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Sample records for activation induced cell

  1. Borrelia burgdorferi Spirochetes Induce Mast Cell Activation and Cytokine Release

    PubMed Central

    Talkington, Jeffrey; Nickell, Steven P.

    1999-01-01

    The Lyme disease spirochete, Borrelia burgdorferi, is introduced into human hosts via tick bites. Among the cell types present in the skin which may initially contact spirochetes are mast cells. Since spirochetes are known to activate a variety of cell types in vitro, we tested whether B. burgdorferi spirochetes could activate mast cells. We report here that freshly isolated rat peritoneal mast cells or mouse MC/9 mast cells cultured in vitro with live or freeze-thawed B. burgdorferi spirochetes undergo low but detectable degranulation, as measured by [5-3H] hydroxytryptamine release, and they synthesize and secrete the proinflammatory cytokine tumor necrosis factor alpha (TNF-α). In contrast to findings in previous studies, where B. burgdorferi-associated activity was shown to be dependent upon protein lipidation, mast cell TNF-α release was not induced by either lipidated or unlipidated recombinant OspA. This activity was additionally shown to be protease sensitive and surface expressed. Finally, comparisons of TNF-α-inducing activity in known low-, intermediate-, and high-passage B. burgdorferi B31 isolates demonstrated passage-dependent loss of activity, indicating that the activity is probably plasmid encoded. These findings document the presence in low-passage B. burgdorferi spirochetes of a novel lipidation-independent activity capable of inducing cytokine release from host cells. PMID:10024550

  2. Increased Mitochondrial Activity in Anthrax-Induced Cell Death

    PubMed Central

    Li, Chi

    2009-01-01

    Pathogenesis of anthrax lethal toxin (LT) is attributed to its ability to cause death of infected cells. New work has demonstrated that increase of mitochondrial F1F0 ATPase activity and subsequent depletion of cellular ATP level are critical early events during LT-induced cell death. PMID:26124679

  3. Activated Factor X Induces Endothelial Cell Senescence Through IGFBP-5

    PubMed Central

    Sanada, Fumihiro; Taniyama, Yoshiaki; Muratsu, Jun; Otsu, Rei; Iwabayashi, Masaaki; Carracedo, Miguel; Rakugi, Hiromi; Morishita, Ryuichi

    2016-01-01

    Uncontrolled coagulation contributes to the pathophysiology of several chronic inflammatory diseases. In these conditions, senescent cells are often observed and is involved in the generation of inflammation. The coincidence of hyper-coagulation, cell senescence, and inflammation suggests the existence of a common underlying mechanism. Recent evidence indicates that activated coagulation factor X (FXa) plays a role in the processes beyond blood coagulation. This non-hematologic function entails the mediation of inflammation and tissue remodeling. We therefore tested the hypothesis that FXa induces cell senescence resulting in tissue inflammation and impaired tissue regeneration. Human umbilical vein endothelial cells were stimulated with FXa for 14 days. The proliferation of cells treated with FXa was significantly smaller, and the fraction of senescence-associated β-galactosidase-positive cells was increased as compared to the control group. RT-qPCR array revealed that FXa increased the expression of IGFBP-5, EGR-1, p53, and p16INK4a. Inhibition of FXa by a direct FXa inhibitor, rivaroxaban, or IGFBP-5 by siRNA decreased FXa-induced cell senescence, restoring cell proliferation. Moreover, in an ischemic hind limb mouse model, FXa inhibited neovascularization by endothelial progenitor cell. However, rivaroxaban significantly restored FXa-induced impaired angiogenesis. In summary, FXa induced endothelial cell senescence through IGFBP-5, resulting in impaired angiogenesis. PMID:27752126

  4. LPS induces pulp progenitor cell recruitment via complement activation.

    PubMed

    Chmilewsky, F; Jeanneau, C; Laurent, P; About, I

    2015-01-01

    Complement system, a major component of the natural immunity, has been recently identified as an important mediator of the dentin-pulp regeneration process through STRO-1 pulp cell recruitment by the C5a active fragment. Moreover, it has been shown recently that under stimulation with lipoteichoic acid, a complex component of the Gram-positive bacteria cell wall, human pulp fibroblasts are able to synthesize all proteins required for complement activation. However, Gram-negative bacteria, which are also involved in tooth decay, are known as powerful activators of complement system and inflammation. Here, we investigated the role of Gram-negative bacteria-induced complement activation on the pulp progenitor cell recruitment using lipopolysaccharide (LPS), a major component of all Gram-negative bacteria. Our results show that incubating pulp fibroblasts with LPS induced membrane attack complex formation and C5a release in serum-free fibroblast cultures. The produced C5a binds to the pulp progenitor cells' membrane and induces their migration toward the LPS stimulation chamber, as revealed by the dynamic transwell migration assays. The inhibition of this migration by the C5aR-specific antagonist W54011 indicates that the pulp progenitor migration is mediated by the interaction between C5a and C5aR. Our findings demonstrate, for the first time, a direct interaction between the recruitment of progenitor pulp cells and the activation of complement system generated by pulp fibroblast stimulation with LPS.

  5. Acetaminophen Induces Human Neuroblastoma Cell Death through NFKB Activation

    PubMed Central

    Posadas, Inmaculada; Santos, Pablo; Ceña, Valentín

    2012-01-01

    Neuroblastoma resistance to apoptosis may contribute to the aggressive behavior of this tumor. Therefore, it would be relevant to activate endogenous cellular death mechanisms as a way to improve neuroblastoma therapy. We used the neuroblastoma SH-SY5Y cell line as a model to study the mechanisms involved in acetaminophen (AAP)-mediated toxicity by measuring CYP2E1 enzymatic activity, NFkB p65 subunit activation and translocation to the nucleus, Bax accumulation into the mitochondria, cytochrome c release and caspase activation. AAP activates the intrinsic death pathway in the SH-SY5Y human neuroblastoma cell line. AAP metabolism is partially responsible for this activation, because blockade of the cytochrome CYP2E1 significantly reduced but did not totally prevent, AAP-induced SH-SY5Y cell death. AAP also induced NFkB p65 activation by phosphorylation and its translocation to the nucleus, where NFkB p65 increased IL-1β production. This increase contributed to neuroblastoma cell death through a mechanism involving Bax accumulation into the mitochondria, cytochrome c release and caspase3 activation. Blockade of NFkB translocation to the nucleus by the peptide SN50 prevented AAP-mediated cell death and IL-1β production. Moreover, overexpression of the antiapoptotic protein Bcl-xL did not decrease AAP-mediated IL-1β production, but prevented both AAP and IL-1β-mediated cell death. We also confirmed the AAP toxic actions on SK-N-MC neuroepithelioma and U87MG glioblastoma cell lines. The results presented here suggest that AAP activates the intrinsic death pathway in neuroblastoma cells through a mechanism involving NFkB and IL-1β. PMID:23166834

  6. Plasma-activated medium induced apoptosis on tumor cells

    NASA Astrophysics Data System (ADS)

    Hori, Masaru; Tanaka, Hiromasa; Mizuno, Masaaki; Nakamura, Kae; Kajiyama, Hiroaki; Takeda, Keigo; Ishikawa, Kenji; Kano, Hiroyuki; Kikkawa, Fumitaka

    2013-09-01

    The non-equilibrium atmospheric pressure plasma (NEAPP) has attracted attention in cancer therapy. In this study, the fresh medium was treated with our developed NEAPP, ultra-high electron density (approximately 2 × 1016 cm-3). The medium called the plasma-activated medium (PAM) killed not normal cells but tumor cells through induction of apoptosis. Cell proliferation assays showed that the tumor cells were selectively killed by the PAM. Those cells induced apoptosis using an apoptotic molecular marker, cleaved Caspase3/7. The molecular mechanisms of PAM-mediated apoptosis in the tumor cells were also found that the PAM downregulated the expression of AKT kinase, a marker molecule in a survival signal transduction pathway. These results suggest that PAM may be a promising tool for tumor therapy by downregulating the survival signals in cancers.

  7. Activation-induced CD154 expression abrogates tolerance induced by apoptotic cells*

    PubMed Central

    Gurung, Prajwal; Kucaba, Tamara A.; Ferguson, Thomas A.; Griffith, Thomas S.

    2009-01-01

    The decision to generate a productive immune response or tolerance often depends on the context in which T cells first see Ag. Using a classical system of tolerance induction, we examined the immunological consequence of Ag encountered in the presence of naïve or activated apoptotic cells. Naïve apoptotic cells induced tolerance when injected i.v.; however, previously activated apoptotic cells induced immunity. Further analysis revealed a key role for CD154, as tolerance resulted after i.v. injection of either naïve or activated apoptotic CD154−/− T cells, while co-injection of an agonistic anti-CD40 mAb with naïve apoptotic T cells induced robust immunity. DC fed activated apoptotic T cells in vitro produced IL-12p40 in a CD154-dependent manner, and the use of IL-12p40−/− mice or mAb-mediated neutralization of IL-12 revealed a link between CD154, IL-12, and the ability of activated apoptotic T cells to induce immunity rather than tolerance. Collectively these results show that CD154 expression on apoptotic T cells can determine the outcome of an immune response to Ag recognized within the context of the apoptotic cells, and suggest the balance between naïve and activated apoptotic T cells may dictate whether a productive immune response is encouraged. PMID:19841180

  8. Vanadium pentoxide induces activation and death of endothelial cells.

    PubMed

    Montiel-Dávalos, Angélica; Gonzalez-Villava, Adriana; Rodriguez-Lara, Vianey; Montaño, Luis Felipe; Fortoul, Teresa I; López-Marure, Rebeca

    2012-01-01

    Vanadium is a transition metal released into the atmosphere, as air-suspended particles, as a result of the combustion of fossil fuels and some metallurgic industry activities. Air-suspended particle pollution causes inflammation-related processes such as thrombosis and other cardiovascular events. Our aim was to evaluate the effect of vanadium pentoxide (V2O5) on endothelial cells since they are key participants in the pathogenesis of several cardiovascular and inflammatory diseases. Cell adhesion, the expression of adhesion molecules and oxidative stress, as well as proliferation, morphology and cell death of human umbilical vein endothelial cells (HUVECs) exposed to V2O5, were evaluated. Vanadium pentoxide at a 3.12 µg cm(-2) concentration induced an enhanced adhesion of the U937 macrophage cell line to HUVECs, owing to an increased expression of late adhesion molecules. HUVECs exposed to V2O5 showed an increase in ROS and nitric oxide production, and a diminished proliferation. These changes in vanadium-treated HUVECs were accompanied by severe morphological changes and apoptotic cell death. Vanadium pentoxide induced serious endothelial cell damage, probably related to the increased cardiovascular morbidity and mortality observed in individuals living in highly air-polluted areas. PMID:21721017

  9. Interleukin-7 and Toll-Like Receptor 7 Induce Synergistic B Cell and T Cell Activation

    PubMed Central

    Bikker, Angela; Kruize, Aike A.; van der Wurff-Jacobs, Kim M. G.; Peters, Rogier P.; Kleinjan, Marije; Redegeld, Frank; de Jager, Wilco; Lafeber, Floris P. J. G.; van Roon, Joël A. G.

    2014-01-01

    Objectives To investigate the potential synergy of IL-7-driven T cell-dependent and TLR7-mediated B cell activation and to assess the additive effects of monocyte/macrophages in this respect. Methods Isolated CD19 B cells and CD4 T cells from healthy donors were co-cultured with TLR7 agonist (TLR7A, Gardiquimod), IL-7, or their combination with or without CD14 monocytes/macrophages (T/B/mono; 1 : 1 : 0,1). Proliferation was measured using 3H-thymidine incorporation and Ki67 expression. Activation marker (CD19, HLA-DR, CD25) expression was measured by FACS analysis. Immunoglobulins were measured by ELISA and release of cytokines was measured by Luminex assay. Results TLR7-induced B cell activation was not associated with T cell activation. IL-7-induced T cell activation alone and together with TLR7A synergistically increased numbers of both proliferating (Ki67+) B cells and T cells, which was further increased in the presence of monocytes/macrophages. This was associated by up regulation of activation markers on B cells and T cells. Additive or synergistic induction of production of immunoglobulins by TLR7 and IL-7 was associated by synergistic induction of T cell cytokines (IFNγ, IL-17A, IL-22), which was only evident in the presence of monocytes/macrophages. Conclusions IL-7-induced CD4 T cell activation and TLR7-induced B cell activation synergistically induce T helper cell cytokine and B cell immunoglobulin production, which is critically dependent on monocytes/macrophages. Our results indicate that previously described increased expression of IL-7 and TLR7 together with increased numbers of macrophages at sites of inflammation in autoimmune diseases like RA and pSS significantly contributes to enhanced lymphocyte activation. PMID:24740301

  10. Activated microglia cause reversible apoptosis of pheochromocytoma cells, inducing their cell death by phagocytosis.

    PubMed

    Hornik, Tamara C; Vilalta, Anna; Brown, Guy C

    2016-01-01

    Some apoptotic processes, such as phosphatidylserine exposure, are potentially reversible and do not necessarily lead to cell death. However, phosphatidylserine exposure can induce phagocytosis of a cell, resulting in cell death by phagocytosis: phagoptosis. Phagoptosis of neurons by microglia might contribute to neuropathology, whereas phagoptosis of tumour cells by macrophages might limit cancer. Here, we examined the mechanisms by which BV-2 microglia killed co-cultured pheochromocytoma (PC12) cells that were either undifferentiated or differentiated into neuronal cells. We found that microglia activated by lipopolysaccharide rapidly phagocytosed PC12 cells. Activated microglia caused reversible phosphatidylserine exposure on and reversible caspase activation in PC12 cells, and caspase inhibition prevented phosphatidylserine exposur and decreased subsequent phagocytosis. Nitric oxide was necessary and sufficient to induce the reversible phosphatidylserine exposure and phagocytosis. The PC12 cells were not dead at the time they were phagocytised, and inhibition of their phagocytosis left viable cells. Cell loss was inhibited by blocking phagocytosis mediated by phosphatidylserine, MFG-E8, vitronectin receptors or P2Y6 receptors. Thus, activated microglia can induce reversible apoptosis of target cells, which is insufficient to cause apoptotic cell death, but sufficient to induce their phagocytosis and therefore cell death by phagoptosis.

  11. Streptococcus induces circulating CLA(+) memory T-cell-dependent epidermal cell activation in psoriasis.

    PubMed

    Ferran, Marta; Galván, Ana B; Rincón, Catalina; Romeu, Ester R; Sacrista, Marc; Barboza, Erika; Giménez-Arnau, Ana; Celada, Antonio; Pujol, Ramon M; Santamaria-Babí, Luis F

    2013-04-01

    Streptococcal throat infection is associated with a specific variant of psoriasis and with HLA-Cw6 expression. In this study, activation of circulating psoriatic cutaneous lymphocyte-associated antigen (CLA)(+) memory T cells cultured together with epidermal cells occurred only when streptococcal throat extracts were added. This triggered the production of Th1, Th17, and Th22 cytokines, as well as epidermal cell mediators (CXCL8, CXCL9, CXCL10, and CXCL11). Streptococcal extracts (SEs) did not induce any activation with either CLA(-) cells or memory T cells cultured together with epidermal cells from healthy subjects. Intradermal injection of activated culture supernatants into mouse skin induced epidermal hyperplasia. SEs also induced activation when we used epidermal cells from nonlesional skin of psoriatic patients with CLA(+) memory T cells. Significant correlations were found between SE induced upregulation of mRNA expression for ifn-γ, il-17, il-22, ip-10, and serum level of antistreptolysin O in psoriatic patients. This study demonstrates the direct involvement of streptococcal infection in pathological mechanisms of psoriasis, such as IL-17 production and epidermal cell activation.

  12. Fractalkine Attenuates Microglial Cell Activation Induced by Prenatal Stress

    PubMed Central

    Ślusarczyk, Joanna; Trojan, Ewa; Głombik, Katarzyna; Chamera, Katarzyna; Roman, Adam; Budziszewska, Bogusława; Basta-Kaim, Agnieszka

    2016-01-01

    The potential contribution of inflammation to the development of neuropsychiatric diseases has recently received substantial attention. In the brain, the main immune cells are the microglia. As they are the main source of inflammatory factors, it is plausible that the regulation of their activation may be a potential therapeutic target. Fractalkine (CX3CL1) and its receptor CX3CR1 play a crucial role in the control of the biological activity of the microglia. In the present study, using microglial cultures we investigated whether fractalkine is able to reverse changes in microglia caused by a prenatal stress procedure. Our study found that the microglia do not express fractalkine. Prenatal stress decreases the expression of the fractalkine receptor, which in turn is enhanced by the administration of exogenous fractalkine. Moreover, treatment with fractalkine diminishes the prenatal stress-induced overproduction of proinflammatory factors such as IL-1β, IL-18, IL-6, TNF-α, CCL2, or NO in the microglial cells derived from prenatally stressed newborns. In conclusion, the present results revealed that the pathological activation of microglia in prenatally stressed newborns may be attenuated by fractalkine administration. Therefore, understanding of the role of the CX3CL1-CX3CR1 system may help to elucidate the mechanisms underlying the neuron-microglia interaction and its role in pathological conditions in the brain. PMID:27239349

  13. Endoplasmic reticulum stress activation mediates Ginseng Rg3-induced anti-gallbladder cancer cell activity.

    PubMed

    Wu, Keren; Li, Ning; Sun, Huaqin; Xu, Tao; Jin, Fa; Nie, Jifeng

    2015-10-23

    In the current study, we examined the potential effect of Ginsenoside Rg3 against gallbladder cancer cells, the underlying signaling mechanisms were also studied. We demonstrated that Rg3 exerted potent cytotoxic and pro-apoptotic activity against established and primary human gallbladder cancer cells. Yet it was safe to non-cancerous gallbladder epithelial cells. At the molecular level, we showed that Rg3 induced endoplasmic reticulum (ER) stress activation, the latter was evidenced by C/EBP homologous protein (CHOP) upregulation, inositol-requiring enzyme 1 (IRE1)/PKR-like endoplasmic reticulum kinase (PERK) phosphorylations, and caspase-12 activation in gallbladder cancer cells. Reversely, the ER stress inhibitor salubrinal, the caspase-12 inhibitor z-ATAD-fmk as well as CHOP shRNA knockdown significantly attenuated Rg3-induced cytotoxicity against gallbladder cancer cells. In vivo, we showed that Rg3 oral administration significantly inhibited GBC-SD gallbladder cancer xenograft growth in nude mice, its activity was, however, compromised with co-administration of the ER stress inhibitor salubrinal. Thus, we suggest that ER stress activation mediates Ginseng Rg3-induced anti-gallbladder cancer cell activity in vitro and in vivo. PMID:26361144

  14. L1 cell adhesion molecule induces melanoma cell motility by activation of mitogen-activated protein kinase pathways.

    PubMed

    Yi, Young-Su; Baek, Kwang-Soo; Cho, Jae Youl

    2014-06-01

    L1 cell adhesion molecule (L1CAM) is highly expressed in various types of cancer cells and has been implicated in the control of cell proliferation and motility. Recently, L1CAM was reported to induce the motility of melanoma cells, but the mechanism of this induction remains poorly understood. In this study, we investigated the molecular mechanisms by which L1CAM induces the motility of melanoma cells. Unlike other types of cancer cells, B16F10 melanoma cells highly expressed L1CAM at both the RNA and protein levels, and the expression of L1CAM induced AP-1 activity. In accordance to AP-1 activation, MAPK signaling pathways were activated by L1CAM. Inhibition of L1CAM expression by L1CAM-specific siRNA suppressed the activation of MAPKs such as ERK and p38. However, no significant change was observed in JNK activation. As expected, upstream MAP2K, MKK3/6, MAP3K, and TAK1 were also deactivated by the inhibition of L1CAM expression. L1CAM induced the motility of B16F10 cells. Inhibition of L1CAM expression suppressed migration and invasion of B16F10 cells, but no suppressive effect was observed on their proliferation and anti-apoptotic resistance. Treatment of B16F10 cells with U0126, an ERK inhibitor, or SB203580, a p38 inhibitor, suppressed the migration and invasion abilities of B16F10 cells. Taken together, our results suggest that L1CAM induces the motility of B16F10 melanoma cells via the activation of MAPK pathways. This finding provides a more detailed molecular mechanism of L1CAM-mediated induction of melanoma cell motility. PMID:24974583

  15. Mycoplasma pneumoniae induces cytotoxic activity in guinea pig bronchoalveolar cells

    SciTech Connect

    Kist, M.; Koester, H.; Bredt, W.

    1985-06-01

    Precultured guinea pig alveolar macrophages (AM) and freshly harvested alveolar cells (FHAC) activated by interaction with Mycoplasma pneumoniae were cytotoxic for xenogeneic /sup 75/selenomethionine-labeled tumor target cells. Phagocytosis of whole opsonized or nonopsonized M. pneumoniae cells was more effective in eliciting cytotoxicity than uptake of sonicated microorganisms. The addition of living mycoplasma cells to the assay system enhanced the cytotoxic effect considerably. Target cells were significantly more susceptible to the cytotoxic action of phagocytes if they were coated with mycoplasma antigen or cocultured together with M. pneumoniae. The activation of the phagocytes could be inhibited by 2-deoxy-D-glucose but not by antimicrobial substances suppressing mycoplasma protein synthesis. It was accompanied by /sup 51/Cr release without detectable signs of cell damage. The supernatants of activated cells were cytotoxic for approximately 24 h. Inhibition, release, and cytotoxic activity indicate the necessity of an intact metabolism of the effector cells and suggest a secretion of cytotoxic substances.

  16. Raloxifene induces autophagy-dependent cell death in breast cancer cells via the activation of AMP-activated protein kinase.

    PubMed

    Kim, Dong Eun; Kim, Yunha; Cho, Dong-Hyung; Jeong, Seong-Yun; Kim, Sung-Bae; Suh, Nayoung; Lee, Jung Shin; Choi, Eun Kyung; Koh, Jae-Young; Hwang, Jung Jin; Kim, Choung-Soo

    2015-01-01

    Raloxifene is a selective estrogen receptor modulator (SERM) that binds to the estrogen receptor (ER), and exhibits potent anti-tumor and autophagy-inducing effects in breast cancer cells. However, the mechanism of raloxifene-induced cell death and autophagy is not well-established. So, we analyzed mechanism underlying death and autophagy induced by raloxifene in MCF-7 breast cancer cells. Treatment with raloxifene significantly induced death in MCF-7 cells. Raloxifene accumulated GFP-LC3 puncta and increased the level of autophagic marker proteins, such as LC3-II, BECN1, and ATG12-ATG5 conjugates, indicating activated autophagy. Raloxifene also increased autophagic flux indicators, the cleavage of GFP from GFP-LC3 and only red fluorescence-positive puncta in mRFP-GFP-LC3-expressing cells. An autophagy inhibitor, 3-methyladenine (3-MA), suppressed the level of LC3-II and blocked the formation of GFP-LC3 puncta. Moreover, siRNA targeting BECN1 markedly reversed cell death and the level of LC3-II increased by raloxifene. Besides, raloxifene-induced cell death was not related to cleavage of caspases-7, -9, and PARP. These results indicate that raloxifene activates autophagy-dependent cell death but not apoptosis. Interestingly, raloxifene decreased the level of intracellular adenosine triphosphate (ATP) and activated the AMPK/ULK1 pathway. However it was not suppressed the AKT/mTOR pathway. Addition of ATP decreased the phosphorylation of AMPK as well as the accumulation of LC3-II, finally attenuating raloxifene-induced cell death. Our current study demonstrates that raloxifene induces autophagy via the activation of AMPK by sensing decreases in ATP, and that the overactivation of autophagy promotes cell death and thereby mediates the anti-cancer effects of raloxifene in breast cancer cells.

  17. Ajoene inhibits the activation of human endothelial cells induced by porcine cells: implications for xenotransplantation.

    PubMed

    Benatuil, Lorenzo; Apitz-Castro, Rafael; Romano, Egidio

    2003-07-01

    Ajoene, is an organosulfur compound derived from garlic that strongly inhibit platelet aggregation, proliferation of human lymphocytes induced by phytohemagglutinin, and in general, blocks membrane-mediated signaling of cell activation. As a thrombotic microangiopathy frequently complicates procedures designed to induce pig-to-baboon chimerism by infusion of large amounts of pig progenitor cells in baboons, it was thought that ajoene might be useful to prevent such complication. For such purpose, we studied the effects of ajoene on the activation of human umbilical vein endothelial cells (HUVEC) induced by pig peripheral blood mononuclear cells (p-PBMC). Co-cultures of p-PBMC with HUVEC results in activation of the HUVEC as shown by over-expression of E-selectin and vascular cells adhesion molecule-1 (VCAM-1). Ajoene (25 microm) strongly inhibits HUVEC activation induced by tumor necrosis factor-alpha (TNF-alpha) or p-PBMC as shown by a down regulation of VCAM-1 and of E-selectin expression. After 5 or 8 h of pre-treatment with Ajoene, HUVEC incubated with TNF and p-PBMC showed an E-selectin or VCAM-1 expression, respectively, at levels similar to the positive control indicating that the inhibitory effect is transient. Ajoene at concentration of 25 microm or lower did not affect HUVEC viability. Based on the finding that Ajoene has a strong, although transient, inhibitory effect on the activation of the endothelium induced by pig cells and its known anti-platelet activity, it is suggested that this garlic compound could be useful to prevent the development of microangiopathy and thrombotic disorders seen in primates infused with pig cells.

  18. The lectin ArtinM binds to mast cells inducing cell activation and mediator release.

    PubMed

    Barbosa-Lorenzi, Valéria Cintra; Buranello, Patrícia Andressa de Almeida; Roque-Barreira, Maria Cristina; Jamur, Maria Célia; Oliver, Constance; Pereira-da-Silva, Gabriela

    2011-12-16

    Mast cells are inflammatory cells that respond to signals of innate and adaptive immunity with immediate and delayed release of mediators. ArtinM, a lectin from Artocarpus integrifolia with immunomodulatory activities, is able to induce mast cell activation, but the mechanisms remain unknown. This study sought to further investigate the effects of the lectin on mast cells. We showed that ArtinM binds to mast cells, possibly to the high affinity receptor for immunoglobulin E (IgE) - FcεRI - and/or to IgE bound to FcεRI. Binding of the lectin resulted in protein tyrosine phosphorylation and release of the pre- and newly-formed mediators, β-hexosaminidase and LTB(4) by mast cells, activities that were potentiated in the presence of IgE. ArtinM also induced the activation of the transcription factors NFκB and NFAT, resulting in expression of some of their target genes such as IL-4 and TNF-α. In view of the established significance of mast cells in many immunological and inflammatory reactions, a better understanding of the mechanisms involved in mast cell activation by ArtinM is crucial to the pharmacological application of the lectin.

  19. Berberine-induced anticancer activities in FaDu head and neck squamous cell carcinoma cells.

    PubMed

    Seo, Yo-Seob; Yim, Min-Ji; Kim, Bok-Hee; Kang, Kyung-Rok; Lee, Sook-Young; Oh, Ji-Su; You, Jae-Seek; Kim, Su-Gwan; Yu, Sang-Joun; Lee, Gyeong-Je; Kim, Do Kyung; Kim, Chun Sung; Kim, Jin-Soo; Kim, Jae-Sung

    2015-12-01

    In the present study, we investigated berberine‑induced apoptosis and the signaling pathways underlying its activity in FaDu head and neck squamous cell carcinoma cells. Berberine did not affect the viability of primary human normal oral keratinocytes. In contrast, the cytotoxicity of berberine was significantly increased in FaDu cells stimulated with berberine for 24 h. Furthermore, berberine increased nuclear condensation and apoptosis rates in FaDu cells than those in untreated control cells. Berberine also induced the upregulation of apoptotic ligands, such as FasL and TNF-related apoptosis-inducing ligand, and triggered the activation of caspase-8, -7 and -3, and poly(ADP ribose) polymerase, characteristic of death receptor-dependent extrinsic apoptosis. Moreover, berberine activated the mitochondria‑dependent apoptotic signaling pathway by upregulating pro-apoptotic factors, such as Bax, Bad, Apaf-1, and the active form of caspase-9, and downregulating anti-apoptotic factors, such as Bcl-2 and Bcl-xL. In addition, berberine increased the expression of the tumor suppressor p53 in FaDu cells. The pan-caspase inhibitor Z-VAD-fmk suppressed the activation of caspase-3 and prevented cytotoxicity in FaDu cells treated with berberine. Interestingly, berberine suppressed cell migration through downregulation of vascular endothelial growth factor (VEGF), matrix metalloproteinase (MMP)-2, and MMP-9. Moreover, the phosphorylation of extracellular signal-regulated kinase (ERK1/2) and p38, components of the mitogen-activated protein kinase pathway that are associated with the expression of MMP and VEGF, was suppressed in FaDu cells treated with berberine for 24 h. Therefore, these data suggested that berberine exerted anticancer effects in FaDu cells through induction of apoptosis and suppression of migration. Berberine may have potential applications as a chemotherapeutic agent for the management of head and neck squamous carcinoma.

  20. Microwave-induced thermogenetic activation of single cells

    SciTech Connect

    Safronov, N. A.; Fedotov, I. V.; Ermakova, Yu. G.; Matlashov, M. E.; Belousov, V. V.; Sidorov-Biryukov, D. A.; Fedotov, A. B.; Zheltikov, A. M.

    2015-04-20

    Exposure to a microwave field is shown to enable thermogenetic activation of individual cells in a culture of cell expressing thermosensitive ion channels. Integration of a microwave transmission line with an optical fiber and a diamond quantum thermometer has been shown to allow thermogenetic single-cell activation to be combined with accurate local online temperature measurements based on an optical detection of electron spin resonance in nitrogen–vacancy centers in diamond.

  1. Activation of AMP-activated protein kinase by tributyltin induces neuronal cell death

    SciTech Connect

    Nakatsu, Yusuke; Kotake, Yaichiro Hino, Atsuko; Ohta, Shigeru

    2008-08-01

    AMP-activated protein kinase (AMPK), a member of the metabolite-sensing protein kinase family, is activated by energy deficiency and is abundantly expressed in neurons. The environmental pollutant, tributyltin chloride (TBT), is a neurotoxin, and has been reported to decrease cellular ATP in some types of cells. Therefore, we investigated whether TBT activates AMPK, and whether its activation contributes to neuronal cell death, using primary cultures of cortical neurons. Cellular ATP levels were decreased 0.5 h after exposure to 500 nM TBT, and the reduction was time-dependent. It was confirmed that most neurons in our culture system express AMPK, and that TBT induced phosphorylation of AMPK. Compound C, an AMPK inhibitor, reduced the neurotoxicity of TBT, suggesting that AMPK is involved in TBT-induced cell death. Next, the downstream target of AMPK activation was investigated. Nitric oxide synthase, p38 phosphorylation and Akt dephosphorylation were not downstream of TBT-induced AMPK activation because these factors were not affected by compound C, but glutamate release was suggested to be controlled by AMPK. Our results suggest that activation of AMPK by TBT causes neuronal death through mediating glutamate release.

  2. Serum Amyloid A Induces Inflammation, Proliferation and Cell Death in Activated Hepatic Stellate Cells

    PubMed Central

    Siegmund, Sören V.; Schlosser, Monika; Schildberg, Frank A.; Seki, Ekihiro; De Minicis, Samuele; Uchinami, Hiroshi; Kuntzen, Christian; Knolle, Percy A.; Strassburg, Christian P.; Schwabe, Robert F.

    2016-01-01

    Serum amyloid A (SAA) is an evolutionary highly conserved acute phase protein that is predominantly secreted by hepatocytes. However, its role in liver injury and fibrogenesis has not been elucidated so far. In this study, we determined the effects of SAA on hepatic stellate cells (HSCs), the main fibrogenic cell type of the liver. Serum amyloid A potently activated IκB kinase, c-Jun N-terminal kinase (JNK), Erk and Akt and enhanced NF-κB-dependent luciferase activity in primary human and rat HSCs. Serum amyloid A induced the transcription of MCP-1, RANTES and MMP9 in an NF-κB- and JNK-dependent manner. Blockade of NF-κB revealed cytotoxic effects of SAA in primary HSCs with signs of apoptosis such as caspase 3 and PARP cleavage and Annexin V staining. Serum amyloid A induced HSC proliferation, which depended on JNK, Erk and Akt activity. In primary hepatocytes, SAA also activated MAP kinases, but did not induce relevant cell death after NF-κB inhibition. In two models of hepatic fibrogenesis, CCl4 treatment and bile duct ligation, hepatic mRNA levels of SAA1 and SAA3 were strongly increased. In conclusion, SAA may modulate fibrogenic responses in the liver in a positive and negative fashion by inducing inflammation, proliferation and cell death in HSCs. PMID:26937641

  3. Activation-induced cytidine deaminase induces reproducible DNA breaks at many non-Ig loci in activated B cells

    PubMed Central

    Staszewski, Ori; Baker, Richard E.; Ucher, Anna J.; Martier, Raygene; Stavnezer, Janet; Guikema, Jeroen E.J.

    2011-01-01

    After immunization or infection, activation-induced cytidine deaminase (AID) initiates diversification of immunoglobulin (Ig) genes in B cells, introducing mutations within the antigen binding V regions (somatic hypermutation, SHM) and double-strand DNA breaks (DSBs) into switch (S) regions, leading to antibody class switch recombination (CSR). We asked if during B cell activation, AID also induces DNA breaks at genes other than IgH genes. Using a non-biased genome-wide approach, we have identified hundreds of reproducible AID-dependent DSBs in mouse splenic B cells shortly after induction of CSR in culture. Most interestingly, AID induces DSBs at sites syntenic with sites of translocations, deletions, and amplifications found in human B cell lymphomas, including within the oncogene B cell lymphoma11a (bcl11a)/evi9. Unlike AID-induced DSBs in Ig genes, genome-wide AID-dependent DSBs are not restricted to transcribed regions, and frequently occur within repeated sequence elements, including CA-repeats and non-CA tandem repeats, and SINEs. PMID:21255732

  4. Activation-induced cytidine deaminase induces reproducible DNA breaks at many non-Ig Loci in activated B cells.

    PubMed

    Staszewski, Ori; Baker, Richard E; Ucher, Anna J; Martier, Raygene; Stavnezer, Janet; Guikema, Jeroen E J

    2011-01-21

    After immunization or infection, activation-induced cytidine deaminase (AID) initiates diversification of immunoglobulin (Ig) genes in B cells, introducing mutations within the antigen-binding V regions (somatic hypermutation, SHM) and double-strand DNA breaks (DSBs) into switch (S) regions, leading to antibody class switch recombination (CSR). We asked if, during B cell activation, AID also induces DNA breaks at genes other than IgH genes. Using a nonbiased genome-wide approach, we have identified hundreds of reproducible, AID-dependent DSBs in mouse splenic B cells shortly after induction of CSR in culture. Most interestingly, AID induces DSBs at sites syntenic with sites of translocations, deletions, and amplifications found in human B cell lymphomas, including within the oncogene B cell lymphoma11a (bcl11a)/evi9. Unlike AID-induced DSBs in Ig genes, genome-wide AID-dependent DSBs are not restricted to transcribed regions and frequently occur within repeated sequence elements, including CA repeats, non-CA tandem repeats, and SINEs.

  5. Antiproliferative and cell apoptosis-inducing activities of compounds from Buddleja davidii in Mgc-803 cells

    PubMed Central

    2012-01-01

    Background Buddleja davidii is widely distributed in the southwestern region of China. We have undertaken a systematic analysis of B. davidii as a Chinese traditional medicine with anticancer activity by isolating natural products for their activity against the human gastric cancer cell line Mgc-803 and the human breast cancer cell line Bcap-37. Results Ten compounds were extracted and isolated from B. davidii, among which colchicine was identified in B. davidii for the first time. The inhibitory activities of these compounds were investigated in Mgc-803, Bcap-37 cells in vitro by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay, and the results showed that luteolin and colchicine had potent inhibitory activities against the growth of Mgc-803 cells. Subsequent fluorescence staining and flow cytometry analysis indicated that these two compounds could induce apoptosis in Mgc-803 cells. The results also showed that the percentages of early apoptotic cells (Annexin V+/PI-, where PI is propidium iodide) and late apoptotic cells (Annexin V+/PI+) increased in a dose- and time-dependent manner. After 36 h of incubation with luteolin at 20 μM, the percentages of cells were approximately 15.4% in early apoptosis and 43.7% in late apoptosis; after 36 h of incubation with colchicine at 20 μM, the corresponding values were 7.7% and 35.2%, respectively. Conclusions Colchicine and luteolin from B. davidii have potential applications as adjuvant therapies for treating human carcinoma cells. These compounds could also induce apoptosis in tumor cells. PMID:22938042

  6. The TACI receptor regulates T-cell-independent marginal zone B cell responses through innate activation-induced cell death.

    PubMed

    Figgett, William A; Fairfax, Kirsten; Vincent, Fabien B; Le Page, Mélanie A; Katik, Indzi; Deliyanti, Devy; Quah, Pin Shie; Verma, Pali; Grumont, Raelene; Gerondakis, Steve; Hertzog, Paul; O'Reilly, Lorraine A; Strasser, Andreas; Mackay, Fabienne

    2013-09-19

    Activation-induced cell death (AICD) plays a critical role in immune homeostasis and tolerance. In T-cell-dependent humoral responses, AICD of B cells is initiated by Fas ligand (FasL) on T cells, stimulating the Fas receptor on B cells. In contrast, T-cell-independent B cell responses involve innate-type B lymphocytes, such as marginal zone (MZ) B cells, and little is known about the mechanisms that control AICD during innate B cell responses to Toll-like receptor (TLR) activation. Here, we show that MZ B cells undergo AICD in response to TLR4 activation in vivo. The transmembrane activator, calcium modulator, and cyclophilin ligand interactor (TACI) receptor and TLR4 cooperate to upregulate expression of both FasL and Fas on MZ B cells and also to repress inhibitors of Fas-induced apoptosis signaling. These findings demonstrate an unappreciated role for TACI and its ligands in the regulation of AICD during T-cell-independent B cell responses.

  7. EPAC activation inhibits acetaldehyde-induced activation and proliferation of hepatic stellate cell via Rap1.

    PubMed

    Yang, Yan; Yang, Feng; Wu, Xiaojuan; Lv, Xiongwen; Li, Jun

    2016-05-01

    Hepatic stellate cells (HSCs) activation represents an essential event during alcoholic liver fibrosis (ALF). Previous studies have demonstrated that the rat HSCs could be significantly activated after exposure to 200 μmol/L acetaldehyde for 48 h, and the cAMP/PKA signaling pathways were also dramatically upregulated in activated HSCs isolated from alcoholic fibrotic rat liver. Exchange protein activated by cAMP (EPAC) is a family of guanine nucleotide exchange factors (GEFs) for the small Ras-like GTPases Rap, and is being considered as a vital mediator of cAMP signaling in parallel with the principal cAMP target protein kinase A (PKA). Our data showed that both cAMP/PKA and cAMP/EPAC signaling pathways were involved in acetaldehyde-induced HSCs. Acetaldehyde could reduce the expression of EPAC1 while enhancing the expression of EPAC2. The cAMP analog Me-cAMP, which stimulates the EPAC/Rap1 pathway, could significantly decrease the proliferation and collagen synthesis of acetaldehyde-induced HSCs. Furthermore, depletion of EPAC2, but not EPAC1, prevented the activation of HSC measured as the production of α-SMA and collagen type I and III, indicating that EPAC1 appears to have protective effects on acetaldehyde-induced HSCs. Curiously, activation of PKA or EPAC perhaps has opposite effects on the synthesis of collagen and α-SMA: EPAC activation by Me-cAMP increased the levels of GTP-bound (activated) Rap1 while PKA activation by Phe-cAMP had no significant effects on such binding. These results suggested that EPAC activation could inhibit the activation and proliferation of acetaldehyde-induced HSCs via Rap1. PMID:26854595

  8. Prodigiosin Induces Autolysins in Actively Grown Bacillus subtilis Cells

    PubMed Central

    Danevčič, Tjaša; Borić Vezjak, Maja; Tabor, Maja; Zorec, Maša; Stopar, David

    2016-01-01

    Prodigiosin produced by marine bacterium Vibrio ruber DSM 14379 exhibits a potent antimicrobial activity against a broad range of Gram positive and Gram negative bacteria. The mechanism of prodigiosin antimicrobial action, however, is not known. In this work, the effect of prodigiosin on Bacillus subtilis growth, cell membrane leakage, and induction of autolysins was studied. Treating B. subtilis with prodigiosin resulted in rapid decline of optical density and increased cell membrane leakage measured by β-galactosidase activity. Cell lysis was initiated immediately after treatment with prodigiosin in the middle exponential phase and was completed within 2 h. Lytic activity of prodigiosin in mutant strains with impaired autolysin genes lytABCD decreased for 80% compared to the wild type strain, while in lytABCDEF mutant strain prodigiosin had no bacteriolytic but only bacteriostatic effect. Fast prodigiosin lytic activity on individual B. subtilis cells was confirmed by a modified comet assay. The results indicate that prodigiosin autolysin induction in B. subtilis is growth phase dependent. PMID:26858704

  9. Prodigiosin Induces Autolysins in Actively Grown Bacillus subtilis Cells.

    PubMed

    Danevčič, Tjaša; Borić Vezjak, Maja; Tabor, Maja; Zorec, Maša; Stopar, David

    2016-01-01

    Prodigiosin produced by marine bacterium Vibrio ruber DSM 14379 exhibits a potent antimicrobial activity against a broad range of Gram positive and Gram negative bacteria. The mechanism of prodigiosin antimicrobial action, however, is not known. In this work, the effect of prodigiosin on Bacillus subtilis growth, cell membrane leakage, and induction of autolysins was studied. Treating B. subtilis with prodigiosin resulted in rapid decline of optical density and increased cell membrane leakage measured by β-galactosidase activity. Cell lysis was initiated immediately after treatment with prodigiosin in the middle exponential phase and was completed within 2 h. Lytic activity of prodigiosin in mutant strains with impaired autolysin genes lytABCD decreased for 80% compared to the wild type strain, while in lytABCDEF mutant strain prodigiosin had no bacteriolytic but only bacteriostatic effect. Fast prodigiosin lytic activity on individual B. subtilis cells was confirmed by a modified comet assay. The results indicate that prodigiosin autolysin induction in B. subtilis is growth phase dependent.

  10. p38 Mitogen-Activated Protein Kinase in beryllium-induced dendritic cell activation.

    PubMed

    Li, L; Huang, Z; Gillespie, M; Mroz, P M; Maier, L A

    2014-12-01

    Dendritic cells (DC) play a role in the regulation of immune responses to haptens, which in turn impact DC maturation. Whether beryllium (Be) is able to induce DC maturation and if this occurs via the MAPK pathway is not known. Primary monocyte-derived DCs (moDCs) models were generated from Be non-exposed healthy volunteers as a non-sensitized cell model, while PBMCs from BeS (Be sensitized) and CBD (chronic beryllium disease) were used as disease models. The response of these cells to Be was evaluated. The expression of CD40 was increased significantly (p<0.05) on HLA-DP Glu69+ moDCs after 100 μM BeSO₄-stimulation. BeSO₄ induced p38MAPK phosphorylation, while IκB-α was degraded in Be-stimulated moDCs. The p38 MAPK inhibitor SB203580 blocked Be-induced NF-κB activation in moDCs, suggesting that p38MAPK and NF-κB are dependently activated by BeSO₄. Furthermore, in BeS and CBD subjects, SB203580 downregulated Be-stimulated proliferation in a dose-dependent manner, and decreased Be-stimulated TNF-α and IFNγ cytokine production. Taken together, this study suggests that Be-induces non-sensitized Glu69+ DCs maturation, and that p38MAPK signaling is important in the Be-stimulated DCs activation as well as subsequent T cell proliferation and cytokine production in BeS and CBD. In total, the MAPK pathway may serve as a potential therapeutic target for human granulomatous lung diseases.

  11. p38 Mitogen-Activated Protein Kinase in beryllium-induced dendritic cell activation

    PubMed Central

    Li, L.; Huang, Z.; Gillespie, M.; Mroz, P.M.; Maier, L.A.

    2014-01-01

    Dendritic cells (DC) play a role in the regulation of immune responses to haptens, which in turn impact DC maturation. Whether beryllium (Be) is able to induce DC maturation and if this occurs via the MAPK pathway is not known. Primary monocyte-derived DCs (moDCs) models were generated from Be non-exposed healthy volunteers as a non-sensitized cell model, while PBMCs from BeS (Be sensitized) and CBD (chronic beryllium disease) were used as disease models. The response of these cells to Be was evaluated. The expression of CD40 was increased significantly (p<0.05) on HLA-DP Glu69+ moDCs after 100 μM BeSO4-stimulation. BeSO4 induced p38MAPK phosphorylation, while IκB-α was degraded in Be-stimulated moDCs. The p38 MAPK inhibitor SB203580 blocked Be-induced NF-κB activation in moDCs, suggesting that p38MAPK and NF-κB are dependently activated by BeSO4. Furthermore, in BeS and CBD subjects, SB203580 downregulated Be-stimulated proliferation in a dose-dependent manner, and decreased Be-stimulated TNF-α and IFNγ cytokine production. Taken together, this study suggests that Be-induces non-sensitized Glu69+ DCs maturation, and that p38MAPK signaling is important in the Be-stimulated DCs activation as well as subsequent T cell proliferation and cytokine production in BeS and CBD. In total, the MAPK pathway may serve as a potential therapeutic target for human granulomatous lung diseases. PMID:25454621

  12. Activation-Induced Cell Death of Dendritic Cells Is Dependent on Sphingosine Kinase 1

    PubMed Central

    Schwiebs, Anja; Friesen, Olga; Katzy, Elisabeth; Ferreirós, Nerea; Pfeilschifter, Josef M.; Radeke, Heinfried H.

    2016-01-01

    Sphingosine 1-phosphate (S1P) is an immune modulatory lipid mediator and has been implicated in numerous pathophysiological processes. S1P is produced by sphingosine kinase 1 (Sphk1) and Sphk2. Dendritic cells (DCs) are central for the direction of immune responses and crucially involved in autoimmunity and cancerogenesis. In this study we examined the function and survival of bone marrow-derived DCs under long-term inflammatory stimulation. We observed that differentiated cells undergo activation-induced cell death (AICD) upon LPS stimulation with an increased metabolic activity shortly after stimulation, followed by a rapid activation of caspase 3 and subsequent augmented apoptosis. Importantly, we highlight a profound role of Sphk1 in secretion of inflammatory cytokines and survival of dendritic cells that might be mediated by a change in sphingolipid levels as well as by a change in STAT3 expression. Cell growth during differentiation of Sphk1-deficient cells treated with the functional S1P receptor antagonist FTYP was reduced. Importantly, in dendritic cells we did not observe a compensatory regulation of Sphk2 mRNA in Sphk1-deficient cells. Instead, we discovered a massive increase in Sphk1 mRNA concentration upon long-term stimulation with LPS in wild type cells that might function as an attempt to rescue from inflammation-caused cell death. Taken together, in this investigation we describe details of a crucial involvement of sphingolipids and Sphk1 in AICD during long-term immunogenic activity of DCs that might play an important role in autoimmunity and might explain the differences in immune response observed in in vivo studies of Sphk1 modulation. PMID:27148053

  13. mTOR inhibition sensitizes ONC201-induced anti-colorectal cancer cell activity.

    PubMed

    Jin, Zhe-Zhu; Wang, Wei; Fang, Di-Long; Jin, Yong-Jun

    2016-09-30

    We here tested the anti-colorectal cancer (CRC) activity by a first-in-class small molecule TRAIL inducer ONC201. The potential effect of mTOR on ONC201's actions was also examined. ONC201 induced moderate cytotoxicity against CRC cell lines (HT-29, HCT-116 and DLD-1) and primary human CRC cells. Significantly, AZD-8055, a mTOR kinase inhibitor, sensitized ONC201-induced cytotoxicity in CRC cells. Meanwhile, ONC201-induced TRAIL/death receptor-5 (DR-5) expression, caspase-8 activation and CRC cell apoptosis were also potentiated with AZD-8055 co-treatment. Reversely, TRAIL sequestering antibody RIK-2 or the caspase-8 specific inhibitor z-IETD-fmk attenuated AZD-8055 plus ONC201-induced CRC cell death. Further, mTOR kinase-dead mutation (Asp-2338-Ala) or shRNA knockdown significantly sensitized ONC201's activity in CRC cells, leading to profound cell death and apoptosis. On the other hand, expression of a constitutively-active S6K1 (T389E) attenuated ONC201-induced CRC cell apoptosis. For the mechanism study, we showed that ONC201 blocked Akt, but only slightly inhibited mTOR in CRC cells. Co-treatment with AZD-8055 also concurrently blocked mTOR activation. These results suggest that mTOR could be a primary resistance factor of ONC201 in CRC cells. PMID:27565731

  14. Sphingosine-1-phosphate mediates epidermal growth factor-induced muscle satellite cell activation

    SciTech Connect

    Nagata, Yosuke Ohashi, Kazuya; Wada, Eiji; Yuasa, Yuki; Shiozuka, Masataka; Nonomura, Yoshiaki; Matsuda, Ryoichi

    2014-08-01

    Skeletal muscle can regenerate repeatedly due to the presence of resident stem cells, called satellite cells. Because satellite cells are usually quiescent, they must be activated before participating in muscle regeneration in response to stimuli such as injury, overloading, and stretch. Although satellite cell activation is a crucial step in muscle regeneration, little is known of the molecular mechanisms controlling this process. Recent work showed that the bioactive lipid sphingosine-1-phosphate (S1P) plays crucial roles in the activation, proliferation, and differentiation of muscle satellite cells. We investigated the role of growth factors in S1P-mediated satellite cell activation. We found that epidermal growth factor (EGF) in combination with insulin induced proliferation of quiescent undifferentiated mouse myoblast C2C12 cells, which are also known as reserve cells, in serum-free conditions. Sphingosine kinase activity increased when reserve cells were stimulated with EGF. Treatment of reserve cells with the D-erythro-N,N-dimethylsphingosine, Sphingosine Kinase Inhibitor, or siRNA duplexes specific for sphingosine kinase 1, suppressed EGF-induced C2C12 activation. We also present the evidence showing the S1P receptor S1P2 is involved in EGF-induced reserve cell activation. Moreover, we demonstrated a combination of insulin and EGF promoted activation of satellite cells on single myofibers in a manner dependent on SPHK and S1P2. Taken together, our observations show that EGF-induced satellite cell activation is mediated by S1P and its receptor. - Highlights: • EGF in combination with insulin induces proliferation of quiescent C2C12 cells. • Sphingosine kinase activity increases when reserve cells are stimulated with EGF. • EGF-induced activation of reserve cells is dependent on sphingosine kinase and ERK. • The S1P receptor S1P2 is involved in EGF-induced reserve cell activation. • EGF-induced reserve cell activation is mediated by S1P and its

  15. Sodium periodate-induced human suppressor cells for polyclonal B cell activation.

    PubMed

    Goust, J M

    1982-09-01

    Sodium periodate (SP) induces proliferation of mature T cells. In this study, human peripheral blood mononuclear cells (MNC) pretreated for 10 minutes at room temperature with increasing concentrations (0.1 to 5 mM) of SP before culture for 7 days in the presence of pokeweed mitogen (PWM) exhibited a dose-dependent inhibition of IgG production, contrasting with an increase in 3H-thymidine uptake. When MNC from 70 normal individuals were pretreated with 1 and 2 mM SP, IgG production in culture was suppressed by 46.8 +/- 4.5% and 60.4 +/- 4.4% (mean +/- S.E.M.), respectively, as compared to IgG synthesis in the presence of PWM alone. Longitudinal studies of MNC obtained from the same normal individuals over 6-10 months showed similar degrees of suppression, indicating that the level of SP-inducible suppressor cell activity remains relatively constant, although the degree of suppression varies among normal persons. Both T cells and monocytes were required for PWM-driven IgG production and for SP-induced suppression. A soluble factor elaborated by SP-treated monocytes was also able to suppress IgG production. This model should provide useful information about abnormal regulation of IgG synthesis in various pathological conditions. PMID:6290661

  16. Direct binding of C1q to apoptotic cells and cell blebs induces complement activation.

    PubMed

    Nauta, Alma J; Trouw, Leendert A; Daha, Mohamed R; Tijsma, Odette; Nieuwland, Rienk; Schwaeble, Wilhelm J; Gingras, Alexandre R; Mantovani, Alberto; Hack, Erik C; Roos, Anja

    2002-06-01

    Deficiency of early components of the classical pathway of complement, particularly C1q, predisposes to the development of systemic lupus erythematosus. Several studies have suggested an association between the classical complement pathway and the clearance of apoptotic cells. Mice with a targeted deletion of the C1q gene develop a lupus-like renal disease, which is associated with the presence of multiple apoptotic bodies in the kidney. In the present study we demonstrate that highly purified C1q binds to apoptotic cells and isolated blebs derived from these apoptotic cells. Binding of C1q to apoptotic cells occurs via the globular heads of C1q and induces activation of the classical complement pathway, as shown by the deposition of C4 and C3 on the surface of these cells and on cell-derived blebs. In addition, for the first time, we demonstrate that surface-bound C1q is present on a subpopulation of microparticles isolated from human plasma. Taken together, these observations demonstrate that C1q binds directly to apoptotic cells and blebs derived therefrom and support a role for C1q, possibly in concert with C4 and C3, in the clearance of apoptotic cells and blebs by the phagocytic system.

  17. Activation of PPAR{gamma} is not involved in butyrate-induced epithelial cell differentiation

    SciTech Connect

    Ulrich, S.; Waechtershaeuser, A.; Loitsch, S.; Knethen, A. von; Bruene, B.; Stein, J. . E-mail: j.stein@em.uni-frankfurt.de

    2005-10-15

    Histone deacetylase-inhibitors affect growth and differentiation of intestinal epithelial cells by inducing expression of several transcription factors, e.g. Peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}) or vitamin D receptor (VDR). While activation of VDR by butyrate mainly seems to be responsible for cellular differentiation, the activation of PPAR{gamma} in intestinal cells remains to be elucidated. The aim of this study was to determine the role of PPAR{gamma} in butyrate-induced cell growth inhibition and differentiation induction in Caco-2 cells. Treatment with PPAR{gamma} ligands ciglitazone and BADGE (bisphenol A diglycidyl) enhanced butyrate-induced cell growth inhibition in a dose- and time-dependent manner, whereas cell differentiation was unaffected after treatment with PPAR{gamma} ligands rosiglitazone and MCC-555. Experiments were further performed in dominant-negative PPAR{gamma} mutant cells leading to an increase in cell growth whereas butyrate-induced cell differentiation was again unaffected. The present study clearly demonstrated that PPAR{gamma} is involved in butyrate-induced inhibition of cell growth, but seems not to play an essential role in butyrate-induced cell differentiation.

  18. Adjuvant-induced antired blood cell activity in CBA mice

    PubMed Central

    McCracken, Ann; McBride, W. H.; Weir, D. M.

    1971-01-01

    Various micro-organisms are known to act as immunological adjuvants and included amongst these are Corynebacteriaceae. Numerous studies on Corynebacterium parvum have shown, in particular, its ability to cause proliferation and enhanced activity of the reticulo-endothelial system. This organism also leads in mice to anaemia and this report describes the simultaneous appearance of a red cell autoantibody in mice injected with C. parvum or another diphtheroid (SF 16) isolated from rheumatoid joint fluid. The significance of this latter observation is considered in relation to the unexplained anaemia of rheumatoid arthritis. PMID:4933319

  19. TGEV nucleocapsid protein induces cell cycle arrest and apoptosis through activation of p53 signaling

    SciTech Connect

    Ding, Li; Huang, Yong; Du, Qian; Dong, Feng; Zhao, Xiaomin; Zhang, Wenlong; Xu, Xingang; Tong, Dewen

    2014-03-07

    Highlights: • TGEV N protein reduces cell viability by inducing cell cycle arrest and apoptosis. • TGEV N protein induces cell cycle arrest and apoptosis by regulating p53 signaling. • TGEV N protein plays important roles in TGEV-induced cell cycle arrest and apoptosis. - Abstract: Our previous studies showed that TGEV infection could induce cell cycle arrest and apoptosis via activation of p53 signaling in cultured host cells. However, it is unclear which viral gene causes these effects. In this study, we investigated the effects of TGEV nucleocapsid (N) protein on PK-15 cells. We found that TGEV N protein suppressed cell proliferation by causing cell cycle arrest at the S and G2/M phases and apoptosis. Characterization of various cellular proteins that are involved in regulating cell cycle progression demonstrated that the expression of N gene resulted in an accumulation of p53 and p21, which suppressed cyclin B1, cdc2 and cdk2 expression. Moreover, the expression of TGEV N gene promoted translocation of Bax to mitochondria, which in turn caused the release of cytochrome c, followed by activation of caspase-3, resulting in cell apoptosis in the transfected PK-15 cells following cell cycle arrest. Further studies showed that p53 inhibitor attenuated TGEV N protein induced cell cycle arrest at S and G2/M phases and apoptosis through reversing the expression changes of cdc2, cdk2 and cyclin B1 and the translocation changes of Bax and cytochrome c induced by TGEV N protein. Taken together, these results demonstrated that TGEV N protein might play an important role in TGEV infection-induced p53 activation and cell cycle arrest at the S and G2/M phases and apoptosis occurrence.

  20. Asymmetric dimethylarginine (ADMA) treatment induces apoptosis in cultured rat mesangial cells via endoplasmic reticulum stress activation.

    PubMed

    Park, Min-Jung; Oh, Ki-Seok; Nho, Jong-Hyun; Kim, Gye-Yeop; Kim, Dong-Il

    2016-06-01

    Asymmetric dimethylarginine (ADMA), a high risk factor for endothelial dysfunction and cardiovascular disease (CVD), has been reported to promote cellular dysfunction via endoplasmic reticulum (ER) stress activation in various cells. Additionally, increased serum ADMA levels have been observed in incipient kidney diseases. Previously, we reported that activated ER stress is associated with mesangial cell apoptosis, observed mainly in overt nephropathy or chronic kidney disease (CKD). However, the effect of ADMA on mesangial cell apoptosis is unknown. Thus, we investigated the effects of ADMA on mesangial cell apoptosis and ER stress signaling. ADMA treatment increased caspase-3 activity and activated three branches of ER stress signaling (PERK, IRE1, and ATF6) that induce mesangial cell apoptosis. Pharmacological inhibitors of ER stress (inhibitors of PERK, IRE1, and S1P) attenuated ADMA-induced cleavage of caspase-3 and induced a decrease in the mitochondrial membrane potential. Furthermore, these inhibitors diminished the number of apoptotic cells induced by ADMA treatment. Taken together, our results indicated that ADMA treatment induces mesangial cell apoptosis via ER stress signaling. These results suggest that ADMA-induced mesangial cell apoptosis could contribute to the progression of overt nephropathy and CKD.

  1. Carcinoma cells induce lumen filling and EMT in epithelial cells through soluble E-cadherin-mediated activation of EGFR.

    PubMed

    Patil, Pratima U; D'Ambrosio, Julia; Inge, Landon J; Mason, Robert W; Rajasekaran, Ayyappan K

    2015-12-01

    In epithelial cancers, carcinoma cells coexist with normal cells. Although it is known that the tumor microenvironment (TME) plays a pivotal role in cancer progression, it is not completely understood how the tumor influences adjacent normal epithelial cells. In this study, a three-dimensional co-culture system comprising non-transformed epithelial cells (MDCK) and transformed carcinoma cells (MSV-MDCK) was used to demonstrate that carcinoma cells sequentially induce preneoplastic lumen filling and epithelial-mesenchymal transition (EMT) in epithelial cysts. MMP-9 secreted by carcinoma cells cleaves cellular E-cadherin (encoded by CDH1) from epithelial cells to generate soluble E-cadherin (sE-cad), a pro-oncogenic protein. We show that sE-cad induces EGFR activation, resulting in lumen filling in MDCK cysts. Long-term sE-cad treatment induced EMT. sE-cad caused lumen filling by induction of the ERK signaling pathway and triggered EMT through the sustained activation of the AKT pathway. Although it is known that sE-cad induces MMP-9 release and consequent EGFR activation in tumor cells, our results, for the first time, demonstrate that carcinoma cells can induce sE-cad shedding in adjacent epithelial cells, which leads to EGFR activation and the eventual transdifferentiation of the normal epithelial cells. PMID:26483386

  2. VEGF secretion during hypoxia depends on free radicals-induced Fyn kinase activity in mast cells

    SciTech Connect

    Garcia-Roman, Jonathan; Ibarra-Sanchez, Alfredo; Lamas, Monica; Gonzalez Espinosa, Claudia

    2010-10-15

    Research highlights: {yields} Bone marrow-derived mast cells (BMMCs) secrete functional VEGF but do not degranulate after Cobalt chloride-induced hypoxia. {yields} CoCl{sub 2}-induced VEGF secretion in mast cells occurs by a Ca{sup 2+}-insensitive but brefeldin A and Tetanus toxin-sensitive mechanism. {yields} Trolox and N-acetylcysteine inhibit hypoxia-induced VEGF secretion but only Trolox inhibits Fc{epsilon}RI-dependent anaphylactic degranulation in mast cells. {yields} Src family kinase Fyn activation after free radical production is necessary for hypoxia-induced VEGF secretion in mast cells. -- Abstract: Mast cells (MC) have an important role in pathologic conditions such as asthma and chronic obstructive pulmonary disease (COPD), where hypoxia conduce to deleterious inflammatory response. MC contribute to hypoxia-induced angiogenesis producing factors such as vascular endothelial growth factor (VEGF), but the mechanisms behind the control of hypoxia-induced VEGF secretion in this cell type is poorly understood. We used the hypoxia-mimicking agent cobalt chloride (CoCl{sub 2}) to analyze VEGF secretion in murine bone marrow-derived mast cells (BMMCs). We found that CoCl{sub 2} promotes a sustained production of functional VEGF, able to induce proliferation of endothelial cells in vitro. CoCl{sub 2}-induced VEGF secretion was independent of calcium rise but dependent on tetanus toxin-sensitive vesicle-associated membrane proteins (VAMPs). VEGF exocytosis required free radicals formation and the activation of Src family kinases. Interestingly, an important deficiency on CoCl{sub 2}-induced VEGF secretion was observed in Fyn kinase-deficient BMMCs. Moreover, Fyn kinase was activated by CoCl{sub 2} in WT cells and this activation was prevented by treatment with antioxidants such as Trolox and N-acetylcysteine. Our results show that BMMCs are able to release VEGF under hypoxic conditions through a tetanus toxin-sensitive mechanism, promoted by free radicals

  3. Sphingosine-1-phosphate mediates epidermal growth factor-induced muscle satellite cell activation.

    PubMed

    Nagata, Yosuke; Ohashi, Kazuya; Wada, Eiji; Yuasa, Yuki; Shiozuka, Masataka; Nonomura, Yoshiaki; Matsuda, Ryoichi

    2014-08-01

    Skeletal muscle can regenerate repeatedly due to the presence of resident stem cells, called satellite cells. Because satellite cells are usually quiescent, they must be activated before participating in muscle regeneration in response to stimuli such as injury, overloading, and stretch. Although satellite cell activation is a crucial step in muscle regeneration, little is known of the molecular mechanisms controlling this process. Recent work showed that the bioactive lipid sphingosine-1-phosphate (S1P) plays crucial roles in the activation, proliferation, and differentiation of muscle satellite cells. We investigated the role of growth factors in S1P-mediated satellite cell activation. We found that epidermal growth factor (EGF) in combination with insulin induced proliferation of quiescent undifferentiated mouse myoblast C2C12 cells, which are also known as reserve cells, in serum-free conditions. Sphingosine kinase activity increased when reserve cells were stimulated with EGF. Treatment of reserve cells with the D-erythro-N,N-dimethylsphingosine, Sphingosine Kinase Inhibitor, or siRNA duplexes specific for sphingosine kinase 1, suppressed EGF-induced C2C12 activation. We also present the evidence showing the S1P receptor S1P2 is involved in EGF-induced reserve cell activation. Moreover, we demonstrated a combination of insulin and EGF promoted activation of satellite cells on single myofibers in a manner dependent on SPHK and S1P2. Taken together, our observations show that EGF-induced satellite cell activation is mediated by S1P and its receptor.

