    PubMed Central

    Tehranirokh, Masoomeh; Kouzani, Abbas Z.; Francis, Paul S.; Kanwar, Jagat R.

    2013-01-01

    Microfluidic technology provides precise, controlled-environment, cost-effective, compact, integrated, and high-throughput microsystems that are promising substitutes for conventional biological laboratory methods. In recent years, microfluidic cell culture devices have been used for applications such as tissue engineering, diagnostics, drug screening, immunology, cancer studies, stem cell proliferation and differentiation, and neurite guidance. Microfluidic technology allows dynamic cell culture in microperfusion systems to deliver continuous nutrient supplies for long term cell culture. It offers many opportunities to mimic the cell-cell and cell-extracellular matrix interactions of tissues by creating gradient concentrations of biochemical signals such as growth factors, chemokines, and hormones. Other applications of cell cultivation in microfluidic systems include high resolution cell patterning on a modified substrate with adhesive patterns and the reconstruction of complicated tissue architectures. In this review, recent advances in microfluidic platforms for cell culturing and proliferation, for both simple monolayer (2D) cell seeding processes and 3D configurations as accurate models of in vivo conditions, are examined. PMID:24273628

  2. Peroxisome proliferators and fatty acids negatively regulate liver X receptor-mediated activity and sterol biosynthesis.

    PubMed

    Johnson, T E; Ledwith, B J

    2001-04-01

    Peroxisome proliferators (PPs) are potent tumor promoters in rodents. The mechanism of hepatocarcinogenesis requires the nuclear receptor peroxisome proliferator activated receptor-alpha (PPARalpha), but might also involve the PPARalpha independent alteration of signaling pathways that regulate cell growth. Here, we studied the effects of PPs on the mevalonate pathway, a critical pathway that controls cell proliferation. Liver X receptors (LXRs) are nuclear receptors that act as sterol sensors in the mevalonate pathway. In gene reporter assays in COS-7 cells, the basal activity of the LXR responsive reporter gene (LXRE-luc) was suppressed by 10 microM lovastatin and zaragozic acid A, suggesting that this activity was attributed to the activation of native LXRs, by endogenously produced mevalonate products. The potent PP and rodent tumor promoter, pirinixic acid (WY-14643) also inhibited LXR-mediated transcription in a dose related manner (approximate IC(50) of 100 microM). As did several other PPs including ciprofibric acid and mono-ethylhexylphthalate. Polyunsaturated and medium to long chain fatty acids at 100 microM were also potent inhibitors; the arachidonic acid analogue eicosatetraynoic acid being the most active (approximate IC(50) of 10 microM). Of the PPs and fatty acids tested, there was a strong correlation between the ability of these agents to suppress de novo sterol synthesis in a rat hepatoma cell line, H4IIEC3, and inhibit LXR-mediated transcription in COS-7 cells, but a discordance between these endpoints and PPARalpha activation and fatty acid acyl-CoA oxidase induction. Taken together, these results suggest that PPs and fatty acids negatively regulate the mevalonate pathway through a mechanism that is not entirely dependent on PPARalpha activation. Because of the importance of the mevalonate pathway in regulating cell proliferation, the modulation of this pathway by PPs and fatty acids might contribute to their actions on cell growth

  3. Regulation of nuclear transport in proliferating and quiescent cells.

    PubMed

    Feldherr, C M; Akin, D

    1993-03-01

    Previously, we compared signal-mediated nuclear transport in proliferating and quiescent BALB/c 3T3 cells and found that both the relative rate of nuclear uptake and the functional size of the transport channels were significantly greater in proliferating cells. In this study, the possible causes of these permeability differences were investigated. To determine if the decrease in transport capacity in quiescent cells was due to a reduction in the availability of soluble cytoplasmic factors (i.e., ATP or receptors for nuclear location sequences), or changes in the properties of the pores themselves, proliferating and quiescent cells were fused, and nuclear import of nucleoplasmin-coated gold (NP-gold) particles was assayed in the heterokaryons 50-60 min later. Significant differences in nuclear uptake were maintained following fusion, even though the two nuclei shared a common cytoplasm, consistent with the view that permeability is regulated at the level of the pores. Cell shape also influenced signal-mediated nuclear import. This was demonstrated by studying transport in rounded and flattened cells attached to different-size palladium domains that were deposited on a nonadhesive substrate. Based on analysis of the nuclear uptake rates of large (110-270 A in diameter) and small (50-80 A in diameter) coated gold particles, it was determined that the functional size of the pores was significantly greater in flattened cells. The effect of growth factors on recovery of nuclear transport capacity following serum depletion was also analyzed. Partial recovery was achieved by treating cells with physiological concentrations of EGF, IGF-1, or PDGF; however, complete recovery required both EGF and IGF-1. PMID:8453991

  4. BCOR regulates myeloid cell proliferation and differentiation.

    PubMed

    Cao, Q; Gearhart, M D; Gery, S; Shojaee, S; Yang, H; Sun, H; Lin, D-C; Bai, J-W; Mead, M; Zhao, Z; Chen, Q; Chien, W-W; Alkan, S; Alpermann, T; Haferlach, T; Müschen, M; Bardwell, V J; Koeffler, H P

    2016-05-01

    BCOR is a component of a variant Polycomb group repressive complex 1 (PRC1). Recently, we and others reported recurrent somatic BCOR loss-of-function mutations in myelodysplastic syndrome and acute myelogenous leukemia (AML). However, the role of BCOR in normal hematopoiesis is largely unknown. Here, we explored the function of BCOR in myeloid cells using myeloid murine models with Bcor conditional loss-of-function or overexpression alleles. Bcor mutant bone marrow cells showed significantly higher proliferation and differentiation rates with upregulated expression of Hox genes. Mutation of Bcor reduced protein levels of RING1B, an H2A ubiquitin ligase subunit of PRC1 family complexes and reduced H2AK119ub upstream of upregulated HoxA genes. Global RNA expression profiling in murine cells and AML patient samples with BCOR loss-of-function mutation suggested that loss of BCOR expression is associated with enhanced cell proliferation and myeloid differentiation. Our results strongly suggest that BCOR plays an indispensable role in hematopoiesis by inhibiting myeloid cell proliferation and differentiation and offer a mechanistic explanation for how BCOR regulates gene expression such as Hox genes. PMID:26847029

  5. Hippo signaling regulates Drosophila intestine stem cell proliferation through multiple pathways

    PubMed Central

    Ren, Fangfang; Wang, Bing; Yue, Tao; Yun, Eun-Young; Ip, Y. Tony; Jiang, Jin

    2010-01-01

    Intestinal stem cells (ISCs) in the Drosophila adult midgut are essential for maintaining tissue homeostasis and replenishing lost cells in response to tissue damage. Here we demonstrate that the Hippo (Hpo) signaling pathway, an evolutionarily conserved pathway implicated in organ size control and tumorigenesis, plays an essential role in regulating ISC proliferation. Loss of Hpo signaling in either midgut precursor cells or epithelial cells stimulates ISC proliferation. We provide evidence that loss of Hpo signaling in epithelial cells increases the production of cytokines of the Upd family and multiple EGFR ligands that activate JAK-STAT and EGFR signaling pathways in ISCs to stimulate their proliferation, thus revealing a unique non–cell-autonomous role of Hpo signaling in blocking ISC proliferation. Finally, we show that the Hpo pathway mediator Yorkie (Yki) is also required in precursor cells for injury-induced ISC proliferation in response to tissue-damaging reagent DSS. PMID:21078993

  6. Extracellular potassium concentration regulates proliferation of immature cerebellar granule cells.

    PubMed

    Borodinsky, L N; Fiszman, M L

    1998-04-17

    The present study examines the effect of depolarizing potassium concentrations on the proliferation of immature rat cerebellar neurons. Cells inoculated in serum free medium and 5 mM KCl (5 K) showed a high degree of 3H-thymidine incorporation that decreased 24-48 h after plating as differentiation began. During the first 24 h after inoculation, cells grown in high potassium (25 K), showed a 34 +/- 3% increase (mean +/- S.E.M., n = 12) in 3H-thymidine incorporation as compared with the values observed in 5 K. After 24 h in vitro, cells grown in 25 K showed 23 +/- 3% (mean +/- S.E.M., n = 3) less DNA synthesis than those inoculated in 5 K. The increase in DNA synthesis due to 25 K was blocked by MgCl2 and nifedipine, but not by omega-conotoxin GVIA, suggesting that it is mediated by a Ca2+ influx via voltage-gated calcium channels (VGCC) of the L-subtype. High potassium-induced cell proliferation was blocked by the mitogen-activated protein kinase kinase (MEK1) inhibitor (PD98059, 75 microM). The number of neurons counted after 48 h in vitro in 25 K was 35-100% above of the number obtained with 5 K and this increase also was blocked by MgCl2 and nifedipine. These data support the hypothesis that depolarizing activity during neurogenesis plays a role in the modulation of cerebellar granule cells proliferation. PMID:9602050

  7. GRP78 is required for cell proliferation and protection from apoptosis in chicken embryo fibroblast cells.

    PubMed

    Jeon, M; Choi, H; Lee, S I; Kim, J S; Park, M; Kim, K; Lee, S; Byun, S J

    2016-05-01

    Chicken serum has been suggested as a supplement to promote chicken cell proliferation and development. However, the molecular mechanisms by which chicken serum stimulates chicken cell proliferation remain unknown. Here, we evaluated the effects of chicken serum supplementation on chicken embryo fibroblast (CEF) and DF-1 cell proliferation. We also sought to elucidate the molecular pathways involved in mediating the effects of chicken serum on fibroblasts and DF-1 cells by overexpression of chicken 78 kDa glucose-regulated protein (chGRP78), which is important for cell growth and the prevention of apoptosis. Our data demonstrated that the addition of 5% chicken serum significantly enhanced fibroblast proliferation. Moreover, knockdown of chGRP78 using siRNA decreased fibroblast proliferation and increased apoptosis. Based on these results, we suggest that the chGRP78-mediated signaling pathway plays a critical role in chicken serum-stimulated fibroblast survival and anti-apoptosis. Therefore, our findings have important implications for the maintenance of chicken fibroblast cells through the inhibition of apoptosis and may lead to the development of new treatments for avian disease. PMID:26944959

  8. The Stochastic Theory of Cell Proliferation

    PubMed Central

    Bronk, Burt V.; Dienes, G. J.; Paskin, Arthur

    1968-01-01

    A stochastic theory of cell kinetics has been developed based on a realistic model of cell proliferation. A characteristic transit time, t̄i, has been assigned to each of the four states (G1, S, G2, M) of the cell cycle. The actual transit time, ti, for any cell is represented by a distribution around t̄i with a variance σi2. Analytic and computer formulations have been used to describe the time development of such characteristics as age distribution, labeling experiments, and response to perturbations of the system by, for example, irradiation and temperature. The decay of synchrony is analyzed in detail and is shown to proceed as a damped wave. From the first few peaks of the synchrony decay one can obtain the distribution function for the cell cycle time. The later peaks decay exponentially with a characteristic decay constant, λ, which depends only on the average cell-cycle time, T̄, and the associated variance. It is shown that the system, upon any sudden disturbance, approaches new “equilibrium” proliferation characteristics via damped periodic transients, the damping being characterized by λ. Thus, the response time of the system, T̄/λ, is as basic a parameter of the system as the cell-cycle time. PMID:5696217

  9. Mitochondrial Regulation of Cell Cycle and Proliferation

    PubMed Central

    Antico Arciuch, Valeria Gabriela; Elguero, María Eugenia; Poderoso, Juan José

    2012-01-01

    Abstract Eukaryotic mitochondria resulted from symbiotic incorporation of α-proteobacteria into ancient archaea species. During evolution, mitochondria lost most of the prokaryotic bacterial genes and only conserved a small fraction including those encoding 13 proteins of the respiratory chain. In this process, many functions were transferred to the host cells, but mitochondria gained a central role in the regulation of cell proliferation and apoptosis, and in the modulation of metabolism; accordingly, defective organelles contribute to cell transformation and cancer, diabetes, and neurodegenerative diseases. Most cell and transcriptional effects of mitochondria depend on the modulation of respiratory rate and on the production of hydrogen peroxide released into the cytosol. The mitochondrial oxidative rate has to remain depressed for cell proliferation; even in the presence of O2, energy is preferentially obtained from increased glycolysis (Warburg effect). In response to stress signals, traffic of pro- and antiapoptotic mitochondrial proteins in the intermembrane space (B-cell lymphoma-extra large, Bcl-2-associated death promoter, Bcl-2 associated X-protein and cytochrome c) is modulated by the redox condition determined by mitochondrial O2 utilization and mitochondrial nitric oxide metabolism. In this article, we highlight the traffic of the different canonical signaling pathways to mitochondria and the contributions of organelles to redox regulation of kinases. Finally, we analyze the dynamics of the mitochondrial population in cell cycle and apoptosis. Antioxid. Redox Signal. 16, 1150–1180. PMID:21967640

  10. Cell proliferation inhibition in reduced gravity

    NASA Technical Reports Server (NTRS)

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

    1994-01-01

    Extended durations of spaceflight have been shown to be deleterious on an organismic level; however, mechanisms underlying cellular sensitivity to the gravitational environment remain to be elucidated. The majority of the gravitational studies to date indicates that cell regulatory pathways may be influenced by their gravitational environment. Still, few cell biology experiments have been performed in space flight and even fewer experiments have been repeated on subsequent flights. With flight opportunities on STS-50, 54, and 57, Sf9 cells were flown in the BioServe Fluids Processing Apparatus and cell proliferation was measured with and without exposure to a cell regulatory sialoglycopeptide (CeReS) inhibitor. Results from these flights indicate that the Sf9 cells grew comparable to ground controls, that the CeReS inhibitor bound to its specific receptor, and that its signal transduction cascade was not gravity sensitive.

  11. The effects of physiological adaptations to calorie restriction on global cell proliferation rates.

    PubMed

    Bruss, Matthew D; Thompson, Airlia C S; Aggarwal, Ishita; Khambatta, Cyrus F; Hellerstein, Marc K

    2011-04-01

    Calorie restriction (CR) reduces the rate of cell proliferation in mitotic tissues. It has been suggested that this reduction in cell proliferation may mediate CR-induced increases in longevity. However, the mechanisms that lead to CR-induced reductions in cell proliferation rates remain unclear. To evaluate the CR-induced physiological adaptations that may mediate reductions in cell proliferation rates, we altered housing temperature and access to voluntary running wheels to determine the effects of food intake, energy expenditure, percent body fat, and body weight on proliferation rates of keratinocytes, liver cells, mammary epithelial cells, and splenic T-cells in C57BL/6 mice. We found that ∼20% CR led to a reduction in cell proliferation rates in all cell types. However, lower cell proliferation rates were not observed with reductions in 1) food intake and energy expenditure in female mice housed at 27°C, 2) percent body fat in female mice provided running wheels, or 3) body weight in male mice provided running wheels compared with ad libitum-fed controls. In contrast, reductions in insulin-like growth factor I were associated with decreased cell proliferation rates. Taken together, these data suggest that CR-induced reductions in food intake, energy expenditure, percent body fat, and body weight do not account for the reductions in global cell proliferation rates observed in CR. In addition, these data are consistent with the hypothesis that reduced cell proliferation rates could be useful as a biomarker of interventions that increase longevity. PMID:21285400

  12. Role of acetylcholine receptors in proliferation and differentiation of P19 embryonal carcinoma cells

    SciTech Connect

    Resende, R.R.; Alves, A.S.; Britto, L.R.G; Ulrich, H.

    2008-04-15

    Coordinated proliferation and differentiation of progenitor cells is the base for production of appropriate numbers of neurons and glia during neuronal development in order to establish normal brain functions. We have used murine embryonal carcinoma P19 cells as an in vitro model for early differentiation to study participation of nicotinic (nAChR) and muscarinic acetylcholine (mAChR) receptors in the proliferation of neural progenitor cells and their differentiation to neurons. We have previously shown that functional nicotinic acetylcholine receptors (nAChRs) already expressed in embryonic cells mediate elevations in cytosolic free calcium concentration ([Ca{sup 2+}]{sub i}) via calcium influx through nAChR channels whereas intracellular stores contribute to nAChR- and mAChR-mediated calcium fluxes in differentiated cells [Resende et al., Cell Calcium 43 (2008) 107-121]. In the present study, we have demonstrated that nicotine provoked inhibition of proliferation in embryonic cells as determined by BrdU labeling. However, in neural progenitor cells nicotine stimulated proliferation which was reversed in the presence of inhibitors of calcium mobilization from intracellular stores, indicating that liberation of intracellular calcium contributed to this proliferation induction. Muscarine induced proliferation stimulation in progenitor cells by activation of G{alpha}{sub q/11}-coupled M{sub 1}, M{sub 3} and M{sub 5} receptors and intracellular calcium stores, whereas G{alpha}{sub i/o}-protein coupled M{sub 2} receptor activity mediated neuronal differentiation.

  13. CCL5 activation of CCR5 regulates cell metabolism to enhance proliferation of breast cancer cells

    PubMed Central

    Gao, Darrin; Rahbar, Ramtin; Fish, Eleanor N.

    2016-01-01

    In earlier studies, we showed that CCL5 enhances proliferation and survival of MCF-7 breast cancer cells in an mTOR-dependent manner and we provided evidence that, for T cells, CCL5 activation of CCR5 results in increased glycolysis and enhanced ATP production. Increases in metabolic activity of cancer cells, specifically increased glycolytic activity and increased expression of glucose transporters, are associated with tumour progression. In this report, we provide evidence that CCL5 enhances the proliferation of human breast cancer cell lines (MDA-MB-231, MCF-7) and mouse mammary tumour cells (MMTV-PyMT), mediated by CCR5 activation. Concomitant with enhanced proliferation we show that CCL5 increases cell surface expression of the glucose transporter GLUT1, and increases glucose uptake and ATP production by these cells. Blocking CCL5-inducible glucose uptake abrogates the enhanced proliferation induced by CCL5. We provide evidence that increased glucose uptake is associated with enhanced glycolysis, as measured by extracellular acidification. Moreover, CCL5 enhances the invasive capacity of these breast cancer cells. Using metabolomics, we demonstrate that the metabolic signature of CCL5-treated primary mouse mammary tumour cells reflects increased anabolic metabolism. The implications are that CCL5–CCR5 interactions in the tumour microenvironment regulate metabolic events, specifically glycolysis, to promote tumour proliferation and invasion. PMID:27335323

  14. CCL5 activation of CCR5 regulates cell metabolism to enhance proliferation of breast cancer cells.

    PubMed

    Gao, Darrin; Rahbar, Ramtin; Fish, Eleanor N

    2016-06-01

    In earlier studies, we showed that CCL5 enhances proliferation and survival of MCF-7 breast cancer cells in an mTOR-dependent manner and we provided evidence that, for T cells, CCL5 activation of CCR5 results in increased glycolysis and enhanced ATP production. Increases in metabolic activity of cancer cells, specifically increased glycolytic activity and increased expression of glucose transporters, are associated with tumour progression. In this report, we provide evidence that CCL5 enhances the proliferation of human breast cancer cell lines (MDA-MB-231, MCF-7) and mouse mammary tumour cells (MMTV-PyMT), mediated by CCR5 activation. Concomitant with enhanced proliferation we show that CCL5 increases cell surface expression of the glucose transporter GLUT1, and increases glucose uptake and ATP production by these cells. Blocking CCL5-inducible glucose uptake abrogates the enhanced proliferation induced by CCL5. We provide evidence that increased glucose uptake is associated with enhanced glycolysis, as measured by extracellular acidification. Moreover, CCL5 enhances the invasive capacity of these breast cancer cells. Using metabolomics, we demonstrate that the metabolic signature of CCL5-treated primary mouse mammary tumour cells reflects increased anabolic metabolism. The implications are that CCL5-CCR5 interactions in the tumour microenvironment regulate metabolic events, specifically glycolysis, to promote tumour proliferation and invasion. PMID:27335323

  15. [Enhanced control of proliferation in telomerized cells].

    PubMed

    Egorov, E E; Moldaver, M V; Vishniakova, Kh S; Terekhov, S M; Dashinimaev, E B; Cheglakov, I B; Toropygin, I Iu; Iarygin, K N; Chumakov, P M; Korochkin, L I; Antonova, G A; Rybalkina, E Iu; Saburina, I N; Burnaevskiĭ, N S; Zelenin, A V

    2007-01-01

    Clones of telomerized fibroblasts of adult human skin have earlier been obtained. It was shown that despite their fast growth in mass cultures, these cells poorly form colonies. Conditioned medium, antioxidants, and reduced partial oxygen pressure enhanced their colony formation, but not to the level characteristic of the initial cells. The conditioned medium of telomerized cells enhanced colony formation to a much greater extent than that of the initial cells. A study of proteome of the telomerized fibroblasts has revealed changes in the activities of tens of genes. A general trend consists in weakening and increased lability of the cytoskeleton and in activation of the mechanisms controlling protein degradation. However, these changes are not very pronounced. During the formation of immortal telomerized cells, selection takes place, which appears to determine changes in the expression of some genes. It was proposed that a decrease in the capacity of telomerized cells for colony formation is due to increased requirements of these cells to cell-cell contacts. The rate of cell growth reached that characteristic of mass cultures only in the largest colonies. In this respect, the telomerized fibroblasts resembled stem cells: they are capable of self-maintenance, but "escape" to differentiation in the absence of the corresponding microenvironment (niche), which is represented by other fibroblasts. Non-dividing cells in the test of colony formation should be regarded as differentiated cells, since they have no features of degradation, preserve their viability, actively move, grow, phagocytized debris, etc. It was also shown that telomerization did not prevent differentiation of myoblasts and human neural stem cells. Thus, the results obtained suggest the existence of normal mechanisms underlying the regulation of proliferation in the telomerized cells, which opens possibilities of their use in cell therapy, especially in the case of autotransplantation to senior people

  16. TLR2 Activation Inhibits Embryonic Neural Progenitor Cell Proliferation

    PubMed Central

    Okun, Eitan; Griffioen, Kathleen J.; Gen-Son, Tae; Lee, Jong-Hwan; Roberts, Nicholas J.; Mughal, Mohamed R.; Hutchison, Emmette; Cheng, Aiwu; Arumugam, Thiruma V.; Lathia, Justin D.; van Praag, Henriette; Mattson, Mark P.

    2010-01-01

    Toll-like receptors (TLRs) play essential roles in innate immunity, and increasing evidence indicates that these receptors are expressed in neurons, astrocytes and microglia in the brain, where they mediate responses to infection, stress and injury. To address the possibility that TLR2 heterodimer activation could affect progenitor cells in the developing brain, we analyzed the expression of TLR2 throughout the mouse cortical development, and assessed the role of TLR2 heterodimer activation in neural progenitor cell (NPC) proliferation. TLR2 mRNA and protein was expressed in the cortex in embryonic and early postnatal stages of development, and in cultured cortical NPC. While NPC from TLR2-deficient and wild type embryos had the same proliferative capacity, TLR2 activation by the synthetic bacterial lipopeptides Pam3CSK4 and FSL1, or low molecular weight hyaluronan, an endogenous ligand for TLR2, inhibited neurosphere formation in vitro. Intracerebral in utero administration of TLR2 ligands resulted in ventricular dysgenesis characterized by increased ventricle size, reduced proliferative area around the ventricles, increased cell density, an increase in PH3+ cells and a decrease in BrdU+ cells in the sub-ventricular zone. Our findings indicate that loss of TLR2 does not result in defects in cerebral development. However, TLR2 is expressed and functional in the developing telencephalon from early embryonic stages and infectious agent-related activation of TLR2 inhibits NPC proliferation. TLR2–mediated inhibition of NPC proliferation may therefore be a mechanism by which infection, ischemia and inflammation adversely affect brain development. PMID:20456021

  17. Collagen coated tantalum substrate for cell proliferation.

    PubMed

    Li, Yinli; Zhang, Shuai; Guo, Lijun; Dong, Mingdong; Liu, Bo; Mamdouh, Wael

    2012-06-15

    The extracellular matrix (ECM) plays a key role in cell culture in various physiological and pathological processes in the field of tissue engineering. Recently, the type I collagen ECM has been widely utilized in vitro model systems for the attachment of many different cell lines since it has multi-functions in human tissues. For example it accounts for 6% of the weight of strong, tendinous muscles. In this paper, we reported a new material by coating tantalum (Ta), one highly biocompatible metal, with type I collagen fibrils. The morphology of the new material was studied by high resolution atomic force microscope. It was shown that the adhesion force between type I collagen fibrils network and Ta was strong enough to overcome surface defects. A possible way to explain the phenomenon is that the longitudinal periodicity of collagen fibrils matches the grain size of the Ta domains, which results in increase of the physical adsorption contact area, thereby inducing the dramatic adhesion enhancement between collagen fibrils and Ta. The obtained material was then employed as a template for cell proliferation. Although the surface of this template is more hydrophobic by comparison with the bare Ta surface, the cells on this material were successfully incubated, indicating that the collagen coated Ta might be used as the buffer layer for proliferating cells in hydrophobic biomaterials. PMID:22494669

  18. ETOH inhibits embryonic neural stem/precursor cell proliferation via PLD signaling

    SciTech Connect

    Fujita, Yuko; Hiroyama, Masami; Sanbe, Atsushi Yamauchi, Junji; Murase, Shoko; Tanoue, Akito

    2008-05-23

    While a mother's excessive alcohol consumption during pregnancy is known to have adverse effects on fetal neural development, little is known about the underlying mechanism of these effects. In order to investigate these mechanisms, we investigated the toxic effect of ethanol (ETOH) on neural stem/precursor cell (NSC) proliferation. In cultures of NSCs, phospholipase D (PLD) is activated following stimulation with epidermal growth factor (EGF) and fibroblast growth factor 2 (FGF2). Exposure of NSCs to ETOH suppresses cell proliferation, while it has no effect on cell death. Phosphatidic acid (PA), which is a signaling messenger produced by PLD, reverses ETOH inhibition of NSC proliferation. Blocking the PLD signal by 1-butanol suppresses the proliferation. ETOH-induced suppression of NSC proliferation and the protective effect of PA for ETOH-induced suppression are mediated through extracellular signal-regulated kinase signaling. These results indicate that exposure to ETOH impairs NSC proliferation by altering the PLD signaling pathway.

  19. Activation of PKC{beta}{sub II} and PKC{theta} is essential for LDL-induced cell proliferation of human aortic smooth muscle cells via Gi-mediated Erk1/2 activation and Egr-1 upregulation

    SciTech Connect

    Heo, Kyung-Sun; Kim, Dong-Uk; Kim, Lila; Nam, Miyoung; Baek, Seung-Tae; Park, Song-Kyu; Park, Youngwoo; Myung, Chang-Seon; Hwang, Sung-Ook Hoe, Kwang-Lae

    2008-03-28

    Native LDL may be a mitogenic stimulus of VSMC proliferation in lesions where endothelial disruption occurs. Recent studies have demonstrated that the mitogenic effects of LDL are accompanied by Erk1/2 activation via an unknown G-protein-coupled receptor (GPCR). In this article, we report that LDL translocated PKC{beta}{sub II} and PKC{theta} from cytosol to plasma membrane, and inhibition of PKC{beta}{sub II} and PKC{theta} decreased LDL effects via the deactivation of Erk1/2. Moreover, pertussis toxin, but not cholera toxin or heparin, inhibited LDL-induced translocation of PKC{beta}{sub II} and PKC{theta}, suggesting that Gi protein plays a role in LDL effects. Of LPA, S1P, and LDL, whose signaling is conveyed via Gi/o proteins, only LDL induced translocation of PKC{beta}{sub II} and PKC{theta}. Inhibition of PKC{beta}{sub II} or PKC{theta}, as well as of Erk1/2 and GPCR, decreases LDL-induced upregulation of Egr-1, which is critical for cell proliferation. This is the first report, to our knowledge, that the participation of PKC{theta} in VSMC proliferation is unique.

  20. Selective cell proliferation can be controlled with CPC particle coatings

    PubMed Central

    Szivek, J.A.; Margolis, D.S.; Schnepp, A.B.; Grana, W.A.; Williams, S.K.