  4. Xestospongin C induces monocytic differentiation of HL60 cells through activation of the ERK pathway.

    PubMed

    Moon, Dong-Oh; Asami, Yukihiro; Kim, Mun-Ock; Jang, Jae-Hyuk; Kim, Bo Yeon; Ahn, Jong Seog; Kim, Gi-Young; Yun, Sung Gyu

    2013-05-01

    Xestospongin C (XC), which is a group of macrocyclic bis-1-oxaquinolizidines, is a potent inhibitor of sarcoendoplasmic reticulum calcium transport ATPase and IP3 receptor. Nevertheless, very less information is available regarding whether XC induces AML differentiation. We investigated the potential role of XC in the differentiation of human leukemia HL60 cells and mechanisms underlying XC actin. XC treatment inhibited proliferation by inducing G1-phase cell cycle arrest in the HL60 cells. In addition, XC induced differentiation of HL60 cells into the CD14(+) monocytic lineage, which was indicated by morphological changes, nitroblue tetrazolium reduction assay, and expressions of CD11b and CD14 surface antigens. Our results also showed that XC promotes phagocytic activity and granularity in HL60 cells, suggesting that the cells are functionally activated. Furthermore, XC enhanced tumor necrosis factor (TNF)-α-mediated cytotoxic effect by increasing the numbers of TNF receptors. Moreover, we showed that XC activates extracellular signal-regulated kinase (ERK) pathway in the differentiation stages. Inhibition of ERK activation using PD98059 significantly decreased NBT+HL60 cells induced by XC treatment. Taken together, the results show that XC promotes monocytic differentiation of HL60 cells via ERK pathway activation, suggesting that XC could be a candidate for use as a differentiation-inducing agent for AML treatment.

  5. A Taiwanese Propolis Derivative Induces Apoptosis through Inducing Endoplasmic Reticular Stress and Activating Transcription Factor-3 in Human Hepatoma Cells

    PubMed Central

    Suk, Fat-Moon; Lien, Gi-Shih; Huang, Wei-Jan; Chen, Chia-Nan; Lu, Shao-Yu; Yan, Ming-De

    2013-01-01

    Activating transcription factor-(ATF-) 3, a stress-inducible transcription factor, is rapidly upregulated under various stress conditions and plays an important role in inducing cancer cell apoptosis. NBM-TP-007-GS-002 (GS-002) is a Taiwanese propolin G (PPG) derivative. In this study, we examined the antitumor effects of GS-002 in human hepatoma Hep3B and HepG2 cells in vitro. First, we found that GS-002 significantly inhibited cell proliferation and induced cell apoptosis in dose-dependent manners. Several main apoptotic indicators were found in GS-002-treated cells, such as the cleaved forms of caspase-3, caspase-9, and poly(ADP-ribose) polymerase (PARP). GS-002 also induced endoplasmic reticular (ER) stress as evidenced by increases in ER stress-responsive proteins including glucose-regulated protein 78 (GRP78), growth arrest- and DNA damage-inducible gene 153 (GADD153), phosphorylated eukaryotic initiation factor 2α (eIF2α), phosphorylated protein endoplasmic-reticular-resident kinase (PERK), and ATF-3. The induction of ATF-3 expression was mediated by mitogen-activated protein kinase (MAPK) signaling pathways in GS-002-treated cells. Furthermore, we found that GS-002 induced more cell apoptosis in ATF-3-overexpressing cells. These results suggest that the induction of apoptosis by the propolis derivative, GS-002, is partially mediated through ER stress and ATF-3-dependent pathways, and GS-002 has the potential for development as an antitumor drug. PMID:24222778

  6. Omacetaxine mepesuccinate induces apoptosis and cell cycle arrest, promotes cell differentiation, and reduces telomerase activity in diffuse large B-cell lymphoma cells

    PubMed Central

    ZHANG, LINA; CHEN, ZHENZHU; ZUO, WENLI; ZHU, XINGHU; LI, YUFU; LIU, XINJIAN; WEI, XUDONG

    2016-01-01

    Clinical studies have demonstrated that omacetaxine mepesuccinate exerts beneficial effects on acute myelogenous leukemia. It has been suggested that omacetaxine mepesuccinate, used alone or with interferon-α or cytarabine, induces remission in patients with chronic myelogenous leukemia. These effects are possibly mediated by its ability to induce apoptosis of leukemia cells and inhibit the activity of telomerase. To determine whether omacetaxine mepesuccinate is beneficial in diffuse large B-cell lymphoma (DLBCL), two DLBCL cell lines [a germinal center B cell-like subtype (GCB) and an activated B cell-like subtype (ABC)] were treated with omacetaxine mepesuccinate at various concentrations for different durations. The present study indicated that omacetaxine mepesuccinate exerts proapoptotic effects in the two cell types in a dose- and time-dependent manner. The ABC subtype demonstrated increased sensitivity compared with the GCB subtype. At 40 ng/ml, omacetaxine mepesuccinate exhibited a marked proapoptotic effect on DLBCL cells compared with the other tumor cells investigated. Furthermore, omacetaxine mepesuccinate induced cell cycle arrest at G0/G1 phase, and promoted cell terminal differentiation of pro-B cells. The present study also demonstrated that omacetaxine mepesuccinate exerted its antitumor effect by reducing telomerase activity. In conclusion, the present study demonstrated that omacetaxine mepesuccinate may induce apoptosis and cell cycle arrest, promote cell differentiation, and reduce telomerase activity in DLBCL cells, thus aiding the development of omacetaxine mepesuccinate-based DLBCL therapeutic strategies. PMID:26935769

  7. Lumican Inhibits SNAIL-Induced Melanoma Cell Migration Specifically by Blocking MMP-14 Activity.

    PubMed

    Stasiak, Marta; Boncela, Joanna; Perreau, Corinne; Karamanou, Konstantina; Chatron-Colliet, Aurore; Proult, Isabelle; Przygodzka, Patrycja; Chakravarti, Shukti; Maquart, François-Xavier; Kowalska, M Anna; Wegrowski, Yanusz; Brézillon, Stéphane

    2016-01-01

    Lumican, a small leucine rich proteoglycan, inhibits MMP-14 activity and melanoma cell migration in vitro and in vivo. Snail triggers epithelial-mesenchymal transitions endowing epithelial cells with migratory and invasive properties during tumor progression. The aim of this work was to investigate lumican effects on MMP-14 activity and migration of Snail overexpressing B16F1 (Snail-B16F1) melanoma cells and HT-29 colon adenocarcinoma cells. Lumican inhibits the Snail induced MMP-14 activity in B16F1 but not in HT-29 cells. In Snail-B16F1 cells, lumican inhibits migration, growth, and melanoma primary tumor development. A lumican-based strategy targeting Snail-induced MMP-14 activity might be useful for melanoma treatment. PMID:26930497

  8. Lumican Inhibits SNAIL-Induced Melanoma Cell Migration Specifically by Blocking MMP-14 Activity

    PubMed Central

    Stasiak, Marta; Boncela, Joanna; Perreau, Corinne; Karamanou, Konstantina; Chatron-Colliet, Aurore; Proult, Isabelle; Przygodzka, Patrycja; Chakravarti, Shukti; Maquart, François-Xavier; Kowalska, M. Anna; Wegrowski, Yanusz; Brézillon, Stéphane

    2016-01-01

    Lumican, a small leucine rich proteoglycan, inhibits MMP-14 activity and melanoma cell migration in vitro and in vivo. Snail triggers epithelial-mesenchymal transitions endowing epithelial cells with migratory and invasive properties during tumor progression. The aim of this work was to investigate lumican effects on MMP-14 activity and migration of Snail overexpressing B16F1 (Snail-B16F1) melanoma cells and HT-29 colon adenocarcinoma cells. Lumican inhibits the Snail induced MMP-14 activity in B16F1 but not in HT-29 cells. In Snail-B16F1 cells, lumican inhibits migration, growth, and melanoma primary tumor development. A lumican-based strategy targeting Snail-induced MMP-14 activity might be useful for melanoma treatment. PMID:26930497

  9. Activation of TIM1 induces colon cancer cell apoptosis via modulating Fas ligand expression.

    PubMed

    Wang, Hao; Zhang, Xueyan; Sun, Wenjing; Hu, Xiaocui; Li, Xiaolin; Fu, Songbin; Liu, Chen

    2016-04-29

    The pathogenesis of colon cancer is unclear. It is proposed that TIM1 has an association with human cancer. The present study aims to investigate the role of TIM1 activation in the inhibition of human colon cancer cells. In this study, human colon cancer cell line, HT29 and T84 cells were cultured. The expression of TIM1 was assessed by real time RT-PCR and Western blotting. The TIM1 on the cancer cells was activated in the culture by adding recombinant TIM4. The chromatin structure at the FasL promoter locus was assessed by chromatin immunoprecipitation. The apoptosis of the cancer cells was assessed by flow cytometry. The results showed that human colon cancer cell lines, HT29 cells and T84 cells, expressed TIM1. Activation of TIM1 by exposing the cells to TIM4 significantly increased the frequency of apoptotic colon cancer cells. The expression of FasL was increased in the cancer cells after treating by TIM4. Blocking Fas or FasL abolished the exposure to TIM4-induced T84 cell apoptosis. In conclusion, HT29 cells and T84 cells express TIM1; activation TIM1 can induce the cancer cell apoptosis. TIM1 may be a novel therapeutic target of colon cancer.

  10. Activation of the IFN-inducible enzyme RNase L causes apoptosis of animal cells.

    PubMed

    Díaz-Guerra, M; Rivas, C; Esteban, M

    1997-09-29

    The interferon (IFN)-induced enzyme RNase L produced by a recombinant vaccinia virus (VV) causes death of mammalian cells with morphological and biochemical characteristics of apoptosis. Coexpression of 2-5A-synthetase enhances apoptosis induced by RNase L Activation of endogenous RNase L by infection with a VV ts mutant (ts22) or with wild-type virus in the presence of the antipoxvirus drug isatin-beta-thiosemicarbazone, a treatment known to significantly increase the amount of double-stranded RNA late during infection, also causes pronounced apoptosis of infected cells. The effects observed with recombinant virus-derived RNase L or with the endogenous enzyme are specific, since apoptosis also occurs in cells derived from mice lacking the IFN-induced protein kinase (PKR). The apoptosis antagonist Bcl-2 prevents induction of cell death by RNase L activation. Apoptosis of mammalian cells by RNase L activation could be a mechanism mediating anticellular actions of IFN.

  11. Transcription factor NFAT1 controls allergic contact hypersensitivity through regulation of activation induced cell death program

    PubMed Central

    Kwon, Ho-Keun; Kim, Gi-Cheon; Hwang, Ji Sun; Kim, Young; Chae, Chang-Suk; Nam, Jong Hee; Jun, Chang-Duk; Rudra, Dipayan; Surh, Charles D.; Im, Sin-Hyeog

    2016-01-01

    Allergic contact hypersensitivity (CHS) is an inflammatory skin disease mediated by allergen specific T cells. In this study, we investigated the role of transcription factor NFAT1 in the pathogenesis of contact hypersensitivity. NFAT1 knock out (KO) mice spontaneously developed CHS-like skin inflammation in old age. Healthy young NFAT1 KO mice displayed enhanced susceptibility to hapten-induced CHS. Both CD4+ and CD8+ T cells from NFAT1 KO mice displayed hyper-activated properties and produced significantly enhanced levels of inflammatory T helper 1(Th1)/Th17 type cytokines. NFAT1 KO T cells were more resistant to activation induced cell death (AICD), and regulatory T cells derived from these mice showed a partial defect in their suppressor activity. NFAT1 KO T cells displayed a reduced expression of apoptosis associated BCL-2/BH3 family members. Ectopic expression of NFAT1 restored the AICD defect in NFAT1 KO T cells and increased AICD in normal T cells. Recipient Rag2−/− mice transferred with NFAT1 KO T cells showed more severe CHS sensitivity due to a defect in activation induced hapten-reactive T cell apoptosis. Collectively, our results suggest the NFAT1 plays a pivotal role as a genetic switch in CD4+/CD8+ T cell tolerance by regulating AICD process in the T cell mediated skin inflammation. PMID:26777750

  12. Glucose Enhances Basal or Melanocortin-Induced cAMP-Response Element Activity in Hypothalamic Cells.

    PubMed

    Breit, Andreas; Wicht, Kristina; Boekhoff, Ingrid; Glas, Evi; Lauffer, Lisa; Mückter, Harald; Gudermann, Thomas

    2016-07-01

    Melanocyte-stimulating hormone (MSH)-induced activation of the cAMP-response element (CRE) via the CRE-binding protein in hypothalamic cells promotes expression of TRH and thereby restricts food intake and increases energy expenditure. Glucose also induces central anorexigenic effects by acting on hypothalamic neurons, but the underlying mechanisms are not completely understood. It has been proposed that glucose activates the CRE-binding protein-regulated transcriptional coactivator 2 (CRTC-2) in hypothalamic neurons by inhibition of AMP-activated protein kinases (AMPKs), but whether glucose directly affects hypothalamic CRE activity has not yet been shown. Hence, we dissected effects of glucose on basal and MSH-induced CRE activation in terms of kinetics, affinity, and desensitization in murine, hypothalamic mHypoA-2/10-CRE cells that stably express a CRE-dependent reporter gene construct. Physiologically relevant increases in extracellular glucose enhanced basal or MSH-induced CRE-dependent gene transcription, whereas prolonged elevated glucose concentrations reduced the sensitivity of mHypoA-2/10-CRE cells towards glucose. Glucose also induced CRCT-2 translocation into the nucleus and the AMPK activator metformin decreased basal and glucose-induced CRE activity, suggesting a role for AMPK/CRTC-2 in glucose-induced CRE activation. Accordingly, small interfering RNA-induced down-regulation of CRTC-2 expression decreased glucose-induced CRE-dependent reporter activation. Of note, glucose also induced expression of TRH, suggesting that glucose might affect the hypothalamic-pituitary-thyroid axis via the regulation of hypothalamic CRE activity. These findings significantly advance our knowledge about the impact of glucose on hypothalamic signaling and suggest that TRH release might account for the central anorexigenic effects of glucose and could represent a new molecular link between hyperglycaemia and thyroid dysfunction. PMID:27144291

  13. Glucose Enhances Basal or Melanocortin-Induced cAMP-Response Element Activity in Hypothalamic Cells.

    PubMed

    Breit, Andreas; Wicht, Kristina; Boekhoff, Ingrid; Glas, Evi; Lauffer, Lisa; Mückter, Harald; Gudermann, Thomas

    2016-07-01

    Melanocyte-stimulating hormone (MSH)-induced activation of the cAMP-response element (CRE) via the CRE-binding protein in hypothalamic cells promotes expression of TRH and thereby restricts food intake and increases energy expenditure. Glucose also induces central anorexigenic effects by acting on hypothalamic neurons, but the underlying mechanisms are not completely understood. It has been proposed that glucose activates the CRE-binding protein-regulated transcriptional coactivator 2 (CRTC-2) in hypothalamic neurons by inhibition of AMP-activated protein kinases (AMPKs), but whether glucose directly affects hypothalamic CRE activity has not yet been shown. Hence, we dissected effects of glucose on basal and MSH-induced CRE activation in terms of kinetics, affinity, and desensitization in murine, hypothalamic mHypoA-2/10-CRE cells that stably express a CRE-dependent reporter gene construct. Physiologically relevant increases in extracellular glucose enhanced basal or MSH-induced CRE-dependent gene transcription, whereas prolonged elevated glucose concentrations reduced the sensitivity of mHypoA-2/10-CRE cells towards glucose. Glucose also induced CRCT-2 translocation into the nucleus and the AMPK activator metformin decreased basal and glucose-induced CRE activity, suggesting a role for AMPK/CRTC-2 in glucose-induced CRE activation. Accordingly, small interfering RNA-induced down-regulation of CRTC-2 expression decreased glucose-induced CRE-dependent reporter activation. Of note, glucose also induced expression of TRH, suggesting that glucose might affect the hypothalamic-pituitary-thyroid axis via the regulation of hypothalamic CRE activity. These findings significantly advance our knowledge about the impact of glucose on hypothalamic signaling and suggest that TRH release might account for the central anorexigenic effects of glucose and could represent a new molecular link between hyperglycaemia and thyroid dysfunction.

  14. Probiotic Lactobacilli Modulate Staphylococcus aureus-Induced Activation of Conventional and Unconventional T cells and NK Cells

    PubMed Central

    Johansson, Maria A.; Björkander, Sophia; Mata Forsberg, Manuel; Qazi, Khaleda Rahman; Salvany Celades, Maria; Bittmann, Julia; Eberl, Matthias; Sverremark-Ekström, Eva

    2016-01-01

    Lactobacilli are probiotic commensal bacteria and potent modulators of immunity. When present in the gut or supplemented as probiotics, they beneficially modulate ex vivo immune responsiveness. Further, factors derived from several lactobacilli strains act immune regulatory in vitro. In contrast, Staphylococcus aureus (S. aureus) is known to induce excessive T cell activation. In this study, we aimed to investigate S. aureus-induced activation of human mucosal-associated invariant T cells (MAIT cells), γδ T cells, NK cells, as well as of conventional CD4+ and CD8+ T cells in vitro. Further, we investigated if lactobacilli-derived factors could modulate their activation. PBMC were cultured with S. aureus 161:2 cell-free supernatants (CFS), staphylococcal enterotoxin A or CD3/CD28-beads alone, or in combination with Lactobacillus rhamnosus GG-CFS or Lactobacillus reuteri DSM 17938-CFS and activation of T and NK cells was evaluated. S. aureus-CFS induced IFN-γ and CD107a expression as well as proliferation. Costimulation with lactobacilli-CFS dampened lymphocyte-activation in all cell types analyzed. Preincubation with lactobacilli-CFS was enough to reduce subsequent activation, and the absence of APC or APC-derived IL-10 did not prevent lactobacilli-mediated dampening. Finally, lactate selectively dampened activation of unconventional T cells and NK cells. In summary, we show that molecules present in the lactobacilli-CFS are able to directly dampen in vitro activation of conventional and unconventional T cells and of NK cells. This study provides novel insights on the immune-modulatory nature of probiotic lactobacilli and suggests a role for lactobacilli in the modulation of induced T and NK cell activation. PMID:27462316

  15. Probiotic Lactobacilli Modulate Staphylococcus aureus-Induced Activation of Conventional and Unconventional T cells and NK Cells.

    PubMed

    Johansson, Maria A; Björkander, Sophia; Mata Forsberg, Manuel; Qazi, Khaleda Rahman; Salvany Celades, Maria; Bittmann, Julia; Eberl, Matthias; Sverremark-Ekström, Eva

    2016-01-01

    Lactobacilli are probiotic commensal bacteria and potent modulators of immunity. When present in the gut or supplemented as probiotics, they beneficially modulate ex vivo immune responsiveness. Further, factors derived from several lactobacilli strains act immune regulatory in vitro. In contrast, Staphylococcus aureus (S. aureus) is known to induce excessive T cell activation. In this study, we aimed to investigate S. aureus-induced activation of human mucosal-associated invariant T cells (MAIT cells), γδ T cells, NK cells, as well as of conventional CD4(+) and CD8(+) T cells in vitro. Further, we investigated if lactobacilli-derived factors could modulate their activation. PBMC were cultured with S. aureus 161:2 cell-free supernatants (CFS), staphylococcal enterotoxin A or CD3/CD28-beads alone, or in combination with Lactobacillus rhamnosus GG-CFS or Lactobacillus reuteri DSM 17938-CFS and activation of T and NK cells was evaluated. S. aureus-CFS induced IFN-γ and CD107a expression as well as proliferation. Costimulation with lactobacilli-CFS dampened lymphocyte-activation in all cell types analyzed. Preincubation with lactobacilli-CFS was enough to reduce subsequent activation, and the absence of APC or APC-derived IL-10 did not prevent lactobacilli-mediated dampening. Finally, lactate selectively dampened activation of unconventional T cells and NK cells. In summary, we show that molecules present in the lactobacilli-CFS are able to directly dampen in vitro activation of conventional and unconventional T cells and of NK cells. This study provides novel insights on the immune-modulatory nature of probiotic lactobacilli and suggests a role for lactobacilli in the modulation of induced T and NK cell activation.

  16. Probiotic Lactobacilli Modulate Staphylococcus aureus-Induced Activation of Conventional and Unconventional T cells and NK Cells.

    PubMed

    Johansson, Maria A; Björkander, Sophia; Mata Forsberg, Manuel; Qazi, Khaleda Rahman; Salvany Celades, Maria; Bittmann, Julia; Eberl, Matthias; Sverremark-Ekström, Eva

    2016-01-01

    Lactobacilli are probiotic commensal bacteria and potent modulators of immunity. When present in the gut or supplemented as probiotics, they beneficially modulate ex vivo immune responsiveness. Further, factors derived from several lactobacilli strains act immune regulatory in vitro. In contrast, Staphylococcus aureus (S. aureus) is known to induce excessive T cell activation. In this study, we aimed to investigate S. aureus-induced activation of human mucosal-associated invariant T cells (MAIT cells), γδ T cells, NK cells, as well as of conventional CD4(+) and CD8(+) T cells in vitro. Further, we investigated if lactobacilli-derived factors could modulate their activation. PBMC were cultured with S. aureus 161:2 cell-free supernatants (CFS), staphylococcal enterotoxin A or CD3/CD28-beads alone, or in combination with Lactobacillus rhamnosus GG-CFS or Lactobacillus reuteri DSM 17938-CFS and activation of T and NK cells was evaluated. S. aureus-CFS induced IFN-γ and CD107a expression as well as proliferation. Costimulation with lactobacilli-CFS dampened lymphocyte-activation in all cell types analyzed. Preincubation with lactobacilli-CFS was enough to reduce subsequent activation, and the absence of APC or APC-derived IL-10 did not prevent lactobacilli-mediated dampening. Finally, lactate selectively dampened activation of unconventional T cells and NK cells. In summary, we show that molecules present in the lactobacilli-CFS are able to directly dampen in vitro activation of conventional and unconventional T cells and of NK cells. This study provides novel insights on the immune-modulatory nature of probiotic lactobacilli and suggests a role for lactobacilli in the modulation of induced T and NK cell activation. PMID:27462316

  17. SB225002 Induces Cell Death and Cell Cycle Arrest in Acute Lymphoblastic Leukemia Cells through the Activation of GLIPR1.

    PubMed

    de Vasconcellos, Jaíra Ferreira; Laranjeira, Angelo Brunelli Albertoni; Leal, Paulo C; Bhasin, Manoj K; Zenatti, Priscila Pini; Nunes, Ricardo J; Yunes, Rosendo A; Nowill, Alexandre E; Libermann, Towia A; Zerbini, Luiz Fernando; Yunes, José Andrés

    2015-01-01

    Acute Lymphoblastic Leukemia (ALL) is the most frequent childhood malignancy. In the effort to find new anti-leukemic agents, we evaluated the small drug SB225002 (N-(2-hydroxy-4-nitrophenyl)-N'-(2-bromophenyl)urea). Although initially described as a selective antagonist of CXCR2, later studies have identified other cellular targets for SB225002, with potential medicinal use in cancer. We found that SB225002 has a significant pro-apoptotic effect against both B- and T-ALL cell lines. Cell cycle analysis demonstrated that treatment with SB225002 induces G2-M cell cycle arrest. Transcriptional profiling revealed that SB225002-mediated apoptosis triggered a transcriptional program typical of tubulin binding agents. Network analysis revealed the activation of genes linked to the JUN and p53 pathways and inhibition of genes linked to the TNF pathway. Early cellular effects activated by SB225002 included the up-regulation of GLIPR1, a p53-target gene shown to have pro-apoptotic activities in prostate and bladder cancer. Silencing of GLIPR1 in B- and T-ALL cell lines resulted in increased resistance to SB225002. Although SB225002 promoted ROS increase in ALL cells, antioxidant N-Acetyl Cysteine pre-treatment only modestly attenuated cell death, implying that the pro-apoptotic effects of SB225002 are not exclusively mediated by ROS. Moreover, GLIPR1 silencing resulted in increased ROS levels both in untreated and SB225002-treated cells. In conclusion, SB225002 induces cell cycle arrest and apoptosis in different B- and T-ALL cell lines. Inhibition of tubulin function with concurrent activation of the p53 pathway, in particular, its downstream target GLIPR1, seems to underlie the anti-leukemic effect of SB225002.

  18. SB225002 Induces Cell Death and Cell Cycle Arrest in Acute Lymphoblastic Leukemia Cells through the Activation of GLIPR1

    PubMed Central

    Leal, Paulo C.; Bhasin, Manoj K.; Zenatti, Priscila Pini; Nunes, Ricardo J.; Yunes, Rosendo A.; Nowill, Alexandre E.; Libermann, Towia A.; Zerbini, Luiz Fernando; Yunes, José Andrés

    2015-01-01

    Acute Lymphoblastic Leukemia (ALL) is the most frequent childhood malignancy. In the effort to find new anti-leukemic agents, we evaluated the small drug SB225002 (N-(2-hydroxy-4-nitrophenyl)-N’-(2-bromophenyl)urea). Although initially described as a selective antagonist of CXCR2, later studies have identified other cellular targets for SB225002, with potential medicinal use in cancer. We found that SB225002 has a significant pro-apoptotic effect against both B- and T-ALL cell lines. Cell cycle analysis demonstrated that treatment with SB225002 induces G2-M cell cycle arrest. Transcriptional profiling revealed that SB225002-mediated apoptosis triggered a transcriptional program typical of tubulin binding agents. Network analysis revealed the activation of genes linked to the JUN and p53 pathways and inhibition of genes linked to the TNF pathway. Early cellular effects activated by SB225002 included the up-regulation of GLIPR1, a p53-target gene shown to have pro-apoptotic activities in prostate and bladder cancer. Silencing of GLIPR1 in B- and T-ALL cell lines resulted in increased resistance to SB225002. Although SB225002 promoted ROS increase in ALL cells, antioxidant N-Acetyl Cysteine pre-treatment only modestly attenuated cell death, implying that the pro-apoptotic effects of SB225002 are not exclusively mediated by ROS. Moreover, GLIPR1 silencing resulted in increased ROS levels both in untreated and SB225002-treated cells. In conclusion, SB225002 induces cell cycle arrest and apoptosis in different B- and T-ALL cell lines. Inhibition of tubulin function with concurrent activation of the p53 pathway, in particular, its downstream target GLIPR1, seems to underlie the anti-leukemic effect of SB225002. PMID:26302043

  19. Mechanism of Activation-Induced Downregulation of Mitofusin 2 in Human Peripheral Blood T Cells.

    PubMed

    Dasgupta, Asish; Chen, Kuang-Hueih; Munk, Rachel B; Sasaki, Carl Y; Curtis, Jessica; Longo, Dan L; Ghosh, Paritosh

    2015-12-15

    Mitofusin 2 (Mfn2), a mitochondrial protein, was shown to have antiproliferative properties when overexpressed. In this article, we show that activation of resting human peripheral blood T cells caused downregulation of Mfn2 levels. This downregulation of Mfn2 was blocked by different inhibitors (mTOR inhibitor rapamycin, PI3K inhibitor LY294002, and Akt inhibitor A443654), producing cells that were arrested in the G0/G1 stage of the cell cycle. Furthermore, the activation-induced downregulation of Mfn2 preceded the entry of the cells into the cell cycle, suggesting that Mfn2 downregulation is a prerequisite for activated T cell entry into the cell cycle. Accordingly, small interfering RNA-mediated knockdown of Mfn2 resulted in increased T cell proliferation. Overexpression of constitutively active AKT resulted in the downregulation of Mfn2, which can be blocked by a proteasome inhibitor. Akt-mediated downregulation of Mfn2 was via the mTORC1 pathway because this downregulation was blocked by rapamycin, and overexpression of wild-type, but not kinase-dead mTOR, caused Mfn2 downregulation. Our data suggested that activation-induced reactive oxygen species production plays an important role in the downregulation of Mfn2. Collectively, our data suggest that the PI3K-AKT-mTOR pathway plays an important role in activation-induced downregulation of Mfn2 and subsequent proliferation of resting human T cells. PMID:26566676

  20. Tetrandrine induces autophagy and differentiation by activating ROS and Notch1 signaling in leukemia cells.

    PubMed

    Liu, Ting; Men, Qiuxu; Wu, Guixian; Yu, Chunrong; Huang, Zan; Liu, Xin; Li, Wenhua

    2015-04-10

    All-trans retinoic acid (ATRA) is a differentiating agent for the treatment of acute promyelocytic leukemia (APL). However, the therapeutic efficacy of ATRA has limitations. Tetrandrine is a traditional Chinese medicinal herb extract with antitumor effects. In this study, we investigated the effects of tetrandrine on human PML-RARα-positive acute promyelocytic leukemia cells. Tetrandrine inhibited tumors in vivo. It induced autophagy and differentiation by triggering ROS generation and activating Notch1 signaling. Tetrandrine induced autophagy and differentiation in M5 type patient primary leukemia cells. The in vivo results indicated that low concentrations of tetrandrine inhibited leukemia cells proliferation and induced autophagy and then facilitated their differentiation, by activating ROS and Notch1 signaling. We suggest that tetrandrine is a potential agent for the treatment of APL by inducing differentiation of leukemia cells. PMID:25797266

  1. Apoptosis of pancreatic adenocarcinoma cells induced by LED-activated pheophorbide a

    NASA Astrophysics Data System (ADS)

    Liu, L.; Leung, A. W. N.; Xia, X. S.; Bai, D. Q.; Lin, H. D.; Xu, C. S.

    2011-02-01

    Apoptosis is an important form of cell death. The present study investigated apoptotic cell death of pancreatic adenocarcinoma cells treated by LED-activated pheophorbide a (Pa) using nuclear staining and flow cytometric analysis with Annexin V/PI staining 8 h after LED-activated Pa. We further investigated the changes of mitochondrial membrane potential 3 h after LED-activated Pa. The results showed that apoptosis significantly occurred and mitochondrial membrane potential markedly decreased in pancreatic adenocarcinoma cells after LED-activated Pa. Our data demonstrated that LED-activated Pa could remarkably induce apoptosis of pancreatic adenocarcinoma cells, and the collapse of mitochondrial membrane potential might be an important event in the apoptosis of pancreatic adenocarcinoma cells after LED-activated Pa.

  2. Differential immunomodulatory activity of tumor cell death induced by cancer therapeutic toll-like receptor ligands.

    PubMed

    Klein, Johanna C; Wild, Clarissa A; Lang, Stephan; Brandau, Sven

    2016-06-01

    Synthetic toll-like receptor (TLR) ligands stimulate defined immune cell subsets and are currently tested as novel immunotherapeutic agents against cancer with, however, varying clinical efficacy. Recent data showed the expression of TLR receptors also on tumor cells. In this study we investigated immunological events associated with the induction of tumor cell death by poly(I:C) and imiquimod. A human head and neck squamous cell carcinoma (HNSCC) cell line was exposed to poly(I:C) and imiquimod, which were delivered exogenously via culture medium or via electroporation. Cell death and cell biological consequences thereof were analyzed. For in vivo analyses, a human xenograft and a syngeneic immunocompetent mouse model were used. Poly(I:C) induced cell death only if delivered by electroporation into the cytosol. Cell death induced by poly(I:C) resulted in cytokine release and activation of monocytes in vitro. Monocytes activated by the supernatant of cancer cells previously exposed to poly(I:C) recruited significantly more Th1 cells than monocytes exposed to control supernatants. If delivered exogenously, imiquimod also induced tumor cell death and some release of interleukin-6, but cell death was not associated with release of Th1 cytokines, interferons, monocyte activation and Th1 recruitment. Interestingly, intratumoral injection of poly(I:C) triggered tumor cell death in tumor-bearing mice and reduced tumor growth independent of TLR signaling on host cells. Imiquimod did not affect tumor size. Our data suggest that common cancer therapeutic RNA compounds can induce functionally diverse types of cell death in tumor cells with implications for the use of TLR ligands in cancer immunotherapy. PMID:27034235

  3. Activation of caspase-8 and Erk-1/2 in domes regulates cell death induced by confluence in MDCK cells.

    PubMed

    Chang, Yung-Heng; Lin, Hsi-Hui; Wang, Yang-Kao; Chiu, Wen-Tai; Su, Hsiao-Wen; Tang, Ming-Jer

    2007-04-01

    Under normal culture conditions, cells adhere to culture dish, spread out, proliferate, and finally cover all areas and reach confluence. During the confluent stage, cell proliferation ceases and differentiation is enhanced. Meanwhile, cell death also appears as the monolayer confluence proceeds. To delineate the mechanism of cell death induced by the confluent process, we employed Madin-Darby canine kidney (MDCK) cells. When approaching confluence, MDCK cells exhibited increase the levels of caspase-2 and enhanced the activity of caspase-8. Using various caspase inhibitors to block apoptosis, we found that only z-VAD-fmk and z-IETD-fmk can inhibit confluent cell death, indicating that confluent cell death is mediated by activation of caspase-8. Overexpression of Bcl-2 inhibited confluent cell death, suggesting the involvement of mitochondria-dependent pathway in confluent cell death. Interestingly, the activity of phospho-Erk (p-Erk) was initially decreased before confluence, but markedly increased after confluence. Immunofluorescence staining studies showed that p-Erk was expressed exclusively on dome-forming cells that underwent apoptosis. Treatment of confluent MDCK cells with PD98059 and UO126, the inhibitors of MEK, enhanced apoptosis as well as activity of caspase-8. These data indicate that elevation of p-Erk activity during confluence may serve to suppress confluent cell death. Taken together, activation of caspase-8 contributes to and results in confluent cell death, whereas elevated p-Erk activity serves to prevent confluent cell death by regulating activation of caspase-8.

  4. Oxidative stress plays a role in high glucose-induced activation of pancreatic stellate cells

    SciTech Connect

    Ryu, Gyeong Ryul; Lee, Esder; Chun, Hyun-Ji; Yoon, Kun-Ho; Ko, Seung-Hyun; Ahn, Yu-Bae; Song, Ki-Ho

    2013-09-20

    Highlights: •High glucose increased production of reactive oxygen species in cultured pancreatic stellate cells. •High glucose facilitated the activation of these cells. •Antioxidant treatment attenuated high glucose-induced activation of these cells. -- Abstract: The activation of pancreatic stellate cells (PSCs) is thought to be a potential mechanism underlying islet fibrosis, which may contribute to progressive β-cell failure in type 2 diabetes. Recently, we demonstrated that antioxidants reduced islet fibrosis in an animal model of type 2 diabetes. However, there is no in vitro study demonstrating that high glucose itself can induce oxidative stress in PSCs. Thus, PSCs were isolated and cultured from Sprague Dawley rats, and treated with high glucose for 72 h. High glucose increased the production of reactive oxygen species. When treated with high glucose, freshly isolated PSCs exhibited myofibroblastic transformation. During early culture (passage 1), PSCs treated with high glucose contained an increased number of α-smooth muscle actin-positive cells. During late culture (passages 2–5), PSCs treated with high glucose exhibited increases in cell proliferation, the expression of fibronectin and connective tissue growth factor, release of interleukin-6, transforming growth factor-β and collagen, and cell migration. Finally, the treatment of PSCs with high glucose and antioxidants attenuated these changes. In conclusion, we demonstrated that high glucose increased oxidative stress in primary rat PSCs, thereby facilitating the activation of these cells, while antioxidant treatment attenuated high glucose-induced PSC activation.

  5. E. adenophorum Induces Cell Cycle and Apoptosis of Renal Cells through Mitochondrial Pathway and Caspase Activation in Saanen Goat

    PubMed Central

    Hu, Yanchun; Luo, Biao; Wu, Lei; Qiao, Yan; Mo, Quan; Xu, Ruiguang; Zhou, Yancheng; Ren, Zhihua; Zuo, Zhicai; Deng, Junliang; Peng, Guangneng; He, Wei; Wei, Yahui

    2015-01-01

    The cytotoxicity effects of E. adenophorum on cell cycle and apoptosis of renal cells in Saanen goat was evaluated by TUNEL, DAPI, AO/EB staining, DNA fragmentation assay, Caspase activity, Western-blot, qRT-PCR and flow cytometry analysis. 16 saanen goats randomly divided into four groups were fed on 0%, 40%, 60% and 80% E. adenophorum diets. The Results showed that E. adenophorum induced typical apoptotic features of renal cells. E. adenophorum significantly suppressed renal cells viability, caused cell cycle activity arrest and induced typical apoptotic features in a dose-dependent manner. However, the protein levels of Fas/FasL, Bid and caspase-8 did not appear significant changes in the process of E. adenophorum-induced apoptosis. Moreover, E. adenophorum administration slightly decreased Bcl-2 expression, promoted Bax translocation to mitochondria, triggered the release of Cyt c from mitochondria into cytosol and activated caspase-9, -3, and cleaved PARP. The mitochondrial p53 translocation was significantly activated, accompanied by a significant increase in the loss of ΔΨm, Cyt c release and caspase-9 activation. Above all, these data suggest that E. adenophorum induces renal cells apoptosis via the activation of mitochondria-mediated apoptosis pathway in renal cells. These findings may provide new insights to understand the mechanisms involved in E. adenophorum-caused cytotoxicity of renal cells. PMID:26382060

  6. Bcl-2 induces cyclin D1 promoter activity in human breast epithelial cells independent of cell anchorage.

    PubMed

    Lin, H M; Lee, Y J; Li, G; Pestell, R G; Kim, H R

    2001-01-01

    Cyclin D1 expression is co-regulated by growth factor and cell adhesion signaling. Cell adhesion to the extracellular matrix activates focal adhesion kinase (FAK), which is essential for cyclin D1 expression. Upon the loss of cell adhesion, cyclin D1 expression is downregulated, followed by apoptosis in normal epithelial cells. Since bcl-2 prevents apoptosis induced by the loss of cell adhesion, we hypothesized that bcl-2 induces survival signaling complementary to cell adhesion-mediated gene regulation. In the present study, we investigated the role of bcl-2 on FAK activity and cyclin D1 expression. We found that bcl-2 overexpression induces cyclin D1 expression in human breast epithelial cell line MCF10A independent of cell anchorage. Increased cyclin D1 expression in stable bcl-2 transfectants is not related to bcl-2-increased G1 duration, but results from cyclin D1 promoter activation. Transient transfection studies confirmed anchorage-independent bcl-2 induction of cyclin D1 promoter activity in human breast epithelial cell lines (MCF10A, BT549, and MCF-7). We provide evidence that bcl-2 induction of cyclin D1 expression involves constitutive activation of focal adhesion kinase, regardless of cell adhesion. The present study suggests a potential oncogenic activity for bcl-2 through cyclin D1 induction, and provides an insight into the distinct proliferation-independent pathway leading to increased cyclin D1 expression in breast cancer.

  7. PTX3 gene activation in EGF-induced head and neck cancer cell metastasis.

    PubMed

    Chang, Wei-Chiao; Wu, Shuo-Lun; Huang, Wan-Chen; Hsu, Jinn-Yuan; Chan, Shih-Hung; Wang, Ju-Ming; Tsai, Jhih-Peng; Chen, Ben-Kuen

    2015-04-10

    Overexpression of the epidermal growth factor (EGF) receptor (EGFR) is associated with enhanced invasion and metastasis in head and neck squamous cell carcinoma (HNSCC). Long Pentraxin PTX3 is involved in immune escape in cancer cells. Here, we identified PTX3 as a promoting factor that mediates EGF-induced HNSCC metastasis. EGF-induced PTX3 transcriptional activation is via the binding of c-Jun to the activator protein (AP)-1 binding site of the PTX3 promoter. PI3K/Akt and NF-κB were essential for the PTX3 activation. EGF-induced PTX3 expression was blocked in c-Jun- and NF-κB-knockdown cells. EGF-mediated PTX3 secretion resulted in the enhancement of cell migration and invasion, and interactions between cancer and endothelial cells. The tail-vein injection animal model revealed that depletion of PTX3 decreased EGF-primed tumor cell metastatic seeding of the lungs. In addition, fibronectin, matrix metalloproteinase-9 (MMP9) and E-cadherin were essential components in EGFR/PTX3-mediated cancer metastasis. In conclusion, PI3K/Akt and NF-κB-dependent regulation of AP-1 mediates PTX3 transcriptional responses to EGF. Autocrine production of EGF-induced PTX3 in turn induces metastatic molecules, activating inflammatory cascades and metastasis.

  8. Biological Character of RetroNectin Activated Cytokine-Induced Killer Cells

    PubMed Central

    2016-01-01

    Adoptive cell therapy (ACT) using autologous cytokine-induced killer (CIK) cells is a promising treatment for metastatic carcinomas. In this study, we investigated the impact of RetroNectin on the proliferation, phenotype alternation, cytokine secretion, and cytotoxic activity of CIK cells from pancreatic cancer patients. Furthermore, we treated 13 patients with metastatic or locally advanced pancreatic cancer using autologous RetroNectin-activated CIK cells (R-CIK cells) alone or in combination with chemotherapy. Compared with only CD3 activated CIK cells (OKT-CIK cells), R-CIK cells showed stronger and faster proliferative ability, with a lower ratio of spontaneous apoptosis. Moreover, this ability continued after IL-2 was withdrawn from the culture system. R-CIK cells could also secrete higher levels of IL-2 and lower levels of IL-4 and IL-5 versus OKT-CIK cells. There was no difference between OKT-CIK and R-CIK cells in cytotoxic ability against lymphoma cell line K562. In patients who received auto-R-CIK cell infusion therapy, the overall objective response rate was 23.1%. Median survival time (mOS) after first R-CIK cell infusion was 10.57 months; the 1-year survival rate was 38.5%. No serious toxicity was associated with R-CIK cell infusion. In conclusion, RetroNectin may enhance antitumor activity of CIK cells: it is safe for use in treating pancreatic cancer. PMID:27433478

  9. Cytotoxic activity of interferon alpha induced dendritic cells as a biomarker of glioblastoma

    NASA Astrophysics Data System (ADS)

    Mishinov, S. V.; Stupak, V. V.; Tyrinova, T. V.; Leplina, O. Yu.; Ostanin, A. A.; Chernykh, E. R.

    2016-08-01

    Dendritic cells (DCs) are the most potent antigen presenting cells that can play direct role in anti-tumor immune response as killer cells. DC tumoricidal activity can be stimulated greatly by type I IFN (IFNα and IFNβ). In the present study, we examined cytostatic and cytotoxic activity of monocyte-derived IFNα-induced DCs generated from patients with brain glioma and evaluated the potential use of these parameters in diagnostics of high-grade gliomas. Herein, we demonstrated that patient DCs do not possess the ability to inhibit the growth of tumor HEp-2 cell line but low-grade and high-grade glioma patients do not differ significantly in DC cytostatic activity. However, glioma patient DCs are characterized by reduced cytotoxic activity against HEp-2 cells. The impairment of DC cytotoxic function is observed mainly in glioblastoma patients. The cytotoxic activity of DCs against HEp-2 cells below 9% is an informative marker for glioblastomas.

  10. Emodin ameliorates cisplatin-induced apoptosis of rat renal tubular cells in vitro by activating autophagy

    PubMed Central

    Liu, Hong; Gu, Liu-bao; Tu, Yue; Hu, Hao; Huang, Yan-ru; Sun, Wei

    2016-01-01

    Aim: A previous report shows that emodin extracted from the Chinese herbs rhubarb and giant knotweed rhizome can ameliorate the anticancer drug cisplatin-induced injury of HEK293 cells. In this study, we investigated whether and how emodin could protect renal tubular epithelial cells against cisplatin-induced nephrotoxicity in vitro. Methods: The viability and apoptosis of normal rat renal tubular epithelial cells (NRK-52E) were detected using formazan assay and flow cytometry analysis, respectively. The expression levels of cleaved caspase-3, autophagy maker LC3 I/II, and AMPK/mTOR signaling pathway-related proteins were measured with Western blot analysis. The changes of morphology and RFP-LC3 fluorescence were observed under microscopy. Results: Cisplatin (10-50 μmol/L) dose-dependently induced cell damage and apoptosis in NRK-52E cells, whereas emodin (10 and 100 μmol/L) significantly ameliorated cisplatin-induced cell damage, apoptosis and caspase-3 cleavage. Emodin dose-dependently increased LC3-II levels and induced RFP-LC3-containing punctate structures in NRK-52E cells. Furthermore, the protective effects of emodin were abolished by bafilomycin A1 (10 nmol/L), and mimicked by rapamycin (100 nmol/L). Moreover, emodin increased the phosphorylation of AMPK and suppressed the phosphorylation of mTOR. The AMPK inhibitor compound C (10 μmol/L) not only abolished emodin-induced autophagy activation, but also emodin-induced anti-apoptotic effects. Conclusion: Emodin ameliorates cisplatin-induced apoptosis of rat renal tubular cells in vitro through modulating the AMPK/mTOR signaling pathways and activating autophagy. Emodin may have therapeutic potential for the prevention of cisplatin-induced nephrotoxicity. PMID:26775661

  11. Activation of muscarinic receptors inhibits glutamate-induced GSK-3β overactivation in PC12 cells

    PubMed Central

    Ma, Ke; Yang, Li-min; Chen, Hong-zhuan; Lu, Yang

    2013-01-01

    Aim: To investigate the actions of the muscarinic agonist carbachol on glutamate-induced neurotoxicity in PC12 cells, and the underlying mechanisms. Methods: PC12 cells were treated with different concentrations of glutamate for 24 or 48 h. The cell viability was measured using MTT assay, and the expression and activation of GSK-3β were detected with Western blot. β-Catenin translocation was detected using immunofluorescence. Luciferase reporter assay and real-time PCR were used to analyze the transcriptional activity of β-catenin. Results: Glutamate (1, 3, and 10 mmol/L) induced PC12 cell death in a dose-dependent manner. Moreover, treatment of the cells with glutamate (1 mmol/L) caused significant overactivation of GSK-3β and prevented β-catenin translocation to the nucleus. Pretreatment with carbachol (0.01 μmol/L) blocked glutamate-induced cell death and GSK-3β overactivation, and markedly enhanced β-catenin transcriptional activity. Conclusion: Activation of muscarinic receptors exerts neuroprotection in PC12 cells by attenuating glutamate-induced GSK-3β overactivation, suggesting potential benefits of muscarinic agonists for Alzheimer's disease. PMID:23685950

  12. Green tea polyphenol epigallocatechin-O-gallate induces cell death by acid sphingomyelinase activation in chronic myeloid leukemia cells

    PubMed Central

    HUANG, YUHUI; KUMAZOE, MOTOFUMI; BAE, JAEHOON; YAMADA, SHUHEI; TAKAI, MIKA; HIDAKA, SHIORI; YAMASHITA, SHUYA; KIM, YOONHEE; WON, YEONGSEON; MURATA, MOTOKI; TSUKAMOTO, SHUNTARO; TACHIBANA, HIROFUMI

    2015-01-01

    An epidemiological study showed that green tea consumption is associated with a reduced risk of hematopoietic malignancy. The major green tea polyphenol epigallocatechin-3-O-gallate (EGCG) is reported to have anticancer effects. Chronic myeloid leukemia (CML) is a major hematopoietic malignancy characterized by expansion of myeloid cells. In the present study, we showed EGCG-induced acid sphingomyelinase (ASM) activation and lipid raft clustering in CML cells. The ASM inhibitor desipramine significantly reduced EGCG-induced cell death. Protein kinase Cδ is a well-known kinase that plays an important role in ASM activation. We observed EGCG-induced phos-phorylation of protein kinase Cδ at Ser664. Importantly, EGCG-induced ASM activation was significantly reduced by pretreatment of CML cells with the soluble guanylate cyclase inhibitor NS2028, suggesting that EGCG induced ASM activation through the cyclic guanosine monophosphate (cGMP)-dependent pathway. Indeed, pharmacological inhibition of a cGMP-negative regulator enhanced the anti-CML effect of EGCG. These results indicate that EGCG-induced cell death via the cGMP/ASM pathway in CML cells. PMID:26135316

  13. atRA-induced apoptosis of mouse embryonic palate mesenchymal cells involves activation of MAPK pathway

    SciTech Connect

    Yu Zengli . E-mail: yuzengli@263.net; Xing Ying . E-mail: xingy@zzu.edu.cn

    2006-08-15

    Our previous studies have shown that atRA treatment resulted in cell-cycle block and growth inhibition in mouse embryonic palatal mesenchymal (MEPM). In the current study, gestation day (GD) 13 MEPM cells were used to test the hypothesis that the growth inhibition by atRA is due to apoptosis. The effects of atRA on apoptosis were assessed by performing MTT assay, Cell Death Detection ELISA and flow cytometry, respectively. Data analysis confirmed that atRA treatment induced apoptosis-like cell death, as shown by decreased cell viability and increased fragmented DNA and sub-G1 fraction. atRA-induced apoptosis was associated with upregulation of bcl-2, translocation of bax protein to the mitochondria from the cytosol, activation of caspase-3 and cytochrome c release into cytosol. atRA-induced apoptosis was abrogated by z-DEVD-fmk, a caspase-3 specific inhibitor, and z-VAD-fmk, a general caspase inhibitor, suggesting that the atRA-induced cell death of MEPM cells occurs through the cytochrome c- and caspase-3-dependent pathways. In addition, atRA treatment caused a strong and sustained activation of c-Jun N-terminal kinase (JNK) and p38 kinase (p38), as well as an early but transient activation of extracellular signal-regulated kinase (ERK). Importantly, atRA-induced DNA fragmentation and capase-3 activation were prevented by pretreatment with the JNK inhibitor (SP600125) and the p38 MAPK inhibitor (SB202190), but not by pretreatment with MEK inhibitor (U0126). From these results, we suggest that mitogen-activated protein kinase-dependent pathways is involved in the atRA-induced apoptosis of MEPM cells.

  14. Activation-induced necroptosis contributes to B-cell lymphopenia in active systemic lupus erythematosus

    PubMed Central

    Fan, H; Liu, F; Dong, G; Ren, D; Xu, Y; Dou, J; Wang, T; Sun, L; Hou, Y

    2014-01-01

    B-cell abnormality including excessive activation and lymphopenia is a central feature of systemic lupus erythematosus (SLE). Although activation threshold, auto-reaction and death of B cells can be affected by intrinsical and/or external signaling, the underlying mechanisms are unclear. Herein, we demonstrate that co-activation of Toll-like receptor 7 (TLR7) and B-cell receptor (BCR) pathways is a core event for the survival/dead states of B cells in SLE. We found that the mortalities of CD19+CD27- and CD19+IgM+ B-cell subsets were increased in the peripheral blood mononuclear cells (PBMCs) of SLE patients. The gene microarray analysis of CD19+ B cells from active SLE patients showed that the differentially expressed genes were closely correlated to TLR7, BCR, apoptosis, necroptosis and immune pathways. We also found that co-activation of TLR7 and BCR could trigger normal B cells to take on SLE-like B-cell characters including the elevated viability, activation and proliferation in the first 3 days and necroptosis in the later days. Moreover, the necroptotic B cells exhibited mitochondrial dysfunction and hypoxia, along with the elevated expression of necroptosis-related genes, consistent with that in both SLE B-cell microarray and real-time PCR verification. Expectedly, pretreatment with the receptor-interacting protein kinase 1 (RIPK1) inhibitor Necrostatin-1, and not the apoptosis inhibitor zVAD, suppressed B-cell death. Importantly, B cells from additional SLE patients also significantly displayed high expression levels of necroptosis-related genes compared with those from healthy donors. These data indicate that co-activation of TLR7 and BCR pathways can promote B cells to hyperactivation and ultimately necroptosis. Our finding provides a new explanation on B-cell lymphopenia in active SLE patients. These data suggest that extrinsic factors may increase the intrinsical abnormality of B cells in SLE patients. PMID:25210799

  15. Activation of Human T-Helper/Inducer Cell, T-Cytotoxic Cell, B-Cell, and Natural Killer (NK)-Cells and induction of Natural Killer Cell Activity against K562 Chronic Myeloid Leukemia Cells with Modified Citrus Pectin

    PubMed Central

    2011-01-01

    Background Modified citrus pectin (MCP) is known for its anti-cancer effects and its ability to be absorbed and circulated in the human body. In this report we tested the ability of MCP to induce the activation of human blood lymphocyte subsets like T, B and NK-cells. Methods MCP treated human blood samples were incubated with specific antibody combinations and analyzed in a flow cytometer using a 3-color protocol. To test functionality of the activated NK-cells, isolated normal lymphocytes were treated with increasing concentrations of MCP. Log-phase PKH26-labeled K562 leukemic cells were added to the lymphocytes and incubated for 4 h. The mixture was stained with FITC-labeled active form of caspase 3 antibody and analyzed by a 2-color flow cytometry protocol. The percentage of K562 cells positive for PKH26 and FITC were calculated as the dead cells induced by NK-cells. Monosaccharide analysis of the MCP was performed by high-performance anion-exchange chromatography with pulse amperometric detection (HPAEC-PAD). Results MCP activated T-cytotoxic cells and B-cell in a dose-dependent manner, and induced significant dose-dependent activation of NK-cells. MCP-activated NK-cells demonstrated functionality in inducing cancer cell death. MCP consisted of oligogalacturonic acids with some containing 4,5-unsaturated non-reducing ends. Conclusions MCP has immunostimulatory properties in human blood samples, including the activation of functional NK cells against K562 leukemic cells in culture. Unsaturated oligogalacturonic acids appear to be the immunostimulatory carbohydrates in MCP. PMID:21816083

  16. Single cell analysis of low-power laser irradiation-induced activation of signaling pathway in cell proliferation

    NASA Astrophysics Data System (ADS)

    Xing, Da; Gao, Xuejuan

    2007-02-01

    Low-power laser irradiation (LPLI) has been shown to promote cell proliferation in various cell types, yet the mechanism of which has not been fully clarified. Investigating the signaling pathways involved in the laser irradiation is important for understanding these processes. The small G protein Ras works as a binary switch in many important intracellular signaling pathways and, therefore, has been one of the focal targets of signal-transduction investigations and drug development. The Ras/Raf/MEK/ERK (extracellular-signal-regulated kinase) signaling pathway is a network that governs proliferation, differentiation and cell survival. Recent studies suggest that Ras/Raf signaling pathway is involved in the LPLI-induced cell proliferation. On the other hand, Protein kinase Cs (PKCs), the Ca 2+ activated, phospholipid-dependent serine/threonine protein kinases, have been recently presumed to be involved in the regulation of cell proliferation induced by LPLI. In this report, to monitor the direct activations of Ras and PKCs after LPLI treatment in living cells in real time, Raichu-Ras reporter and C kinase activity reporter (CKAR) were utilized, both of which were constructed based on fluorescence resonance energy transfer (FRET) technique. The direct activation of Ras is predominantly initiated from the different microdomains of the plasma membrane. The results are monitored during cell proliferation induced by LPLI (0.8 J/cm2) in serum-starved COS-7 cells expressing Raichu-Ras reporter using FRET imaging on laser scanning confocal microscope. Furthermore, the increasing activation of PKCs is also monitored during cell proliferation induced by LPLI (0.8 J/cm2) in serum-starved human lung adenocarcinoma cells (ASTC-a-1) expressing CKAR reporter using the similar way. Taken together, the dynamic increases of H-Ras and PKCs activities are observed during the processes of cell proliferation induced by LPLI.

  17. Activation of human T-helper/inducer cell, T-cytotoxic/suppressor cell, B-cell, and natural killer (NK)-cells and induction of NK cell activity against K562 chronic myeloid leukemia cells with modified citrus pectin

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Background Modified citrus pectin (MCP) is known for its anti-cancer effects and its ability to be absorbed and circulated in the human body. In this report we tested the ability of MCP to induce the activation of human blood lymphocyte subsets including T-helper/inducer cell, Tcytotoxic/suppres...

  18. Effects of cell heterogeneity on production of polypeptide growth factors and mesoderm-inducing activity by Xenopus laevis XTC cells.

    PubMed

    Snoek, G T; Koster, C H; de Laat, S W; Heideveld, M; Durston, A J; van Zoelen, E J

    1990-04-01

    The Xenopus laevis XTC cell line has been analyzed for the production of polypeptide growth factors and mesoderm-inducing activity. By the use of specific biological assays, it is shown that XTC cells produce a growth factor functionally related to the platelet-derived growth factor (PDGF) and two transforming growth factor (TGF) beta-like activities. Mesoderm-inducing activity, as measured on X. laevis ectodermal explants from stage 10 embryos, was found to coelute on a Bio-Gel P-100 column with one of the TGF beta-like activities at an apparent molecular weight of 6-10 kDa. Analysis of the DNA content from XTC cells by flow cytometry demonstrated that the cell line is heterogeneous and consists of both tetraploid and diploid cells. Cloning of the XTC cells and selecting single-cell colonies on the basis of their ability to grow in soft agar resulted in the isolation of several homogeneous, morphologically different clonal derivatives. Analysis of conditioned medium from these clonal derivatives showed that only one of them, the only diploid line among six investigated, produced a strong heat- and acid-stable mesoderm-inducing activity that induced notochord and muscle formation in stage 10 X. laevis ectodermal explants. The relation between this activity and a recently described TGF beta-like mesoderm-inducing factor obtained from XTC-conditioned medium will be discussed. In conclusion, a clonal cell line derived from X. laevis XTC cells which provides a good source for further characterization of mesoderm-inducing factors has been established.

  19. Alternaria extract activates autophagy that induces IL-18 release from airway epithelial cells.

    PubMed

    Murai, Hiroki; Okazaki, Shintaro; Hayashi, Hisako; Kawakita, Akiko; Hosoki, Koa; Yasutomi, Motoko; Sur, Sanjiv; Ohshima, Yusei

    2015-09-01

    Alternaria alternata is a major outdoor allergen that causes allergic airway diseases. Alternaria extract (ALT-E) has been shown to induce airway epithelial cells to release IL-18 and thereby initiate Th2-type responses. We investigated the underlying mechanisms involved in IL-18 release from ALT-E-stimulated airway epithelial cells. Normal human bronchial epithelial cells and A549 human lung adenocarcinoma cells were stimulated with ALT-E in the presence of different inhibitors of autophagy or caspases. IL-18 levels in culture supernatants were measured by ELISA. The numbers of autophagosomes, an LC3-I to LC3-II conversion, and p62 degradation were determined by immunofluorescence staining and immunoblotting. 3-methyladenine and bafilomycin, which inhibit the formation of preautophagosomal structures and autolysosomes, respectively, suppressed ALT-E-induced IL-18 release by cells, whereas caspase 1 and 8 inhibitors did not. ALT-E-stimulation increased autophagosome formation, LC-3 conversion, and p62 degradation in airway epithelial cells. LPS-stimulation induced the LC3 conversion in A549 cells, but did not induce IL-18 release or p62 degradation. Unlike LPS, ALT-E induced airway epithelial cells to release IL-18 via an autophagy dependent, caspase 1 and 8 independent pathway. Although autophagy has been shown to negatively regulate canonical inflammasome activity in TLR-stimulated macrophages, our data indicates that this process is an unconventional mechanism of IL-18 secretion by airway epithelial cells.

  20. Rebuilding cytoskeleton roads: Active-transport-induced polarization of cells

    NASA Astrophysics Data System (ADS)

    Hawkins, R. J.; Bénichou, O.; Piel, M.; Voituriez, R.

    2009-10-01

    Many cellular processes require a polarization axis which generally initially emerges as an inhomogeneous distribution of molecular markers in the cell. We present a simple analytical model of a general mechanism of cell polarization taking into account the positive feedback due to the coupled dynamics of molecular markers and cytoskeleton filaments. We find that the geometry of the organization of cytoskeleton filaments, nucleated on the membrane (e.g., cortical actin) or from a center in the cytoplasm (e.g., microtubule asters), dictates whether the system is capable of spontaneous polarization or polarizes only in response to external asymmetric signals. Our model also captures the main features of recent experiments of cell polarization in two considerably different biological systems, namely, mating budding yeast and neuron growth cones.

  1. Curcumin-induced recovery from hepatic injury involves induction of apoptosis of activated hepatic stellate cells.

    PubMed

    Priya, S; Sudhakaran, P R

    2008-10-01

    Hepatic stellate cells (HSCs) undergo activation and transdifferentiation to myofibroblast like cells in liver injury, leading to liver fibrosis. During recovery from injury, activated HSCs may either revert back to quiescent state or undergo apoptosis or both. In the present study, we have examined whether recovery from hepatic injury involves apoptosis of activated HSCs and tested whether curcumin (the yellow pigment from Curcuma longa Linn.) promotes recovery from hepatic injury by inducing apoptosis of these cells. Hepatic injury was induced by CCl4 and apoptosis was studied in HSCs isolated from liver by MTT assay, DNA fragmentation, and DAPI and annexin staining. Hepatic recovery was assessed by measuring hepatic marker activities, such as serum GOT, GPT and protein. Hepatic recovery occurred within 4 weeks after inducing injury in untreated control, whereas curcumin treatment caused hepatic recovery within 2 weeks, as evidenced by the reduction of hepatic marker activities to near normal levels. HSCs isolated from liver of animals treated with curcumin showed maximum apoptotic marker activities in 2nd week, whereas in HSCs from untreated control recovering from injury, maximum apoptosis was observed in 4th week. Induction of apoptosis in vivo during hepatic recovery was also suggested by increase in caspase-3 activity. Treatment of isolated HSCs in culture with curcumin caused apoptosis during later stages confirming that curcumin induced apoptosis of activated HSCs and not in unactivated quiescent HSCs. These results suggested that hepatoprotective effect of curcumin causing recovery from injury involved apoptosis of activated HSCs. PMID:19069843

  2. Leptin activates STAT and ERK2 pathways and induces gastric cancer cell proliferation

    SciTech Connect

    Pai, Rama . E-mail: rpai@uci.edu; Lin Cal; Tran, Teresa; Tarnawski, Andrzej . E-mail: atarnawski@yahoo.com

    2005-06-17

    Although leptin is known to induce proliferative response in gastric cancer cells, the mechanism(s) underlying this action remains poorly understood. Here, we provide evidence that leptin-induced gastric cancer cell proliferation involves activation of STAT and ERK2 signaling pathways. Leptin-induced STAT3 phosphorylation is independent of ERK2 activation. Leptin increases SHP2 phosphorylation and enhances binding of Grb2 to SHP2. Inhibition of SHP2 expression with siRNA but not SHP2 phosphatase activity abolished leptin-induced ERK2 activation. While JAK inhibition with AG490 significantly reduced leptin-induced ERK2, STAT3 phosphorylation, and cell proliferation, SHP2 inhibition only partially reduced cancer cell proliferation. Immunostaining of gastric cancer tissues displayed local overexpression of leptin and its receptor indicating that leptin might be produced and act locally in a paracrine or autocrine manner. These findings indicate that leptin promotes cancer growth by activating multiple signaling pathways and therefore blocking its action at the receptor level could be a rational therapeutic strategy.

  3. Structure-activity relationship of 9-methylstreptimidone, a compound that induces apoptosis selectively in adult T-cell leukemia cells.

    PubMed

    Takeiri, Masatoshi; Ota, Eisuke; Nishiyama, Shigeru; Kiyota, Hiromasa; Umezawa, Kazuo

    2012-01-01

    We previously reported that 9-methylstreptimidone, a piperidine compound isolated from a culture filtrate of Streptomyces, induces apoptosis selectively in adult T-cell leukemia cells. It was screened for a compound that inhibits LPS-induced NF-kappaB and NO production in mouse macrophages. However, 9-methystreptimidone is poorly obtained from the producing microorganism and difficult to synthesize. Therefore, in the present research, we studied the structure-activity relationship to look for new selective inhibitors. We found that the structure of the unsaturated hydrophobic portion of 9-methylstreptimidone was essential for the inhibition of LPS-induced NO production. Among the 9-methylstreptimidone-related compounds tested, (+/-)-4,alpha-diepi-streptovitacin A inhibited NO production in macrophage-like cells as potently as 9-methylstreptimidone and without cellular toxicity. Moreover, this compound selectively induced apoptosis in adult T-cell leukemia MT-1 cells.

  4. Interleukin-13 interferes with activation-induced t-cell apoptosis by repressing p53 expression

    PubMed Central

    Yang, Li; Xu, Ling-Zhi; Liu, Zhi-Qiang; Yang, Gui; Geng, Xiao-Rui; Mo, Li-Hua; Liu, Zhi-Gang; Zheng, Peng-Yuan; Yang, Ping-Chang

    2016-01-01

    The etiology and the underlying mechanism of CD4+ T-cell polarization are unclear. This study sought to investigate the mechanism by which interleukin (IL)-13 prevents the activation-induced apoptosis of CD4+ T cells. Here we report that CD4+ T cells expressed IL-13 receptor α2 in the intestine of sensitized mice. IL-13 suppressed both the activation-induced apoptosis of CD4+ T cells and the expression of p53 and FasL. Exposure to recombinant IL-13 inhibited activation-induced cell death (AICD) along with the expression of p53, caspase 3, and tumor necrosis factor-α in CD4+ T cells. Administration of an anti-IL-13 antibody enhanced the effect of specific immunotherapy on allergic inflammation in the mouse intestine, enforced the expression of p53 in intestinal CD4+ T cells, and enhanced the frequency of CD4+ T-cell apoptosis upon challenge with specific antigens. In summary, blocking IL-13 enhances the therapeutic effect of antigen-specific immunotherapy by regulating apoptosis and thereby enforcing AICD in CD4+ T cells. PMID:26189367

  5. Influence of autologous dendritic cells on cytokine-induced killer cell proliferation, cell phenotype and antitumor activity in vitro

    PubMed Central

    Cao, Jingsong; Chen, Cong; Wang, Yuhuan; Chen, Xuecheng; Chen, Zeying; Luo, Xiaoling

    2016-01-01

    Dendritic cell (DCs) are essential antigen processing and presentation cells that play a key role in the immune response. In this study, DCs were co-cultured with cytokine-induced killer cells (DC-CIKs) in vitro to detect changes in cell proliferation, cell phenotype and cell cytotoxicity. The results revealed that the DCs were suitable for co-culture with CIKs at day 7, and that cell quantity of DC-CIKs was lower than that of CIKs until day 11, but it was significantly improved to 1.17-fold that of CIKs at day 13. Flow cytometry was used to detect the cell phenotype of CIKs and DC-CIKs. Compared with CIKs at day 13, the percentage of CD3+, CD3+CD4+, CD3+CD8+ and CD3+CD56+ T cells in DC-CIKs was significantly improved 1.02, 1.79, 1.26 and 2.44-fold, respectively. In addition, trypan blue staining analysis demonstrated that the cell viability of CIKs and DC-CIKs was 96% and 98%, respectively. Furthermore, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) analysis verified that CIK and DC-CIK cytotoxicity in Hela cells was 58% and 80%, respectively, with a significant difference. Taken together, our results indicate that the cell proliferation, cell phenotype and antitumor activity of CIKs were all enhanced following co-culture with DCs in vitro. These results are likely to be useful for DC-CIK application in antitumor therapies. PMID:27602134

  6. Influence of autologous dendritic cells on cytokine-induced killer cell proliferation, cell phenotype and antitumor activity in vitro

    PubMed Central

    Cao, Jingsong; Chen, Cong; Wang, Yuhuan; Chen, Xuecheng; Chen, Zeying; Luo, Xiaoling

    2016-01-01

    Dendritic cell (DCs) are essential antigen processing and presentation cells that play a key role in the immune response. In this study, DCs were co-cultured with cytokine-induced killer cells (DC-CIKs) in vitro to detect changes in cell proliferation, cell phenotype and cell cytotoxicity. The results revealed that the DCs were suitable for co-culture with CIKs at day 7, and that cell quantity of DC-CIKs was lower than that of CIKs until day 11, but it was significantly improved to 1.17-fold that of CIKs at day 13. Flow cytometry was used to detect the cell phenotype of CIKs and DC-CIKs. Compared with CIKs at day 13, the percentage of CD3+, CD3+CD4+, CD3+CD8+ and CD3+CD56+ T cells in DC-CIKs was significantly improved 1.02, 1.79, 1.26 and 2.44-fold, respectively. In addition, trypan blue staining analysis demonstrated that the cell viability of CIKs and DC-CIKs was 96% and 98%, respectively. Furthermore, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) analysis verified that CIK and DC-CIK cytotoxicity in Hela cells was 58% and 80%, respectively, with a significant difference. Taken together, our results indicate that the cell proliferation, cell phenotype and antitumor activity of CIKs were all enhanced following co-culture with DCs in vitro. These results are likely to be useful for DC-CIK application in antitumor therapies.

  7. Porphyromonas gingivalis Lipopolysaccharide Induced Proliferation and Activation of Natural Killer Cells in Vivo.

    PubMed

    Wang, Yuhua; Zhang, Wei; Xu, Li; Jin, Jun-O

    2016-01-01

    Porphyromonas gingivalis (P. gingivalis) lipopolysaccharide (LPS) promoted different innate immune activation than that promoted by Escherichia coli (E. coli) LPS. In this study, we examined the effect of P. gingivalis LPS on the proliferation and activation of natural killer (NK) cells in vivo and compared that function with that of E. coli LPS. Administration of P. gingivalis LPS to C57BL/6 mice induced stronger proliferation of NK cells in the spleen and submandibular lymph nodes (sLNs) and increased the number of circulating NK cells in blood compared to those treated with E. coli LPS. However, P. gingivalis LPS did not induce interferon-gamma (IFN-γ) production and CD69 expression in the spleen and sLN NK cells in vivo, and this was attributed to the minimal activation of the spleen and sLN dendritic cells (DCs), including low levels of co-stimulatory molecule expression and pro-inflammatory cytokine production. Furthermore, P. gingivalis LPS-treated NK cells showed less cytotoxic activity against Yac-1 target cells than E. coli LPS-treated NK cells. Hence, these data demonstrated that P. gingivalis LPS promoted limited activation of spleen and sLN NK cells in vivo, and this may play a role in the chronic inflammatory state observed in periodontal disease. PMID:27548133

  8. Antiproliferative and apoptosis-inducing activity of Brucea javanica extract on human carcinoma cells.

    PubMed

    Lau, Fung Yi; Chui, Chung Hin; Gambari, Roberto; Kok, Stanton Hon Lung; Kan, Kin Luen; Cheng, Gregory Yin Ming; Wong, Raymond Siu Ming; Teo, Ivy Tuang Ngo; Cheng, Chor Hing; Wan, Thomas Shek Kwong; Chan, Albert Sun Chi; Tang, Johnny Cheuk On

    2005-12-01

    We have recently demonstrated the antiproliferative and apoptotic activities of herbal traditional Chinese medicines, including the analomous fruit extract of Gleditsia sinensis, the fresh juice of Scutellaria barbata and the warmed water extract of Radix Sophorae Tonkinensis on a series of human carcinoma cells. Here, we further report the potential anti-cancer activity of the warmed water extract of Brucea javanica (BJE). Four cancer cell lines, including A549 non-small cell lung cancer, Hep3B hepatocellular carcinoma, MDA-MB231 breast cancer and SLMT-1 oesophageal squamous cell carcinoma, were incubated with BJE and strong apoptotic induction was observed under inverted microscopic investigation for all of the four cell lines tested. Using the MDA-MB231 breast cancer cell line as an experimental model, additional analyses supported the hypothesis that the mitochondrial membrane potential depolarization pathway was induced by BJE. The APO-1/Fas receptor death induction pathway was not activated under the influence of BJE, as studied by staining with Fas ligand and Fas receptor specific antibodies. Accordingly, only weak activation of caspase 8 was observed upon BJE treatment. On the other hand, caspase 3 activity was stimulated up to five-fold in BJE-treated cells compared to untreated controls. Oligonucleosomal DNA fragmentation formation was detected by labelling the nucleic acid ladders with TdT-mediated dUTP nick end labelling. Collectively, BJE-induced cancer cell death proceeds through a mitochondrial dependent pathway associated with caspase 3 activation. PMID:16273300

  9. Apoptosome activation, an important molecular instigator in 6-mercaptopurine induced Leydig cell death

    PubMed Central

    Morgan, Jessica A.; Lynch, John; Panetta, John C.; Wang, Yao; Frase, Sharon; Bao, Ju; Zheng, Jie; Opferman, Joseph T.; Janke, Laura; Green, Daniel M.; Chemaitilly, Wassim; Schuetz, John D.

    2015-01-01

    Leydig cells are crucial to the production of testosterone in males. It is unknown if the cancer chemotherapeutic drug, 6-mercaptopurine (6 MP), produces Leydig cell failure among adult survivors of childhood acute lymphoblastic leukemia. Moreover, it is not known whether Leydig cell failure is due to either a loss of cells or an impairment in their function. Herein, we show, in a subset of childhood cancer survivors, that Leydig cell failure is related to the dose of 6 MP. This was extended, in a murine model, to demonstrate that 6 MP exposure induced caspase 3 activation, and the loss of Leydig cells was independent of Bak and Bax activation. The death of these non-proliferating cells was triggered by 6 MP metabolism, requiring formation of both cytosolic reactive oxygen species and thiopurine nucleotide triphosphates. The thiopurine nucleotide triphosphates (with physiological amounts of dATP) uniquely activated the apoptosome. An ABC transporter (Abcc4/Mrp4) reduced the amount of thiopurines, thereby providing protection for Leydig cells. The studies reported here demonstrate that the apoptosome is uniquely activated by thiopurine nucleotides and suggest that 6 MP induced Leydig cell death is likely a cause of Leydig cell failure in some survivors of childhood cancer. PMID:26576726

  10. Cisplatin induces production of reactive oxygen species via NADPH oxidase activation in human prostate cancer cells.

    PubMed

    Itoh, Tomohiro; Terazawa, Riyako; Kojima, Keitaro; Nakane, Keita; Deguchi, Takashi; Ando, Masashi; Tsukamasa, Yasuyuki; Ito, Masafumi; Nozawa, Yoshinori

    2011-09-01

    This study aimed to examine the roles of reactive oxygen species (ROS) in cisplatin treatment of human prostate cancer cells; hormone-sensitive LNCaP and hormone-refractory PC3 and DU145 cells. Intracellular levels of ROS and H(2)O(2) were measured and visualized using specific fluorescent probes. NADPH oxidase (NOX) activity was detected by lucigenin chemiluminescence assay. Expression levels of NOX isoforms were determined by semi-quantitative RT-PCR. Cisplatin treatment increased the intracellular levels of ROS and H(2)O(2) in three prostate cancer cell lines. The increase was transient and robust in hormone-sensitive LNCaP cells compared with hormone-refractory PC3 and DU145 cells. Consistent with these findings, the NOX activity induced by cisplatin was higher in LNCaP cells than in PC3 and DU145 cells. Expression pattern of NOX isoforms varied among three cell lines and the NOX activity was independent of NOX expression. Taken together, we have shown that cisplatin induces production of ROS and H(2)O(2) via NOX activation in human prostate cancer cell lines, which is most prominent in hormone-sensitive LNCaP cells. PMID:21682664

  11. Morphine Modulates Interleukin-4- or Breast Cancer Cell-induced Pro-metastatic Activation of Macrophages.

    PubMed

    Khabbazi, Samira; Goumon, Yannick; Parat, Marie-Odile

    2015-01-01

    Interactions between cancer cells and stromal cells in the tumour microenvironment play a key role in the control of invasiveness, metastasis and angiogenesis. Macrophages display a range of activation states in specific pathological contexts and alternatively activated (M2) macrophages can promote tumour aggressiveness. Opioids are able to modulate tumour growth and metastasis. We tested whether morphine modulates the activation of macrophages induced by (i) interleukin-4 (IL-4), the prototypical M2 polarization-inducing cytokine, or (ii) coculture with breast cancer cells. We showed that IL-4 causes increased MMP-9 production and expression of the alternative activation markers arginase-1 and MRC-1. Morphine prevented IL-4-induced increase in MMP-9 in a naloxone- and methylnaltrexone-reversible fashion. Morphine also prevented IL-4-elicited alternative activation of RAW264.7 macrophages. Expression of MMP-9 and arginase-1 were increased when RAW264.7 were subjected to paracrine activation by 4T1 cells, and this effect was prevented by morphine via an opioid receptor-mediated mechanism. Morphine further decreased 4T1 breast cancer cell invasion elicited by co-culture with RAW264.7. Reduction of MMP-9 expression and alternative activation of macrophages by morphine was confirmed using mouse bone marrow-derived macrophages. Taken together, our results indicate that morphine may modulate tumour aggressiveness by regulating macrophage protease production and M2 polarization within the tumour microenvironment.

  12. TGF-{beta}2 inhibits AKT activation and FGF-2-induced corneal endothelial cell proliferation

    SciTech Connect

    Lu Jiawei; Lu Zhenyu; Reinach, Peter

    2006-11-01

    The corneal endothelial cells form a boundary layer between anterior chamber and cornea. This single cell layer is important to maintain cornea transparency by eliciting net fluid transport into the anterior chamber. Injuries of the corneal endothelial layer in humans lead to corneal swelling and translucence. This hindrance is thought to be due to limited proliferative capacity of the endothelial layer. Fibroblast growth factor 2 (FGF-2) and transforming growth factor-beta 2 (TGF-{beta}2) are both found in aqueous humor, and these two cytokines promote and inhibit cell growth, respectively. The intracellular signaling mechanisms by which TGF-{beta}2 suppresses the mitogenic response to FGF-2, however, remain unclear. We have addressed this question by investigating potential crosstalk between FGF-2-induced and TGF-{beta}2-regulated intracellular signaling events in cultured bovine corneal endothelial (BCE) cells. We found that TGF-{beta}2 and FGF-2 oppositely affect BCE cell proliferation and TGF-{beta}2 can override the stimulating effects of FGF-2 by increasing COX-2 expression in these cells. Consistent with these findings, overexpression of COX-2 significantly reduced FGF-2-induced cell proliferation whereas a COX-2 specific inhibitor NS398 reversed the effect of TGF-{beta}2 on FGF-2-induced cell proliferation. The COX-2 product prostaglandin E2 (PGE-2) blocks FGF-2-induced cell proliferation. Whereas FGF-2 stimulates cell proliferation by activating the AKT pathway, TGF-{beta}2 and PGE-2 both inhibit this pathway. In accordance with the effect of PGE-2, cAMP also inhibits FGF-2-induced AKT activation. These findings suggest that the mitogenic response to FGF-2 in vivo in the corneal endothelial layer may be inhibited by TGF-{beta}2-induced suppression of the PI3-kinase/AKT signaling pathway.

  13. Activation-Induced Cytidine Deaminase Expression in Human B Cell Precursors Is Essential for Central B Cell Tolerance.

    PubMed

    Cantaert, Tineke; Schickel, Jean-Nicolas; Bannock, Jason M; Ng, Yen-Shing; Massad, Christopher; Oe, Tyler; Wu, Renee; Lavoie, Aubert; Walter, Jolan E; Notarangelo, Luigi D; Al-Herz, Waleed; Kilic, Sara Sebnem; Ochs, Hans D; Nonoyama, Shigeaki; Durandy, Anne; Meffre, Eric

    2015-11-17

    Activation-induced cytidine deaminase (AID), the enzyme-mediating class-switch recombination (CSR) and somatic hypermutation (SHM) of immunoglobulin genes, is essential for the removal of developing autoreactive B cells. How AID mediates central B cell tolerance remains unknown. We report that AID enzymes were produced in a discrete population of immature B cells that expressed recombination-activating gene 2 (RAG2), suggesting that they undergo secondary recombination to edit autoreactive antibodies. However, most AID+ immature B cells lacked anti-apoptotic MCL-1 and were deleted by apoptosis. AID inhibition using lentiviral-encoded short hairpin (sh)RNA in B cells developing in humanized mice resulted in a failure to remove autoreactive clones. Hence, B cell intrinsic AID expression mediates central B cell tolerance potentially through its RAG-coupled genotoxic activity in self-reactive immature B cells.

  14. Basal and inducible anti-inflammatory epoxygenase activity in endothelial cells

    SciTech Connect

    Askari, Ara A.; Thomson, Scott; Edin, Matthew L.; Lih, Fred B.; Zeldin, Darryl C.; Bishop-Bailey, David

    2014-04-04

    Highlights: • We examined epoxygenase product formation and regulation in endothelial cells. • The epoxygenase CYP2J2 is an LPS (TLR-4) inducible enzyme in endothelial cells. • The endothelial cell line EA.Hy926 synthesises epoxygenase products. • Inhibition of endothelial epoxygenases increases TNFα secretion. • Soluble epoxide hydrolase inhibitors reduce inflammation-induced TNFα and NFκB. - Abstract: The roles of CYP lipid-metabolizing pathways in endothelial cells are poorly understood. Human endothelial cells expressed CYP2J2 and soluble epoxide hydrolase (sEH) mRNA and protein. The TLR-4 agonist LPS (1 μg/ml; 24 h) induced CYP2J2 but not sEH mRNA and protein. LC–MS/MS analysis of the stable commonly used human endothelial cell line EA.Hy926 showed active epoxygenase and epoxide hydrolase activity: with arachidonic acid (stable epoxide products 5,6-DHET, and 14,15-DHET), linoleic acid (9,10-EPOME and 12,13-EPOME and their stable epoxide hydrolase products 9,10-DHOME and 12,13-DHOME), docosahexaenoic acid (stable epoxide hydrolase product 19,20-DiHDPA) and eicosapentaenoic acid (stable epoxide hydrolase product 17,18-DHET) being formed. Inhibition of epoxygenases using either SKF525A or MS-PPOH induced TNFα release, but did not affect LPS, IL-1β, or phorbol-12-myristate-13-acetate (PMA)-induced TNFα release. In contrast, inhibition of soluble epoxide hydrolase by AUDA or TPPU inhibited basal, LPS, IL-1β and PMA induced TNFα release, and LPS-induced NFκB p65 nuclear translocation. In conclusion, human endothelial cells contain a TLR-4 regulated epoxygenase CYP2J2 and metabolize linoleic acid > eicosapentaenoic acid > arachidonic acid > docosahexaenoic acid to products with anti-inflammatory activity.

  15. Nuclear Factor of Activated T Cells Transcription Factor Nfatp Controls Superantigen-Induced Lethal Shock

    PubMed Central

    Tsytsykova, Alla V.; Goldfeld, Anne E.

    2000-01-01

    Tumor necrosis factor α (TNF-α) is the key mediator of superantigen-induced T cell lethal shock. Here, we show that nuclear factor of activated T cells transcription factor, NFATp, controls susceptibility to superantigen-induced lethal shock in mice through its activation of TNF-α gene transcription. In NFATp-deficient mice, T cell stimulation leads to delayed induction and attenuation of TNF-α mRNA levels, decreased TNF-α serum levels, and resistance to superantigen-induced lethal shock. By contrast, after lipopolysaccharide (LPS) challenge, serum levels of TNF-α and susceptibility to shock are unaffected. These results demonstrate that NFATp is an essential activator of immediate early TNF-α gene expression in T cells and they present in vivo evidence of the inducer- and cell type–specific regulation of TNF-α gene expression. Furthermore, they suggest NFATp as a potential selective target in the treatment of superantigen-induced lethal shock. PMID:10952728

  16. Neferine induces autophagy of human ovarian cancer cells via p38 MAPK/ JNK activation.

    PubMed

    Xu, Limei; Zhang, Xiyu; Li, Yinuo; Lu, Shuhua; Lu, Shan; Li, Jieyin; Wang, Yuqiong; Tian, Xiaoxue; Wei, Jian-Jun; Shao, Changshun; Liu, Zhaojian

    2016-07-01

    Ovarian cancer is the most lethal gynecological malignancy. Patients usually have poor prognosis because of late diagnosis, relapse, and chemoresistance. It is pressing to seek novel agent for the treatment of ovarian cancer. Neferine is a bisbenzylisoquinoline alkaloid isolated from the embryos of lotus (Nelumbo nucifera). In this study, we investigated the antitumor effect of neferine on ovarian cancer cells. We found that neferine exhibited growth-inhibitory effect on human ovarian cancer cells, whereas showing less cytotoxic to non-malignant fallopian tube epithelial cells. Furthermore, we demonstrated that neferine induced autophagy and inactivated the mTOR pathway. Finally, we found that both p38 MAPK and JNK signaling pathways were activated by neferine treatment and contributed to the induction of autophagy in ovarian cancer cells. In conclusion, our findings showed that neferine induced autophagy of human ovarian cancer cells via p38 MAPK/JNK activation. Neferine may be explored as a promising antitumoral agent in ovarian cancer. PMID:26738868

  17. Light-activated hypericin induces cellular destruction of nasopharyngeal carcinoma cells

    NASA Astrophysics Data System (ADS)

    Xu, C. S.; Leung, A. W. N.

    2010-01-01

    Hypericin from Hypericum perforatum plants shows an important promise in the photodynamic therapy on malignant tumor. The present study investigated that light-activated hypericin induced the cellular destruction of nasopharyngeal carcinoma cells. The result showed that hypericin resulted in a drug- and light-dose dependent cytotoxicity in the CNE-2 cells, meaning the photocytotoxicity of hypericin depends on both of the drug concentration (0 - 2.5 μM) and light-doses (1 - 8 J/cm2). We further investigated the apoptosis of the CNE-2 cells 8 hours after photosensitization of hypericin using fluorescence microscopy with Hoechst 33258 staining. Flow cytometry with annexin V-FITC and PI staining was used to analyze early and late apoptosis. These data demonstrated that light-activated hypericin could significantly lead to the cellular destruction of the CNE-2 cells and induce early apoptosis as a prominent mode of cell death.

  18. Salidroside Suppresses HUVECs Cell Injury Induced by Oxidative Stress through Activating the Nrf2 Signaling Pathway.

    PubMed

    Zhu, Yao; Zhang, Ya-Jie; Liu, Wei-Wei; Shi, Ai-Wu; Gu, Ning

    2016-01-01

    Oxidative stress plays an important role in the pathogenesis of cardiovascular diseases. Salidroside (SAL), one of the main effective constituents of Rhodiola rosea, has been reported to suppress oxidative stress-induced cardiomyocyte injury and necrosis by promoting transcription of nuclear factor E2-related factor 2 (Nrf2)-regulated genes such as heme oxygenase-1 (HO-1) and NAD(P)H dehydrogenase (quinone1) (NQO1). However, it has not been indicated whether SAL might ameliorate endothelial injury induced by oxidative stress. Here, our study demonstrated that SAL might suppress HUVEC cell injury induced by oxidative stress through activating the Nrf2 signaling pathway. The results of our study indicated that SAL decreased the levels of intercellular reactive oxygen species (ROS) and malondialdehyde (MDA), and improved the activities of superoxide dismutase (SOD) and catalase (CAT), resulting in protective effects against oxidative stress-induced cell damage in HUVECs. It suppressed oxidative stress damage by inducing Nrf2 nuclear translocation and activating the expression of Nrf2-regulated antioxidant enzyme genes such as HO-1 and NQO1 in HUVECs. Knockdown of Nrf2 with siRNA abolished the cytoprotective effects against oxidative stress, decreased the expression of Nrf2, HO-1, and NQO1, and inhibited the nucleus translocation of Nrf2 in HUVECs. This study is the first to demonstrate that SAL suppresses HUVECs cell injury induced by oxidative stress through activating the Nrf2 signaling pathway. PMID:27517893

  19. Salidroside Suppresses HUVECs Cell Injury Induced by Oxidative Stress through Activating the Nrf2 Signaling Pathway.

    PubMed

    Zhu, Yao; Zhang, Ya-Jie; Liu, Wei-Wei; Shi, Ai-Wu; Gu, Ning

    2016-01-01

    Oxidative stress plays an important role in the pathogenesis of cardiovascular diseases. Salidroside (SAL), one of the main effective constituents of Rhodiola rosea, has been reported to suppress oxidative stress-induced cardiomyocyte injury and necrosis by promoting transcription of nuclear factor E2-related factor 2 (Nrf2)-regulated genes such as heme oxygenase-1 (HO-1) and NAD(P)H dehydrogenase (quinone1) (NQO1). However, it has not been indicated whether SAL might ameliorate endothelial injury induced by oxidative stress. Here, our study demonstrated that SAL might suppress HUVEC cell injury induced by oxidative stress through activating the Nrf2 signaling pathway. The results of our study indicated that SAL decreased the levels of intercellular reactive oxygen species (ROS) and malondialdehyde (MDA), and improved the activities of superoxide dismutase (SOD) and catalase (CAT), resulting in protective effects against oxidative stress-induced cell damage in HUVECs. It suppressed oxidative stress damage by inducing Nrf2 nuclear translocation and activating the expression of Nrf2-regulated antioxidant enzyme genes such as HO-1 and NQO1 in HUVECs. Knockdown of Nrf2 with siRNA abolished the cytoprotective effects against oxidative stress, decreased the expression of Nrf2, HO-1, and NQO1, and inhibited the nucleus translocation of Nrf2 in HUVECs. This study is the first to demonstrate that SAL suppresses HUVECs cell injury induced by oxidative stress through activating the Nrf2 signaling pathway.

  20. Activation of peroxisome proliferator-activated receptor-{gamma} (PPAR{gamma}) induces cell death through MAPK-dependent mechanism in osteoblastic cells

    SciTech Connect

    Kim, Sung Hun; Yoo, Chong Il; Kim, Hui Taek; Park, Ji Yeon; Kwon, Chae Hwa; Keun Kim, Yong . E-mail: kim430@pusan.ac.kr

    2006-09-01

    The present study was undertaken to determine the role of the mitogen-activated protein kinase (MAPK) subfamilies in cell death induced by PPAR{gamma} agonists in osteoblastic cells. Ciglitazone and troglitazone, PPAR{gamma} agonists, resulted in a concentration- and time-dependent cell death, which was largely attributed to apoptosis. But a PPAR{alpha} agonist ciprofibrate did not affect the cell death. Ciglitazone caused reactive oxygen species (ROS) generation and ciglitazone-induced cell death was prevented by antioxidants, suggesting an important role of ROS generation in the ciglitazone-induced cell death. ROS generation and cell death induced by ciglitazone were inhibited by the PPAR{gamma} antagonist GW9662. Ciglitazone treatment caused activation of extracellular signal-regulated kinase (ERK) and p38. Activation of ERK was dependent on epidermal growth factor receptor (EGFR) and that of p38 was independent. Ciglitazone-induced cell death was significantly prevented by PD98059, an inhibitor of ERK upstream kinase MEK1/2, and SB203580, a p38 inhibitor. Ciglitazone treatment increased Bax expression and caused a loss of mitochondrial membrane potential, and its effect was prevented by N-acetylcysteine, PD98059, and SB203580. Ciglitazone induced caspase activation, which was prevented by PD98059 and SB203580. The general caspase inhibitor z-DEVD-FMK and the specific inhibitor of caspases-3 DEVD-CHO exerted the protective effect against the ciglitazone-induced cell death. The EGFR inhibitors AG1478 and suramin protected against the ciglitazone-induced cell death. Taken together, these findings suggest that the MAPK signaling pathways play an active role in mediating the ciglitazone-induced cell death of osteoblasts and function upstream of a mitochondria-dependent mechanism. These data may provide a novel insight into potential therapeutic strategies for treatment of osteoporosis.

  1. Th-1 lymphocytes induce dendritic cell tumor killing activity by an IFN-γ-dependent mechanism.

    PubMed

    LaCasse, Collin J; Janikashvili, Nona; Larmonier, Claire B; Alizadeh, Darya; Hanke, Neale; Kartchner, Jessica; Situ, Elaine; Centuori, Sara; Har-Noy, Michael; Bonnotte, Bernard; Katsanis, Emmanuel; Larmonier, Nicolas

    2011-12-15

    Dendritic cells (DCs) encompass a heterogeneous population of cells capable of orchestrating innate and adaptive immune responses. The ability of DCs to act as professional APCs has been the foundation for the development and use of these cells as vaccines in cancer immunotherapy. DCs are also endowed with the nonconventional property of directly killing tumor cells. The current study investigates the regulation of murine DC cytotoxic function by T lymphocytes. We provide evidence that CD4(+) Th-1, but not Th-2, Th-17 cells, or regulatory T cells, are capable of inducing DC cytotoxic function. IFN-γ was identified as the major factor responsible for Th-1-induced DC tumoricidal activity. Tumor cell killing mediated by Th-1-activated killer DCs was dependent on inducible NO synthase expression and NO production. Importantly, Th-1-activated killer DCs were capable of presenting the acquired Ags from the killed tumor cells to T lymphocytes in vitro or in vivo. These observations offer new possibilities for the application of killer DCs in cancer immunotherapy.

  2. Platelet-Activating Factor Induces Epigenetic Modifications in Human Mast Cells

    PubMed Central

    Gorbea, Enrique; Ullrich, Stephen E.

    2015-01-01

    Ultraviolet (UV) radiation-induced systemic immune suppression is a major risk factor for skin cancer induction. The migration of dermal mast cells from the skin to the draining lymph nodes plays a prominent role in activating systemic immune suppression. UV-induced keratinocyte-derived platelet-activating factor (PAF) activates mast cell migration, in part by up regulating the expression of CXCR4 on the surface of mast cells. Others have indicated that epigenetic mechanisms regulate CXCR4 expression, so we asked whether PAF activates epigenetic mechanisms in mast cells. Human mast cells were treated with PAF and the effect on DNA methylation and/or acetylation was measured. PAF suppressed the expression of DNA methyltransferase (DNMT) 1 and 3b. On the other hand, PAF increased p300 histone acetyltransferase expression, and the acetylation of histone H3, which coincided with a decreased expression of the histone deacetylase HDAC2. Chromatin immunoprecipitation assays indicated that PAF-treatment activated the acetylation of the CXCR4 promoter. Finally, inhibiting histone acetylation blocked p300 up-regulation and suppressed PAF-induced surface expression of CXCR4. Our findings suggest a novel molecular mechanism for PAF, activation of epigenetic modifications. We suggest that PAF may serve as an endogenous molecular mediator that links the environment (UV radiation) with the epigenome. PMID:26316070

  3. Monocarboxylate transporter 1 contributes to growth factor-induced tumor cell migration independent of transporter activity

    PubMed Central

    Gray, Alana L.; Coleman, David T.; Shi, Runhua; Cardelli, James A.

    2016-01-01

    Tumor progression to metastatic disease contributes to the vast majority of incurable cancer. Understanding the processes leading to advanced stage cancer is important for the development of future therapeutic strategies. Here, we establish a connection between tumor cell migration, a prerequisite to metastasis, and monocarboxylate transporter 1 (MCT1). MCT1 transporter activity is known to regulate aspects of tumor progression and, as such, is a clinically relevant target for treating cancer. Knockdown of MCT1 expression caused decreased hepatocyte growth factor (HGF)-induced as well as epidermal growth factor (EGF)-induced tumor cell scattering and wound healing. Western blot analysis suggested that MCT1 knockdown (KD) hinders signaling through the HGF receptor (c-Met) but not the EGF receptor. Exogenous, membrane-permeable MCT1 substrates were not able to rescue motility in MCT1 KD cells, nor was pharmacologic inhibition of MCT1 able to recapitulate decreased cell motility as seen with MCT1 KD cells, indicating transporter activity of MCT1 was dispensable for EGF- and HGF-induced motility. These results indicate MCT1 expression, independent of transporter activity, is required for growth factor-induced tumor cell motility. The findings presented herein suggest a novel function for MCT1 in tumor progression independent of its role as a monocarboxylate transporter. PMID:27127175

  4. Euphorbia mauritanica and Kedrostis hirtella extracts can induce anti-proliferative activities in lung cancer cells.

    PubMed

    Thafeni, Makhosazana A; Sayed, Yasien; Motadi, Lesetja R

    2012-12-01

    Cancer is a public health problem in the world accounting for most of the deaths. Currently, common treatment of cancer such as chemotherapy works by killing fast-growing cancer cells. Unfortunately, chemotherapy cannot tell the difference between cancer cells and fast-growing healthy cells, including red and white blood cells. As a result, one of the most serious potential side effects of some types of chemotherapy is a low white blood cell count that makes it unreliable (Parkin et al. [34]; Pauk et al. [3]). Even though intense research has been going on in recent years, successful therapeutic targets against this disease have been elusive. In this study, we evaluate the anti-proliferative activity of Euphorbia mauritanica and Kedrostis hirtella in lung cancer. In our assessment it was observed that E. mauritanica and K. hirtella were able to induce cell death at 5 μg/ml in A549 cells over 22 h and at 10 μg/ml over 24 h in the Lqr1 cell line. Molecular analysis of DNA fragmentation and Annexin V were used to examine the type of cell death induced by E. mauritanica and K. hirtella extracts. These results showed an increase in necrotic and apoptotic characteristics with both nuclear DNA fragmentation and smear. Therefore, these results suggest that E. mauritanica and K. hirtella may play a role in inducing cell death in lung cancer cells. However, further studies need to be conducted to ascertain these results. PMID:23086267

  5. Lipopolysaccharide induces multinuclear cell from RAW264.7 line with increased phagocytosis activity

    SciTech Connect

    Nakanishi-Matsui, Mayumi; Yano, Shio; Matsumoto, Naomi; Futai, Masamitsu

    2012-08-24

    Highlights: Black-Right-Pointing-Pointer LPS induces multinuclear cells from murine macrophage-derived RAW264.7 cells. Black-Right-Pointing-Pointer The multinuclear cells are formed through cell-cell fusion in the presence of Ca{sup 2+}. Black-Right-Pointing-Pointer The multinuclear cells do not express osteoclast-specific enzymes. Black-Right-Pointing-Pointer They internalized more and larger beads than mononuclear cells and osteoclasts. -- Abstract: Lipopolysaccharide (LPS), an outer membrane component of Gram-negative bacteria, induces strong proinflammatory responses, including the release of cytokines and nitric oxide from macrophage. In this study, we found that a murine macrophage-derived line, RAW264.7, became multinuclear through cell-cell fusion after incubation with highly purified LPS or synthetic lipid A in the presence of Ca{sup 2+}. The same cell line is known to differentiate into multinuclear osteoclast, which expresses a specific proton pumping ATPase together with osteoclast markers on stimulation by the extracellular domain of receptor activator of nuclear factor {kappa}B ligand (Toyomura, T., Murata, Y., Yamamoto, A., Oka, T., Sun-Wada, G.-H., Wada, Y. and Futai, M., 2003). The LPS-induced multinuclear cells did not express osteoclast-specific enzymes including tartrate-resistant acid phosphatase and cathepsin K. During multinuclear cell formation, the cells internalized more and larger polystyrene beads (diameter 6-15 {mu}m) than mononuclear cells and osteoclasts. The internalized beads were located in lysosome-marker positive organelles, which were probably phagolysosomes. The LPS-induced multinuclear cell could be a good model system to study phagocytosis of large foreign bodies.

  6. TRPV1 Activation in Primary Cortical Neurons Induces Calcium-Dependent Programmed Cell Death.

    PubMed

    Song, Juhyun; Lee, Jun Hong; Lee, Sung Ho; Park, Kyung Ah; Lee, Won Taek; Lee, Jong Eun

    2013-03-01

    Transient receptor potential cation channel, subfamily V, member 1 (TRPV1, also known as vanilloid receptor 1) is a receptor that detects capsaicin, a pungent component of chili peppers, and noxious heat. Although its function in the primary nociceptor as a pain receptor is well established, whether TRPV1 is expressed in the brain is still under debate. In this study, the responses of primary cortical neurons were investigated. Here, we report that 1) capsaicin induces caspase-3-dependent programmed cell death, which coincides with increased production of nitric oxide and peroxynitrite ; that 2) the prolonged capsaicin treatment induces a steady increase in the degree of capase-3 activation, which is prevented by the removal of capsaicin; 3) and that blocking calcium entry and calcium-mediated signaling prevents capsaicin-induced cell death. These results indicate that cortical neurons express TRPV1 whose prolonged activation causes cell death. PMID:23585723

  7. Primary WWOX phosphorylation and JNK activation during etoposide induces cytotoxicity in HEK293 cells

    PubMed Central

    Jamshidiha, M.; Habibollahi, P.; Ostad, S.N.; Ghahremani, M.H.

    2010-01-01

    Background and the purpose of the study Etoposide is an antineoplastic agent used in multiple cancers. It is known that etoposide induce cell death via interaction with topoisomerase II; however, the etopoisde cellular response is poorly understood. Upon etoposide induced DNA damage, many stress signaling pathways including JNK are activated. In response to DNA damage, it has been shown that WWOX, a recently introduced tumor suppressor, can be activated. In this study the activation of WWOX and JNK and their interaction following etoposide treatment were evaluated. Materials and Methods HEK293 cells treated with etoposide were lysed in a time course manner. The whole cell lysates were used to evaluate JNK and WWOX activation pattern using Phospho specific antibodies on western blots. The viability of cells treated with etoposide, JNK specific inhibitor and their combination was examined using MTT assay. Results Findings of this study indicate that WWOX and JNK are activated in a simultaneous way in response to DNA damage. Moreover, JNK inhibition enhances etoposide induced cytotoxicity in HEK293. Conclusion Taken together, our results indicate that etoposide induces cytotoxicity and WWOX phosphorylation and the cytotoxicty is augmented by blocking JNK pathway. PMID:22615609

  8. Lipopolysaccharide (LPS)-Induced Biliary Epithelial Cell NRas Activation Requires Epidermal Growth Factor Receptor (EGFR).

    PubMed

    Trussoni, Christy E; Tabibian, James H; Splinter, Patrick L; O'Hara, Steven P

    2015-01-01

    Cholangiocytes (biliary epithelial cells) actively participate in microbe-induced proinflammatory responses in the liver and contribute to inflammatory and infectious cholangiopathies. We previously demonstrated that cholangiocyte TLR-dependent NRas activation contributes to proinflammatory/ proliferative responses. We test the hypothesis that LPS-induced activation of NRas requires the EGFR. SV40-transformed human cholangiocytes (H69 cells), or low passage normal human cholangiocytes (NHC), were treated with LPS in the presence or absence of EGFR or ADAM metallopeptidase domain 17 (TACE) inhibitors. Ras activation assays, quantitative RT-PCR, and proliferation assays were performed in cells cultured with or without inhibitors or an siRNA to Grb2. Immunofluorescence for phospho-EGFR was performed on LPS-treated mouse samples and specimens from patients with primary sclerosing cholangitis, primary biliary cirrhosis, hepatitis C, and normal livers. LPS-treatment induced an association between the TLR/MyD88 and EGFR/Grb2 signaling apparatus, NRas activation, and EGFR phosphorylation. NRas activation was sensitive to EGFR and TACE inhibitors and correlated with EGFR phosphorylation. The TACE inhibitor and Grb2 depletion prevented LPS-induced IL6 expression (p<0.05) and proliferation (p<0.01). Additionally, cholangiocytes from LPS-treated mouse livers and human primary sclerosing cholangitis (PSC) livers exhibited increased phospho-EGFR (p<0.01). Moreover, LPS-induced mouse cholangiocyte proliferation was inhibited by concurrent treatment with the EGFR inhibitor, Erlotinib. Our results suggest that EGFR is essential for LPS-induced, TLR4/MyD88-mediated NRas activation and induction of a robust proinflammatory cholangiocyte response. These findings have implications not only for revealing the signaling potential of TLRs, but also implicate EGFR as an integral component of cholangiocyte TLR-induced proinflammatory processes.

  9. Lack of p53 augments thymoquinone-induced apoptosis and caspase activation in human osteosarcoma cells.

    PubMed

    Roepke, Martin; Diestel, Antje; Bajbouj, Khuloud; Walluscheck, Diana; Schonfeld, Peter; Roessner, Albert; Schneider-Stock, Regine; Gali-Muhtasib, Hala

    2007-02-01

    We have recently shown that thymoquinone (TQ) is an antineoplastic drug that induces p53-dependent apoptosis in human colon cancer cells. This study evaluated the antiproliferative and pro-apoptotic effects of TQ in two human osteosarcoma cell lines with different p53 mutation status. TQ decreased cell survival dose-dependently and, more significantly, in p53-null MG63 cells (IC(50) = 17 muM) than in p53-mutant MNNG/HOS cells (IC(50) = 38 muM). Cell viability was reduced more selectively in MG63 tumor cells than in normal human osteoblasts. Flow cytometric analysis showed that TQ induced a much greater increase in the PreG(1) (apoptotic) cell population, but no cell cycle arrest in MG63. G(2)/M arrest in MNNG/HOS cells was associated with p21(WAF1) upregulation. Using three DNA damage assays, TQ was confirmed to result in a significantly greater extent of apoptosis in p53 null MG63 cells. Although the Bax/Bcl-2 ratios were not differentially modulated in both cell lines, the mitochondrial pathway appeared to be involved in TQ-induced apoptosis in MG63 by showing the cleavage of caspases-9 and -3. Oxidative stress and mitochondrial O(2)(*-) generation in isolated rat mitochondria were enhanced by TQ as measured by the dose-dependent reduction in aconitase enzyme activity and Amplex Red oxidation respectively. TQ-induced oxidative damage, reflected by an increase in gamma-H2AX foci and increased protein expression levels of gamma-H2AX and the DNA repair enzyme, NBS1, was more pronounced in MNNG/HOS than in MG63. We suggest that the resistance of MNNG/HOS cells to drug-induced apoptosis is caused by the up-regulation of p21(WAF1) by the mutant p53 (transcriptional activity was shown by p53 siRNA treatment) which induces cell cycle arrest and allows to repair DNA damage. Collectively, these findings show that TQ induces p53-independent apoptosis in human osteosarcoma cells. As the loss of p53 function is frequently observed in osteosarcoma patients, our data suggest

  10. Glucocorticoid Receptor Activation Inhibits Chemotherapy-induced Cell Death in High-grade Serous Ovarian Carcinoma

    PubMed Central

    Stringer-Reasor, Erica M.; Baker, Gabrielle M.; Skor, Maxwell N.; Kocherginsky, Masha; Lengyel, Ernst; Fleming, Gini F.; Conzen, Suzanne D.

    2015-01-01

    Objectives To test the hypothesis that glucocorticoid receptor (GR) activation increases resistance to chemotherapy in high-grade serous ovarian cancer (HGS-OvCa) and that treatment with a GR antagonist will improve sensitivity to chemotherapy. Methods GR expression was assessed in OvCa cell lines by qRT-PCR and Western blot analysis and in xenografts and primary human tumors using immunohistochemistry (IHC). We also examined the effect of GR activation versus inhibition on chemotherapy-induced cytotoxicity in OvCa cell lines and in a xenograft model. Results With the exception of IGROV-1 cells, all OvCa cell lines tested had detectable GR expression by Western blot and qRT-PCR analysis. Twenty-five out of the 27 human primary HGS-OvCas examined expressed GR by IHC. No cell line expressed detectable progesterone receptor (PR) or androgen receptor (AR) by Western blot analysis. In vitro assays showed that in GR-positive HeyA8 and SKOV3 cells, dexamethasone (100 nM) treatment upregulated the pro-survival genes SGK1 and MKP1/DUSP1 and inhibited carboplatin/gemcitabine-induced cell death. Concurrent treatment with two GR antagonists, either mifepristone (100 nM) or CORT125134 (100 nM), partially reversed these effects. There was no anti-apoptotic effect of dexamethasone on chemotherapy-induced cell death in IGROV-1 cells, which did not have detectable GR protein. Mifepristone treatment alone was not cytotoxic in any cell line. HeyA8 OvCa xenograft studies demonstrated that adding mifepristone to carboplatin/gemcitabine increased tumor shrinkage by 48% compared to carboplatin/gemcitabine treatment alone (P=0.0004). Conclusions These results suggest that GR antagonism sensitizes GR+ OvCa to chemotherapy-induced cell death through inhibition of GR-mediated cell survival pathways. PMID:26115975

  11. Dasatinib inhibits both osteoclast activation and prostate cancer PC-3 cell-induced osteoclast formation

    PubMed Central

    Araujo, John C.; Poblenz, Ann; Corn, Paul G.; Parikh, Nila U.; Starbuck, Michael W.; Thompson, Jerry T.; Lee, Francis; Logothetis, Christopher J.; Darnay, Bryant G.

    2013-01-01

    Purpose Therapies to target prostate cancer bone metastases have only limited effects. New treatments are focused on the interaction between cancer cells, bone marrow cells and the bone matrix. Osteoclasts play an important role in the development of bone tumors caused by prostate cancer. Since Src kinase has been shown to be necessary for osteoclast function, we hypothesized that dasatinib, a Src family kinase inhibitor, would reduce osteoclast activity and prostate cancer (PC-3) cell-induced osteoclast formation. Results Dasatinib inhibited RANKL-induced osteoclast differentiation of bone marrow-derived monocytes with an EC50 of 7.5 nM. PC-3 cells, a human prostate cancer cell line, were able to differentiate RAW 264.7 cells, a murine monocytic cell line, into osteoclasts and dasatinib inhibited this differentiation. In addition, conditioned medium from PC-3 cell cultures was able to differentiate RAW 264.7 cells into osteoclasts and this too, was inhibited by dasatinib. Even the lowest concentration of dasatinib, 1.25 nmol, inhibited osteoclast differentiation by 29%. Moreover, dasatinib inhibited osteoclast activity by 58% as measured by collagen 1 release. Experimental design We performed in vitro experiments utilizing the Src family kinase inhibitor dasatinib to target osteoclast activation as a means of inhibiting prostate cancer bone metastases. Conclusion Dasatinib inhibits osteoclast differentiation of mouse primary bone marrow-derived monocytes and PC-3 cell-induced osteoclast differentiation. Dasatinib also inhibits osteoclast degradation activity. Inhibiting osteoclast differentiation and activity may be an effective targeted therapy in patients with prostate cancer bone metastases. PMID:19855158

  12. Activated CD8+ T cells induce expansion of Vβ5+ regulatory T cells via TNFR2 signaling

    PubMed Central

    Joedicke, Jara J; Myers, Lara; Carmody, Aaron B; Messer, Ronald J; Wajant, Harald; Lang, Karl S; Lang, Philipp A; Mak, Tak W; Hasenkrug, Kim J; Dittmer, Ulf

    2014-01-01

    Vβ5+ regulatory T cells (Tregs), which are specific for a mouse endogenous retroviral superantigen, become activated and proliferate in response to Friend retrovirus (FV) infection. We previously reported that FV-induced expansion of this Treg subset was dependent on CD8+ T cells and TNFα, but independent of IL-2. We now show that the inflammatory milieu associated with FV infection is not necessary for induction of Vβ5+ Treg expansion. Rather, it is the presence of activated CD8+ T cells that is critical for their expansion. The data indicate that the mechanism involves signaling between the membrane-bound form of TNFα (memTNFα) on activated CD8+ T cells and TNF receptor 2 (TNFR2) on Tregs. CD8+ T cells expressing memTNFα but no soluble TNFα (solTNFα) remained competent to induce strong Vβ5+ Treg expansion in vivo. In addition, Vβ5+ Tregs expressing only TNFR2 but no TNFR1 were still responsive to expansion. Finally, treatment of naïve mice with solTNFα did not induce Vβ5+ Treg expansion, but treatment with a TNFR2-specific agonist did. These results reveal a new mechanism of intercellular communication between activated CD8+ T cell effectors and Tregs that results in the activation and expansion of a Treg subset that subsequently suppresses CD8+ T cell functions. PMID:25098294

  13. Cocaine induces cell death and activates the transcription nuclear factor kappa-B in PC12 cells.

    PubMed

    Lepsch, Lucilia B; Munhoz, Carolina D; Kawamoto, Elisa M; Yshii, Lidia M; Lima, Larissa S; Curi-Boaventura, Maria F; Salgado, Thais M L; Curi, Rui; Planeta, Cleopatra S; Scavone, Cristoforo

    2009-01-01

    Cocaine is a worldwide used drug and its abuse is associated with physical, psychiatric and social problems. The mechanism by which cocaine causes neurological damage is very complex and involves several neurotransmitter systems. For example, cocaine increases extracellular levels of dopamine and free radicals, and modulates several transcription factors. NF-kappaB is a transcription factor that regulates gene expression involved in cellular death. Our aim was to investigate the toxicity and modulation of NF-kappaB activity by cocaine in PC 12 cells. Treatment with cocaine (1 mM) for 24 hours induced DNA fragmentation, cellular membrane rupture and reduction of mitochondrial activity. A decrease in Bcl-2 protein and mRNA levels, and an increase in caspase 3 activity and cleavage were also observed. In addition, cocaine (after 6 hours treatment) activated the p50/p65 subunit of NF-kappaB complex and the pretreatment of the cells with SCH 23390, a D1 receptor antagonist, attenuated the NF-kappaB activation. Inhibition of NF-kappaB activity by using PDTC and Sodium Salicilate increased cell death caused by cocaine. These results suggest that cocaine induces cell death (apoptosis and necrosis) and activates NF-kappaB in PC12 cells. This activation occurs, at least partially, due to activation of D1 receptors and seems to have an anti-apoptotic effect on these cells. PMID:19183502

  14. Constitutive Activation of PI3K in Oocyte Induces Ovarian Granulosa Cell Tumors.

    PubMed

    Kim, So-Youn; Ebbert, Katherine; Cordeiro, Marilia H; Romero, Megan M; Whelan, Kelly A; Suarez, Adrian A; Woodruff, Teresa K; Kurita, Takeshi

    2016-07-01

    Cell-cell interactions play crucial roles in the maintenance of tissue homeostasis, a loss of which often leads to varying diseases, including cancer. Here, we report that uncontrolled PI3K activity within oocytes irreversibly transforms granulosa cells (GC), causing GC tumors (GCT) through perturbed local cell communication. Previously, we reported reproductive phenotypes of transgenic mice, in which expression of constitutively active mutant PI3K was induced in primordial oocytes by Gdf9-iCre. The transgenic mice (Cre(+)) demonstrated severe ovarian phenotypes, including the overgrowth of excess ovarian follicles and anovulation. Surprisingly, the Cre(+) mice became cachectic by postnatal day 80 due to bilateral GCT. Although GCT cells proliferated independently of oocytes, local interactions with mutant PI3K-positive oocytes during early folliculogenesis were essential for the GC transformation. Growing GCT cells expressed high levels of inhibin βA and nuclear SMAD3, and the proliferation rate was positively correlated with a high activin A to inhibin A ratio. These results suggested that the tumor cells stimulated their growth through an activin A autocrine signaling pathway, a hypothesis confirmed by activin A secretion in cultured GCT cells, which proliferated in response. Although communication between the oocyte and surrounding somatic cells is critical for the normal development of ovarian follicles, perturbations in oocyte-GC communication during early folliculogenesis can induce GCT by activating an autocrine growth circuit program in GC. Cancer Res; 76(13); 3851-61. ©2016 AACR. PMID:27197196

  15. Conditioned medium from alternatively activated macrophages induce mesangial cell apoptosis via the effect of Fas

    SciTech Connect

    Huang, Yuan; Luo, Fangjun; Li, Hui; Jiang, Tao; Zhang, Nong

    2013-11-15

    During inflammation in the glomerulus, the proliferation of myofiroblast-like mesangial cells is commonly associated with the pathological process. Macrophages play an important role in regulating the growth of resident mesangial cells in the glomeruli. Alternatively activated macrophage (M2 macrophage) is a subset of macrophages induced by IL-13/IL-4, which is shown to play a repair role in glomerulonephritis. Prompted by studies of development, we performed bone marrow derived macrophage and rat mesangial cell co-culture study. Conditioned medium from IL-4 primed M2 macrophages induced rat mesangial cell apoptosis. The pro-apoptotic effect of M2 macrophages was demonstrated by condensed nuclei stained with Hoechst 33258, increased apoptosis rates by flow cytometry analysis and enhanced caspase-3 activation by western blot. Fas protein was up-regulated in rat mesangial cells, and its neutralizing antibody ZB4 partly inhibited M2 macrophage-induced apoptosis. The up-regulated arginase-1 expression in M2 macrophage also contributed to this apoptotic effect. These results indicated that the process of apoptosis triggered by conditioned medium from M2 macrophages, at least is partly conducted through Fas in rat mesangial cells. Our findings provide compelling evidence that M2 macrophages control the growth of mesangial cells in renal inflammatory conditions. - Highlights: • Conditioned-medium from M2 macrophages induces rat mesangial cell (MsC) apoptosis. • M2 macrophage conditioned medium exerts its pro-apoptotic effects via Fas ligand. • Arginase-1 activity in M2 macrophages plays a role in inducing apoptosis in rat MsC.

  16. Natural Product Vibsanin A Induces Differentiation of Myeloid Leukemia Cells through PKC Activation.

    PubMed

    Yu, Zu-Yin; Xiao, He; Wang, Li-Mei; Shen, Xing; Jing, Yu; Wang, Lin; Sun, Wen-Feng; Zhang, Yan-Feng; Cui, Yu; Shan, Ya-Jun; Zhou, Wen-Bing; Xing, Shuang; Xiong, Guo-Lin; Liu, Xiao-Lan; Dong, Bo; Feng, Jian-Nan; Wang, Li-Sheng; Luo, Qing-Liang; Zhao, Qin-Shi; Cong, Yu-Wen

    2016-05-01

    All-trans retinoic acid (ATRA)-based cell differentiation therapy has been successful in treating acute promyelocytic leukemia, a unique subtype of acute myeloid leukemia (AML). However, other subtypes of AML display resistance to ATRA-based treatment. In this study, we screened natural, plant-derived vibsane-type diterpenoids for their ability to induce differentiation of myeloid leukemia cells, discovering that vibsanin A potently induced differentiation of AML cell lines and primary blasts. The differentiation-inducing activity of vibsanin A was mediated through direct interaction with and activation of protein kinase C (PKC). Consistent with these findings, pharmacological blockade of PKC activity suppressed vibsanin A-induced differentiation. Mechanistically, vibsanin A-mediated activation of PKC led to induction of the ERK pathway and decreased c-Myc expression. In mouse xenograft models of AML, vibsanin A administration prolonged host survival and inhibited PKC-mediated inflammatory responses correlated with promotion of skin tumors in mice. Collectively, our results offer a preclinical proof of concept for vibsanin A as a myeloid differentiation-inducing compound, with potential application as an antileukemic agent. Cancer Res; 76(9); 2698-709. ©2016 AACR.

  17. Artemisinin induces ROS-mediated caspase3 activation in ASTC-a-1 cells

    NASA Astrophysics Data System (ADS)

    Xiao, Feng-Lian; Chen, Tong-Sheng; Qu, Jun-Le; Liu, Cheng-Yi

    2010-02-01

    Artemisinin (ART), an antimalarial phytochemical from the sweet wormwood plant or a naturally occurring component of Artemisia annua, has been shown a potential anticancer activity by apoptotic pathways. In our report, cell counting kit (CCK-8) assay showed that treatment of human lung adenocarcinoma (ASTC-a-1) cells with ART effectively increase cell death by inducing apoptosis in a time- and dose-dependent fashion. Hoechst 33258 staining was used to detect apoptosis as well. Reactive oxygen species (ROS) generation was observed in cells exposed to ART at concentrations of 400 μM for 48 h. N-acetyl-L-cysteine (NAC), an oxygen radical scavenger, suppressed the rate of ROS generation and inhibited the ART-induced apoptosis. Moreover, AFC assay (Fluorometric assay for Caspase3 activity) showed that ROS was involved in ART-induced caspase3 acitvation. Taken together, our data indicate that ART induces ROS-mediated caspase3 activation in a time-and dose-dependent way in ASCT-a-1 cells.

  18. Liver X receptor β activation induces pyroptosis of human and murine colon cancer cells

    PubMed Central

    Derangère, V; Chevriaux, A; Courtaut, F; Bruchard, M; Berger, H; Chalmin, F; Causse, S Z; Limagne, E; Végran, F; Ladoire, S; Simon, B; Boireau, W; Hichami, A; Apetoh, L; Mignot, G; Ghiringhelli, F; Rébé, C

    2014-01-01

    Liver X receptors (LXRs) have been proposed to have some anticancer properties, through molecular mechanisms that remain elusive. Here we report for the first time that LXR ligands induce caspase-1-dependent cell death of colon cancer cells. Caspase-1 activation requires Nod-like-receptor pyrin domain containing 3 (NLRP3) inflammasome and ATP-mediated P2 × 7 receptor activation. Surprisingly, LXRβ is mainly located in the cytoplasm and has a non-genomic role by interacting with pannexin 1 leading to ATP secretion. Finally, LXR ligands have an antitumoral effect in a mouse colon cancer model, dependent on the presence of LXRβ, pannexin 1, NLRP3 and caspase-1 within the tumor cells. Our results demonstrate that LXRβ, through pannexin 1 interaction, can specifically induce caspase-1-dependent colon cancer cell death by pyroptosis. PMID:25124554

  19. LED-activated pheophorbide a induces cellular destruction of colon cancer cells

    NASA Astrophysics Data System (ADS)

    Xu, C. S.; Leung, A. W. N.; Liu, L.; Xia, X. S.

    2010-07-01

    Pheophorbide a (Pa) from Chinese herbal medicine Scutellaria Barbata and Silkworm Excreta shows an important promise in the photodynamic therapy on malignant tumor. The present study investigated that LED-activated Pa induced the cellular destruction of colon cancer HT-29 cells. The results showed that Pa resulted in a drug-dose dependent photocytotoxicity in the HT-29 cells, meaning the photocytotoxicity of Pa depends on the drug concentration (0 - 2 μM). We further investigated the apoptosis of the HT-29 cells 18 hours after photosensitization of Pa using a confocal laser scanning microscopy with Hoechst 33258 staining. These data demonstrated that LED-activated Pa could significantly induce the cellular destruction of the HT-29 cells.