    2008-01-01

    To develop implantable, engineered, cartilage constructs supported by a scaffold, techniques to encourage rapid tissue growth into, and on the scaffold are essential. Preliminary studies indicated that human endothelial cells proliferated at different rates on different calcium phosphate ceramic (CPC) particles. Judicious selection of particles may encourage specific cell proliferation, leading to an ordered growth of tissues for angiogenesis, osteogenesis, and chondrogenesis. The goal of this study was to identify CPC surfaces that encourage bone and vascular cell growth, and other surfaces that support chondrocyte growth while inhibiting proliferation of vascular cells. Differences in bone and vascular cell proliferation were observed when using epoxy without embedded CPCs to encourage bone cells, and when three CPCs were tested, which encouraged vascular cell proliferation. One of these (CPC 7) also substantially depressed cartilage cell proliferation. Only one small-diameter crystalline CPC (CPC 2) supported rapid chondrocyte proliferation, and maintained the cartilage cell phenotype. PMID:17252549

  1. Targeting Homeostatic T Cell Proliferation to Control Beta-Cell Autoimmunity.

    PubMed

    Vignali, Debora; Monti, Paolo

    2016-05-01

    Immunomodulation of the autoreactive T cell response is considered a major strategy to control beta-cell autoimmunity, both in the natural history of type 1 diabetes and in islet transplantation, which can be affected by autoimmunity recurrence. So far, these strategies have had modest results, prompting efforts to define novel cellular and molecular targets to control autoreactive T cell expansion and activation. Novel findings highlighted the important role of the homeostatic cytokine interleukin-7 in inducing proliferation and differentiation of autoreactive T cell clones that causes beta-cell autoimmunity. In this review, we discuss recent evidences and novel findings on the role of IL-7 mediated homeostatic T cell proliferation in the process of beta-cell destruction and evidences of how targeting IL-7 and its receptor could be an innovative and effective strategy to control beta-cell autoimmunity. PMID:26983628

  2. Mast cells and histamine enhance the proliferation of non-small cell lung cancer cells.

    PubMed

    Stoyanov, Evgeniy; Uddin, Mohib; Mankuta, David; Dubinett, Steven M; Levi-Schaffer, Francesca

    2012-01-01

    Non-small cell lung cancer (NSCLC) is the most common form of lung cancer with an extremely low survival rate. It is characterized by a chronic inflammatory process with intense mast cell infiltrate that is associated with reduced survival. The aim of this study was to test the hypothesis that mast cells have an enhancing effect on NSCLC proliferation. To assess the tumor-promoting potential of mast cells, we used the human alveolar basal adenocarcinoma (A549) and the mouse Lewis lung carcinoma (LLC) cell lines, umbilical cord blood-derived mast cells (CBMC) and the mast cell-deficient mouse Sash model. The proliferation rate of A549/LLC cells was markedly increased by mast cells and histamine. Histamine proliferating activity was mediated via H(1), H(2) and H(4) receptors and caused ERK phosphorylation. LLC induced in Sash mice or in wild-type mice treated with the mast cell stabilizer nedocromil sodium displayed an accelerated growth (number of metastic colonies in the lungs, total lung area and lung/total mice weight ratio). In summary, we have shown a significant effect of mast cells and histamine in enhancing NSCLC/LLCX growth in vitro, while in a mouse LLC model in vivo we have found that mast cells are important negative regulators of cancer development. Therefore our results would indicate a pro-tumorogenic effect of the mast cells in vitro on established lung tumor cell lines, and anti-tumorogenic effect in mice at lung cancer induction. In conclusion, mast cell/anti-histamine targeted therapies should carefully consider this dual effect. PMID:21733595

  3. PXR stimulates growth factor-mediated hepatocyte proliferation by cross-talk with the FOXO transcription factor.

    PubMed

    Shizu, Ryota; Abe, Taiki; Benoki, Satoshi; Takahashi, Miki; Kodama, Susumu; Miayata, Masaaki; Matsuzawa, Atsushi; Yoshinari, Kouichi

    2016-02-01

    Growth factor-mediated hepatocyte proliferation is crucial in liver regeneration and the recovery of liver function after injury. The nuclear receptor, pregnane X receptor (PXR), is a key transcription factor for the xenobiotic-induced expression of genes associated with various liver functions. Recently, we reported that PXR activation stimulates xenobiotic-induced hepatocyte proliferation. In the present study, we investigated whether PXR activation also stimulates growth factor-mediated hepatocyte proliferation. In G0 phase-synchronized, immortalized mouse hepatocytes, serum or epidermal growth factor treatment increased cell growth and this growth was augmented by the expression of mouse PXR and co-treatment with pregnenolone 16α-carbonitrile (PCN), a PXR ligand. In a liver regeneration model using carbon tetrachloride, PCN treatment enhanced the injury-induced increase in the number of Ki-67-positive nuclei as well as Ccna2 and Ccnb1 mRNA levels in wild-type (WT) but not Pxr-null mice. Chronological analysis of this model demonstrated that PCN treatment shifted the maximum cell proliferation to an earlier time point and increased the number of M-phase cells at those time points. In WT but not Pxr-null mice, PCN treatment reduced hepatic mRNA levels of genes involved in the suppression of G0/G1- and G1/S-phase transition, e.g. Rbl2, Cdkn1a and Cdkn1b. Analysis of the Rbl2 promoter revealed that PXR activation inhibited its Forkhead box O3 (FOXO3)-mediated transcription. Finally, the PXR-mediated enhancement of hepatocyte proliferation was inhibited by the expression of dominant active FOXO3 in vitro. The results of the present study suggest that PXR activation stimulates growth factor-mediated hepatocyte proliferation in mice, at least in part, through inhibiting FOXO3 from accelerating cell-cycle progression. PMID:26574435

  4. Adventitial transplantation of blood outgrowth endothelial cells in porcine haemodialysis grafts alleviates hypoxia and decreases neointimal proliferation through a matrix metalloproteinase-9-mediated pathway—a pilot study

    PubMed Central

    Hughes, Deborah; Fu, Alex A.; Puggioni, Alessandra; Glockner, James F.; Anwer, Bilal; McGuire, Antonio M.; Mukhopadhyay, Debabrata; Misra, Sanjay

    2009-01-01

    Purpose. We hypothesized that adventitial transplantation of blood outgrowth endothelial cells (BOEC) to the vein-to-graft anastomosis of polytetrafluoroethylene grafts will reduce neointimal hyperplasia by reducing hypoxia inducible factor-1α (HIF-1α), by increasing angiogenesis in a porcine model of chronic renal insufficiency with haemodialysis polytetrafluoroethylene grafts. Because matrix metalloproteinases (MMPs) have been shown to be involved with angiogenesis, the expression of MMPs and their inhibitors was determined. Methods. Chronic renal insufficiency was created by subtotal renal infarction and 28 days later, arteriovenous PTFE grafts were placed bilaterally from the carotid artery to the jugular vein. Autologous blood outgrowth endothelial cells labeled with Lac Z were transplanted to the adventitia of the vein-to-graft anastomosis using polyglycolic acid scaffolding and scaffolding only to other side (control). Animals were killed 14 days later and vessels were explanted from the vein-to-graft anastomosis of both sides and underwent immunohistochemical analysis, western blotting and zymography for HIF-1α, MMP-2, MMP-9, TIMP-1 and TIMP-2. BOEC were also made hypoxic and normoxic for 12, 24 and 48 h to determine protein expression for MMPs and TIMPs. Results. Under hypoxia, BOEC significantly increased the expression of pro MMP-2 by 12 h and TIMP-2 by 24 h when compared to normoxic cells (P < 0.05). Transplantation of BOEC resulted in a significant decrease in both HIF-1α and intima-to-media ratio with a significant increase in both pro and active MMP-9 when compared to control vessels (P < 0.05). MMP-9 activity was localized to the neointima of the transplanted vessels by immunohistochemistry. There was increased CD31 density with engraftment of BOEC cells into the neointima of both the transplanted vessels compared to controls (P = NS). Conclusion. Transplantation of BOEC resulted in a significant decrease in intimal hyperplasia and HIF-1α with

  5. Leptin promotes cell proliferation and survival of trophoblastic cells.

    PubMed

    Magariños, María Paula; Sánchez-Margalet, Víctor; Kotler, Mónica; Calvo, Juan Carlos; Varone, Cecilia L

    2007-02-01

    Leptin, the 16-kDa protein product of the obese gene, was originally considered as an adipocyte-derived signaling molecule for the central control of metabolism. However, leptin has been suggested to be involved in other functions during pregnancy, particularly in placenta. In the present work, we studied a possible effect of leptin on trophoblastic cell proliferation, survival, and apoptosis. Recombinant human leptin added to JEG-3 and BeWo choriocarcinoma cell lines showed a stimulatory effect on cell proliferation up to 3 and 2.4 times, respectively, measured by (3)H-thymidine incorporation and cell counting. These effects were time and dose dependent. Maximal effect was achieved at 250 ng leptin/ml for JEG-3 cells and 50 ng leptin/ml for BeWo cells. Moreover, by inhibiting endogenous leptin expression with 2 microM of an antisense oligonucleotide (AS), cell proliferation was diminished. We analyzed cell population distribution during the different stages of cell cycle by fluorescence-activated cell sorting, and we found that leptin treatment displaced the cells towards a G2/M phase. We also found that leptin upregulated cyclin D1 expression, one of the key cell cycle-signaling proteins. Since proliferation and death processes are intimately related, the effect of leptin on cell apoptosis was investigated. Treatment with 2 microM leptin AS increased the number of apoptotic cells 60 times, as assessed by annexin V-fluorescein isothiocyanate/propidium iodide staining, and the caspase-3 activity was increased more than 2 fold. This effect was prevented by the addition of 100 ng leptin/ml. In conclusion, we provide evidence that suggests that leptin is a trophic and mitogenic factor for trophoblastic cells by virtue of its inhibiting apoptosis and promoting proliferation. PMID:17021346

  6. Rnd3 regulates lung cancer cell proliferation through notch signaling.

    PubMed

    Tang, Yongjun; Hu, Chengping; Yang, Huaping; Cao, Liming; Li, Yuanyuan; Deng, Pengbo; Huang, Li

    2014-01-01

    Rnd3/RhoE is a small Rho GTPase involved in the regulation of different cell behaviors. Dysregulation of Rnd3 has been linked to tumorigenesis and metastasis. Lung cancers are the leading cause of cancer-related death in the West and around the world. The expression of Rnd3 and its ectopic role in non-small cell lung cancer (NSCLC) remain to be explored. Here, we reported that Rnd3 was down-regulated in three NSCLC cell lines: H358, H520 and A549. The down-regulation of Rnd3 led to hyper-activation of Rho Kinase and Notch signaling. The reintroduction of Rnd3 or selective inhibition of Notch signaling, but not Rho Kinase signaling, blocked the proliferation of H358 and H520 cells. Mechanistically, Notch intracellular domain (NICD) protein abundance in H358 cells was regulated by Rnd3-mediated NICD proteasome degradation. Rnd3 regulated H358 and H520 cell proliferation through a Notch1/NICD/Hes1 signaling axis independent of Rho Kinase. PMID:25372032

  7. Rnd3 Regulates Lung Cancer Cell Proliferation through Notch Signaling

    PubMed Central

    Tang, Yongjun; Hu, Chengping; Yang, Huaping; Cao, Liming; Li, Yuanyuan; Deng, Pengbo; Huang, Li

    2014-01-01

    Rnd3/RhoE is a small Rho GTPase involved in the regulation of different cell behaviors. Dysregulation of Rnd3 has been linked to tumorigenesis and metastasis. Lung cancers are the leading cause of cancer-related death in the West and around the world. The expression of Rnd3 and its ectopic role in non-small cell lung cancer (NSCLC) remain to be explored. Here, we reported that Rnd3 was down-regulated in three NSCLC cell lines: H358, H520 and A549. The down-regulation of Rnd3 led to hyper-activation of Rho Kinase and Notch signaling. The reintroduction of Rnd3 or selective inhibition of Notch signaling, but not Rho Kinase signaling, blocked the proliferation of H358 and H520 cells. Mechanistically, Notch intracellular domain (NICD) protein abundance in H358 cells was regulated by Rnd3-mediated NICD proteasome degradation. Rnd3 regulated H358 and H520 cell proliferation through a Notch1/NICD/Hes1 signaling axis independent of Rho Kinase. PMID:25372032

  8. Cell proliferation is a key determinant of the outcome of FOXO3a activation

    SciTech Connect

    Poulsen, Raewyn C. Carr, Andrew J.; Hulley, Philippa A.

    2015-06-19

    The FOXO family of forkhead transcription factors have a pivotal role in determining cell fate in response to oxidative stress. FOXO activity can either promote cell survival or induce cell death. Increased FOXO-mediated cell death has been implicated in the pathogenesis of degenerative diseases affecting musculoskeletal tissues. The aim of this study was to determine the conditions under which one member of the FOXO family, FOXO3a, promotes cell survival as opposed to cell death. Treatment of primary human tenocytes with 1 pM hydrogen peroxide for 18 h resulted in increased protein levels of FOXO3a. In peroxide-treated cells cultured in low serum media, FOXO3a inhibited cell proliferation and protected against apoptosis. However in peroxide treated cells cultured in high serum media, cell proliferation was unchanged but level of apoptosis significantly increased. Similarly, in tenocytes transduced to over-express FOXO3a, cell proliferation was inhibited and level of apoptosis unchanged in cells cultured in low serum. However there was a robust increase in cell death in FOXO3a-expressing cells cultured in high serum. Inhibition of cell proliferation in either peroxide-treated or FOXO3a-expressing cells cultured in high serum protected against apoptosis induction. Conversely, addition of a Chk2 inhibitor to peroxide-treated or FOXO3a-expressing cells overrode the inhibitory effect of FOXO3a on cell proliferation and led to increased apoptosis in cells cultured in low serum. This study demonstrates that proliferating cells may be particularly susceptible to the apoptosis-inducing actions of FOXO3a. Inhibition of cell proliferation by FOXO3a may be a critical event in allowing the pro-survival rather than the pro-apoptotic activity of FOXO3a to prevail. - Highlights: • FOXO3a activity can result in either promotion of cell survival or apoptosis. • The outcome of FOXO3a activation differs in proliferating compared to non-proliferating cells. • Proliferating

  9. The rapamycin sensitivity of human T-cell leukaemia virus type I-induced T-cell proliferation is mediated independently of the polypyrimidine motifs in the 5' long terminal repeat.

    PubMed

    Rose, N; Lever, A

    2001-02-01

    The immunosuppressant rapamycin can regulate the translation of a subset of messenger RNAs, a phenotype which has been linked to the presence of a polypyrimidine motif [C(N)(4-14)] downstream of the mRNA cap structure. T-cell clones naturally infected with transcriptionally active human T-cell leukaemia virus, type I (HTLV-I) undergo autologous proliferation; this phenotype is inhibited by rapamycin but not FK506, which reverses the rapamycin effect. Within the R region of the HTLV-I 5' long terminal repeat (LTR) there are seven polypyrimidine motifs. We sought to determine if these were involved in the sensitivity of proliferation to the presence of rapamycin. Here we illustrate the generation of an in vitro model of this rapamycin-sensitivity and the analysis of LTR mutants which were created to determine the importance of the polypyrimidine motifs. Reporter gene assays suggest the effect is independent of the polypyrimidine motifs in the virus leader sequence. PMID:11161283

  10. Symbiotic lactobacilli stimulate gut epithelial proliferation via Nox-mediated generation of reactive oxygen species

    PubMed Central

    Jones, Rheinallt M; Luo, Liping; Ardita, Courtney S; Richardson, Arena N; Kwon, Young Man; Mercante, Jeffrey W; Alam, Ashfaqul; Gates, Cymone L; Wu, Huixia; Swanson, Phillip A; Lambeth, J David; Denning, Patricia W; Neish, Andrew S

    2013-01-01

    The resident prokaryotic microbiota of the metazoan gut elicits profound effects on the growth and development of the intestine. However, the molecular mechanisms of symbiotic prokaryotic–eukaryotic cross-talk in the gut are largely unknown. It is increasingly recognized that physiologically generated reactive oxygen species (ROS) function as signalling secondary messengers that influence cellular proliferation and differentiation in a variety of biological systems. Here, we report that commensal bacteria, particularly members of the genus Lactobacillus, can stimulate NADPH oxidase 1 (Nox1)-dependent ROS generation and consequent cellular proliferation in intestinal stem cells upon initial ingestion into the murine or Drosophila intestine. Our data identify and highlight a highly conserved mechanism that symbiotic microorganisms utilize in eukaryotic growth and development. Additionally, the work suggests that specific redox-mediated functions may be assigned to specific bacterial taxa and may contribute to the identification of microbes with probiotic potential. PMID:24141879

  11. Liver epithelial cells inhibit proliferation and invasiveness of hepatoma cells.

    PubMed

    Jeng, Kuo-Shyang; Jeng, Chi-Juei; Jeng, Wen-Juei; Sheen, I-Shyan; Li, Shih-Yun; Hung, Zih-Hang; Hsiau, Hsin-I; Yu, Ming-Che; Chang, Chiung-Fang

    2016-03-01

    Hepatocellular carcinoma (HCC) is a worldwide malignancy with poor prognosis. Liver progenitors or stem cells could be a potential therapy for HCC treatment since they migrate toward tumors. Rat liver epithelial (RLE) cells have both progenitor and stem cell-like properties. Therefore, our study elucidated the therapeutic effect of RLE cells in rat hepatoma cells. RLE cells were isolated from 10-day old rats and characterized for stem cell marker expression. RLE cells and rat hepatoma cells (H4-IIE-C3 cells) were co-cultured and divided into four groups with different ratios of RLE and hepatoma cells. Group A had only rat hepatoma cells as a control group. The ratios of rat hepatoma and RLE cells in group B, C and D were 5:1, 1:1 and 1:5, respectively. Effective inhibition of cell proliferation and migration was found in group D when compared to group A. There was a significant decrease in Bcl2 expression and increase in late apoptosis of rat hepatoma cells when adding more RLE cells. RLE cells reduced cell proliferation and migration of rat hepatoma cells. These results suggested that RLE cells could be used as a potential cell therapy. PMID:26647726

  12. Site-specific Proteasome Phosphorylation Controls Cell Proliferation and Tumorigenesis

    PubMed Central

    Guo, Xing; Wang, Xiaorong; Wang, Zhiping; Banerjee, Sourav; Yang, Jing; Huang, Lan; Dixon, Jack E.

    2015-01-01

    Despite the fundamental importance of proteasomal degradation in cells, little is known about whether and how the 26S proteasome itself is regulated in coordination with various physiological processes. Here we show that the proteasome is dynamically phosphorylated during cell cycle at Thr25 of the 19S subunit Rpt3. CRISPR/Cas9-mediated genome editing, RNA interference and biochemical studies demonstrate that blocking Rpt3-Thr25 phosphorylation markedly impairs proteasome activity and impedes cell proliferation. Through a kinome-wide screen, we have identified dual-specificity tyrosine-regulated kinase 2 (DYRK2) as the primary kinase that phosphorylates Rpt3-Thr25, leading to enhanced substrate translocation and degradation. Importantly, loss of the single phosphorylation of Rpt3-Thr25 or knockout of DYRK2 significantly inhibits tumor formation by proteasome-addicted human breast cancer cells in mice. These findings define an important mechanism for proteasome regulation and demonstrate the biological significance of proteasome phosphorylation in regulating cell proliferation and tumorigenesis. PMID:26655835

  13. Biodiesel from soybean promotes cell proliferation in vitro.

    PubMed

    Gioda, Adriana; Rodríguez-Cotto, Rosa I; Amaral, Beatriz Silva; Encarnación-Medina, Jarline; Ortiz-Martínez, Mario G; Jiménez-Vélez, Braulio D

    2016-08-01

    Toxicological responses of exhaust emissions of biodiesel are different due to variation in methods of generation and the tested biological models. A chemical profile was generated using ICP-MS and GC-MS for the biodiesel samples obtained in Brazil. A cytotoxicity assay and cytokine secretion experiments were evaluated in human bronchial epithelial cells (BEAS-2B). Cells were exposed to polar (acetone) and nonpolar (hexane) extracts from particles obtained from fuel exhaust: fossil diesel (B5), pure soybean biodiesel (B100), soybean biodiesel with additive (B100A) and ethanol additive (EtOH). Biodiesel and its additives exhibited higher organic and inorganic constituents on particles when compared to B5. The biodiesel extracts did not exert any toxic effect at concentrations 10, 25, 50, 75, and 100μgmL(-1). In fact quite the opposite, a cell proliferation effect induced by the B100 and B100A extracts is reported. A small increase in concentrations of inflammatory mediators (Interleukin-6, IL-6; and Interleukin-8, IL-8) in the medium of biodiesel-treated cells was observed, however, no statistical difference was found. An interesting finding indicates that the presence of metals in the nonpolar (hexane) fraction of biodiesel fuel (B100) represses cytokine release in lung cells. This was revealed by the use of the metal chelator. Results suggest that metals associated with biodiesel's organic constituents might play a significant role in molecular mechanisms associated to cellular proliferation and immune responses. PMID:27179667

  14. Survivin expression is associated with lens epithelial cell proliferation and fiber cell differentiation

    PubMed Central

    Mansergh, Fiona C.; Boulton, Michael E.; Gunhaga, Lena

    2012-01-01

    Purpose Survivin (Birc5) is the smallest member of the inhibitor of apoptosis (IAP) protein family, which regulates the cell cycle/apoptosis balance. The purpose of this study was to examine Survivin expression in the embryonic chick lens, in chick lens epithelial cell cultures, and in the postnatal mouse lens. Methods Survivin expression was examined using a combination of quantitative real-time polymerase chain reaction, western blotting, and immunocytochemistry. To correlate Survivin expression with the timing of proliferation, we determined the profile of cell proliferation in the developing lens using the cell cycle marker proliferating cell nuclear antigen (PCNA) in quantitative western blotting and immunocytochemistry studies. We also examined the expression of PCNA and the extent of denucleation using terminal deoxynucleotidyl transferase (TdT)-mediated biotin-dUTP nick-end labeling (TUNEL) of lentoids (lens fiber-like cells) during chick lens epithelial cell differentiation in vitro. Results At embryonic day (ED) 4, Survivin immunostaining was present in two pools in lens epithelial cells and fiber cells: cytoplasmic and nuclear. The nuclear staining became more pronounced as the lens epithelial cells differentiated into lens fiber cells. At ED12, Survivin staining was observed in lens fiber cell nuclei containing marginalized chromatin, indicative of early denucleation events. Using western blotting, Survivin expression peaked at ED6, diminishing thereafter. This profile of expression correlated with the events in chick lens epithelial cell cultures: i) increased Survivin expression was associated with an increase in PCNA staining up to day 6 of culture and ii) downregulation of Survivin expression at day 8 of culture was coincident with a dramatic decrease in PCNA staining and an increase in TdT-mediated biotin-dUTP nick-end labeling in lentoids. In early postnatal mouse lenses, Survivin and PCNA were highly expressed and decreased thereafter during

  15. Post microtextures accelerate cell proliferation and osteogenesis.

    PubMed

    Kim, Eun Jung; Boehm, Cynthia A; Mata, Alvaro; Fleischman, Aaron J; Muschler, George F; Roy, Shuvo

    2010-01-01

    The influence of surface microtexture on osteogenesis was investigated in vitro by examining the proliferation and differentiation characteristics of a class of adult stem cells and their progeny, collectively known as connective tissue progenitor cells (CTPs). Human bone marrow-derived CTPs were cultured for up to 60 days on smooth polydimethylsiloxane (PDMS) surfaces and on PDMS with post microtextures that were 10 microm in diameter and 6 microm in height, with 10 microm separation. DNA quantification revealed that the numbers of CTPs initially attached to both substrates were similar. However, cells on microtextured PDMS transitioned from lag phase after 4 days of culture, in contrast to 6 days for cells on smooth surfaces. By day 9 cells on the smooth surfaces exhibited arbitrary flattened shapes and migrated without any preferred orientation. In contrast, cells on the microtextured PDMS grew along the array of posts in an orthogonal manner. By days 30 and 60 cells grew and covered all surfaces with extracellular matrix. Western blot analysis revealed that the expression of integrin alpha5 was greater on the microtextured PDMS compared with smooth surfaces. Real time reverse transcription-polymerase chain reaction revealed that gene expression of alkaline phosphatase had decreased by days 30 and 60, compared with that on day 9, for both substrates. Gene expression of collagen I and osteocalcin was consistently greater on post microtextures relative to smooth surfaces at all time points. PMID:19539062

  16. Post microtextures accelerate cell proliferation and osteogenesis

    PubMed Central

    Kim, Eun Jung; Boehm, Cynthia A.; Mata, Alvaro; Fleischman, Aaron J.; Muschler, George F.; Roy, Shuvo

    2013-01-01

    The influence of surface microtexture on osteogenesis was investigated in vitro by examining the proliferation and differentiation characteristics of a class of adult stem cells and their progeny, collectively known as connective tissue progenitor cells (CTPs). Human bone marrow-derived CTPs were cultured for up to 60 days on smooth polydimethylsiloxane (PDMS) surfaces and on PDMS with post microtextures that were 10 µm in diameter and 6 µm in height, with 10 µm separation. DNA quantification revealed that the numbers of CTPs initially attached to both substrates were similar. However, cells on microtextured PDMS transitioned from lag phase after 4 days of culture, in contrast to 6 days for cells on smooth surfaces. By day 9 cells on the smooth surfaces exhibited arbitrary flattened shapes and migrated without any preferred orientation. In contrast, cells on the microtextured PDMS grew along the array of posts in an orthogonal manner. By days 30 and 60 cells grew and covered all surfaces with extracellular matrix. Western blot analysis revealed that the expression of integrin α5 was greater on the microtextured PDMS compared with smooth surfaces. Real time reverse transcription-polymerase chain reaction revealed that gene expression of alkaline phosphatase had decreased by days 30 and 60, compared with that on day 9, for both substrates. Gene expression of collagen I and osteocalcin was consistently greater on post microtextures relative to smooth surfaces at all time points. PMID:19539062

  17. Quantitative analysis of in vivo cell proliferation.

    PubMed

    Cameron, Heather A

    2006-11-01

    Injection and immunohistochemical detection of 5-bromo-2'-deoxyuridine (BrdU) has become the standard method for studying the birth and survival of neurons, glia, and other cell types in the nervous system. BrdU, a thymidine analog, becomes stably incorporated into DNA during the S-phase of mitosis. Because DNA containing BrdU can be specifically recognized by antibodies, this method allows dividing cells to be marked at any given time and then identified at time points from a few minutes to several years later. BrdU immunohistochemistry is suitable for cell counting to examine the regulation of cell proliferation and cell fate. It can be combined with labeling by other antibodies, allowing confocal analysis of cell phenotype or expression of other proteins. The potential for nonspecific labeling and toxicity are discussed. Although BrdU immunohistochemistry has almost completely replaced tritiated thymidine autoradiography for labeling dividing cells, this method and situations in which it is still useful are also described. PMID:18428635

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

    SciTech Connect

    Zhang, Yu; Cheng, Jung-Chien; Huang, He-Feng; Leung, Peter C.K.

    2013-11-01

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

  19. Numb-deficient satellite cells have regeneration and proliferation defects.

    PubMed

    George, Rajani M; Biressi, Stefano; Beres, Brian J; Rogers, Erik; Mulia, Amanda K; Allen, Ronald E; Rawls, Alan; Rando, Thomas A; Wilson-Rawls, Jeanne

    2013-11-12

    The adaptor protein Numb has been implicated in the switch between cell proliferation and differentiation made by satellite cells during muscle repair. Using two genetic approaches to ablate Numb, we determined that, in its absence, muscle regeneration in response to injury was impaired. Single myofiber cultures demonstrated a lack of satellite cell proliferation in the absence of Numb, and the proliferation defect was confirmed in satellite cell cultures. Quantitative RT-PCR from Numb-deficient satellite cells demonstrated highly up-regulated expression of p21 and Myostatin, both inhibitors of myoblast proliferation. Transfection with Myostatin-specific siRNA rescued the proliferation defect of Numb-deficient satellite cells. Furthermore, overexpression of Numb in satellite cells inhibited Myostatin expression. These data indicate a unique function for Numb during the initial activation and proliferation of satellite cells in response to muscle injury. PMID:24170859

  20. Impact of Mesenchymal Stem Cell secreted PAI-1 on colon cancer cell migration and proliferation

    SciTech Connect

    Hogan, Niamh M.; Joyce, Myles R.; Murphy, J. Mary; Barry, Frank P.; O’Brien, Timothy; Kerin, Michael J.; Dwyer, Roisin M.

    2013-06-14

    Highlights: •MSCs were directly co-cultured with colorectal cancer (CRC) cells on 3D scaffolds. •MSCs influence CRC protein/gene expression, proliferation and migration. •We report a significant functional role of MSC-secreted PAI-1 in colon cancer. -- Abstract: Mesenchymal Stem Cells are known to engraft and integrate into the architecture of colorectal tumours, with little known regarding their fate following engraftment. This study aimed to investigate mediators of Mesenchymal Stem Cell (MSC) and colon cancer cell (CCC) interactions. Mesenchymal Stem Cells and colon cancer cells (HT29 and HCT-116) were cultured individually or in co-culture on 3-dimensional scaffolds. Conditioned media containing all secreted factors was harvested at day 1, 3 and 7. Chemokine secretion and expression were analyzed by Chemi-array, ELISA (Macrophage migration inhibitory factor (MIF), plasminogen activator inhibitor type 1 (PAI-1)) and RQ-PCR. Colon cancer cell migration and proliferation in response to recombinant PAI-1, MSCs and MSCs + antibody to PAI-1 was analyzed using Transwell inserts and an MTS proliferation assay respectively. Chemi-array revealed secretion of a wide range of factors by each cell population, including PAI-1and MIF. ELISA analysis revealed Mesenchymal Stem Cells to secrete the highest levels of PAI-1 (MSC mean 10.6 ng/mL, CCC mean 1.01 ng/mL), while colon cancer cells were the principal source of MIF. MSC-secreted PAI-1 stimulated significant migration of both CCC lines, with an antibody to the chemokine shown to block this effect (67–88% blocking,). A cell-line dependant effect on CCC proliferation was shown for Mesenchymal Stem Cell-secreted PAI-1 with HCT-116 cells showing decreased proliferation at all concentrations, and HT29 cells showing increased proliferation in the presence of higher PAI-1 levels. This is the first study to identify PAI-1 as an important mediator of Mesenchymal Stem Cell/colon cancer cell interactions and highlights the

  1. Folic Acid Supplementation Stimulates Notch Signaling and Cell Proliferation in Embryonic Neural Stem Cells

    PubMed Central

    Liu, Huan; Huang, Guo-wei; Zhang, Xu-mei; Ren, Da-lin; X. Wilson, John

    2010-01-01

    The present study investigated the effect of folic acid supplementation on the Notch signaling pathway and cell proliferation in rat embryonic neural stem cells (NSCs). The NSCs were isolated from E14–16 rat brain and grown as neurospheres in serum-free suspension culture. Individual cultures were assigned to one of 3 treatment groups that differed according to the concentration of folic acid in the medium: Control (baseline folic acid concentration of 4 mg/l), low folic acid supplementation (4 mg/l above baseline, Folate-L) and high folic acid supplementation (40 mg/l above baseline, Folate-H). NSCs were identified by their expression of immunoreactive nestin and proliferating cells by incorporation of 5'bromo-2'deoxyuridine. Cell proliferation was also assessed by methyl thiazolyl tetrazolium assay. Notch signaling was analyzed by real-time PCR and western blot analyses of the expression of Notch1 and hairy and enhancer of split 5 (Hes5). Supplementation of NSCs with folic acid increased the mRNA and protein expression levels of Notch1 and Hes5. Folic acid supplementation also stimulated NSC proliferation dose-dependently. Embryonic NSCs respond to folic acid supplementation with increased Notch signaling and cell proliferation. This mechanism may mediate the effects of folic acid supplementation on neurogenesis in the embryonic nervous system. PMID:20838574

  2. Lysyl oxidase propeptide inhibits smooth muscle cell signaling and proliferation

    SciTech Connect

    Hurtado, Paola A.; Vora, Siddharth; Sume, Siddika Selva; Yang, Dan; Hilaire, Cynthia St.; Guo Ying; Palamakumbura, Amitha H.; Schreiber, Barbara M.; Ravid, Katya; Trackman, Philip C.

    2008-02-01

    Lysyl oxidase is required for the normal biosynthesis and maturation of collagen and elastin. It is expressed by vascular smooth muscle cells, and its increased expression has been previously found in atherosclerosis and in models of balloon angioplasty. The lysyl oxidase propeptide (LOX-PP) has more recently been found to have biological activity as a tumor suppressor, and it inhibits Erk1/2 Map kinase activation. We reasoned that LOX-PP may have functions in normal non-transformed cells. We, therefore, investigated its effects on smooth muscle cells, focusing on important biological processes mediated by Erk1/2-dependent signaling pathways including proliferation and matrix metalloproteinase-9 (MMP-9) expression. In addition, we investigated whether evidence for accumulation of LOX-PP could be found in vivo in a femoral artery injury model. Recombinant LOX-PP was expressed and purified, and was found to inhibit primary rat aorta smooth muscle cell proliferation and DNA synthesis by more than 50%. TNF-{alpha}-stimulated MMP-9 expression and Erk1/2 activation were both significantly inhibited by LOX-PP. Immunohistochemistry studies carried out with affinity purified anti-LOX-PP antibody showed that LOX-PP epitopes were expressed at elevated levels in vascular lesions of injured arteries. These novel data suggest that LOX-PP may provide a feedback control mechanism that serves to inhibit properties associated with the development of vascular pathology.

  3. Potassium channels in cell cycle and cell proliferation

    PubMed Central

    Urrego, Diana; Tomczak, Adam P.; Zahed, Farrah; Stühmer, Walter; Pardo, Luis A.

    2014-01-01

    Normal cell-cycle progression is a crucial task for every multicellular organism, as it determines body size and shape, tissue renewal and senescence, and is also crucial for reproduction. On the other hand, dysregulation of the cell-cycle progression leading to uncontrolled cell proliferation is the hallmark of cancer. Therefore, it is not surprising that it is a tightly regulated process, with multifaceted and very complex control mechanisms. It is now well established that one of those mechanisms relies on ion channels, and in many cases specifically on potassium channels. Here, we summarize the possible mechanisms underlying the importance of potassium channels in cell-cycle control and briefly review some of the identified channels that illustrate the multiple ways in which this group of proteins can influence cell proliferation and modulate cell-cycle progression. PMID:24493742

  4. Biliary repair and carcinogenesis are mediated by IL-33–dependent cholangiocyte proliferation

    PubMed Central

    Li, Jun; Razumilava, Nataliya; Gores, Gregory J.; Walters, Stephanie; Mizuochi, Tatsuki; Mourya, Reena; Bessho, Kazuhiko; Wang, Yui-Hsi; Glaser, Shannon S.; Shivakumar, Pranavkumar; Bezerra, Jorge A.

    2014-01-01

    Injury to the biliary epithelium triggers inflammation and fibrosis, which can result in severe liver diseases and may progress to malignancy. Development of a type 1 immune response has been linked to biliary injury pathogenesis; however, a subset of patients with biliary atresia, the most common childhood cholangiopathy, exhibit increased levels of Th2-promoting cytokines. The relationship among different inflammatory drivers, epithelial repair, and carcinogenesis remains unclear. Here, we determined that the Th2-activating cytokine IL-33 is elevated in biliary atresia patient serum and in the livers and bile ducts of mice with experimental biliary atresia. Administration of IL-33 to WT mice markedly increased cholangiocyte proliferation and promoted sustained cell growth, resulting in dramatic and rapid enlargement of extrahepatic bile ducts. The IL-33–dependent proliferative response was mediated by an increase in the number of type 2 innate lymphoid cells (ILC2s), which released high levels of IL-13 that in turn promoted cholangiocyte hyperplasia. Induction of the IL-33/ILC2/IL-13 circuit in a murine biliary injury model promoted epithelial repair; however, induction of this circuit in mice with constitutive activation of AKT and YAP in bile ducts induced cholangiocarcinoma with liver metastases. These findings reveal that IL-33 mediates epithelial proliferation and suggest that activation of IL-33/ILC2/IL-13 may improve biliary repair and disruption of the circuit may block progression of carcinogenesis. PMID:24892809

  5. Angiotensin receptor I stimulates osteoprogenitor proliferation through TGFβ-mediated signaling.

    PubMed

    Querques, Francesca; Cantilena, Bruno; Cozzolino, Carmine; Esposito, Maria Teresa; Passaro, Fabiana; Parisi, Silvia; Lombardo, Barbara; Russo, Tommaso; Pastore, Lucio

    2015-07-01

    Clinical studies of large human populations and pharmacological interventions in rodent models have recently suggested that anti-hypertensive drugs that target angiotensin II (Ang II) activity may also reduce loss of bone mineral density. Here, we identified in a genetic screening the Ang II type I receptor (AT1R) as a potential determinant of osteogenic differentiation and, implicitly, bone formation. Silencing of AT1R expression by RNA interference severely impaired the maturation of a multipotent mesenchymal cell line (W20-17) along the osteoblastic lineage. The same effect was also observed after the addition of the AT1R antagonist losartan but not the AT2R inhibitor PD123,319. Additional cell culture assays traced the time of greatest losartan action to the early stages of W20-17 differentiation, namely during cell proliferation. Indeed, addition of Ang II increased proliferation of differentiating W20-17 and primary mesenchymal stem cells and this stimulation was reversed by losartan treatment. Cells treated with losartan also displayed an appreciable decrease of activated (phosphorylated)-Smad2/3 proteins. Moreover, Ang II treatment elevated endogenous transforming growth factor β (TGFβ) expression considerably and in an AT1R-dependent manner. Finally, exogenous TGFβ was able to restore high proliferative activity to W20-17 cells that were treated with both Ang II and losartan. Collectively, these results suggest a novel mechanism of Ang II action in bone metabolism that is mediated by TGFβ and targets proliferation of osteoblast progenitors. PMID:25556973

  6. EPO gene expression induces the proliferation, migration and invasion of bladder cancer cells through the p21WAF1‑mediated ERK1/2/NF-κB/MMP-9 pathway.

    PubMed

    Park, Sung Lyea; Won, Se Yeon; Song, Jun-Hui; Kim, Wun-Jae; Moon, Sung-Kwon

    2014-11-01

    Erythropoietin (EPO) is a cytokine that modulates the production of red blood cells. Previous studies have contradicted the assumed role of EPO in tumor cell proliferation. In the present study, we investigated the effect of EPO in the proliferation, migration and invasion that is involved in the signaling pathways and cell-cycle regulation of bladder cancer 5637 cells. The results showed that an overexpression of the EPO gene has a potent stimulatory effect on DNA synthesis, migration and invasion. EPO gene expression increased the expression of matrix metalloproteinase (MMP)-9 via the binding activity of NF-κB, AP-1 and Sp-1 in 5637 cells. The transfection of 5637 cells with the EPO gene induced the phosphorylation of ERK1/2. Treatment with ERK1/2 inhibitor U0126 significantly inhibited the increased proliferation, migration and invasion of EPO gene-transfected cells. U0126 treatment suppressed the induction of MMP-9 expression through NF-κB binding activity in EPO gene transfectants. In addition, EPO gene expression was correlated with the upregulation of cyclins/CDKs and the upregulation of the CDK inhibitor p21WAF1 expression. Finally, the inhibition of p21WAF1 function by siRNA blocked the proliferation, migration, invasion and phosphorylation of ERK1/2 signaling, as well as MMP-9 expression and activation of NF-κB in EPO gene-transfected cells. These novel findings suggest that the molecular mechanisms of EPO contribute to the progression and development of bladder tumors. PMID:25175278

  7. Secretome identification of immune cell factors mediating metastatic cell homing

    PubMed Central

    Aguado, Brian A.; Wu, Jia J.; Azarin, Samira M.; Nanavati, Dhaval; Rao, Shreyas S.; Bushnell, Grace G.; Medicherla, Chaitanya B.; Shea, Lonnie D.

    2015-01-01

    Metastatic cell homing is a complex process mediated in part by diffusible factors secreted from immune cells found at a pre-metastatic niche. We report on connecting secretomics and TRanscriptional Activity CEll aRray (TRACER) data to identify functional paracrine interactions between immune cells and metastatic cells as novel mediators of homing. Metastatic breast cancer mouse models were used to generate a diseased splenocyte conditioned media (D-SCM) containing immune cell secreted factors. MDA-MB-231 metastatic cell activity including cell invasion, migration, transendothelial migration, and proliferation were increased in D-SCM relative to control media. Our D-SCM secretome analysis yielded 144 secreted factor candidates that contribute to increased metastatic cell activity. The functional mediators of homing were identified using MetaCore software to determine interactions between the immune cell secretome and the TRACER-identified active transcription factors within metastatic cells. Among the 5 candidate homing factors identified, haptoglobin was selected and validated in vitro and in vivo as a key mediator of homing. Our studies demonstrate a novel systems biology approach to identify functional signaling factors associated with a cellular phenotype, which provides an enabling tool that complements large-scale protein identification provided by proteomics. PMID:26634905

  8. Neisseria lactamica selectively induces mitogenic proliferation of the naive B cell pool via cell surface Ig.

    PubMed

    Vaughan, Andrew T; Brackenbury, Louise S; Massari, Paola; Davenport, Victoria; Gorringe, Andrew; Heyderman, Robert S; Williams, Neil A

    2010-09-15

    Neisseria lactamica is a commensal bacteria that colonizes the human upper respiratory tract mucosa during early childhood. In contrast to the closely related opportunistic pathogen Neisseria meningitidis, there is an absence of adaptive cell-mediated immunity to N. lactamica during the peak age of carriage. Instead, outer membrane vesicles derived from N. lactamica mediate a B cell-dependent proliferative response in mucosal mononuclear cells that is associated with the production of polyclonal IgM. We demonstrate in this study that this is a mitogenic human B cell response that occurs independently of T cell help and any other accessory cell population. The ability to drive B cell proliferation is a highly conserved property and is present in N. lactamica strains derived from diverse clonal complexes. CFSE staining of purified human tonsillar B cells demonstrated that naive IgD(+) and CD27(-) B cells are selectively induced to proliferate by outer membrane vesicles, including the innate CD5(+) subset. Neither purified lipooligosaccharide nor PorB from N. lactamica is likely to be responsible for this activity. Prior treatment of B cells with pronase to remove cell-surface Ig or treatment with BCR-specific Abs abrogated the proliferative response to N. lactamica outer membrane vesicles, suggesting that this mitogenic response is dependent upon the BCR. PMID:20709949

  9. Myelin-phagocytosing macrophages modulate autoreactive T cell proliferation

    PubMed Central

    2011-01-01

    Introduction Multiple sclerosis (MS) is a chronic, inflammatory, demyelinating disease of the central nervous system (CNS) in which macrophages play a central role. Initially, macrophages where thought to be merely detrimental in MS, however, recent evidence suggests that their functional phenotype is altered following myelin phagocytosis. Macrophages that have phagocytosed myelin may be less inflammatory and may exert beneficial effects. The presence of myelin-containing macrophages in CNS-draining lymph nodes and perivascular spaces of MS patients suggests that these cells are ideally positioned to exert an immune regulatory role. Therefore we evaluated in this study the effect of myelin-phagocytosing macrophages on lymphocyte reactivity. Methods Thioglycolate-elicited rat peritoneal macrophages were loaded with myelin and cocultured with myelin-basic protein (MBP) or ovalbumin (OVA) reactive lymphocytes. Lymphocyte proliferation was determined by CFSE-labeling. The role of nitric oxide in regulating lymphocyte proliferation was assessed by addition of an inhibitor of inducible nitric oxide synthase to the coculture. In vivo immune regulation was investigated by treating MBP- and OVA-immunized animals subcutaneously with myelin. Cognate antigen specific lymphocyte proliferation and nitric oxide production were determined 9d post-immunization. Results In this study we demonstrate that myelin-phagocytosing macrophages inhibit TCR-triggered lymphocyte proliferation in an antigen-independent manner. The observed immune suppression is mediated by an increase in NO production by myelin-phagocytosing macrophages upon contact with lymphocytes. Additionally, myelin delivery to primarily CD169+ macrophages in popliteal lymph nodes of OVA-immunized animals results in a reduced cognate antigen specific proliferation. In contrast to OVA-immunized animals, lymphocytes from MBP-immunized animals displayed an increased proliferation after stimulation with their cognate antigen

  10. Slow and sustained nitric oxide releasing compounds inhibit multipotent vascular stem cell proliferation and differentiation without causing cell death

    SciTech Connect

    Curtis, Brandon M.; Leix, Kyle Alexander; Ji, Yajing; Glaves, Richard Samuel Elliot; Ash, David E.; Mohanty, Dillip K.

    2014-07-18

    Highlights: • Multipotent vascular stem cells (MVSCs) proliferate and differentiate. • Nitric oxide inhibits proliferation of MVSCs. • Nitric oxide inhibits MVSC differentiation to mesenchymal-like stem cells (MSCs). • Smooth muscle cells (SMCs) neither de-differentiate nor proliferate. - Abstract: Atherosclerosis is the leading cause of cerebral and myocardial infarction. It is believed that neointimal growth common in the later stages of atherosclerosis is a result of vascular smooth muscle cell (SMC) de-differentiation in response to endothelial injury. However, the claims of the SMC de-differentiation theory have not been substantiated by monitoring the fate of mature SMCs in response to such injuries. A recent study suggests that atherosclerosis is a consequence of multipotent vascular stem cell (MVSC) differentiation. Nitric oxide (NO) is a well-known mediator against atherosclerosis, in part because of its inhibitory effect on SMC proliferation. Using three different NO-donors, we have investigated the effects of NO on MVSC proliferation. Results indicate that NO inhibits MVSC proliferation in a concentration dependent manner. A slow and sustained delivery of NO proved to inhibit proliferation without causing cell death. On the other hand, larger, single-burst NO concentrations, inhibits proliferation, with concurrent significant cell death. Furthermore, our results indicate that endogenously produced NO inhibits MVSC differentiation to mesenchymal-like stem cells (MSCs) and subsequently to SMC as well.

  11. RNA interference targeting raptor inhibits proliferation of gastric cancer cells

    SciTech Connect

    Wu, William Ka Kei; Lee, Chung Wa; Cho, Chi Hin; Chan, Francis Ka Leung; Yu, Jun; Sung, Joseph Jao Yiu

    2011-06-10

    Mammalian target of rapamycin complex 1 (mTORC1) is dysregulated in gastric cancer. The biologic function of mTORC1 in gastric carcinogenesis is unclear. Here, we demonstrate that disruption of mTORC1 function by RNA interference-mediated downregulation of raptor substantially inhibited gastric cancer cell proliferation through induction of G{sub 0}/G{sub 1}-phase cell cycle arrest. The anti-proliferative effect was accompanied by concomitant downregulation of activator protein-1 and upregulation of Smad2/3 transcriptional activities. In addition, the expression of cyclin D{sub 3} and p21{sup Waf1}, which stabilizes cyclin D/cdk4 complex for G{sub 1}-S transition, was reduced by raptor knockdown. In conclusion, disruption of mTORC1 inhibits gastric cancer cell proliferation through multiple pathways. This discovery may have an implication in the application of mTORC1-directed therapy for the treatment of gastric cancer.

  12. Piperlongumine Suppresses Proliferation of Human Oral Squamous Cell Carcinoma through Cell Cycle Arrest, Apoptosis and Senescence.

    PubMed

    Chen, San-Yuan; Liu, Geng-Hung; Chao, Wen-Ying; Shi, Chung-Sheng; Lin, Ching-Yen; Lim, Yun-Ping; Lu, Chieh-Hsiang; Lai, Peng-Yeh; Chen, Hau-Ren; Lee, Ying-Ray

    2016-01-01

    Oral squamous cell carcinoma (OSCC), an aggressive cancer originating in the oral cavity, is one of the leading causes of cancer deaths in males worldwide. This study investigated the antitumor activity and mechanisms of piperlongumine (PL), a natural compound isolated from Piper longum L., in human OSCC cells. The effects of PL on cell proliferation, the cell cycle, apoptosis, senescence and reactive oxygen species (ROS) levels in human OSCC cells were investigated. PL effectively inhibited cell growth, caused cell cycle arrest and induced apoptosis and senescence in OSCC cells. Moreover, PL-mediated anti-human OSCC behavior was inhibited by an ROS scavenger N-acetyl-l-cysteine (NAC) treatment, suggesting that regulation of ROS was involved in the mechanism of the anticancer activity of PL. These findings suggest that PL suppresses tumor growth by regulating the cell cycle and inducing apoptosis and senescence and is a potential chemotherapy agent for human OSCC cells. PMID:27120594

  13. Piperlongumine Suppresses Proliferation of Human Oral Squamous Cell Carcinoma through Cell Cycle Arrest, Apoptosis and Senescence

    PubMed Central

    Chen, San-Yuan; Liu, Geng-Hung; Chao, Wen-Ying; Shi, Chung-Sheng; Lin, Ching-Yen; Lim, Yun-Ping; Lu, Chieh-Hsiang; Lai, Peng-Yeh; Chen, Hau-Ren; Lee, Ying-Ray

    2016-01-01

    Oral squamous cell carcinoma (OSCC), an aggressive cancer originating in the oral cavity, is one of the leading causes of cancer deaths in males worldwide. This study investigated the antitumor activity and mechanisms of piperlongumine (PL), a natural compound isolated from Piper longum L., in human OSCC cells. The effects of PL on cell proliferation, the cell cycle, apoptosis, senescence and reactive oxygen species (ROS) levels in human OSCC cells were investigated. PL effectively inhibited cell growth, caused cell cycle arrest and induced apoptosis and senescence in OSCC cells. Moreover, PL-mediated anti-human OSCC behavior was inhibited by an ROS scavenger N-acetyl-l-cysteine (NAC) treatment, suggesting that regulation of ROS was involved in the mechanism of the anticancer activity of PL. These findings suggest that PL suppresses tumor growth by regulating the cell cycle and inducing apoptosis and senescence and is a potential chemotherapy agent for human OSCC cells. PMID:27120594

  14. Snail controls proliferation of Drosophila ovarian epithelial follicle stem cells, independently of E-cadherin.

    PubMed

    Tseng, Chen-Yuan; Kao, Shih-Han; Hsu, Hwei-Jan

    2016-06-15

    Epithelial stem cells undergo constant self-renewal and differentiation to maintain the homeostasis of epithelial tissues that undergo rapid turnover. Recent studies have shown that the epithelial-mesenchymal transition (EMT), which is primarily mediated by Snail via the suppression of E-cadherin, is able to generate cells with stem cell properties. However, the role of Snail in epithelial stem cells remains unclear. Here, we report that Snail directly controls proliferation of follicle stem cells (FSCs) in Drosophila females. Disruption of Snail expression in FSCs compromises their proliferation, but not their maintenance. Conversely, FSCs with excessive Snail expression display increased proliferation and lifespan, which is accompanied by a moderate decrease in the expression of E-cadherin (required for adhesion of FSCs to their niche) at the junction between their adjacent cells, indicating a conserved role of Snail in E-cadherin inhibition, which promote epithelial cell proliferation. Interestingly, a decrease in E-cadherin in snail-knock down FSCs does not restore the decreased proliferation of snail-knock down FSCs, suggesting that adhesion strength of FSCs to their niche is dispensable for Snail-mediated FSC division. Our results demonstrate that Snail controls epithelial stem cell division independently of its known role in the EMT, which contributes to induction of cancer stem cells. PMID:27141871

  15. [The effects of actovegin on cell proliferation of permanent lines].

    PubMed

    Gulevskiĭ, A K; Trifonova, A V; Lavrik, A A

    2008-01-01

    The influence of Actovegin on proliferation activity and mitotic regimen of cells of permanent lines PK-15-IEKVM and BHK-21 clone 13/04 was investigated. Addition of Actovegin into growth media containing bovine serums of different components and concentrations stimulates cell proliferation. Conclusion has been made that Actovegin can be used in cell culture biotechnology. PMID:18411759

  16. Proliferation of mutators in A cell population.

    PubMed Central

    Mao, E F; Lane, L; Lee, J; Miller, J H

    1997-01-01

    A Lac- strain of Escherichia coli that reverts by the addition of a G to a G-G-G-G-G-G sequence was used to study the proliferation of mutators in a bacterial culture. Selection for the Lac+ phenotype, which is greatly stimulated in mismatch repair-deficient strains, results in an increase in the percentage of mutators in the selected population from less than 1 per 100,000 cells to 1 per 200 cells. All the mutators detected were deficient in the mismatch repair system. Mutagenesis results in a similar increase in the percentage of mutators. Mutagenesis combined with a single selection can result in a population of more than 50% mutators when a sample of several thousand cells is grown out and selected. Mutagenesis combined with two or more successive selections can generate a population that is 100% mutator. These experiments are discussed in relation to ideas that an early step in carcinogenesis is the creation of a mutator phenotype. PMID:8990293

  17. Proliferating cell nuclear antigen in neutrophil fate.

    PubMed

    Witko-Sarsat, Véronique; Ohayon, Delphine

    2016-09-01

    The life span of a neutrophil is a tightly regulated process as extended survival is beneficial for pathogen elimination and cell death necessary to prevent cytotoxic content release from activated neutrophils at the inflammatory site. Therefore, the control between survival and death must be a dynamic process. We have previously described that proliferating cell nuclear antigen (PCNA) which is known as a nuclear protein pivotal in DNA synthesis, is a key element in controlling neutrophil survival through its association with procaspases. Contrary to the dogma which asserted that PCNA has a strictly nuclear function, in mature neutrophils, PCNA is present exclusively within the cytosol due to its nuclear export at the end of the granulocytic differentiation. More recent studies are consistent with the notion that the cytosolic scaffold of PCNA is aimed at modulating neutrophil fate rather than simply preventing death. Ultimately, targeting neutrophil survival might have important applications not just in the field of immunology and inflammation, but also in hematology and transfusion. The neutrophil emerges as a unique and powerful cellular model to unravel the basic mechanisms governing the cell cycle-independent functions of PCNA and should be considered as a leader of the pack. PMID:27558345

  18. Dental pulp stem cells suppress the proliferation of lymphocytes via transforming growth factor-β1.

    PubMed

    Ding, Gang; Niu, Jianyi; Liu, Yi

    2015-04-01

    Dental pulp stem cells (DPSCs) possess self-renewal capability, multi-lineage differentiation potential, and can generate a dentin-pulp-like tissue in vivo, which is promising for tooth regeneration. To enlarge the cells resource of DPSCs and explore the feasibility of DPSCs-mediated immune therapy, it is prerequisite to investigate the immunological properties of DPSCs and the underlying mechanisms. Human DPSCs and peripheral blood mononuclear cells were isolated and cultured. Then we used lymphocytes proliferation assays, cytokines detection, Transwell cultures, neutralization experiments, and flow cytometry to examine the in vitro immune characteristics of DPSCs. We found that DPSCs failed to stimulate allogeneic T cells proliferation and suppressed T cells proliferation, B cells proliferation, and mixed lymphocyte reaction. In addition, DPSCs could up-regulate IL-10, down-regulate the production of IL-2, IL-17, and IFN-γ, and did not affect the production of IL-6. Monoclonal antibody against transforming growth factor-β1 restored the T cells proliferation inhibited by DPSCs. Moreover, the population of regulatory T cells increased significantly and T-helper 17 cells decreased significantly in peripheral blood mononuclear cells co-cultured with DPSCs. These data confirmed that DPSCs are low immunogenic, could inhibit the proliferation of lymphocytes, regulate the production of cytokines in vitro, and the secretion of transforming growth factor-β1 may be involved in this event. PMID:25605036

  19. Topography induces differential sensitivity on cancer cell proliferation via Rho-ROCK-Myosin contractility

    PubMed Central

    Chaudhuri, Parthiv Kant; Pan, Catherine Qiurong; Low, Boon Chuan; Lim, Chwee Teck

    2016-01-01

    Although the role of stiffness on proliferative response of cancer cells has been well studied, little is known about the effect of topographic cues in guiding cancer cell proliferation. Here, we examined the effect of topographic cues on cancer cell proliferation using micron scale topographic features and observed that anisotropic features like microgratings at specific dimension could reduce proliferation of non-cancer breast epithelial cells (MCF-10A) but not that for malignant breast cancer cells (MDA-MB-231 and MCF-7). However, isotropic features such as micropillars did not affect proliferation of MCF-10A, indicating that the anisotropic environmental cues are essential for this process. Interestingly, acto-myosin contraction inhibitory drugs, Y-27632 and blebbistatin prevented micrograting-mediated inhibition on proliferation. Here, we propose the concept of Mechanically-Induced Dormancy (MID) where topographic cues could activate Rho-ROCK-Myosin signaling to suppress non-cancerous cells proliferation whereas malignant cells are resistant to this inhibitory barrier and therefore continue uncontrolled proliferation. PMID:26795068

  20. Epithelial Cell Proliferation Contributes to Airway Remodeling in Severe Asthma

    PubMed Central

    Cohen, Lance; E, Xueping; Tarsi, Jaime; Ramkumar, Thiruvamoor; Horiuchi, Todd K.; Cochran, Rebecca; DeMartino, Steve; Schechtman, Kenneth B.; Hussain, Iftikhar; Holtzman, Michael J.; Castro, Mario

    2007-01-01

    Rationale: Despite long-term therapy with corticosteroids, patients with severe asthma develop irreversible airway obstruction. Objectives: To evaluate if there are structural and functional differences in the airway epithelium in severe asthma associated with airway remodeling. Methods: In bronchial biopsies from 21 normal subjects, 11 subjects with chronic bronchitis, 9 subjects with mild asthma, and 31 subjects with severe asthma, we evaluated epithelial cell morphology: epithelial thickness, lamina reticularis (LR) thickness, and epithelial desquamation. Levels of retinoblastoma protein (Rb), Ki67, and Bcl-2 were measured, reflecting cellular proliferation and death. Terminal deoxynucleotidyl-mediated dUTP nick end labeling (TUNEL) was used to study cellular apoptosis. Measurements and Main Results: Airway epithelial and LR thickness was greater in subjects with severe asthma compared with those with mild asthma, normal subjects, and diseased control subjects (p = 0.009 and 0.033, respectively). There was no significant difference in epithelial desquamation between groups. Active, hypophosphorylated Rb expression was decreased (p = 0.002) and Ki67 was increased (p < 0.01) in the epithelium of subjects with severe asthma as compared with normal subjects, indicating increased cellular proliferation. Bcl-2 expression was decreased (p < 0.001), indicating decreased cell death suppression. There was a greater level of apoptotic activity in the airway biopsy in subjects with severe asthma as compared with the normal subjects using the TUNEL assay (p = 0.002), suggesting increased cell death. Conclusions: In subjects with severe asthma, as compared with subjects with mild asthma, normal subjects, and diseased control subjects, we found novel evidence of increased cellular proliferation in the airway contributing to a thickened epithelium and LR. These changes may contribute to the progressive decline in lung function and airway remodeling in patients with severe

  1. Matrix Metalloproteinase-3 in Odontoblastic Cells Derived from Ips Cells: Unique Proliferation Response as Odontoblastic Cells Derived from ES Cells

    PubMed Central

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

    2013-01-01

    We previously reported that matrix metalloproteinase (MMP)-3 accelerates wound healing following dental pulp injury. In addition, we reported that a proinflammatory cytokine mixture (tumor necrosis factor-α, interleukin (IL)-1β and interferon-γ) induced MMP-3 activity in odontoblast-like cells derived from mouse embryonic stem (ES) cells, suggesting that MMP-3 plays a potential unique physiological role in wound healing and regeneration of dental pulp in odontoblast-like cells. In this study, we tested the hypothesis that upregulation of MMP-3 activity by IL-1β promotes proliferation and apoptosis of purified odontoblast-like cells derived from induced pluripotent stem (iPS) and ES cells. Each odontoblast-like cell was isolated and incubated with different concentrations of IL-1β. MMP-3 mRNA and protein expression were assessed using RT-PCR and western blotting, respectively. MMP-3 activity was measured using immunoprecipitation and a fluorescence substrate. Cell proliferation and apoptosis were determined using ELISA for BrdU and DNA fragmentation, respectively. siRNA was used to reduce MMP-3 transcripts in these cells. Treatment with IL-1β increased MMP-3 mRNA and protein levels, and MMP-3 activity in odontoblast-like cells. Cell proliferation was found to markedly increase with no changes in apoptosis. Endogenous tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-2 were constitutively expressed during all experiments. The exocytosis inhibitor, Exo1, potently suppressed the appearance of MMP-3 in the conditioned medium. Treatment with siRNA against MMP-3 suppressed an IL-1β-induced increase in MMP-3 expression and activity, and also suppressed cell proliferation, but unexpectedly increased apoptosis in these cells (P<0.05). Exogenous MMP-3 was found to induce cell proliferation in odontoblast-like cells derived from iPS cells and ES cells. This siRNA-mediated increase in apoptosis could be reversed with exogenous MMP-3 stimulation (P<0.05). Taken

  2. Regulation of cell proliferation by hypoxia-inducible factors.

    PubMed

    Hubbi, Maimon E; Semenza, Gregg L

    2015-12-15

    Hypoxia is a physiological cue that impacts diverse physiological processes, including energy metabolism, autophagy, cell motility, angiogenesis, and erythropoiesis. One of the key cell-autonomous effects of hypoxia is as a modulator of cell proliferation. For most cell types, hypoxia induces decreased cell proliferation, since an increased number of cells, with a consequent increase in O2 demand, would only exacerbate hypoxic stress. However, certain cell populations maintain cell proliferation in the face of hypoxia. This is a common pathological hallmark of cancers, but can also serve a physiological function, as in the maintenance of stem cell populations that reside in a hypoxic niche. This review will discuss major molecular mechanisms by which hypoxia regulates cell proliferation in different cell populations, with a particular focus on the role of hypoxia-inducible factors. PMID:26491052

  3. Cocaine- and Amphetamine-regulated Transcript (CART) Protects Beta Cells against Glucotoxicity and Increases Cell Proliferation*

    PubMed Central

    Sathanoori, Ramasri; Olde, Björn; Erlinge, David; Göransson, Olga; Wierup, Nils

    2013-01-01

    Cocaine- and amphetamine-regulated transcript (CART) is an islet peptide that promotes glucose-stimulated insulin secretion in beta cells via cAMP/PKA-dependent pathways. In addition, CART is a regulator of neuronal survival. In this study, we examined the effect of exogenous CART 55–102 on beta cell viability and dissected its signaling mechanisms. Evaluation of DNA fragmentation and chromatin condensation revealed that CART 55–102 reduced glucotoxicity-induced apoptosis in both INS-1 (832/13) cells and isolated rat islets. Glucotoxicity in INS-1 (832/13) cells also caused a 50% reduction of endogenous CART protein. We show that CART increased proliferation in INS-1 (832/13) cells, an effect that was blocked by PKA, PKB, and MEK1 inhibitors. In addition, CART induced phosphorylation of CREB, IRS, PKB, FoxO1, p44/42 MAPK, and p90RSK in INS-1 (832/13) cells and isolated rat islets, all key mediators of cell survival and proliferation. Thus, we demonstrate that CART 55-102 protects beta cells against glucotoxicity and promotes proliferation. Taken together our data point to the potential use of CART in therapeutic interventions targeted at enhancing functional beta cell mass and long-term insulin secretion in T2D. PMID:23250745

  4. Cocaine- and amphetamine-regulated transcript (CART) protects beta cells against glucotoxicity and increases cell proliferation.