  20. Human intestinal epithelial cell-induced CD8+ T cell activation is mediated through CD8 and the activation of CD8-associated p56lck

    PubMed Central

    1995-01-01

    The activation of CD8+ suppressor T cells by normal intestinal epithelial cells in antigen-specific or allogeneic mixed cell culture systems has significant implications for the regulation of mucosal immune responses. In this study, we found that the capacity of epithelial cells to induce CD8+ suppressor T cell activation appeared to be linked to the binding of CD8 molecules on the T cell surface. This appears to be mediated by a non-class I molecule expressed on the epithelial cell surface, which binds to CD8 and results in the activation of the CD8-associated src-like tyrosine kinase, p56lck. Epithelial cell-stimulated p56lck activation is an early event (in contrast to monocytes) and is essential for T cell activation, since proliferation could be completely abrogated by pretreatment of T cells with genestein or herbamycin, both of which are protein tyrosine kinase inhibitors. Pretreatment of T cells with anti-CD8 or of intestinal epithelial cells with an anti-epithelial cell mAb B9 inhibited p56lck activation and further confirmed that CD8 on the T cell and a CD8 ligand on the epithelial cell were involved in this T cell activation event. The specificity of this reaction was confirmed in experiments in which murine transfectants 3G4 and 3G8, expressing CD4 or CD8, respectively, were used. Coculture of 3G8 with epithelial cells but not with monocytes activated p56lck in this cell line, whereas p56lck was preferentially activated in 3G4 cells when monocytes were used as the stimulator cells. Although stimulation through CD8- and CD8-associated p56lck was important for epithelial cell-induced T cell activation, T cell proliferation could not be induced by cross-linking CD8 alone with monoclonal antibody anti-CD8. These data suggest that a second signal, possibly through the T cell antigen receptor since activation of the T cell receptor-associated kinase fyn was also seen, is required for epithelial cell-driven T cell proliferation. PMID:7561681

  1. ASBESTOS-INDUCED ACTIVATION OF SIGNALING PATHWAYS IN HUMAN BRONCHIAL EPITHELIAL CELLS

    EPA Science Inventory

    Title: Asbestos-Induced Activation of Signaling Pathways in Human
    Bronchial Epithelial Cells

    X. Wang, MD 1, J. M. Samet, PhD 2 and A. J. Ghio, MD 2. 1 Center for
    Environmental Medicine, Asthma and Lung Biology, University of North
    Carolina, Chapel Hill, NC, Uni...

  2. Copper compound induces autophagy and apoptosis of glioma cells by reactive oxygen species and jnk activation

    PubMed Central

    2012-01-01

    Background Glioblastoma multiforme (GBM) is the most aggressive of the primary brain tumors, with a grim prognosis despite intensive treatment. In the past decades, progress in research has not significantly increased overall survival rate. Methods The in vitro antineoplastic effect and mechanism of action of Casiopeina III-ia (Cas III-ia), a copper compound, on rat malignant glioma C6 cells was investigated. Results Cas III-ia significantly inhibited cell proliferation, inducing autophagy and apoptosis, which correlated with the formation of autophagic vacuoles, overexpression of LC3, Beclin 1, Atg 7, Bax and Bid proteins. A decrease was detected in the mitochondrial membrane potential and in the activity of caspase 3 and 8, together with the generation of intracellular reactive oxygen species (ROS) and increased activity of c-jun NH2-terminal kinase (JNK). The presence of 3-methyladenine (as selective autophagy inhibitor) increased the antineoplastic effect of Cas III-ia, while Z-VAD-FMK only showed partial protection from the antineoplastic effect induced by Cas III-ia, and ROS antioxidants (N-acetylcysteine) decreased apoptosis, autophagy and JNK activity. Moreover, the JNK –specific inhibitor SP600125 prevented Cas III-ia-induced cell death. Conclusions Our data suggest that Cas III-ia induces cell death by autophagy and apoptosis, in part due to the activation of ROS –dependent JNK signaling. These findings support further studies of Cas III-ia as candidate for treatment of human malignant glioma. PMID:22540380

  3. Cytotoxin-induced NADPH oxides activation: roles in regulation of cell death.

    PubMed

    Zhang, Yongtao; Bi, Xiaolei; Jiang, Fan

    2015-07-01

    Numerous studies have shown that a variety of cytotoxic agents can activate the NADPH oxidase system and induce redox-dependent regulation of cellular functions. Cytotoxin-induced NADPH oxidase activation may either exert cytoprotective actions (e.g., survival, proliferation, and stress tolerance) or cause cell death. Here we summarize the experimental evidence showing the context-dependent dichotomous effects of NADPH oxidase on cell fate under cytotoxic stress conditions and the potential redox signaling mechanisms underlying this phenomenon. Clearly, it is difficult to create a unified paradigm on the toxicological implications of NADPH oxidase activation in response to cytotoxic stimuli. We suggest that interventional strategies targeting the NADPH oxidase system to prevent the adverse impacts of cytotoxins need to be contemplated in a stimuli- and cell type-specific manner.

  4. Trimethyltin-Induced Microglial Activation via NADPH Oxidase and MAPKs Pathway in BV-2 Microglial Cells

    PubMed Central

    Kim, Da Jung; Kim, Yong Sik

    2015-01-01

    Trimethyltin (TMT) is known as a potent neurotoxicant that causes neuronal cell death and neuroinflammation, particularly in the hippocampus. Microglial activation is one of the prominent pathological features of TMT neurotoxicity. Nevertheless, it remains unclear how microglial activation occurs in TMT intoxication. In this study, we aimed to investigate the signaling pathways in TMT-induced microglial activation using BV-2 murine microglial cells. Our results revealed that TMT generates reactive oxygen species (ROS) and increases the expression of CD11b and nuclear factor-κB- (NF-κB-) mediated nitric oxide (NO) and tumor necrosis factor- (TNF-) α in BV-2 cells. We also observed that NF-κB activation was controlled by p38 and JNK phosphorylation. Moreover, TMT-induced ROS generation occurred via nicotinamide adenine dinucleotide phosphate (NADPH) oxidase in BV-2 cells. Interestingly, treatment with the NADPH oxidase inhibitor apocynin significantly suppressed p38 and JNK phosphorylation and NF-κB activation and ultimately the production of proinflammatory mediators upon TMT exposure. These findings indicate that NADPH oxidase-dependent ROS generation activated p38 and JNK mitogen-activated protein kinases (MAPKs), which then stimulated NF-κB to release proinflammatory mediators in the TMT-treated BV-2 cells. PMID:26221064

  5. Resveratrol protects ROS-induced cell death by activating AMPK in H9c2 cardiac muscle cells

    PubMed Central

    Hwang, Jin-Taek; Kwon, Dae Young; Park, Ock Jin

    2007-01-01

    Resveratrol, one of polyphenols derived from red wine, has been shown to protect against cell death, possibly through the association with several signaling pathways. Currently numerous studies indicate that cardiovascular diseases are linked to the release of intracellular reactive oxygen species (ROS) often generated in states such as ischemia/reperfusion injury. In the present study, we investigated whether resveratrol has the capability to control intracellular survival signaling cascades involving AMP-activated kinase (AMPK) in the inhibitory process of cardiac injury. We hypothesized that resveratrol may exert a protective effect on damage to heart muscle through modulating of the AMPK signaling pathway. We mimicked ischemic conditions by inducing cell death with H2O2 in H9c2 muscle cells. In this experiment, resveratrol induced strong activation of AMPK and inhibited the occurrence of cell death caused by treatment with H2O2. Under the same conditions, inhibition of AMPK using dominant negative AMPK constructs dramatically abolished the effect of resveratrol on cell survival in H2O2-treated cardiac muscle cells. These results indicate that resveratrol-induced cell survival is mediated by AMPK in H9c2 cells and may exert a novel therapeutic effect on oxidative stress induced in cardiac disorders. PMID:18850225

  6. Necrosis-induced TLR3 Activation Promotes TLR2 Expression in Gingival Cells.

    PubMed

    Mori, K; Yanagita, M; Hasegawa, S; Kubota, M; Yamashita, M; Yamada, S; Kitamura, M; Murakami, S

    2015-08-01

    Damage-associated molecular patterns (DAMPs), endogenous molecules released from injured or dying cells, evoke sterile inflammation that is not induced by microbial pathogens. Periodontal diseases are infectious diseases caused by oral microorganisms; however, in some circumstances, DAMPs might initiate inflammatory responses before host cells recognize pathogen-associated molecular patterns. Here, we showed that the necrotic cell supernatant (NCS) functioned as an endogenous danger signal when released from necrotic epithelial cells exposed to repeat freeze thawing. The NCS contained RNA and stimulated the production of inflammatory cytokines interleukin 6 (IL-6) and IL-8 from gingival epithelial cells and gingival fibroblasts. Targeted knockdown of Toll-like receptor 3 (TLR3) in these cells significantly suppressed the ability of the NCS to induce IL-6 and IL-8 production. Epithelial cells and fibroblasts recognized the NCS from heterologous cells. Interestingly, the activation of TLR3, rather than other TLRs, induced TLR2 mRNA expression and proteins in gingival epithelial cells, and pretreatment with the NCS or polyinosinic:polycytidylic acid (Poly(I:C)), a strong TLR3 activator, enhanced inflammatory cytokine production induced by subsequent stimulation with Porphyromonas gingivalis (P. gingivalis) lipopolysaccharide, a TLR2 agonist. Moreover, the NCS reduced the expression of epithelial tight junction molecules zona occludens 1 and occludin and increased the permeability of epithelial tight junctions. These findings suggest that endogenous danger signal molecules such as self-RNA released from necrotic cells are recognized by TLR3 and that a subsequent increase of TLR2 expression in periodontal compartments such as gingival epithelial cells and gingival fibroblasts may enhance the inflammatory response to periodontopathic microbes recognized by TLR2 such as P. gingivalis, which also disrupts epithelial barrier functions. Thus, DAMPs may be involved in the

  7. RCC1-dependent activation of Ran accelerates cell cycle and DNA repair, inhibiting DNA damage–induced cell senescence

    PubMed Central

    Cekan, Pavol; Hasegawa, Keisuke; Pan, Yu; Tubman, Emily; Odde, David; Chen, Jin-Qiu; Herrmann, Michelle A.; Kumar, Sheetal; Kalab, Petr

    2016-01-01

    The coordination of cell cycle progression with the repair of DNA damage supports the genomic integrity of dividing cells. The function of many factors involved in DNA damage response (DDR) and the cell cycle depends on their Ran GTPase–regulated nuclear–cytoplasmic transport (NCT). The loading of Ran with GTP, which is mediated by RCC1, the guanine nucleotide exchange factor for Ran, is critical for NCT activity. However, the role of RCC1 or Ran⋅GTP in promoting cell proliferation or DDR is not clear. We show that RCC1 overexpression in normal cells increased cellular Ran⋅GTP levels and accelerated the cell cycle and DNA damage repair. As a result, normal cells overexpressing RCC1 evaded DNA damage–induced cell cycle arrest and senescence, mimicking colorectal carcinoma cells with high endogenous RCC1 levels. The RCC1-induced inhibition of senescence required Ran and exportin 1 and involved the activation of importin β–dependent nuclear import of 53BP1, a large NCT cargo. Our results indicate that changes in the activity of the Ran⋅GTP–regulated NCT modulate the rate of the cell cycle and the efficiency of DNA repair. Through the essential role of RCC1 in regulation of cellular Ran⋅GTP levels and NCT, RCC1 expression enables the proliferation of cells that sustain DNA damage. PMID:26864624

  8. Resveratrol inhibits the hydrogen dioxide-induced apoptosis via Sirt 1 activation in osteoblast cells.

    PubMed

    He, Na; Zhu, Xuewei; He, Wei; Zhao, Shiwei; Zhao, Weiyan; Zhu, Chunlei

    2015-01-01

    Sirt 1 plays a critical role in stress responses. We determined the deregulation of Sirt 1 activity, p53 acetylation, Bcl-2 expression, and mitochondria-dependent apoptosis in mouse osteoblast MC3T3-E1 cells which were exposed to H2O2. And then we investigated the protective role of Sirt 1 activator, Resveratrol (RSV), against the H2O2-induced apoptosis. Results demonstrated that Sirt 1 and Bcl-2 were inhibited, whereas p53 acetylation, Bax, and caspase 9 were promoted by H2O2, as was aggravated by the Sirt 1 inhibitor, EX-527. Instead, RSV inhibited the H2O2-induced both p53 acetylation and the caspase 9 activation, whereas ameliorated the H2O2-induced Bcl-2 inhibition and apoptosis. In conclusion, Sirt 1 was downregulated during the H2O2-induced apoptosis in MC3T3-E1 cells. And the chemical activation of Sirt 1 inhibited the H2O2-induced apoptosis via the downregulation of p53 acetylation. Our results suggest that Sirt 1 upregulation appears to be an important strategy to inhibit the oxidative stress-induced apoptosis.

  9. Silver nanoparticles-induced cytotoxicity requires ERK activation in human bladder carcinoma cells.

    PubMed

    Castiglioni, Sara; Cazzaniga, Alessandra; Perrotta, Cristiana; Maier, Jeanette A M

    2015-09-17

    Silver nanoparticles are toxic both in vitro and in vivo. We have investigated the possibility to exploit the cytotoxic potential of silver nanoparticles in T24 bladder carcinoma cells using both bare and PolyVinylPyrrolidone-coated silver nanoparticles. We show that the two types of silver nanoparticles promote morphological changes and cytoskeletal disorganization, are cytotoxic and induce cell death. These effects are due to the increased production of reactive oxygen species which are responsible, at least in part, for the sustained activation of ERK1/2. Indeed, both cytotoxicity and ERK1/2 activation are prevented by exposing the cells to the anti-oxidant N-acetylcysteine. Also blocking the ERK1/2 pathway with the MEK inhibitor PD98059 protects the cells from nanoparticles' cytotoxicity. Our findings suggest that ERK activation plays a role in silver nanoparticle-mediated cytotoxicity in T24 cells.

  10. Hypoxia-induced cell death and changes in hypoxia-inducible factor-1 activity in PC12 cells upon exposure to nerve growth factor.

    PubMed

    Charlier, Nico; Leclere, Norbert; Felderhoff, Ursula; Heldt, Julia; Kietzmann, Thomas; Obladen, Michael; Gross, Johann

    2002-07-15

    The transcription factor hypoxia-inducible factor-1 (HIF-1) strongly contributes to the expression of adaptive genes under hypoxic conditions. In addition, HIF-1 has been implicated in the regulation of delayed neuronal cell death. Suspension-grown and adherent PC12 cells treated with NGF were used as an experimental model for studying the relationship between hypoxia-induced cell death and activation of HIF-1. Cell damage was assessed by flow cytometry of double-stained (Annexin V and propidiumiodide) cells, and by analysis of the overall death parameters LDH and mitochondrial dehydrogenase. In parallel, cells were transfected with a control and a three-hypoxia-responsive-elements (HRE)-containing vector and HIF-1-driven luciferase activity was determined. Exposure of NGF-treated PC12 cells to hypoxia resulted in a higher cell death rate when compared to untreated controls. PC12 cells exposed for 2 days to NGF exhibited a decrease of HIF-1 activity up to a factor of ten. This decrease may contribute to the enhanced hypoxia-induced cell death via reduced expression of HIF-1alpha-regulated genes responsible for adaptation to hypoxia, like those for glucose transport proteins and enzymes of the glycolytic chain. The decrease in HIF-1 activity and the increase in hypoxia sensitivity may suggest that NGF act as an hierarchically organized signaling molecule.

  11. Curcumin induces apoptotic cell death of activated human CD4+ T cells via increasing endoplasmic reticulum stress and mitochondrial dysfunction.

    PubMed

    Zheng, Min; Zhang, Qinggao; Joe, Yeonsoo; Lee, Bong Hee; Ryu, Do Gon; Kwon, Kang Beom; Ryter, Stefan W; Chung, Hun Taeg

    2013-03-01

    Curcumin, a natural polyphenolic antioxidant compound, exerts well-known anti-inflammatory and immunomodulatory effects, the latter which can influence the activation of immune cells including T cells. Furthermore, curcumin can inhibit the expression of pro-inflammatory cytokines and chemokines, through suppression of the NF-κB signaling pathway. The beneficial effects of curcumin in diseases such as arthritis, allergy, asthma, atherosclerosis, diabetes and cancer may be due to its immunomodulatory properties. We studied the potential of curcumin to modulate CD4+ T cells-mediated autoimmune disease, by examining the effects of this compound on human CD4+ lymphocyte activation. Stimulation of human T cells with PHA or CD3/CD28 induced IL-2 mRNA expression and activated the endoplasmic reticulum (ER) stress response. The treatment of T cells with curcumin induced the unfolded protein response (UPR) signaling pathway, initiated by the phosphorylation of PERK and IRE1. Furthermore, curcumin increased the expression of the ER stress associated transcriptional factors XBP-1, cleaved p50ATF6α and C/EBP homologous protein (CHOP) in human CD4+ and Jurkat T cells. In PHA-activated T cells, curcumin further enhanced PHA-induced CHOP expression and reduced the expression of the anti-apoptotic protein Bcl-2. Finally, curcumin treatment induced apoptotic cell death in activated T cells via eliciting an excessive ER stress response, which was reversed by the ER-stress inhibitor 4-phenylbutyric acid or transfection with CHOP-specific siRNA. These results suggest that curcumin can impact both ER stress and mitochondria functional pathways, and thereby could be used as a promising therapy in the context of Th1-mediated autoimmune diseases. PMID:23415873

  12. Accumulation of tissue factor in endothelial cells induces cell apoptosis, mediated through p38 and p53 activation.

    PubMed

    ElKeeb, A M; Collier, M E W; Maraveyas, A; Ettelaie, C

    2015-08-01

    We previously reported that high levels of tissue factor (TF) can induce cellular apoptosis in endothelial cells. In this study, TF-mediated mechanisms of induction of apoptosis were explored. Endothelial cells were transfected to express wild-type TF. Additionally, cells were transfected to express Asp253-substituted, or Ala253-substitued TF to enhance or prevent TF release, respectively. Alternatively, cells were pre-incubated with TF-rich and TF-poor microvesicles. Cell proliferation, apoptosis and the expression of cyclin D1, p53, bax and p21 were measured following activation of cells with PAR2-agonist peptide. Greatest levels of cell proliferation and cyclin D1 expression were observed in cells expressing wild-type or Asp253-substituted TF. In contrast, increased cellular apoptosis was observed in cells expressing Ala253-substituted TF, or cells pre-incubated with TF-rich microvesicles. The level of p53 protein, p53-phosphorylation at ser33, p53 nuclear localisation and transcriptional activity, but not p53 mRNA, were increased in cells expressing wild-type and Ala253-substituted TF, or in cells pre-incubated with TF-rich microvesicles. However, the expression of bax and p21 mRNA, and Bax protein were only increased in cells pre-incubated with TF-rich microvesicle and in cells expressing Ala253-substituted TF. Inhibition of the transcriptional activity of p53 using pifithrin-α suppressed the expression of Bax. Finally, siRNA-mediated suppression of p38α, or inhibition using SB202190 significantly reduced the p53 protein levels, p53 nuclear localisation and transcriptional activity, suppressed Bax expression and prevented cellular apoptosis. In conclusion, accumulation of TF within endothelial cells, or sequestered from the surrounding can induce cellular apoptosis through mechanisms mediated by p38, and involves the stabilisation of p53. PMID:25903973

  13. Monocytes Induce STAT3 Activation in Human Mesenchymal Stem Cells to Promote Osteoblast Formation

    PubMed Central

    Nicolaidou, Vicky; Wong, Mei Mei; Redpath, Andia N.; Ersek, Adel; Baban, Dilair F.; Williams, Lynn M.; Cope, Andrew P.; Horwood, Nicole J.

    2012-01-01

    A major therapeutic challenge is how to replace bone once it is lost. Bone loss is a characteristic of chronic inflammatory and degenerative diseases such as rheumatoid arthritis and osteoporosis. Cells and cytokines of the immune system are known to regulate bone turnover by controlling the differentiation and activity of osteoclasts, the bone resorbing cells. However, less is known about the regulation of osteoblasts (OB), the bone forming cells. This study aimed to investigate whether immune cells also regulate OB differentiation. Using in vitro cell cultures of human bone marrow-derived mesenchymal stem cells (MSC), it was shown that monocytes/macrophages potently induced MSC differentiation into OBs. This was evident by increased alkaline phosphatase (ALP) after 7 days and the formation of mineralised bone nodules at 21 days. This monocyte-induced osteogenic effect was mediated by cell contact with MSCs leading to the production of soluble factor(s) by the monocytes. As a consequence of these interactions we observed a rapid activation of STAT3 in the MSCs. Gene profiling of STAT3 constitutively active (STAT3C) infected MSCs using Illumina whole human genome arrays showed that Runx2 and ALP were up-regulated whilst DKK1 was down-regulated in response to STAT3 signalling. STAT3C also led to the up-regulation of the oncostatin M (OSM) and LIF receptors. In the co-cultures, OSM that was produced by monocytes activated STAT3 in MSCs, and neutralising antibodies to OSM reduced ALP by 50%. These data indicate that OSM, in conjunction with other mediators, can drive MSC differentiation into OB. This study establishes a role for monocyte/macrophages as critical regulators of osteogenic differentiation via OSM production and the induction of STAT3 signalling in MSCs. Inducing the local activation of STAT3 in bone cells may be a valuable tool to increase bone formation in osteoporosis and arthritis, and in localised bone remodelling during fracture repair. PMID:22802946

  14. Enhancement of activation-induced cell death by fibronectin in murine CD4+ CD8+ thymocytes.

    PubMed Central

    Takayama, E; Kina, T; Katsura, Y; Tadakuma, T

    1998-01-01

    Development of T cells in the thymus is achieved through the interactions of thymocytes with their microenvironments. This study focused on the function of fibronectin (FN), a major extracellular matrix molecule in the thymus, in the cell death induced by activation via the T-cell antigen receptor. FN alone did not increase cell death in murine thymocytes above the baseline level, but it significantly enhanced the cell death induced by fixed anti-CD3 monoclonal antibody (mAb), especially when a high concentration of anti-CD3 mAb was used. DNA fragmentation increased in parallel with cell death, indicating that cell death was a result of the apoptosis. Fluorescence-activated cell sorter (FACS) analysis revealed that the activation-induced cell death (AICD) caused by anti-CD3 mAb alone, or by a combination of anti-CD3 mAb and FN, occurred selectively in CD4+ CD8+ thymocytes. Very late activation antigen (VLA)-4 and VLA-5 are two major ligands to FN on thymocytes. The expression of both ligands was investigated at different stages of thymocyte development. VLA-4 was predominantly expressed at the CD4- CD8- stage, and thereafter the expression was reduced, whereas VLA-5 was constantly expressed during maturation. Furthermore, the enhancing effect by FN was inhibited in the presence of the Gly-Arg-Gly-Asp-Ser-Pro (GRGDSP) peptide but not in the presence of the connecting segment-1 (CS-1) peptide, suggesting that enhancement of AICD observed in CD4+ CD8+ thymocytes is mediated through VLA-5. Images Figure 1 Figure 5 PMID:9893044

  15. ASBESTOS-INDUCED ACTIVATION OF CELL SIGNALING PATHWAYS IN HUMAN BRONCHIAL EPITHELIAL CELLS

    EPA Science Inventory

    Using respiratory epithelial cells transfected with either superoxide dismutase (SOD) or catalase, the authors tested the hypothesis that the activation of the epidermal growth factor (EGF) receptor signal pathway after asbestos exposure involves an oxidative stress. Western blot...

  16. Hepatitis B virus e antigen induces activation of rat hepatic stellate cells

    SciTech Connect

    Zan, Yanlu; Zhang, Yuxia; Tien, Po

    2013-06-07

    Highlights: •HBeAg expression in HSCs induced production of ECM protein and liver fibrotic markers. •The activation and proliferation of HSCs were mediated by TGF-β. •HBeAg protein purified from cell medium directly activated HSCs. -- Abstract: Chronic hepatitis B virus infection is a major cause of hepatic fibrosis, leading to liver cirrhosis and hepatocellular carcinoma. Hepatitis B virus e antigen (HBeAg) is an accessory protein of HBV, not required for viral replication but important for natural infection in vivo. Hepatic stellate cells (HSCs) are the major producers of excessive extracellular matrix during liver fibrogenesis. Therefore, we examined the influence of HBeAg on HSCs. The rat HSC line HSC-T6 was transfected with HBeAg plasmids, and expression of α-smooth muscle actin, collagen I, transforming growth factor-β1 (TGF-β), and tissue inhibitors of metalloproteinase 1 (TIMP-1) was investigated by quantitative real-time PCR. The proliferation of HSCs was determined by MTS analysis. HBeAg transduction induced up-regulation of these fibrogenic genes and proliferation of HSCs. We found that HBeAg induced TGF-β secretion in HSCs, and the activation of HSCs was prevented by a neutralizing anti-TGF-β antibody. Depletion and addition of HBeAg protein in conditioned medium from HSC-T6 cells transduced with HBeAg indicated that HBeAg directly induced the activation and proliferation of rat primary HSCs. Taken together, HBeAg induces the activation and proliferation of HSCs, mainly mediated by TGF-β, and HBeAg protein purified from cell medium can directly activate HSCs.

  17. Proteinase-activated receptor-1 mediates allogeneic CD8(+) T cell-induced apoptosis of vascular endothelial cells.

    PubMed

    Quan, Li; Jian, Zhang; Ping, Zou; Weiming, Li

    2009-12-01

    Vascular endothelial-cells injury plays a pivotal role in the pathogenesis of graft-versus-host disease (GVHD) and transplant-associated endothelial injury syndrome. Vascular endothelial cells are an exposed target tissue for immune-mediated injury during GVHD. Early endothelial injury syndromes share common features with acute GVHD. Chronic GVHD leads to a rarefaction of microvessels caused by the infiltration of alloreactive cytotoxic T lymphocytes. In this context, allogeneic reactive cytotoxic T cell may contribute to apoptosis of vascular endothelial cells. The involvement of proteinase-activated receptor (PAR-1) in regulation of apoptosis has been recently recognized in many cell types. We hypothesized that apoptosis of vascular endothelial cells induced by allogeneic cytotoxic T cell are mediated via the PAR-1. Allogeneic CD8(+) T cell, PAR-1 agonist peptide (SFLLRN) induced apoptosis of human umbilical vein endothelial cells (HUVECs) and human dermal microvascular endothelial cells (HDMECs) as assessed by AnnexinV-FITC labeling. To ascertain the mechanism of endothelial apoptosis, we determined that allogeneic CD8(+) T cell, SFLLRN enhanced cleavage of caspase-3 and led to p38MAPK activation as assessed by Western blot. The effects of allogeneic CD8(+) T cell and SFLLRN on apoptosis of vascular endothelial cells were largely prevented by a cleavage-blocking anti-human PAR-1-antibody (ATAP2) and a specific inhibitor of p38MAPK. In concert, these observations provide strong evidence that allogeneic CD8(+) T cell induces apoptosis of human vascular endothelial cells through PAR-1-dependent modulation of intrinsic apoptotic pathway via alterations of p38MAPK and caspase-3. PMID:19082770

  18. Platelet activating factor induces dopamine release in PC-12 cell line

    SciTech Connect

    Bussolino, F.; Tessari, F.; Turrini, F.; Braquet, P.; Camussi, G.; Prosdocimi, M.; Bosia, A. Institut Henri Beaufour, Le Plessis Robinson )

    1988-10-01

    The ability of platelet activating factor (PAF) to stimulate dopamine release and modify calcium homeostasis in PC-12 cell line was studied. PAF-induced dopamine release is related to its molecular form, with only the R-form steric configuration ((R)PAF), but not its S-form or its 2-lyso derivative, effective at being active. In addition, PAF acts at very low concentrations in a dose-dependent manner (0.1-30 nM). Preincubation with PAF receptor antagonists (CV-3988 and BN52021) as well as the specific desensitization of PC-12 cells to (R)PAF abolish the (R)PAF-induced dopamine release. Several lines of evidence suggest that dopamine release is dependent on a (R)PAF-induced calcium influx and efflux modulation. Dopamine release by PC-12 cells challenged with (R)PAF is associated with a rapid {sup 45}Ca influx and efflux and a rise in cytoplasmic calcium concentrations ((Ca{sup 2+}){sub i}) evaluated by using the calcium indicators fura-2 and quin2. At 30 nM (R)PAF, the absence of extracellular calcium inhibits the dopamine release but not the rise of (Ca{sup 2+}){sub i} from the internal stores, suggesting the importance of calcium influx in (R)PAF-induced dopamine release. PAF, which has been reported to be synthesized by stimulated neuronal cells may thus have a physiological modulatory role on cells with neurosecretory properties.

  19. Bufotalin-induced apoptosis in osteoblastoma cells is associated with endoplasmic reticulum stress activation.

    PubMed

    Zhu, Yun-Rong; Xu, Yong; Fang, Jian-Feng; Zhou, Feng; Deng, Xiong-Wei; Zhang, Yun-Qing

    2014-08-15

    The search for novel and more efficient chemo-agents against malignant osteoblastoma is important. In this study, we examined the potential anti-osteoblastoma function of bufotalin, and studied the underlying mechanisms. Our results showed that bufotalin induced osteoblastoma cell death and apoptosis in dose- and time-dependent manners. Further, bufotalin induced endoplasmic reticulum (ER) stress activation in osteoblastoma cells, the latter was detected by the induction of C/EBP homologous protein (CHOP), phosphorylation of inositol-requiring enzyme 1 (IRE1) and PKR-like endoplasmic reticulum kinase (PERK), as well as caspase-12 activation. Conversely, the ER stress inhibitor salubrinal, the caspase-12 inhibitor z-ATAD-fmk as well as CHOP depletion by shRNA significantly inhibited bufotalin-induced osteoblastoma cell death and apoptosis. Finally, by using a mice xenograft model, we demonstrated that bufotalin inhibited U2OS osteoblastoma cell growth in vivo. In summary, our results suggest that ER stress contributes to bufotalin-induced apoptosis in osteoblastoma cells. Bufotalin might be investigated as a novel anti-osteoblastoma agent. PMID:25068992

  20. Activation of decidual invariant natural killer T cells promotes lipopolysaccharide-induced preterm birth.

    PubMed

    Li, Liping; Yang, Jing; Jiang, Yao; Tu, Jiaoqin; Schust, Danny J

    2015-04-01

    Invariant natural killer T (iNKT) cells are crucial for host defense against a variety of microbial pathogens, but the underlying mechanisms of iNKT cells activation by microbes are not fully explained. In this study, we investigated the molecular mechanisms of iNKT cell activation in lipopolysaccharide (LPS)-stimulated preterm birth using an adoptive transfer system and diverse neutralizing antibodies (Abs) and inhibitors. We found that adoptive transfer of decidual iNKT cells to LPS-stimulated iNKT cell deficient Jα18(-/-) mice that lack invariant Vα14Jα281T cell receptor (TCR) expression significantly decreased the time to delivery and increased the percentage of decidual iNKT cells. Neutralizing Abs against Toll-like receptor 4 (TLR-4), CD1d, interleukin (IL)-12 and IL-18, and inhibitors blocking the activation of nuclear factor κB (NF-κB), mitogen-activated protein kinase (MAPK) p38 and extracellular signal-regulated kinase (ERK) significantly reduced in vivo percentages of decidual iNKT cells, their intracellular interferon (IFN)-γ production and surface CD69 expression. In vitro, in the presence of the same Abs and inhibitors used as in vivo, decidual iNKT cells co-cultured with LPS-pulsed dendritic cells (DCs) showed significantly decreased extracellular and intracellular IFN-γ secretion and surface CD69 expression. Our data demonstrate that the activation of decidual iNKT cells plays an important role in inflammation-induced preterm birth. Activation of decidual iNKT cells also requires TLR4-mediated NF-κB, MAPK p38 and ERK pathways, the proinflammatory cytokines IL-12 and IL-18, and endogenous glycolipid antigens presented by CD1d.

  1. Simvastatin induces caspase-independent apoptosis in LPS-activated RAW264.7 macrophage cells

    SciTech Connect

    Kim, Yong Chan; Song, Seok Bean; Lee, Mi Hee; Kang, Kwang Il; Lee, Hayyoung; Paik, Sang-Gi; Kim, Kyoon Eon; Kim, Young Sang . E-mail: young@cnu.ac.kr

    2006-01-20

    Macrophages participate in several inflammatory pathologies such as sepsis and arthritis. We examined the effect of simvastatin on the LPS-induced proinflammatory macrophage RAW264.7 cells. Co-treatment of LPS and a non-toxic dose of simvastatin induced cell death in RAW264.7 cells. The cell death was accompanied by disruption of mitochondrial membrane potential (MMP), genomic DNA fragmentation, and caspase-3 activation. Surprisingly, despite caspase-dependent apoptotic cascade being completely blocked by Z-VAD-fmk, a pan-caspase inhibitor, the cell death was only partially repressed. In the presence of Z-VAD-fmk, DNA fragmentation was blocked, but DNA condensation, disruption of MMP, and nuclear translocation of apoptosis inducing factor were obvious. The cell death by simvastatin and LPS was effectively decreased by both the FPP and GGPP treatments as well as mevalonate. Our findings indicate that simvastatin triggers the cell death of LPS-treated RAW264.7 cells through both caspase-dependent and -independent apoptotic pathways, suggesting a novel mechanism of statins for the severe inflammatory disease therapy.

  2. CRFR1 activation protects against cytokine-induced β-cell death.

    PubMed

    Blaabjerg, Lykke; Christensen, Gitte L; Matsumoto, Masahito; van der Meulen, Talitha; Huising, Mark O; Billestrup, Nils; Vale, Wylie W

    2014-12-01

    During the development of diabetes β-cells are exposed to elevated concentrations of proinflammatory cytokines, TNFα and IL1β, which in vitro induce β-cell death. The class B G-protein-coupled receptors (GPCRs): corticotropin-releasing factor receptor 1 (CRFR1) and CRFR2 are expressed in pancreatic islets. As downstream signaling by other class B GPCRs can protect against cytokine-induced β-cell apoptosis, we evaluated the protective potential of CRFR activation in β-cells in a pro-inflammatory setting. CRFR1/CRFR2 ligands activated AKT and CRFR1 signaling and reduced apoptosis in human islets. In rat and mouse insulin-secreting cell lines (INS-1 and MIN6), CRFR1 agonists upregulated insulin receptor substrate 2 (IRS2) expression, increased AKT activation, counteracted the cytokine-mediated decrease in BAD phosphorylation, and inhibited apoptosis. The anti-apoptotic signaling was dependent on prolonged exposure to corticotropin-releasing factor family peptides and followed PKA-mediated IRS2 upregulation. This indicates that CRFR signaling counteracts proinflammatory cytokine-mediated apoptotic pathways through upregulation of survival signaling in β-cells. Interestingly, CRFR signaling also counteracted basal apoptosis in both cultured INS-1 cells and intact human islets. PMID:25324488

  3. Immune activation induces immortalization of HTLV-1 LTR-Tax transgenic CD4+ T cells.

    PubMed

    Swaims, Alison Y; Khani, Francesca; Zhang, Yingyu; Roberts, Arthur I; Devadas, Satish; Shi, Yufang; Rabson, Arnold B

    2010-10-21

    Infection with the human T-cell leukemia virus-1 (HTLV-1) results in a variety of diseases including adult T-cell leukemia/lymphoma (ATL). Although the pathogenesis of these disorders is poorly understood, it involves complex interactions with the host immune system. Activation of infected T cells may play an important role in disease pathogenesis through induction of the oncogenic HTLV-1 Tax transactivator protein. To test this hypothesis, we employed transgenic mice in which Tax is regulated by the HTLV-1 LTR. T-cell receptor stimulation of LTR-Tax CD4(+) T cells induced Tax expression, hyper-proliferation, and immortalization in culture. The transition to cellular immortalization was accompanied by markedly increased expression of the antiapoptotic gene, mcl-1, previously implicated as important in T-cell survival. Immortalized cells exhibited a CD4(+)CD25(+)CD3(-) phenotype commonly observed in ATL. Engraftment of activated LTR-Tax CD4(+) T cells into NOD/Shi-scid/IL-2Rγ null mice resulted in a leukemia-like phenotype with expansion and tissue infiltration of Tax(+), CD4(+) lymphocytes. We suggest that immune activation of infected CD4(+) T cells plays an important role in the induction of Tax expression, T-cell proliferation, and pathogenesis of ATL in HTLV-1-infected individuals. PMID:20634377

  4. AMP-activated protein kinase induces actin cytoskeleton reorganization in epithelial cells

    SciTech Connect

    Miranda, Lisa; Carpentier, Sarah; Platek, Anna; Hussain, Nusrat; Gueuning, Marie-Agnes; Vertommen, Didier; Ozkan, Yurda; Sid, Brice; Hue, Louis; Courtoy, Pierre J.; Rider, Mark H.; Horman, Sandrine

    2010-06-04

    AMP-activated protein kinase (AMPK), a known regulator of cellular and systemic energy balance, is now recognized to control cell division, cell polarity and cell migration, all of which depend on the actin cytoskeleton. Here we report the effects of A769662, a pharmacological activator of AMPK, on cytoskeletal organization and signalling in epithelial Madin-Darby canine kidney (MDCK) cells. We show that AMPK activation induced shortening or radiation of stress fibers, uncoupling from paxillin and predominance of cortical F-actin. In parallel, Rho-kinase downstream targets, namely myosin regulatory light chain and cofilin, were phosphorylated. These effects resembled the morphological changes in MDCK cells exposed to hyperosmotic shock, which led to Ca{sup 2+}-dependent AMPK activation via calmodulin-dependent protein kinase kinase-{beta}(CaMKK{beta}), a known upstream kinase of AMPK. Indeed, hypertonicity-induced AMPK activation was markedly reduced by the STO-609 CaMKK{beta} inhibitor, as was the increase in MLC and cofilin phosphorylation. We suggest that AMPK links osmotic stress to the reorganization of the actin cytoskeleton.

  5. Activation-induced apoptosis in peripheral blood mononuclear cells during hepatosplenic Schistosoma mansoni infections.

    PubMed

    Ghoneim, H M; Demian, S R; Heshmat, M G; Ismail, N S; El-Sayed, Laila H

    2008-01-01

    It is well established that programmed cell death (apoptosis) is an important regulator of host responses during infection with a variety of intra- and extra-cellular pathogens. The present work aimed at assessment of in vitro spontaneous and phytohemagglutinin (PHA)-induced apoptosis in mononuclear cells isolated from patients with hepatosplenic form of S. mansoni infections. Cell death data were correlated to the degree of lymphoproliferative responses to PHA as well as to the serum anti-schistosomal antibody titers. A markedly significant increase in PHA-induced apoptosis in lymphocytes isolated from S. mansoni-infected patients was seen when compared to the corresponding healthy controls. However, a slight difference was recorded between the two studied groups regarding the spontaneous apoptosis. This was accompanied with a significant impairment of in vitro PHA-induced lymphoproliferation of T cells from S. mansoni patients. Data of the present study supports the hypothesis that activation-induced cell death (AICD) is a potentially contributing factor in T helper (Th) cell regulation during chronic stages of schistosomiasis, which represents a critically determinant factor in the host-parasite interaction and might influence the destiny of parasitic infections either towards establishment of chronic infection or towards host death.

  6. Activation-induced apoptosis in peripheral blood mononuclear cells during hepatosplenic Schistosoma mansoni infections.

    PubMed

    Ghoneim, H M; Demian, S R; Heshmat, M G; Ismail, N S; El-Sayed, Laila H

    2008-01-01

    It is well established that programmed cell death (apoptosis) is an important regulator of host responses during infection with a variety of intra- and extra-cellular pathogens. The present work aimed at assessment of in vitro spontaneous and phytohemagglutinin (PHA)-induced apoptosis in mononuclear cells isolated from patients with hepatosplenic form of S. mansoni infections. Cell death data were correlated to the degree of lymphoproliferative responses to PHA as well as to the serum anti-schistosomal antibody titers. A markedly significant increase in PHA-induced apoptosis in lymphocytes isolated from S. mansoni-infected patients was seen when compared to the corresponding healthy controls. However, a slight difference was recorded between the two studied groups regarding the spontaneous apoptosis. This was accompanied with a significant impairment of in vitro PHA-induced lymphoproliferation of T cells from S. mansoni patients. Data of the present study supports the hypothesis that activation-induced cell death (AICD) is a potentially contributing factor in T helper (Th) cell regulation during chronic stages of schistosomiasis, which represents a critically determinant factor in the host-parasite interaction and might influence the destiny of parasitic infections either towards establishment of chronic infection or towards host death. PMID:20306689

  7. Hernandezine, a novel AMPK activator induces autophagic cell death in drug-resistant cancers

    PubMed Central

    Law, Betty Yuen Kwan; Mok, Simon Wing Fai; Chan, Wai Kit; Xu, Su Wei; Wu, An Guo; Yao, Xiao Jun; Wang, Jing Rong; Liu, Liang; Wong, Vincent Kam Wai

    2016-01-01

    Drug resistance hinder most cancer chemotherapies and leads to disease recurrence and poor survival of patients. Resistance of cancer cells towards apoptosis is the major cause of these symptomatic behaviours. Here, we showed that isoquinoline alkaloids, including liensinine, isoliensinine, dauricine, cepharanthine and hernandezine, putatively induce cytotoxicity against a repertoire of cancer cell lines (HeLa, A549, MCF-7, PC3, HepG2, Hep3B and H1299). Proven by the use of apoptosis-resistant cellular models and autophagic assays, such isoquinoline alkaloid-induced cytotoxic effect involves energy- and autophagy-related gene 7 (Atg7)-dependent autophagy that resulted from direct activation of AMP activated protein kinase (AMPK). Hernandezine possess the highest efficacy in provoking such cell death when compared with other examined compounds. We confirmed that isoquinoline alkaloid is structurally varied from the existing direct AMPK activators. In conclusion, isoquinoline alkaloid is a new class of compound that induce autophagic cell death in drug-resistant fibroblasts or cancers by exhibiting its direct activation on AMPK. PMID:26811496

  8. Target sequence accessibility limits activation-induced cytidine deaminase activity in primary mediastinal B-cell lymphoma.

    PubMed

    Popov, Sergey W; Moldenhauer, Gerhard; Wotschke, Beate; Brüderlein, Silke; Barth, Thomas F; Dorsch, Karola; Ritz, Olga; Möller, Peter; Leithäuser, Frank

    2007-07-15

    Activation-induced cytidine deaminase (AID) initiates somatic hypermutation (SHM) and class switch recombination (CSR) in activated B lymphocytes and is potentially implicated in genomic instability of B-cell malignancies. For unknown reasons, B-cell neoplasms often lack SHM and CSR in spite of high AID expression. Here, we show that primary mediastinal B-cell lymphoma (PMBL), an immunoglobulin (Ig)-negative lymphoma that possesses hypermutated, class-switched Ig genes, expresses high levels of AID with an intact primary structure but does not do CSR in 14 of 16 cases analyzed. Absence of CSR coincided with low Ig germ-line transcription, whereas high level germ-line transcription was observed only in those two cases with active CSR. Interleukin-4/CD40L costimulation induced CSR and a marked up-regulation of germ-line transcription in the PMBL-derived cell line MedB-1. In the PMBL cell line Karpas 1106P, CSR was not inducible and germ-line transcription remained low on stimulation. However, Karpas 1106P, but not MedB-1, had ongoing SHM of the Ig gene and BCL6. These genes were transcribed in Karpas 1106P, whereas transcription was undetectable or low in MedB-1 cells. Thus, accessibility of the target sequences seems to be a major limiting factor for AID-dependent somatic gene diversification in PMBL. PMID:17638864

  9. Increased Anion Channel Activity Is an Unavoidable Event in Ozone-Induced Programmed Cell Death

    PubMed Central

    Errakhi, Rafik; Hiramatsu, Takuya; Meimoun, Patrice; Briand, Joël; Iwaya-Inoue, Mari; Kawano, Tomonori; Bouteau, François

    2010-01-01

    Background Ozone is a major secondary air pollutant often reaching high concentrations in urban areas under strong daylight, high temperature and stagnant high-pressure systems. Ozone in the troposphere is a pollutant that is harmful to the plant. Principal Findings By exposing cells to a strong pulse of ozonized air, an acute cell death was observed in suspension cells of Arabidopsis thaliana used as a model. We demonstrated that O3 treatment induced the activation of a plasma membrane anion channel that is an early prerequisite of O3-induced cell death in A. thaliana. Our data further suggest interplay of anion channel activation with well known plant responses to O3, Ca2+ influx and NADPH-oxidase generated reactive oxygen species (ROS) in mediating the oxidative cell death. This interplay might be fuelled by several mechanisms in addition to the direct ROS generation by O3; namely, H2O2 generation by salicylic and abscisic acids. Anion channel activation was also shown to promote the accumulation of transcripts encoding vacuolar processing enzymes, a family of proteases previously reported to contribute to the disruption of vacuole integrity observed during programmed cell death. Significance Collectively, our data indicate that anion efflux is an early key component of morphological and biochemical events leading to O3-induced programmed cell death. Because ion channels and more specifically anion channels assume a crucial position in cells, an understanding about the underlying role(s) for ion channels in the signalling pathway leading to programmed cell death is a subject that warrants future investigation. PMID:20967217

  10. Iron stimulates plasma-activated medium-induced A549 cell injury

    PubMed Central

    Adachi, Tetsuo; Nonomura, Saho; Horiba, Minori; Hirayama, Tasuku; Kamiya, Tetsuro; Nagasawa, Hideko; Hara, Hirokazu

    2016-01-01

    Non-thermal atmospheric pressure plasma is applicable to living cells and has emerged as a novel technology for cancer therapy. Plasma has recently been shown to affect cells not only by direct irradiation, but also by indirect treatments with previously prepared plasma-activated medium (PAM). Iron is an indispensable element but is also potentially toxic because it generates the hydroxyl radical (•OH) in the presence of hydrogen peroxide (H2O2) via the Fenton reaction. The aim of the present study was to demonstrate the contribution of iron to PAM-induced A549 adenocarcinoma cell apoptosis. We detected the generation of •OH and elevation of intracellular ferrous ions in PAM-treated cells and found that they were inhibited by iron chelator. The elevations observed in ferrous ions may have been due to their release from the intracellular iron store, ferritin. Hydroxyl radical-induced DNA injury was followed by the activation of poly(ADP-ribose) polymerase-1, depletion of NAD+ and ATP, and elevations in intracellular Ca2+. The sensitivities of normal cells such as smooth muscle cells and keratinocytes to PAM were less than that of A549 cells. These results demonstrated that H2O2 in PAM and/or •OH generated in the presence of iron ions disturbed the mitochondrial-nuclear network in cancer cells. PMID:26865334

  11. Magnetic Field-Induced T Cell Receptor Clustering by Nanoparticles Enhances T Cell Activation and Stimulates Antitumor Activity

    PubMed Central

    2015-01-01

    Iron–dextran nanoparticles functionalized with T cell activating proteins have been used to study T cell receptor (TCR) signaling. However, nanoparticle triggering of membrane receptors is poorly understood and may be sensitive to physiologically regulated changes in TCR clustering that occur after T cell activation. Nano-aAPC bound 2-fold more TCR on activated T cells, which have clustered TCR, than on naive T cells, resulting in a lower threshold for activation. To enhance T cell activation, a magnetic field was used to drive aggregation of paramagnetic nano-aAPC, resulting in a doubling of TCR cluster size and increased T cell expansion in vitro and after adoptive transfer in vivo. T cells activated by nano-aAPC in a magnetic field inhibited growth of B16 melanoma, showing that this novel approach, using magnetic field-enhanced nano-aAPC stimulation, can generate large numbers of activated antigen-specific T cells and has clinically relevant applications for adoptive immunotherapy. PMID:24564881

  12. Colchicum autumnale agglutinin activates all murine T-lymphocytes but does not induce the proliferation of all activated cells.

    PubMed

    Bemer, V; Van Damme, E J; Peumans, W J; Perret, R; Truffa-Bachi, P

    1996-08-25

    Plant lectins with mitogenic properties for T-lymphocytes have been particularly useful for the study of T-cell activation and effector functions. In the search for mitogenic lectins possessing activation features different from the ones associated with the already known mitogens, we found that an agglutinin isolated from Colchicum autumnale tubers, Colchicum autumnale agglutinin (CAA), possesses interesting properties. First, contrasting with the classical mitogens, CAA induces the proliferation of a fraction of the CD4+ and CD8+ mouse T-lymphocytes. Second, the CAA-induced proliferation requires MHC class II and CD4 molecules. Third, although only a fraction of T-cells enters into the cell cycle, all T-lymphocytes are activated and express high levels of the activation markers CD69 and CD44. Finally, CAA-stimulation is characterized by a particular pattern of the cytokine gene expression, reflected by the transcription of the IL2, IL5, and IFN-gamma genes, while the IL4 and IL10 genes remained silent. Taken together these data demonstrate that CAA activation does not conform to the pathway of T-cell triggering observed with classical mitogenes and represents a new tool for the analysis of T-cell activation.

  13. Phyllostachys edulis extract induces apoptosis signaling in osteosarcoma cells, associated with AMPK activation

    PubMed Central

    Chou, Chi-Wen; Cheng, Ya-Wen; Tsai, Chung-Hung

    2014-01-01

    Objective Bamboo is distributed worldwide, and its different parts are used as foods or as a traditional herb. Recently, antitumoral effects of bamboo extracts on several tumors have been increasingly reported; however, antitumoral activity of bamboo extracts on osteosarcoma remains unclear. In the present study, we investigated effects of an aqueous Phyllostachys edulis leaf extract (PEE) on osteosarcoma cells and the underlying mechanism of inhibition. Methods The growth of human osteosarcoma cell lines 143B and MG-63 and lung fibroblast MRC-5 cells was determined by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. Apoptosis was demonstrated using TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) assay and flow cytometric analysis. Phosphorylation and protein levels were determined by immunoblotting. Results After treatment with PEE, viability of 143B and MG-63 cells was dose-dependently reduced to 36.3%±1.6% of control values, which were similar to AICAR (5-aminoimidazole-4-carboxamide 1-β-D-ribofuranoside) treatments. In parallel, ratios of apoptotic cells and cells in the sub-G1 phase were significantly increased. Further investigation showed that PEE treatments led to activation of caspase cascades and changes of apoptotic mediators Bcl2, Bax, and p53. Consistently, our results revealed that PEE activated adenosine monophosphate-activated protein kinase (AMPK) signaling, and the AMPK activation was associated with the induction of apoptotic signaling. Conclusion Our results indicated that PEE suppressed the growth of 143B and MG-63 cells but moderately affected MRC-5 cells. PEE-induced apoptosis may attribute to AMPK activation and the following activation of apoptotic signaling cascades. These findings revealed that PEE possesses antitumoral activity on human osteosarcoma cells by manipulating AMPK signaling, suggesting that PEE alone or combined with regular antitumor drugs may be beneficial as osteosarcoma

  14. Platelet activating factor produced in vitro by Kaposi's sarcoma cells induces and sustains in vivo angiogenesis.

    PubMed Central

    Bussolino, F; Arese, M; Montrucchio, G; Barra, L; Primo, L; Benelli, R; Sanavio, F; Aglietta, M; Ghigo, D; Rola-Pleszczynski, M R

    1995-01-01

    Imbalance in the network of soluble mediators may play a pivotal role in the pathogenesis of Kaposi's sarcoma (KS). In this study, we demonstrated that KS cells grown in vitro produced and in part released platelet activating factor (PAF), a powerful lipid mediator of inflammation and cell-to-cell communication. IL-1, TNF, and thrombin enhanced the synthesis of PAF. PAF receptor mRNA and specific, high affinity binding site for PAF were present in KS cells. Nanomolar concentration of PAF stimulated the chemotaxis and chemokinesis of KS cells, endothelial cells, and vascular smooth muscle cells. The migration response to PAF was inhibited by WEB 2170, a hetrazepinoic PAF receptor antagonist. Because neoangiogenesis is essential for the growth and progression of KS and since PAF can activate vascular endothelial cells, we examined the potential role of PAF as an instrumental mediator of angiogenesis associated with KS. Conditioned medium (CM) from KS cells (KS-CM) or KS cells themselves induced angiogenesis and macrophage recruitment in a murine model in which Matrigel was injected subcutaneously. These effects were inhibited by treating mice with WEB 2170. Synthetic PAF or natural PAF extracted from plasma of patients with classical KS also induced angiogenesis, which in turn was inhibited by WEB 2170. The action of PAF was amplified by expression of other angiogenic factors and chemokines: these included basic and acidic fibroblast growth factor, placental growth factor, vascular endothelial growth factor and its specific receptor flk-1, hepatocyte growth factor, KC, and macrophage inflammatory protein-2. Treatment with WEB 2170 abolished the expression of the transcripts of these molecules within Matrigel containing KS-CM. These results indicate that PAF may cooperate with other angiogenic molecules and chemokines in inducing vascular development in KS. Images PMID:7543496

  15. A Trypanosoma cruzi alkaline antigen induces polyclonal B-cell activation of normal murine spleen cells by T-cell-independent, BCR-directed stimulation.

    PubMed

    Montes, C L; Zuñiga, E; Minoprio, P; Vottero-Cima, E; Gruppi, A

    1999-08-01

    We have previously reported that a cytosolic alkaline fraction (FI) obtained from epimastigotes of Trypanosoma cruzi promotes the activation, proliferation and differentiation of normal murine B cells into antibody-secreting plasmocytes. Neither the mechanism nor the cells involved in the FI-induced polyclonal B-cell activation were established. In this work we report that accessory cells are required for FI-induced polyclonal B-cell activation as no proliferative responses were obtained following treatment of normal spleen mononuclear cells (NSMC) with L-leucine methyl ester. Furthermore, FI did not induce the expression of CD25 on T cells and it promoted the proliferation of a T-cell-depleted population, indicating that it acts in a T-independent manner. We observed that NSMC were stimulated in vitro by FI-released cytokines, such as interleukin (IL)-4, IL-6 and IL-10, which are involved in B-cell proliferation and differentiation. Interestingly, while significant amounts of interferon-gamma (IFN-gamma) were found in culture supernatants we did not observe detectable levels of IL-2. Additionally, we found that B-cell receptor (BCR) and major histocompatibility complex (MHC) class II antigens were involved in the proliferative response induced by FI because antibodies directed against cell-surface immunoglobulin M (IgM), CD45 and MHC class II molecules inhibited the FI-induced B-cell proliferation. CD40 ligand (CD40L) did not participate in such a phenomenon.

  16. Coculture with intraocular lens material-activated macrophages induces an inflammatory phenotype in lens epithelial cells.

    PubMed

    Pintwala, Robert; Postnikoff, Cameron; Molladavoodi, Sara; Gorbet, Maud

    2015-03-01

    Cataracts are the leading cause of blindness worldwide, requiring surgical implantation of an intraocular lens. Despite evidence of leukocyte ingress into the postoperative lens, few studies have investigated the leukocyte response to intraocular lens materials. A novel coculture model was developed to examine macrophage activation by hydrophilic acrylic (poly(2-hydroxyethyl methacrylate)) and hydrophobic acrylic (polymethylmethacrylate) commercial intraocular lens. The human monocytic cell line THP-1 was differentiated into macrophages and cocultured with human lens epithelial cell line (HLE-B3) with or without an intraocular lens for one, two, four, or six days. Using flow cytometry and confocal microscopy, expression of the macrophage activation marker CD54 (intercellular adhesion molecule-1) and production of reactive oxygen species via the fluorogenic probe 2',7'-dichlorodihydrofluorescein diacetate were examined in macrophages. α-Smooth muscle actin, a transdifferentiation marker, was characterized in lens epithelial cells. The poly(2-hydroxyethyl methacrylate) intraocular lens prevented adhesion but induced significant macrophage activation (p < 0.03) versus control (no intraocular lens), while the polymethylmethacrylate intraocular lens enabled adhesion and multinucleated fusion, but induced no significant activation. Coculture with either intraocular lens increased reactive oxygen species production in macrophages after one day (p < 0.03) and increased expression of α-smooth muscle actin in HLE B-3 after six days, although only poly(2-hydroxyethyl methacrylate) induced a significant difference versus control (p < 0.01). Our results imply that-contrary to prior uveal biocompatibility understanding-macrophage adherence is not necessary for a strong inflammatory response to an intraocular lens, with hydrophilic surfaces inducing higher activation than hydrophobic surfaces. These findings provide a new method of inquiry into uveal

  17. Coculture with intraocular lens material-activated macrophages induces an inflammatory phenotype in lens epithelial cells.

    PubMed

    Pintwala, Robert; Postnikoff, Cameron; Molladavoodi, Sara; Gorbet, Maud

    2015-03-01

    Cataracts are the leading cause of blindness worldwide, requiring surgical implantation of an intraocular lens. Despite evidence of leukocyte ingress into the postoperative lens, few studies have investigated the leukocyte response to intraocular lens materials. A novel coculture model was developed to examine macrophage activation by hydrophilic acrylic (poly(2-hydroxyethyl methacrylate)) and hydrophobic acrylic (polymethylmethacrylate) commercial intraocular lens. The human monocytic cell line THP-1 was differentiated into macrophages and cocultured with human lens epithelial cell line (HLE-B3) with or without an intraocular lens for one, two, four, or six days. Using flow cytometry and confocal microscopy, expression of the macrophage activation marker CD54 (intercellular adhesion molecule-1) and production of reactive oxygen species via the fluorogenic probe 2',7'-dichlorodihydrofluorescein diacetate were examined in macrophages. α-Smooth muscle actin, a transdifferentiation marker, was characterized in lens epithelial cells. The poly(2-hydroxyethyl methacrylate) intraocular lens prevented adhesion but induced significant macrophage activation (p < 0.03) versus control (no intraocular lens), while the polymethylmethacrylate intraocular lens enabled adhesion and multinucleated fusion, but induced no significant activation. Coculture with either intraocular lens increased reactive oxygen species production in macrophages after one day (p < 0.03) and increased expression of α-smooth muscle actin in HLE B-3 after six days, although only poly(2-hydroxyethyl methacrylate) induced a significant difference versus control (p < 0.01). Our results imply that-contrary to prior uveal biocompatibility understanding-macrophage adherence is not necessary for a strong inflammatory response to an intraocular lens, with hydrophilic surfaces inducing higher activation than hydrophobic surfaces. These findings provide a new method of inquiry into uveal

  18. Loss of interferon-induced Stat1 phosphorylation in activated T cells.

    PubMed

    Van De Wiele, C Justin; Marino, Julie H; Whetsell, Michael E; Vo, Stephen S; Masengale, Rhonda M; Teague, T Kent

    2004-03-01

    Modulation of cytokine responsiveness following T cell activation represents an important mechanism that shapes the fate of T cells after encounters with antigens. We activated T cells in mice with superantigen and assessed their ability to phosphorylate Stat1 in response to interferon-gamma (IFN-gamma) and IFN-alpha. After 4 h of activation in vivo, T cells became deficient in their ability to phosphorylate Stat1 in response to either cytokine. The loss of IFN sensitivity was accompanied by increased mRNA transcription for multiple suppressors of cytokine signaling (SOCS) genes (SOCS1, SOCS3, and SOCS7). The transcript levels of these SOCS were elevated only during the early hours after activation and were at or below normal levels by 60 h. Likewise, the activation-induced inhibition of IFN-alpha signaling was transient, and sensitivity was restored by 3 days postactivation. The loss of sensitivity to IFN-gamma persisted, however, and was still evident at 3 days. These data suggest that SOCS-independent mechanisms specific for inhibition of IFN-gamma signaling may be present at later stages of the T cell response. The loss of Stat1 signaling may be a factor in differentiation of T cells during and after activation, and it could also represent a protective mechanism against the toxic effects of IFN-gamma during immune responses.

  19. Biscoumarin derivatives: Synthesis, crystal structure, theoretical studies and induced apoptosis activity on bladder urothelial cancer cell

    NASA Astrophysics Data System (ADS)

    Xin, Jia-jia; Li, Jing; Zhang, Zi-dan; Hu, Xing-bin; Li, Ming-kai

    2015-03-01

    In this study, five new biscoumarin derivatives (1-5) were synthesized and compound 4 inhibited the proliferation of the bladder urothelial cells (J82 cell line) obviously after 48 h treatment at different concentration (1, 5 and 10 μmol/L), and J82 cells were predominantly induced to apoptotic cell death after compound 4 treatment. Morphologic changes of bladder urothelial cancer cells were also observed under transmission electron microscopy (TEM) after compound 4 treatment. In addition, compound 4 had much less toxicity to human umbilical vein endothelial cells. To explore the possible anti-cancer mechanism of compound 4, two classical intramolecular Osbnd H⋯O hydrogen bonds (HBs) in their structures and the corresponding HB energies were performed with the density functional theory (DFT) [B3LYP/6-31G∗] method. Anti-bladder cancer activity of compound 4 is possible due to the intramolecular weakest HB energies.

  20. Dihydroartemisinin induces endothelial cell anoikis through the activation of the JNK signaling pathway

    PubMed Central

    Zhang, Jiao; Guo, Ling; Zhou, Xia; Dong, Fengyun; Li, Liqun; Cheng, Zuowang; Xu, Yinghua; Liang, Jiyong; Xie, Qi; Liu, Ju

    2016-01-01

    Angiogenesis is required for the growth and metastasis of solid tumors. The anti-malarial agent dihydroartemisinin (DHA) demonstrates potent anti-angiogenic activity, but the underlying molecular mechanisms are not yet fully understood. During the process of angiogenesis, endothelial cells migrating from existing capillaries may undergo programmed cell death after detaching from the extracellular matrix, a process that is defined as anchorage-dependent apoptosis or anoikis. In the present study, DHA-induced cell death was compared in human umbilical vein endothelial cells (HUVECs) cultured in suspension and attached to culture plates. In suspended HUVECs, the cell viability was decreased and apoptosis was increased with the treatment of 50 µM DHA for 5 h, while the same treatment did not affect the attached HUVECs. In addition, 50 µM DHA increased the phosphorylation of c-Jun N-terminal kinase (JNK) in suspended HUVECs, but not in attached HUVECs, for up to 5 h of treatment. The JNK inhibitor, SP600125, reversed DHA-induced cell death in suspended HUVECs, suggesting that the JNK pathway may mediate DHA-induced endothelial cell anoikis. The data from the present study indicates a novel mechanism for understanding the anti-angiogenic effects of DHA, which may be used as a component for chemotherapy. PMID:27602117

  1. 2-Methoxyestradiol induced Bax phosphorylation and apoptosis in human retinoblastoma cells via p38 MAPK activation.