    PubMed

    Sathanoori, Ramasri; Olde, Björn; Erlinge, David; Göransson, Olga; Wierup, Nils

    2013-02-01

    Cocaine- and amphetamine-regulated transcript (CART) is an islet peptide that promotes glucose-stimulated insulin secretion in beta cells via cAMP/PKA-dependent pathways. In addition, CART is a regulator of neuronal survival. In this study, we examined the effect of exogenous CART 55-102 on beta cell viability and dissected its signaling mechanisms. Evaluation of DNA fragmentation and chromatin condensation revealed that CART 55-102 reduced glucotoxicity-induced apoptosis in both INS-1 (832/13) cells and isolated rat islets. Glucotoxicity in INS-1 (832/13) cells also caused a 50% reduction of endogenous CART protein. We show that CART increased proliferation in INS-1 (832/13) cells, an effect that was blocked by PKA, PKB, and MEK1 inhibitors. In addition, CART induced phosphorylation of CREB, IRS, PKB, FoxO1, p44/42 MAPK, and p90RSK in INS-1 (832/13) cells and isolated rat islets, all key mediators of cell survival and proliferation. Thus, we demonstrate that CART 55-102 protects beta cells against glucotoxicity and promotes proliferation. Taken together our data point to the potential use of CART in therapeutic interventions targeted at enhancing functional beta cell mass and long-term insulin secretion in T2D. PMID:23250745

  5. Ganghwaljetongyeum, an anti-arthritic remedy, attenuates synoviocyte proliferation and reduces the production of proinflammatory mediators in macrophages: the therapeutic effect of GHJTY on rheumatoid arthritis

    PubMed Central

    2013-01-01

    Background Ganghwaljetongyeum (GHJTY), a complex herbal decoction, is used to treat rheumatoid arthritis. However, the action mechanism of GHJTY is not still unclear on rheumatoid arthritis. In this study, we examined the beneficial effects and the action mechanisms of GHJTY on synoviocyte proliferation and inflammatory mediators. Methods To test the effect of GHJTY on synoviocyte proliferation, HIG-82 cells, rabbit knee synovial membrane cells, were treated with GHJTY under IL-1β. To evaluate the effects of GHJTY on proinflammatory mediators, we tested cytokine levels in RAW264.7 cells. Results Proliferation of HIG-82 cells was significantly inhibited by GHJTY treatment. We found that GHJTY caused cytoskeleton damage to HIG-82 cells. In contrast, treatment of GHJTY did not show any cytotoxicity to other different origin cell lines, HeLa and RAW264.7 cells. GHJTY inhibited IL-1β-mediated NF-κB activation in HIG-82 cells and reduced the LPS-mediated production of proinflammatory cytokines, TNF-α, IL-12, and NO in RAW264.7 cells. In addition, the expression of cyclooxygenase in LPS-activated RAW264.7 cells was also decreased by GHJTY treatment. Conclusions These results suggest that GHJTY might effectively attenuate rheumatoid arthritis by inhibiting the production of proinflammatory mediators and the proliferation of synoviocytes. PMID:23442977

  6. Vasopressin regulation of epithelial colonic proliferation and permeability is mediated by pericryptal platelet-derived growth factor A.

    PubMed

    Miró, Lluïsa; Pérez-Bosque, Anna; Maijó, Mònica; Naftalin, Richard J; Moretó, Miquel

    2014-10-01

    Arginine vasopressin (AVP) has trophic effects on the rat distal colon, increasing the growth of pericryptal myofibroblasts and reducing the colonic crypt wall permeability. This study aimed to reproduce in vitro the effects of AVP observed in vivo using cultures of human CCD-18Co myofibroblasts and T84 colonic epithelial cells. Proliferation of myofibroblasts was quantified by bromodeoxyuridine incorporation; the expression of platelet-derived growth factor A (PDGFA), platelet-derived growth factor B, epidermal growth factor, transforming growth factor-β and vascular endothelial growth factor was measured by PCR and the expression of epithelial junction proteins by Western blot. Arginine vasopressin stimulated myofibroblast proliferation and the expression of PDGFA without affecting the expression of platelet-derived growth factor B, epidermal growth factor, transforming growth factor-β or vascular endothelial growth factor. These effects were prevented when AVP receptor inhibitors were present in the medium. Pre-incubation of CCD-18Co cells with anti-PDGF antibody or with an inhibitor of the PDGF receptor abolished the effects of AVP. When colonocytes were incubated with medium obtained from myofibroblasts incubated with AVP, both cell proliferation and the expression of epithelial junction proteins increased; however, direct incubation of colonocytes with AVP did not modify these variables. These results demonstrate that AVP stimulates myofibroblast proliferation and induces PDGFA secretion, implying that PDGFA mediates local myofibroblast proliferation by an autocrine feedback loop and regulates epithelial proliferation and permeability by a paracrine mechanism. PMID:25085844

  7. Isolinderalactone inhibits proliferation of A549 human non‑small cell lung cancer cells by arresting the cell cycle at the G0/G1 phase and inducing a Fas receptor and soluble Fas ligand-mediated apoptotic pathway.

    PubMed

    Chang, Wei-An; Lin, En-Shyh; Tsai, Ming-Ju; Huang, Ming-Shyan; Kuo, Po-Lin

    2014-05-01

    Lung cancer is currently the leading cause of cancer-related mortality worldwide. In Taiwan, lung cancer is also the type of malignancy that is the major cause of cancer-mortality. Investigating the mechanism of apoptosis of lung cancer cells is important in the treatment of lung cancer. In the present study, isolinderalactone was demonstrated to exhibit anticancer effects in A549 human non-small cell lung cancer cells. The effect of isolinderalactone on apoptosis, cell cycle distribution p21 levels and the Fas receptor and soluble Fas ligand (sFasL) were assayed in order to determine the mechanism underlying the anticancer effect of isolinderalactone. It was demonstrated that isolinderalactone may induce p21 expression and then cause the cell cycle arrest of A549 cells. The data of the present study also revealed that the Fas/sFasL apoptotic system is significant in the mechanism of isolinderalactone‑induced apoptosis of A549 cells. These novel findings demonstrated that isolinderalactone may cause the cell cycle arrest of A549 cells by induction of p21, and induce apoptosis of A549 human non-small-cell lung carcinoma cells through the Fas/sFasL apoptotic system. PMID:24604009

  8. Wnt9A Induction Linked to Suppression of Human Colorectal Cancer Cell Proliferation

    PubMed Central

    Ali, Irshad; Medegan, Bani; Braun, Donald P.

    2016-01-01

    Most studies of Wnt signaling in malignant tissues have focused on the canonical Wnt pathway (CWP) due to its role in stimulating cellular proliferation. The role of the non-canonical Wnt pathway (NCWP) in tissues with dysregulated Wnt signaling is not fully understood. Understanding NCWP’s role is important since these opposing pathways act in concert to maintain homeostasis in healthy tissues. Our preliminary studies demonstrated that LiCl inhibited proliferation of primary cells derived from colorectal cancer (CRC). Since LiCl stimulates cell proliferation in normal tissues and NCWP suppresses it, the present study was designed to investigate the impact of NCWP components in LiCl-mediated effects. LiCl-mediated inhibition of CRC cell proliferation (p < 0.001) and increased apoptosis (p < 0.01) coincided with 23-fold increase (p < 0.025) in the expression of the NCWP ligand, Wnt9A. LiCl also suppressed β-catenin mRNA (p < 0.03), total β-catenin protein (p < 0.025) and the active form of β-catenin. LiCl-mediated inhibition of CRC cell proliferation was partially reversed by IWP-2, and Wnt9A antibody. Recombinant Wnt9A protein emulated LiCl effects by suppressing β-catenin protein (p < 0.001), inhibiting proliferation (p < 0.001) and increasing apoptosis (p < 0.03). This is the first study to demonstrate induction of a NCWP ligand, Wnt9A as part of a mechanism for LiCl-mediated suppression of CRC cell proliferation. PMID:27049382

  9. Differential Glutamate Metabolism in Proliferating and Quiescent Mammary Epithelial Cells.

    PubMed

    Coloff, Jonathan L; Murphy, J Patrick; Braun, Craig R; Harris, Isaac S; Shelton, Laura M; Kami, Kenjiro; Gygi, Steven P; Selfors, Laura M; Brugge, Joan S

    2016-05-10

    Mammary epithelial cells transition between periods of proliferation and quiescence during development, menstrual cycles, and pregnancy, and as a result of oncogenic transformation. Utilizing an organotypic 3D tissue culture model coupled with quantitative metabolomics and proteomics, we identified significant differences in glutamate utilization between proliferating and quiescent cells. Relative to quiescent cells, proliferating cells catabolized more glutamate via transaminases to couple non-essential amino acid (NEAA) synthesis to α-ketoglutarate generation and tricarboxylic acid (TCA) cycle anaplerosis. As cells transitioned to quiescence, glutamine consumption and transaminase expression were reduced, while glutamate dehydrogenase (GLUD) was induced, leading to decreased NEAA synthesis. Highly proliferative human tumors display high transaminase and low GLUD expression, suggesting that proliferating cancer cells couple glutamine consumption to NEAA synthesis to promote biosynthesis. These findings describe a competitive and partially redundant relationship between transaminases and GLUD, and they reveal how coupling of glutamate-derived carbon and nitrogen metabolism can be regulated to support cell proliferation. PMID:27133130

  10. Skin cell proliferation stimulated by microneedles.

    PubMed

    Liebl, Horst; Kloth, Luther C

    2012-03-01

    A classical wound may be defined as a disruption of tissue integrity. Wounds, caused by trauma from accidents or surgery, that close via secondary intention rely on the biological phases of healing, i.e., hemostasis, inflammation, proliferation, and remodeling (HIPR). Depending on the wound type and severity, the inflammation phase begins immediately after injury and may last for an average of 7-14 days. Concurrent with the inflammation phase or slightly delayed, cell proliferation is stimulated followed by the activation of the remodeling (maturation) phase. The latter phase can last as long as 1 year or more, and the final healed state is represented by a scar tissue, a cross-linked collagen formation that usually aligns collagen fibers in a single direction. One may assume that skin microneedling that involves the use of dozens or as many as 200 needles that limit penetration to 1.5 mm over 1 cm(2) of skin would cause trauma and bleeding followed by the classical HIPR. However, this is not the case or at least the HIPR phases are significantly curtailed and healing never ends in a scar formation. Conversely dermabrasion used in aesthetic medicine for improving skin quality is based on "ablation" (destruction or wounding of superficial skin layers), which requires several weeks for healing that involves formation of new skin layers. Such procedures provoke an acute inflammatory response. We believe that a less intense inflammatory response occurs following microneedle perforation of the skin. However, the mechanism of action of microneedling appears to be different. Here we review the potential mechanisms by which microneedling of the skin facilitates skin repair without scarring after the treatment of superficial burns, acne, hyperpigmentation, and the non-advancing periwound skin surrounding the chronic ulcerations of the integument. PMID:24527373

  11. Skin Cell Proliferation Stimulated by Microneedles

    PubMed Central

    Liebl, Horst; Kloth, Luther C.

    2012-01-01

    A classical wound may be defined as a disruption of tissue integrity. Wounds, caused by trauma from accidents or surgery, that close via secondary intention rely on the biological phases of healing, i.e., hemostasis, inflammation, proliferation, and remodeling (HIPR). Depending on the wound type and severity, the inflammation phase begins immediately after injury and may last for an average of 7–14 days. Concurrent with the inflammation phase or slightly delayed, cell proliferation is stimulated followed by the activation of the remodeling (maturation) phase. The latter phase can last as long as 1 year or more, and the final healed state is represented by a scar tissue, a cross-linked collagen formation that usually aligns collagen fibers in a single direction. One may assume that skin microneedling that involves the use of dozens or as many as 200 needles that limit penetration to 1.5 mm over 1 cm2 of skin would cause trauma and bleeding followed by the classical HIPR. However, this is not the case or at least the HIPR phases are significantly curtailed and healing never ends in a scar formation. Conversely dermabrasion used in aesthetic medicine for improving skin quality is based on “ablation” (destruction or wounding of superficial skin layers), which requires several weeks for healing that involves formation of new skin layers. Such procedures provoke an acute inflammatory response. We believe that a less intense inflammatory response occurs following microneedle perforation of the skin. However, the mechanism of action of microneedling appears to be different. Here we review the potential mechanisms by which microneedling of the skin facilitates skin repair without scarring after the treatment of superficial burns, acne, hyperpigmentation, and the non-advancing periwound skin surrounding the chronic ulcerations of the integument. PMID:24527373

  12. Histone variant H2A.Z.2 mediates proliferation and drug sensitivity of malignant melanoma

    PubMed Central

    Vardabasso, Chiara; Gaspar-Maia, Alexandre; Hasson, Dan; Pünzeler, Sebastian; Valle-Garcia, David; Straub, Tobias; Keilhauer, Eva C.; Strub, Thomas; Dong, Joanna; Panda, Taniya; Chung, Chi-Yeh; Yao, Jonathan L.; Singh, Rajendra; Segura, Miguel F.; Fontanals-Cirera, Barbara; Verma, Amit; Mann, Matthias; Hernando, Eva; Hake, Sandra B.; Bernstein, Emily

    2015-01-01

    SUMMARY Histone variants are emerging as key regulatory molecules in cancer. Here we report a novel role for the H2A.Z isoform H2A.Z.2 as a driver of malignant melanoma. H2A.Z.2 is highly expressed in metastatic melanoma, correlates with decreased patient survival, and is required for cellular proliferation. Our integrated genomic analyses reveal that H2A.Z.2 controls the transcriptional output of E2F target genes in melanoma cells. These genes are highly expressed and display a distinct signature of H2A.Z occupancy. We identify BRD2 as an H2A.Z interacting protein, whose levels are also elevated in melanoma. We further demonstrate that H2A.Z.2 regulated genes are bound by BRD2 and E2F1 in a H2A.Z.2-dependent manner. Importantly, H2A.Z.2 deficiency sensitizes melanoma cells to chemotherapy and targeted therapies. Collectively, our findings implicate H2A.Z.2 as a mediator of cell proliferation and drug sensitivity in malignant melanoma, holding translational potential for novel therapeutic strategies. PMID:26051178

  13. Uterine epithelial cell proliferation and endometrial hyperplasia: evidence from a mouse model.

    PubMed

    Gao, Yang; Li, Shu; Li, Qinglei

    2014-08-01

    In the uterus, epithelial cell proliferation changes during the estrous cycle and pregnancy. Uncontrolled epithelial cell proliferation results in implantation failure and/or cancer development. Transforming growth factor-β (TGF-β) signaling is a fundamental regulator of diverse biological processes and is indispensable for multiple reproductive functions. However, the in vivo role of TGF-β signaling in uterine epithelial cells remains poorly defined. We have shown that in the uterus, conditional deletion of the Type 1 receptor for TGF-β (Tgfbr1) using anti-Müllerian hormone receptor type 2 (Amhr2) Cre leads to myometrial defects. Here, we describe enhanced epithelial cell proliferation by immunostaining of Ki67 in the uteri of these mice. The aberration culminated in endometrial hyperplasia in aged females. To exclude the potential influence of ovarian steroid hormones, the proliferative status of uterine epithelial cells was assessed following ovariectomy. Increased uterine epithelial cell proliferation was also revealed in ovariectomized Tgfbr1 Amhr2-Cre conditional knockout mice. We further demonstrated that transcript levels for fibroblast growth factor 10 (Fgf10) were markedly up-regulated in Tgfbr1 Amhr2-Cre conditional knockout uteri. Consistently, treatment of primary uterine stromal cells with TGF-β1 significantly reduced Fgf10 mRNA expression. Thus, our findings suggest a potential involvement of TGFBR1-mediated signaling in the regulation of uterine epithelial cell proliferation, and provide genetic evidence supporting the role of uterine epithelial cell proliferation in the pathogenesis of endometrial hyperplasia. PMID:24770950

  14. TRAIL-receptor costimulation inhibits proximal TCR signaling and suppresses human T cell activation and proliferation.

    PubMed

    Lehnert, Corinna; Weiswange, Maxi; Jeremias, Irmela; Bayer, Carina; Grunert, Michaela; Debatin, Klaus-Michael; Strauss, Gudrun

    2014-10-15

    The TRAIL-receptor/TRAIL system originally described to induce apoptosis preferentially in malignant cells is also known to be involved in T cell homeostasis and the response to viral infections and autoimmune diseases. Whereas the expression of TRAIL on activated NK and T cells increases their cytotoxicity, induction of TRAIL on APCs can turn them into apoptosis inducers but might also change their immunostimulatory capacity. Therefore, we analyzed how TRAIL-receptor (TRAIL-R) costimulation is modulating TCR-mediated activation of human T cells. T cells triggered by rTRAIL in combination with anti-CD3 and -CD28 Abs exhibited a strong decrease in the expression of activation markers and Th1 and Th2 cytokines compared with CD3/CD28-activated T cells. Most importantly, proliferation of TRAIL-R costimulated T cells was strongly impaired, but no apoptosis was induced. Addition of exogenous IL-2 could not rescue T cells silenced by TRAIL-R costimulation, and TRAIL-mediated inhibition of T cell proliferation only prevented TCR-triggered proliferation but was ineffective if T cells were activated downstream of the TCR. Inhibition of T cell proliferation was associated with abrogation of proximal TCR signaling by inhibiting recruitment of TCR-associated signaling molecules to lipid rafts, followed by abrogation of protein tyrosine phosphorylation of ZAP70, phospholipase C-γ1, and protein kinase C-θ, and impaired nuclear translocation of NFAT, AP-1, and NF-κB. Most importantly, TRAIL-R costimulation efficiently inhibited alloantigen-induced T cell proliferation and CD3/28-induced activation and proliferation of autoreactive T cells derived from patients with Omenn syndrome, indicating that coactivation of TRAIL-R and TCR represents a mechanism to downmodulate T cell immune responses. PMID:25217163

  15. Armet, a UPR-upregulated protein, inhibits cell proliferation and ER stress-induced cell death

    SciTech Connect

    Apostolou, Andria; Shen Yuxian; Liang Yan; Luo Jun; Fang Shengyun

    2008-08-01

    The accumulation of misfolded proteins in the endoplasmic reticulum (ER) causes ER stress that initiates the unfolded protein response (UPR). UPR activates both adaptive and apoptotic pathways, which contribute differently to disease pathogenesis. To further understand the functional mechanisms of UPR, we identified 12 commonly UPR-upregulated genes by expression microarray analysis. Here, we describe characterization of Armet/MANF, one of the 12 genes whose function was not clear. We demonstrated that the Armet/MANF protein was upregulated by various forms of ER stress in several cell lines as well as by cerebral ischemia of rat. Armet/MANF was localized in the ER and Golgi and was also a secreted protein. Silencing Armet/MANF by siRNA oligos in HeLa cells rendered cells more susceptible to ER stress-induced death, but surprisingly increased cell proliferation and reduced cell size. Overexpression of Armet/MANF inhibited cell proliferation and improved cell viability under glucose-free conditions and tunicamycin treatment. Based on its inhibitory properties for both proliferation and cell death we have demonstrated, Armet is, thus, a novel secreted mediator of the adaptive pathway of UPR.

  16. Surfactant protein A integrates activation signal strength to differentially modulate T cell proliferation.

    PubMed

    Mukherjee, Sambuddho; Giamberardino, Charles; Thomas, Joseph; Evans, Kathy; Goto, Hisatsugu; Ledford, Julie G; Hsia, Bethany; Pastva, Amy M; Wright, Jo Rae

    2012-02-01

    Pulmonary surfactant lipoproteins lower the surface tension at the alveolar-airway interface of the lung and participate in host defense. Previous studies reported that surfactant protein A (SP-A) inhibits lymphocyte proliferation. We hypothesized that SP-A-mediated modulation of T cell activation depends upon the strength, duration, and type of lymphocyte activating signals. Modulation of T cell signal strength imparted by different activating agents ex vivo and in vivo in different mouse models and in vitro with human T cells shows a strong correlation between strength of signal (SoS) and functional effects of SP-A interactions. T cell proliferation is enhanced in the presence of SP-A at low SoS imparted by exogenous mitogens, specific Abs, APCs, or in homeostatic proliferation. Proliferation is inhibited at higher SoS imparted by different doses of the same T cell mitogens or indirect stimuli such as LPS. Importantly, reconstitution with exogenous SP-A into the lungs of SP-A(-/-) mice stimulated with a strong signal also resulted in suppression of T cell proliferation while elevating baseline proliferation in unstimulated T cells. These signal strength and SP-A-dependent effects are mediated by changes in intracellular Ca(2+) levels over time, involving extrinsic Ca(2+)-activated channels late during activation. These effects are intrinsic to the global T cell population and are manifested in vivo in naive as well as memory phenotype T cells. Thus, SP-A appears to integrate signal thresholds to control T cell proliferation. PMID:22219327

  17. YAP Regulates Cell Proliferation, Migration, and Steroidogenesis in Adult Granulosa Cell Tumors

    PubMed Central

    Fu, David; Lv, Xiangmin; Hua, Guohua; He, Chunbo; Dong, Jixin; Lele, Subodh M.; Li, David Wan-Cheng; Zhai, Qiongli; Davis, John S.; Wang, Cheng

    2014-01-01

    The Hippo signaling pathway has been implicated as a conserved regulator of organ size in both Drosophila and mammals. Yes associated protein (YAP), the central component of the Hippo signaling cascade, functions as an oncogene in several malignancies. Ovarian granulosa cell tumors (GCT) are characterized by enlargement of ovary, excess production of estrogen, high frequency of recurrence and potential of malignancy and metastasis. Whether the Hippo pathway plays a role in the pathogenesis of GCT is unknown. This study was conducted to examine the expression of YAP in human adult GCTs and to determine the role of YAP in the proliferation and steroidogenesis of GCT cells. Compared with age-matched normal human ovaries, GCT tissues exhibited higher levels of YAP expression. YAP protein was predominantly expressed in the nucleus of tumor cells, whereas the non-tumor ovarian stromal cells expressed very low levels of YAP. YAP was also expressed in cultured primary human granulosa cells and in KGN and COV434 GCT cell lines. siRNA-mediated knockdown of YAP in KGN cells resulted in a significant reduction in cell proliferation (P<0.001). Conversely, overexpression of wild-type YAP or a constitutively active YAP mutant resulted in a significant increase in KGN cell proliferation and migration. Moreover, YAP knockdown reduced FSH-induced aromatase (CYP19A1) protein expression and estrogen production in KGN cells. These results demonstrate that YAP plays an important role in regulating GCT cell proliferation, migration and steroidogenesis. Targeting the Hippo/YAP pathway may provide a novel therapeutic approach for GCT. PMID:24389730

  18. Lipopolysaccharide induces cholangiocyte proliferation via an interleukin-6-mediated activation of p44/p42 mitogen-activated protein kinase.