    PubMed

    Min, Hongbo; Ghatnekar, Gautam S; Ghatnekar, Angela V; You, Xiaohong; Bu, Min; Guo, Xinyi; Bu, Shizhong; Shen, Bo; Huang, Qin

    2012-07-01

    Retinoblastoma (Rb) is a common childhood intraocular cancer that affects approximately 300 children each year in the United States alone. 2-Methoxyestradiol (2ME), an endogenous metabolite of 17-β-estradiol that dose not bind to nuclear estrogen receptor, exhibits potent apoptotic activity against rapidly growing tumor cells. Here, we report that 2ME induction of apoptosis was demonstrated by early fragmented DNA after 48 h of incubation with 10 µM 2ME in Rb cell lines. Subsequently, a decrease of proliferation was observed in a time- and dose-dependent manner. Further analysis of the mechanism indicates that p38 kinase plays a critical role in 2ME-induced apoptosis in Y79 cells, even though ERK was also activated by 2ME under the same conditions. Activation of p38 kinase also mediates 2ME induced Bax phosphorylated at Thr(167) after a 6 h treatment of 2ME, which in turn prevents formation of the Bcl-2-Bax heterodimer. Both p38 specific inhibitor, SB 203580, or p38 knockdown by specific siRNA, blocked 2ME induction of Bax phosphorylation. Furthermore, only transiently transfected mutant BaxT167A, but not Bax S163A, inhibited 2ME-induced apoptosis. In summary, our data suggest that 2ME induces apoptosis in human Rb cells by causing phosphorylation of p38 Mitogen-activated protein kinase (MAPK), which appears to be correlated with phosphorlation of Bax. This understanding of 2ME's ability may help develop it as a promising therapeutic candidate by inducing apoptosis in a Rb. PMID:21769948

  2. 12-Deoxyphorbols Promote Adult Neurogenesis by Inducing Neural Progenitor Cell Proliferation via PKC Activation

    PubMed Central

    Geribaldi-Doldán, Noelia; Flores-Giubi, Eugenia; Murillo-Carretero, Maribel; García-Bernal, Francisco; Carrasco, Manuel; Macías-Sánchez, Antonio J.; Domínguez-Riscart, Jesús; Verástegui, Cristina; Hernández-Galán, Rosario

    2016-01-01

    Background: Neuropsychiatric and neurological disorders frequently occur after brain insults associated with neuronal loss. Strategies aimed to facilitate neuronal renewal by promoting neurogenesis constitute a promising therapeutic option to treat neuronal death-associated disorders. In the adult brain, generation of new neurons occurs physiologically throughout the entire life controlled by extracellular molecules coupled to intracellular signaling cascades. Proteins participating in these cascades within neurogenic regions constitute potential pharmacological targets to promote neuronal regeneration of injured areas of the central nervous system. Methodology: We have performed in vitro and in vivo approaches to determine neural progenitor cell proliferation to understand whether activation of kinases of the protein kinase C family facilitates neurogenesis in the adult brain. Results: We have demonstrated that protein kinase C activation by phorbol-12-myristate-13-acetate induces neural progenitor cell proliferation in vitro. We also show that the nontumorogenic protein kinase C activator prostratin exerts a proliferative effect on neural progenitor cells in vitro. This effect can be reverted by addition of the protein kinase C inhibitor G06850, demonstrating that the effect of prostratin is mediated by protein kinase C activation. Additionally, we show that prostratin treatment in vivo induces proliferation of neural progenitor cells within the dentate gyrus of the hippocampus and the subventricular zone. Finally, we describe a library of diterpenes with a 12-deoxyphorbol structure similar to that of prostratin that induces a stronger effect than prostratin on neural progenitor cell proliferation both in vitro and in vivo. Conclusions: This work suggests that protein kinase C activation is a promising strategy to expand the endogenous neural progenitor cell population to promote neurogenesis and highlights the potential of 12-deoxyphorbols as pharmaceutical

  3. Antibodies That Block or Activate Mouse B Cell Activating Factor of the Tumor Necrosis Factor (TNF) Family (BAFF), Respectively, Induce B Cell Depletion or B Cell Hyperplasia.

    PubMed

    Kowalczyk-Quintas, Christine; Schuepbach-Mallepell, Sonia; Vigolo, Michele; Willen, Laure; Tardivel, Aubry; Smulski, Cristian R; Zheng, Timothy S; Gommerman, Jennifer; Hess, Henry; Gottenberg, Jacques-Eric; Mackay, Fabienne; Donzé, Olivier; Schneider, Pascal

    2016-09-16

    B cell activating factor of the TNF family (BAFF), also known as B lymphocyte stimulator, is a ligand required for the generation and maintenance of B lymphocytes. In this study, the ability of different monoclonal antibodies to recognize, inhibit, or activate mouse BAFF was investigated. One of them, a mouse IgG1 named Sandy-2, prevented the binding of BAFF to all of its receptors, BAFF receptor, transmembrane activator and calcium modulating ligand interactor, and B cell maturation antigen, at a stoichiometric ratio; blocked the activity of mouse BAFF on a variety of cell-based reporter assays; and antagonized the prosurvival action of BAFF on primary mouse B cells in vitro A single administration of Sandy-2 in mice induced B cell depletion within 2 weeks, down to levels close to those observed in BAFF-deficient mice. This depletion could then be maintained with a chronic treatment. Sandy-2 and a previously described rat IgG1 antibody, 5A8, also formed a pair suitable for the sensitive detection of endogenous circulating BAFF by ELISA or using a homogenous assay. Interestingly, 5A8 and Sandy-5 displayed activities opposite to that of Sandy-2 by stimulating recombinant BAFF in vitro and endogenous BAFF in vivo These tools will prove useful for the detection and functional manipulation of endogenous mouse BAFF and provide an alternative to the widely used BAFF receptor-Fc decoy receptor for the specific depletion of BAFF in mice. PMID:27451394

  4. Cadmium induces the activation of cell wall integrity pathway in budding yeast.

    PubMed

    Xiong, Bing; Zhang, Lilin; Xu, Huihui; Yang, Yi; Jiang, Linghuo

    2015-10-01

    MAP kinases are important signaling molecules regulating cell survival, proliferation and differentiation, and can be activated by cadmium stress. In this study, we demonstrate that cadmium induces phosphorylation of the yeast cell wall integrity (CWI) pathway_MAP kinase Slt2, and this cadmium-induced CWI activation is mediated by the cell surface sensor Mid2 through the GEF Rom1, the central regulator Rho1 and Bck1. Nevertheless, cadmium stress does not affect the subcellular localization of Slt2 proteins. In addition, this cadmium-induced CWI activation is independent on the calcium/calcineurin signaling and the high osmolarity glycerol (HOG) signaling pathways in yeast cells. Furthermore, we tested the cadmium sensitivity of 42 paired double-gene deletion mutants between six CWI components and seven components of the HOG pathway. Our results indicate that the CWI pathway is epistatic to the HOG pathway in cadmium sensitivity. However, gene deletion mutations for the Swi4/Swi6 transcription factor complex show synergistic effects with mutations of HOG components in cadmium sensitivity.

  5. Retargeting cytokine-induced killer cell activity by CD16 engagement with clinical-grade antibodies

    PubMed Central

    Cappuzzello, Elisa; Tosi, Anna; Zanovello, Paola; Sommaggio, Roberta; Rosato, Antonio

    2016-01-01

    ABSTRACT Cytokine-induced Killer (CIK) cells are a heterogeneous population of ex vivo expanded T lymphocytes capable of MHC-unrestricted antitumor activity, which share phenotypic and functional features with both NK and T cells. Preclinical data and initial clinical studies demonstrated their high tolerability in vivo, supporting CIK cells as a promising cell population for adoptive cell immunotherapy. In this study, we report for the first time that CIK cells display a donor-dependent expression of CD16, which can be engaged by trastuzumab or cetuximab to exert a potent antibody-dependent cell-mediated cytotoxicity (ADCC) against ovarian and breast cancer cell lines, leading to an increased lytic activity in vitro, and an enhanced therapeutic efficacy in vivo. Thus, an efficient tumor antigen-specific retargeting can be achieved by a combination therapy with clinical-grade monoclonal antibodies already widely used in cancer therapy, and CIK cell populations that are easily expandable in very large numbers, inexpensive, safe and do not require genetic manipulations. Overall, these data provide a new therapeutic strategy for the treatment of Her2 and EGFR expressing tumors by adoptive cell therapy, which could find wide implementation and application, and could also be expanded to the use of additional therapeutic antibodies. PMID:27622068

  6. Retargeting cytokine-induced killer cell activity by CD16 engagement with clinical-grade antibodies.

    PubMed

    Cappuzzello, Elisa; Tosi, Anna; Zanovello, Paola; Sommaggio, Roberta; Rosato, Antonio

    2016-08-01

    Cytokine-induced Killer (CIK) cells are a heterogeneous population of ex vivo expanded T lymphocytes capable of MHC-unrestricted antitumor activity, which share phenotypic and functional features with both NK and T cells. Preclinical data and initial clinical studies demonstrated their high tolerability in vivo, supporting CIK cells as a promising cell population for adoptive cell immunotherapy. In this study, we report for the first time that CIK cells display a donor-dependent expression of CD16, which can be engaged by trastuzumab or cetuximab to exert a potent antibody-dependent cell-mediated cytotoxicity (ADCC) against ovarian and breast cancer cell lines, leading to an increased lytic activity in vitro, and an enhanced therapeutic efficacy in vivo. Thus, an efficient tumor antigen-specific retargeting can be achieved by a combination therapy with clinical-grade monoclonal antibodies already widely used in cancer therapy, and CIK cell populations that are easily expandable in very large numbers, inexpensive, safe and do not require genetic manipulations. Overall, these data provide a new therapeutic strategy for the treatment of Her2 and EGFR expressing tumors by adoptive cell therapy, which could find wide implementation and application, and could also be expanded to the use of additional therapeutic antibodies. PMID:27622068

  7. BMP9-Induced Survival Effect in Liver Tumor Cells Requires p38MAPK Activation

    PubMed Central

    García-Álvaro, María; Addante, Annalisa; Roncero, Cesáreo; Fernández, Margarita; Fabregat, Isabel; Sánchez, Aránzazu; Herrera, Blanca

    2015-01-01

    The study of bone morphogenetic proteins (BMPs) role in tumorigenic processes, and specifically in the liver, has gathered importance in the last few years. Previous studies have shown that BMP9 is overexpressed in about 40% of hepatocellular carcinoma (HCC) patients. In vitro data have also shown evidence that BMP9 has a pro-tumorigenic action, not only by inducing epithelial to mesenchymal transition (EMT) and migration, but also by promoting proliferation and survival in liver cancer cells. However, the precise mechanisms driving these effects have not yet been established. In the present work, we deepened our studies into the intracellular mechanisms implicated in the BMP9 proliferative and pro-survival effect on liver tumor cells. In HepG2 cells, BMP9 induces both Smad and non-Smad signaling cascades, specifically PI3K/AKT and p38MAPK. However, only the p38MAPK pathway contributes to the BMP9 growth-promoting effect on these cells. Using genetic and pharmacological approaches, we demonstrate that p38MAPK activation, although dispensable for the BMP9 proliferative activity, is required for the BMP9 protective effect on serum withdrawal-induced apoptosis. These findings contribute to a better understanding of the signaling pathways involved in the BMP9 pro-tumorigenic role in liver tumor cells. PMID:26343646

  8. Endothelial cell apoptosis induced by bacteria-activated platelets requires caspase-8 and -9 and generation of reactive oxygen species.

    PubMed

    Kuckleburg, Christopher J; Tiwari, Raksha; Czuprynski, Charles J

    2008-02-01

    A common feature of severe sepsis is vascular inflammation and damage to the endothelium. Because platelets can be directly activated by bacteria and endotoxin, these cells may play an important role in determining the outcome of sepsis. For example, inhibiting platelet interactions with the endothelium has been shown to attenuate endothelial cell damage and improve survival during sepsis. Although not entirely understood, the interactions between bacteria-activated platelets and the endothelium may play a key role in the vascular pathology of bacterial sepsis. Haemophilus somnus is a bacterial pathogen that causes diffuse vascular inflammation and endothelial damage. In some cases H. somnus infection results in an acute and fatal form of vasculitis in the cerebral microvasculature known as thrombotic meningoencephalitis (TME). In this study, we have characterized the mechanisms involved in endothelial cell apoptosis induced by activated platelets. We observed that direct contact between H. somnus-activated platelets and endothelial cells induced significant levels of apoptosis; however, Fas receptor activation on bovine endothelial cells was not able to induce apoptosis unless protein synthesis was disrupted. Endothelial cell apoptosis by H. somnus-activated platelets required activation of both caspase-8 and caspase-9, as inhibitors of either caspase inhibited apoptosis. Furthermore, activated platelets induced endothelial cell production of reactive oxygen species (ROS) and disrupting ROS activity in endothelial cells significantly inhibited apoptosis. These findings suggest that bacterial activation of platelets may contribute to endothelial cell dysfunction observed during sepsis, specifically by inducing endothelial cell apoptosis.

  9. Low-power laser irradiation inhibits Aβ25-35-induced cell apoptosis through Akt activation

    NASA Astrophysics Data System (ADS)

    Zhang, Zhigang; Tang, Yonghong

    2009-08-01

    Low-power laser irradiation (LPLI) can modulate various cellular processes such as proliferation, differentiation and apoptosis. Recently, LPLI has been applied to moderate Alzheimer's disease (AD), but the underlying mechanism remains unknown. The protective role of LPLI against the amyloid beta peptide (Aβ), a major constituent of AD plaques, has not been studied. PI3K/Akt pathway is extremely important in protecting cells from apoptosis caused by diverse stress stimuli. However, whether LPLI can inhibit Aβ-induced apoptosis through Akt activation is still unclear. In current study, using FRET (fluorescence resonance energy transfer) technique, we investigated the activity of Akt in response to LPLI treatment. B kinase activity reporter (BKAR), a recombinant FRET probe of Akt, was utilized to dynamically detect the activation of Akt after LPLI treatment. The results show that LPLI promoted the activation of Akt. Moreover, LPLI inhibits apoptosis induced by Aβ25-35 and the apoptosis inhibition can be abolished by wortmannin, a specific inhibitor of PI3K/Akt. Taken together, these results suggest that LPLI can inhibit Aβ25-35-induced cell apoptosis through Akt activation.

  10. FK866-induced NAMPT inhibition activates AMPK and downregulates mTOR signaling in hepatocarcinoma cells

    SciTech Connect

    Schuster, Susanne; Penke, Melanie; Gorski, Theresa; Gebhardt, Rolf; Weiss, Thomas S.; Kiess, Wieland; Garten, Antje

    2015-03-06

    Background: Nicotinamide phosphoribosyltransferase (NAMPT) is the key enzyme of the NAD salvage pathway starting from nicotinamide. Cancer cells have an increased demand for NAD due to their high proliferation and DNA repair rate. Consequently, NAMPT is considered as a putative target for anti-cancer therapies. There is evidence that AMP-activated protein kinase (AMPK) and mammalian target of rapamycin (mTOR) become dysregulated during the development of hepatocellular carcinoma (HCC). Here, we investigated the effects of NAMPT inhibition by its specific inhibitor FK866 on the viability of hepatocarcinoma cells and analyzed the effects of FK866 on the nutrient sensor AMPK and mTOR complex1 (mTORC1) signaling. Results: FK866 markedly decreased NAMPT activity and NAD content in hepatocarcinoma cells (Huh7 cells, Hep3B cells) and led to delayed ATP reduction which was associated with increased cell death. These effects could be abrogated by administration of nicotinamide mononucleotide (NMN), the enzyme product of NAMPT. Our results demonstrated a dysregulation of the AMPK/mTOR pathway in hepatocarcinoma cells compared to non-cancerous hepatocytes with a higher expression of mTOR and a lower AMPKα activation in hepatocarcinoma cells. We found that NAMPT inhibition by FK866 significantly activated AMPKα and inhibited the activation of mTOR and its downstream targets p70S6 kinase and 4E-BP1 in hepatocarcinoma cells. Non-cancerous hepatocytes were less sensitive to FK866 and did not show changes in AMPK/mTOR signaling after FK866 treatment. Conclusion: Taken together, these findings reveal an important role of the NAMPT-mediated NAD salvage pathway in the energy homeostasis of hepatocarcinoma cells and suggest NAMPT inhibition as a potential treatment option for HCC. - Highlights: • FK866 increases cell death in p53-deficient hepatocarcinoma cells. • AMPK/mTOR signaling is dysregulated in hepatocarcinoma cells. • FK866-induced NAMPT inhibition activates AMPK

  11. mAChRs activation induces epithelial-mesenchymal transition on lung epithelial cells

    PubMed Central

    2014-01-01

    Background Epithelial-mesenchymal transition (EMT) has been proposed as a mechanism in the progression of airway diseases and cancer. Here, we explored the role of acetylcholine (ACh) and the pathway involved in the process of EMT, as well as the effects of mAChRs antagonist. Methods Human lung epithelial cells were stimulated with carbachol, an analogue of ACh, and epithelial and mesenchymal marker proteins were evaluated using western blot and immunofluorescence analyses. Results Decreased E-cadherin expression and increased vimentin and α-SMA expression induced by TGF-β1 in alveolar epithelial cell (A549) were significantly abrogated by the non-selective mAChR antagonist atropine and enhanced by the acetylcholinesterase inhibitor physostigmine. An EMT event also occurred in response to physostigmine alone. Furthermore, ChAT express and ACh release by A549 cells were enhanced by TGF-β1. Interestingly, ACh analogue carbachol also induced EMT in A549 cells as well as in bronchial epithelial cells (16HBE) in a time- and concentration-dependent manner, the induction of carbachol was abrogated by selective antagonist of M1 (pirenzepine) and M3 (4-DAMP) mAChRs, but not by M2 (methoctramine) antagonist. Moreover, carbachol induced TGF-β1 production from A549 cells concomitantly with the EMT process. Carbachol-induced EMT occurred through phosphorylation of Smad2/3 and ERK, which was inhibited by pirenzepine and 4-DAMP. Conclusions Our findings for the first time indicated that mAChR activation, perhaps via M1 and M3 mAChR, induced lung epithelial cells to undergo EMT and provided insights into novel therapeutic strategies for airway diseases in which lung remodeling occurs. PMID:24678619

  12. Sphingosine induces phospholipase D and mitogen activated protein kinase in vascular smooth muscle cells.

    PubMed

    Taher, M M; Abd-Elfattah, A S; Sholley, M M

    1998-12-01

    The enzymes phospholipase D and diacylglycerol kinase generate phosphatidic acid which is considered to be a mitogen. Here we report that sphingosine produced a significant amount of phosphatidic acid in vascular smooth muscle cells from the rat aorta. The diacylglycerol kinase inhibitor R59 949 partially depressed sphingosine induced phosphatidic acid formation, suggesting that activation of phospholipase C and diacylglycerol kinase can not account for the bulk of phosphatidic acid produced and that additional pathways such as phospholipase D may contribute to this. Further, we have shown that phosphatidylethanol was produced by sphingosine when vascular smooth muscle cells were stimulated in the presence of ethanol. Finally, as previously shown for other cell types, sphingosine stimulated mitogen-activated protein kinase in vascular smooth muscle cells.

  13. Metastable primordial germ cell-like state induced from mouse embryonic stem cells by Akt activation

    SciTech Connect

    Yamano, Noriko; Kimura, Tohru; Watanabe-Kushima, Shoko; Shinohara, Takashi; Nakano, Toru

    2010-02-12

    Specification to primordial germ cells (PGCs) is mediated by mesoderm-induction signals during gastrulation. We found that Akt activation during in vitro mesodermal differentiation of embryonic stem cells (ESCs) generated self-renewing spheres with differentiation states between those of ESCs and PGCs. Essential regulators for PGC specification and their downstream germ cell-specific genes were expressed in the spheres, indicating that the sphere cells had commenced differentiation to the germ lineage. However, the spheres did not proceed to spermatogenesis after transplantation into testes. Sphere cell transfer to the original feeder-free ESC cultures resulted in chaotic differentiation. In contrast, when the spheres were cultured on mouse embryonic fibroblasts or in the presence of ERK-cascade and GSK3 inhibitors, reversion to the ESC-like state was observed. These results indicate that Akt signaling promotes a novel metastable and pluripotent state that is intermediate to those of ESCs and PGCs.

  14. N-nitroso-N-ethylurea activates DNA damage surveillance pathways and induces transformation in mammalian cells

    PubMed Central

    2014-01-01

    Background The DNA damage checkpoint signalling cascade sense damaged DNA and coordinates cell cycle arrest, DNA repair, and/or apoptosis. However, it is still not well understood how the signalling system differentiates between different kinds of DNA damage. N-nitroso-N-ethylurea (NEU), a DNA ethylating agent induces both transversions and transition mutations. Methods Immunoblot and comet assays were performed to detect DNA breaks and activation of the canonical checkpoint signalling kinases following NEU damage upto 2 hours. To investigate whether mismatch repair played a role in checkpoint activation, knock-down studies were performed while flow cytometry analysis was done to understand whether the activation of the checkpoint kinases was cell cycle phase specific. Finally, breast epithelial cells were grown as 3-dimensional spheroid cultures to study whether NEU can induce upregulation of vimentin as well as disrupt cell polarity of the breast acini, thus causing transformation of epithelial cells in culture. Results We report a novel finding that NEU causes activation of major checkpoint signalling kinases, Chk1 and Chk2. This activation is temporally controlled with Chk2 activation preceding Chk1 phosphorylation, and absence of cross talk between the two parallel signalling pathways, ATM and ATR. Damage caused by NEU leads to the temporal formation of both double strand and single strand breaks. Activation of checkpoints following NEU damage is cell cycle phase dependent wherein Chk2 is primarily activated during G2-M phase whilst in S phase, there is immediate Chk1 phosphorylation and delayed Chk2 response. Surprisingly, the mismatch repair system does not play a role in checkpoint activation, at doses and duration of NEU used in the experiments. Interestingly, NEU caused disruption of the well-formed polarised spheroid archithecture and upregulation of vimentin in three-dimensional breast acini cultures of non-malignant breast epithelial cells upon NEU

  15. Th2-Associated Alternative Kupffer Cell Activation Promotes Liver Fibrosis without Inducing Local Inflammation

    PubMed Central

    López-Navarrete, Giuliana; Ramos-Martínez, Espiridión; Suárez-Álvarez, Karina; Aguirre-García, Jesús; Ledezma-Soto, Yadira; León-Cabrera, Sonia; Gudiño-Zayas, Marco; Guzmán, Carolina; Gutiérrez-Reyes, Gabriela; Hernández-Ruíz, Joselín; Camacho-Arroyo, Ignacio; Robles-Díaz, Guillermo; Kershenobich, David; Terrazas, Luis I.; Escobedo, Galileo

    2011-01-01

    Cirrhosis is the final outcome of liver fibrosis. Kupffer cell-mediated hepatic inflammation is considered to aggravate liver injury and fibrosis. Alternatively-activated macrophages are able to control chronic inflammatory events and trigger wound healing processes. Nevertheless, the role of alternative Kupffer cell activation in liver harm is largely unclear. Thus, we evaluated the participation of alternatively-activated Kupffer cells during liver inflammation and fibrosis in the murine model of carbon tetrachloride-induced hepatic damage. To stimulate alternative activation in Kupffer cells, 20 Taenia crassiceps (Tc) larvae were inoculated into BALBc/AnN female mice. Six weeks post-inoculation, carbon tetrachloride or olive oil were orally administered to Tc-inoculated and non-inoculated mice twice per week during other six weeks. The initial exposure of animals to T. crassiceps resulted in high serum concentrations of IL-4 accompanied by a significant increase in the hepatic mRNA levels of Ym-1, with no alteration in iNOS expression. In response to carbon tetrachloride, recruitment of inflammatory cell populations into the hepatic parenchyma was 5-fold higher in non-inoculated animals than Tc-inoculated mice. In contrast, carbon tetrachloride-induced liver fibrosis was significantly less in non-inoculated animals than in the Tc-inoculated group. The latter showed elevated IL-4 serum levels and low IFN-γ concentrations during the whole experiment, associated with hepatic expression of IL-4, TGF-β, desmin and α-sma, as well as increased mRNA levels of Arg-1, Ym-1, FIZZ-1 and MMR in Kupffer cells. These results suggest that alternative Kupffer cell activation is favored in a Th2 microenvironment, whereby such liver resident macrophages could exhibit a dichotomic role during chronic hepatic damage, being involved in attenuation of the inflammatory response but at the same time exacerbation of liver fibrosis. PMID:22110380

  16. SV40 enhancer activation during retinoic acid-induced differentiation of F9 embryonal carcinoma cells.

    PubMed Central

    Sleigh, M J; Lockett, T J

    1985-01-01

    The transient expression vector pSV2CAT, which carries the bacterial chloramphenicol acetyl transferase (CAT) gene under the control of the SV40 early promoter, was used to transfect the murine embryonal carcinoma cell line F9 at various times during the retinoic acid-induced differentiation of these cells. Expression of the CAT gene under SV40 promoter control was found to increase markedly on F9 cell differentiation, measured relative to expression from the thymidine kinase promoter in the same cells. A series of constructs was prepared to identify the features of the SV40 early promoter required for transcription in differentiated and undifferentiated cells, as well as the factors limiting transcription in each case. The increased transcription seen on F9 cell differentiation was not observed when cells were transfected with molecules lacking a functional enhancer. It appears that as embryonal carcinoma cells differentiate, increased SV40 transcription results from enhancer sequence activation. In both differentiated and undifferentiated cell types the level of transcription was found to be limited by the availability and/or activity of cellular factors necessary for enhancer function. Images Fig. 1. PMID:3004973

  17. A Novel Resveratrol Based Tubulin Inhibitor Induces Mitotic Arrest and Activates Apoptosis in Cancer Cells

    PubMed Central

    Thomas, Elizabeth; Gopalakrishnan, Vidya; Hegde, Mahesh; Kumar, Sujeet; Karki, Subhas S.; Raghavan, Sathees C.; Choudhary, Bibha

    2016-01-01

    Resveratrol is one of the most widely studied bioactive plant polyphenols which possesses anticancer properties. Previously we have reported synthesis, characterization and identification of a novel resveratrol analog, SS28. In the present study, we show that SS28 induced cytotoxicity in several cancer cell lines ex vivo with an IC50 value of 3–5 μM. Mechanistic evaluation of effect of SS28 in non-small cell lung cancer cell line (A549) and T-cell leukemic cell line (CEM) showed that it inhibited Tubulin polymerization during cell division to cause cell cycle arrest at G2/M phase of the cell cycle at 12–18 h time period. Immunofluorescence studies confirmed the mitotic arrest upon treatment with SS28. Besides, we show that SS28 binds to Tubulin with a dissociation constant of 0.414 ± 0.11 μM. Further, SS28 treatment resulted in loss of mitochondrial membrane potential, activation of Caspase 9 and Caspase 3, leading to PARP-1 cleavage and finally cell death via intrinsic pathway of apoptosis. Importantly, treatment with SS28 resulted in regression of tumor in mice. Hence, our study reveals the antiproliferative activity of SS28 by disrupting microtubule dynamics by binding to its cellular target Tubulin and its potential to be developed as an anticancer molecule. PMID:27748367

  18. Platelet-activating factor induces cell cycle arrest and disrupts the DNA damage response in mast cells

    PubMed Central

    Puebla-Osorio, N; Damiani, E; Bover, L; Ullrich, S E

    2015-01-01

    Platelet-activating factor (PAF) is a potent phospholipid modulator of inflammation that has diverse physiological and pathological functions. Previously, we demonstrated that PAF has an essential role in ultraviolet (UV)-induced immunosuppression and reduces the repair of damaged DNA, suggesting that UV-induced PAF is contributing to skin cancer initiation by inducing immune suppression and also affecting a proper DNA damage response. The exact role of PAF in modulating cell proliferation, differentiation or transformation is unclear. Here, we investigated the mechanism(s) by which PAF affects the cell cycle and impairs early DNA damage response. PAF arrests proliferation in transformed and nontransformed human mast cells by reducing the expression of cyclin-B1 and promoting the expression of p21. PAF-treated cells show a dose-dependent cell cycle arrest mainly at G2–M, and a decrease in the DNA damage response elements MCPH1/BRIT-1 and ataxia telangiectasia and rad related (ATR). In addition, PAF disrupts the localization of p-ataxia telangiectasia mutated (p-ATM), and phosphorylated-ataxia telangiectasia and rad related (p-ATR) at the site of DNA damage. Whereas the potent effect on cell cycle arrest may imply a tumor suppressor activity for PAF, the impairment of proper DNA damage response might implicate PAF as a tumor promoter. The outcome of these diverse effects may be dependent on specific cues in the microenvironment. PMID:25950475

  19. Belinostat-induced apoptosis and growth inhibition in pancreatic cancer cells involve activation of TAK1-AMPK signaling axis

    SciTech Connect

    Wang, Bing Wang, Xin-bao; Chen, Li-yu; Huang, Ling; Dong, Rui-zen

    2013-07-19

    Highlights: •Belinostat activates AMPK in cultured pancreatic cancer cells. •Activation of AMPK is important for belinostat-induced cytotoxic effects. •ROS and TAK1 are involved in belinostat-induced AMPK activation. •AMPK activation mediates mTOR inhibition by belinostat. -- Abstract: Pancreatic cancer accounts for more than 250,000 deaths worldwide each year. Recent studies have shown that belinostat, a novel pan histone deacetylases inhibitor (HDACi) induces apoptosis and growth inhibition in pancreatic cancer cells. However, the underlying mechanisms are not fully understood. In the current study, we found that AMP-activated protein kinase (AMPK) activation was required for belinostat-induced apoptosis and anti-proliferation in PANC-1 pancreatic cancer cells. A significant AMPK activation was induced by belinostat in PANC-1 cells. Inhibition of AMPK by RNAi knockdown or dominant negative (DN) mutation significantly inhibited belinostat-induced apoptosis in PANC-1 cells. Reversely, AMPK activator AICAR and A-769662 exerted strong cytotoxicity in PANC-1 cells. Belinostat promoted reactive oxygen species (ROS) production in PANC-1 cells, increased ROS induced transforming growth factor-β-activating kinase 1 (TAK1)/AMPK association to activate AMPK. Meanwhile, anti-oxidants N-Acetyl-Cysteine (NAC) and MnTBAP as well as TAK1 shRNA knockdown suppressed belinostat-induced AMPK activation and PANC-1 cell apoptosis. In conclusion, we propose that belinostat-induced apoptosis and growth inhibition require the activation of ROS-TAK1-AMPK signaling axis in cultured pancreatic cancer cells.

  20. Linalool Inhibits LPS-Induced Inflammation in BV2 Microglia Cells by Activating Nrf2.

    PubMed

    Li, Yang; Lv, Ou; Zhou, Fenggang; Li, Qingsong; Wu, Zhichao; Zheng, Yongri

    2015-07-01

    Linalool, a natural compound of the essential oils, has been reported to have anti-inflammatory effects. This study aimed to investigate the anti-inflammatory effects and mechanism of linalool in LPS-stimulated BV2 microglia cells. BV2 microglia cells were stimulated with LPS in the presence or absence of linalool. The production of inflammatory mediators TNF-α, IL-1β, NO, and PGE2 as well as Nrf2, HO-1 expression were detected. Our results showed that linalool inhibited LPS-induced TNF-α, IL-1β, NO, and PGE2 production in a dose-dependent manner. Linalool also inhibited LPS-induced NF-κB activation. Treatment of linalool induced nuclear translocation of Nrf2 and expression of HO-1. In addition, our results showed that the anti-inflammatory effect of linalool was attenuated by transfection with Nrf2 siRNA. In conclusion, these results suggested that linalool inhibits LPS-induced inflammation in BV2 microglia cells by activating Nrf2/HO-1 signaling pathway.

  1. An inducible transcription factor activates expression of human immunodeficiency virus in T cells

    NASA Astrophysics Data System (ADS)

    Nabel, Gary; Baltimore, David

    1987-04-01

    Human immunodeficiency virus (HIV) production from latently infected T lymphocytes can be induced with compounds that activate the cells to secrete lymphokines1,2. The elements in the HIV genome which control activation are not known but expression might be regulated through a variety of DNA elements. The cis-acting control elements of the viral genome are enhancer and promoter regions. The virus also encodes trans-acting factors specified by the tat-III (refs 3-6) and art genes7. We have examined whether products specific to activated T cells might stimulate viral transcription by binding to regions on viral DNA. Activation of T cells, which increases HIV expression up to 50-fold, correlated with induction of a DNA binding protein indistinguishable from a recognized transcription factor, called NF-κB (ref. 8), with binding sites in the viral enhancer. Mutation of these binding sites abolished inducibility. That NF-κB acts in synergy with the viral tat-III gene product to enhance HIV expression in T cells may have implications for the pathogenesis of AIDS (acquired immune deficiency syndrome).

  2. Sweetness-induced activation of membrane dipole potential in STC-1 taste cells.

    PubMed

    Chen, Li-Chun; Xie, Ning-Ning; Deng, Shao-Ping

    2016-12-01

    The biological functions of cell membranes strongly influence the binding and transport of molecular species. We developed STC-1 cell line stably expressing the sweet taste receptor (T1R2/T1R3), and explored the possible correlation between sweeteners and membrane dipole potential of STC-1 cells. In this study, sweetener-induced dipole potential activation was elucidated using a fluorescence-based measurement technique, by monitoring the voltage sensitive probe Di-8-ANEPPS using a dual wavelength ratiometric approach. It indicated that the presence of sweeteners resulted in cell membrane dipole potential change, and interaction of artificial sweeteners with taste cells resulted in a greater reduction in potential compared with natural sweeteners. Our work presents a newly developed approach using a fluorescence-based measurement technique to study sweetener-induced dipole potential activation of STC-1 cells. This new approach could be used as a complementary tool to study the function of sweet taste receptors or other GPCRs and helps to understand the basis sweetness mechanism. PMID:27374594

  3. Zinc deficiency enhanced inflammatory response by increasing immune cell activation and inducing IL6 promoter demethylation

    PubMed Central

    Wong, Carmen P.; Rinaldi, Nicole A.; Ho, Emily

    2015-01-01

    Scope Zinc deficiency results in immune dysfunction and promotes systemic inflammation. The objective of this study was to examine the effects of zinc deficiency on cellular immune activation and epigenetic mechanisms that promote inflammation. This work is potentially relevant to the aging population given that age-related immune defects, including chronic inflammation, coincide with declining zinc status. Methods and results An in vitro cell culture system and the aged mouse model were used to characterize immune activation and DNA methylation profiles that may contribute to the enhanced proinflammatory response mediated by zinc deficiency. Zinc deficiency up-regulated cell activation markers ICAM1, MHC class II, and CD86 in THP1 cells, that coincided with increased IL1β and IL6 responses following LPS stimulation. A decreased zinc status in aged mice was similarly associated with increased ICAM1 and IL6 gene expression. Reduced IL6 promoter methylation was observed in zinc deficient THP1 cells, as well as in aged mice and human lymphoblastoid cell lines derived from aged individuals. Conclusion Zinc deficiency induced inflammatory response in part by eliciting aberrant immune cell activation and altered promoter methylation. Our results suggested potential interactions between zinc status, epigenetics, and immune function, and how their dysregulation could contribute to chronic inflammation. PMID:25656040

  4. Genotoxicity induced by a shale oil byproduct in Chinese hamster cells following metabolic activation

    SciTech Connect

    Okinaka, R.T.; Nickols, J.W.; Chen, D.J.; Strniste, G.F.

    1982-01-01

    A process water obtained from a holding tank during the surface retorting of oil shale has been shown to induce a linear dose response of 100 histidine revertants/sub ..mu../1 in the Ames/Salmonella test. The complex mixture has also previously been shown to induce genotoxicity in mammalian cells following activation by near ultraviolet light and natural sunlight. This report focuses on the effects of a particular oil shale retort process water on cultured Chinese hamster cells following metabolic activation by either rat liver homogenate or lethally irradiated but metabolically competent Syrian hamster embryonic cells. Cytotoxic and mutagenic responses induced by the process water and a fractionated sample from it containing the majority of the mutagenic activity (as assessed by the Salmonella test) were measured under conditions designed to optimally measure the mutagenic potency of the promutagen, benzo(a)pyrene. These results suggest a possible discrepancy in the genotoxic potential of this complex mixture when various methods are utilized to measure its potential.

  5. Boletus edulis biologically active biopolymers induce cell cycle arrest in human colon adenocarcinoma cells.

    PubMed

    Lemieszek, Marta Kinga; Cardoso, Claudia; Ferreira Milheiro Nunes, Fernando Hermínio; Ramos Novo Amorim de Barros, Ana Isabel; Marques, Guilhermina; Pożarowski, Piotr; Rzeski, Wojciech

    2013-04-25

    The use of biologically active compounds isolated from edible mushrooms against cancer raises global interest. Anticancer properties are mainly attributed to biopolymers including mainly polysaccharides, polysaccharopeptides, polysaccharide proteins, glycoproteins and proteins. In spite of the fact that Boletus edulis is one of the widely occurring and most consumed edible mushrooms, antitumor biopolymers isolated from it have not been exactly defined and studied so far. The present study is an attempt to extend this knowledge on molecular mechanisms of their anticancer action. The mushroom biopolymers (polysaccharides and glycoproteins) were extracted with hot water and purified by anion-exchange chromatography. The antiproliferative activity in human colon adenocarcinoma cells (LS180) was screened by means of MTT and BrdU assays. At the same time fractions' cytotoxicity was examined on the human colon epithelial cells (CCD 841 CoTr) by means of the LDH assay. Flow cytometry and Western blotting were applied to cell cycle analysis and protein expression involved in anticancer activity of the selected biopolymer fraction. In vitro studies have shown that fractions isolated from Boletus edulis were not toxic against normal colon epithelial cells and in the same concentration range elicited a very prominent antiproliferative effect in colon cancer cells. The best results were obtained in the case of the fraction designated as BE3. The tested compound inhibited cancer cell proliferation which was accompanied by cell cycle arrest in the G0/G1-phase. Growth inhibition was associated with modulation of the p16/cyclin D1/CDK4-6/pRb pathway, an aberration of which is a critical step in the development of many human cancers including colon cancer. Our results indicate that a biopolymer BE3 from Boletus edulis possesses anticancer potential and may provide a new therapeutic/preventive option in colon cancer chemoprevention.

  6. Renal Denervation Prevents Immune Cell Activation and Renal Inflammation in Angiotensin II–Induced Hypertension

    PubMed Central

    Xiao, Liang; Kirabo, Annet; Wu, Jing; Saleh, Mohamed A.; Zhu, Linjue; Wang, Feng; Takahashi, Takamune; Loperena, Roxana; Foss, Jason D.; Mernaugh, Raymond L.; Chen, Wei; Roberts, Jackson; Osborn, John W.; Itani, Hana A.; Harrison, David G.

    2015-01-01

    Rationale Inflammation and adaptive immunity plays a crucial role in the development of hypertension. Angiotensin II and likely other hypertensive stimuli activate the central nervous system and promote T cell activation and end-organ damage in peripheral tissues. Objective To determine if renal sympathetic nerves mediate renal inflammation and T cell activation in hypertension. Methods and Results Bilateral renal denervation (RDN) using phenol application to the renal arteries reduced renal norepinephrine (NE) levels and blunted angiotensin II induced hypertension. Bilateral RDN also reduced inflammation, as reflected by decreased accumulation of total leukocytes, T cells and both CD4+ and CD8+ T cells in the kidney. This was associated with a marked reduction in renal fibrosis, albuminuria and nephrinuria. Unilateral RDN, which partly attenuated blood pressure, only reduced inflammation in the denervated kidney, suggesting that this effect is pressure independent. Angiotensin II also increased immunogenic isoketal-protein adducts in renal dendritic cells (DCs) and increased surface expression of costimulation markers and production of IL-1α, IL-1β, and IL-6 from splenic dendritic cells. NE also dose dependently stimulated isoketal formation in cultured DCs. Adoptive transfer of splenic DCs from angiotensin II-treated mice primed T cell activation and hypertension in recipient mice. RDN prevented these effects of hypertension on DCs. In contrast to these beneficial effects of ablating all renal nerves, renal afferent disruption with capsaicin had no effect on blood pressure or renal inflammation. Conclusions Renal sympathetic nerves contribute to dendritic cell activation, subsequent T cell infiltration and end-organ damage in the kidney in the development of hypertension. PMID:26156232

  7. Liquiritigenin Induces Tumor Cell Death through Mitogen-Activated Protein Kinase- (MPAKs-) Mediated Pathway in Hepatocellular Carcinoma Cells

    PubMed Central

    Lu, Jiahui; Liu, Yan; Meng, Qingfan; Xie, Jing; Wang, Zhenzuo

    2014-01-01

    Liquiritigenin (LQ), separated from Glycyrrhiza radix, possesses anti-inflammatory, antihyperlipidemic, and antiallergic effects. Our present study aims to investigate the antihepatocellular carcinoma effects of LQ both in cell and animal models. LQ strikingly reduced cell viability, enhanced apoptotic rate, induced lactate dehydrogenase over-release, and increased intracellular reactive oxygen species (ROS) level and caspase 3 activity in both PLC/PRL/5 and HepG2 cells. The expression of cleaved PARP, the hall-marker of apoptosis, was enhanced by LQ. LQ treatment resulted in a reduction of the expressions of B-cell lymphoma 2 (Bcl-2) and B-cell lymphoma-extra large (Bcl-xL), and an increase of the phosphorylation of c-Jun N-terminal kinases (JNK) and P38. LQ-mediated cell viability reduction, mitochondrial dysfunction, apoptosis related protein abnormal expressions, and JNK and P38 activation were partially abolished by N-Acetyl-L-cysteine (a ROS inhibitor) pretreatment. Moreover, LQ suppressed the activation of extracellular signaling-regulated kinase (ERKs) and reduced the translocation of phosphor-ERKs from cytoplasm to nucleus. This antitumor activity was further confirmed in PLC/PRL/5-xenografted mice model. All these data indicate that the antihepatocellular carcinoma effects of LQ are related to its modulation of the activations of mitogen-activated protein kinase (MAPKs). The study provides experimental evidence supporting LQ as a potential therapeutic agent for hepatocellular carcinoma treatment. PMID:24738081

  8. Gallic Acid Induces Necroptosis via TNF–α Signaling Pathway in Activated Hepatic Stellate Cells

    PubMed Central

    Chang, Ya Ju; Hsu, Shih Lan; Liu, Yi Ting; Lin, Yu Hsuan; Lin, Ming Hui; Huang, Shu Jung; Ho, Ja-an Annie; Wu, Li-Chen

    2015-01-01

    Gallic acid (3, 4, 5-trihydroxybenzoic acid, GA), a natural phenolic acid widely found in gallnuts, tea leaves and various fruits, possesses several bioactivities against inflammation, oxidation, and carcinogenicity. The beneficial effect of GA on the reduction of animal hepatofibrosis has been indicated due to its antioxidative property. However, the cytotoxicity of GA autoxidation causing cell death has also been reported. Herein, we postulated that GA might target activated hepatic stellate cells (aHSCs), the cell type responsible for hepatofibrosis, to mitigate the process of fibrosis. The molecular cytotoxic mechanisms that GA exerted on aHSCs were then analyzed. The results indicated that GA elicited aHSC programmed cell death through TNF–α–mediated necroptosis. GA induced significant oxidative stress through the suppression of catalase activity and the depletion of glutathione (GSH). Elevated oxidative stress triggered the production of TNF–α facilitating the undergoing of necroptosis through the up-regulation of key necroptotic regulatory proteins TRADD and receptor-interacting protein 3 (RIP3), and the inactivation of caspase–8. Calmodulin and calpain–1 activation were engaged, which promoted subsequent lysosomal membrane permeabilization (LMP). The TNF–α antagonist (SPD–304) and the RIP1 inhibitor (necrostatin–1, Nec–1) confirmed GA-induced TNFR1–mediated necroptosis. The inhibition of RIP1 by Nec–1 diverted the cell death from necroptosis to apoptosis, as the activation of caspase 3 and the increase of cytochrome c. Collectively, this is the first report indicating that GA induces TNF signaling–triggered necroptosis in aHSCs, which may offer an alternative strategy for the amelioration of liver fibrosis. PMID:25816210

  9. Gelsolin Induces Colorectal Tumor Cell Invasion via Modulation of the Urokinase-Type Plasminogen Activator Cascade

    PubMed Central

    Zhuo, Jingli; Tan, Ee Hong; Yan, Benedict; Tochhawng, Lalchhandami; Jayapal, Manikandan; Koh, Shiuan; Tay, Hwee Kee; Maciver, Sutherland K.; Hooi, Shing Chuan; Salto-Tellez, Manuel; Kumar, Alan Prem; Goh, Yaw Chong; Lim, Yaw Chyn; Yap, Celestial T.

    2012-01-01

    Gelsolin is a cytoskeletal protein which participates in actin filament dynamics and promotes cell motility and plasticity. Although initially regarded as a tumor suppressor, gelsolin expression in certain tumors correlates with poor prognosis and therapy-resistance. In vitro, gelsolin has anti-apoptotic and pro-migratory functions and is critical for invasion of some types of tumor cells. We found that gelsolin was highly expressed at tumor borders infiltrating into adjacent liver tissues, as examined by immunohistochemistry. Although gelsolin contributes to lamellipodia formation in migrating cells, the mechanisms by which it induces tumor invasion are unclear. Gelsolin’s influence on the invasive activity of colorectal cancer cells was investigated using overexpression and small interfering RNA knockdown. We show that gelsolin is required for invasion of colorectal cancer cells through matrigel. Microarray analysis and quantitative PCR indicate that gelsolin overexpression induces the upregulation of invasion-promoting genes in colorectal cancer cells, including the matrix-degrading urokinase-type plasminogen activator (uPA). Conversely, gelsolin knockdown reduces uPA levels, as well as uPA secretion. The enhanced invasiveness of gelsolin-overexpressing cells was attenuated by treatment with function-blocking antibodies to either uPA or its receptor uPAR, indicating that uPA/uPAR activity is crucial for gelsolin-dependent invasion. In summary, our data reveals novel functions of gelsolin in colorectal tumor cell invasion through its modulation of the uPA/uPAR cascade, with potentially important roles in colorectal tumor dissemination to metastatic sites. PMID:22927998

  10. Kurarinol induces hepatocellular carcinoma cell apoptosis through suppressing cellular signal transducer and activator of transcription 3 signaling

    SciTech Connect

    Shu, Guangwen; Yang, Jing; Zhao, Wenhao; Xu, Chan; Hong, Zongguo; Mei, Zhinan; Yang, Xinzhou

    2014-12-01

    Kurarinol is a flavonoid isolated from roots of the medical plant Sophora flavescens. However, its cytotoxic activity against hepatocellular carcinoma (HCC) cells and toxic effects on mammalians remain largely unexplored. Here, the pro-apoptotic activities of kurarinol on HCC cells and its toxic impacts on tumor-bearing mice were evaluated. The molecular mechanisms underlying kurarinol-induced HCC cell apoptosis were also investigated. We found that kurarinol dose-dependently provoked HepG2, Huh-7 and H22 HCC cell apoptosis. In addition, kurarinol gave rise to a considerable decrease in the transcriptional activity of signal transducer and activator of transcription 3 (STAT3) in HCC cells. Suppression of STAT3 signaling is involved in kurarinol-induced HCC cell apoptosis. In vivo studies showed that kurarinol injection substantially induced transplanted H22 cell apoptosis with low toxic impacts on tumor-bearing mice. Similarly, the transcriptional activity of STAT3 in transplanted tumor tissues was significantly suppressed after kurarinol treatment. Collectively, our current research demonstrated that kurarinol has the capacity of inducing HCC cell apoptosis both in vitro and in vivo with undetectable toxic impacts on the host. Suppressing STAT3 signaling is implicated in kurarinol-mediated HCC cell apoptosis. - Highlights: • Kurarinol induces hepatocellular carcinoma (HCC) cell apoptosis. • Kurarinol induces HCC cell apoptosis via inhibiting STAT3. • Kurarinol exhibits low toxic effects on tumor-bearing animals.

  11. PGC-1α-Dependent Mitochondrial Adaptation Is Necessary to Sustain IL-2-Induced Activities in Human NK Cells

    PubMed Central

    Jara, Claudia; Ibañez, Jorge; Ahumada, Viviana; Acuña-Castillo, Claudio; Martin, Adrian; Córdova, Alexandra

    2016-01-01

    Human Natural Killer (NK) cells are a specialized heterogeneous subpopulation of lymphocytes involved in antitumor defense reactions. NK cell effector functions are critically dependent on cytokines and metabolic activity. Among various cytokines modulating NK cell function, interleukin-2 (IL-2) can induce a more potent cytotoxic activity defined as lymphokine activated killer activity (LAK). Our aim was to determine if IL-2 induces changes at the mitochondrial level in NK cells to support the bioenergetic demand for performing this enhanced cytotoxic activity more efficiently. Purified human NK cells were cultured with high IL-2 concentrations to develop LAK activity, which was assessed by the ability of NK cells to lyse NK-resistant Daudi cells. Here we show that, after 72 h of culture of purified human NK cells with enough IL-2 to induce LAK activity, both the mitochondrial mass and the mitochondrial membrane potential increased in a PGC-1α-dependent manner. In addition, oligomycin, an inhibitor of ATP synthase, inhibited IL-2-induced LAK activity at 48 and 72 h of culture. Moreover, the secretion of IFN-γ from NK cells with LAK activity was also partially dependent on PGC-1α expression. These results indicate that PGC-1α plays a crucial role in regulating mitochondrial function involved in the maintenance of LAK activity in human NK cells stimulated with IL-2. PMID:27413259

  12. Cobalt protoporphyrin induces differentiation of monocytic THP-1 cells through regulation of cytoplasmic Ref-1-related NADPH oxidase activity.

    PubMed

    Song, Ju Dong; Lee, Sang Kwon; Park, Si Eun; Kim, Kang Mi; Kim, Koanhoi; Park, Yeong Min; Park, Young Chul

    2011-11-01

    Cobalt protoporphyrin (CoPP) is a potent and effective metalloporphyrin inducer of heme oxygenase-1 (HO-1) activity in many tissues. Here, we report that CoPP induces differentiation of monocytic THP-1 cells into macrophage-like cells. CoPP induced a marked growth inhibition with a slight reduction in viability, and increased adhesion and spreading of THP-1 cells. However, other protoporphyrins did not. CoPP also resulted in expression of CD11b, MMP9, MSR1, CD14 and ICAM-1, which are differentiation markers for macrophages. Interestingly, we observed a decrease of cytoplasmic redox factor-1 (Ref-1) levels in the process of CoPP-induced differentiation of THP-1 cells. In addition, knockdown of Ref-1 by siRNA enhanced cell adhesion induced by CoPP. Furthermore, an inhibitor of NADPH oxidase, diphenyleneiodonium (DPI), completely abolished CoPP-induced adhesion of Ref-1-deficient cells using an siRNA. A cytosolic factor for NADPH oxidase activity, p47phox, was significantly increased in THP-1 cells by CoPP treatment. Κnockdown of Ref-1 increased CoPP-induced p47phox expression in THP-1 cells. Taken together, these results suggest that CoPP induces differentiation of monocytic THP-1 cells, and that the CoPP-induced differentiation is associated with cytoplasmic Ref-1-related NADPH oxidase activity.

  13. Fasciola hepatica Kunitz Type Molecule Decreases Dendritic Cell Activation and Their Ability to Induce Inflammatory Responses

    PubMed Central

    Falcón, Cristian R.; Masih, Diana; Gatti, Gerardo; Sanchez, María Cecilia; Motrán, Claudia C.; Cervi, Laura

    2014-01-01

    The complete repertoire of proteins with immunomodulatory activity in Fasciola hepatica (Fh) has not yet been fully described. Here, we demonstrated that Fh total extract (TE) reduced LPS-induced DC maturation, and the DC ability to induce allogeneic responses. After TE fractionating, a fraction lower than 10 kDa (F<10 kDa) was able to maintain the TE properties to modulate the DC pro- and anti-inflammatory cytokine production induced by LPS. In addition, TE or F<10 kDa treatment decreased the ability of immature DC to stimulate the allogeneic responses and induced a novo allogeneic CD4+CD25+Foxp3+ T cells. In contrast, treatment of DC with T/L or F<10 kDa plus LPS (F<10/L) induced a regulatory IL-27 dependent mechanism that diminished the proliferative and Th1 and Th17 allogeneic responses. Finally, we showed that a Kunitz type molecule (Fh-KTM), present in F<10 kDa, was responsible for suppressing pro-inflammatory cytokine production in LPS-activated DC, by printing tolerogenic features on DC that impaired their ability to induce inflammatory responses. These results suggest a modulatory role for this protein, which may be involved in the immune evasion mechanisms of the parasite. PMID:25486609

  14. Protein kinase C activation induces phosphatidylserine exposure on red blood cells.

    PubMed

    de Jong, Kitty; Rettig, Michael P; Low, Philip S; Kuypers, Frans A

    2002-10-15

    We have shown previously that red blood cells (RBCs) can be induced to influx Ca(2+) when treated with lipid mediators, such as lysophosphatidic acid and prostaglandin E(2), that are released during clot formation. Since calcium loading of RBCs can lead to both protein kinase C (PKC) activation and phosphatidylserine (PS) exposure, we decided to investigate the possible linkage between PKC activation and membrane PS scrambling using phorbol 12-myristate-13-acetate (PMA), a commonly used activator of PKC. Treatment of RBCs with PMA in a calcium-containing buffer caused immediate PS exposure in an RBC subpopulation. The size of the subpopulation did not change upon further incubation, indicating that not all RBCs are equally susceptible to this treatment. Using a fluorescent indicator, we found a subpopulation of RBCs with elevated intracellular calcium levels. In the absence of extracellular calcium, no PS exposure was found. However, we did find cells with high levels of calcium that did not expose PS, and a variable percentage of PS-exposing cells that did not show elevated calcium concentrations. Inhibition of PKC with either calphostin C, a blocker of the PMA binding site, or chelerythrine chloride, an inhibitor of the active site, diminished the level of formation of PS-exposing cells. However, the inhibitors had different effects on calcium internalization, indicating that a high calcium concentration alone was not responsible for inducing PS exposure in the absence of PKC activity. Moreover, PKC inhibition could prevent PS exposure induced by calcium and ionophore treatment of RBCs. We conclude that PKC is implicated in the mechanism of membrane phospholipid scrambling.

  15. Baicalein induces apoptosis via ROS-dependent activation of caspases in human bladder cancer 5637 cells.

    PubMed

    Choi, Eun-Ok; Park, Cheol; Hwang, Hye-Jin; Hong, Su Hyun; Kim, Gi-Young; Cho, Eun-Ju; Kim, Wun-Jae; Choi, Yung Hyun

    2016-09-01

    Baicalein is a flavonoid derived originally from the root of Scutellaria baicalensis Georgi, which has been used in Oriental medicines for treating various diseases. Although this compound has been reported to have anticancer activities in several human cancer cell lines, the therapeutic effects of baicalein on human bladder cancer and its mechanisms of action have not been extensively studied. This study investigated the proapoptotic effects of baicalein in human bladder cancer 5637 cells. For this study, cell viability and apoptosis were evaluated using the 3-(4,5-dimethylthia-zol-2-yl)-2,5-diphenyltetrazolium bromide assay, trypan blue dye exclusion assay 4,6-diamidino-2-phenylindole staining, and flow cytometry. Measurements of the mitochondrial membrane potential (MMP), caspase activity assays and western blots were conducted to determine whether 5637 cell death occurred by apoptosis. Treatment with baicalein resulted in a concentration-dependent growth inhibition coupled with apoptosis induction, as indicated by the results of nuclei morphology examination and flow cytometry analyses. The induction of the apoptotic cell death of 5637 cells by baicalein exhibited a correlation with the downregulation of members of the inhibitor of apoptosis protein (IAP) family, including cIAP-1 and cIAP-2, and the activation of caspase-9 and -3 accompanied by proteolytic degradation of poly(ADP-ribose)-polymerase. The study also showed that baicalein decreases the expression of the proapoptotic protein Bax, increases antiapoptotic Bcl-2 expression, and noticeably aggravates the loss of MMP. Concomitantly, the data showed that baicalein increases the levels of death receptors and their associated ligands and enhances the activation of caspase-8 and truncation of Bid. However, the pan-caspase inhibitor can reverse baicalein-induced apoptosis, demonstrating that it is a caspase-dependent pathway. Moreover, it was found that baicalein can induce the production of reactive oxygen

  16. Baicalein induces apoptosis via ROS-dependent activation of caspases in human bladder cancer 5637 cells.

    PubMed

    Choi, Eun-Ok; Park, Cheol; Hwang, Hye-Jin; Hong, Su Hyun; Kim, Gi-Young; Cho, Eun-Ju; Kim, Wun-Jae; Choi, Yung Hyun

    2016-09-01

    Baicalein is a flavonoid derived originally from the root of Scutellaria baicalensis Georgi, which has been used in Oriental medicines for treating various diseases. Although this compound has been reported to have anticancer activities in several human cancer cell lines, the therapeutic effects of baicalein on human bladder cancer and its mechanisms of action have not been extensively studied. This study investigated the proapoptotic effects of baicalein in human bladder cancer 5637 cells. For this study, cell viability and apoptosis were evaluated using the 3-(4,5-dimethylthia-zol-2-yl)-2,5-diphenyltetrazolium bromide assay, trypan blue dye exclusion assay 4,6-diamidino-2-phenylindole staining, and flow cytometry. Measurements of the mitochondrial membrane potential (MMP), caspase activity assays and western blots were conducted to determine whether 5637 cell death occurred by apoptosis. Treatment with baicalein resulted in a concentration-dependent growth inhibition coupled with apoptosis induction, as indicated by the results of nuclei morphology examination and flow cytometry analyses. The induction of the apoptotic cell death of 5637 cells by baicalein exhibited a correlation with the downregulation of members of the inhibitor of apoptosis protein (IAP) family, including cIAP-1 and cIAP-2, and the activation of caspase-9 and -3 accompanied by proteolytic degradation of poly(ADP-ribose)-polymerase. The study also showed that baicalein decreases the expression of the proapoptotic protein Bax, increases antiapoptotic Bcl-2 expression, and noticeably aggravates the loss of MMP. Concomitantly, the data showed that baicalein increases the levels of death receptors and their associated ligands and enhances the activation of caspase-8 and truncation of Bid. However, the pan-caspase inhibitor can reverse baicalein-induced apoptosis, demonstrating that it is a caspase-dependent pathway. Moreover, it was found that baicalein can induce the production of reactive oxygen

  17. Procaspase-activating compound 1 induces a caspase-3-dependent cell death in cerebellar granule neurons

    SciTech Connect

    Aziz, Gulzeb; Akselsen, Oyvind W.; Hansen, Trond V.; Paulsen, Ragnhild E.

    2010-09-15

    Procaspase-activating compound 1, PAC-1, has been introduced as a direct activator of procaspase-3 and has been suggested as a therapeutic agent against cancer. Its activation of procaspase-3 is dependent on the chelation of zinc. We have tested PAC-1 and an analogue of PAC-1 as zinc chelators in vitro as well as their ability to activate caspase-3 and induce cell death in chicken cerebellar granule neuron cultures. These neurons are non-dividing, primary cells with normal caspase-3. The results reported herein show that PAC-1 chelates zinc, activates procaspase-3, and leads to caspase-3-dependent cell death in neurons, as the specific caspase-3-inhibitor Ac-DEVD-cmk inhibited both the caspase-3 activity and cell death. Thus, chicken cerebellar granule neurons is a suitable model to study mechanisms of interference with apoptosis of PAC-1 and similar compounds. Furthermore, the present study also raises concern about potential neurotoxicity of PAC-1 if used in cancer therapy.

  18. Osteopontin induces {beta}-catenin signaling through activation of Akt in prostate cancer cells

    SciTech Connect

    Robertson, Brian W.; Chellaiah, Meenakshi A.

    2010-01-01

    Secretion of osteopontin (OPN) by cancer cells is a known mediator of tumorigenesis and cancer progression in both experimental and clinical studies. Our work demonstrates that OPN can activate Akt, an important step in cancer progression. Both ILK and PI3K are integral proteins in the OPN/Akt pathway, as inhibition of either kinase leads to a loss of OPN-mediated Akt activation. Subsequent to OPN-induced Akt activation, we observe inactivation of GSK-3{beta}, a regulator of {beta}-catenin. Osteopontin stimulation leads to an overall increase in {beta}-catenin protein levels with a resultant transfer of {beta}-catenin to the nucleus. Through the nuclear import of {beta}-catenin, OPN increases both the transcription and protein levels of MMP-7 and CD44, which are known TCF/LEF transcription targets. This work describes an important aspect of cancer progression induced by OPN.

  19. Lysophosphatidic acid induces cell migration through the selective activation of Akt1

    PubMed Central

    Kim, Eun Kyoung; Yun, Sung Ji; Do, Kee Hun; Kim, Min Sung; Cho, Mong; Suh, Dong-Soo; Kim, Chi Dae; Kim, Jae Ho; Birnbaum, Morris J.

    2008-01-01

    Akt plays pivotal roles in many physiological responses including growth, proliferation, survival, metabolism, and migration. In the current studies, we have evaluated the isoform-specific role of akt in lysophosphatidic acid (LPA)-induced cell migration. Ascites from ovarian cancer patients (AOCP) induced mouse embryo fibroblast (MEF) cell migration in a dose-dependent manner. On the other hand, ascites from liver cirrhosis patients (ALCP) did not induce MEF cell migration. AOCP-induced MEF cell migration was completely blocked by pre-treatment of cells with LPA receptor antagonist, Ki16425. Both LPA- and AOCP-induced MEF cell migration was completely attenuated by PI3K inhibitor, LY294002. Furthermore, cells lacking Akt1 displayed defect in LPA-induced cell migration. Re-expression of Akt1 in DKO (Akt1-/-Akt2-/-) cells restored LPA-induced cell migration, whereas re-expression of Akt2 in DKO cells could not restore the LPA-induced cell migration. Finally, Akt1 was selectively phosphorylated by LPA and AOCP stimulation. These results suggest that LPA is a major factor responsible for AOCP-induced cell migration and signaling specificity of Akt1 may dictate LPA-induced cell migration. PMID:18779657

  20. Chemotherapy induces stemness in osteosarcoma cells through activation of Wnt/β-catenin signaling.

    PubMed

    Martins-Neves, Sara R; Paiva-Oliveira, Daniela I; Wijers-Koster, Pauline M; Abrunhosa, Antero J; Fontes-Ribeiro, Carlos; Bovée, Judith V M G; Cleton-Jansen, Anne-Marie; Gomes, Célia M F

    2016-01-28

    Development of resistance represents a major drawback in osteosarcoma treatment, despite improvements in overall survival. Treatment failure and tumor progression have been attributed to pre-existing drug-resistant clones commonly assigned to a cancer stem-like phenotype. Evidence suggests that non stem-like cells, when submitted to certain microenvironmental stimuli, can acquire a stemness phenotype thereby strengthening their capacity to handle with stressful conditions. Here, using osteosarcoma cell lines and a mouse xenograft model, we show that exposure to conventional chemotherapeutics induces a phenotypic cell transition toward a stem-like phenotype. This associates with activation of Wnt/β-catenin signaling, up-regulation of pluripotency factors and detoxification systems (ABC transporters and Aldefluor activity) that ultimately leads to chemotherapy failure. Wnt/β-catenin inhibition combined with doxorubicin, in the MNNG-HOS cells, prevented the up-regulation of factors linked to transition into a stem-like state and can be envisaged as a way to overcome adaptive resistance. Finally, the analysis of the public R2 database, containing microarray data information from diverse osteosarcoma tissues, revealed a correlation between expression of stemness markers and a worse response to chemotherapy, which provides evidence for drug-induced phenotypic stem cell state transitions in osteosarcoma. PMID:26577806

  1. Histamine induces activation of protein kinase D that mediates tissue factor expression and activity in human aortic smooth muscle cells.

    PubMed

    Hao, Feng; Wu, Daniel Dongwei; Xu, Xuemin; Cui, Mei-Zhen

    2012-12-01

    Histamine, an inflammatory mediator, has been shown to influence the pathogenesis of vascular wall cells. However, the molecular basis of its influence is not well understood. Our data reveal that histamine markedly induces protein kinase D (PKD) activation in human aortic smooth muscle cells. PKD belongs to a family of serine/threonine protein kinases, and its function in vascular disease is largely unknown. Our data show that histamine-induced PKD phosphorylation is dependent on the activation of histamine receptor 1 and protein kinase C (PKC). To determine the role of PKD in the histamine pathway, we employed a small-interfering RNA approach to downregulate PKD expression and found that PKD1 and PKD2 are key mediators for expression of tissue factor (TF), which is the key initiator of blood coagulation and is important for thrombosis. Our results show that PKD2 predominantly mediates histamine-induced TF expression via the p38 mitogen-activated protein kinase (MAPK) pathway, whereas PKD1 mediates histamine-induced TF expression through a p38 MAPK-independent pathway. We demonstrate that histamine induces TF expression via the PKC-dependent PKD activation. Our data provide the first evidence that PKD is a new component in histamine signaling in live cells and that PKD has a novel function in the histamine signaling pathway leading to gene expression, as evidenced by TF expression. Importantly, our data reveal a regulatory link from histamine to PKD and TF, providing new insights into the mechanisms of coagulation and the development of atherothrombosis.

  2. NOD2 activation induces muscle cell-autonomous innate immune responses and insulin resistance.

    PubMed

    Tamrakar, Akhilesh K; Schertzer, Jonathan D; Chiu, Tim T; Foley, Kevin P; Bilan, Philip J; Philpott, Dana J; Klip, Amira

    2010-12-01

    Insulin resistance is associated with chronic low-grade inflammation in vivo, largely mediated by activated innate immune cells. Cytokines and pathogen-derived ligands of surface toll-like receptors can directly cause insulin resistance in muscle cells. However, it is not known if intracellular pathogen sensors can, on their own, provoke insulin resistance. Here, we show that the cytosolic pattern recognition receptors nucleotide-binding oligomerization domain-containing protein (NOD)1 and NOD2 are expressed in immune and metabolic tissues and hypothesize that their activation in muscle cells would result in cell-autonomous responses leading to insulin resistance. Bacterial peptidoglycan motifs that selectively activate NOD2 were directly administered to L6- GLUT4myc myotubes in culture. Within 3 h, insulin resistance arose, characterized by reductions in each insulin-stimulated glucose uptake, GLUT4 translocation, Akt Ser(473) phosphorylation, and insulin receptor substrate 1 tyrosine phosphorylation. Muscle cell-autonomous responses to NOD2 ligand included activation of the stress/inflammation markers c-Jun N-terminal kinase, ERK1/2, p38 MAPK, degradation of inhibitor of κBα, and production of proinflammatory cytokines. These results show that NOD2 alone is capable of acutely inducing insulin resistance within muscle cells, possibly by activating endogenous inflammatory signals and/or through cytokine production, curbing upstream insulin signals. NOD2 is hence a new inflammation target connected to insulin resistance, and this link occurs without the need of additional contributing cell types. This study provides supporting evidence for the integration of innate immune and metabolic responses through the involvement of NOD proteins and suggests the possible participation of cell autonomous immune responses in the development of insulin resistance in skeletal muscle, the major depot for postprandial glucose utilization.

  3. Activation of K+ channel by 1-EBIO rescues the head and neck squamous cell carcinoma cells from Ca2+ ionophore-induced cell death

    PubMed Central

    Yin, Ming Zhe; Park, Seok-Woo; Kang, Tae Wook; Kim, Kyung Soo; Yoo, Hae Young; Lee, Junho; Hah, J. Hun; Sung, Myung Hun

    2016-01-01

    Ion channels in carcinoma and their roles in cell proliferation are drawing attention. Intracellular Ca2+ ([Ca2+]i)-dependent signaling affects the fate of cancer cells. Here we investigate the role of Ca2+-activated K+ channel (SK4) in head and neck squamous cell carcinoma cells (HNSCCs) of different cell lines; SNU-1076, OSC-19 and HN5. Treatment with 1 µM ionomycin induced cell death in all the three cell lines. Whole-cell patch clamp study suggested common expressions of Ca2+-activated Cl- channels (Ano-1) and Ca2+-activated nonselective cation channels (CAN). 1-EBIO, an activator of SK4, induced outward K+ current (ISK4) in SNU-1076 and OSC-19. In HN5, ISK4 was not observed or negligible. The 1-EBIO-induced current was abolished by TRAM-34, a selective SK4 blocker. Interestingly, the ionomycin-induced cell death was effectively prevented by 1-EBIO in SNU-1076 and OSC-19, and the rescue effect was annihilated by combined TRAM-34. Consistent with the lower level of ISK4, the rescue by 1-EBIO was least effective in HN5. The results newly demonstrate the role of SK4 in the fate of HNSCCs under the Ca2+ overloaded condition. Pharmacological modulation of SK4 might provide an intriguing novel tool for the anti-cancer strategy in HNSCC. PMID:26807020

  4. Ethanolic extract of dandelion (Taraxacum mongolicum) induces estrogenic activity in MCF-7 cells and immature rats.

    PubMed

    Oh, Seung Min; Kim, Ha Ryong; Park, Yong Joo; Lee, Yong Hwa; Chung, Kyu Hyuck

    2015-11-01

    Plants of the genus Taraxacum, commonly known as dandelions, are used to treat breast cancer in traditional folk medicine. However, their use has mainly been based on empirical findings without sufficient scientific evidence. Therefore, we hypothesized that dandelions would behave as a Selective estrogen receptor modulator (SERM) and be effective as hormone replacement therapy (HRT) in the postmenopausal women. In the present study, in vitro assay systems, including cell proliferation assay, reporter gene assay, and RT-PCR to evaluate the mRNA expression of estrogen-related genes (pS2 and progesterone receptor, PR), were performed in human breast cancer cells. Dandelion ethanol extract (DEE) significantly increased cell proliferation and estrogen response element (ERE)-driven luciferase activity. DEE significantly induced the expression of estrogen related genes such as pS2 and PR, which was inhibited by tamoxifen at 1 μmol·L(-1). These results indicated that DEE could induce estrogenic activities mediated by a classical estrogen receptor pathway. In addition, immature rat uterotrophic assay was carried out to identify estrogenic activity of DEE in vivo. The lowest concentration of DEE slightly increased the uterine wet weight, but there was no significant effect with the highest concentration of DEE. The results demonstrate the potential estrogenic activities of DEE, providing scientific evidence supporting their use in traditional medicine. PMID:26614455

  5. Ethanolic extract of dandelion (Taraxacum mongolicum) induces estrogenic activity in MCF-7 cells and immature rats.

    PubMed

    Oh, Seung Min; Kim, Ha Ryong; Park, Yong Joo; Lee, Yong Hwa; Chung, Kyu Hyuck

    2015-11-01

    Plants of the genus Taraxacum, commonly known as dandelions, are used to treat breast cancer in traditional folk medicine. However, their use has mainly been based on empirical findings without sufficient scientific evidence. Therefore, we hypothesized that dandelions would behave as a Selective estrogen receptor modulator (SERM) and be effective as hormone replacement therapy (HRT) in the postmenopausal women. In the present study, in vitro assay systems, including cell proliferation assay, reporter gene assay, and RT-PCR to evaluate the mRNA expression of estrogen-related genes (pS2 and progesterone receptor, PR), were performed in human breast cancer cells. Dandelion ethanol extract (DEE) significantly increased cell proliferation and estrogen response element (ERE)-driven luciferase activity. DEE significantly induced the expression of estrogen related genes such as pS2 and PR, which was inhibited by tamoxifen at 1 μmol·L(-1). These results indicated that DEE could induce estrogenic activities mediated by a classical estrogen receptor pathway. In addition, immature rat uterotrophic assay was carried out to identify estrogenic activity of DEE in vivo. The lowest concentration of DEE slightly increased the uterine wet weight, but there was no significant effect with the highest concentration of DEE. The results demonstrate the potential estrogenic activities of DEE, providing scientific evidence supporting their use in traditional medicine.

  6. NANOG alone induces germ cells in primed epiblast in vitro by activation of enhancers

    PubMed Central

    Zylicz, Jan J.; Tang, Walfred W. C.; Sengupta, Roopsha; Kobayashi, Toshihiro; Kim, Shinseog; Butler, Richard; Dietmann, Sabine; Surani, M. Azim

    2015-01-01

    Nanog, a core pluripotency factor in the inner cell mass of blastocysts, is also expressed in unipotent primordial germ cells (PGC) in mice1, where its precise role is yet unclear2-4. We investigated this in an in vitro model, where naïve pluripotent embryonic stem cells (ESCs) cultured in bFGF/ActivinA develop as epiblast-like cells (EpiLCs), and gain competence for PGC-like fate5. Consequently, bone morphogenetic protein (BMP4), or ectopic expression of key germline transcription factors Prdm1/ Prdm14/ Tfap2c, directly induce PGC-like cells (PGCLCs) in EpiLCs, but not in ESCs6-8. Here we report an unexpected discovery that Nanog alone can induce PGCLCs in EpiLCs, independently of BMP4. We propose that following the dissolution of the naïve ESC pluripotency network during establishment of EpiLCs9,10, the epigenome is reset for cell fate determination. Indeed, we found genome-wide changes in NANOG binding pattern between ESCs and EpiLCs, indicating epigenetic resetting of regulatory elements. Accordingly, we show that NANOG can bind and activate enhancers of Prdm1 and Prdm14 in EpiLCs in vitro; BLIMP1 (encoded by Prdm1) then directly induces Tfap2c. Furthermore, while SOX2 and NANOG promote the pluripotent state in ESCs, they show contrasting roles in EpiLCs since Sox2 specifically represses PGCLC induction by Nanog. This study demonstrates a broadly applicable mechanistic principle for how cells acquire competence for cell fate determination, resulting in the context-dependent roles of key transcription factors during development. PMID:26751055

  7. Troglitazone enhances tamoxifen-induced growth inhibitory activity of MCF-7 cells

    SciTech Connect

    Yu, Hong-Nu; Noh, Eun-Mi; Lee, Young-Rae; Roh, Si-Gyun; Song, Eun-Kyung; Han, Myung-Kwan; Lee, Yong-Chul; Shim, In Kyong; Lee, Seung Jin; Jung, Sung Hoo; Kim, Jong-Suk Youn, Hyun Jo

    2008-12-05

    Peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}) ligands have been identified as a potential source of therapy for human cancers. However, PPAR{gamma} ligands have a limitation for breast cancer therapy, since estrogen receptor {alpha} (ER{sub {alpha}}) negatively interferes with PPAR{gamma} signaling in breast cancer cells. Here we show that ER{sub {alpha}} inhihits PPAR{gamma} transactivity and ER{sub {alpha}}-mediated inhibition of PPAR{gamma} transactivity is blocked by tamoxifen, an estrogen receptor blocker. The activation of ER{sub {alpha}} with 17-{beta}-estradiol blocked PPRE transactivity induced by troglitazone, a PPAR{gamma} ligand, indicating the resistance of ER{sub {alpha}}-positive breast cancer cells to troglitazone. Indeed, troglitazone inhibited the growth of ER{sub {alpha}}-negative MDA-MB-231 cells more than that of ER{sub {alpha}}-positive MCF-7 cells. Combination of troglitazone with tamoxifen led to a marked increase in growth inhibition of ER{sub {alpha}}-positive MCF-7 cells compared to either agent alone. Our data indicates that troglitazone enhances the growth inhibitory activity of tamoxifen in ER{sub {alpha}}-positive MCF-7 cells.

  8. Mechanisms of cell death pathway activation following drug-induced inhibition of mitochondrial complex I

    PubMed Central

    Imaizumi, Naoki; Kwang Lee, Kang; Zhang, Carmen; Boelsterli, Urs A.

    2015-01-01

    Respiratory complex I inhibition by drugs and other chemicals has been implicated as a frequent mode of mitochondria-mediated cell injury. However, the exact mechanisms leading to the activation of cell death pathways are incompletely understood. This study was designed to explore the relative contributions to cell injury of three distinct consequences of complex I inhibition, i.e., impairment of ATP biosynthesis, increased formation of superoxide and, hence, peroxynitrite, and inhibition of the mitochondrial protein deacetylase, Sirt3, due to imbalance of the NADH/NAD+ ratio. We used the antiviral drug efavirenz (EFV) to model drug-induced complex I inhibition. Exposure of cultured mouse hepatocytes to EFV resulted in a rapid onset of cell injury, featuring a no-effect level at 30 µM EFV and submaximal effects at 50 µM EFV. EFV caused a concentration-dependent decrease in cellular ATP levels. Furthermore, EFV resulted in increased formation of peroxynitrite and oxidation of mitochondrial protein thiols, including cyclophilin D (CypD). This was prevented by the superoxide scavenger, Fe-TCP, or the peroxynitrite decomposition catalyst, Fe-TMPyP. Both ferroporphyrins completely protected from EFV-induced cell injury, suggesting that peroxynitrite contributed to the cell injury. Finally, EFV increased the NADH/NAD+ ratio, inhibited Sirt3 activity, and led to hyperacetylated lysine residues, including those in CypD. However, hepatocytes isolated from Sirt3-null mice were protected against 40 µM EFV as compared to their wild-type controls. In conclusion, these data are compatible with the concept that chemical inhibition of complex I activates multiple pathways leading to cell injury; among these, peroxynitrite formation may be the most critical. PMID:25625582

  9. Rejuvenation of MPTP-induced human neural precursor cell senescence by activating autophagy

    SciTech Connect

    Zhu, Liang; Dong, Chuanming; Sun, Chenxi; Ma, Rongjie; Yang, Danjing; Zhu, Hongwen; Xu, Jun

    2015-08-21

    Aging of neural stem cell, which can affect brain homeostasis, may be caused by many cellular mechanisms. Autophagy dysfunction was found in aged and neurodegenerative brains. However, little is known about the relationship between autophagy and human neural stem cell (hNSC) aging. The present study used 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP) to treat neural precursor cells (NPCs) derived from human embryonic stem cell (hESC) line H9 and investigate related molecular mechanisms involved in this process. MPTP-treated NPCs were found to undergo premature senescence [determined by increased senescence-associated-β-galactosidase (SA-β-gal) activity, elevated intracellular reactive oxygen species level, and decreased proliferation] and were associated with impaired autophagy. Additionally, the cellular senescence phenotypes were manifested at the molecular level by a significant increase in p21 and p53 expression, a decrease in SOD2 expression, and a decrease in expression of some key autophagy-related genes such as Atg5, Atg7, Atg12, and Beclin 1. Furthermore, we found that the senescence-like phenotype of MPTP-treated hNPCs was rejuvenated through treatment with a well-known autophagy enhancer rapamycin, which was blocked by suppression of essential autophagy gene Beclin 1. Taken together, these findings reveal the critical role of autophagy in the process of hNSC aging, and this process can be reversed by activating autophagy. - Highlights: • We successfully establish hESC-derived neural precursor cells. • MPTP treatment induced senescence-like state in hESC-derived NPCs. • MPTP treatment induced impaired autophagy of hESC-derived NPCs. • MPTP-induced hESC-derived NPC senescence was rejuvenated by activating autophagy.

  10. Role of mitochondria ROS generation in ethanol-induced NLRP3 inflammasome activation and cell death in astroglial cells

    PubMed Central

    Alfonso-Loeches, Silvia; Ureña-Peralta, Juan R.; Morillo-Bargues, Maria José; Oliver-De La Cruz, Jorge; Guerri, Consuelo

    2014-01-01

    Toll-like receptors (TLRs) and NOD-like receptors (NLRs) are innate immunity sensors that provide an early/effective response to pathogenic or injury conditions. We have reported that ethanol-induced TLR4 activation triggers signaling inflammatory responses in glial cells, causing neuroinflammation and brain damage. However, it is uncertain if ethanol is able to activate NLRs/inflammasome in astroglial cells, which is the mechanism of activation, and whether there is crosstalk between both immune sensors in glial cells. Here we show that chronic ethanol treatment increases the co-localization of caspase-1 with GFAP+ cells, and up-regulates IL-1β and IL-18 in the frontal medial cortex in WT, but not in TLR4 knockout mice. We further show that cultured cortical astrocytes expressed several inflammasomes (NLRP3, AIM2, NLRP1, and IPAF), although NLRP3 mRNA is the predominant form. Ethanol, as ATP and LPS treatments, up-regulates NLRP3 expression, and causes caspase-1 cleavage and the release of IL-1β and IL-18 in astrocytes supernatant. Ethanol-induced NLRP3/caspase-1 activation is mediated by mitochondrial (m) reactive oxygen species (ROS) generation because when using a specific mitochondria ROS scavenger, the mito-TEMPO (500 μM) or NLRP3 blocking peptide (4 μg/ml) or a specific caspase-1 inhibitor, Z-YVAD-FMK (10 μM), abrogates mROS release and reduces the up-regulation of IL-1β and IL-18 induced by ethanol or LPS or ATP. Confocal microscopy studies further confirm that ethanol, ATP or LPS promotes NLRP3/caspase-1 complex recruitment within the mitochondria to promote cell death by caspase-1-mediated pyroptosis, which accounts for ≈73% of total cell death (≈22%) and the remaining (≈25%) die by caspase-3-dependent apoptosis. Suppression of the TLR4 function abrogates most ethanol effects on NLRP3 activation and reduces cell death. These findings suggest that NLRP3 participates, in ethanol-induced neuroinflammation and highlight the NLRP3/TLR4 crosstalk in

  11. Cell cycle regulation of breast cancer cells through estrogen-induced activities of ERK and Akt protein kinases.

    PubMed

    Geffroy, Nancy; Guédin, Aurore; Dacquet, Catherine; Lefebvre, Philippe

    2005-06-15

    The proliferative effect of estrogens on breast cancer cell (BCC) is mainly mediated through estrogen receptors (ER). Non-transcriptional effects of estrogens, exerted through activation of several protein kinases, may also contribute to BCC proliferation. However, the relative contribution of these two responses to BCC proliferation is not known. We characterized a novel estrogenic receptor ligand which possess Akt and ERK activating properties distinct from that of 17beta-estradiol. Early and delayed waves of activation of these kinases were detected upon estrogenic challenge of BCC, but only molecules able to promote a significant, delayed activation of ERK-induced BCC proliferation. Estrogen-induced cell cycle progression was not sensitive to the inhibition of ERK-regulating kinases MEK1 and 2. ERalpha was found to be necessary, but not sufficient for kinases activation. Thus, estrogens elicit a distinct pattern of early and delayed activation of ERK and Akt, and early protein kinase activation is probably not involved in BCC proliferation. Structural variations in the estrogen molecule may confer novel biological properties unrelated to estrogen-dependent transcriptional activation.

  12. beta-Naphthoflavone protects from peritonitis by reducing TNF-alpha-induced endothelial cell activation.

    PubMed

    Hsu, Sheng-Yao; Liou, Je-Wen; Cheng, Tsung-Lin; Peng, Shih-Yi; Lin, Chi-Chen; Chu, Yuan-Yuan; Luo, Wei-Cheng; Huang, Zheng-Kai; Jiang, Shinn-Jong

    2015-12-01

    β-Naphthoflavone (β-NF), a ligand of the aryl hydrocarbon receptor, has been shown to possess anti-oxidative properties. We investigated the anti-oxidative and anti-inflammatory potential of β-NF in human microvascular endothelial cells treated with tumor necrosis factor-alpha (TNF-α). Pretreatment with β-NF significantly inhibited TNF-α-induced intracellular reactive oxygen species, translocation of p67(phox), and TNF-α-induced monocyte binding and transmigration. In addition, β-NF significantly inhibited TNF-α-induced ICAM-1 and VCAM-1 expression. The mRNA expression levels of the inflammatory cytokines TNF-α and IL-6 were reduced by β-NF, as was the infiltration of white blood cells, in a peritonitis model. The inhibition of adhesion molecules was associated with suppressed nuclear translocation of NF-κB p65 and Akt, and suppressed phosphorylation of ERK1/2 and p38. The translocation of Egr-1, a downstream transcription factor involved in the MEK-ERK signaling pathway, was suppressed by β-NF treatment. Our findings show that β-NF inhibits TNF-α-induced NF-kB and ERK1/2 activation and ROS generation, thereby suppressing the expression of adhesion molecules. This results in reduced adhesion and transmigration of leukocytes in vitro and prevents the infiltration of leukocytes in a peritonitis model. Our findings also suggest that β-NF might prevent TNF-α-induced inflammation.

  13. YAP induces cisplatin resistance through activation of autophagy in human ovarian carcinoma cells

    PubMed Central

    Xiao, Lan; Shi, Xiao-Yan; Zhang, Ying; Zhu, Ying; Zhu, Lin; Tian, Wang; Zhu, Bing-Kun; Wei, Zhao-Lian

    2016-01-01

    Objective To identify the role of YAP in cisplatin resistance in human ovarian cancer cells and in the regulation of autophagy in these cancer cells. Materials and methods The cisplatin-sensitive OV2008 parental cell line and its cisplatin-resistant variant C13K were cultured. RNA interference was used to knock down the YAP gene. Accumulation of GFP-LC3 puncta was performed by fluorescence microscopy. The formation of autophagosomes was observed by transmission electron microscopy. Drug sensitivity was examined using CCK-8 assay, while apoptosis, the level of intracellular rhodamine 123 and lysosomal acidification were analyzed by fluorescence-activated cell sorting. Acid phosphatase activity was measured using an acid phosphatase-assay kit. Real-time polymerase chain reaction, Western blotting, and immunofluorescence detection were used to detect the protein and messenger RNA expression of YAP, YAP target genes, CCND1, cleaved PARP, and caspase 3, Atg-3 and -5, and the LC3B protein. Results YAP signaling may regulate cisplatin resistance in ovarian cancer cells by augmenting cellular autophagic flux. After knockdown of YAP-sensitized C13K cells to cisplatin by inducing a decrease in autophagy, YAP led to an increase in autophagy via enhancement of autolysosome degradation. Conclusion YAP-mediated autophagy may play a protective role in cisplatin-resistant human ovarian cancer cells. Therefore, YAP-mediated autophagy should be explored as a new target for enhancing the efficacy of cisplatin against ovarian cancer and other types of malignancies. PMID:27073322

  14. Sapphyrins induce apoptosis in hematopoietic tumor-derived cell lines and show in vivo antitumor activity.

    PubMed

    Naumovski, Louie; Ramos, Jason; Sirisawad, Mint; Chen, Jun; Thiemann, Patti; Lecane, Philip; Magda, Darren; Wang, Zhong; Cortez, Cecilia; Boswell, Garry; Gyu Cho, Dong; Sessler, Jonathan; Miller, Richard

    2005-06-01

    Sapphyrins are pentapyrrolic, metal-free, expanded porphyrins. In the present study, the activity of sapphyrins as anticancer agents in hematopoietic-derived tumor cells was explored. It was found that a dihydroxylated water-soluble sapphyrin derivative (PCI-2000) is a potent inducer of apoptosis in a wide variety of tumor cell lines including lymphoma (Ramos, DHL-4, and HF-1), leukemia (Jurkat and HL-60), and myeloma (8226/S, 1-310, C2E3, and 1-414). PCI-2000 triggers an apoptotic pathway in these tumor cells as shown by release of cytochrome c from mitochondria; activation of caspases 9, 8, and 3; cleavage of the caspase substrate poly(ADP-ribose) polymerase; and Annexin V binding. Apoptosis can be partially inhibited by overexpression of the antiapoptotic protein Bcl-2 or treatment with benzyloxycarbonyl-valine-alanine-aspartic acid-fluoromethylketone, a cell-permeable caspase inhibitor. Both PCI-2000 and PCI-2010, a tetrahydroxy bis-carbamate derivative of PCI-2000, result in increased levels of phosphorylated p38 mitogen-activated protein kinase. Inhibition of p38 mitogen-activated protein kinase phosphorylation resulted in a synergistic increase of PCI-2000 cytotoxicity. PCI-2010 showed less toxicity in mice than PCI-2000 and was active in slowing the growth of Ramos and HL-60 tumor xenografts in nude mice. These results provide preclinical rationale for the further study of sapphyrins for potential use in the treatment of hematopoietic-derived tumors.

  15. Lipopolysaccharide-activated microglia induce death of oligodendrocyte progenitor cells and impede their development.

    PubMed

    Pang, Y; Campbell, L; Zheng, B; Fan, L; Cai, Z; Rhodes, P

    2010-03-17

    Damage to oligodendrocyte (OL) progenitor cells (OPCs) and hypomyelination are two hallmark features of periventricular leukomalacia (PVL), the most common form of brain damage in premature infants. Clinical and animal studies have linked the incidence of PVL to maternal infection/inflammation, and activated microglia have been proposed to play a central role. However, the precise mechanism of how activated microglia adversely affects the survival and development of OPCs is still not clear. Here we demonstrate that lipopolysaccharide (LPS)-activated microglia are deleterious to OPCs, that is, impeding OL lineage progression, reducing the production of myelin basic protein (MBP), and mediating OPC death. We further demonstrate that LPS-activated microglia mediate OPC death by two distinct mechanisms in a time-dependent manner. The early phase of cell damage occurs within 24 h after LPS treatment, which is mediated by nitric oxide (NO)-dependent oxidative damage and is prevented by N(G)-nitro-l-arginine methyl ester (l-NAME), a general inhibitor of nitric oxide synthase. The delayed cell death is evident at 48 h after LPS treatment, is mediated by cytokines, and is prevented by blocking the activity of tumor necrosis factor-alpha (TNF-alpha) and pro-nerve growth factor (proNGF), but not by l-NAME. Furthermore, microglia-derived insulin-like growth factor-1 (IGF-1) and ciliary neurotrophic factor (CNTF) were significantly suppressed by LPS, and exogenous IGF-1 and CNTF synergistically protected OLs from death induced by LPS-treated microglia conditioned medium, indicating that a deficiency in trophic support may also be involved in OL death. Our finding that LPS-activated microglia not only induce two waves of cell death but also greatly impair OL development may shed some light on the mechanisms underlying selective white matter damage and hypomyelination in PVL.

  16. Calpain inhibition induces activation of the distinct signalling pathways and cell migration in human monocytes.

    PubMed

    Noma, Haruyoshi; Kato, Takayuki; Fujita, Hisakazu; Kitagawa, Maki; Yamano, Tsunekazu; Kitagawa, Seiichi

    2009-09-01

    We have recently reported that constitutively active calpain negatively regulates activation of the distinct signalling pathways and cell migration in human neutrophils. Here, we report that a similar regulatory system is also functioning in human monocytes, but not lymphocytes. Calpain was constitutively active in resting human monocytes, but not lymphocytes. Mitogen-activated protein kinases, including extracellular signal-regulated kinase (ERK), p38 and c-Jun N-terminal kinase (JNK), phosphatidylinositol 3-kinase (PI3K)/Akt and p21-activated kinase (PAK, an effector molecule of Rac) were rapidly (within 1 min) activated in monocytes, but not lymphocytes, upon exposure to calpain inhibitors (PD150606 and N-acetyl-Leu-Leu-Nle-CHO), but not PD145305 (the inactive analogue of PD150606). Following activation of these signalling pathways, monocytes displayed active migration within 5 min after exposure to calpain inhibitors, and active migration was sustained for more than 45 min. The micropipette method revealed that calpain inhibition-mediated monocyte migration was chemotaxis, not random migration. The studies with pharmacological inhibitors suggest that calpain inhibition-mediated monocyte migration is mediated by activation of ERK, p38, JNK, PI3K/Akt and Rac. NSC23766 (Rac inhibitor) and pertussis toxin (PTX) suppressed calpain inhibitor-induced phosphorylation of distinct signalling molecules (PAK, ERK, p38, JNK and Akt) as well as cell migration, suggesting that the PTX-sensitive G protein and Rac axis may be a possible key target of calpain inhibitors. These findings suggest that constitutively active calpain negatively regulates activation of the distinct signalling pathways and cell migration in resting monocytes, but not lymphocytes.

  17. Ziyuglycoside II induces cell cycle arrest and apoptosis through activation of ROS/JNK pathway in human breast cancer cells.

    PubMed

    Zhu, Xue; Wang, Ke; Zhang, Kai; Zhu, Ling; Zhou, Fanfan

    2014-05-16

    Ziyuglycoside II, a triterpenoid saponin compound extracted from Sanguisorba officinalis L., has been reported to have a wide range of clinical applications including anti-cancer effect. In this study, the anti-proliferative effect of ziyuglycoside II in two classic human breast cancer cell lines, MCF-7 and MDA-MB-231, was extensively investigated. Our study indicated that ziyuglycoside II could effectively induce G2/M phase arrest and apoptosis in both cell lines. Cell cycle blocking was associated with the down-regulation of Cdc25C, Cdc2, cyclin A and cyclin B1 as well as the up-regulation of p21/WAF1, phospho-Cdc25C and phospho-Cdc2. Ziyuglycoside II treatment also induced reactive oxygen species (ROS) production and apoptosis by activating the extrinsic/Fas/FasL pathway as well as the intrinsic/mitochondrial pathway. More importantly, the c-Jun NH2-terminal kinase (JNK), a downstream target of ROS, was found to be a critical mediator of ziyuglycoside II-induced cell apoptosis. Further knockdown of JNK by siRNA could inhibit ziyuglycoside II-mediated apoptosis with attenuating the up-regulation of Bax and Fas/FasL as well as the down-regulation of Bcl-2. Taken together, the cell death of breast cancer cells in response to ziyuglycoside II was dependent upon cell cycle arrest and cell apoptosis via a ROS-dependent JNK activation pathway. Our findings may significantly contribute to the understanding of the anti-proliferative effect of ziyuglycoside II, in particular to breast carcinoma and provide novel insights into the potential application of such compound in breast cancer therapy. PMID:24680927

  18. Novel Platinum(II) compounds modulate insulin-degrading enzyme activity and induce cell death in neuroblastoma cells.

    PubMed

    Tundo, Grazia R; Sbardella, Diego; De Pascali, Sandra A; Ciaccio, Chiara; Coletta, Massimo; Fanizzi, Francesco P; Marini, Stefano

    2015-01-01

    The properties of three novel Platinum(II) compounds toward the insulin-degrading enzyme (IDE) enzymatic activity have been investigated under physiological conditions. The rationale of this study resides on previous observations that these compounds, specifically designed and synthesized by some of us, induce apoptosis in various cancer cell lines, whereas IDE has been proposed as a putative oncogene involved in neuroblastoma onset and progression. Two of these compounds, namely [PtCl(O,O'-acac)(DMSO)] and [Pt(O,O'-acac)(γ-acac)(DMS)], display a modulatory behavior, wherefore activation or inhibition of IDE activity occurs over different concentration ranges (suggesting the existence of two binding sites on the enzyme). On the other hand, [Pt(O,O'-acac)(γ-acac)(DMSO)] shows a typical competitive inhibitory pattern, characterized by a meaningful affinity constant (K i  = 0.95 ± 0.21 μM). Although all three compounds induce cell death in neuroblastoma SHSY5Y cells at concentrations exceeding 2 μM, the two modulators facilitate cells' proliferation at concentrations ≤ 1.5 μM, whereas the competitive inhibitor [Pt(O,O'-acac)(γ-acac)(DMSO)] only shows a pro-apoptotic activity at all investigated concentrations. These features render the [Pt(O,O'-acac)(γ-acac)(DMSO)] a promising "lead compound" for the synthesis of IDE-specific inhibitors (not characterized yet) with therapeutic potentiality.

  19. The novel HDAC inhibitor AR-42-induced anti-colon cancer cell activity is associated with ceramide production

    SciTech Connect

    Xu, Weihong; Xu, Bin; Yao, Yiting; Yu, Xiaoling; Shen, Jie

    2015-08-07

    In the current study, we investigated the potential activity of AR-42, a novel histone deacetylase (HDAC) inhibitor, against colon cancer cells. Our in vitro results showed that AR-42 induced ceramide production, exerted potent anti-proliferative and pro-apoptotic activities in established (SW-620 and HCT-116 lines) and primary human colon cancer cells. Exogenously-added sphingosine 1-phosphate (S1P) suppressed AR-42-induced activity, yet a cell-permeable ceramide (C4) facilitated AR-42-induced cytotoxicity against colon cancer cells. In addition, AR-42-induced ceramide production and anti-colon cancer cell activity were inhibited by the ceramide synthase inhibitor fumonisin B1, but were exacerbated by PDMP, which is a ceramide glucosylation inhibitor. In vivo, oral administration of a single dose of AR-42 dramatically inhibited SW-620 xenograft growth in severe combined immunodeficient (SCID) mice, without inducing overt toxicities. Together, these results show that AR-42 dramatically inhibits colon cancer cell proliferation in vitro and in vivo, and ceramide production might be the key mechanism responsible for its actions. - Highlights: • AR-42 is anti-proliferative against primary/established colon cancer cells. • AR-42 induces significant apoptotic death in primary/established colon cancer cells. • Ceramide production mediates AR-42-induced cytotoxicity in colon cancer cells. • AR-42 oral administration potently inhibits SW-620 xenograft growth in SCID mice.

  20. Kindlin-2 cooperates with talin to activate integrins and induces cell spreading by directly binding paxillin

    PubMed Central

    Theodosiou, Marina; Widmaier, Moritz; Böttcher, Ralph T; Rognoni, Emanuel; Veelders, Maik; Bharadwaj, Mitasha; Lambacher, Armin; Austen, Katharina; Müller, Daniel J; Zent, Roy; Fässler, Reinhard

    2016-01-01

    Integrins require an activation step prior to ligand binding and signaling. How talin and kindlin contribute to these events in non-hematopoietic cells is poorly understood. Here we report that fibroblasts lacking either talin or kindlin failed to activate β1 integrins, adhere to fibronectin (FN) or maintain their integrins in a high affinity conformation induced by Mn2+. Despite compromised integrin activation and adhesion, Mn2+ enabled talin- but not kindlin-deficient cells to initiate spreading on FN. This isotropic spreading was induced by the ability of kindlin to directly bind paxillin, which in turn bound focal adhesion kinase (FAK) resulting in FAK activation and the formation of lamellipodia. Our findings show that talin and kindlin cooperatively activate integrins leading to FN binding and adhesion, and that kindlin subsequently assembles an essential signaling node at newly formed adhesion sites in a talin-independent manner. DOI: http://dx.doi.org/10.7554/eLife.10130.001 PMID:26821125

  1. MAPKAPK-2-mediated LIM-kinase activation is critical for VEGF-induced actin remodeling and cell migration

    PubMed Central

    Kobayashi, Miho; Nishita, Michiru; Mishima, Toshiaki; Ohashi, Kazumasa; Mizuno, Kensaku

    2006-01-01

    Vascular endothelial growth factor-A (VEGF-A) induces actin reorganization and migration of endothelial cells through a p38 mitogen-activated protein kinase (MAPK) pathway. LIM-kinase 1 (LIMK1) induces actin remodeling by phosphorylating and inactivating cofilin, an actin-depolymerizing factor. In this study, we demonstrate that activation of LIMK1 by MAPKAPK-2 (MK2; a downstream kinase of p38 MAPK) represents a novel signaling pathway in VEGF-A-induced cell migration. VEGF-A induced LIMK1 activation and cofilin phosphorylation, and this was inhibited by the p38 MAPK inhibitor SB203580. Although p38 phosphorylated LIMK1 at Ser-310, it failed to activate LIMK1 directly; however, MK2 activated LIMK1 by phosphorylation at Ser-323. Expression of a Ser-323-non-phosphorylatable mutant of LIMK1 suppressed VEGF-A-induced stress fiber formation and cell migration; however, expression of a Ser-323-phosphorylation-mimic mutant enhanced these processes. Knockdown of MK2 by siRNA suppressed VEGF-A-induced LIMK1 activation, stress fiber formation, and cell migration. Expression of kinase-dead LIMK1 suppressed VEGF-A-induced tubule formation. These findings suggest that MK2-mediated LIMK1 phosphorylation/activation plays an essential role in VEGF-A-induced actin reorganization, migration, and tubule formation of endothelial cells. PMID:16456544

  2. Involvement of mast cells and proteinase-activated receptor 2 in oxaliplatin-induced mechanical allodynia in mice.

    PubMed

    Sakamoto, Ayumi; Andoh, Tsugunobu; Kuraishi, Yasushi

    2016-03-01

    The chemotherapeutic agent oxaliplatin induces neuropathic pain, a dose-limiting side effect, but the underlying mechanisms are not fully understood. Here, we show the potential involvement of cutaneous mast cells in oxaliplatin-induced mechanical allodynia in mice. A single intraperitoneal injection of oxaliplatin induced mechanical allodynia, which peaked on day 10 after injection. Oxaliplatin-induced mechanical allodynia was almost completely prevented by congenital mast cell deficiency. The numbers of total and degranulated mast cells was significantly increased in the skin after oxaliplatin administration. Repetitive topical application of the mast cell stabilizer azelastine hydrochloride inhibited mechanical allodynia and the degranulation of mast cells without affecting the number of mast cells in oxaliplatin-treated mice. The serine protease inhibitor camostat mesilate and the proteinase-activated receptor 2 (PAR2) antagonist FSLLRY-NH2 significantly inhibited oxaliplatin-induced mechanical allodynia. However, it was not inhibited by the H1 histamine receptor antagonist terfenadine. Single oxaliplatin administration increased the activity of cutaneous serine proteases, which was attenuated by camostat and mast cell deficiency. Depletion of the capsaicin-sensitive primary afferents by neonatal capsaicin treatment almost completely prevented oxaliplatin-induced mechanical allodynia, the increase in the number of mast cells, and the activity of cutaneous serine proteases. These results suggest that serine protease(s) released from mast cells and PAR2 are involved in oxaliplatin-induced mechanical allodynia. Therefore, oxaliplatin may indirectly affect the functions of mast cells through its action on capsaicin-sensitive primary afferents.

  3. Involvement of mast cells and proteinase-activated receptor 2 in oxaliplatin-induced mechanical allodynia in mice.

    PubMed

    Sakamoto, Ayumi; Andoh, Tsugunobu; Kuraishi, Yasushi

    2016-03-01

    The chemotherapeutic agent oxaliplatin induces neuropathic pain, a dose-limiting side effect, but the underlying mechanisms are not fully understood. Here, we show the potential involvement of cutaneous mast cells in oxaliplatin-induced mechanical allodynia in mice. A single intraperitoneal injection of oxaliplatin induced mechanical allodynia, which peaked on day 10 after injection. Oxaliplatin-induced mechanical allodynia was almost completely prevented by congenital mast cell deficiency. The numbers of total and degranulated mast cells was significantly increased in the skin after oxaliplatin administration. Repetitive topical application of the mast cell stabilizer azelastine hydrochloride inhibited mechanical allodynia and the degranulation of mast cells without affecting the number of mast cells in oxaliplatin-treated mice. The serine protease inhibitor camostat mesilate and the proteinase-activated receptor 2 (PAR2) antagonist FSLLRY-NH2 significantly inhibited oxaliplatin-induced mechanical allodynia. However, it was not inhibited by the H1 histamine receptor antagonist terfenadine. Single oxaliplatin administration increased the activity of cutaneous serine proteases, which was attenuated by camostat and mast cell deficiency. Depletion of the capsaicin-sensitive primary afferents by neonatal capsaicin treatment almost completely prevented oxaliplatin-induced mechanical allodynia, the increase in the number of mast cells, and the activity of cutaneous serine proteases. These results suggest that serine protease(s) released from mast cells and PAR2 are involved in oxaliplatin-induced mechanical allodynia. Therefore, oxaliplatin may indirectly affect the functions of mast cells through its action on capsaicin-sensitive primary afferents. PMID:26804251

  4. Benzo[a]pyrene-induced cell cycle progression occurs via ERK-induced Chk1 pathway activation in human lung cancer cells.

    PubMed

    Wang, Bing-Yen; Wu, Sung-Yu; Tang, Sheau-Chung; Lai, Chien-Hung; Ou, Chu-Chyn; Wu, Ming-Fang; Hsiao, Yi-Min; Ko, Jiunn-Liang

    2015-03-01

    Benzo[a]pyrene (B[a]P) is a potent lung carcinogen derived from tobacco smoking and environmental contamination. This study aimed to investigate the signal transduction pathway responsible for B[a]P-induced non-small cell lung cancer (NSCLC) development. We exposed the human NSCLC cell lines Calu-1, CL3, H1299, CH27, H23, and H1355 to B[a]P and assessed cell cycle progression using flow cytometry. Expression of cell cycle mediators was measured using Western blot analyses and electrophoretic mobility shift assays (EMSAs). B[a]P exposure dramatically induced S-phase accumulation in H1355 cells. Phospho-p53 (Ser15 and Ser20), phospho-ERK, phospho-p38, and Bax were significantly increased in H1355 cells whereas phospho-Rb was decreased in these cells. In addition, B[a]P induced phosphorylation of checkpoint kinase-1 (Chk1) but not Chk2. EMSA experiments revealed a slower migrating band after c-Myc bound the E-box in response to B[a]P treatment, which was abolished upon the addition of the ERK inhibitor PD98059 in H1355 cells. Phospho-ERK inhibition and dominant negative mutant Chk1 expression reversed B[a]P-induced S phase accumulation and downregulated phospho-Chk1 and phospho-ERK expression. Taken together, these results suggest that activation of ERK and its downstream mediator Chk1 may contribute to B[a]P-induced S phase accumulation in H1355 cells. The results could help in the development of lung cancer treatments that target the Chk1 pathway through ERK.

  5. Ribosome Synthesis and MAPK Activity Modulate Ionizing Radiation-Induced Germ Cell Apoptosis in Caenorhabditis elegans

    PubMed Central

    Eberhard, Ralf; Stergiou, Lilli; Hofmann, E. Randal; Hofmann, Jen; Haenni, Simon; Teo, Youjin; Furger, André; Hengartner, Michael O.

    2013-01-01

    Synthesis of ribosomal RNA by RNA polymerase I (RNA pol I) is an elemental biological process and is key for cellular homeostasis. In a forward genetic screen in C. elegans designed to identify DNA damage-response factors, we isolated a point mutation of RNA pol I, rpoa-2(op259), that leads to altered rRNA synthesis and a concomitant resistance to ionizing radiation (IR)-induced germ cell apoptosis. This weak apoptotic IR response could be phenocopied when interfering with other factors of ribosome synthesis. Surprisingly, despite their resistance to DNA damage, rpoa-2(op259) mutants present a normal CEP-1/p53 response to IR and increased basal CEP-1 activity under normal growth conditions. In parallel, rpoa-2(op259) leads to reduced Ras/MAPK pathway activity, which is required for germ cell progression and physiological germ cell death. Ras/MAPK gain-of-function conditions could rescue the IR response defect in rpoa-2(op259), pointing to a function for Ras/MAPK in modulating DNA damage-induced apoptosis downstream of CEP-1. Our data demonstrate that a single point mutation in an RNA pol I subunit can interfere with multiple key signalling pathways. Ribosome synthesis and growth-factor signalling are perturbed in many cancer cells; such an interplay between basic cellular processes and signalling might be critical for how tumours evolve or respond to treatment. PMID:24278030

  6. Particulate matter (PM₁₀) induces metalloprotease activity and invasion in airway epithelial cells.

    PubMed

    Morales-Bárcenas, Rocío; Chirino, Yolanda I; Sánchez-Pérez, Yesennia; Osornio-Vargas, Álvaro Román; Melendez-Zajgla, Jorge; Rosas, Irma; García-Cuellar, Claudia María

    2015-09-17

    Airborne particulate matter with an aerodynamic diameter ≤ 10 μm (PM10) is a risk factor for the development of lung diseases and cancer. The aim of this work was to identify alterations in airway epithelial (A549) cells induced by PM10 that could explain how subtoxic exposure (10 μg/cm(2)) promotes a more aggressive in vitro phenotype. Our results showed that cells exposed to PM10 from an industrial zone (IZ) and an urban commercial zone (CZ) induced an increase in protease activity and invasiveness; however, the cell mechanism is different, as only PM10 from CZ up-regulated the activity of metalloproteases MMP-2 and MMP-9 and disrupted E-cadherin/β-catenin expression after 48 h of exposure. These in vitro findings are relevant in terms of the mechanism action of PM10 in lung epithelial cells, which could be helpful in understanding the pathogenesis of some human illness associated with highly polluted cities.

  7. Activated Wnt signaling induces myofibroblast differentiation of mesenchymal stem cells, contributing to pulmonary fibrosis.

    PubMed

    Sun, Zhaorui; Wang, Cong; Shi, Chaowen; Sun, Fangfang; Xu, Xiaomeng; Qian, Weiping; Nie, Shinan; Han, Xiaodong

    2014-05-01

    Acute lung injury may lead to fibrogenesis. However, no treatment is currently available. This study was conducted to determine the effects of bone marrow-derived mesenchymal stem cells (MSCs) in a model of HCl-induced acute lung injury in Sprague-Dawley (SD) rats. Stromal cell-derived factor (SDF)-1 and its receptor CXC chemokine receptor (CXCR)4 have been shown to participate in mobilizing MSCs. Adenovirus carrying the CXCR4 gene was used to transfect MSCs in order to increase the engraftment numbers of MSCs at injured sites. Histological examination data demonstrated that the engraftment of MSCs did not attenuate lung injury and pulmonary fibrosis. The results showed that engraftment of MSCs almost differentiated into myofibroblasts, but rarely differentiated into lung epithelial cells. Additionally, it was demonstrated that activated canonical Wnt/β-catenin signaling in injured lung tissue regulated the myofibroblast differentiation of MSCs in vivo. The in vitro study results demonstrated that activation of the Wnt/β-catenin signaling stimulated MSCs to express myofibroblast markers; however, this process was attenuated by Wnt antagonist DKK1. Therefore, the results demonstrated that the aberrant activation of Wnt signaling induces the myofibroblast differentiation of engrafted MSCs, thus contributing to pulmonary fibrosis following lung injury. PMID:24573542

  8. Conditional inactivation of PDCD2 induces p53 activation and cell cycle arrest

    PubMed Central

    Granier, Celine J.; Wang, Wei; Tsang, Tiffany; Steward, Ruth; Sabaawy, Hatem E.; Bhaumik, Mantu; Rabson, Arnold B.

    2014-01-01

    ABSTRACT PDCD2 (programmed cell death domain 2) is a highly conserved, zinc finger MYND domain-containing protein essential for normal development in the fly, zebrafish and mouse. The molecular functions and cellular activities of PDCD2 remain unclear. In order to better understand the functions of PDCD2 in mammalian development, we have examined PDCD2 activity in mouse blastocyst embryos, as well as in mouse embryonic stem cells (ESCs) and embryonic fibroblasts (MEFs). We have studied mice bearing a targeted PDCD2 locus functioning as a null allele through a splicing gene trap, or as a conditional knockout, by deletion of exon2 containing the MYND domain. Tamoxifen-induced knockout of PDCD2 in MEFs, as well as in ESCs, leads to defects in progression from the G1 to the S phase of cell cycle, associated with increased levels of p53 protein and p53 target genes. G1 prolongation in ESCs was not associated with induction of differentiation. Loss of entry into S phase of the cell cycle and marked induction of nuclear p53 were also observed in PDCD2 knockout blastocysts. These results demonstrate a unique role for PDCD2 in regulating the cell cycle and p53 activation during early embryonic development of the mouse. PMID:25150276

  9. Chloroquine activates the p53 pathway and induces apoptosis in human glioma cells

    PubMed Central

    Kim, Ella L.; Wüstenberg, Robin; Rübsam, Anne; Schmitz-Salue, Christoph; Warnecke, Gabriele; Bücker, Eva-Maria; Pettkus, Nadine; Speidel, Daniel; Rohde, Veit; Schulz-Schaeffer, Walter; Deppert, Wolfgang; Giese, Alf

    2010-01-01

    Glioblastoma is the most common malignant brain tumor in adults. The currently available treatments offer only a palliative survival advantage and the need for effective treatments remains an urgent priority. Activation of the p53 growth suppression/apoptotic pathway is one of the promising strategies in targeting glioma cells. We show that the quinoline derivative chloroquine activates the p53 pathway and suppresses growth of glioma cells in vitro and in vivo in an orthotopic (U87MG) human glioblastoma mouse model. Induction of apoptosis is one of the mechanisms underlying the effects of chloroquine on suppressing glioma cell growth and viability. siRNA-mediated downregulation of p53 in wild-type but not mutant p53 glioblastoma cells substantially impaired chloroquine-induced apoptosis. In addition to its p53-activating effects, chloroquine may also inhibit glioma cell growth via p53-independent mechanisms. Our results clarify the mechanistic basis underlying the antineoplastic effect of chloroquine and reveal its therapeutic potential as an adjunct to glioma chemotherapy. PMID:20308316

  10. HIV-1 trans-activator of transcription substitutes for oxidative signaling in activation-induced T cell death.

    PubMed

    Gülow, Karsten; Kaminski, Marcin; Darvas, Katalin; Süss, Dorothee; Li-Weber, Min; Krammer, Peter H

    2005-05-01

    Termination of an immune response requires elimination of activated T lymphocytes by activation-induced cell death (AICD). In AICD, CD95 (Apo-1/Fas) ligand (L) triggers apoptosis of CD95-positive activated T lymphocytes. In AIDS patients, AICD is strongly enhanced and accelerated. We and others have previously shown that HIV-1 trans-activator of transcription (HIV-1 Tat) sensitizes T cells toward CD95-mediated apoptosis and up-regulates CD95L expression by affecting the cellular redox balance. In this study, we show that it is hydrogen peroxide (H(2)O(2)) that functions as an essential second messenger in TCR signaling. The H(2)O(2) signal combined with simultaneous calcium (Ca(2+)) influx into the cytosol constitutes the minimal requirement for induction of CD95L expression. Either signal alone is insufficient. We further show that HIV-1 Tat interferes with TCR signaling and induces a H(2)O(2) signal. H(2)O(2) generated by HIV-1 Tat combines with CD4-dependent calcium influx and causes massive T cell apoptosis. Thus, our data provide an explanation for CD4(+) T lymphocyte depletion during progression of AIDS.

  11. Tat-APE1/ref-1 protein inhibits TNF-alpha-induced endothelial cell activation.

    PubMed

    Song, Yun Jeong; Lee, Ji Young; Joo, Hee Kyoung; Kim, Hyo Shin; Lee, Sang Ki; Lee, Kwon Ho; Cho, Chung-Hyun; Park, Jin Bong; Jeon, Byeong Hwa

    2008-03-28

    Apurinic/apyrimidinic endonuclease 1/redox factor-1 (APE1/ref-1) is a multifunctional protein involved both in DNA base excision repair and redox regulation. In this study we evaluated the protective role of Tat-mediated APE1/ref-1 transduction on the tumor necrosis factor (TNF)-alpha-activated endothelial activation in cultured human umbilical vein endothelial cells. To construct Tat-APE1/ref-1 fusion protein, human full length of APE1/ref-1 was fused with Tat-protein transduction domain. Purified Tat-APE1/ref-1 fusion protein efficiently transduced cultured endothelial cells in a dose-dependent manner and reached maximum expression at 1h after incubation. Transduced Tat-APE1/ref-1 showed inhibitory activity on the TNF-alpha-induced monocyte adhesion and vascular cell adhesion molecule-1 expression in cultured endothelial cells. These results suggest Tat-APE1/ref-1 might be useful to reduce vascular endothelial activation or vascular inflammatory disorders.

  12. Activation of the Nrf2-regulated antioxidant cell response inhibits HEMA-induced oxidative stress and supports cell viability.

    PubMed

    Gallorini, Marialucia; Petzel, Christine; Bolay, Carola; Hiller, Karl-Anton; Cataldi, Amelia; Buchalla, Wolfgang; Krifka, Stephanie; Schweikl, Helmut

    2015-07-01

    Oxidative stress due to increased formation of reactive oxygen species (ROS) in target cells of dental resin monomers like 2-hydroxyethyl methacrylate (HEMA) is a major mechanism underlying the disturbance of vital cell functions including mineralization and differentiation, responses of the innate immune system, and the induction of cell death via apoptosis. Although a shift in the equilibrium between cell viability and apoptosis is related to the non-enzymatic antioxidant glutathione (GSH) in HEMA-exposed cells, the major mechanisms of adaptive antioxidant cell responses to maintain cellular redox homeostasis are still unknown. The present study provides insight into the induction of a communicating network of pathways under the control of the redox-sensitive transcription factor Nrf2, a major transcriptional activator of genes coding for enzymatic antioxidants. Here, oxidative stress was indicated by DCF fluorescence in cells after a short exposure (1 h) to HEMA, while DHR123 fluorescence significantly increased about 1.8-fold after a long exposure period (24 h) showing the formation of hydrogen peroxide (H2O2). The corresponding expression of Nrf2 was activated immediately after HEMA exposure (1 h) and remained constant up to 24 h. Nrf2-regulated expression of enzymes of the glutathione metabolism (glutathione peroxidase 1/2, glutathione reductase) decreased in HEMA-exposed cells as a result of GSH depletion, and superoxide dismutase expression was downregulated after H2O2 overproduction. However, the expression of Nrf2-controlled enzymatic antioxidants (catalase, peroxiredoxin, thioredoxin 1, thioredoxin reductase, heme oxygenase-1) and the NADPH-regenerating system (glucose 6-phosphate dehydrogenase, transaldolase) was increased. Phenolic tert-butylhydroquinone (tBHQ), a classic inducer of the Nrf2 pathway, reduced oxidative stress and protected cells from HEMA-induced cell death through a shift in the number of cells in necrosis to apoptosis. The

  13. Aluminum induces oxidative burst, cell wall NADH peroxidase activity, and DNA damage in root cells of Allium cepa L.

    PubMed

    Achary, V Mohan M; Parinandi, Narasimham L; Panda, Brahma B

    2012-08-01

    Plants under stress incur an oxidative burst that involves a rapid and transient overproduction of reactive oxygen species (ROS: O(2) (•-) , H(2) O(2) , (•) OH). We hypothesized that aluminum (Al), an established soil pollutant that causes plant stress, would induce an oxidative burst through the activation of cell wall-NADH peroxidase (NADH-PX) and/or plasma membrane-associated NADPH oxidase (NADPH-OX), leading to DNA damage in the root cells of Allium cepa L. Growing roots of A. cepa were treated with Al(3+) (800 μM of AlCl(3) ) for 3 or 6 hr without or with the pretreatment of inhibitors specific to NADH-PX and NADPH-OX for 2 hr. At the end of the treatment, the extent of ROS generation, cell death, and DNA damage were determined. The cell wall-bound protein (CWP) fractions extracted from the untreated control and the Al-treated roots under the aforementioned experimental conditions were also subjected to in vitro studies, which measured the extent of activation of peroxidase/oxidase, generation of (•) OH, and DNA damage. Overall, the present study demonstrates that the cell wall-bound NADH-PX contributes to the Al-induced oxidative burst through the generation of ROS that lead to cell death and DNA damage in the root cells of A. cepa. Furthermore, the in vitro studies revealed that the CWP fraction by itself caused DNA damage in the presence of NADH, supporting a role for NADH-PX in the stress response. Altogether, this study underscores the crucial function of the cell wall-bound NADH-PX in the oxidative burst-mediated cell death and DNA damage in plants under Al stress.

  14. Inactivation of glutathione peroxidase activity contributes to UV-induced squamous cell carcinoma formation.

    PubMed

    Walshe, Jennifer; Serewko-Auret, Magdalena M; Teakle, Ngari; Cameron, Sarina; Minto, Kelly; Smith, Louise; Burcham, Philip C; Russell, Terry; Strutton, Geoffrey; Griffin, Anthony; Chu, Fong-Fong; Esworthy, Stephen; Reeve, Vivienne; Saunders, Nicholas A

    2007-05-15

    Cutaneous squamous cell carcinomas (CSCC) are a common malignancy of keratinocytes that arise in sites of the skin exposed to excessive UV radiation. In the present study, we show that human SCC cell lines, preneoplastic solar keratoses (SK), and CSCC are associated with perturbations in glutathione peroxidase (GPX) activity and peroxide levels. Specifically, we found that two of three SKs and four of five CSCCs, in vivo, were associated with decreased GPX activity and all SKs and CSCCs were associated with an elevated peroxide burden. Given the association of decreased GPX activity with CSCC, we examined the basis for the GPX deficiency in the CSCCs. Our data indicated that GPX was inactivated by a post-translational mechanism and that GPX could be inactivated by increases in intracellular peroxide levels. We next tested whether the decreased peroxidase activity coupled with an elevated peroxidative burden might contribute to CSCC formation in vivo. This was tested in Gpx1(-/-) and Gpx2(-/-) mice exposed to solar-simulated UV radiation. These studies showed that Gpx2 deficiency predisposed mice to UV-induced CSCC formation. These results suggest that inactivation of GPX2 in human skin may be an early event in UV-induced SCC formation.

  15. Statins activate GATA-6 and induce differentiated vascular smooth muscle cells

    SciTech Connect

    Wada, Hiromichi Abe, Mitsuru; Ono, Koh; Morimoto, Tatsuya; Kawamura, Teruhisa; Takaya, Tomohide; Satoh, Noriko; Fujita, Masatoshi; Kita, Toru; Shimatsu, Akira; Hasegawa, Koji

    2008-10-03

    The beneficial effects of 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (statins) beyond cholesterol lowering involve their direct actions on vascular smooth muscle cells (VSMCs). However, the effects of statins on phenotypic modulation of VSMCs are unknown. We herein show that simvastatin (Sm) and atorvastatin (At) inhibited DNA synthesis in human aortic VSMCs dose-dependently, while cell toxicity was not observed below the concentration of 1 {mu}M of Sm or 100 nM of At. Stimulating proliferative VSMCs with Sm or At induced the expression of SM-{alpha}-actin and SM-MHC, highly specific markers of differentiated phenotype. Sm up-regulated the binding activity of GATA-6 to SM-MHC GATA site and activated the transfected SM-MHC promoter in proliferative VSMCs, while mutating the GATA-6 binding site abolished this activation. Geranylgeranylpyrophosphate (10 {mu}M), an inhibitor of Rho family proteins, abolished the statin-mediated induction of the differentiated phenotype in VSMCs. These findings suggest that statins activate GATA-6 and induce differentiated VSMCs.

  16. Activation of Peroxisome Proliferator-activated Receptor α Induces Lysosomal Biogenesis in Brain Cells

    PubMed Central

    Ghosh, Arunava; Jana, Malabendu; Modi, Khushbu; Gonzalez, Frank J.; Sims, Katherine B.; Berry-Kravis, Elizabeth; Pahan, Kalipada

    2015-01-01

    Lysosomes are ubiquitous membrane-enclosed organelles filled with an acidic interior and are central to the autophagic, endocytic, or phagocytic pathway. In contrast to its classical function as the waste management machinery, lysosomes are now considered to be an integral part of various cellular signaling processes. The diverse functionality of this single organelle requires a very complex and coordinated regulation of its activity with transcription factor EB (TFEB), a master regulator of lysosomal biogenesis, at its core. However, mechanisms by which TFEB is regulated are poorly understood. This study demonstrates that gemfibrozil, an agonist of peroxisome proliferator-activated receptor (PPAR) α, alone and in conjunction with all-trans-retinoic acid is capable of enhancing TFEB in brain cells. We also observed that PPARα, but not PPARβ and PPARγ, is involved in gemfibrozil-mediated up-regulation of TFEB. Reporter assay and chromatin immunoprecipitation studies confirmed the recruitment of retinoid X receptor α, PPARα, and PGC1α on the PPAR-binding site on the Tfeb promoter as well. Subsequently, the drug-mediated induction of TFEB caused an increase in lysosomal protein and the lysosomal abundance in cell. Collectively, this study reinforces the link between lysosomal biogenesis and lipid metabolism with TFEB at the crossroads. Furthermore, gemfibrozil may be of therapeutic value in the treatment of lysosomal storage disorders in which autophagy-lysosome pathway plays an important role. PMID:25750174

  17. Neutralization of (NK-cell-derived) B-cell activating factor by Belimumab restores sensitivity of chronic lymphoid leukemia cells to direct and Rituximab-induced NK lysis.