    PubMed

    Park, J; Gores, G J; Patel, T

    1999-04-01

    The biliary epithelium is exposed to mediators of inflammation such as bacterial endotoxin or lipopolysaccharide (LPS) in a variety of inflammatory conditions. These conditions are also characterized by cholangiocyte proliferation and a predisposition to malignancy. Furthermore, LPS can enhance the expression of interleukin-6 (IL-6), a known biliary mitogen. However, the effects of LPS on cholangiocyte proliferation or IL-6 secretion are unknown. Thus, our aims were to determine if LPS stimulates cholangiocyte proliferation by IL-6-dependent signaling pathways. H69 cells derived from normal human intrahepatic cholangiocytes proliferated in response to LPS. Cholangiocytes responded to LPS (and other inflammatory cytokines such as tumor necrosis factor alpha [TNF-alpha] and IL-1beta) by increased secretion of IL-6, which had a mitogenic effect on H69 cells. Preincubation with anti-IL-6 neutralizing antibodies inhibited LPS-induced proliferation. Furthermore, cholangiocytes possessed the IL-6 receptor complex subunits and intact signaling mechanisms leading to activation of signal transducers and activators of transcription (STAT) factors. Although both p38 and p44/p42 mitogen-activated protein kinases (MAPKs) were constitutively present and active in cholangiocytes, IL-6 increased p44/p42, but not p38 MAPK activity. PD098059 inhibited activation of p44/p42 MAPK in cholangiocytes and completely blocked DNA synthesis in response to IL-6 or LPS. These studies identify a critical role for the p44/p42 MAPK in cholangiocyte proliferation and demonstrate that the proliferative response of cholangiocytes to inflammatory mediators such as LPS involves IL-6-mediated activation of the p44/p42 MAPK pathway. PMID:10094943

  19. ROS, MAPK/ERK and PKC play distinct roles in EGF-stimulated human corneal cell proliferation and migration.

    PubMed

    Huo, Y-N; Chen, W; Zheng, X-X

    2015-01-01

    Cornea is at the outermost surface of eye globe, and it easily receives damage from ultraviolet light exposure, physiology wounding, and infections. It is essential to understand the mechanisms controlling human corneal epithelial (HCE) cell proliferation and wound healing. Epidermal growth factor (EGF) could stimulate cell proliferation and migration in various cell types. Therefore, we investigated the roles and mechanisms of EGF on HCE cell proliferation and migration. CCK-8 kit and wound healing experiment were used to investigate HCE cell proliferation and cell migration, respectively. ROS activity was quantified by DCFDA and flow cytometry. Western blot and Q-PCR were performed to examine protein and RNA levels. EGF could promote HCE cell proliferation and migration in both physiology status and UV irradiation conditions, which is used to mimic the disease condition in human corneal epithelial cells. Interestingly, the promotion effect of EGF on HCE cell proliferation is mainly mediated by activated ROS signaling under disease condition. However, the EGF function is mediated by ROS and MAPK/ERK pathway in EGF-treated corneal epithelial cells in physiology status, in which ROS and MAPK/ERK pathway have no mutual influence on the other signaling pathway in EGF-stimulated corneal epithelial cells. We also revealed that MAPK/ERK pathway instead of ROS mediates EGF-stimulated HCE cell migration. Interestingly, we found that PKC proteins were downregulated by EGF in HCE cells that is partially mediated by ROS signaling, while PKC pathway was not involved in EGF-stimulated corneal cell proliferation and migration. EGF promotes human corneal cell proliferation and migration both in physiology and disease conditions, and ROS, MAPK/ERK and PKC pathways play different roles in these processes. PMID:26567598

  20. NDRG1 links p53 with proliferation-mediated centrosome homeostasis and genome stability

    PubMed Central

    Croessmann, Sarah; Wong, Hong Yuen; Zabransky, Daniel J.; Chu, David; Mendonca, Janet; Sharma, Anup; Mohseni, Morassa; Rosen, D. Marc; Scharpf, Robert B.; Cidado, Justin; Cochran, Rory L.; Parsons, Heather A.; Dalton, W. Brian; Erlanger, Bracha; Button, Berry; Cravero, Karen; Kyker-Snowman, Kelly; Beaver, Julia A.; Kachhap, Sushant; Hurley, Paula J.; Lauring, Josh; Park, Ben Ho

    2015-01-01

    The tumor protein 53 (TP53) tumor suppressor gene is the most frequently somatically altered gene in human cancers. Here we show expression of N-Myc down-regulated gene 1 (NDRG1) is induced by p53 during physiologic low proliferative states, and mediates centrosome homeostasis, thus maintaining genome stability. When placed in physiologic low-proliferating conditions, human TP53 null cells fail to increase expression of NDRG1 compared with isogenic wild-type controls and TP53 R248W knockin cells. Overexpression and RNA interference studies demonstrate that NDRG1 regulates centrosome number and amplification. Mechanistically, NDRG1 physically associates with γ-tubulin, a key component of the centrosome, with reduced association in p53 null cells. Strikingly, TP53 homozygous loss was mutually exclusive of NDRG1 overexpression in over 96% of human cancers, supporting the broad applicability of these results. Our study elucidates a mechanism of how TP53 loss leads to abnormal centrosome numbers and genomic instability mediated by NDRG1. PMID:26324937

  1. DNA Replication Licensing Affects Cell Proliferation or Endoreplication in a Cell Type–Specific Manner

    PubMed Central

    del Mar Castellano, María; Boniotti, María Beatrice; Caro, Elena; Schnittger, Arp; Gutierrez, Crisanto

    2004-01-01

    In eukaryotic cells, the function of DNA replication licensing components (Cdc6 and Cdt1, among others) is crucial for cell proliferation and genome stability. However, little is known about their role in whole organisms and whether licensing control interfaces with differentiation and developmental programs. Here, we study Arabidopsis thaliana CDT1, its regulation, and the consequences of overriding licensing control. The availability of AtCDT1 is strictly regulated at two levels: (1) at the transcription level, by E2F and growth-arresting signals, and (2) posttranscriptionally, by CDK phosphorylation, a step that is required for its proteasome-mediated degradation. We also show that CDC6 and CDT1 are key targets for the coordination of cell proliferation, differentiation, and development. Indeed, altered CDT1 or CDC6 levels have cell type–specific effects in developing Arabidopsis plants: in leaf cells competent to divide, cell proliferation is stimulated, whereas in cells programmed to undergo differentiation-associated endoreplication rounds, extra endocycles are triggered. Thus, we propose that DNA replication licensing control is critical for the proper maintenance of proliferative potential, developmental programs, and morphogenetic patterns. PMID:15316110

  2. Simultaneous cross-linking of CD6 and CD28 induces cell proliferation in resting T cells.

    PubMed Central

    Osorio, L M; Rottenberg, M; Jondal, M; Chow, S C

    1998-01-01

    In the present study, we showed that simultaneous ligation of the monoclonal antibodies (mAb) against CD6 and CD28 induces T-cell proliferation in purified resting T lymphocytes in the absence of T-cell receptor (TCR) occupancy. No cell proliferation was observed when the mAb were cross-linked alone or used simultaneously in the soluble form. T-cell proliferation mediated through CD6/CD28 is accompanied by the up-regulation of interleukin-2 (IL-2) mRNA and expression of IL-2 receptors on the cell surface. In the presence of IL-2-neutralizing mAb the proliferative response of the T cell induced through CD6/CD28 was inhibited dose dependently. Cross-linking mAb to CD6 and CD28 alone or together did not down-regulate the CD3/TCR complex. T-cell proliferation mediated through CD6/CD28 was only partially blocked by the immunosuppressive drug, cyclosporin A (CsA), whereas anti-CD28-induced T-cell proliferation in the presence of the phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA), was unaffected. In sharp contrast T-cell proliferation mediated by anti-CD6 in the presence of TPA was efficiently blocked by CsA. In addition, two protein kinase C (PKC) inhibitors, GF 109203X and H-7 dose-dependently inhibited T-cell proliferation mediated through CD6/CD28, suggesting that PKC activation may be involved. Furthermore, there was a marked differential dose-dependent inhibitory effect of the PKC inhibitors on T-cell proliferation mediated by the co-ligation of anti-CD6 or anti-CD28 in the presence of anti-CD3, with the former being more sensitive to PKC inhibition. Taken collectively, our results suggest that T-cell activation can occur through an antigen-independent pathway by cross-linking the accessory molecules, CD6 and CD28, and that these two cell surface antigens may have distinct signalling pathways. Images Figure 3 PMID:9640246

  3. Micronutrient supplementation and T-cell mediated immune responses in patients with tuberculosis in Tanzania

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Limited studies exist regarding whether incorporating micronutrient supplements during tuberculosis (TB) treatment may improve cell-mediated immune response. We examine the effect of micronutrient supplementation on lymphocyte proliferation response to mycobacteria or T cell mitogens in a randomize...

  4. Autocrine regulation of human urothelial cell proliferation and migration during regenerative responses in vitro

    SciTech Connect

    Varley, Claire; Hill, Gemma; Pellegrin, Stephanie; Shaw, Nicola J.; Selby, Peter J.; Trejdosiewicz, Ludwik K.; Southgate, Jennifer . E-mail: js35@york.ac.uk

    2005-05-15

    Regeneration of the urothelium is rapid and effective in order to maintain a barrier to urine following tissue injury. Whereas normal human urothelial (NHU) cells are mitotically quiescent and G0 arrested in situ, they rapidly enter the cell cycle upon seeding in primary culture and show reversible growth arrest at confluency. We have used this as a model to investigate the role of EGF receptor signaling in urothelial regeneration and wound-healing. Transcripts for HER-1, HER-2, and HER-3 were expressed by quiescent human urothelium in situ. Expression of HER-1 was upregulated in proliferating cultures, whereas HER-2 and HER-3 were more associated with a growth-arrested phenotype. NHU cells could be propagated in the absence of exogenous EGF, but autocrine signaling through HER-1 via the MAPK and PI3-kinase pathways was essential for proliferation and migration during urothelial wound repair. HB-EGF was expressed by urothelium in situ and HB-EGF, epiregulin, TGF-{alpha}, and amphiregulin were expressed by proliferating NHU cells. Urothelial wound repair in vitro was attenuated by neutralizing antibodies against HER-1 ligands, particularly amphiregulin. By contrast, the same ligands applied exogenously promoted migration, but inhibited proliferation, implying that HER-1 ligands provoke differential effects in NHU cells depending upon whether they are presented as soluble or juxtacrine ligands. We conclude that proliferation and migration during wound healing in NHU cells are mediated through an EGFR autocrine signalling loop and our results implicate amphiregulin as a key mediator.

  5. Spontaneous Proliferation of H2M-/- CD4 T Cells Results in Unusual Acute Hepatocellular Necrosis

    PubMed Central

    Do, Jeong-su; Baldwin, William M.; Min, Booki

    2014-01-01

    Naïve CD4 T cells are triggered to undergo spontaneous proliferation, a proliferative response induced in response to homeostatic stimulation, when exposed to severe lymphopenic environments. They spontaneously acquire proinflammatory effector phenotypes, playing a major role in inducing chronic inflammation in the intestine that is believed to be induced by T cell recognition of commensal antigens. While the antigens inducing the T cell responses and inflammation are being extensively investigated, the role of clonality of T cells involved in this process remains poorly understood. In this study, we utilized naïve CD4 T cells isolated from B6 H2M−/− mice, in which MHCII molecules are complexed with a single CLIP molecule, and examined spontaneous proliferation and intestinal inflammation of CD4 T cells expressing limited T cell receptor repertoire diversity. We found that H2M−/− CD4 T cells undergo robust spontaneous proliferation, differentiate into IFNγ-producing Th1 type effector cells, and, most unexpectedly, induce severe acute hepatocellular necrosis. T cell interaction with MHCII molecule on cells of hematopoietic origin was essential to induce the pathology. Interestingly, B cells are fully capable of preventing necrotic inflammation via IL-10-independent and B7-H1-dependent mechanism. This could be a useful animal model to examine T cell-mediated liver inflammation and B cell-mediated immune regulation. PMID:25313460

  6. Dual effect of LPS on murine myeloid leukemia cells: Pro-proliferation and anti-proliferation.

    PubMed

    Yu, Lingling; Zhao, Yingmin; Gu, Xin; Wang, Jijun; Pang, Lei; Zhang, Yanqing; Li, Yaoyao; Jia, Xiaoqin; Wang, Xin; Gu, Jian; Yu, Duonan

    2016-06-10

    Modification of the bone marrow microenvironment is considered as a promising strategy to control leukemic cell proliferation, diseases progression and relapse after treatment. However, due to the diversity and complexity of the cellular and molecular compartments in the leukemic microenvironment, it is extremely difficult to dissect the role of each individual molecule or cell type in vivo. Here we established an in vitro system to dissect the role of lipopolysaccharide (LPS), stromal cells and endothelial cells in the growth of mouse myeloid tumor cells and B-lymphoma cells. We found that either LPS or bone marrow stromal cells as a feeder layer in culture is required for the proliferation of myeloid tumor cells. Surprisingly, the growth of myeloid leukemic cells on stromal cells is strongly inhibited when coupled with LPS in culture. This opposing effect of LPS, a complete switch from pro-proliferation to antitumor growth is due, at least in part, to the rapidly increased production of interleukin 12, Fas ligand and tissue inhibitor of metalloproteinases-2 from stromal cells stimulated by LPS. These results demonstrate that LPS can either facilitate or attenuate tumor cell proliferation, thus changing the disease course of myeloid leukemias through its direct effect or modulation of the tumor microenvironment. PMID:26968634

  7. Human regulatory T cells suppress proliferation of B lymphoma cells.

    PubMed

    Grygorowicz, Monika Anna; Biernacka, Marzena; Bujko, Mateusz; Nowak, Eliza; Rymkiewicz, Grzegorz; Paszkiewicz-Kozik, Ewa; Borycka, Ilona Sara; Bystydzienski, Zbigniew; Walewski, Jan; Markowicz, Sergiusz

    2016-08-01

    Activated regulatory T cells (Tregs) suppress proliferation and differentiation of normal B cells. In our study, allogeneic polyclonal CD4 (+) CD25 (+) Tregs and CD4 (+) CD25 (+) CD127(lo)Tregs expanded in vitro in the presence of rapamycin and low dose IL-2 suppressed proliferation of 11 out of 12 established lymphoma B-cell lines. The effect of expanded CD4 (+) CD25 (+) Tregs on survival of freshly isolated lymphoma B cells maintained in culture with soluble multimeric CD40L and IL-4 was variable across lymphoma entities. The survival of freshly isolated follicular lymphoma cells usually decreased in cocultures with CD4 (+) CD25 (+) Tregs. Treg effect on chronic lymphocytic leukemia/small lymphocytic lymphoma cells ranged from suppression to help in individual patients. CD4 (+) CD25 (+) Tregs or CD4 (+) CD25 (+) CD127(lo)Tregs expanded ex vivo with rapamycin could be used to suppress regrowth of residual lymphoma after autologous hematopoietic cell transplantation (HCT), and to counteract both graft-versus-host disease and lymphoma re-growth after allogeneic HCT in select patients with lymphoma susceptible to the regulation by Tregs. PMID:26758248

  8. Knockdown of integrin α3β1 expression induces proliferation and migration of non-small cell lung cancer cells.

    PubMed

    Yoon, Hyun Jae; Cho, Young-Rak; Joo, Ji-Hye; Seo, Dong-Wan

    2013-02-01

    Integrin α3β1 is expressed on many types of cancer cells and can regulate tumor growth and progression. In the present study, we examined the roles and molecular mechanism of integrin α3β1 in modulating cell proliferation and migration of p53-deficient non-small cell lung cancer (NSCLC) cells. Reduced expression of integrin α3 by RNA silencing clearly induces cell proliferation and migration in H1299 cells, compared with those in control cells. Enhanced proliferation in integrin α3-silenced cells is mediated by upregulation and nuclear localization of cyclin-dependent kinases, and these effects require the activation of Akt and ERK as evidenced by treatment with LY294002 and PD98059, respectively. Furthermore, suppression of integrin α3 expression induces the expression of nuclear factor-κB and Bcl-2 as well as epidermal growth factor receptor, which are positively correlated with cell proliferation and survival. In contrast, increase in cell migration of integrin α3-silenced cells is found to be independent of Akt or ERK signaling pathways. Collectively, these findings suggest that integrin α3β1 plays pivotal roles in regulating cell proliferation and migration that enhance the invasive type of p53-deficient NSCLC cells. PMID:23233127

  9. Apoptosis and proliferation of oligodendrocyte progenitor cells in the irradiated rodent spinal cord

    SciTech Connect

    Atkinson, Shelley L.; Li Yuqing; Wong, C. Shun . E-mail: shun.wong@sw.ca

    2005-06-01

    Purpose: Oligodendrocytes undergo early apoptosis after irradiation. The aim of this study was to determine the relationship between oligodendroglial apoptosis and proliferation of oligodendrocyte progenitor cells (OPC) in the irradiated central nervous system. Methods and Materials: Adult rats and p53 transgenic mice were given single doses of 2 Gy, 8 Gy, or 22 Gy to the cervical spinal cord. Apoptosis was assessed using TUNEL (Tdt-mediated dUTP terminal nick-end labeling) staining or by examining nuclear morphology. Oligodendrocyte progenitor cells were identified with an NG2 antibody or by in situ hybridization for platelet-derived growth factor receptor {alpha}. Proliferation of OPC was assessed by in vivo bromodeoxyuridine (BrdU) labeling and subsequent immunohistochemistry. Because radiation-induced apoptosis of oligodendroglial cells is p53 dependent, p53 transgenic mice were used to study the relationship between apoptosis and cell proliferation. Results: Oligodendrocyte progenitor cells underwent apoptosis within 24 h of irradiation in the rat. That did not result in a change in OPC density at 24 h. Oligodendrocyte progenitor cell density was significantly reduced by 2-4 weeks, but showed recovery by 6 weeks after irradiation. An increase in BrdU-labeled cells was observed at 2 weeks after 8 Gy or 22 Gy, and proliferating cells in the rat spinal cord were immunoreactive for NG2. The mouse spinal cord showed a similar early cell proliferation after irradiation. No difference was observed in the proliferation response in the spinal cord of p53 -/- mice compared with wild type animals. Conclusions: Oligodendroglial cells undergo early apoptosis and OPC undergo early proliferation after ionizing radiation. However, apoptosis is not likely to be the trigger for early proliferation of OPC in the irradiated central nervous system.

  10. Regulation of global gene expression and cell proliferation by APP.

    PubMed

    Wu, Yili; Zhang, Si; Xu, Qin; Zou, Haiyan; Zhou, Weihui; Cai, Fang; Li, Tingyu; Song, Weihong

    2016-01-01

    Down syndrome (DS), caused by trisomy of chromosome 21, is one of the most common genetic disorders. Patients with DS display growth retardation and inevitably develop characteristic Alzheimer's disease (AD) neuropathology, including neurofibrillary tangles and neuritic plaques. The expression of amyloid precursor protein (APP) is increased in both DS and AD patients. To reveal the function of APP and elucidate the pathogenic role of increased APP expression in DS and AD, we performed gene expression profiling using microarray method in human cells overexpressing APP. A set of genes are significantly altered, which are involved in cell cycle, cell proliferation and p53 signaling. We found that overexpression of APP inhibits cell proliferation. Furthermore, we confirmed that the downregulation of two validated genes, PSMA5 and PSMB7, inhibits cell proliferation, suggesting that the downregulation of PSMA5 and PSMB7 is involved in APP-induced cell proliferation impairment. Taken together, this study suggests that APP regulates global gene expression and increased APP expression inhibits cell proliferation. Our study provides a novel insight that APP overexpression may contribute to the growth impairment in DS patients and promote AD pathogenesis by inhibiting cell proliferation including neural stem cell proliferation and neurogenesis. PMID:26936520

  11. Regulation of global gene expression and cell proliferation by APP

    PubMed Central

    Wu, Yili; Zhang, Si; Xu, Qin; Zou, Haiyan; Zhou, Weihui; Cai, Fang; Li, Tingyu; Song, Weihong

    2016-01-01

    Down syndrome (DS), caused by trisomy of chromosome 21, is one of the most common genetic disorders. Patients with DS display growth retardation and inevitably develop characteristic Alzheimer’s disease (AD) neuropathology, including neurofibrillary tangles and neuritic plaques. The expression of amyloid precursor protein (APP) is increased in both DS and AD patients. To reveal the function of APP and elucidate the pathogenic role of increased APP expression in DS and AD, we performed gene expression profiling using microarray method in human cells overexpressing APP. A set of genes are significantly altered, which are involved in cell cycle, cell proliferation and p53 signaling. We found that overexpression of APP inhibits cell proliferation. Furthermore, we confirmed that the downregulation of two validated genes, PSMA5 and PSMB7, inhibits cell proliferation, suggesting that the downregulation of PSMA5 and PSMB7 is involved in APP-induced cell proliferation impairment. Taken together, this study suggests that APP regulates global gene expression and increased APP expression inhibits cell proliferation. Our study provides a novel insight that APP overexpression may contribute to the growth impairment in DS patients and promote AD pathogenesis by inhibiting cell proliferation including neural stem cell proliferation and neurogenesis. PMID:26936520

  12. Knockdown of DDX46 inhibits proliferation and induces apoptosis in esophageal squamous cell carcinoma cells.

    PubMed

    Li, Bin; Li, Yu-Min; He, Wen-Ting; Chen, Hao; Zhu, Hong-Wen; Liu, Tao; Zhang, Jian-Hua; Song, Tie-Niu; Zhou, Ya-Li

    2016-07-01

    Esophageal squamous cell carcinoma (ESCC) is the most common type of esophageal carcinoma and remains the leading cause of cancer-related death worldwide. DEAD-box RNA helicases play critical roles in cellular metabolism and in many cases have been implicated in cellular proliferation and neoplastic transformation. DDX46 belongs to DEAD-box helicase family, the expression pattern of DDX46 in ESCC tissues and the biologic role in ESCC progression have not been implicated previously. In this study, DDX46 expression in human ESCC and adjacent normal tissues were explored using immunohistochemistry, and ESCC cell lines compared with normal esophageal epithelium cell were quantified using real‑time PCR. Next, lentivirus-mediated RNA interference was applied to silence DDX46 in TE-1 and Eca-109 cells. Cell growth was monitored using high content screening. Cell viability was measured by MTT assay. Cell colony-forming capacity was measured by colony formation assay. Cell cycle progression and apoptosis were determined by flow cytometry. Further, the stress and apoptosis signaling antibody array kit was used to detect the changes of signaling molecules in TE-1 cells after DDX46 knockdown. We found that DDX46 was significantly upregulated in ESCC tissues and cells compared with normal tissues and cells. DDX46 knockdown led to decreased proliferation and increased apoptosis in TE-1 and Eca-109 cells. Moreover, DDX46 silencing resulted in apoptotic induction via decreased phosphorylation of Akt and IκBα, as well as negative regulation of NF-κB signaling. In conclusion, these results demonstrate that DDX46 knockdown inhibited cell growth, and induced apoptosis, suggest that DDX46 is critical for ESCC cells proliferation. In addition, this study provides a foundation for further study into the clinical potential diagnosis and novel therapeutic target for ESCC. PMID:27176873

  13. Electrospun poly(ε-caprolactone)-based skin substitutes: In vivo evaluation of wound healing and the mechanism of cell proliferation.

    PubMed

    Augustine, Robin; Dominic, Edwin Anto; Reju, Indu; Kaimal, Balarama; Kalarikkal, Nandakumar; Thomas, Sabu

    2015-10-01

    In the present study, we have fabricated electrospun poly(ε-caprolactone)-based membranes, characterized and studied the in vivo cell migration and proliferation and wound healing activity. Moreover, we did not seed any cells prior to the animal implantation and we could observe excellent fibroblast attachment and cell proliferation. Further full thickness excision wound on guinea pig completely healed within 35 days. We could reach in an assumption that the enhanced cell proliferation and wound healing might be due to the surface degradation of the polymer under physiological conditions and the formation of functional groups like hydroxyl and carboxyl groups that promoted cell proliferation in a cell adhesion protein mediated mechanism. This study is a novel tissue engineering concept for the reconstruction of a damaged tissue without the in vitro cell seeding and proliferation prior to the in vivo implantation. PMID:25418134

  14. Upregulation of KPNβ1 in gastric cancer cell promotes tumor cell proliferation and predicts poor prognosis.

    PubMed

    Zhu, Jia; Wang, Yingying; Huang, Hua; Yang, Qichang; Cai, Jing; Wang, Qiuhong; Gu, Xiaoling; Xu, Pan; Zhang, Shusen; Li, Manhua; Ding, Haifang; Yang, Lei

    2016-01-01

    KPNβ1, also known as importin β, P97, is reported as one of soluble transport factors that mediates transportion of proteins and RNAs between the nucleus and cytoplasm in cellular process. Recent studies show that KPNβ1 is a tumor gene which is highly expressed in several malignant tumors such as ovarian cancer, cervical tumor, neck cancer, and lung cancer via promoting cell proliferation or inhibiting cell apoptotic pathways. However, the the role of KPNβ1 in gastric cancer remains unclear. In this study, Western blot and immunohistochemistrical analyses showed that KPNβ1 was significantly upregulated in clinical gastric cancer specimens compared with adjacent noncancerous tissues. KPNβ1 was positively correlated with tumor grade, Ki-67, and predicted poor prognosis of gastric cancer. More importantly, through starvation-refeeding model, CCK8 assay, flow cytometry, colony formation assays, the vitro studies demonstrated that KPNβ1 promoted proliferation of gastric cancer cells, while KPNβ1 knockdown led to decreased cell proliferation and arrested cell cycle at G1 phase. Furthermore, our results also indicated that KPNβ1 expression could result in docetaxel resistance. And, KPNβ1 could interact with Stat1, contributed to its nucleus import in gastric cancer cells. These findings provided a novel promising therapeutic targets for clinical treatment against human gastric cancer. PMID:26242264

  15. Simvastatin suppresses breast cancer cell proliferation induced by senescent cells

    PubMed Central

    Liu, Su; Uppal, Harpreet; Demaria, Marco; Desprez, Pierre-Yves; Campisi, Judith; Kapahi, Pankaj

    2015-01-01

    Cellular senescence suppresses cancer by preventing the proliferation of damaged cells, but senescent cells can also promote cancer though the pro-inflammatory senescence-associated secretory phenotype (SASP). Simvastatin, an HMG-coA reductase inhibitor, is known to attenuate inflammation and prevent certain cancers. Here, we show that simvastatin decreases the SASP of senescent human fibroblasts by inhibiting protein prenylation, without affecting the senescent growth arrest. The Rho family GTPases Rac1 and Cdc42 were activated in senescent cells, and simvastatin reduced both activities. Further, geranylgeranyl transferase, Rac1 or Cdc42 depletion reduced IL-6 secretion by senescent cells. We also show that simvastatin mitigates the effects of senescent conditioned media on breast cancer cell proliferation and endocrine resistance. Our findings identify a novel activity of simvastatin and mechanism of SASP regulation. They also suggest that senescent cells, which accumulate after radio/chemo therapy, promote endocrine resistance in breast cancer and that simvastatin might suppress this resistance. PMID:26658759

  16. HDAC6 promotes cell proliferation and confers resistance to temozolomide in glioblastoma.

    PubMed

    Wang, Zhihao; Hu, Pengchao; Tang, Fang; Lian, Haiwei; Chen, Xiong; Zhang, Yingying; He, Xiaohua; Liu, Wanhong; Xie, Conghua

    2016-08-28

    Histone deacetylases are considered to be among the most promising targets in drug development for cancer therapy. Histone deacetylase 6 (HDAC6) is a unique cytoplasmic enzyme that regulates many biological processes involved in tumorigenesis through its deacetylase and ubiquitin-binding activities. Here, we report that HDAC6 is overexpressed in glioblastoma tissues and cell lines. Overexpression of HDAC6 promotes the proliferation and spheroid formation of glioblastoma cells. HDAC6 overexpression confers resistance to temozolomide (TMZ) mediated cell proliferation inhibition and apoptosis induction. Conversely, knockdown of HDAC6 inhibits cell proliferation, impairs spheroid formation and sensitizes glioblastoma cells to TMZ. The inhibition of HDAC6 deacetylase activity by selective inhibitors inhibits the proliferation of glioblastoma cells and induces apoptosis. HDAC6 selective inhibitors can sensitize glioblastoma cells to TMZ. Moreover, we showed that HDAC6 mediated EGFR stabilization might partly account for its oncogenic role in glioblastoma. TMZ resistant glioblastoma cells showed higher expression of HDAC6 and more activation of EGFR. HDAC6 inhibitors decrease EGFR protein levels and impair the activation of the EGFR pathway. Taken together, our results suggest that the inhibition of HDAC6 may be a promising strategy for the treatment of glioblastoma. PMID:27267806

  17. A Septin Requirement Differentiates Autonomous- and Contact-Facilitated T Cell Proliferation

    PubMed Central

    Mujal, Adriana M.; Gilden, Julia K.; Gérard, Audrey; Kinoshita, Makoto; Krummel, Matthew F.

    2015-01-01

    T cell proliferation is initiated by T cell antigen receptor (TCR) triggering and/or by soluble growth factors. In characterizing T cells lacking the septin cytoskeleton, we found that successful cell division has discrete septin-dependent and -independent pathways. Septin-deficient T cells failed cytokinesis when prompted by pharmacological activation or cytokines. In contrast, cell division was independent of septins when cell-cell contacts, such as those from antigen-presenting cells, provided a niche. This septin-independent pathway was mediated by phosphatidylinositol-3-OH kinase activation through a combination of integrins and co-stimulatory signals. We could differentiate cytokine- versus antigen-driven expansion in vivo and thus demonstrate that targeting septins has strong potential to moderate detrimental bystander or homeostatic cytokine-driven proliferation without influencing expansion driven by conventional antigen-presentation. PMID:26692174

  18. Direct interactions with both p27 and Cdk2 regulate Spy1-mediated proliferation in vivo and in vitro.

    PubMed

    Al Sorkhy, Mohammad; Fifield, Bre-Anne; Myers, Dorothy; Porter, Lisa A

    2016-01-01

    Families of cyclin-like proteins have emerged that bind and activate cyclin dependent kinases (Cdk)s, directing the phosphorylation of noncanonical Cdk substrates. One of these proteins, Spy1, has demonstrated the unique ability to directly bind and activate both Cdk1 and Cdk2, as well as binding and promoting the degradation of at least one Cdk inhibitor, p27(Kip1). Spy1 accelerates somatic cell growth and proliferation and is implicated in a number of human cancers including the breast, brain and liver. Herein we isolate key residues mediating the direct interaction with p27. We use mutants of Spy1 to determine the physiological role of direct interactions with distinct binding partners Cdk2 and p27. We demonstrate that disrupting the direct interaction with either Spy1 binding partner decreased endogenous activity of Cdk2, as well as Spy1-mediated proliferation. However, only the direct interaction with p27 was essential for Spy1-mediated effects on p27 stability. In vivo neither mutation completely prevented tumorigenesis, although each mutation slowed the rate of Spy1-mediated tumorigenesis and decreased overall tumor volumes. This work supports the conclusion that direct interaction with both p27 and Cdk2 contribute to Spy1-mediated effects on cell growth. It is important to elucidate the dynamics of these interactions and to consider these data when assessing functional outcomes. PMID:26771716

  19. Cholinergic Enhancement of Cell Proliferation in the Postnatal Neurogenic Niche of the Mammalian Spinal Cord

    PubMed Central

    Corns, Laura F.; Atkinson, Lucy; Daniel, Jill; Edwards, Ian J.; New, Lauryn

    2015-01-01

    Abstract The region surrounding the central canal (CC) of the spinal cord is a highly plastic area, defined as a postnatal neurogenic niche. Within this region are ependymal cells that can proliferate and differentiate to form new astrocytes and oligodendrocytes following injury and cerebrospinal fluid contacting cells (CSFcCs). The specific environmental conditions, including the modulation by neurotransmitters that influence these cells and their ability to proliferate, are unknown. Here, we show that acetylcholine promotes the proliferation of ependymal cells in mice under both in vitro and in vivo conditions. Using whole cell patch clamp in acute spinal cord slices, acetylcholine directly depolarized ependymal cells and CSFcCs. Antagonism by specific nicotinic acetylcholine receptor (nAChR) antagonists or potentiation by the α7 containing nAChR (α7*nAChR) modulator PNU 120596 revealed that both α7*nAChRs and non‐α7*nAChRs mediated the cholinergic responses. Using the nucleoside analogue EdU (5‐ethynyl‐2'‐deoxyuridine) as a marker of cell proliferation, application of α7*nAChR modulators in spinal cord cultures or in vivo induced proliferation in the CC region, producing Sox‐2 expressing ependymal cells. Proliferation also increased in the white and grey matter. PNU 120596 administration also increased the proportion of cells coexpressing oligodendrocyte markers. Thus, variation in the availability of acetylcholine can modulate the rate of proliferation of cells in the ependymal cell layer and white and grey matter through α7*nAChRs. This study highlights the need for further investigation into how neurotransmitters regulate the response of the spinal cord to injury or during aging. Stem Cells 2015;33:2864–2876 PMID:26038197

  20. Telmisartan Induced Inhibition of Vascular Cell Proliferation beyond Angiotensin Receptor Blockade and PPARγ Activation

    PubMed Central

    Yamamoto, Koichi; Ohishi, Mitsuru; Ho, Christopher; Kurtz, Theodore W; Rakugi, Hiromi

    2010-01-01

    We investigated the ability of ARBs with PPARγ agonist activity (telmisartan and irbesartan), and ARBs devoid of PPARγ agonist activity (eprosartan and valsartan), to inhibit vascular cell proliferation studied in the absence of angiotensin II stimulation. Telmisartan and to a lesser extent irbesartan, inhibited proliferation of human aortic vascular smooth muscle cells in a dose dependent fashion whereas eprosartan and valsartan did not. To investigate the role of PPARγ in the antiproliferative effects of telmisartan, we studied genetically engineered NIH3T3 cells that express PPARγ. Pioglitazone inhibited proliferation of NIH3T3 cells expressing PPARγ, but had little effect on control NIH3T3 cells that lack PPARγ. In contrast, telmisartan inhibited proliferation equally in NIH3T3 with and without PPARγ. Valsartan failed to inhibit proliferation of either cell line. In addition, telmisartan inhibited proliferation equally in aortic smooth muscle cells derived from mice with targeted knockout of PPARγ in smooth muscle and from control mice whereas valsartan had no effect on cell proliferation. Telmisartan but not valsartan, reduced phosphorylation of AKT but not ERK otherwise induced by exposure to serum of either quiescent human smooth muscle cells, quiescent mice smooth muscle cells lacking PPARγ or quiescent CHO-K1 cells lacking AT1 receptor. In summary, the antiproliferative effect of telmisartan in the absence of exogenously supplemented angiotensin II involve more than just AT1 receptor blockade and do not require activation of PPARγ. It might be postulated that inhibition of AKT activation is a mechanism mediating the antiproliferative effects of telmisartan including in cells lacking AT1 receptors or PPARγ. PMID:19822796

  1. EGFR-mediated Akt and MAPKs signal pathways play a crucial role in patulin-induced cell proliferation in primary murine keratinocytes via modulation of Cyclin D1 and COX-2 expression.

    PubMed

    Alam, Shamshad; Pal, Anu; Kumar, Rahul; Dwivedi, Premendra D; Das, Mukul; Ansari, Kausar M

    2014-12-01

    Patulin (PAT), a present day major contaminant of commercial apple and apple products is reported to be carcinogenic, embryotoxic, and immunotoxic. While oral and inhalation are considered to be the most prevalent routes of exposure to this toxin, exposure through skin is now being extensively investigated. Our previous study showed that short-term dermal exposure to PAT resulted in toxicological injury to the skin, while long-term exposure induced skin tumorigenesis. In this study, we explore the mechanism involve in proliferation of mouse keratinocytes by PAT. Our study revealed that PAT rapidly induces phosphorylation of EGFR, activation of the Ras/MAPKs, and Akt pathways. This in-turn leads to the activation of NF-κB/AP-1 transcription factors which then binds to the promoter region of the cell growth regulatory genes Cyclin D1 and COX-2 inducing their expression leading ultimately to PMKs proliferation. Inhibition of EGFR or the Ras/MAPKs, PI3/Akt pathways with different pharmacological inhibitors or knockdown of NF-κB, c-jun, c-fos, Cyclin D1, and COX-2 with siRNA inhibited PAT-induced PMKs proliferation. PMID:23813870

  2. FOG-2 mediated recruitment of the NuRD complex regulates cardiomyocyte proliferation during heart development.

    PubMed

    Garnatz, Audrey S; Gao, Zhiguang; Broman, Michael; Martens, Spencer; Earley, Judy U; Svensson, Eric C

    2014-11-01

    FOG-2 is a multi-zinc finger protein that binds the transcriptional activator GATA4 and modulates GATA4-mediated regulation of target genes during heart development. Our previous work has demonstrated that the Nucleosome Remodeling and Deacetylase (NuRD) complex physically interacts with FOG-2 and is necessary for FOG-2 mediated repression of GATA4 activity in vitro. However, the relevance of this interaction for FOG-2 function in vivo has remained unclear. In this report, we demonstrate the importance of FOG-2/NuRD interaction through the generation and characterization of mice homozygous for a mutation in FOG-2 that disrupts NuRD binding (FOG-2(R3K5A)). These mice exhibit a perinatal lethality and have multiple cardiac malformations, including ventricular and atrial septal defects and a thin ventricular myocardium. To investigate the etiology of the thin myocardium, we measured the rate of cardiomyocyte proliferation in wild-type and FOG-2(R3K5A) developing hearts. We found cardiomyocyte proliferation was reduced by 31±8% in FOG-2(R3K5A) mice. Gene expression analysis indicated that the cell cycle inhibitor Cdkn1a (p21(cip1)) is up-regulated 2.0±0.2-fold in FOG-2(R3K5A) hearts. In addition, we demonstrate that FOG-2 can directly repress the activity of the Cdkn1a gene promoter, suggesting a model by which FOG-2/NuRD promotes ventricular wall thickening by repression of this cell cycle inhibitor. Consistent with this notion, the genetic ablation of Cdkn1a in FOG-2(R3K5A) mice leads to an improvement in left ventricular function and a partial rescue of left ventricular wall thickness. Taken together, our results define a novel mechanism in which FOG-2/NuRD interaction is required for cardiomyocyte proliferation by directly down-regulating the cell cycle inhibitor Cdkn1a during heart development. PMID:25196150

  3. FOG-2 Mediated Recruitment of the NuRD Complex Regulates Cardiomyocyte Proliferation during Heart Development

    PubMed Central

    Garnatz, Audrey S.; Gao, Zhiguang; Broman, Michael; Martens, Spencer; Earley, Judy U.; Svensson, Eric C.

    2014-01-01

    SUMMARY FOG-2 is a multi-zinc finger protein that binds the transcriptional activator GATA4 and modulates GATA4-mediated regulation of target genes during heart development. Our previous work has demonstrated that the Nucleosome Remodeling and Deacetylase (NuRD) complex physically interacts with FOG-2 and is necessary for FOG-2 mediated repression of GATA4 activity in vitro. However, the relevance of this interaction for FOG-2 function in vivo has remained unclear. In this report, we demonstrate the importance of FOG-2/NuRD interaction through the generation and characterization of mice homozygous for a mutation in FOG-2 that disrupts NuRD binding (FOG-2R3K5A). These mice exhibit a perinatal lethality and have multiple cardiac malformations, including ventricular and atrial septal defects and a thin ventricular myocardium. To investigate the etiology of the thin myocardium, we measured the rate of cardiomyocyte proliferation in wild-type and FOG-2R3K5A developing hearts. We found cardiomyocyte proliferation was reduced by 31 ± 8% in FOG-2R3K5A mice. Gene expression analysis indicated that the cell cycle inhibitor Cdkn1a (p21cip1) is up-regulated 2.0 ± 0.2-fold in FOG-2R3K5A hearts. In addition, we demonstrate that FOG-2 can directly repress the activity of the Cdkn1a gene promoter, suggesting a model by which FOG-2/NuRD promotes ventricular wall thickening by repression of this cell cycle inhibitor. Consistent with this notion, the genetic ablation of Cdkn1a in FOG-2R3K5A mice leads to an improvement in left ventricular function and a partial rescue of left ventricular wall thickness. Taken together, our results define a novel mechanism in which FOG-2/NuRD interaction is required for cardiomyocyte proliferation by directly down-regulating the cell cycle inhibitor Cdkn1a during heart development. PMID:25196150

  4. Gallic acid suppresses cell viability, proliferation, invasion and angiogenesis in human glioma cells

    PubMed Central

    Lu, Yong; Jiang, Feng; Jiang, Hao; Wu, Kalina; Zheng, Xuguang; Cai, Yizhong; Katakowski, Mark; Chopp, Michael; To, Shing-Shun Tony

    2010-01-01

    Gallic acid, an organic acid, also known as 3,4,5-trihydroxybenzoic acid, is cytotoxic against certain cancer cells, without harming normal cells. The objective of this study is to evaluate whether gallic acid can inhibit glioma cell viability, proliferation, invasion and reduce glioma cell mediated angiogenesis. Treatment of U87 and U251n glioma cells with gallic acid inhibited cell viability in a dose- and time-dependent manner. BrdU and tube formation assays indicated that gallic acid significantly decreased glioma cell proliferation and tube formation in mouse brain endothelial cells, respectively. In addition, gallic acid decreased U87 cell invasion in vitro. Western blot analysis showed that expression of ADAM17, p-Akt and p-Erk was suppressed by gallic acid in both U87 and U251n cell lines. These data suggest that suppression of ADAM17 and downregulation of PI3K/Akt and Ras/MAPK signaling pathways may contribute to gallic acid-induced decrease of invasiveness. Gallic acid may be a valuable candidate for treatment of brain tumor. PMID:20553913

  5. Dehydroepiandrosterone inhibits cell proliferation and improves viability by regulating S phase and mitochondrial permeability in primary rat Leydig cells

    PubMed Central

    LIU, LIN; WANG, DIAN; LI, LONGLONG; DING, XIAO; MA, HAITIAN

    2016-01-01

    Dehydroepiandrosterone (DHEA) is widely used as a nutritional supplement and exhibits putative anti-aging properties. However, the molecular basis of the actions of DHEA, particularly on the biological characteristics of target cells, remain unclear. The aim of the current study was to investigate the effects of DHEA on cell viability, cell proliferation, cell cycle and mitochondrial function in primary rat Leydig cells. Adult Leydig cells were purified by Percoll gradient centrifugation, and cell proliferation was detected using a Click-iT® EdU Assay kit and cell cycle assessment performed using flow cytometry. Mitochondrial membrane potential was detected using JC-1 staining assay. The results of the current study demonstrate that DHEA decreased cell proliferation in a dose-dependent manner, whereas it improved cell viability in a time-dependent and dose-dependent manner. Flow cytometry analysis demonstrated that DHEA treatment increased the S phase cell population and decreased the G2/M cell population. Cyclin A and CDK2 mRNA levels were decreased in primary rat Leydig cells following DHEA treatment. DHEA treatment decreased the transmembrane electrical gradient in primary Leydig cells, whereas treatment significantly increased succinate dehydrogenase activity. These results indicated that DHEA inhibits primary rat Leydig cell proliferation by decreasing cyclin mRNA level, whereas it improves cells viability by modulating the permeability of the mitochondrial membrane and succinate dehydrogenase activity. These findings may demonstrate an important molecular mechanism by which DHEA activity is mediated. PMID:27220727

  6. Dehydroepiandrosterone inhibits cell proliferation and improves viability by regulating S phase and mitochondrial permeability in primary rat Leydig cells.

    PubMed

    Liu, Lin; Wang, Dian; Li, Longlong; Ding, Xiao; Ma, Haitian

    2016-07-01

    Dehydroepiandrosterone (DHEA) is widely used as a nutritional supplement and exhibits putative anti‑aging properties. However, the molecular basis of the actions of DHEA, particularly on the biological characteristics of target cells, remain unclear. The aim of the current study was to investigate the effects of DHEA on cell viability, cell proliferation, cell cycle and mitochondrial function in primary rat Leydig cells. Adult Leydig cells were purified by Percoll gradient centrifugation, and cell proliferation was detected using a Click-iT® EdU Assay kit and cell cycle assessment performed using flow cytometry. Mitochondrial membrane potential was detected using JC-1 staining assay. The results of the current study demonstrate that DHEA decreased cell proliferation in a dose‑dependent manner, whereas it improved cell viability in a time‑dependent and dose‑dependent manner. Flow cytometry analysis demonstrated that DHEA treatment increased the S phase cell population and decreased the G2/M cell population. Cyclin A and CDK2 mRNA levels were decreased in primary rat Leydig cells following DHEA treatment. DHEA treatment decreased the transmembrane electrical gradient in primary Leydig cells, whereas treatment significantly increased succinate dehydrogenase activity. These results indicated that DHEA inhibits primary rat Leydig cell proliferation by decreasing cyclin mRNA level, whereas it improves cells viability by modulating the permeability of the mitochondrial membrane and succinate dehydrogenase activity. These findings may demonstrate an important molecular mechanism by which DHEA activity is mediated. PMID:27220727

  7. P44/WDR77 restricts the sensitivity of proliferating cells to TGFβ signaling

    SciTech Connect

    Yi, Pengfei; Gao, Shen; Gu, Zhongping; Huang, Tao; Wang, Zhengxin

    2014-07-18

    Highlights: • P44/WDR77 causes proliferating cells to become non-responsive to TGFβ signaling. • P44/WDR77 down-regulates TβRII and TβR2 expression. • P44/WDR77 down-regulated TGFβ signaling correlates with lung tumorigenesis. - Abstract: We previously reported that a novel WD-40 domain-containing protein, p44/WDR77, drives quiescent epithelial cells to re-enter the cell cycle and plays an essential role for growth of lung and prostate cancer cells. Transforming growth factor beta (TGFβ) signaling is important in the maintenance of non-transformed cells in the quiescent or slowly cycling stage. However, both non-transformed proliferating cells and human cancer cells are non-responsive to endogenous TGFβ signaling. The mechanism by which proliferating cells become refractory to TGFβ inhibition is not well established. Here, we found that silencing p44/WDR77 increased cellular sensitivity to TGFβ signaling and that this was inversely correlated with decreased cell proliferation. Smad2 or 3 phosphorylation, TGFβ-mediated transcription, and TGFβ2 and TGFβ receptor type II (TβRII) expression were dramatically induced by silencing of p44/WDR77. These data support the hypothesis that p44/WDR77 down-regulates the expression of the TGFβ ligand and its receptor, thereby leading to a cellular non-response to TGFβ signaling. Finally, we found that p44/WDR77 expression was correlated with cell proliferation and decreased TGFβ signaling during lung tumorigenesis. Together, these results suggest that p44/WDR77 expression causes the non-sensitivity of proliferating cells to TGFβ signaling, thereby contributing to cellular proliferation during lung tumorigenesis.

  8. Notch-Mediated Cell Adhesion.

    PubMed

    Murata, Akihiko; Hayashi, Shin-Ichi

    2016-01-01

    Notch family members are generally recognized as signaling molecules that control various cellular responses in metazoan organisms. Early fly studies and our mammalian studies demonstrated that Notch family members are also cell adhesion molecules; however, information on the physiological roles of this function and its origin is limited. In this review, we discuss the potential present and ancestral roles of Notch-mediated cell adhesion in order to explore its origin and the initial roles of Notch family members dating back to metazoan evolution. We hypothesize that Notch family members may have initially emerged as cell adhesion molecules in order to mediate multicellularity in the last common ancestor of metazoan organisms. PMID:26784245

  9. Notch-Mediated Cell Adhesion

    PubMed Central

    Murata, Akihiko; Hayashi, Shin-Ichi

    2016-01-01

    Notch family members are generally recognized as signaling molecules that control various cellular responses in metazoan organisms. Early fly studies and our mammalian studies demonstrated that Notch family members are also cell adhesion molecules; however, information on the physiological roles of this function and its origin is limited. In this review, we discuss the potential present and ancestral roles of Notch-mediated cell adhesion in order to explore its origin and the initial roles of Notch family members dating back to metazoan evolution. We hypothesize that Notch family members may have initially emerged as cell adhesion molecules in order to mediate multicellularity in the last common ancestor of metazoan organisms. PMID:26784245

  10. Microenvironmental stimuli for proliferation of functional islet β-cells

    PubMed Central

    2014-01-01

    Diabetes is characterized by high blood glucose level due to either autoimmune destruction of islet β-cells or insufficient insulin secretion or glucose non-responsive production of insulin by β-cells. It is highly desired to replace biological functional β-cells for the treatment of diabetes. Unfortunately, β-cells proliferate with an extremely low rate. This cellular property hinders cell-based therapy for clinical application. Many attempts have been made to develop techniques that allow production of large quantities of clinically relevant islet β-cells in vitro. A line of studies evidently demonstrate that β-cells can proliferate under certain circumstances, giving the hopes for generating and expanding these cells in vitro and transplanting them to the recipient. In this review, we discuss the requirements of microenvironmental stimuli that stimulate β-cell proliferation in cell cultures. We highlight advanced approaches for augmentation of β-cell expansion that have recently emerged in this field. Furthermore, knowing the signaling pathways and molecular mechanisms would enable manipulating cell proliferation and optimizing its insulin secretory function. Thus, signaling pathways involved in the enhancement of cell proliferation are discussed as well. PMID:24594290

  11. Exercise-induced promotion of hippocampal cell proliferation requires beta-endorphin.

    PubMed

    Koehl, M; Meerlo, P; Gonzales, D; Rontal, A; Turek, F W; Abrous, D N

    2008-07-01

    Adult hippocampal neurogenesis is influenced by a variety of stimuli, including exercise, but the mechanisms by which running affects neurogenesis are not yet fully understood. Because beta-endorphin, which is released in response to exercise, increases cell proliferation in vitro, we hypothesized that it could exert a similar effect in vivo and mediate the stimulatory effects of running on neurogenesis. We thus analyzed the effects of voluntary wheel-running on adult neurogenesis (proliferation, differentiation, survival/death) in wild-type and beta-endorphin-deficient mice. In wild-type mice, exercise promoted cell proliferation evaluated by sacrificing animals 24 h after the last 5-bromo-2'-deoxyuridine (BrdU) pulse and by using endogenous cell cycle markers (Ki67 and pH(3)). This was accompanied by an increased survival of 4-wk-old BrdU-labeled cells, leading to a net increase of neurogenesis. Beta-endorphin deficiency had no effect in sedentary mice, but it completely blocked the running-induced increase in cell proliferation; this blockade was accompanied by an increased survival of 4-wk-old cells and a decreased cell death. Altogether, adult neurogenesis was increased in response to exercise in knockout mice. We conclude that beta-endorphin released during running is a key factor for exercise-induced cell proliferation and that a homeostatic balance may regulate the final number of new neurons. PMID:18263701

  12. Estrogen mediated epithelial proliferation in the uterus is directed by stromal Fgf10 and Bmp8a

    PubMed Central

    Chung, Daesuk; Gao, Fei; Jegga, Anil G.; Das, Sanjoy K.

    2014-01-01

    To define endometrial stromal-derived paracrine mediators that participate in estradiol-17β (E2)-induced epithelial proliferation, microarray analysis of gene expression was carried out in mouse uterine epithelial–stromal co-culture systems under the condition of E2 or vehicle (control). Our results demonstrated gene alteration by E2: in epithelial cells, we found up-regulation of 119 genes and down-regulation of 28 genes, while in stroma cells we found up-regulation of 144 genes and down-regulation of 184 genes. A functional enrichment analysis of the upregulated epithelial genes implicated them for proliferation, while upregulated stromal genes were associated with extracellular functions. Quantitative RT-PCR and in situ hybridization results confirmed differential gene expression in both cell cultures and ovariectomized uteri after the above treatments. Based on our identification of stromal secretory factors, we found evidence that suppression by siRNA specifically for Bmp8a and/or Fgf10 in the stromal layer caused significant inhibition of proliferation by E2 in the co-culture system, suggesting Bmp8a and Fgf10 act as paracrine mediators during E2-dependent control of uterine proliferation. The localization of receptors and receptor activation signaling in epithelial cells in both the co-culture system and uteri was consistent with their involvement in ligand–receptor signaling. Interestingly, loss of Bmp8a or Fgf10 also caused abrogation of E2-regulated epithelial receptor signaling in co-culture systems, suggesting that stroma-derived Fgf10 and Bmp8a are responsible for epithelial communication. Overall, stromal Fgf10 and Bmp8a serve as potential paracrine factors for E2-dependent regulation of epithelial proliferation in the uterus. PMID:25451979

  13. Silencing Aurora-A with siRNA inhibits cell proliferation in human lung adenocarcinoma cells.

    PubMed

    Zhong, Ning; Shi, Shunbin; Wang, Hongzhen; Wu, Guangzhou; Wang, Yunliang; Ma, Qiang; Wang, Hongwei; Liu, Yuanhua; Wang, Jinzhi

    2016-09-01

    Aurora kinase A (AURKA) is an oncogenic serine/threonine kinase, it plays important roles in tumorigenesis and chemoresistance. In this study, we investigated the expression of AURKA in lung adenocarcinoma tissues, the role of small interference RNA targeting AURKA on growth, cell cycle, and apoptosis of lung adenocarcinoma cell lines in vitro. The AURKA is highly expressed in lung adenocarcinoma tissues and human lung adenocarcinoma cell lines. Lentivirus-mediated short hairpin RNA (shRNA) was used to knock down AURKA expression in human lung adenocarcinoma cell lines H1299 and A549. The results indicated that depletion of AURKA could inhibit cell growth, cause cell cycle arrest and apoptosis. The potential mechanisms of AURKA inhibition induced cell cycle arrest and apoptosis are associated with downregulated RAF-1, CCND2, CCND3, CDK4, PAK4, EGFR and upregulated WEE1 expression. Furthermore, AURKA knockdown cooperated with vincristine (VCR) to repress A549 cell proliferation. Therefore, AURKA plays important roles in the proliferation of human lung adenocarcinoma cells, which suggests that AURKA could be a promising tool for lung adenocarcinoma therapy. PMID:27571708

  14. Vitamin D inhibition of lung adenocarcinoma cell proliferation in vitro.

    PubMed

    Li, Rong; Lou, Yuqing; Zhang, Weiyan; Dong, Qianggang; Han, Baohui

    2014-11-01

    Vitamin D has the capability to inhibit tumor cell proliferation and promote tumor cell apoptosis but whether this mechanism exists in lung adenocarcinoma cells remains to be studied. Our objective is to explore whether vitamin D has the capability to inhibit lung adenocarcinoma cell proliferation and synergize with cisplatin. Our method was to explore the effect of different concentrations of 1,25(OH)2D3 with or without cisplatin on lung adenocarcinoma cells by detecting cell proliferation rates at different time points. 1,25(OH)2D3 was capsulated with nanomaterial before acting on lung adenocarcinoma cells, and cell proliferation rates at different time points were detected with the CCK-8 method. When vitamin D was applied at a concentration of 1 × 10(-7) and 1 × 10(-6) mol/L on A549, PC9, SPC-A1, and H1650 cells for 72 h, no inhibition occurred on cell proliferation. Between the concentrations of 1 × 10(-5) and 0.5 × 10(-5) mol/L, inhibition on cell proliferation increased with drug action time. Between the concentration of 2.5 × 10(-5) and 0.03 × 10(-5) mol/L, inhibition on cell proliferation increased with increasing drug concentration. Analysis using bivariate correlations showed that the correlation coefficient of the proliferation inhibition rate and drug content was 0.580 (p < 0.0001). The correlation coefficient of proliferation inhibition rate and the drug action time was 0.379 (p = 0.01). The combined use of vitamin D and dichlorodiammine-platinum(II) (DDP) significantly increased the inhibition rate on A549 cell proliferation, which peaked after culturing for 96 h (Table 4). Further analysis using bivariate correlations showed that the correlation coefficient between proliferation inhibition rate and DDP concentration was 0.319 (p < 0.0001). The correlation coefficient of the proliferation inhibition rate and vitamin D concentration was 0.269 (p < 0.0001). The correlation coefficient of proliferation inhibition and drug action time was 0.221(p

  15. WISP1 mediates IL-6-dependent proliferation in primary human lung fibroblasts

    PubMed Central

    Klee, S.; Lehmann, M.; Wagner, D. E.; Baarsma, H. A.; Königshoff, M.

    2016-01-01

    Idiopathic pulmonary fibrosis (IPF) is a progressive and fatal interstitial lung disease. IPF is characterized by epithelial cell injury and reprogramming, increases in (myo)fibroblasts, and altered deposition of extracellular matrix. The Wnt1-inducible signaling protein 1 (WISP1) is involved in impaired epithelial-mesenchymal crosstalk in pulmonary fibrosis. Here, we aimed to further investigate WISP1 regulation and function in primary human lung fibroblasts (phLFs). We demonstrate that WISP1 is directly upregulated by Transforming growth factor β1 (TGFβ1) and Tumor necrosis factor α (TNFα) in phLFs, using a luciferase-based reporter system. WISP1 mRNA and protein secretion increased in a time- and concentration-dependent manner by TGFβ1 and TNFα in phLFs, as analysed by qPCR and ELISA, respectively. Notably, WISP1 is required for TGFβ1- and TNFα-dependent induction of interleukin 6 (IL-6), a mechanism that is conserved in IPF phLFs. The siRNA-mediated WISP1 knockdown led to a significant IL-6 reduction after TGFβ1 or TNFα stimulation. Furthermore, siRNA-mediated downregulation or antibody-mediated neutralization of WISP1 reduced phLFs proliferation, a process that was in part rescued by IL-6. Taken together, these results strongly indicate that WISP1-induced IL-6 expression contributes to the pro-proliferative effect on fibroblasts, which is likely orchestrated by a variety of profibrotic mediators, including Wnts, TGFβ1 and TNFα. PMID:26867691

  16. Peroxisome proliferator-activated receptor gamma overexpression suppresses proliferation of human colon cancer cells

    SciTech Connect

    Tsukahara, Tamotsu; Haniu, Hisao

    2012-08-03

    Highlights: Black-Right-Pointing-Pointer We examined the correlation between PPAR{gamma} expression and cell proliferation. Black-Right-Pointing-Pointer PPAR{gamma} overexpression reduces cell viability. Black-Right-Pointing-Pointer We show the synergistic effect of cell growth inhibition by a PPAR{gamma} agonist. -- Abstract: Peroxisome proliferator-activated receptor gamma (PPAR{gamma}) plays an important role in the differentiation of intestinal cells and tissues. Our previous reports indicate that PPAR{gamma} is expressed at considerable levels in human colon cancer cells. This suggests that PPAR{gamma} expression may be an important factor for cell growth regulation in colon cancer. In this study, we investigated PPAR{gamma} expression in 4 human colon cancer cell lines, HT-29, LOVO, DLD-1, and Caco-2. Real-time polymerase chain reaction (PCR) and Western blot analysis revealed that the relative levels of PPAR{gamma} mRNA and protein in these cells were in the order HT-29 > LOVO > Caco-2 > DLD-1. We also found that PPAR{gamma} overexpression promoted cell growth inhibition in PPAR{gamma} lower-expressing cell lines (Caco-2 and DLD-1), but not in higher-expressing cells (HT-29 and LOVO). We observed a correlation between the level of PPAR{gamma} expression and the cells' sensitivity for proliferation.