    PubMed

    Wild, J; Schmiedel, B J; Maurer, A; Raab, S; Prokop, L; Stevanović, S; Dörfel, D; Schneider, P; Salih, H R

    2015-08-01

    Natural killer (NK) cells are cytotoxic lymphocytes that substantially contribute to the therapeutic benefit of antitumor antibodies like Rituximab, a crucial component in the treatment of B-cell malignancies. In chronic lymphocytic leukemia (CLL), the ability of NK cells to lyse the malignant cells and to mediate antibody-dependent cellular cytotoxicity upon Fc receptor stimulation is compromised, but the underlying mechanisms are largely unclear. We report here that NK-cells activation-dependently produce the tumor necrosis factor family member 'B-cell activating factor' (BAFF) in soluble form with no detectable surface expression, also in response to Fc receptor triggering by therapeutic CD20-antibodies. BAFF in turn enhanced the metabolic activity of primary CLL cells and impaired direct and Rituximab-induced lysis of CLL cells without affecting NK reactivity per se. The neutralizing BAFF antibody Belimumab, which is approved for treatment of systemic lupus erythematosus, prevented the effects of BAFF on the metabolism of CLL cells and restored their susceptibility to direct and Rituximab-induced NK-cell killing in allogeneic and autologous experimental systems. Our findings unravel the involvement of BAFF in the resistance of CLL cells to NK-cell antitumor immunity and Rituximab treatment and point to a benefit of combinatory approaches employing BAFF-neutralizing drugs in B-cell malignancies.

  18. Prion-induced neurotoxicity: Possible role for cell cycle activity and DNA damage response

    PubMed Central

    Bujdoso, Raymond; Landgraf, Matthias; Jackson, Walker S; Thackray, Alana M

    2015-01-01

    Protein misfolding neurodegenerative diseases arise through neurotoxicity induced by aggregation of host proteins. These conditions include Alzheimer’s disease, Huntington’s disease, Parkinson’s disease, motor neuron disease, tauopathies and prion diseases. Collectively, these conditions are a challenge to society because of the increasing aged population and through the real threat to human food security by animal prion diseases. It is therefore important to understand the cellular and molecular mechanisms that underlie protein misfolding-induced neurotoxicity as this will form the basis for designing strategies to alleviate their burden. Prion diseases are an important paradigm for neurodegenerative conditions in general since several of these maladies have now been shown to display prion-like phenomena. Increasingly, cell cycle activity and the DNA damage response are recognised as cellular events that participate in the neurotoxic process of various neurodegenerative diseases, and their associated animal models, which suggests they are truly involved in the pathogenic process and are not merely epiphenomena. Here we review the role of cell cycle activity and the DNA damage response in neurodegeneration associated with protein misfolding diseases, and suggest that these events contribute towards prion-induced neurotoxicity. In doing so, we highlight PrP transgenic Drosophila as a tractable model for the genetic analysis of transmissible mammalian prion disease. PMID:26279981

  19. Nilotinib induces apoptosis and autophagic cell death of activated hepatic stellate cells via inhibition of histone deacetylases.

    PubMed

    Shaker, Mohamed E; Ghani, Ayaz; Shiha, Gamal E; Ibrahim, Tarek M; Mehal, Wajahat Z

    2013-08-01

    Increasing hepatic stellate cell (HSC) death is a very attractive approach for limiting liver fibrosis. Tyrosine kinase inhibitors have been shown to have anti-fibrotic properties, but the mechanisms are poorly understood. Here, we identified the mechanism of action of the second-generation tyrosine kinase inhibitor nilotinib in inducing HSC death. Human HSC line (LX-2) and rat HSCs were treated with nilotinib and its predecessor, imatinib, in the absence or presence of various blockers, known to interfere with death signaling pathways. Nilotinib, but not imatinib, induced progressive cell death of activated, but not quiescent, HSCs in a dose-dependent manner. Activated HSCs died through apoptosis, as denoted by increased DNA fragmentation and caspase activation, and through autophagy, as indicated by the accumulation of autophagic markers, light chain (LC)3A-II and LC3B-II. Although inhibition of caspases with Z-VAD-FMK suppressed nilotinib-induced HSCs' apoptosis, there was no increase in HSCs' survival, because autophagy was exacerbated. However, blocking the mitochondrial permeability transition pore (mPTP) opening with cyclosporin A completely abolished both apoptosis and autophagy due to nilotinib. Moreover, nilotinib treatment decreased the protein expression of histone deacetylases 1, 2 and 4. Interestingly, pretreament with C646, a selective p300/CBP histone acetyl transferase inhibitor, resulted in diverting nilotinib-induced apoptosis and autophagy towards necrosis. In conclusion, the identification of mPTP as a target of nilotinib in activated HSCs suggests coordination with histone deacetylases inhibition to induce apoptosis and autophagy. Thus, our study provides novel insights into the anti-fibrotic effects of nilotinib.

  20. LPS-induced cytokine production in human dendritic cells is regulated by sialidase activity

    PubMed Central

    Stamatos, Nicholas M.; Carubelli, Ivan; van de Vlekkert, Diantha; Bonten, Erik J.; Papini, Nadia; Feng, Chiguang; Venerando, Bruno; d'Azzo, Alessandra; Cross, Alan S.; Wang, Lai-Xi; Gomatos, Peter J.

    2010-01-01

    Removal of sialic acid from glycoconjugates on the surface of monocytes enhances their response to bacterial LPS. We tested the hypothesis that endogenous sialidase activity creates a permissive state for LPS-induced cytokine production in human monocyte-derived DCs. Of the four genetically distinct sialidases (Neu1–4), Neu1, Neu3, and Neu4 are expressed in human monocytes, but only Neu1 and Neu3 are up-regulated as cells differentiate into DCs. Neu1 and Neu3 are present on the surface of monocytes and DCs and are also present intracellularly. DCs contain a greater amount of sialic acid than monocytes, but the amount of sialic acid/mg total protein declines during differentiation to DCs. This relative hyposialylation of cells does not occur in mature DCs grown in the presence of zanamivir, a pharmacologic inhibitor of Neu3 but not Neu1, or DANA, an inhibitor of Neu1 and Neu3. Inhibition of sialidase activity during differentiation to DCs causes no detectable change in cell viability or expression of DC surface markers. Differentiation of monocytes into DCs in the presence of zanamivir results in reduced LPS- induced expression of IL-6, IL-12p40, and TNF-α by mature DCs, demonstrating a role for Neu3 in cytokine production. A role for Neu3 is supported by inhibition of cytokine production by DANA in DCs from Neu1–/– and WT mice. We conclude that sialidase-mediated change in sialic acid content of specific cell surface glycoconjugates in DCs regulates LPS-induced cytokine production, thereby contributing to development of adaptive immune responses. PMID:20826611

  1. YAP activation protects urothelial cell carcinoma from treatment-induced DNA damage

    PubMed Central

    Ciamporcero, Eric; Shen, He; Ramakrishnan, Swathi; Ku, Sheng Yu; Chintala, Sreenivasulu; Shen, Li; Adelaiye, Remi; Miles, Kiersten Marie; Ullio, Chiara; Pizzimenti, Stefania; Daga, Martina; Azabdaftari, Gissou; Attwood, Kris; Johnson, Candace; Zhang, Jianmin; Barrera, Giuseppina; Pili, Roberto

    2015-01-01

    Current standard of care for muscle-invasive urothelial cell carcinoma (UCC) is surgery along with perioperative platinum-based chemotherapy. UCC is sensitive to cisplatin-based regimens, but acquired resistance eventually occurs, and a subset of tumors is intrinsically resistant. Thus, there is an unmet need for new therapeutic approaches to target chemotherapy-resistant UCC. Yes-associated protein (YAP) is a transcriptional co-activator that has been associated with bladder cancer progression and cisplatin resistance in ovarian cancer. In contrast, YAP has been shown to induce DNA damage associated apoptosis in non-small cell lung carcinoma. However, no data have been reported on the YAP role in UCC chemo-resistance. Thus, we have investigated the potential dichotomous role of YAP in UCC response to chemotherapy utilizing two patient-derived xenograft models recently established. Constitutive expression and activation of YAP inversely correlated with in vitro and in vivo cisplatin sensitivity. YAP overexpression protected while YAP knock-down sensitized UCC cells to chemotherapy and radiation effects via increased accumulation of DNA damage and apoptosis. Furthermore, pharmacological YAP inhibition with verteporfin inhibited tumor cell proliferation and restored sensitivity to cisplatin. In addition, nuclear YAP expression was associated with poor outcome in UCC patients who received perioperative chemotherapy. In conclusion, these results suggest that YAP activation exerts a protective role and represents a pharmacological target to enhance the anti-tumor effects of DNA damaging modalities in the treatment of UCC. PMID:26119935

  2. Quantifying signaling pathway activation to monitor the quality of induced pluripotent stem cells.

    PubMed

    Makarev, Eugene; Fortney, Kristen; Litovchenko, Maria; Braunewell, Karl H; Zhavoronkov, Alex; Atala, Anthony

    2015-09-15

    Many attempts have been made to evaluate the safety and potency of human induced pluripotent stem cells (iPSCs) for clinical applications using transcriptome data, but results so far have been ambiguous or even contradictory. Here, we characterized stem cells at the pathway level, rather than at the gene level as has been the focus of previous work. We meta-analyzed publically-available gene expression data sets and evaluated signaling and metabolic pathway activation profiles for 20 human embryonic stem cell (ESC) lines, 12 human iPSC lines, five embryonic body lines, and six fibroblast cell lines. We demonstrated the close resemblance of iPSCs with ESCs at the pathway level, and provided examples of how pathway activity can be applied to identify iPSC line abnormalities or to predict in vitro differentiation potential. Our results indicate that pathway activation profiling is a promising strategy for evaluating the safety and potency of iPSC lines in translational medicine applications.

  3. Quantifying signaling pathway activation to monitor the quality of induced pluripotent stem cells

    PubMed Central

    Makarev, Eugene; Fortney, Kristen; Litovchenko, Maria; Braunewell, Karl H.; Zhavoronkov, Alex; Atala, Anthony

    2015-01-01

    Many attempts have been made to evaluate the safety and potency of human induced pluripotent stem cells (iPSCs) for clinical applications using transcriptome data, but results so far have been ambiguous or even contradictory. Here, we characterized stem cells at the pathway level, rather than at the gene level as has been the focus of previous work. We meta-analyzed publically-available gene expression data sets and evaluated signaling and metabolic pathway activation profiles for 20 human embryonic stem cell (ESC) lines, 12 human iPSC lines, five embryonic body lines, and six fibroblast cell lines. We demonstrated the close resemblance of iPSCs with ESCs at the pathway level, and provided examples of how pathway activity can be applied to identify iPSC line abnormalities or to predict in vitro differentiation potential. Our results indicate that pathway activation profiling is a promising strategy for evaluating the safety and potency of iPSC lines in translational medicine applications. PMID:26327604

  4. Quantifying signaling pathway activation to monitor the quality of induced pluripotent stem cells.

    PubMed

    Makarev, Eugene; Fortney, Kristen; Litovchenko, Maria; Braunewell, Karl H; Zhavoronkov, Alex; Atala, Anthony

    2015-09-15

    Many attempts have been made to evaluate the safety and potency of human induced pluripotent stem cells (iPSCs) for clinical applications using transcriptome data, but results so far have been ambiguous or even contradictory. Here, we characterized stem cells at the pathway level, rather than at the gene level as has been the focus of previous work. We meta-analyzed publically-available gene expression data sets and evaluated signaling and metabolic pathway activation profiles for 20 human embryonic stem cell (ESC) lines, 12 human iPSC lines, five embryonic body lines, and six fibroblast cell lines. We demonstrated the close resemblance of iPSCs with ESCs at the pathway level, and provided examples of how pathway activity can be applied to identify iPSC line abnormalities or to predict in vitro differentiation potential. Our results indicate that pathway activation profiling is a promising strategy for evaluating the safety and potency of iPSC lines in translational medicine applications. PMID:26327604

  5. Active Components of Fungus Shiraia bambusiscola Can Specifically Induce BGC823 Gastric Cancer Cell Apoptosis

    PubMed Central

    Zhang, Shubing; Qiu, Dewen; Liu, Jingjiang; Li, Zhijian

    2016-01-01

    Objective Gastric cancer is a major health issue worldwide. Using a therapeutic approach, with minor side-effects, is very essential for the treatment of the gastric cancer. Shiraia bambusicola is a parasitic fungus which is widely used in China for curing several diseases with little side-effects. However, the mechanisms are not well understood yet. The aim of this study was to further understand the pharmacological mechanisms of Shiraia bambusicola and investigate whether it can be used for curing gastric cancer. Materials and Methods In this experimental study, we mainly tested the effect of active components extracted from Shiraia bambusicola on BGC823, A549 and HepG2 cells. We used MTT assay to test cell viability. We also analyzed morphologic changes caused by apoptosis using Hoechst 33342 fluorescence staining, as well as cell cycle status and apoptosis ratio using flow-cytometer. In addition, protein expression level was tested by Western-blotting assay. Results BGC-823 cell proliferation was specifically inhibited by active components of Shiraia bambusicola. Meanwhile, these active components could induce BGC-823 cells apoptosis and retard the cell cycle in S/G2 phase. We also determined that two critical protein markers cleaved Poly(ADP-ribose) polymerase-1 (PARP-1) and FLICE-inhibitory protein (FLIP), involved in apoptosis process, were regulated by these active components. Conclusion These data shed light on the treatment of human gastric cancer and conclude that Shiraia bambusicola can be a good therapeutic candidate for treatment of this malignancy. PMID:27540519

  6. A vascular injury model using focal heat-induced activation of endothelial cells

    PubMed Central

    Sylman, J.L.; Artzer, D.T.; Rana, K.; Neeves, K.B.

    2015-01-01

    Endothelial cells (EC) both inhibit and promote platelet function depending on their activation state. Quiescent EC inhibit platelet activation by constitutive secretion of platelet inhibitors. Activated EC promote platelet adhesion by secretion of von Willebrand factor (vWF). EC also secrete an extracellular matrix that support platelet adhesion when exposed following vascular injury. Previous studies of EC-platelet interactions under flow activate entire monolayers of cells by chemical activation. In this study, EC cultured in microfluidic channels were focally activated by heat from an underlying microelectrode. Based on finite element modeling, microelectrodes induced peak temperature increases of 10–40 °C above 37 °C after applying 5–9 V for 30 s resulting in three zones: (1) A quiescent zone corresponded to peak temperatures of less than 15 °C characterized by no EC activation or platelet accumulation. (2) An activation zone corresponding to an increase of 16–22 °C yielded EC that were viable, secreted elevated levels of vWF, and were P-selectin positive. Platelets accumulated in the retracted spaces between EC in the activation zone at a wall shear rate of 150 s−1. Experiments with blocking antibodies show that platelets adhere via GPIbα-vWF and α6β1-laminin interactions. (3) A kill zone corresponded to peak temperatures of greater than 23 °C where EC were not viable and did not support platelet adhesion. These data define heating conditions for the activation of EC, causing the secretion of vWF and the exposure of a subendothelial matrix that support platelet adhesion and aggregation. This model provides for spatially defined zones of EC activation that could be a useful tool for measuring the relative roles of anti- and prothrombotic roles of EC at the site of vascular injury. PMID:26087748

  7. Syrbactin Structural Analog TIR-199 Blocks Proteasome Activity and Induces Tumor Cell Death.

    PubMed

    Bachmann, André S; Opoku-Ansah, John; Ibarra-Rivera, Tannya R; Yco, Lisette P; Ambadi, Sudhakar; Roberts, Christopher C; Chang, Chia-En A; Pirrung, Michael C

    2016-04-15

    Multiple myeloma is an aggressive hematopoietic cancer of plasma cells. The recent emergence of three effective FDA-approved proteasome-inhibiting drugs, bortezomib (Velcade®), carfilzomib (Kyprolis®), and ixazomib (Ninlaro®), confirms that proteasome inhibitors are therapeutically useful against neoplastic disease, in particular refractory multiple myeloma and mantle cell lymphoma. This study describes the synthesis, computational affinity assessment, and preclinical evaluation of TIR-199, a natural product-derived syrbactin structural analog. Molecular modeling and simulation suggested that TIR-199 covalently binds each of the three catalytic subunits (β1, β2, and β5) and revealed key interaction sites. In vitro and cell culture-based proteasome activity measurements confirmed that TIR-199 inhibits the proteasome in a dose-dependent manner and induces tumor cell death in multiple myeloma and neuroblastoma cells as well as other cancer types in the NCI-60 cell panel. It is particularly effective against kidney tumor cell lines, with >250-fold higher anti-tumor activities than observed with the natural product syringolin A. In vivo studies in mice revealed a maximum tolerated dose of TIR-199 at 25 mg/kg. The anti-tumor activity of TIR-199 was confirmed in hollow fiber assays in mice. Adverse drug reaction screens in a kidney panel revealed no off-targets of concern. This is the first study to examine the efficacy of a syrbactin in animals. Taken together, the results suggest that TIR-199 is a potent new proteasome inhibitor with promise for further development into a clinical drug for the treatment of multiple myeloma and other forms of cancer.

  8. Absence of missense mutations in activated c-myc genes in avian leukosis virus-induced B-cell lymphomas

    SciTech Connect

    Hahn, M.; Hayward, W.S.

    1988-06-01

    The authors determined the nucleotide sequences of two independent DNA clones which contained the activated c-myc genes from avian leukosis virus-induced B-cell lymphomas. Neither of these c-myce genes contained missense mutations. This strongly supports the notion that the c-myc photo-oncogene in avian leukosis virus-induced B-cell lymphomas can be oncogenically activated by altered expression of the gene without a change in the primary structure of the gene product.

  9. Evodiamine Attenuates PDGF-BB-Induced Migration of Rat Vascular Smooth Muscle Cells through Activating PPARγ.

    PubMed

    Ge, Xie; Chen, Siyu; Liu, Mei; Liang, Tingming; Liu, Chang

    2015-11-26

    The uncontrolled migration of vascular smooth muscle cells (VSMCs) into the intima is a critical process in the development of atherosclerosis. Evodiamine, an indole alkaloid extracted from the Chinese medicine evodia, has been shown to inhibit tumor cell invasion and protect the cardiovascular system, but its effects on VSMCs remain unknown. In the present study, we investigated the inhibitory effects of evodiamine on the platelet-derived growth factor-BB (PDGF-BB)-induced VSMC migration using wound healing and transwell assays, and assessed its role in decreasing the protein levels of matrix metalloproteinases and cell adhesion molecules. More importantly, we found that evodiamine activated the expression and nuclear translocation of peroxisome proliferator-activated receptor γ (PPARγ). Inhibition of PPARγ activity by using its antagonist T0070907 and its specific siRNA oligonucleotides significantly attenuated the inhibitory effects of evodiamine on VSMC migration. Taken together, our results indicate a promising anti-atherogenic effect of evodiamine through attenuation of VSMC migration by activating PPARγ.

  10. Insights into the mechanism of human papillomavirus E2-induced procaspase-8 activation and cell death

    PubMed Central

    Singh, Nitu; Senapati, Sanjib; Bose, Kakoli

    2016-01-01

    High-risk human papillomavirus (HR-HPV) E2 protein, the master regulator of viral life cycle, induces apoptosis of host cell that is independent of its virus-associated regulatory functions. E2 protein of HR-HPV18 has been found to be involved in novel FADD-independent activation of caspase-8, however, the molecular basis of this unique non-death-fold E2-mediated apoptosis is poorly understood. Here, with an interdisciplinary approach that involves in silico, mutational, biochemical and biophysical probes, we dissected and characterized the E2-procasapse-8 binding interface. Our data demonstrate direct non-homotypic interaction of HPV18 E2 transactivation domain (TAD) with α2/α5 helices of procaspase-8 death effector domain-B (DED-B). The observed interaction mimics the homotypic DED-DED complexes, wherein the conserved hydrophobic motif of procaspase-8 DED-B (F122/L123) occupies a groove between α2/α3 helices of E2 TAD. This interaction possibly drives DED oligomerization leading to caspase-8 activation and subsequent cell death. Furthermore, our data establish a model for E2-induced apoptosis in HR-HPV types and provide important clues for designing E2 analogs that might modulate procaspase-8 activation and hence apoptosis. PMID:26906543

  11. Activated T cells induce expression of B7/BB1 on normal or leukemic B cells through a CD40-dependent signal

    PubMed Central

    1993-01-01

    Cognate interactions between antigen-presenting B and T cells play crucial roles in immunologic responses. T cells that have been activated via the crosslinking of CD3 are able to induce B cell proliferation and immunoglobulin secretion in a major histocompatibility complex-unrestricted and contact-dependent manner. We find that such activated human CD4+ T cells, but not control Ig- treated T cells, may induce normal or leukemic B cells to express B7/BB1 and significantly higher levels of CD54 intercellular adhesion molecule 1 via a process that also requires direct cell-cell contact. To discern what cell surface molecule(s) may be responsible for signalling B cells to express B7/BB1, we added various monoclonal antibodies (mAbs) specific for T or B cell accessory molecules or control mAbs to cocultures of alpha-CD3-activated T cells and resting B cells. We find that only alpha-CD40 mAbs can significantly inhibit the increased expression of B7/BB1, suggesting that the ligand for CD40 expressed on activated T cells may be an important inducer of B7/BB1 expression. Subsequent experiments in fact demonstrate that alpha-CD40 mAbs, but not control mAbs, induce changes in B cell phenotype similar to those induced by activated T cells when the mAbs are presented on Fc gamma RII (CDw32)-expressing L cells. These phenotypic changes have significant effects on B cell function. Whereas chronic lymphocytic leukemia (CLL) B cells normally are very poor stimulators in allogeneic mixed lymphocyte reactions (MLRs), CLL-B cells preactivated via CD40 crosslinking are significantly better presenters of alloantigen, affecting up to 30-fold-greater stimulation of T cell proliferation than that induced by control treated or nontreated CLL-B cells. Similarly, the MLR of T cells stimulated by allogeneic nonleukemic B cells can be enhanced significantly if the stimulator B cells are preactivated via CD40 crosslinking. The enhanced MLR generated by such preactivated B cells may be inhibited

  12. Ionizing Radiation Induces Macrophage Foam Cell Formation and Aggregation Through JNK-Dependent Activation of CD36 Scavenger Receptors

    SciTech Connect

    Katayama, Ikuo; Hotokezaka, Yuka; Matsuyama, Toshifumi; Sumi, Tadateru; Nakamura, Takashi

    2008-03-01

    Purpose: Irradiated arteries of cancer patients can be associated with atherosclerosis-like lesions containing cholesterol-laden macrophages (foam cells). Endothelial cell damage by irradiation does not completely explain the foam cell formation. We investigated the possible underlying mechanisms for ionizing radiation (IR)-induced foam cell formation. Methods and Materials: Human peripheral blood monocytes were activated by macrophage colony-stimulating factor and then treated with varying doses of IR in vitro in the absence of endothelial cells. Scavenger receptor expression and foam cell formation of IR-treated macrophages were investigated in the presence or absence of oxidized low-density lipoprotein. We also assessed the importance of mitogen-activated protein kinase activity in the macrophage colony-stimulating factor-activated human monocytes (macrophages) for the foam cell formation. Results: We found that IR treatment of macrophage colony-stimulating factor-activated human peripheral blood monocytes resulted in the enhanced expression of CD36 scavenger receptors and that cholesterol accumulated in the irradiated macrophages with resultant foam cell formation in the presence of oxidized low-density lipoprotein. Furthermore, when cultured on collagen gels, human macrophages formed large foam cell aggregates in response to IR. Antibodies against CD36 inhibited the IR-induced foam cell formation and aggregation, indicating that the IR-induced foam cell formation and the subsequent aggregation are dependent on functional CD36. In addition, we found that IR of human macrophages resulted in c-Jun N-terminal kinase activation and that c-Jun N-terminal kinase inhibition suppressed IR-induced CD36 expression and the subsequent foam cell formation and aggregation. Conclusion: Taken together, these results suggest that IR-induced foam cell formation is mediated by c-Jun N-terminal kinase-dependent CD36 activation.

  13. Dynamic Assessment of Fibroblast Mechanical Activity during Rac-induced Cell Spreading in 3-D Culture

    PubMed Central

    Petroll, W. Matthew; Ma, Lisha; Kim, Areum; Ly, Linda; Vishwanath, Mridula

    2009-01-01

    The goal of this study was to determine the morphological and sub-cellular mechanical effects of Rac activation on fibroblasts within 3-D collagen matrices. Corneal fibroblasts were plated at low density inside 100 μm thick fibrillar collagen matrices and cultured for 1 to 2 days in serum-free media. Time-lapse imaging was then performed using Nomarski DIC. After an acclimation period, perfusion was switched to media containing PDGF. In some experiments, Y-27632 or blebbistatin were used to inhibit Rho-kinase (ROCK) or myosin II, respectively. PDGF activated Rac and induced cell spreading, which resulted in an increase in cell length, cell area, and the number of pseudopodial processes. Tractional forces were generated by extending pseudopodia, as indicated by centripetal displacement and realignment of collagen fibrils. Interestingly, the pattern of pseudopodial extension and local collagen fibril realignment was highly dependent upon the initial orientation of fibrils at the leading edge. Following ROCK or myosin II inhibition, significant ECM relaxation was observed, but small displacements of collagen fibrils continued to be detected at the tips of pseudopodia. Taken together, the data suggests that during Rac-induced cell spreading within 3-D matrices, there is a shift in the distribution of forces from the center to the periphery of corneal fibroblasts. ROCK mediates the generation of large myosin II-based tractional forces during cell spreading within 3-D collagen matrices, however residual forces can be generated at the tips of extending pseudopodia that are both ROCK and myosin II-independent. PMID:18452153

  14. GD2-specific CAR T Cells Undergo Potent Activation and Deletion Following Antigen Encounter but can be Protected From Activation-induced Cell Death by PD-1 Blockade.

    PubMed

    Gargett, Tessa; Yu, Wenbo; Dotti, Gianpietro; Yvon, Eric S; Christo, Susan N; Hayball, John D; Lewis, Ian D; Brenner, Malcolm K; Brown, Michael P

    2016-06-01

    Chimeric antigen receptor (CAR) T cells have shown great promise in the treatment of hematologic malignancies but more variable results in the treatment of solid tumors and the persistence and expansion of CAR T cells within patients has been identified as a key correlate of antitumor efficacy. Lack of immunological "space", functional exhaustion, and deletion have all been proposed as mechanisms that hamper CAR T-cell persistence. Here we describe the events following activation of third-generation CAR T cells specific for GD2. CAR T cells had highly potent immediate effector functions without evidence of functional exhaustion in vitro, although reduced cytokine production reversible by PD-1 blockade was observed after longer-term culture. Significant activation-induced cell death (AICD) of CAR T cells was observed after repeated antigen stimulation, and PD-1 blockade enhanced both CAR T-cell survival and promoted killing of PD-L1(+) tumor cell lines. Finally, we assessed CAR T-cell persistence in patients enrolled in the CARPETS phase 1 clinical trial of GD2-specific CAR T cells in the treatment of metastatic melanoma. Together, these data suggest that deletion also occurs in vivo and that PD-1-targeted combination therapy approaches may be useful to augment CAR T-cell efficacy and persistence in patients.

  15. (−)-Epicatechin induces calcium and translocation independent eNOS activation in arterial endothelial cells

    PubMed Central

    Ramirez-Sanchez, Israel; Maya, Lisandro; Ceballos, Guillermo

    2011-01-01

    The consumption of cacao-derived (i.e., cocoa) products provides beneficial cardiovascular effects in healthy subjects as well as individuals with endothelial dysfunction such as smokers, diabetics, and postmenopausal women. The vascular actions of cocoa are related to enhanced nitric oxide (NO) production. These actions can be reproduced by the administration of the cacao flavanol (−)-epicatechin (EPI). To further understand the mechanisms behind the vascular action of EPI, we investigated the effects of Ca2+ depletion on endothelial nitric oxide (NO) synthase (eNOS) activation/phosphorylation and translocation. Human coronary artery endothelial cells were treated with EPI or with bradykinin (BK), a well-known Ca2+-dependent eNOS activator. Results demonstrate that both EPI and BK induce increases in intracellular calcium and NO levels. However, under Ca2+-free conditions, EPI (but not BK) is still capable of inducing NO production through eNOS phosphorylation at serine 615, 633, and 1177. Interestingly, EPI-induced translocation of eNOS from the plasmalemma was abolished upon Ca2+ depletion. Thus, under Ca2+-free conditions, EPI can stimulate NO synthesis independent of calmodulin binding to eNOS and of its translocation into the cytoplasm. We also examined the effect of EPI on the NO/cGMP/vasodilator-stimulated phosphoprotein (VASP) pathway activation in isolated Ca2+-deprived canine mesenteric arteries. Results demonstrate that under these conditions, EPI induces the activation of this vasorelaxation-related pathway and that this effect is inhibited by pretreatment with nitro-l-arginine methyl ester, suggesting a functional relevance for this phenomenon. PMID:21209365

  16. Cell death is induced by ciglitazone, a peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}) agonist, independently of PPAR{gamma} in human glioma cells

    SciTech Connect

    Lee, Myoung Woo; Kim, Dae Seong; Kim, Hye Ryung; Kim, Hye Jin; Yang, Jin Mo; Ryu, Somi; Noh, Yoo Hun; Lee, Soo Hyun; Son, Meong Hi; Jung, Hye Lim; Yoo, Keon Hee; Koo, Hong Hoe; Sung, Ki Woong

    2012-01-06

    Highlights: Black-Right-Pointing-Pointer Greater than 30 {mu}M ciglitazone induces cell death in glioma cells. Black-Right-Pointing-Pointer Cell death by ciglitazone is independent of PPAR{gamma} in glioma cells. Black-Right-Pointing-Pointer CGZ induces cell death by the loss of MMP via decreased Akt. -- Abstract: Peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}) regulates multiple signaling pathways, and its agonists induce apoptosis in various cancer cells. However, their role in cell death is unclear. In this study, the relationship between ciglitazone (CGZ) and PPAR{gamma} in CGZ-induced cell death was examined. At concentrations of greater than 30 {mu}M, CGZ, a synthetic PPAR{gamma} agonist, activated caspase-3 and induced apoptosis in T98G cells. Treatment of T98G cells with less than 30 {mu}M CGZ effectively induced cell death after pretreatment with 30 {mu}M of the PPAR{gamma} antagonist GW9662, although GW9662 alone did not induce cell death. This cell death was also observed when cells were co-treated with CGZ and GW9662, but was not observed when cells were treated with CGZ prior to GW9662. In cells in which PPAR{gamma} was down-regulated cells by siRNA, lower concentrations of CGZ (<30 {mu}M) were sufficient to induce cell death, although higher concentrations of CGZ ( Greater-Than-Or-Slanted-Equal-To 30 {mu}M) were required to induce cell death in control T98G cells, indicating that CGZ effectively induces cell death in T98G cells independently of PPAR{gamma}. Treatment with GW9662 followed by CGZ resulted in a down-regulation of Akt activity and the loss of mitochondrial membrane potential (MMP), which was accompanied by a decrease in Bcl-2 expression and an increase in Bid cleavage. These data suggest that CGZ is capable of inducing apoptotic cell death independently of PPAR{gamma} in glioma cells, by down-regulating Akt activity and inducing MMP collapse.

  17. Loss of EBP50 stimulates EGFR activity to induce EMT phenotypic features in biliary cancer cells.

    PubMed

    Clapéron, A; Guedj, N; Mergey, M; Vignjevic, D; Desbois-Mouthon, C; Boissan, M; Saubaméa, B; Paradis, V; Housset, C; Fouassier, L

    2012-03-15

    Scaffold proteins form multiprotein complexes that are central to the regulation of intracellular signaling. The scaffold protein ezrin-radixin-moesin-binding phosphoprotein 50 (EBP50) is highly expressed at the plasma membrane of normal biliary epithelial cells and binds epidermal growth factor receptor (EGFR), a tyrosine kinase receptor with oncogenic properties. This study investigated EBP50-EGFR interplay in biliary cancer. We report that in a collection of 106 cholangiocarcinomas, EBP50 was delocalized to the cytoplasm of tumor cells in 66% of the cases. Ectopic expression of EBP50 was correlated with the presence of satellite nodules and with the expression of EGFR, which was at the plasma membrane, implying a loss of interaction with EBP50 in these cases. In vitro, loss of interaction between EBP50 and EGFR was mimicked by EBP50 depletion using a small interfering RNA approach in human biliary carcinoma cells co-expressing the two proteins at their plasma membrane, and in which interaction between EBP50 and EGFR was validated. EBP50 depletion caused an increase in EGFR expression at their surface, and a sustained activation of the receptor and of its downstream effectors (extracellular signal-regulated kinase 1/2, signal transducer and activator of transcription 3) in both basal and EGF-stimulated conditions. Cells lacking EBP50 showed epithelial-to-mesenchymal transition-associated features, including reduction in E-cadherin and cytokeratin-19 expression, induction of S100A4 and of the E-cadherin transcriptional repressor, Slug, and loss of cell polarity. Accordingly, depletion of EBP50 induced the disruption of adherens junctional complexes, the development of lamellipodia structures and the subsequent acquisition of motility properties. All these phenotypic changes were prevented upon inhibition of EGFR tyrosine kinase by gefitinib. These findings indicate that loss of EBP50 at the plasma membrane in tumor cells may contribute to biliary carcinogenesis

  18. Crocodile blood extract induces the apoptosis of lung cancer cells through PTEN activity.

    PubMed

    Ou, Yuqian; Ho, Wing Shing

    2016-09-01

    Current treatment strategies for lung cancer cause undesirable side‑effects. Integrated medicine with a curative approach has become a common approach to the treatment strategy. Recent studies suggest that American alligator blood is effective in reducing colorectal cancer cell viability in vitro, but the mechanism remains unclear. In the present study, we aimed to study the anticancer activity of crocodile blood extracts on lung cancer cell line A549 and investigate the possible mechanisms involved. In vitro studies were utilized to investigate the effects on the cancer cells after incubation with the blood extracts. The active fraction that showed more efficacy in inhibiting cell growth was characterized in the supernatant (S2) from whole blood extracts. High performance liquid chromatography (HPLC) analysis revealed that S2 contained more polar moiety from whole blood. S2 induced DNA fragmentation. Cell cycle arrest in the G1/M phase was demonstrated and mitochondrial membrane permeability was disrupted. An increase in the generation of reactive oxygen species (ROS) and increased activities of caspase-3 and caspase-7 were detected. Furthermore, release of cytochrome c, upregulation of expression of Bax, p53, p21, Bid, cleaved forms of the caspase family and PARP along with downregulation of Bcl-2, PCNA, MDM2, caspase‑8, wild types of caspase family proteins and PARP were recorded after treatment with S2 fractions. Moreover, the PI3K/AKT survival pathway was downregulated by S2 fractions in the lung cancer cell line. PMID:27431918

  19. Interaction between LPS-induced NO production and IDO activity in mouse peritoneal cells in the presence of activated Valpha14 NKT cells.

    PubMed

    Ohtaki, Hirofumi; Ito, Hiroyasu; Ando, Kazuki; Ishikawa, Tetsuya; Hoshi, Masato; Tanaka, Ryo; Osawa, Yosuke; Yokochi, Takashi; Moriwaki, Hisataka; Saito, Kuniaki; Seishima, Mitsuru

    2009-11-13

    In this study, we demonstrated that lipopolysaccharide (LPS) markedly increased nitric oxide (NO) production and indoleamine 2,3-dioxygenase (IDO) activity in mouse peritoneal cells in the presence of activated Valpha14 natural killer T cells. Moreover, LPS-induced NO production in peritoneal cells from IDO-knockout (KO) mice was more increased than that from wild-type mice. However, there was no significant difference in the expression of inducible nitric oxide synthase (iNOS) mRNA and protein between the wild-type and IDO-KO mice. No significant difference was also observed in the ratio of CD3- and DX5-positive cells and F4/80- and TLR4-positive cells in peritoneal cells between the wild-type and IDO-KO mice. Since the IDO activity was enhanced by an NO inhibitor, NO may be post-translationally consumed by inhibiting the IDO activity. IDO is well known to play an important role in immunosuppression during inflammatory disease. Therefore, the inhibition of IDO by NO may exacerbate inflammation in the peritoneal cavity.

  20. Monocyte-Induced Prostate Cancer Cell Invasion is Mediated by Chemokine ligand 2 and Nuclear Factor-κB Activity

    PubMed Central

    Lindholm, Paul F; Sivapurapu, Neela; Jovanovic, Borko; Kajdacsy-Balla, André

    2015-01-01

    Study Background The tumor microenvironment contains inflammatory cells which can influence cancer growth and progression; however the mediators of these effects vary with different cancer types. The mechanisms by which prostate cancer cells communicate with monocytes to promote cancer progression are incompletely understood. This study tested prostate cancer cell and monocyte interactions that lead to increased prostate cancer cell invasion. Methods We analyzed the prostate cancer cell invasion and NF-κB activity and cytokine expression during interaction with monocyte-lineage cells in co-cultures. The roles of monocyte chemotactic factor (MCP-1/CCL2) and NF-κB activity for co-culture induced prostate cancer invasion were tested. Clinical prostate cancer NF-κB expression was analyzed by immunohistochemistry. Results In co-cultures of prostate cancer cell lines with monocyte-lineage cells, (C-C motif) ligand 2 (CCL2) levels were significantly increased when compared with monocytes or cancer cells cultured alone. Prostate cancer cell invasion was induced by recombinant CCL2 in a dose dependent manner, similar to co-cultures with monocytes. The monocyte-induced prostate cancer cell invasion was inhibited by CCL2 neutralizing antibodies and by the CCR2 inhibitor, RS102895. Prostate cancer cell invasion and CCL2 expression induced in the co-cultures was inhibited by Lactacystin and Bay11-7082 NF-κB inhibitors. Prostate cancer cell NF-κB DNA binding activity depended on CCL2 dose and was inhibited by CCL2 neutralizing antibodies. Clinical prostate cancer NF-κB expression correlated with tumor grade. Conclusions Co-cultures with monocyte-lineage cell lines stimulated increased prostate cancer cell invasion through increased CCL2 expression and increased prostate cancer cell NF-κB activity. CCL2 and NF-κB may be useful therapeutic targets to interfere with inflammation-induced prostate cancer invasion. PMID:26317041

  1. Agonist-Mediated Activation of STING Induces Apoptosis in Malignant B Cells.

    PubMed

    Tang, Chih-Hang Anthony; Zundell, Joseph A; Ranatunga, Sujeewa; Lin, Cindy; Nefedova, Yulia; Del Valle, Juan R; Hu, Chih-Chi Andrew

    2016-04-15

    Endoplasmic reticulum (ER) stress responses through the IRE-1/XBP-1 pathway are required for the function of STING (TMEM173), an ER-resident transmembrane protein critical for cytoplasmic DNA sensing, IFN production, and cancer control. Here we show that the IRE-1/XBP-1 pathway functions downstream of STING and that STING agonists selectively trigger mitochondria-mediated apoptosis in normal and malignant B cells. Upon stimulation, STING was degraded less efficiently in B cells, implying that prolonged activation of STING can lead to apoptosis. Transient activation of the IRE-1/XBP-1 pathway partially protected agonist-stimulated malignant B cells from undergoing apoptosis. In Eμ-TCL1 mice with chronic lymphocytic leukemia, injection of the STING agonist 3'3'-cGAMP induced apoptosis and tumor regression. Similarly efficacious effects were elicited by 3'3'-cGAMP injection in syngeneic or immunodeficient mice grafted with multiple myeloma. Thus, in addition to their established ability to boost antitumoral immune responses, STING agonists can also directly eradicate malignant B cells. Cancer Res; 76(8); 2137-52. ©2016 AACR. PMID:26951929

  2. Etoposide Induces Apoptosis in Activated Human Hepatic Stellate Cells via ER Stress

    PubMed Central

    Wang, Chen; Zhang, Feng; Cao, Yu; Zhang, Mingming; Wang, Aixiu; Xu, Mingcui; Su, Min; Zhang, Ming; Zhuge, Yuzheng

    2016-01-01

    The activation of hepatic stellate cells (HSCs) plays a vital role in the progression of liver fibrosis, and the induction of HSCs apoptosis may attenuate or reverse fibrogenesis. The therapeutic effects of etoposide(VP-16), a widely used anticancer agent, on HSCs apoptosis and liver fibrosis resolution are still unclear. Here, we report that VP-16 reduced the proliferation of LX-2 cells and led to significantly high levels of apoptosis, as indicated by Annexin V staining and the proteolytic cleavage of the executioner caspase-3 and PARP. Additionally, the unfolded protein response regulators CHOP, BIP, caspase-12, p-eIF2α and IRE1α, which are considered endoplasmic reticulum (ER) stress markers, were upregulated by VP-16. The strong inhibitory effect of VP-16 on LX-2 cells was mainly dependent on ER stress, which activated JNK signaling pathway. Remarkably, VP-16 treatment decreased the expression of α-SMA and type I collagen and simultaneously increased the ratio of matrix metalloproteinases (MMPs) to tissue inhibitor of matrix metalloproteinases (TIMPs). In contrast, VP-16 induced significantly more apoptosis in HSCs than in normal hepatocytes. Taken together, our findings demonstrate that VP-16 exerts a proapoptotic effect on LX-2 cells and has an antifibrogenic effect on collagen deposition, suggesting a new strategy for the treatment of liver fibrosis. PMID:27680712

  3. Abrupt reflow enhances cytokine-induced proinflammatory activation of endothelial cells during simulated shock and resuscitation.

    PubMed

    Li, Ranran; Zijlstra, Jan G; Kamps, Jan A A M; van Meurs, Matijs; Molema, Grietje

    2014-10-01

    Circulatory shock and resuscitation are associated with systemic hemodynamic changes, which may contribute to the development of MODS (multiple organ dysfunction syndrome). In this study, we used an in vitro flow system to simulate the consecutive changes in blood flow as occurring during hemorrhagic shock and resuscitation in vivo. We examined the kinetic responses of different endothelial genes in human umbilical vein endothelial cells preconditioned to 20 dyne/cm unidirectional laminar shear stress for 48 h to flow cessation and abrupt reflow, respectively, as well as the effect of flow cessation and reflow on tumor necrosis factor-α (TNF-α)-induced endothelial proinflammatory activation. Endothelial CD31 and VE-cadherin were not affected by the changes in flow in the absence or presence of TNF-α. The messenger RNA levels of proinflammatory molecules E-selectin, VCAM-1 (vascular cell adhesion molecule 1), and IL-8 (interleukin 8) were significantly induced by flow cessation respectively acute reflow, whereas ICAM-1 (intercellular adhesion molecule 1) was downregulated on flow cessation and induced by subsequent acute reflow. Flow cessation also affected the Ang/Tie2 (Angiopoietin/Tie2 receptor tyrosine kinase) system by downregulating Tie2 and inducing its endothelial ligand Ang2, an effect that was further extended on acute reflow. Furthermore, the induction of proinflammatory adhesion molecules by TNF-α under flow cessation was significantly enhanced on subsequent acute reflow. This study demonstrated that flow alterations per se during shock and resuscitation contribute to endothelial activation and that these alterations interact with proinflammatory factors coexisting in vivo such as TNF-α. The abrupt reflow-related enhancement of cytokine-induced endothelial proinflammatory activation supports the concept that sudden regain of flow during resuscitation has an aggravating effect on endothelial activation, which may play a significant role in vascular

  4. Alantolactone Induces Apoptosis in HepG2 Cells through GSH Depletion, Inhibition of STAT3 Activation, and Mitochondrial Dysfunction

    PubMed Central

    Khan, Muhammad; Li, Ting; Ahmad Khan, Muhammad Khalil; Rasul, Azhar; Nawaz, Faisal; Sun, Meiyan; Zheng, Yongchen; Ma, Tonghui

    2013-01-01

    Signal transducer and activator of transcription 3 (STAT3) constitutively expresses in human liver cancer cells and has been implicated in apoptosis resistance and tumorigenesis. Alantolactone, a sesquiterpene lactone, has been shown to possess anticancer activities in various cancer cell lines. In our previous report, we showed that alantolactone induced apoptosis in U87 glioblastoma cells via GSH depletion and ROS generation. However, the molecular mechanism of GSH depletion remained unexplored. The present study was conducted to envisage the molecular mechanism of alantolactone-induced apoptosis in HepG2 cells by focusing on the molecular mechanism of GSH depletion and its effect on STAT3 activation. We found that alantolactone induced apoptosis in HepG2 cells in a dose-dependent manner. This alantolactone-induced apoptosis was found to be associated with GSH depletion, inhibition of STAT3 activation, ROS generation, mitochondrial transmembrane potential dissipation, and increased Bax/Bcl-2 ratio and caspase-3 activation. This alantolactone-induced apoptosis and GSH depletion were effectively inhibited or abrogated by a thiol antioxidant, N-acetyl-L-cysteine (NAC). The data demonstrate clearly that intracellular GSH plays a central role in alantolactone-induced apoptosis in HepG2 cells. Thus, alantolactone may become a lead chemotherapeutic candidate for the treatment of liver cancer. PMID:23533997

  5. Leptin induces CYP1B1 expression in MCF-7 cells through ligand-independent activation of the ERα pathway

    SciTech Connect

    Khanal, Tilak; Kim, Hyung Gyun; Do, Minh Truong; Choi, Jae Ho; Won, Seong Su; Kang, Wonku; Chung, Young Chul; Jeong, Tae Cheon; Jeong, Hye Gwang

    2014-05-15

    Leptin, a hormone with multiple biological actions, is produced predominantly by adipose tissue. Among its functions, leptin can stimulate tumour cell growth. Oestrogen receptor α (ERα), which plays an essential role in breast cancer development, can be transcriptionally activated in a ligand-independent manner. In this study, we investigated the effect of leptin on CYP1B1 expression and its mechanism in breast cancer cells. Leptin induced CYP1B1 protein, messenger RNA expression and promoter activity in ERα-positive MCF-7 cells but not in ERα-negative MDA-MB-231 cells. Additionally, leptin increased 4-hydroxyoestradiol in MCF-7 cells. Also, ERα knockdown by siRNA significantly blocked the induction of CYP1B1 expression by leptin, indicating that leptin induced CYP1B1 expression via an ERα-dependent mechanism. Transient transfection with CYP1B1 deletion promoter constructs revealed that the oestrogen response element (ERE) plays important role in the up-regulation of CYP1B1 by leptin. Furthermore, leptin stimulated phosphorylation of ERα at serine residues 118 and 167 and increased ERE-luciferase activity, indicating that leptin induced CYP1B1 expression by ERα activation. Finally, we found that leptin activated ERK and Akt signalling pathways, which are upstream kinases related to ERα phosphorylation induced by leptin. Taken together, our results indicate that leptin-induced CYP1B1 expression is mediated by ligand-independent activation of the ERα pathway as a result of the activation of ERK and Akt in MCF-7 cells. - Highlights: • Leptin increased 4-hydroxyoestradiol in MCF-7 breast cancer cells. • Leptin activated ERK and Akt kinases related to ERα phosphorylation. • Leptin induces phosphorylation of ERα at serine residues 118 and 167. • Leptin induces ERE-luciferase activity.

  6. Trypanosoma cruzi cytosolic alkaline antigens (FI) induce polyclonal activation in murine normal B cells.

    PubMed

    Montes, C L; Vottero-Cima, E; Gruppi, A

    1996-08-01

    Several reports have described polyclonal activation in mice acutely infected with Trypanosoma cruzi. The aim of this work was to analyse the participation of one T. cruzi antigenic fraction in this immunological event. The antigen selected was FI, an antigenic fraction of pI 7-9 obtained from T. cruzi cytosol separated by isoelectricfocusing. FI is constituted by molecules with molecular weights of around 60 and 20 KDa. The authors assayed the ability of this antigenic fraction to induce polyclonal activation of spleen mononuclear cells from normal (NSMC) BALB/c mice. NSMC showed a marked lymphoproliferative response measured by 3H-thymidine incorporation after 3 days of culture in presence of FI. The values reached by FI-stimulated cells were 10 times higher than the controls (non-stimulated cells). This effect was dose-dependent. Furthermore, the authors observed that a purified T-cell population in the presence of adherent cells was unaffected by FI. Additionally, in a culture of NSMC, FI stimulated the proliferation of B cells as observed by the increase of the percentage of B220+ cells determined by FACS using FITC-conjugated anti-mouse B220. The authors noticed that the percentage of B220+Ly1+(CD5) populations in the presence of FI did not change with respect to the control (non-stimulated cells), indicating that FI expanded both conventional and CD5+ B cells. The isotypic pattern of the antibodies produced after 6 days of culture of NSMC in the presence of FI was predominantly IgM, which reacted with highly conserved antigens such as actin, myosin, myoglobin, thyroglobulin and carbonic anhydrase, but did not react with FI. A slight increase of IgG1 and IgG3 with respect to the control was observed but no changes on the levels of IgG2 was noticed. These results indicate that FI promotes activation, proliferation and differentiation in antibody-secreting cells of normal murine B lymphocytes.

  7. IL-8 induces the epithelial-mesenchymal transition of renal cell carcinoma cells through the activation of AKT signaling

    PubMed Central

    Zhou, Nan; Lu, Fuding; Liu, Cheng; Xu, Kewei; Huang, Jian; Yu, Dexin; Bi, Liangkuan

    2016-01-01

    The epithelial-mesenchymal transition (EMT) process has increasingly been examined due to its role in the progression of human tumors. Renal cell carcinoma (RCC) is one of the most common urological tumors that results in patient mortality. Previous studies have demonstrated that the EMT process is closely associated with the metastasis of RCC; however, the underlying molecular mechanism has not been determined yet. The present study revealed that interleukin (IL)-8 was highly expressed in metastatic RCC. IL-8 could induce the EMT of an RCC cell line by enhancing N-cadherin expression and decreasing E-cadherin expression. Furthermore, IL-8 could induce AKT phosphorylation, and the phosphatidylinositol-4,5-bisphosphate 3-kinase inhibitor LY294002 could inhibit the EMT of RCC cells that was induced by IL-8. Therefore, these results suggest that IL-8 is able to promote the EMT of RCC through the activation of the AKT signal transduction pathway, and this may provide a possible molecular mechanism for RCC metastasis. PMID:27588140

  8. Polydatin induces bone marrow stromal cells migration by activation of ERK1/2.

    PubMed

    Chen, ZhenQiu; Wei, QiuShi; Hong, GuoJu; Chen, Da; Liang, Jiang; He, Wei; Chen, Mei Hui

    2016-08-01

    Bone marrow stromal cells (BMSCs) have proven to be useful for the treatment of numerous human diseases. However, the reparative ability of BMSCs is limited by their poor migration. Polydatin, widely used in traditional Chinese remedies, has proven to exert protective effects to BMSCs. However, little is known about its role in BMSCs migration. In this study, we studied the effects of polydatin on rat BMSCs migration using the scratch wound healing and transwell migration assays. Our results showed polydatin could promote BMSCs migration. Further experiments showed activation of ERK 1/2, but not JNK, was required for polydatin-induced BMSCs migration, suggesting that polydatin may promote BMSCs migration via the ERK 1/2 signaling pathways. Taken together, our results indicate that polydatin might be beneficial for stem cell replacement therapy by improving BMSCs migration.

  9. CCL2 enhances pluripotency of human induced pluripotent stem cells by activating hypoxia related genes.

    PubMed

    Hasegawa, Yuki; Tang, Dave; Takahashi, Naoko; Hayashizaki, Yoshihide; Forrest, Alistair R R; Suzuki, Harukazu

    2014-01-01

    Standard culture of human induced pluripotent stem cells (hiPSCs) requires basic Fibroblast Growth Factor (bFGF) to maintain the pluripotent state, whereas hiPSC more closely resemble epiblast stem cells than true naïve state ES which requires LIF to maintain pluripotency. Here we show that chemokine (C-C motif) ligand 2 (CCL2) enhances the expression of pluripotent marker genes through the phosphorylation of the signal transducer and activator of transcription 3 (STAT3) protein. Moreover, comparison of transcriptomes between hiPSCs cultured with CCL2 versus with bFGF, we found that CCL2 activates hypoxia related genes, suggesting that CCL2 enhanced pluripotency by inducing a hypoxic-like response.Further, we show that hiPSCs cultured with CCL2 can differentiate at a higher efficiency than culturing withjust bFGF and we show CCL2 can be used in feeder-free conditions [corrected]. Taken together, our finding indicates the novel functions of CCL2 in enhancing its pluripotency in hiPSCs. PMID:24957798

  10. In vitro anticancer activity of loquat tea by inducing apoptosis in human leukemia cells.

    PubMed

    Zar, Phyu Phyu Khine; Yano, Satoshi; Sakao, Kozue; Hashimoto, Fumio; Nakano, Takayuki; Fujii, Makoto; Hou, De-Xing

    2014-01-01

    Fresh loquat leaves have been used as folk health herb in Asian countries for long time, although the evidence supporting their functions is still minimal. This study aimed to clarify the chemopreventive effect of loquat tea extract (LTE) by investigating the inhibition on proliferation, and underlying mechanisms in human promyelocytic leukemia cells (HL-60). LTE inhibited proliferation of HL-60 in a dose-dependent manner. Molecular data showed that the isolated fraction of LTE induced apoptosis of HL-60 as characterized by DNA fragmentation; activation of caspase-3, -8, and -9; and inactivation of poly(ADP)ribose polymerase. Moreover, LTE fraction increased the ratio of pro-apoptotic Bcl-2-associated X protein (Bax)/anti-apoptotic myeloid cell leukemia 1 (Mcl-1) that caused mitochondrial membrane potential loss and cytochrome c released to cytosol. Thus, our data indicate that LTE might induce apoptosis in HL-60 cells through a mitochondrial dysfunction pathway. These findings enhance our understanding for chemopreventive function of loquat tea.

  11. mTOR inhibitors counteract tamoxifen-induced activation of breast cancer stem cells.

    PubMed

    Karthik, Govindasamy-Muralidharan; Ma, Ran; Lövrot, John; Kis, Lorand Levente; Lindh, Claes; Blomquist, Lennart; Fredriksson, Irma; Bergh, Jonas; Hartman, Johan

    2015-10-10

    Breast cancer cells with stem cell characteristics (CSC) are a distinct cell population with phenotypic similarities to mammary stem cells. CSCs are important drivers of tumorigenesis and the metastatic process. Tamoxifen is the most widely used hormonal therapy for estrogen receptor (ER) positive cancers. In our study, tamoxifen was effective in reducing proliferation of ER + adherent cancer cells, but not their CSC population. We isolated, expanded and incubated CSC from seven breast cancers with or without tamoxifen. By genome-wide transcriptional analysis we identified tamoxifen-induced transcriptional pathways associated with ribosomal biogenesis and mRNA translation, both regulated by the mTOR-pathway. We observed induction of the key mTOR downstream targets S6K1, S6RP and 4E-BP1 in-patient derived CSCs by tamoxifen on protein level. Using the mTOR inhibitors rapamycin, everolimus and PF-04691502 (a dual PI3K/mTOR inhibitor) and in combination with tamoxifen, significant reduction in mammosphere formation was observed. Hence, we suggest that the CSC population play a significant role during endocrine resistance through activity of the mTOR pathway. In addition, tamoxifen further stimulates the mTOR-pathway but can be antagonized using mTOR-inhibitors. PMID:26208432

  12. Hypoxia activates Akt and induces phosphorylation of GSK-3 in PC12 cells.

    PubMed

    Beitner-Johnson, D; Rust, R T; Hsieh, T C; Millhorn, D E

    2001-01-01

    Akt is a serine/threonine kinase that has been shown to play a central role in promoting cell survival and opposing apoptosis. We evaluated the effect of hypoxia on Akt in rat pheochromocytoma (PC12) cells. PC12 cells were exposed to varying levels of hypoxia, including 21%, 15%, 10%, 5%, and 1% O(2). Hypoxia dramatically increased phosphorylation of Akt (Ser(473)). This effect peaked after 6 h exposure to hypoxia, but persisted strongly for up to 24 h. Phosphorylation of Akt was paralleled with a progressive increase in phosphorylation of glycogen synthase kinase-3 (GSK-3), one of its downstream substrates. The effect of hypoxia on phosphorylation of Akt was completely blocked by pretreatment of the cells with wortmannin (100 nM), indicating that this effect is mediated by phosphatidylinositol 3-kinase (P13K). In contrast, whereas hypoxia also strongly induced phosphorylation of the transcription factors CREB and EPAS1, these effects persisted in the presence of wortmannin. Thus, hypoxia regulates both P13K-dependent and P13K-independent signaling pathways. Furthermore, activation of the P13K and Akt signaling pathways may be one mechanism by which cells adapt and survive under conditions of hypoxia. PMID:11257444

  13. EP2 Induces p38 Phosphorylation via the Activation of Src in HEK 293 Cells

    PubMed Central

    Chun, Kyung-Soo; Shim, Minsub

    2015-01-01

    Prostaglandin E2 (PGE2), a major product of cyclooxygenase, binds to four different prostaglandin E2 receptors (EP1, EP2, EP3, and EP4) which are G-protein coupled transmembrane receptors (GPCRs). Although GPCRs including EP receptors have been shown to be associated with their specific G proteins, recent evidences suggest that GPCRs can regulate MAPK signaling via non-G protein coupled pathways including Src. EP2 is differentially expressed in various tissues and the expression of EP2 is induced by extracellular stimuli. We hypothesized that an increased level of EP2 expression may affect MAPK signaling. The overexpression of EP2 in HEK 293 cells resulted in significant increase in intracellular cAMP levels response to treatment with butaprost, a specific EP2 agonist, while overexpression of EP2 alone did not increase intracellular cAMP levels. However, EP2 overexpression in the absence of PGE2 induced an increase in the level of p38 phosphorylation as well as the kinase activity of p38, suggesting that up-regulation of EP2 may promote p38 activation via non-G protein coupled pathway. Inhibition of Src completely blocked EP2-induced p38 phosphorylation and overexpression of Src increased the level of p38 phosphorylation, indicating that Src is upstream kinase for EP2-induced p38 phosphorylation. EP2 overexpression also increased the Src activity and EP2 protein was co-immunoprecipitated with Src. Furthermore, sequential co-immunoprecipitation studies showed that EP2, Src, and β-arrestin can form a complex. Our study found a novel pathway in which EP2 is associated with Src, regulating p38 pathway. PMID:26535079

  14. Microglia activated by IL-4 or IFN-gamma differentially induce neurogenesis and oligodendrogenesis from adult stem/progenitor cells.

    PubMed

    Butovsky, Oleg; Ziv, Yaniv; Schwartz, Adi; Landa, Gennady; Talpalar, Adolfo E; Pluchino, Stefano; Martino, Gianvito; Schwartz, Michal

    2006-01-01

    Cell renewal in the adult central nervous system (CNS) is limited, and is blocked in inflammatory brain conditions. We show that both neurogenesis and oligodendrogenesis of adult neural progenitor cells in mice are blocked by inflammation-associated (endotoxin-activated) microglia, but induced by microglia activated by cytokines (IL-4 or low level of IFN-gamma) associated with T-helper cells. Blockage was correlated with up-regulation of microglial production of tumor necrosis factor-alpha. The effect induced by IL-4-activated microglia was mediated, at least in part, by insulin-like growth factor-I. The IL-4-activated microglia showed a bias towards oligodendrogenesis whereas the IFN-gamma-activated microglia showed a bias towards neurogenesis. It thus appears that microglial phenotype critically affects their ability to support or impair cell renewal from adult stem cell.

  15. A novel cell-penetrating peptide derived from WT1 enhances p53 activity, induces cell senescence and displays antimelanoma activity in xeno- and syngeneic systems☆

    PubMed Central

    Massaoka, Mariana H.; Matsuo, Alisson L.; Figueiredo, Carlos R.; Girola, Natalia; Faria, Camyla F.; Azevedo, Ricardo A.; Travassos, Luiz R.

    2014-01-01

    The Wilms tumor protein 1 (WT1) transcription factor has been associated in malignant melanoma with cell survival and metastasis, thus emerging as a candidate for targeted therapy. A lysine–arginine rich peptide, WT1-pTj, derived from the ZF domain of WT1 was evaluated as an antitumor agent against A2058 human melanoma cells and B16F10-Nex2 syngeneic murine melanoma. Peptide WT1-pTj quickly penetrated human melanoma cells and induced senescence, recognized by increased SA-β-galactosidase activity, enhanced transcriptional activity of p53, and induction of the cell cycle inhibitors p21 and p27. Moreover, the peptide bound to p53 and competed with WT1 protein for binding to p53. WT1-pTj treatment led to sustained cell growth suppression, abrogation of clonogenicity and G2/M cell cycle arrest. Notably, in vivo studies showed that WT1-pTj inhibited both the metastases and subcutaneous growth of murine melanoma in syngeneic mice, and prolonged the survival of nude mice challenged with human melanoma cells. The 27-amino acid cell-penetrating WT1-derived peptide, depends on C3 and H16 for effective antimelanoma activity, inhibits proliferation of WT1-expressing human tumor cell lines, and may have an effective role in the treatment of WT1-expressing malignancies. PMID:24490140

  16. Juglanthraquinone C Induces Intracellular ROS Increase and Apoptosis by Activating the Akt/Foxo Signal Pathway in HCC Cells

    PubMed Central

    2016-01-01

    Juglanthraquinone C (JC), a naturally occurring anthraquinone extracted from Juglans mandshurica, could induce apoptosis of cancer cells. This study aims to investigate the detailed cytotoxicity mechanism of JC in HepG2 and BEL-7402 cells. The Affymetrix HG-U133 Plus 2.0 arrays were first used to analyze the mRNA expression exposed to JC or DMSO in HepG2 cells. Consistent with the previous results, the data indicated that JC could induce apoptosis and hyperactivated Akt. The Western blot analysis further revealed that Akt, a well-known survival protein, was strongly activated in HepG2 and BEL-7402 cells. Furthermore, an obvious inhibitory effect on JC-induced apoptosis was observed when the Akt levels were decreased, while the overexpression of constitutively active mutant Akt greatly accelerated JC-induced apoptosis. The subsequent results suggested that JC treatment suppressed nuclear localization and increased phosphorylated levels of Foxo3a, and the overexpression of Foxo3a abrogated JC-induced apoptosis. Most importantly, the inactivation of Foxo3a induced by JC further led to an increase of intracellular ROS levels by suppressing ROS scavenging enzymes, and the antioxidant N-acetyl-L-cysteine and catalase successfully decreased JC-induced apoptosis. Collectively, this study demonstrated that JC induced the apoptosis of hepatocellular carcinoma (HCC) cells by activating Akt/Foxo signaling pathway and increasing intracellular ROS levels. PMID:26682007

  17. Sinomenine induces the generation of intestinal Treg cells and attenuates arthritis via activation of aryl hydrocarbon receptor.