  17. APE/Ref-1 makes fine-tuning of CD40-induced B cell proliferation

    PubMed Central

    Merluzzi, Sonia; Gri, Giorgia; Gattei, Valter; Pagano, Michele; Pucillo, Carlo

    2009-01-01

    Apurinic/apyrimidinic endonuclease-1/Redox factor-1, a multifunctional DNA base excision repair and redox regulation enzyme, plays an important role in oxidative signalling, transcription factor regulation, and cell cycle control. Recently, we have demonstrated that following the triggering of CD40 on B cells, APE/Ref-1 translocates from the cytoplasm to the nucleus and regulates the activity of B cell-specific transcription factors. In the present paper we investigate whether APE/Ref-1 plays a role in controlling CD40-mediated B cell proliferation too. We demonstrate a concurrent increase in proliferation and decrease in apoptosis of primary mouse B cells activated by CD40 cross-linking and transfected with functional APE/Ref-1 antisense oligonucleotide. Moreover, we provide evidence that a redox-mediated signalling mechanism is involved in this process and we propose that APE/Ref-1, controlling the intracellular redox state, may also affect the cell cycle by inducing nucleus-cytoplasm redistribution of p21. Together, these findings suggest that APE/Ref-1 could act as a negative regulator in an adaptive response to elevated ROS levels following CD40 cross-linking. Considering the important role of ROS and APE/Ref-1 in CD40-mediated B cell proliferation, our data will contribute to understand the mechanisms of tumor escape and suggest APE/Ref-1 as a novel target for tumor therapeutic approaches. PMID:18617267

  18. Effect of advanced glycation end-products on cell proliferation and cell death.

    PubMed

    Peterszegi, G; Molinari, J; Ravelojaona, V; Robert, L

    2006-09-01

    The effect of advanced glycation end products (AGE-s) was studied on the proliferation and cell death of human skin fibroblasts in culture. Several AGE-products were prepared from proteins, a peptide and amino acids, using Glucose or Fructose, with or without Fe2+. The AGE preparations increased cell death at the 7th day, after only 72 hours of incubation. Some of these glycation products modified also proliferation. This effect of AGE-s was even maintained without these products in fresh medium for a second period of incubation up to 10 days from the start of the experiment. In order to explore the role of AGE-receptors, especially of AGE-receptor and of growth factor receptors (fibroblast and epidermal growth factors receptors), antibodies to these receptors were added to cell cultures and their effect on both cell death and proliferation were determined as for the AGE-s. These anti-receptor antibodies imitated to some extent the results obtained with AGE-s, producing increase of cell death and proliferation, followed above a certain concentration of antibodies by a decrease and a new increase or plateau. This might correspond to the internalization of the receptors followed by a re-expression on the cell membrane. The role of receptor-mediated Reactive Oxygen Species-production was also explored using scavengers: N-acetyl-cysteine (NAC), L-Carnosine, superoxide dismutase (SOD) and Catalase. Several of these scavengers decreased cell death, suggesting that Reactive Oxygen Species-production is partially involved in the observed phenomena. PMID:16919894

  19. Cholangiocyte N-Ras Protein Mediates Lipopolysaccharide-induced Interleukin 6 Secretion and Proliferation*

    PubMed Central

    O'Hara, Steven P.; Splinter, Patrick L.; Trussoni, Christy E.; Gajdos, Gabriella B.; Lineswala, Pooja N.; LaRusso, Nicholas F.

    2011-01-01

    Cholangiocytes, the epithelial cells lining the bile ducts in the liver, are periodically exposed to potentially injurious microbes and/or microbial products. As a result, cholangiocytes actively participate in microbe-associated, hepatic proinflammatory responses. We previously showed that infection of cultured human cholangiocytes with the protozoan parasite, Cryptosporidium parvum, or treatment with Gram-negative bacteria-derived LPS, activates NFκB in a myeloid differentiation 88 (MyD88)-dependent manner. Here, we describe a novel signaling pathway initiated by Toll-like receptors (TLRs) involving the small GTPase, Ras, that mediates cholangiocyte proinflammatory cytokine production and induction of cholangiocyte proliferation. Using cultured human cholangiocytes and a Ras activation assay, we found that agonists of plasma membrane TLRs (TLR 1, 2, 4, 5, and 6) rapidly (<10 min) activated N-Ras, but not other p21 Ras isoforms, resulting in the rapid (<15 min) phosphorylation of the downstream Ras effector, ERK1/2. RNA interference-induced depletion of TRAF6, a downstream effector of MyD88 and known activator of MAPK signaling, had no effect on N-Ras activation. Following N-Ras activation the proinflammatory cytokine, IL6, is rapidly secreted. Using a luciferase reporter, we demonstrated that LPS treatment induced IL6 promoter-driven luciferase which was suppressed using MEK/ERK pharmacologic inhibitors (PD98059 or U0126) and RNAi-induced depletion of N-Ras. Finally, we showed that LPS increased cholangiocyte proliferation (1.5-fold), which was inhibited by depletion of N-Ras; TLR agonist-induced proliferation was also inhibited following pretreatment with an IL6 receptor-blocking antibody. Together, our results support a novel signaling axis involving microbial activation of N-Ras likely involved in the cholangiocyte pathogen-induced proinflammatory response. PMID:21757746

  20. Bromoenol Lactone Attenuates Nicotine-Induced Breast Cancer Cell Proliferation and Migration

    PubMed Central

    Calderon, Lindsay E.; Liu, Shu; Arnold, Nova; Breakall, Bethany; Rollins, Joseph; Ndinguri, Margaret

    2015-01-01

    Objectives Calcium independent group VIA phospholipase A2 (iPLA2β) and Matrix Metalloproteinase-9 (MMP-9) are upregulated in many disease states; their involvement with cancer cell migration has been a recent subject for study. Further, the molecular mechanisms mediating nicotine-induced breast cancer cell progression have not been fully investigated. This study aims to investigate whether iPLA2β mediates nicotine-induced breast cancer cell proliferation and migration through both in-vitro and in-vivo techniques. Subsequently, the ability of Bromoenol Lactone (BEL) to attenuate the severity of nicotine-induced breast cancer was examined. Method and Results We found that BEL significantly attenuated both basal and nicotine-induced 4T1 breast cancer cell proliferation, via an MTT proliferation assay. Breast cancer cell migration was examined by both a scratch and transwell assay, in which, BEL was found to significantly decrease both basal and nicotine-induced migration. Additionally, nicotine-induced MMP-9 expression was found to be mediated in an iPLA2β dependent manner. These results suggest that iPLA2β plays a critical role in mediating both basal and nicotine-induced breast cancer cell proliferation and migration in-vitro. In an in-vivo mouse breast cancer model, BEL treatment was found to significantly reduce both basal (p<0.05) and nicotine-induced tumor growth (p<0.01). Immunohistochemical analysis showed BEL decreased nicotine-induced MMP-9, HIF-1alpha, and CD31 tumor tissue expression. Subsequently, BEL was observed to reduce nicotine-induced lung metastasis. Conclusion The present study indicates that nicotine-induced migration is mediated by MMP-9 production in an iPLA2β dependent manner. Our data suggests that BEL is a possible chemotherapeutic agent as it was found to reduce both nicotine-induced breast cancer tumor growth and lung metastasis. PMID:26588686

  1. Cell proliferation of Paramecium tetraurelia on a slow rotating clinostat

    NASA Astrophysics Data System (ADS)

    Sawai, Satoe; Mogami, Yoshihiro; Baba, Shoji A.

    Paramecium is known to proliferate faster under microgravity conditions, and slower under hypergravity. Experiments using axenic culture medium have demonstrated that hypergravity affected directly on the proliferation of Paramecium itself. In order to assess the mechanisms underlying the physiological effects of gravity on cell proliferation, Paramecium tetraurelia was grown under clinorotation (2.5 rpm) and the time course of the proliferation was investigated in detail on the basis of the logistic analysis. On the basis of the mechanical properties of Paramecium, this slow rate of the rotation appears to be enough to simulate microgravity in terms of the randomization of the cell orientation with respect to gravity. P. tetraurelia was cultivated in a closed chamber in which cells were confined without air bubbles, reducing the shear forces and turbulences under clinorotation. The chamber is made of quartz and silicone rubber film; the former is for the optically-flat walls for the measurement of cell density by means of a non-invasive laser optical-slice method, and the latter for gas exchange. Because of the small dimension for culture space, Paramecium does not accumulate at the top of the chamber in spite of its known negative gravitactic behavior. We measured the cell density at regular time intervals without breaking the configuration of the chamber, and analyzed the proliferation parameters by fitting the data to a logistic equation. As a result, P. tetraurelia showed reduced proliferation under slow clinorotation. The saturation of the cell density as well as the maximum proliferation rate decreased, although we found no significant changes on the half maximal time for proliferation. We also found that the mean swimming velocity decreased under slow clinorotation. These results were not consistent with those under microgravity and fast rotating clinostat. This may suggest that randomization of the cell orientation performed by slow rotating clinostat has

  2. Cell proliferation of Paramecium tetraurelia under simulated microgravity

    NASA Astrophysics Data System (ADS)

    Sawai, S.; Mogami, Y.; Baba, S. A.

    Paramecium is known to proliferate faster under microgravity in space and slower under hypergravity Experiments using axenic culture medium have demonstrated that the hypergravity affected directly on the proliferation of Paramecium itself Kato et al 2003 In order to assess the mechanisms underlying the physiological effects of gravity on cell proliferation Paramecium tetraurelia was grown under simulated microgravity performed by clinorotation and the time course of the proliferation was investigated in detail on the basis of the logistic analysis P tetraurelia was cultivated in a closed chamber in which cells were confined without air babbles reducing the shear stresses and turbulence under the rotation The chamber is made of quartz and silicone rubber film the former is for the optically-flat walls for the measurement of cell density by means of a non-invasive laser optical-slice method and the latter for gas exchange Because the closed chamber has an inner dimension of 3 times 3 times 60 mm Paramecium does not accumulate at the top of the chamber despite its negative gravitactic behavior We measured the cell density at regular time intervals without breaking the configuration of the chamber and analyzed the proliferation parameters by fitting the data to a logistic equation Clinorotation had the effects of reducing the proliferation of P tetraurelia It reduced both the saturation cell density and the maximum proliferation rate although it had little effect on the

  3. Cell Surface GRP78 Accelerated Breast Cancer Cell Proliferation and Migration by Activating STAT3.

    PubMed

    Yao, Xiaoli; Liu, Hua; Zhang, Xinghua; Zhang, Liang; Li, Xiang; Wang, Changhua; Sun, Shengrong

    2015-01-01

    High levels of cell surface glucose regulated protein 78 (sGRP78) have been implicated in cancer growth, survival, metastasis, and chemotherapy resistance. However, the underlying mechanism remains largely unknown. Here we report that the level of sGRP78 expression in human breast tumors gradually increases during cancer progression. Overexpression of GRP78 significantly enhanced its membrane distribution in human MCF-7 breast cancer cells, but had no effect on endoplasmic reticulum (ER) stress. High levels of sGRP78 facilitated cell proliferation and migration, as well as suppressed cell apoptosis. Neutralization of sGRP78 by a specific antibody against GRP78 alleviated sGRP78-induced cell growth and migration. Importantly, high phosphorylation levels of the signal transducer and activator of transcription 3 (STAT3) were found in human breast tumors that express sGRP78 and MCF-7 cells infected with adenovirus encoding human GRP78. Pretreatment with a GRP78 antibody suppressed STAT3 phosphorylation. Furthermore, genetic and pharmacological inhibition of STAT3 reversed the impacts of GRP78 on cell proliferation, apoptosis, and migration. These findings indicate that STAT3 mediates sGRP78-promoted breast cancer cell growth and migration. PMID:25973748

  4. Increased proliferation and gliogenesis of cultured rat neural progenitor cells by lipopolysaccharide-stimulated astrocytes.

    PubMed

    Go, Hyo Sang; Shin, Chan Young; Lee, Sung Hoon; Jeon, Se-Jin; Kim, Ki Chan; Choi, Chang Soon; Ko, Kwang Ho

    2009-01-01

    Neural progenitor cells (NPC) are self-renewing multipotent cells that generate neurons and glial cells in the brain. NPCs generate neurons and glia not only during development but also after neural injury. Recent studies have shown that endogenous NPCs are activated after brain injury and migrate toward damaged areas where astrocyte activation occurs. Considering the massive proliferation of astrocytes as well as the production of several kinds of cytoactive molecules after brain injury, such as NO, growth factors and cytokines, it is tempting to think that cytoactive molecules released by activated glial cells regulate neural progenitor differentiation and proliferation through inflammatory mediators. To test this hypothesis, we stimulated rat primary astrocytes with lipopolysaccharide (LPS) to induce the activation of astrocytes. After addition of the conditioned media from LPS-stimulated astrocytes to NPC culture, proliferation was examined by MTT assay and bromodeoxyuridine (BrdU) incorporation. The differentiation of NPC into neurons and astrocytes was examined by Western blot, ELISA and immunocytochemical staining with cell-type-specific markers. Conditioned media from LPS-stimulated astrocytes increased NPC proliferation as well as gliogenesis as compared with control conditioned media from astrocytes without LPS stimulation. In contrast, neurogenesis was decreased by LPS-conditioned media. To investigate the molecular mechanism mediating glial differentiation and proliferation of NPC by reactive astrocytes, we added inhibitors of the Erk and JNK pathways during LPS stimulation. These inhibitors - except for a p38 inhibitor - decreased NPC proliferation and glial differentiation. These results suggest that LPS stimulated astrocytes generate factors regulating NPC proliferation and gliogenesis via the Erk and JNK pathways. PMID:19609085

  5. Mesenchymal precursor cells maintain the differentiation and proliferation potentials of breast epithelial cells

    PubMed Central

    2014-01-01

    Introduction Stromal-epithelial interactions play a fundamental role in tissue homeostasis, controlling cell proliferation and differentiation. Not surprisingly, aberrant stromal-epithelial interactions contribute to malignancies. Studies of the cellular and molecular mechanisms underlying these interactions require ex vivo experimental model systems that recapitulate the complexity of human tissue without compromising the differentiation and proliferation potentials of human primary cells. Methods We isolated and characterized human breast epithelial and mesenchymal precursors from reduction mammoplasty tissue and tagged them with lentiviral vectors. We assembled heterotypic co-cultures and compared mesenchymal and epithelial cells to cells in corresponding monocultures by analyzing growth, differentiation potentials, and gene expression profiles. Results We show that heterotypic culture of non-immortalized human primary breast epithelial and mesenchymal precursors maintains their proliferation and differentiation potentials and constrains their growth. We further describe the gene expression profiles of stromal and epithelial cells in co-cultures and monocultures and show increased expression of the tumor growth factor beta (TGFβ) family member inhibin beta A (INHBA) in mesenchymal cells grown as co-cultures compared with monocultures. Notably, overexpression of INHBA in mesenchymal cells increases colony formation potential of epithelial cells, suggesting that it contributes to the dynamic reciprocity between breast mesenchymal and epithelial cells. Conclusions The described heterotypic co-culture system will prove useful for further characterization of the molecular mechanisms mediating interactions between human normal or neoplastic breast epithelial cells and the stroma, and will provide a framework to test the relevance of the ever-increasing number of oncogenomic alterations identified in human breast cancer. PMID:24916766

  6. Cdk5r1 Overexpression Induces Primary β-Cell Proliferation

    PubMed Central

    Draney, Carrie; Hobson, Amanda E.; Grover, Samuel G.; Jack, Benjamin O.; Tessem, Jeffery S.

    2016-01-01

    Decreased β-cell mass is a hallmark of type 1 and type 2 diabetes. Islet transplantation as a method of diabetes therapy is hampered by the paucity of transplant ready islets. Understanding the pathways controlling islet proliferation may be used to increase functional β-cell mass through transplantation or by enhanced growth of endogenous β-cells. We have shown that the transcription factor Nkx6.1 induces β-cell proliferation by upregulating the orphan nuclear hormone receptors Nr4a1 and Nr4a3. Using expression analysis to define Nkx6.1-independent mechanisms by which Nr4a1 and Nr4a3 induce β-cell proliferation, we demonstrated that cyclin-dependent kinase 5 regulatory subunit 1 (Cdk5r1) is upregulated by Nr4a1 and Nr4a3 but not by Nkx6.1. Overexpression of Cdk5r1 is sufficient to induce primary rat β-cell proliferation while maintaining glucose stimulated insulin secretion. Overexpression of Cdk5r1 in β-cells confers protection against apoptosis induced by etoposide and thapsigargin, but not camptothecin. The Cdk5 kinase complex inhibitor roscovitine blocks islet proliferation, suggesting that Cdk5r1 mediated β-cell proliferation is a kinase dependent event. Overexpression of Cdk5r1 results in pRb phosphorylation, which is inhibited by roscovitine treatment. These data demonstrate that activation of the Cdk5 kinase complex is sufficient to induce β-cell proliferation while maintaining glucose stimulated insulin secretion. PMID:26788519

  7. Laminin 5 regulates polycystic kidney cell proliferation and cyst formation.

    PubMed

    Joly, Dominique; Berissi, Sophie; Bertrand, Amélie; Strehl, Laetitia; Patey, Natacha; Knebelmann, Bertrand

    2006-09-29

    Renal cyst formation is the hallmark of autosomal dominant polycystic kidney disease (ADPKD). ADPKD cyst-lining cells have an increased proliferation rate and are surrounded by an abnormal extracellular matrix (ECM). We have previously shown that Laminin 5 (Ln-5, a alpha(3)beta(3)gamma(2) trimer) is aberrantly expressed in the pericystic ECM of ADPKD kidneys. We report that ADPKD cells in primary cultures produce and secrete Ln-5 that is incorporated to the pericystic ECM in an in vitro model of cystogenesis. In monolayers, purified Ln-5 induces ERK activation and proliferation of ADPKD cells, whereas upon epidermal growth factor stimulation blocking endogenously produced Ln-5 with anti-gamma(2) chain antibody reduces the sustained ERK activation and inhibits proliferation. In three-dimensional gel culture, addition of purified Ln-5 stimulates cell proliferation and cyst formation, whereas blocking endogenous Ln-5 strongly inhibits cyst formation. Ligation of alpha(6)beta(4) integrin, a major Ln-5 receptor aberrantly expressed by ADPKD cells, induces beta(4) integrin phosphorylation, ERK activation, cell proliferation, and cyst formation. These findings indicate that Ln-5 is an important regulator of ADPKD cell proliferation and cystogenesis and suggest that Ln-5 gamma(2) chain and Ln-5-alpha(6)beta(4) integrin interaction both contribute to these phenotypic changes. PMID:16870608

  8. UNUSUAL EOSINOPHILIC GRANULE CELL PROLIFERATION IN COHO SALMON (ONCHORHYNCHUS KISUTCH)

    EPA Science Inventory

    Proliferative lesions comprised of eosinophilic granule cells (EGCs) extended throughout the gastrointestinal tract of several mature, spawning coho salmon Oncorhynchus kisutch (Walbaum). istological examination of the tumour showed extensive proliferation and infiltration of EGC...

  9. In vitro study on shRNA-mediated reduction of testis developmental related gene 1 expression and its effects on the proliferation, invasion and apoptosis of NTERA-2 cells

    PubMed Central

    GAN, YU; YANG, JIANFU; WANG, YONG; TAN, ZHENGYU; JIANG, XIANZHEN; TANG, YUXIN

    2015-01-01

    Testis developmental related gene 1 (TDRG1) is a novel human testis-specific gene. TDRG1 is differentially expressed in cancerous tissue compared with normal testicular tissue and demonstrates a unique expression pattern in normal testes; therefore, this gene may be involved in the occurrence and development of testicular germ cell tumors (TGCT). In the present study, the expression level of TDRG1 was downregulated in human TGCT NTERA-2 cells by RNA interference (RNAi) in order to investigate the association between TDRG1 and TGCT. The TDRG1 mRNA and protein expression levels in NTERA-2 cells were significantly inhibited following transfection with specific RNAi plasmids. The ability to proliferate (inhibited by 15.4% at day 3 and 26.1% at day 5; P<0.001) and invade (reduced by 49.1%; P=0.01) in vitro was suppressed in cells in which the expression level of TDRG1 was reduced, and a corresponding increase in the apoptotic potential was observed (the early apoptotic potential and total apoptotic potential were increased by 75%; P=0.019 and 54.8%; P=0.009, respectively). The results of the present study indicated that the biological behavior of NTERA-2 cells is associated with TDRG1 expression levels, and that this gene may be a novel target candidate in the treatment of TGCT. PMID:26170977

  10. Differential migration and proliferation of geometrical ensembles of cell clusters

    SciTech Connect

    Kumar, Girish; Chen, Bo; Co, Carlos C.; Ho, Chia-Chi

    2011-06-10

    Differential cell migration and growth drives the organization of specific tissue forms and plays a critical role in embryonic development, tissue morphogenesis, and tumor invasion. Localized gradients of soluble factors and extracellular matrix have been shown to modulate cell migration and proliferation. Here we show that in addition to these factors, initial tissue geometry can feedback to generate differential proliferation, cell polarity, and migration patterns. We apply layer by layer polyelectrolyte assembly to confine multicellular organization and subsequently release cells to demonstrate the spatial patterns of cell migration and growth. The cell shapes, spreading areas, and cell-cell contacts are influenced strongly by the confining geometry. Cells within geometric ensembles are morphologically polarized. Symmetry breaking was observed for cells on the circular pattern and cells migrate toward the corners and in the direction parallel to the longest dimension of the geometric shapes. This migration pattern is disrupted when actomyosin based tension was inhibited. Cells near the edge or corner of geometric shapes proliferate while cells within do not. Regions of higher rate of cell migration corresponded to regions of concentrated growth. These findings demonstrate that multicellular organization can result in spatial patterns of migration and proliferation.

  11. Paracrine Wnt/β-catenin signaling mediates proliferation of undifferentiated spermatogonia in the adult mouse testis

    PubMed Central

    Takase, Hinako M.; Nusse, Roeland

    2016-01-01

    Spermatogonial stem cells (SSCs) fuel the production of male germ cells but the mechanisms behind SSC self-renewal, proliferation, and differentiation are still poorly understood. Using the Wnt target gene Axin2 and genetic lineage-tracing experiments, we found that undifferentiated spermatogonia, comprising SSCs and transit amplifying progenitor cells, respond to Wnt/β-catenin signals. Genetic elimination of β-catenin indicates that Wnt/β-catenin signaling promotes the proliferation of these cells. Signaling is likely initiated by Wnt6, which is uniquely expressed by neighboring Sertoli cells, the only somatic cells in the seminiferous tubule that support germ cells and act as a niche for SSCs. Therefore, unlike other stem cell systems where Wnt/β-catenin signaling is implicated in self-renewal, the Wnt pathway in the testis specifically contributes to the proliferation of SSCs and progenitor cells. PMID:26929341

  12. Knockdown of stromal interaction molecule 1 attenuates hepatocyte growth factor-induced endothelial progenitor cell proliferation.

    PubMed

    Shi, Yankun; Song, Mingbao; Guo, Ruiwei; Wang, Hong; Gao, Pan; Shi, Weibin; Huang, Lan

    2010-03-01

    Increased Ca(2+) entry through store-operated Ca(2+) channels (SOCCs) plays an essential role in the regulation of hepatocyte growth factor (HGF)-induced cell proliferation. Stromal interaction molecule 1 (STIM1) is thought to transmit endoplasmic reticulum (ER) Ca(2+) store depletion signals to the plasma membrane (PM), causing the opening of SOCCs in the PM. However, the relationship between HGF and STIM1 in endothelial progenitor cell (EPC) proliferation remains uncharacterized. The objective of this study was to evaluate the potential involvement of STIM1 in HGF-induced EPC proliferation. For this purpose, we used cultured rat bone marrow-derived EPCs and found that HGF-induced EPC proliferation at low concentrations. Store-operated Ca(2+) entry (SOCE) was elevated in HGF-treated EPCs, and the SOCC inhibitors 2-aminoethoxydiphenyl borate (2-APB) and BTP-2 inhibited the HGF-induced proliferation response. Moreover, STIM1 mRNA and protein expression levels were increased in response to HGF stimulation and knockdown of STMI1 decreased SOCE and prevented HGF-induced EPC proliferation. In conclusion, our data suggest that HGF-induced EPC proliferation is mediated partly via activation of STIM1. PMID:20404049

  13. Scaffold architecture and fibrin gels promote meniscal cell proliferation

    SciTech Connect

    Pawelec, K. M. E-mail: jw626@cam.ac.uk; Best, S. M.; Cameron, R. E.; Wardale, R. J. E-mail: jw626@cam.ac.uk

    2015-01-01

    Stability of the knee relies on the meniscus, a complex connective tissue with poor healing ability. Current meniscal tissue engineering is inadequate, as the signals for increasing meniscal cell proliferation have not been established. In this study, collagen scaffold structure, isotropic or aligned, and fibrin gel addition were tested. Metabolic activity was promoted by fibrin addition. Cellular proliferation, however, was significantly increased by both aligned architectures and fibrin addition. None of the constructs impaired collagen type I production or triggered adverse inflammatory responses. It was demonstrated that both fibrin gel addition and optimized scaffold architecture effectively promote meniscal cell proliferation.

  14. Melatonin improves reprogramming efficiency and proliferation of bovine-induced pluripotent stem cells.

    PubMed

    Bai, Chunyu; Li, Xiangchen; Gao, Yuhua; Yuan, Ziao; Hu, Pengfei; Wang, Hui; Liu, Changqing; Guan, Weijun; Ma, Yuehui

    2016-09-01

    Melatonin can modulate neural stem cell (NSC) functions such as proliferation and differentiation into NSC-derived pluripotent stem cells (N-iPS) in brain tissue, but the effect and mechanism underlying this are unclear. Thus, we studied how primary cultured bovine NSCs isolated from the retinal neural layer could transform into N-iPS cell. NSCs were exposed to 0.01, 0.1, 1, 10, or 100 μm melatonin, and cell viability studies indicated that 10 μm melatonin can significantly increase cell viability and promote cell proliferation in NSCs in vitro. Thus, 10 μm melatonin was used to study miR-302/367-mediated cell reprogramming of NSCs. We noted that this concentration of melatonin increased reprogramming efficiency of N-iPS cell generation from primary cultured bovine NSCs and that this was mediated by downregulation of apoptosis-related genes p53 and p21. Then, N-iPS cells were treated with 1, 10, 100, or 500 μm melatonin, and N-iPS (M-N-iPS) cell proliferation was measured. We noted that 100 μm melatonin increased proliferation of N-iPS cells via increased phosphorylation of intracellular ERK1/2 via activation of its pathway in M-N-iPS via melatonin receptors 1 (MT1). Finally, we verified that N-iPS cells and M-N-iPS cells are similar to typical embryonic stem cells including the expression of pluripotency markers (Oct4 and Nanog), the ability to form teratomas in vivo, and the capacity to differentiate into all three embryonic germ layers. PMID:27090494

  15. Inhibition of brain tumor cell proliferation by alternating electric fields

    SciTech Connect

    Jeong, Hyesun; Oh, Seung-ick; Hong, Sunghoi E-mail: radioyoon@korea.ac.kr; Sung, Jiwon; Jeong, Seonghoon; Yoon, Myonggeun E-mail: radioyoon@korea.ac.kr; Koh, Eui Kwan

    2014-11-17

    This study was designed to investigate the mechanism by which electric fields affect cell function, and to determine the optimal conditions for electric field inhibition of cancer cell proliferation. Low-intensity (<2 V/cm) and intermediate-frequency (100–300 kHz) alternating electric fields were applied to glioblastoma cell lines. These electric fields inhibited cell proliferation by inducing cell cycle arrest and abnormal mitosis due to the malformation of microtubules. These effects were significantly dependent on the intensity and frequency of applied electric fields.

  16. EDA-containing fibronectin increases proliferation of embryonic stem cells.

    PubMed

    Losino, Noelia; Waisman, Ariel; Solari, Claudia; Luzzani, Carlos; Espinosa, Darío Fernández; Sassone, Alina; Muro, Andrés F; Miriuka, Santiago; Sevlever, Gustavo; Barañao, Lino; Guberman, Alejandra

    2013-01-01

    Embryonic stem cells (ESC) need a set of specific factors to be propagated. They can also grow in conditioned medium (CM) derived from a bovine granulosa cell line BGC (BGC-CM), a medium that not only preserves their main features but also increases ESC´s proliferation rate. The mitogenic properties of this medium were previously reported, ascribing this effect to an alternative spliced generated fibronectin isoform that contains the extra domain A (FN EDA(+)). Here, we investigated if the FN EDA(+) isoform increased proliferation of mouse and human ES cells. We analyzed cell proliferation using conditioned media produced by different mouse embryonic fibroblast (MEF) lines genetically engineered to express FN constitutively including or excluding the EDA domain (FN EDA(-)), and in media supplemented with recombinant peptides containing or not the EDA. We found that the presence of EDA in the medium increased mouse and human ESC's proliferation rate. Here we showed for the first time that this FN isoform enhances ESC's proliferation. These findings suggest a possible conserved behavior for regulation of ES cells proliferation by this FN isoform and could contribute to improve their culturing conditions both for research and cell therapy. PMID:24244705

  17. EDA-Containing Fibronectin Increases Proliferation of Embryonic Stem Cells

    PubMed Central

    Losino, Noelia; Waisman, Ariel; Solari, Claudia; Luzzani, Carlos; Espinosa, Darío Fernández; Sassone, Alina; Muro, Andrés F.; Miriuka, Santiago; Sevlever, Gustavo; Barañao, Lino; Guberman, Alejandra

    2013-01-01

    Embryonic stem cells (ESC) need a set of specific factors to be propagated. They can also grow in conditioned medium (CM) derived from a bovine granulosa cell line BGC (BGC-CM), a medium that not only preserves their main features but also increases ESC´s proliferation rate. The mitogenic properties of this medium were previously reported, ascribing this effect to an alternative spliced generated fibronectin isoform that contains the extra domain A (FN EDA+). Here, we investigated if the FN EDA+ isoform increased proliferation of mouse and human ES cells. We analyzed cell proliferation using conditioned media produced by different mouse embryonic fibroblast (MEF) lines genetically engineered to express FN constitutively including or excluding the EDA domain (FN EDA-), and in media supplemented with recombinant peptides containing or not the EDA. We found that the presence of EDA in the medium increased mouse and human ESC’s proliferation rate. Here we showed for the first time that this FN isoform enhances ESC’s proliferation. These findings suggest a possible conserved behavior for regulation of ES cells proliferation by this FN isoform and could contribute to improve their culturing conditions both for research and cell therapy. PMID:24244705

  18. Curcumin and docosahexaenoic acid block insulin-induced colon carcinoma cell proliferation.

    PubMed

    Fenton, Jenifer I; McCaskey, Sarah J

    2013-03-01

    Diets high in fish and curcumin are associated with a decreased risk of CRC. Insulin resistance and obesity are associated with increased CRC risk and higher reoccurrence rates. We utilized cell culture to determine if dietary compounds could reduce insulin-induced cell proliferation comparing the response in normal and metastatic colon epithelial cells. We treated model normal murine colon epithelial cells (YAMC) and adenocarcinoma cells (MC38) with docosahexaenoic acid (DHA) or curcumin alone and then co-treatments of the diet-derived compound and insulin were combined. Cell proliferation was stimulated with insulin (1 ug/mL) to model insulin resistance in obesity. Despite the presence of insulin, proliferation was reduced in the MC38 cells treated with 10 μM curcumin (p<0.001) and 50 μM DHA (p<0.001). Insulin stimulated MAPK and MEK phosphorylation was inhibited by DHA and curcumin in MC38 cancer cells. Here we show that curcumin and DHA can block insulin-induced colon cancer cell proliferation in vitro via a MEK mediated mechanism. PMID:23266210

  19. Restriction of mast cell proliferation through hyaluronan synthesis by co-cultured fibroblasts.

    PubMed

    Takano, Hirotsugu; Furuta, Kazuyuki; Yamashita, Kazuhito; Sakanaka, Mariko; Itano, Naoki; Gohda, Eiichi; Nakayama, Kazuhisa; Kimata, Koji; Sugimoto, Yukihiko; Ichikawa, Atsushi; Tanaka, Satoshi

    2012-01-01

    Appropriate culture models for tissue mast cells are required to determine how they are involved in regulation of local immune responses. We previously established a culture model for cutaneous mast cells, in which bone marrow-derived immature mast cells were co-cultured with Swiss 3T3 fibroblasts in the presence of stem cell factor. In this study, we focused on the roles of hyaluronan, which is produced by the feeder fibroblasts and forms the extracellular matrix during the co-culture period. Hyaluronan synthesis was found to be mediated by hyaluronan synthase 2 (HAS2) expressed in Swiss 3T3 cells. A decreases in the amount of hyaluronan, which was achieved by retroviral expression of short hairpin RNA for Has2 or by addition of hyaluronidase, significantly enhanced the proliferation of the cultured mast cells without any obvious effects on their maturation. Although we previously demonstrated that CD44 is required for proliferation of cutaneous mast cells, the deficiency of hyaluronan did not affect the proliferation of the cultured mast cells that lack CD44. These findings suggest that the extracellular matrix containing hyaluronan may have a potential to restrict proliferation of cutaneous mast cells in a CD44-independent manner. PMID:22382329

  20. Control of cell proliferation and elongation by miR396.

    PubMed

    Ercoli, María Florencia; Rojas, Arantxa M L; Debernardi, Juan Manuel; Palatnik, Javier F; Rodriguez, Ramiro E

    2016-06-01

    The combinatory effects of cell proliferation and cell elongation determines the rate at which organs growth. In the root meristematic zone cells both divide and expand, while post-mitotic cells in the elongation zone only expands until they reach their final size. The transcription factors of the GROWTH-REGULATING FACTOR (GRF) class promote cell proliferation in various plant organs. Their expression is restricted to cells with a high proliferative capacity, yet strong downregulation of the GRF activity compromise the plant survival. Part of expression pattern of the GRFs is ensured by the post-transcriptional repression mediated by the conserved microRNA miR396. Here we show the quantitative effects in root growth caused by GRF depletion in a series of transgenic lines with different miR396 levels. We show that high miRNA levels affect cell elongation and proliferation in roots. Detailed analysis suggests that cell proliferation is restricted due to a reduction in cell cycle speed that might result from defects in the accumulation of mitotic cyclins. The results provide insights into the participation of the miRNA-GRF regulatory network in root development. PMID:27172373

  1. Role of Calmodulin in Cell Proliferation

    NASA Technical Reports Server (NTRS)

    Chafouleas, J.

    1983-01-01

    Calmodulin levels were found to increase as cells enter plateau. The data suggest that the cells are exiting the cell cycle late in the G sub 1 phase, or that the calmodulin levels in plateau cells are uncoupled to progression into S phase in plateau cells. Upon release, calmodulin levels rapidly decrease. Following this decrease, there is a increase prior to S phase.

  2. M2 Muscarinic acetylcholine receptor modulates rat airway smooth muscle cell proliferation

    PubMed Central

    2013-01-01

    Airways chronic inflammatory conditions in asthma and COPD are characterized by tissue remodeling, being smooth muscle hyperplasia, the most important feature. Non-neuronal and neuronal Acetylcholine acting on muscarinic receptors (MAChRs) has been postulated as determinant of tissue remodeling in asthma and COPD by promoting proliferation and phenotypic changes of airway smooth muscle cells (ASMC). The objective was to evaluate proliferative responses to muscarinic agonist as carbamylcholine (Cch) and to identify the MAchR subtype involved. ASMC were isolated from tracheal fragments of Sprague–Dawley rats by enzymatic digestion. Proliferation assays were performed by MTS-PMS method. Viability was confirmed by trypan blue exclusion method. Mitogens as, epidermal growth factor (EGF), Tumor necrosis factor-alpha (TNF-α) and fetal bovine serum (FBS) increased ASMC proliferation (p < 0.05, n = 5). Cch alone increased ASMC proliferation at 24 and 48 hrs. However, combination of Cch with other mitogens exhibited a dual effect, synergistic proliferation effect in the presence of EGF (5 ng/mL) and 5% FBS and inhibiting the proliferation induced by 10% FBS, EGF (10 ng/mL) and TNF-α (10 ng/mL). To determine the MAChR subtype involved in these biological responses, a titration curve of selective muscarinic antagonists were performed. The Cch stimulatory and inhibitory effects on ASCM proliferation was blocked by AF-DX-116 (M2AChR selective antagonist), in greater proportion than 4-DAMP (M3AChR selective antagonist), suggesting that the modulation of muscarinic agonist-induced proliferation is M2AChR mediated responses. Thus, M2AChR can activate multiple signal transduction systems and mediate both effects on ASMC proliferation depending on the plethora and variable airway microenvironments existing in asthma and COPD. PMID:24377382

  3. SPINDLY, ERECTA, and Its Ligand STOMAGEN Have a Role in Redox-Mediated Cortex Proliferation in the Arabidopsis Root

    PubMed Central

    Cui, Hongchang; Kong, Danyu; Wei, Pengcheng; Hao, Yueling; Torii, Keiko U.; Lee, Jin Suk; Li, Jie

    2014-01-01

    Reactive oxygen species (ROS) are harmful to all living organisms and therefore they must be removed to ensure normal growth and development. ROS are also signaling molecules, but so far little is known about the mechanisms of ROS perception and developmental response in plants. We here report that hydrogen peroxide induces cortex proliferation in the Arabidopsis root and that SPINDLY (SPY), an O-linked glucosamine acetyltransferase, regulates cortex proliferation by maintaining cellular redox homeostasis. We also found that mutation in the leucine-rich receptor kinase ERECTA and its putative peptide ligand STOMAGEN block the effect of hydrogen peroxide on root cortex proliferation. However, ERECTA and STOMAGEN are expressed in the vascular tissue, whereas extra cortex cells are produced from the endodermis, suggesting the involvement of intercellular signaling. SPY appears to act downstream of ERECTA, because the spy mutation still caused cortex proliferation in the erecta mutant background. We therefore have not only gained insight into the mechanism by which SPY regulates root development but also uncovered a novel pathway for ROS signaling in plants. The importance of redox-mediated cortex proliferation as a protective mechanism against oxidative stress is also discussed. PMID:25267734

  4. SPINDLY, ERECTA, and its ligand STOMAGEN have a role in redox-mediated cortex proliferation in the Arabidopsis root.

    PubMed

    Cui, Hongchang; Kong, Danyu; Wei, Pengcheng; Hao, Yueling; Torii, Keiko U; Lee, Jin Suk; Li, Jie

    2014-12-01

    Reactive oxygen species (ROS) are harmful to all living organisms and therefore they must be removed to ensure normal growth and development. ROS are also signaling molecules, but so far little is known about the mechanisms of ROS perception and developmental response in plants. We here report that hydrogen peroxide induces cortex proliferation in the Arabidopsis root and that SPINDLY (SPY), an O-linked glucosamine acetyltransferase, regulates cortex proliferation by maintaining cellular redox homeostasis. We also found that mutation in the leucine-rich receptor kinase ERECTA and its putative peptide ligand STOMAGEN block the effect of hydrogen peroxide on root cortex proliferation. However, ERECTA and STOMAGEN are expressed in the vascular tissue, whereas extra cortex cells are produced from the endodermis, suggesting the involvement of intercellular signaling. SPY appears to act downstream of ERECTA, because the spy mutation still caused cortex proliferation in the erecta mutant background. We therefore have not only gained insight into the mechanism by which SPY regulates root development but also uncovered a novel pathway for ROS signaling in plants. The importance of redox-mediated cortex proliferation as a protective mechanism against oxidative stress is also discussed. PMID:25267734

  5. Proliferation of differentiated glial cells in the brain stem.

    PubMed

    Barradas, P C; Cavalcante, L A

    1998-02-01

    Classical studies of macroglial proliferation in muride rodents have provided conflicting evidence concerning the proliferating capabilities of oligodendrocytes and microglia. Furthermore, little information has been obtained in other mammalian orders and very little is known about glial cell proliferation and differentiation in the subclass Metatheria although valuable knowledge may be obtained from the protracted period of central nervous system maturation in these forms. Thus, we have studied the proliferative capacity of phenotypically identified brain stem oligodendrocytes by tritiated thymidine radioautography and have compared it with known features of oligodendroglial differentiation as well as with proliferation of microglia in the opossum Didelphis marsupialis. We have detected a previously undescribed ephemeral, regionally heterogeneous proliferation of oligodendrocytes expressing the actin-binding, ensheathment-related protein 2'3'-cyclic nucleotide 3'-phosphodiesterase (CNPase), that is not necessarily related to the known regional and temporal heterogeneity of expression of CNPase in cell bodies. On the other hand, proliferation of microglia tagged by the binding of Griffonia simplicifolia B4 isolectin, which recognizes an alpha-D-galactosyl-bearing glycoprotein of the plasma membrane of macrophages/microglia, is known to be long lasting, showing no regional heterogeneity and being found amongst both ameboid and differentiated ramified cells, although at different rates. The functional significance of the proliferative behavior of these differentiated cells is unknown but may provide a low-grade cell renewal in the normal brain and may be augmented under pathological conditions. PMID:9686148

  6. Drosophila Myc integrates multiple signaling pathways to regulate intestinal stem cell proliferation during midgut regeneration

    PubMed Central

    Ren, Fangfang; Shi, Qing; Chen, Yongbin; Jiang, Alice; Ip, Y Tony; Jiang, Huaqi; Jiang, Jin

    2013-01-01

    Intestinal stem cells (ISCs) in the Drosophila adult midgut are essential for maintaining tissue homeostasis, and their proliferation and differentiation speed up in order to meet the demand for replenishing the lost cells in response to injury. Several signaling pathways including JAK-STAT, EGFR and Hippo (Hpo) pathways have been implicated in damage-induced ISC proliferation, but the mechanisms that integrate these pathways have remained elusive. Here, we demonstrate that the Drosophila homolog of the oncoprotein Myc (dMyc) functions downstream of these signaling pathways to mediate their effects on ISC proliferation. dMyc expression in precursor cells is stimulated in response to tissue damage, and dMyc is essential for accelerated ISC proliferation and midgut regeneration. We show that tissue damage caused by dextran sulfate sodium feeding stimulates dMyc expression via the Hpo pathway, whereas bleomycin feeding activates dMyc through the JAK-STAT and EGFR pathways. We provide evidence that dMyc expression is transcriptionally upregulated by multiple signaling pathways, which is required for optimal ISC proliferation in response to tissue damage. We have also obtained evidence that tissue damage can upregulate dMyc expression post-transcriptionally. Finally, we show that a basal level of dMyc expression is required for ISC maintenance, proliferation and lineage differentiation during normal tissue homeostasis. PMID:23896988

  7. The Antiaging Gene Klotho Regulates Proliferation and Differentiation of Adipose-Derived Stem Cells.

    PubMed

    Fan, Jun; Sun, Zhongjie

    2016-06-01

    Klotho was originally discovered as an aging-suppressor gene. The purpose of this study was to investigate whether secreted Klotho (SKL) affects the proliferation and differentiation of adipose-derived stem cells (ADSCs). RT-PCR and Western blot analysis showed that short-form Klotho was expressed in mouse ADSCs. The Klotho gene mutation KL(-/-) significantly decreased proliferation of ADSCs and expression of pluripotent transcription factors (Nanog, Sox-2, and Oct-4) in mice. The adipogenic differentiation of ADSCs was also decreased in KL(-/-) mice. Incubation with Klotho-deficient medium decreased ADSC proliferation, pluripotent transcription factor levels, and adipogenic differentiation, which is similar to what was found in KL(-/-) mice. These results indicate that Klotho deficiency suppresses ADSC proliferation and differentiation. Interestingly, treatment with recombinant SKL protein rescued the Klotho deficiency-induced impairment in ADSC proliferation and adipogenic differentiation. SKL also regulated ADSCs' differentiation to other cell lineages (osteoblasts, myofibroblasts), indicating that SKL maintains stemness of ADSCs. It is intriguing that overexpression of SKL significantly increased PPAR-γ expression and lipid formation in ADSCs following adipogenic induction, indicating enhanced adipogenic differentiation. Overexpression of SKL inhibited expression of TGFβ1 and its downstream signaling mediator Smad2/3. This study demonstrates, for the first time, that SKL is essential to the maintenance of normal proliferation and differentiation in ADSCs. Klotho regulates adipogenic differentiation in ADSCs, likely via inhibition of TGFβ1 and activation of PPAR-γ. Stem Cells 2016;34:1615-1625. PMID:26865060

  8. The role of peroxisome proliferator-activated receptor-{beta}/{delta} in epidermal growth factor-induced HaCaT cell proliferation

    SciTech Connect

    Liang Pengfei; Jiang Bimei; Yang Xinghua; Xiao Xianzhong Huang Xu; Long Jianhong; Zhang Pihong; Zhang Minghua; Xiao Muzhang; Xie Tinghong; Huang Xiaoyuan

    2008-10-15

    Epidermal growth factor (EGF) has been shown to be a potent mitogen for epidermal cells both in vitro and in vivo, thus contributing to the development of an organism. It has recently become clear that peroxisome proliferator-activated receptor-{beta}/{delta} (PPAR{beta}/{delta}) expression and activation is involved in the cell proliferation. However, little is known about the role of PPAR{beta}/{delta} in EGF-induced proliferation of HaCaT keratinocytes. In this study, HaCaT cells were cultured in the presence and absence of EGF and we identified that EGF induced an increase of PPAR{beta}/{delta} mRNA and protein level expression in time-dependent and dose-dependent manner, and AG1487, an EGF receptor (EGFR) special inhibitor, caused attenuation of PPAR{beta}/{delta} protein expression. Electrophoretic mobility shift assay (EMSA) revealed that EGF significantly increased PPAR{beta}/{delta} binding activity in HaCaT keratinocytes. Antisense phosphorothioate oligonucleotides (asODNs) against PPAR{beta}/{delta} caused selectively inhibition of PPAR{beta}/{delta} protein content induced by EGF and significantly attenuated EGF-mediated cell proliferation. Treatment of the cells with L165041, a specific synthetic ligand for PPAR{beta}/{delta}, significantly enhanced EGF-mediated cell proliferation. Finally, c-Jun ablation inhibited PPAR{beta}/{delta} up-regulation induced by EGF, and chromatin immunoprecipitation (ChIP) showed that c-Jun bound to the PPAR{beta}/{delta} promoter and the binding increased in EGF-stimulated cells. These results demonstrate that EGF induces PPAR{beta}/{delta} expression in a c-Jun-dependent manner and PPAR{beta}/{delta} plays a vital role in EGF-stimulated proliferation of HaCaT cells.

  9. Cepharanthine inhibits in vitro VSMC proliferation and migration and vascular inflammatory responses mediated by RAW264.7.

    PubMed

    Paudel, Keshav Raj; Karki, Rajendra; Kim, Dong-Wook

    2016-08-01

    Pathogenesis of atherosclerosis involves vascular smooth muscle cell (VSMC) migration and proliferation followed by an inflammation mediated by activated macrophages in the tunica intima of blood vessels. Cepharanthine (CEP) belongs to bisbenzylisoquinoline alkaloids found in the plant Stephania cepharantha, which has been used for various diseases like cancer, alopecia areata, venomous snakebites, and malaria. In this study, we investigated whether CEP suppresses VSMC migration and proliferation and inhibits inflammatory mediator production in macrophage (RAW264.7). Our results showed that CEP possessed significant DPPH scavenging and metal chelating activities. It also markedly inhibited lipid peroxidation. Similarly, CEP suppressed the nitric oxide (NO) production and expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX-2) in RAW264.7 cells. Moreover, the level of prostaglandin E2 was also suppressed and the formation of macrophage derived foam cell was attenuated in RAW264.7 cells. Likewise, NO production in isolated peritoneal macrophage and VSMC migration in response to LPS stimulated RAW264.7 was also halted by CEP treatment. Also, VSMC migration induced by platelet-derived growth factor (PDGF-BB) was inhibited by CEP dose dependently. The anti-migratory effect of CEP on VSMCs was due to its inhibitory effect on metalloproteinase-9 (MMP-9) expression, preventing the degradation of extracellular matrix (ECM) component. Furthermore, CEP suppressed PDGF-BB induced VSMC proliferation by down-regulation of mitogen activated protein kinase (MAPK) signaling molecules. CEP also inhibited the translocation of NF-κB from cytosol to nucleus. Thus, our results suggest that CEP exerts potent anti-atherosclerotic effect through attenuation of inflammation, lipid peroxidation and VSMC migration and proliferation. PMID:27021874

  10. Adipose-derived mesenchymal stem cells promote cell proliferation and invasion of epithelial ovarian cancer

    SciTech Connect

    Chu, Yijing; Tang, Huijuan; Guo, Yan; Guo, Jing; Huang, Bangxing; Fang, Fang; Cai, Jing Wang, Zehua

    2015-09-10

    Adipose-derived mesenchymal stem cell (ADSC) is an important component of tumor microenvironment. However, whether ADSCs have a hand in ovarian cancer progression remains unclear. In this study, we investigated the impact of human ADSCs derived from the omentum of normal donors on human epithelial ovarian cancer (EOC) cells in vitro and in vivo. Direct and indirect co-culture models including ADSCs and human EOC cell lines were established and the effects of ADSCs on EOC cell proliferation were evaluated by EdU incorporation and flow cytometry. Transwell migration assays and detection of MMPs were performed to assess the invasion activity of EOC cells in vitro. Mouse models were established by intraperitoneal injection of EOC cells with or without concomitant ADSCs to investigate the role of ADSCs in tumor progression in vivo. We found that ADSCs significantly promoted proliferation and invasion of EOC cells in both direct and indirect co-culture assays. In addition, after co-culture with ADSCs, EOC cells secreted higher levels of matrix metalloproteinases (MMPs), and inhibition of MMP2 and MMP9 partially relieved the tumor-promoting effects of ADSCs in vitro. In mouse xenograft models, we confirmed that ADSCs promoted EOC growth and metastasis and elevated the expression of MMP2 and MMP9. Our findings indicate that omental ADSCs play a promotive role during ovarian cancer progression. - Highlights: • Omental adipose derived stem cells enhanced growth and invasion properties of ovarian cancer cells. • Adipose derived stem cells promoted the growth and metastasis of ovarian cancer in mice models. • Adipose derived stem cells promoted MMPs expression and secretion of ovarian cancer cells. • Elevated MMPs mediated the tumor promoting effects of ADSCs.

  11. Overexpression of heme oxygenase-1 protects smooth muscle cells against oxidative injury and inhibits cell proliferation.

    PubMed

    Zhang, Min; Zhang, Bao Hui; Chen, Li; An, Wei

    2002-06-01

    To investigate whether the expression of exogenous heme oxygenase-1 (HO-1) gene within vascular smooth muscle cells (VSMC) could protect the cells from free radical attack and inhibit cell proliferation, we established an in vitro transfection of human HO-1 gene into rat VSMC mediated by a retroviral vector. The results showed that the profound expression of HO-1 protein as well as HO activity was 1.8- and 2.0-fold increased respectively in the transfected cells compared to the non-transfected ones. The treatment of VSMC with different concentrations of H2O2 led to the remarkable cell damage as indicated by survival rate and LDH leakage. However, the resistance of the HO-1 transfected VSMC against H2O2 was significantly raised. This protective effect was dramatically diminished when the transfected VSMC were pretreated with ZnPP-IX, a specific inhibitor of HO, for 24 h. In addition, we found that the growth potential of the transfected cells was significantly inhibited directly by increased activity of HO-1, and this effect might be related to decreased phosphorylation of MAPK. These results suggest that the overexpression of introduced hHO-1 is potentially able to reduce the risk factors of atherosclerosis, partially due to its cellular protection against oxidative injury and to its inhibitory effect on cellular proliferation. PMID:12118938

  12. The role of Rac1 in the regulation of NF-kB activity, cell proliferation, and cell migration in non-small cell lung carcinoma

    PubMed Central

    Gastonguay, Adam; Berg, Tracy; Hauser, Andrew D.; Schuld, Nathan; Lorimer, Ellen; Williams, Carol L.

    2012-01-01

    The small GTPase Rac1 regulates many cellular processes, including cytoskeletal reorganization, cell migration, proliferation, and survival. Additionally, Rac1 plays a major role in activating NF-κB-mediated transcription. Both Rac1 and NF-κB regulate many properties of the malignant phenotype, including anchorage-independent proliferation and survival, metastasis, and angiogenesis. Despite these findings, the roles of Rac1and NF-κB in non-small cell lung carcinoma, a leading cause of cancer deaths, have not been thoroughly investigated. Here, we compared the effects of Rac1 siRNA to that of the Rac1 inhibitor NSC23766 on multiple features of the NSCLC malignant phenotype, including NF-κB activity. We show that the siRNA-mediated silencing of Rac1 in lung cancer cells results in decreased cell proliferation and migration. The decrease in proliferation was observed in both anchorage-dependent and anchorage-independent assays. Furthermore, cells with decreased Rac1 expression have a slowed progression through the G1 phase of the cell cycle. These effects induced by Rac1 siRNA correlated with a decrease in NF-κB transcriptional activity. Additionally, inhibition of NF-κB signaling with BAY 11–7082 inhibited proliferation; indicating that the loss of cell proliferation and migration induced by the silencing of Rac1 expression may be attributed in part to loss of NF-κB activity. Interestingly, treatment with the Rac1 inhibitor NSC23766 strongly inhibits cell proliferation, cell cycle progression, and NF-κB activity in lung cancer cells, to an even greater extent than the inhibition induced by Rac1 siRNA. These findings indicate that Rac1 plays an important role in lung cancer cell proliferation and migration, most likely through its ability to promote NF-κB activity, and highlight Rac1 pathways as therapeutic targets for the treatment of lung cancer. PMID:22549160

  13. Downregulation of cell division cycle 25 homolog C reduces the radiosensitivity and proliferation activity of esophageal squamous cell carcinoma.

    PubMed

    Yin, Yachao; Dou, Xiaoyan; Duan, Shimiao; Zhang, Lei; Xu, Quanjing; Li, Hongwei; Li, Duojie

    2016-09-30

    Radiation therapy is one of the most important methods of contemporary cancer treatment. Cells in the G2 and M phases are more sensitive to radiation therapy, and cell division cycle 25 homolog C (CDC25C) is essential in shifting the cell cycle between these two phases. In this study, the knockdown of CDC25C in human esophageal squamous carcinoma EC9706 cells was mediated by transfecting shRNA against human CDC25C-subcloning into pGV248. The levels of CDC25C mRNA and protein expression were assessed by reverse transcription-polymerase chain reaction (RT-PCR) and western blotting, respectively. Moreover, cell proliferation and radiosensitivity were measured. Stable CDC25C-knockdown EC9706 cell lines were successfully established. Furthermore, the proliferation of both control and CDC25C-shRNA-EC9706 cells was inhibited after the cells were treated with increasing X-ray doses, and the proliferation of the control cells was affected more significantly (p<0.05). Moreover, cell colony formation assays allowed us to reach the same conclusion. Taken together, our experiments demonstrated that the knockdown of CDC25C can reduce both the radiotherapy sensitivity and the proliferation activity of EC9706 cells. Thus, CDC25C might be a potential biomarker for radiotherapy treatment. PMID:27188256

  14. Mechanisms of Edible Bird's Nest Extract-Induced Proliferation of Human Adipose-Derived Stem Cells.

    PubMed

    Roh, Kyung-Baeg; Lee, Jienny; Kim, Young-Soo; Park, Junho; Kim, Jang-Hyun; Lee, Jongsung; Park, Deokhoon

    2012-01-01

    Although edible bird's nest (EBN) has been shown to potentiate mitogenic responses, scientific evidence of its efficacy is still limited. In addition, human adipose-derived stem cells (hADSCs) are increasingly accepted as a source for stem cell therapy. Therefore, the aim of this study was to investigate the effects of the EBN extract (EBNE) on the proliferation of hADSCs and its action mechanisms. We found that EBNE strongly promoted the proliferation of hADSCs. In addition, EBNE-induced proliferation was found to be mediated through the production of IL-6 and VEGF, which was induced by activation of AP-1 and NF-κB. Specially, we found that production of IL-6 and VEGF was induced by EBNE. In addition, EBNE-induced production of IL-6 and VEGF was inhibited by PD98059 (a p44/42 MAPK inhibitor), SB203580 (a p38 MAPK inhibitor), and PDTC (a NF-κB inhibitor), but not SP600125 (a JNK inhibitor). Similarly, EBNE-induced proliferation of hADSCs was also attenuated by PD98059, SB203580, and PDTC but not SP600125. Taken together, these findings suggest that the EBNE-induced proliferation of hADSCs primarily occurs through increased expression of IL-6 and VEGF genes, which is mediated by the activation of NF-κB and AP-1 through p44/42 MAPK and p38 MAPK. PMID:22110547

  15. Integrin-Mediated Adhesion and Proliferation of Human MCs Elicited by A Hydroxyproline-Lacking, Collagen-like Peptide

    PubMed Central

    Krishna, Ohm D.; Jha, Amit K.; Jia, Xinqiao; Kiick, Kristi L.

    2011-01-01

    In this study, we evaluated the competence of a rationally designed collagen-like peptide (CLP-Cys) sequence - containing the minimal essential Glycine-Glutamic acid-Arginine (GER) triplet but lacking the hydroxyproline residue - for supporting human mesenchymal stem cell (hMSC) adhesion, spreading and proliferation. Cellular responses to the CLP-Cys sequence were analyzed by conjugating the peptide to two different substrates – a hard, planar glass surface and a soft hyaluronic acid (HA) particle-based hydrogel. Integrin-mediated cell spreading and adhesion were observed for hMSCs cultivated on the CLP-Cys functionalized surfaces, whereas on control surfaces lacking the peptide motif, cells either did not adhere or maintained a round morphology. On the glass surface, CLP-Cys-mediated spreading led to the formation of extended and well developed stress fibers composed of F-actin bundles and focal adhesion complexes while on the soft gel surface, less cytoskeletal reorganization was observed. The hMSCs proliferated significantly on the surfaces presenting CLP-Cys, compared to the control surfaces lacking CLP-Cys. Competitive binding assay employing soluble CLP-Cys revealed a dose-dependent inhibition of hMSC adhesion to the CLP-Cys-presenting surfaces. Blocking the α2β1 receptor on hMSC also resulted in a reduction of cell adhesion on both types of CLP-Cys surfaces, confirming the affinity of CLP-Cys to α2β1 receptors. These results established the competence of the hydroxyproline-free CLP-Cys for eliciting integrin-mediated cellular responses including adhesion, spreading and proliferation. Thus, CLP-Cys-modified HA hydrogels are attractive candidates as bioactive scaffolds for tissue engineering applications. PMID:21658756

  16. Integrin-mediated adhesion and proliferation of human MSCs elicited by a hydroxyproline-lacking, collagen-like peptide.

    PubMed

    Krishna, Ohm D; Jha, Amit K; Jia, Xinqiao; Kiick, Kristi L

    2011-09-01

    In this study, we evaluated the competence of a rationally designed collagen-like peptide (CLP-Cys) sequence - containing the minimal essential Glycine-Glutamic acid-Arginine (GER) triplet but lacking the hydroxyproline residue - for supporting human mesenchymal stem cell (hMSC) adhesion, spreading and proliferation. Cellular responses to the CLP-Cys sequence were analyzed by conjugating the peptide to two different substrates - a hard, planar glass surface and a soft hyaluronic acid (HA) particle-based hydrogel. Integrin-mediated cell spreading and adhesion were observed for hMSCs cultivated on the CLP-Cys functionalized surfaces, whereas on control surfaces lacking the peptide motif, cells either did not adhere or maintained a round morphology. On the glass surface, CLP-Cys-mediated spreading led to the formation of extended and well developed stress fibers composed of F-actin bundles and focal adhesion complexes while on the soft gel surface, less cytoskeletal reorganization organiz