    PubMed

    Tong, Bei; Yuan, Xusheng; Dou, Yannong; Wu, Xin; Wang, Yuhui; Xia, Yufeng; Dai, Yue

    2016-10-01

    Sinomenine (SIN), an anti-arthritis drug, has previously been proven to exert immunomodulatory activity in rats by inducing intestinal regulatory T-cells (Treg cells). Here, we assessed the effect of SIN on the generation and function of Treg cells in autoimmune arthritis, and the underlying mechanisms in view of aryl hydrocarbon receptor (AhR). The proportions of Treg cells and IL-17-producing T-cells (Th17 cells) differentiated from naive T-cells were analyzed by flow cytometric analysis. The AhR agonistic effect of SIN was tested by analyzing the activation of downstream signaling pathways and target genes. The dependence of intestinal Treg cell induction and arthritis alleviation by SIN on AhR activation was confirmed in a mouse collagen-induced arthritis (CIA) model. SIN promoted the differentiation and function of intestinal Treg cells in vitro. It induced the expression and activity of AhR target gene, promoted AhR/Hsp90 dissociation and AhR nuclear translocation, induced XRE reporter activity, and facilitated AhR/XRE binding in vitro, displaying the potential to be an agonist of AhR. In CIA mice, SIN induced the generation of intestinal Treg cells, and facilitated the immunosuppressive function of these Treg cells as shown by an adoptive transfer test. In addition, the induction of intestinal Treg cells and the anti-arthritic effect of SIN in CIA mice could be largely diminished by the AhR antagonist resveratrol. SIN attenuates arthritis by promoting the generation and function of Treg cells in an AhR-dependent manner. PMID:27617398

  18. Porcine parvovirus infection induces apoptosis in PK-15 cells through activation of p53 and mitochondria-mediated pathway

    SciTech Connect

    Zhang, Hongling; Huang, Yong; Du, Qian; Luo, Xiaomao; Zhang, Liang; Zhao, Xiaomin; Tong, Dewen

    2015-01-09

    Highlights: • PPV reduces PK-15 cells viability by inducing apoptosis. • PPV infection induces apoptosis through mitochondria-mediated pathway. • PPV infection activates p53 to regulate the mitochondria apoptotic signaling. - Abstract: Porcine parvovirus (PPV) infection has been reported to induce the cytopathic effects (CPE) in some special host cells and contribute the occurrence of porcine parvovirus disease, but the molecular mechanisms underlying PPV-induced CPE are not clear. In this study, we investigated the morphological and molecular changes of porcine kidney cell line (PK-15 cells) infected with PPV. The results showed that PPV infection inhibited the viability of PK-15 cells in a time and concentration dependent manner. PPV infection induced typical apoptotic features including chromatin condensation, apoptotic body formation, nuclear fragmentation, and Annexin V-binding activity. Further studies showed that Bax was increased and translocated to mitochondria, whereas Bcl-2 was decreased in PPV-infected cells, which caused mitochondrial outer-membrane permeabilization, resulting in the release of mitochondrial cytochrome c, followed by caspase-9 and caspase-3 activation. However, the expression of Fas and Fas ligand (FasL) did not appear significant changes in the process of PPV-induced apoptosis. Moreover, PPV infection activated p53 signaling, which was involved in the activation of apoptotic signaling induced by PPV infection via regulation of Bax and Bcl-2. Taken together, our results demonstrated that PPV infection induced apoptosis in PK-15 cells through activation of p53 and mitochondria-mediated apoptosis pathway. This study may contribute to shed light on the molecular pathogenesis of PPV infection.

  19. Interferon-γ-induced activation of JAK1 and JAK2 suppresses tumor cell susceptibility to NK cells through upregulation of PD-L1 expression

    PubMed Central

    Bellucci, Roberto; Martin, Allison; Bommarito, Davide; Wang, Kathy; Hansen, Steen H; Freeman, Gordon J; Ritz, Jerome

    2015-01-01

    Inhibition of JAK1 or JAK2 in human tumor cells was previously shown to increase susceptibility of these cells to NK cell lysis. In the present study, we examined the cellular mechanisms that mediate this effect in hematopoietic tumor cell lines and primary tumor cells. Incubation of tumor cells with supernatant from activated NK cells or interferon-gamma (IFNγ)-induced activation of pSTAT1 and increased expression of PD-L1 without altering expression of other activating or inhibitory NK cell ligands. These functional effects were blocked by chemical JAK inhibition or shRNAs targeting JAK1, JAK2 or STAT1. Inhibition of IFNγ signaling also prevented the upregulation of PD-L1 and blocking PD-L1 resulted in increased tumor lysis by NK cells. These results show that NK cell activation and secretion of IFNγ results in activation of JAK1, JAK2 and STAT1 in tumor cells, resulting in rapid up-regulation of PD-L1 expression. Increased expression of PD-L1 results in increased resistance to NK cell lysis. Blockade of JAK pathway activation prevents increased PD-L1 expression resulting in increased susceptibility of tumor cells to NK cell activity. These observations suggest that JAK pathway inhibitors as well as PD-1 and PD-L1 antibodies may work synergistically with other immune therapies by preventing IFN-induced inhibition of NK cell-mediated tumor cell lysis. PMID:26155422

  20. On involvement of transcription factors nuclear factor kappa-light-chain-enhancer of activated B cells, activator protein-1 and signal transducer and activator of transcription-3 in photodynamic therapy-induced death of crayfish neurons and satellite glial cells.

    PubMed

    Berezhnaya, Elena; Neginskaya, Marya; Kovaleva, Vera; Sharifulina, Svetlana; Ischenko, Irina; Komandirov, Maxim; Rudkovskii, Mikhail; Uzdensky, Anatoly B

    2015-07-01

    Photodynamic therapy (PDT) is currently used in the treatment of brain tumors. However, not only malignant cells but also neighboring normal neurons and glial cells are damaged during PDT. In order to study the potential role of transcription factors-nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), activator protein (AP-1), and signal transducer and activator of transcription-3 (STAT-3)-in photodynamic injury of normal neurons and glia, we photosensitized the isolated crayfish mechanoreceptor consisting of a single sensory neuron enveloped by glial cells. Application of different inhibitors and activators showed that transcription factors NF-κB (inhibitors caffeic acid phenethyl ester and parthenolide, activator betulinic acid), AP-1 (inhibitor SR11302), and STAT-3 (inhibitors stattic and cucurbitacine) influenced PDT-induced death and survival of neurons and glial cells in different ways. These experiments indicated involvement of NF-κB in PDT-induced necrosis of neurons and apoptosis of glial cells. However, in glial cells, it played the antinecrotic role. AP-1 was not involved in PDT-induced necrosis of neurons and glia, but mediated glial apoptosis. STAT-3 was involved in PDT-induced apoptosis of glial cells and necrosis of neurons and glia. Therefore, signaling pathways that regulate cell death and survival in neurons and glial cells are different. Using various inhibitors or activators of transcription factors, one can differently influence the sensitivity and resistance of neurons and glial cells to PDT. PMID:26160345

  1. On involvement of transcription factors nuclear factor kappa-light-chain-enhancer of activated B cells, activator protein-1 and signal transducer and activator of transcription-3 in photodynamic therapy-induced death of crayfish neurons and satellite glial cells

    NASA Astrophysics Data System (ADS)

    Berezhnaya, Elena; Neginskaya, Marya; Kovaleva, Vera; Sharifulina, Svetlana; Ischenko, Irina; Komandirov, Maxim; Rudkovskii, Mikhail; Uzdensky, Anatoly B.

    2015-07-01

    Photodynamic therapy (PDT) is currently used in the treatment of brain tumors. However, not only malignant cells but also neighboring normal neurons and glial cells are damaged during PDT. In order to study the potential role of transcription factors-nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), activator protein (AP-1), and signal transducer and activator of transcription-3 (STAT-3)-in photodynamic injury of normal neurons and glia, we photosensitized the isolated crayfish mechanoreceptor consisting of a single sensory neuron enveloped by glial cells. Application of different inhibitors and activators showed that transcription factors NF-κB (inhibitors caffeic acid phenethyl ester and parthenolide, activator betulinic acid), AP-1 (inhibitor SR11302), and STAT-3 (inhibitors stattic and cucurbitacine) influenced PDT-induced death and survival of neurons and glial cells in different ways. These experiments indicated involvement of NF-κB in PDT-induced necrosis of neurons and apoptosis of glial cells. However, in glial cells, it played the antinecrotic role. AP-1 was not involved in PDT-induced necrosis of neurons and glia, but mediated glial apoptosis. STAT-3 was involved in PDT-induced apoptosis of glial cells and necrosis of neurons and glia. Therefore, signaling pathways that regulate cell death and survival in neurons and glial cells are different. Using various inhibitors or activators of transcription factors, one can differently influence the sensitivity and resistance of neurons and glial cells to PDT.

  2. Activated Human Mast Cells Induce LOX-1-Specific Scavenger Receptor Expression in Human Monocyte-Derived Macrophages

    PubMed Central

    Alanne-Kinnunen, Mervi; Lappalainen, Jani; Öörni, Katariina; Kovanen, Petri T.

    2014-01-01

    Objective Activated mast cells in atherosclerotic lesions degranulate and release bioactive compounds capable of regulating atherogenesis. Here we examined the ability of activated human primary mast cells to regulate the expression of the major scavenger receptors in cultured human primary monocyte-derived macrophages (HMDMs). Results Components released by immunologically activated human primary mast cells induced a transient expression of lectin-like oxidized LDL receptor (LOX-1) mRNA in HMDMs, while the expression of two other scavenger receptors, MSR1 and CD36, remained unaffected. The LOX-1-inducing secretory components were identified as histamine, tumor necrosis factor alpha (TNF-α), and transforming growth factor beta (TGF-β1), which exhibited a synergistic effect on LOX-1 mRNA expression. Histamine induced a transient expression of LOX-1 protein. Mast cellinduced increase in LOX-1 expression was not associated with increased uptake of oxidized LDL by the macrophages. Conclusions Mast cell-derived histamine, TNF-α, and TGF-β1 act in concert to induce a transient increase in LOX-1 expression in human primary monocyte-derived macrophages. The LOX-1-inducing activity potentially endows mast cells a hitherto unrecognized role in the regulation of innate immune reactions in atherogenesis. PMID:25250731

  3. Targeting protein neddylation with an NEDD8-activating enzyme inhibitor MLN4924 induced apoptosis or senescence in human lymphoma cells

    PubMed Central

    Wang, Yanchun; Luo, Zhongguang; Pan, Yongfu; Wang, Weige; Zhou, Xiaoyan; Jeong, Lak Shin; Chu, Yiwei; Liu, Jie; Jia, Lijun

    2015-01-01

    Recent studies indicate that post-translational protein neddylation is required for the maintenance of cell viability in several lymphoma cell lines, while inhibition of the neddylation pathway with an NEDD8-activating enzyme (NAE) inhibitor MLN4924 induces apoptosis in lymphoma cells. However, the mechanism by which neddylation inhibition induces apoptosis in lymphoma cells has not been fully elucidated. Moreover, it is unknown whether neddylation inhibition triggers non-apoptotic cell-killing responses, such as cell senescence, in lymphoma cells. Here, we report that MLN4924 specifically inhibited protein neddylation, inactivated cullin-RING E3 ligase (CRL), the best-known neddylation substrate, and induced the accumulation of tumor-suppressive CRL substrates in lymphoma cells. Moreover, MLN4924 potently suppressed the growth of lymphoma cells by inducing G2 cell-cycle arrest, followed by apoptosis or senescence in a cell line-dependent manner. MLN4924-induced apoptosis was mediated by intrinsic apoptotic signaling with substantial up-regulation of pro-apoptotic Bik and Noxa as well as down-regulation of anti-apoptotic XIAP, c-IAP1 and c-IAP2, while senescence induction upon neddylation inhibition seemed dependent on the expression of tumor suppressor p21/p27. Together, these findings expand our understanding on how lymphoma cells respond to neddylation inhibition and support the development of neddylation inhibitors (e.g. MLN4924) for the treatment of lymphoma. PMID:25782162

  4. Cadmium induces autophagy through ROS-dependent activation of the LKB1-AMPK signaling in skin epidermal cells

    SciTech Connect

    Son, Young-Ok; Wang Xin; Hitron, John Andrew; Zhang Zhuo; Cheng Senping; Budhraja, Amit; Ding Songze; Lee, Jeong-Chae; Shi Xianglin

    2011-09-15

    Cadmium is a toxic heavy metal which is environmentally and occupationally relevant. The mechanisms underlying cadmium-induced autophagy are not yet completely understood. The present study shows that cadmium induces autophagy, as demonstrated by the increase of LC3-II formation and the GFP-LC3 puncta cells. The induction of autophagosomes was directly visualized by electron microscopy in cadmium-exposed skin epidermal cells. Blockage of LKB1 or AMPK by siRNA transfection suppressed cadmium-induced autophagy. Cadmium-induced autophagy was inhibited in dominant-negative AMPK-transfected cells, whereas it was accelerated in cells transfected with the constitutively active form of AMPK. mTOR signaling, a negative regulator of autophagy, was downregulated in cadmium-exposed cells. In addition, cadmium generated reactive oxygen species (ROS) at relatively low levels, and caused poly(ADP-ribose) polymerase-1 (PARP) activation and ATP depletion. Inhibition of PARP by pharmacological inhibitors or its siRNA transfection suppressed ATP reduction and autophagy in cadmium-exposed cells. Furthermore, cadmium-induced autophagy signaling was attenuated by either exogenous addition of catalase and superoxide dismutase, or by overexpression of these enzymes. Consequently, these results suggest that cadmium-mediated ROS generation causes PARP activation and energy depletion, and eventually induces autophagy through the activation of LKB1-AMPK signaling and the down-regulation of mTOR in skin epidermal cells. - Highlights: > Cadmium, a toxic heavy metal, induces autophagic cell death through ROS-dependent activation of the LKB1-AMPK signaling. > Cadmium generates intracellular ROS at low levels and this leads to severe DNA damage and PARP activation, resulting in ATP depletion, which are the upstream events of LKB1-AMPK-mediated autophagy. > This novel finding may contribute to further understanding of cadmium-mediated diseases.

  5. A sycamore cell wall polysaccharide and a chemically related tomato leaf polysaccharide possess similar proteinase inhibitor-inducing activities.

    PubMed

    Ryan, C A; Bishop, P; Pearce, G

    1981-09-01

    A large pectic polysaccharide, called rhamnogalacturonan I, that is solubilized by a fungal endo-alpha-1,4-polygalacturonase from the purified walls of suspension-cultured sycamore cells possesses proteinase inhibitor-inducing activity similar to that of the proteinase inhibitor-inducing factor, a pectic-like oligosaccharide fraction isolated from tomato leaves. This suggests that the proteinase inhibitor-inducing activity resides in particular polysaccharide fragments which can be released when plant cell walls are exposed to appropriate enzyme degradation as a result of either wounding or pest attack.

  6. MYC-nick promotes cell migration by inducing fascin expression and Cdc42 activation

    PubMed Central

    Anderson, Sarah; Poudel, Kumud Raj; Roh-Johnson, Minna; Brabletz, Thomas; Yu, Ming; Borenstein-Auerbach, Nofit; Grady, William N.; Bai, Jihong; Moens, Cecilia B.; Eisenman, Robert N.; Conacci-Sorrell, Maralice

    2016-01-01

    MYC-nick is a cytoplasmic, transcriptionally inactive member of the MYC oncoprotein family, generated by a proteolytic cleavage of full-length MYC. MYC-nick promotes migration and survival of cells in response to chemotherapeutic agents or withdrawal of glucose. Here we report that MYC-nick is abundant in colonic and intestinal tumors derived from mouse models with mutations in the Wnt, TGF-β, and PI3K pathways. Moreover, MYC-nick is elevated in colon cancer cells deleted for FBWX7, which encodes the major E3 ligase of full-length MYC frequently mutated in colorectal cancers. MYC-nick promotes the migration of colon cancer cells assayed in 3D cultures or grown as xenografts in a zebrafish metastasis model. MYC-nick accelerates migration by activating the Rho GTPase Cdc42 and inducing fascin expression. MYC-nick, fascin, and Cdc42 are frequently up-regulated in cells present at the invasive front of human colorectal tumors, suggesting a coordinated role for these proteins in tumor migration. PMID:27566402

  7. MYC-nick promotes cell migration by inducing fascin expression and Cdc42 activation.

    PubMed

    Anderson, Sarah; Poudel, Kumud Raj; Roh-Johnson, Minna; Brabletz, Thomas; Yu, Ming; Borenstein-Auerbach, Nofit; Grady, William N; Bai, Jihong; Moens, Cecilia B; Eisenman, Robert N; Conacci-Sorrell, Maralice

    2016-09-13

    MYC-nick is a cytoplasmic, transcriptionally inactive member of the MYC oncoprotein family, generated by a proteolytic cleavage of full-length MYC. MYC-nick promotes migration and survival of cells in response to chemotherapeutic agents or withdrawal of glucose. Here we report that MYC-nick is abundant in colonic and intestinal tumors derived from mouse models with mutations in the Wnt, TGF-β, and PI3K pathways. Moreover, MYC-nick is elevated in colon cancer cells deleted for FBWX7, which encodes the major E3 ligase of full-length MYC frequently mutated in colorectal cancers. MYC-nick promotes the migration of colon cancer cells assayed in 3D cultures or grown as xenografts in a zebrafish metastasis model. MYC-nick accelerates migration by activating the Rho GTPase Cdc42 and inducing fascin expression. MYC-nick, fascin, and Cdc42 are frequently up-regulated in cells present at the invasive front of human colorectal tumors, suggesting a coordinated role for these proteins in tumor migration. PMID:27566402

  8. Pyruvate and cilostazol protect cultured rat cortical pericytes against tissue plasminogen activator (tPA)-induced cell death.

    PubMed

    Kim, Ha Na; Kim, Tae-Youn; Yoon, Young Hee; Koh, Jae-Young

    2015-12-01

    Since even a brief ischemia can cause permanent brain damage, rapid restoration of blood flow is critical to limiting damage. Although intravenous tPA during the acute stage is the treatment of choice for achieving reperfusion, this treatment is sometimes associated with brain hemorrhage. Agents that reduce tPA-related bleeding risk may help expand its therapeutic window. This study assessed whether zinc dyshomeostasis underlies the toxic effect of tPA on brain vascular pericytes; whether pyruvate, an inhibitor of zinc toxicity, protects pericytes against tPA-induced cell death; and whether cilostazol, which protects pericytes against tPA-induced cell death, affects zinc dyshomeostasis associated with tPA toxicity. Cultured pericytes from newborn rat brains were treated with 10-200 μg/ml tPA for 24 h, inducing cell death in a concentration-dependent manner. tPA-induced cell death was preceded by increases in intracellular free zinc levels, and was substantially attenuated by plasminogen activator inhibitor-1 (PAI-1) or TPEN. Pyruvate completely blocked direct zinc toxicity and tPA-induced pericyte cell death. Both cAMP and cilostazol, a PDE3 inhibitor that attenuates tPA-induced pericyte cell death in vitro and tPA-induced brain hemorrhage in vivo, reduced zinc- and tPA-induced pericyte cell death, suggesting that zinc dyshomeostasis may be targeted by cilostazol in tPA toxicity. These findings show that tPA-induced pericyte cell death may involve zinc dyshomeostasis, and that pyruvate and cilostazol attenuate tPA-induced cell death by reducing the toxic cascade triggered by zinc dyshomeostasis. Since pyruvate is an endogenous metabolite and cilostazol is an FDA-approved drug, in vivo testing of both as protectors against tPA-induced brain hemorrhage may be warranted. This article is part of a Special Issue entitled SI: Neuroprotection.

  9. Wnt activation protects against neomycin-induced hair cell damage in the mouse cochlea.

    PubMed

    Liu, L; Chen, Y; Qi, J; Zhang, Y; He, Y; Ni, W; Li, W; Zhang, S; Sun, S; Taketo, M M; Wang, L; Chai, R; Li, H

    2016-01-01

    Recent studies have reported the role of Wnt/β-catenin signaling in hair cell (HC) development, regeneration, and differentiation in the mouse cochlea; however, the role of Wnt/β-catenin signaling in HC protection remains unknown. In this study, we took advantage of transgenic mice to specifically knockout or overactivate the canonical Wnt signaling mediator β-catenin in HCs, which allowed us to investigate the role of Wnt/β-catenin signaling in protecting HCs against neomycin-induced damage. We first showed that loss of β-catenin in HCs made them more vulnerable to neomycin-induced injury, while constitutive activation of β-catenin in HCs reduced HC loss both in vivo and in vitro. We then showed that loss of β-catenin in HCs increased caspase-mediated apoptosis induced by neomycin injury, while β-catenin overexpression inhibited caspase-mediated apoptosis. Finally, we demonstrated that loss of β-catenin in HCs led to increased expression of forkhead box O3 transcription factor (Foxo3) and Bim along with decreased expression of antioxidant enzymes; thus, there were increased levels of reactive oxygen species (ROS) after neomycin treatment that might be responsible for the increased aminoglycoside sensitivity of HCs. In contrast, β-catenin overexpression reduced Foxo3 and Bim expression and ROS levels, suggesting that β-catenin is protective against neomycin-induced HC loss. Our findings demonstrate that Wnt/β-catenin signaling has an important role in protecting HCs against neomycin-induced HC loss and thus might be a new therapeutic target for the prevention of HC death.

  10. Wnt activation protects against neomycin-induced hair cell damage in the mouse cochlea

    PubMed Central

    Liu, L; Chen, Y; Qi, J; Zhang, Y; He, Y; Ni, W; Li, W; Zhang, S; Sun, S; Taketo, M M; Wang, L; Chai, R; Li, H

    2016-01-01

    Recent studies have reported the role of Wnt/β-catenin signaling in hair cell (HC) development, regeneration, and differentiation in the mouse cochlea; however, the role of Wnt/β-catenin signaling in HC protection remains unknown. In this study, we took advantage of transgenic mice to specifically knockout or overactivate the canonical Wnt signaling mediator β-catenin in HCs, which allowed us to investigate the role of Wnt/β-catenin signaling in protecting HCs against neomycin-induced damage. We first showed that loss of β-catenin in HCs made them more vulnerable to neomycin-induced injury, while constitutive activation of β-catenin in HCs reduced HC loss both in vivo and in vitro. We then showed that loss of β-catenin in HCs increased caspase-mediated apoptosis induced by neomycin injury, while β-catenin overexpression inhibited caspase-mediated apoptosis. Finally, we demonstrated that loss of β-catenin in HCs led to increased expression of forkhead box O3 transcription factor (Foxo3) and Bim along with decreased expression of antioxidant enzymes; thus, there were increased levels of reactive oxygen species (ROS) after neomycin treatment that might be responsible for the increased aminoglycoside sensitivity of HCs. In contrast, β-catenin overexpression reduced Foxo3 and Bim expression and ROS levels, suggesting that β-catenin is protective against neomycin-induced HC loss. Our findings demonstrate that Wnt/β-catenin signaling has an important role in protecting HCs against neomycin-induced HC loss and thus might be a new therapeutic target for the prevention of HC death. PMID:26962686

  11. Methamphetamine activates nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and induces human immunodeficiency virus (HIV) transcription in human microglial cells.

    PubMed

    Wires, Emily S; Alvarez, David; Dobrowolski, Curtis; Wang, Yun; Morales, Marisela; Karn, Jonathan; Harvey, Brandon K

    2012-10-01

    Human immunodeficiency virus (HIV) primarily infects glial cells in the central nervous system (CNS). Recent evidence suggests that HIV-infected individuals who abuse drugs such as methamphetamine (METH) have higher viral loads and experience more severe neurological complications than HIV-infected individuals who do not abuse drugs. The aim of this study was to determine the effect of METH on HIV expression from the HIV long terminal repeat (LTR) promoter and on an HIV integrated provirus in microglial cells, the primary host cells for HIV in the CNS. Primary human microglial cells immortalized with SV40 T antigen (CHME-5 cells) were cotransfected with an HIV LTR reporter and the HIV Tat gene, a key regulator of viral replication and gene expression, and exposed to METH. Our results demonstrate that METH treatment induced LTR activation, an effect potentiated in the presence of Tat. We also found that METH increased the nuclear translocation of the nuclear factor kappa B (NF-κB), a key cellular transcriptional regulator of the LTR promoter, and the activity of an NF-κB-specific reporter plasmid in CHME-5 cells. The presence of a dominant-negative regulator of NF-κB blocked METH-related activation of the HIV LTR. Furthermore, treatment of HIV-latently infected CHME-5 (CHME-5/HIV) cells with METH induced HIV expression and nuclear translocation of the p65 subunit of NF-κB. These results suggest that METH can stimulate HIV gene expression in microglia cells through activation of the NF-κB signaling pathway. This mechanism may outline the initial biochemical events leading to the observed increased neurodegeneration in HIV-positive individuals who use METH.

  12. Streptozotocin induced activation of oxidative stress responsive splenic cell signaling pathways: Protective role of arjunolic acid

    SciTech Connect

    Manna, Prasenjit; Ghosh, Jyotirmoy; Das, Joydeep

    2010-04-15

    Present study investigates the beneficial role of arjunolic acid (AA) against the alteration in the cytokine levels and simultaneous activation of oxidative stress responsive signaling pathways in spleen under hyperglycemic condition. Diabetes was induced by injection of streptozotocin (STZ) (at a dose of 70 mg/kg body weight, injected in the tail vain). STZ administration elevated the levels of IL-2 as well as IFN-gamma and attenuated the level of TNF-alpha in the sera of diabetic animals. In addition, hyperglycemia is also associated with the increased production of intracellular reactive intermediates resulting with the elevation in lipid peroxidation, protein carbonylation and reduction in intracellular antioxidant defense. Investigating the oxidative stress responsive cell signaling pathways, increased expressions (immunoreactive concentrations) of phosphorylated p65 as well as its inhibitor protein phospho IkappaBalpha and phosphorylated mitogen activated protein kinases (MAPKs) have been observed in diabetic spleen tissue. Studies on isolated splenocytes revealed that hyperglycemia caused disruption of mitochondrial membrane potential, elevation in the concentration of cytosolic cytochrome c as well as activation of caspase 3 leading to apoptotic cell death. Histological examination revealed that diabetic induction depleted the white pulp scoring which is in agreement with the reduced immunological response. Treatment with AA prevented the hyperglycemia and its associated pathogenesis in spleen tissue. Results suggest that AA might act as an anti-diabetic and immunomodulatory agent against hyperglycemia.

  13. Trientine, a copper-chelating agent, induced apoptosis in murine fibrosarcoma cells by activation of the p38 MAPK pathway.

    PubMed

    KADOWAKI, Shingo; ENDOH, Daiji; OKUI, Toyo; HAYASHI, Masanobu

    2009-11-01

    We have reported that treatment with trientine, Cu-chelating agent, inhibits tumor growth in a murine transplantation model using fibrosarcoma and induces apoptosis in tumor cells in vivo and in vitro. When fibrosarcoma cells were treated with 10 mM trientine, the activities of p38 MAPK in treated cells were approximately 3-4 times higher than those in untreated cells. Proportions of cells in which apoptosis was induced by trientine increased in an incubation time-dependent manner from days 2 to 6. The proportions of apoptotic cells in the cells treated with trientine and SB203580, an inhibitor of p38 MAPK, were approximately 50% in those of cells treated with trientine alone. The present results showed that the p38 MAPK pathway may play an important role in induction of apoptosis in fibrosarcoma cells by trientine.

  14. Lupiwighteone induces cell cycle arrest and apoptosis and activates the Nrf2/ARE pathway in human neuroblastoma cells.

    PubMed

    Ren, Jie; Yang, Jie; Xu, Yuanyuan; Huang, Qianhui; Yang, Meng; Hu, Kun

    2015-02-01

    Lupiwighteone (Lup) is a kind of natural isoflavone, but its pharmacological effect and active mechanism are rarely reported. This study aimed to investigate the anticancer and cancer preventive effects of Lup on human neuroblastoma (SH-SY5Y) cells. We found that Lup could inhibit SH-SY5Y cells growth in a concentration- and time-dependent manner. Further studies suggested that Lup could induce G2/M phase arrest associated with an evident decrease in cyclin B1/D1 and cyclin dependent kinase (CDK) 1/2/4/6 protein expressions. Moreover, Lup could regulate the changes of mitochondrial membrane potential and increase intracellular reactive oxygen species (ROS) production. After the cells were treated with Lup, topical morphological characteristics were observed; apoptosis-related protein expressions, such as Bax, cytochrome c, cleaved caspase-9, cleaved caspase-3 and cleaved PARP-1 were increased; and protein expressions, such as Bcl-2, procaspase-9, PARP-1 and P-Akt were decreased. These changes were observed simultaneously. In addition, Nrf2 transcription factor activation was detected by an ARE-GFP reporter assay. Nrf2 nuclear localization was then investigated using a fluorescence microscope. Furthermore, Nrf2 and Keap1 protein levels were determined by western blot. Our results may provide a scientific basis for the application of the anticancer and cancer preventive effects of Lup on SH-SY5Y cells.

  15. Activation of anaphase-promoting complex by p53 induces a state of dormancy in cancer cells against chemotherapeutic stress

    PubMed Central

    Dai, Yafei; Wang, Lujuan; Tang, Jingqun; Cao, Pengfei; Luo, Zhaohui; Sun, Jun; Kiflu, Abraha; Sai, Buqing; Zhang, Meili; Wang, Fan; Li, Guiyuan; Xiang, Juanjuan

    2016-01-01

    Cancer dormancy is a stage in tumor progression in which residual disease remains occult and asymptomatic for a prolonged period. Cancer cell dormancy is the main cause of cancer recurrence and failure of therapy. However, cancer dormancy is poorly characterized and the mechanisms of how cancer cells develop dormancy and relapse remain elusive. In this study, 5- fluorouracil (5-FU) was used to induce cancer cell dormancy. We found that cancer cells escape the cytotoxicity of 5-FU by becoming “dormant”. After exposure to 5-FU, residual non-small cell lung cancer (NSCLC) cells underwent epithelial-mesenchymal transition (EMT), followed by mesenchymal-epithelial transition (MET). These EMT-transformed NSCLC cells were in the state of cell quiescence where cells were not dividing and were arrested in the cell cycle in G0-G1. The dormant cells underwent an EMT showed characteristics of cancer stem cells. P53 is strongly accumulated in response to 5-FU-induced dormant cells through the activation of ubiquitin ligase anaphase-promoting complex (APC/C) and TGF-β/Smad signaling. In contrast to the EMT-transformed cells, MET-transformed cells showed an increased ability to proliferate, suggesting that dormant EMT cells were reactivated in the MET process. During the EMT-MET process, DNA repair including nonhomologous end joining (NHEJ) and homologous recombination (HR) is critical to dormant cell reactivation. Our findings provide a mechanism to unravel cancer cell dormancy and reactivation of the cancer cell population. PMID:27009858

  16. INSULIN INDUCED EPIDERMAL GROWTH FACTOR ACTIVATION IN VASCULAR SMOOTH MUSCLE CELLS IS ADAM-DEPENDENT

    PubMed Central

    Roztocil, Elisa; Nicholl, Suzanne M.; Davies, Mark G.

    2008-01-01

    Background With the rise in metabolic syndrome, understanding the role of insulin signaling within the cells of vasculature has become more important but yet remains poorly defined. The study examines the role of insulin actions on a pivotal cross-talk receptor, Epidermal Growth Factor Receptor (EGFR). EGFR is transactivated by both G-protein-coupled receptors and receptor linked tyrosine kinases and is key to many of their responses. Objective To determine the pathway of EGFR transactivation by insulin in human coronary smooth muscle cells (VSMC) Methods VSMC were cultured in vitro. Assays of EGFR phosphorylation were examined in response to insulin in the presence and absence of the plasmin inhibitors (e-aminocaproic acid and aprotinin) matrix metalloprotease (MMP) inhibitor GM6001, the ADAM (A Disintegrin And Metalloproteinase Domain) inhibitors TAPI-0 and TAPI-1, Heparin binding epidermal growth factor (HB-EGF) inhibitor, CRM197, HB-EGF inhibitory antibodies, EGF inhibitory antibodies and the EGFR inhibitor AG1478. Results Insulin induced time-dependent EGFR phosphorylation, which was inhibited by AG1478 in a concentration dependent manner. Application of the plasmin inhibitors did not block the response. EGFR phosphorylation by insulin was blocked by inhibition of MMP activity and the ligand HB-EGF. The presence of the ADAM inhibitors, TAPI-0 and TAPI-1 significantly decreased EGFR activation. EGFR phosphorylation by EGF was not interrupted by inhibition of plasmin, MMPs TAPIs, or HB-EGF. Direct blockade of the EGFR prevented activation by both insulin and EGF. Conclusion Insulin can induce transactivation of EGFR by an ADAM-mediated, HB-EGF dependent process. This is the first description of crosstalk via ADAM between insulin and EGFR in vascular SMC. Targeting a pivotal cross-talk receptor such as EGFR, which can be transactivated by both G-protein-coupled receptors and receptor tyrosine kinases is an attractive molecular target. PMID:18656632

  17. Transforming growth factor-beta1 induces activation of Ras, Raf-1, MEK and MAPK in rat hepatic stellate cells.

    PubMed

    Reimann, T; Hempel, U; Krautwald, S; Axmann, A; Scheibe, R; Seidel, D; Wenzel, K W

    1997-02-10

    The transdifferentiation of hepatic stellate cells into myofibroblast-like cells and the proliferation of the transdifferentiated cells are controlled by TGF-beta1. Little is known about the intracellular signal transducers of TGF-beta1. In this paper we show that in cultured hepatic stellate cells TGF-beta1 induces activation of Ras, Raf-1, MEK and MAPK p42 and p44. The activation of MAPK depends on the activation of MEK. Our data exclude that the observed effects are mediated by a bFGF or PDGF autocrine loop. PMID:9038360

  18. Hypoxia counteracts taxol-induced apoptosis in MDA-MB-231 breast cancer cells: role of autophagy and JNK activation.

    PubMed

    Notte, A; Ninane, N; Arnould, T; Michiels, C

    2013-05-16

    Cancer cell resistance against chemotherapy is still a heavy burden to improve anticancer treatments. Autophagy activation and the development of hypoxic regions within the tumors are known to promote cancer cell resistance. Therefore, we sought to evaluate the role of autophagy and hypoxia on the taxol-induced apoptosis in MDA-MB-231 breast cancer cells. The results showed that taxol induced apoptosis after 16 h of incubation, and that hypoxia protected MDA-MB-231 cells from taxol-induced apoptosis. In parallel, taxol induced autophagy activation already after 2 h of incubation both under normoxia and hypoxia. Autophagy activation after taxol exposure was shown to be a protective mechanism against taxol-induced cell death both under normoxia and hypoxia. However, at longer incubation time, the autophagic process reached a saturation point under normoxia leading to cell death, whereas under hypoxia, autophagy flow still correctly took place allowing the cells to survive. Autophagy induction is induced after taxol exposure via mechanistic target of rapamycin (mTOR) inhibition, which is more important in cells exposed to hypoxia. Taxol also induced c-Jun N-terminal kinase (JNK) activation and phosphorylation of its substrates B-cell CLL/lymphoma 2 (Bcl2) and BCL2-like 1 (BclXL) under normoxia and hypoxia very early after taxol exposure. Bcl2 and BclXL phosphorylation was decreased more importantly under hypoxia after long incubation time. The role of JNK in autophagy and apoptosis induction was studied using siRNAs. The results showed that JNK activation promotes resistance against taxol-induced apoptosis under normoxia and hypoxia without being involved in induction of autophagy. In conclusion, the resistance against taxol-induced cell death observed under hypoxia can be explained by a more effective autophagic flow activated via the classical mTOR pathway and by a mechanism involving JNK, which could be dependent on Bcl2 and BclXL phosphorylation but independent of

  19. Hypoxia counteracts taxol-induced apoptosis in MDA-MB-231 breast cancer cells: role of autophagy and JNK activation

    PubMed Central

    Notte, A; Ninane, N; Arnould, T; Michiels, C

    2013-01-01

    Cancer cell resistance against chemotherapy is still a heavy burden to improve anticancer treatments. Autophagy activation and the development of hypoxic regions within the tumors are known to promote cancer cell resistance. Therefore, we sought to evaluate the role of autophagy and hypoxia on the taxol-induced apoptosis in MDA-MB-231 breast cancer cells. The results showed that taxol induced apoptosis after 16 h of incubation, and that hypoxia protected MDA-MB-231 cells from taxol-induced apoptosis. In parallel, taxol induced autophagy activation already after 2 h of incubation both under normoxia and hypoxia. Autophagy activation after taxol exposure was shown to be a protective mechanism against taxol-induced cell death both under normoxia and hypoxia. However, at longer incubation time, the autophagic process reached a saturation point under normoxia leading to cell death, whereas under hypoxia, autophagy flow still correctly took place allowing the cells to survive. Autophagy induction is induced after taxol exposure via mechanistic target of rapamycin (mTOR) inhibition, which is more important in cells exposed to hypoxia. Taxol also induced c-Jun N-terminal kinase (JNK) activation and phosphorylation of its substrates B-cell CLL/lymphoma 2 (Bcl2) and BCL2-like 1 (BclXL) under normoxia and hypoxia very early after taxol exposure. Bcl2 and BclXL phosphorylation was decreased more importantly under hypoxia after long incubation time. The role of JNK in autophagy and apoptosis induction was studied using siRNAs. The results showed that JNK activation promotes resistance against taxol-induced apoptosis under normoxia and hypoxia without being involved in induction of autophagy. In conclusion, the resistance against taxol-induced cell death observed under hypoxia can be explained by a more effective autophagic flow activated via the classical mTOR pathway and by a mechanism involving JNK, which could be dependent on Bcl2 and BclXL phosphorylation but

  20. Dectin-1 agonist selectively induces IgG1 class switching by LPS-activated mouse B cells.

    PubMed

    Seo, Beom-Seok; Park, Ha-Yan; Yoon, Hee-Kyung; Yoo, Yung-Choon; Lee, Junglim; Park, Seok-Rae

    2016-10-01

    Heat-killed Saccharomyces cerevisiae (HKSC) is an agonist for Dectin-1, a major fungal cell wall β-glucan receptor. We previously reported that HKSC selectively enhances IgG1 production by LPS-activated mouse B cells. To determine if this IgG1 selectivity is caused by selective IgG1 class switching, we performed RT-PCRs for measuring germline transcripts (GLTs), flow cytometric analyses for detecting Ig-expressing cells, and ELISPOT assays for measuring the number of Ig-secreting cells in HKSC/LPS-stimulated mouse B cell cultures. HKSC selectively enhanced expression of GLTγ1, the number of IgG1-expressing cells, and the number of IgG1-secreting B cells in the presence of LPS stimulation. In addition, HKSC induced the expression of CD69, an activation marker for B lymphocytes, and the expression of surface Dectin-1. Two Dectin-1 antagonists, laminarin and a neutralizing Dectin-1 antibody, selectively diminished HKSC-reinforced IgG1 production by LPS-stimulated B cells. Furthermore, depleted zymosan (dzn), a Dectin-1 agonist with increased selectivity, also selectively enhanced GLTγ1 transcription. The Dectin-1 antagonists blocked dzn-induced IgG1 production by LPS-activated B cells. Collectively, these results suggest that Dectin-1 agonists selectively induce IgG1 class switching by direct stimulation of Dectin-1 on LPS-activated B cells resulting in selective production of IgG1. PMID:27568820

  1. Dectin-1 agonist selectively induces IgG1 class switching by LPS-activated mouse B cells.

    PubMed

    Seo, Beom-Seok; Park, Ha-Yan; Yoon, Hee-Kyung; Yoo, Yung-Choon; Lee, Junglim; Park, Seok-Rae

    2016-10-01

    Heat-killed Saccharomyces cerevisiae (HKSC) is an agonist for Dectin-1, a major fungal cell wall β-glucan receptor. We previously reported that HKSC selectively enhances IgG1 production by LPS-activated mouse B cells. To determine if this IgG1 selectivity is caused by selective IgG1 class switching, we performed RT-PCRs for measuring germline transcripts (GLTs), flow cytometric analyses for detecting Ig-expressing cells, and ELISPOT assays for measuring the number of Ig-secreting cells in HKSC/LPS-stimulated mouse B cell cultures. HKSC selectively enhanced expression of GLTγ1, the number of IgG1-expressing cells, and the number of IgG1-secreting B cells in the presence of LPS stimulation. In addition, HKSC induced the expression of CD69, an activation marker for B lymphocytes, and the expression of surface Dectin-1. Two Dectin-1 antagonists, laminarin and a neutralizing Dectin-1 antibody, selectively diminished HKSC-reinforced IgG1 production by LPS-stimulated B cells. Furthermore, depleted zymosan (dzn), a Dectin-1 agonist with increased selectivity, also selectively enhanced GLTγ1 transcription. The Dectin-1 antagonists blocked dzn-induced IgG1 production by LPS-activated B cells. Collectively, these results suggest that Dectin-1 agonists selectively induce IgG1 class switching by direct stimulation of Dectin-1 on LPS-activated B cells resulting in selective production of IgG1.

  2. Curcumin induces apoptosis by inhibiting sarco/endoplasmic reticulum Ca2+ ATPase activity in ovarian cancer cells.

    PubMed

    Seo, Jeong-Ah; Kim, Boyun; Dhanasekaran, Danny N; Tsang, Benjamin K; Song, Yong Sang

    2016-02-01

    Aberrant increase in the expression levels of sarco/endoplasmic reticulum calcium ATPase (SERCA), which regulates Ca(2+) homeostasis, has been observed in ovarian cancers. In this study, we demonstrated that curcumin increases cytosolic Ca(2+) concentration through inhibition of SERCA activity, causing apoptosis in ovarian cancer cells but not in normal cells, including peripheral blood mononuclear cells (PBMCs) and ovarian surface epithelial cells (OSE). Curcumin induced apoptosis in ovarian cancer cells in a concentration- and time-dependent manner. Cytosolic Ca(2+) flux was evident after the curcumin treatment (15 µM). Treatment with Ca(2+) chelator reduced curcumin-induced apoptosis, confirming the possible involvement of increased cytosolic Ca(2+) concentration in this response. Basal mRNA and protein levels of SERCA2 were significantly higher in ovarian cancer cells than in OSE. SERCA activity was suppressed by curcumin, with no effect on protein expression. Forced expression of the SERCA2b gene in ovarian cancer cells prevented curcumin-induced cytosolic Ca(2+) elevation and subsequent apoptosis, supporting an important role of SERCA in curcumin-induced apoptosis of ovarian cancer cells. Taken together, inhibition of SERCA activity by curcumin disrupts the Ca(2+) homeostasis and thereby promotes apoptosis in ovarian cancer cells.

  3. Curcumin induces apoptosis by inhibiting sarco/endoplasmic reticulum Ca2+ ATPase activity in ovarian cancer cells.

    PubMed

    Seo, Jeong-Ah; Kim, Boyun; Dhanasekaran, Danny N; Tsang, Benjamin K; Song, Yong Sang

    2016-02-01

    Aberrant increase in the expression levels of sarco/endoplasmic reticulum calcium ATPase (SERCA), which regulates Ca(2+) homeostasis, has been observed in ovarian cancers. In this study, we demonstrated that curcumin increases cytosolic Ca(2+) concentration through inhibition of SERCA activity, causing apoptosis in ovarian cancer cells but not in normal cells, including peripheral blood mononuclear cells (PBMCs) and ovarian surface epithelial cells (OSE). Curcumin induced apoptosis in ovarian cancer cells in a concentration- and time-dependent manner. Cytosolic Ca(2+) flux was evident after the curcumin treatment (15 µM). Treatment with Ca(2+) chelator reduced curcumin-induced apoptosis, confirming the possible involvement of increased cytosolic Ca(2+) concentration in this response. Basal mRNA and protein levels of SERCA2 were significantly higher in ovarian cancer cells than in OSE. SERCA activity was suppressed by curcumin, with no effect on protein expression. Forced expression of the SERCA2b gene in ovarian cancer cells prevented curcumin-induced cytosolic Ca(2+) elevation and subsequent apoptosis, supporting an important role of SERCA in curcumin-induced apoptosis of ovarian cancer cells. Taken together, inhibition of SERCA activity by curcumin disrupts the Ca(2+) homeostasis and thereby promotes apoptosis in ovarian cancer cells. PMID:26607901

  4. Real-time monitoring of cell mechanical changes induced by endothelial cell activation and their subsequent binding with leukemic cell lines.

    PubMed

    Tan, Liang; Lin, Peiling; Pezeshkian, Bahareh; Rehman, Abdul; Madlambayan, Gerard; Zeng, Xiangqun

    2014-06-15

    Endothelial cell (EC) activation and their subsequent binding with different cells have various mechanical consequences that, if monitored real time, can serve as a functional biomarker of many pathophysiological response mechanisms. This work presents an innovative and facile strategy to conduct such monitoring using quartz crystal microbalance (QCM), thereby relating the shifts in its frequency and motional resistance to morphological changes upon cell-cell and cell-substrate interactions. By activating ECs with TNF-α and then characterizing their binding with HL-60 and KG-1 leukemia cells, we are able to induce the mechanical changes in ECs especially in the region of cell-substrate contact which resulted in dynamically coupled mass and viscoelastic changes representing the extent of both activation and binding. The activated ECs suffered a decrease of cellular contact area, leading to positive frequency shift and decreased motional resistance. The binding of leukemia cells onto pre-activated ECs exerted a mechanical force to regain the cell surface contact which resulted in the obvious QCM responses opposite to that of activation, and proportional to the number of cells added, in spite of the fact that these added cells are extremely outside the extinction boundary of the shear wave generated by QCM. Different cell lines demonstrate different attachment behavior, which was detected by the QCM. Despite these variations are quite subtle, yet the sensitivity of the technique for dynamic changes at the interface makes them detectable. Moreover, the reproducibility of the generated data determined at each step by deviation measurements (<10%) in response plot was very high despite the high possible heterogeneity in cell populations. The results are explained on the basis of simple theoretical and physical models, although, the development of a more quantitative and precise model is underway in our laboratory.

  5. Real-Time Monitoring of Cell Mechanical Changes Induced by Endothelial Cell Activation and their Subsequent Binding with Leukemic Cell Lines

    PubMed Central

    Tan, Liang; Lin, Peiling; Pezeshkian, Bahareh; Rehman, Abdul; Madlambayan, Gerard; Zeng, Xiangqun

    2014-01-01

    Endothelial cell (EC) activation and their subsequent binding with different cells have various mechanical consequences that, if monitored real time, can serve as a functional biomarker of many pathophysiological response mechanisms. This work presents an innovative and facile strategy to conduct such monitoring using quartz crystal microbalance (QCM), thereby relating the shifts in its frequency and motional resistance to morphological changes upon cell-cell and cell-substrate interactions. By activating ECs with TNF-α and then characterizing their binding with HL-60 and KG-1 leukemia cells, we are able to induce the mechanical changes in ECs especially in the region of cell-substrate contact which resulted in dynamically coupled mass and viscoelastic changes representing the extent of both activation and binding. The activated ECs suffered a decrease of cellular contact area, leading to positive frequency shift and decreased motional resistance. The binding of leukemia cells onto pre-activated ECs exerted a mechanical force to regain the cell surface contact which resulted in the obvious QCM responses opposite to that of activation, and proportional to the number of cells added, in spite of the fact that these added cells are extremely outside the extinction depth of the shear wave generated by QCM. Different cell lines demonstrate different attachment behavior, which was detected by the QCM. Despite these variations are quite subtle, yet the sensitivity of the technique for dynamic changes at the interface makes them detectable. Moreover, the reproducibility of the generated data determined at each step by deviation measurements (<10%) in response plot was very high despite the high possible heterogeneity in cell populations. The results are explained on the basis of simple theoretical and physical models, although, the development of a more quantitative and precise model is underway in our laboratory. PMID:24487102

  6. Curcumin Attenuated Bupivacaine-Induced Neurotoxicity in SH-SY5Y Cells Via Activation of the Akt Signaling Pathway.

    PubMed

    Fan, You-Ling; Li, Heng-Chang; Zhao, Wei; Peng, Hui-Hua; Huang, Fang; Jiang, Wei-Hang; Xu, Shi-Yuan

    2016-09-01

    Bupivacaine is widely used for regional anesthesia, spinal anesthesia, and pain management. However, bupivacaine could cause neuronal injury. Curcumin, a low molecular weight polyphenol, has a variety of bioactivities and may exert neuroprotective effects against damage induced by some stimuli. In the present study, we tested whether curcumin could attenuate bupivacaine-induced neurotoxicity in SH-SY5Y cells. Cell injury was evaluated by examining cell viability, mitochondrial damage and apoptosis. We also investigated the levels of activation of the Akt signaling pathway and the effect of Akt inhibition by triciribine on cell injury following bupivacaine and curcumin treatment. Our findings showed that the bupivacaine treatment could induce neurotoxicity. Pretreatment of the SH-SY5Y cells with curcumin significantly attenuated bupivacaine-induced neurotoxicity. Interestingly, the curcumin treatment increased the levels of Akt phosphorylation. More significantly, the pharmacological inhibition of Akt abolished the cytoprotective effect of curcumin against bupivacaine-induced cell injury. Our data suggest that pretreating SH-SY5Y cells with curcumin provides a protective effect on bupivacaine-induced neuronal injury via activation of the Akt signaling pathway.

  7. Insulin receptor signaling activated by penta-O-galloyl-α-D: -glucopyranose induces p53 and apoptosis in cancer cells.

    PubMed

    Cao, Yanyan; Evans, Susan C; Soans, Eroica; Malki, Ahmed; Liu, Yi; Liu, Yan; Chen, Xiaozhuo

    2011-09-01

    p53 is essential for cell cycle arrest and apoptosis induction while insulin receptor (IR) signaling is important for cell metabolism and proliferation and found upregulated in cancers. While IR has recently been found to be involved in apoptosis, p53 induction or apoptosis mediated through IR signaling activation has never been documented. Here, we report that the IR signaling pathway, particularly the IR-MEK pathway, mediates biological and biochemical changes in p53 and apoptosis in tumor cells. Specifically, natural compound penta-O-galloyl-α-D: -glucopyranose (α-PGG), a previously characterized IR signaling activator, induced apoptosis in RKO cells without significantly affecting its normal counterpart FHC cells. α-PGG induced apoptosis in RKO cells through p53, Bax and caspase 3. Importantly, α-PGG's ability to elevate p53 was diminished by IR inhibitor and IR-siRNA, suggesting a non-conventional role of IR as being involved in p53 induction. Further studies revealed that α-PGG activated MEK, a downstream signaling factor of IR. Blocking MEK significantly suppressed α-PGG-induced p53 and Bax elevation. All these results suggested that α-PGG induced p53, Bax, and apoptosis through the IR-MEK signaling pathway. The unique activity of α-PGG, a novel IR phosphorylation and apoptosis inducer, may offer a new therapeutic strategy for eliciting apoptotic signal and inhibiting cancer growth.

  8. The novel HDAC inhibitor AR-42-induced anti-colon cancer cell activity is associated with ceramide production.

    PubMed

    Xu, Weihong; Xu, Bin; Yao, Yiting; Yu, Xiaoling; Shen, Jie

    2015-08-01

    In the current study, we investigated the potential activity of AR-42, a novel histone deacetylase (HDAC) inhibitor, against colon cancer cells. Our in vitro results showed that AR-42 induced ceramide production, exerted potent anti-proliferative and pro-apoptotic activities in established (SW-620 and HCT-116 lines) and primary human colon cancer cells. Exogenously-added sphingosine 1-phosphate (S1P) suppressed AR-42-induced activity, yet a cell-permeable ceramide (C4) facilitated AR-42-induced cytotoxicity against colon cancer cells. In addition, AR-42-induced ceramide production and anti-colon cancer cell activity were inhibited by the ceramide synthase inhibitor fumonisin B1, but were exacerbated by PDMP, which is a ceramide glucosylation inhibitor. In vivo, oral administration of a single dose of AR-42 dramatically inhibited SW-620 xenograft growth in severe combined immunodeficient (SCID) mice, without inducing overt toxicities. Together, these results show that AR-42 dramatically inhibits colon cancer cell proliferation in vitro and in vivo, and ceramide production might be the key mechanism responsible for its actions.

  9. BAY 11-7085 induces glucocorticoid receptor activation and autophagy that collaborate with apoptosis to induce human synovial fibroblast cell death

    PubMed Central

    Relic, Biserka; Charlier, Edith; Deroyer, Celine; Malaise, Olivier; Neuville, Sophie; Desoroux, Aline; Gillet, Philippe; de Seny, Dominique; Malaise, Michel G.

    2016-01-01

    Inhibition of proapoptotic pathways in synovial fibroblasts is one of the major causes of synovial proliferation and hyperplasia in rheumatic diseases. We have shown previously that NF-κB inhibitor BAY 11-7085, through inactivation of PPAR-γ, induces apoptosis in human synovial fibroblasts. In this work we showed that BAY 11-7085 induced autophagy that preceded BAY 11-7085-induced apoptosis. Of interest, BAY 11-7085 induced Serine 211 phosphorylation and degradation of glucocorticoid receptor (GR). Glucocorticoid prednisolone induced both activation and degradation of GR, as well as autophagy in synovial fibroblasts. BAY 11-7085-induced cell death was significantly decreased with glucocorticoid inhibitor mifepristone and with inhibitors of autophagy. Both BAY 11-7085-induced autophagy and GR activation were down regulated with PPAR-γ agonist, 15d-PGJ2 and MEK/ERK inhibitor UO126. Inhibition of autophagy markedly decreased endogenous and BAY 11-7085-induced ERK phosphorylation, suggesting a positive feed back loop between ERK activation and autophagy in synovial fibroblasts. Co-transfection of MEK1 with PPAR-γ1 in HEK293 cells caused known inhibitory phosphorylation of PPAR-γ1 (Serine 112) and enhanced GR degradation, in the absence or presence of prednisolone. Furthermore, GR was both phosphorylated on Serine 211 and down regulated in synovial fibroblasts during serum starvation induced autophagy. These results showed that GR activation and PPAR-γ inactivation mediated BAY 11-7085-induced autophagy. PMID:26993765

  10. Early LPS-induced ERK activation in retinal pigment epithelium cells is dependent on PIP 2 -PLC.

    PubMed

    Mateos, Melina V; Kamerbeek, Constanza B; Giusto, Norma M; Salvador, Gabriela A

    2016-06-01

    This article presents additional data regarding the study "The phospholipase D pathway mediates the inflammatory response of the retinal pigment epithelium" [1]. The new data presented here show that short exposure of RPE cells to lipopolysaccharide (LPS) induces an early and transient activation of the extracellular signal-regulated kinase (ERK1/2). This early ERK1/2 activation is dependent on phosphatidylinositol bisphosphate-phospholipase C (PIP2-PLC). On the contrary, neither the phospholipase D 1 (PLD1) nor the PLD2 inhibition is able to modulate the early ERK1/2 activation induced by LPS in RPE cells.

  11. Activated Müller Cells Involved in ATP-Induced Upregulation of P2X7 Receptor Expression and Retinal Ganglion Cell Death

    PubMed Central

    Xue, Ying; Xie, Yuting; Xue, Bo; Guan, Huaijin

    2016-01-01

    P2X7 receptor (P2X7R), an ATP-gated ion channel, plays an important role in glaucomatous retinal ganglion cell (RGC) apoptotic death, in which activated retinal Müller glial cells may be involved by releasing ATP. In the present study, we investigated whether and how activated Müller cells may induce changes in P2X7R expression in RGCs by using immunohistochemistry and Western blot techniques. Intravitreal injection of DHPG, a group I metabotropic glutamate receptor (mGluR I) agonist, induced upregulation of GFAP expression, suggestive of Müller cell activation (gliosis), as we previously reported. Accompanying Müller cell activation, P2X7R protein expression was upregulated, especially in the cells of ganglion cell layer (GCL), which was reversed by coinjection of brilliant blue G (BBG), a P2X7R blocker. In addition, intravitreal injection of ATP also induced upregulation of P2X7R protein expression. Similar results were observed in cultured retinal neurons by ATP treatment. Moreover, both DHPG and ATP intravitreal injection induced a reduction in the number of fluorogold retrogradely labeled RGCs, and the DHPG effect was partially rescued by coinjection of BBG. All these results suggest that activated Müller cells may release ATP and, in turn, induce upregulation of P2X7R expression in the cells of GCL, thus contributing to RGC death. PMID:27738636

  12. REST reduction is essential for hypoxia-induced neuroendocrine differentiation of prostate cancer cells by activating autophagy signaling

    PubMed Central

    Lin, Tzu-Ping; Chang, Yi-Ting; Lee, Sung-Yuan; Campbell, Mel; Wang, Tien-Chiao; Shen, Shu-Huei; Chung, Hsiao-Jen; Chang, Yen-Hwa; Chiu, Allen W.; Pan, Chin-Chen; Lin, Chi-Hung; Chu, Cheng-Ying; Kung, Hsing-Jien; Cheng, Chia-Yang; Chang, Pei-Ching

    2016-01-01

    Prostate cancer (PCa) with neuroendocrine differentiation (NED) is tightly associated with hormone refractory PCa (HRPC), an aggressive form of cancer that is nearly impossible to treat. Determining the mechanism of the development of NED may yield novel therapeutic strategies for HRPC. Here, we first demonstrate that repressor element-1 silencing transcription factor (REST), a transcriptional repressor of neuronal genes that has been implicated in androgen-deprivation and IL-6 induced NED, is essential for hypoxia-induced NED of PCa cells. Bioinformatics analysis of transcriptome profiles of REST knockdown during hypoxia treatment demonstrated that REST is a master regulator of hypoxia-induced genes. Gene set enrichment analysis (GSEA) of hypoxia and REST knockdown co-upregulated genes revealed their correlation with HRPC. Consistently, gene ontology (GO) analysis showed that REST reduction potential associated with hypoxia-induced tumorigenesis, NE development, and AMPK pathway activation. Emerging reports have revealed that AMPK activation is a potential mechanism for hypoxia-induced autophagy. In line with this, we demonstrate that REST knockdown alone is capable of activating AMPK and autophagy activation is essential for hypoxia-induced NED of PCa cells. Here, making using of in vitro cell-based assay for NED, we reveal a new role for the transcriptional repressor REST in hypoxia-induced NED and characterized a sequential molecular mechanism downstream of REST resulting in AMPK phosphorylation and autophagy activation, which may be a common signaling pathway leading to NED of PCa. PMID:27034167

  13. Terpenoid tetrahydroisoquinoline alkaloids emetine, klugine, and isocephaeline inhibit the activation of hypoxia-inducible factor-1 in breast tumor cells.

    PubMed

    Zhou, Yu-Dong; Kim, Yong-Pil; Mohammed, Kaleem Asjad; Jones, Deborah K; Muhammad, Ilias; Dunbar, D Chuck; Nagle, Dale G

    2005-06-01

    Klugine (1), isocephaeline (2), and emetine (4) inhibited hypoxia-inducible factor-1 (HIF-1) activation by hypoxia in T47D breast tumor cells (IC(50) values 0.2, 1.1, and 0.11 muM, respectively). Compounds 1, 2, and 4 inhibited both hypoxia- and iron chelator-induced HIF-1 activation by blocking HIF-1alpha protein accumulation. PMID:15974627

  14. Excessive L-cysteine induces vacuole-like cell death by activating endoplasmic reticulum stress and mitogen-activated protein kinase signaling in intestinal porcine epithelial cells.

    PubMed

    Ji, Yun; Wu, Zhenlong; Dai, Zhaolai; Sun, Kaiji; Zhang, Qing; Wu, Guoyao

    2016-01-01

    High intake of dietary cysteine is extremely toxic to animals and the underlying mechanism remains largely unknown. This study was conducted to test the hypothesis that excessive L-cysteine induces cell death by activating endoplasmic reticulum (ER) stress and mitogen-activated protein kinase (MAPK) signaling in intestinal porcine epithelial cells. Jejunal enterocytes were cultured in the presence of 0-10 mmol/L L-cysteine. Cell viability, morphologic alterations, mRNA levels for genes involved in ER stress, protein abundances for glucose-regulated protein 78, C/EBP homologous protein (CHOP), alpha subunit of eukaryotic initiation factor-2 (eIF2α), extracellular signal-regulated kinase (ERK1/2), p38 MAPK, and c-Jun N-terminal protein kinase (JNK1/2) were determined. The results showed that L-cysteine (5-10 mmol/L) reduced cell viability (P < 0.05) and led to vacuole-like cell death in intestinal porcine epithelial cells. These adverse effects of L-cysteine were not affected by the autophagy inhibitor 3-methyladenine. The protein abundances for CHOP, phosphorylated (p)-eIF2α, p-JNK1/2, p-p38 MAPK, and the spliced form of XBP-1 mRNA were enhanced (P < 0.05), whereas those for p-ERK1/2 were reduced (P < 0.05). Collectively, excessive L-cysteine induces vacuole-like cell death via the activation of ER stress and MAPK signaling in small intestinal epithelial cells. These signaling pathways may be potential targets for developing effective strategies to prevent the toxicity of dietary cysteine.

  15. Principal cell activity induces spine relocation of adult-born interneurons in the olfactory bulb

    PubMed Central

    Breton-Provencher, Vincent; Bakhshetyan, Karen; Hardy, Delphine; Bammann, Rodrigo Roberto; Cavarretta, Francesco; Snapyan, Marina; Côté, Daniel; Migliore, Michele; Saghatelyan, Armen

    2016-01-01

    Adult-born neurons adjust olfactory bulb (OB) network functioning in response to changing environmental conditions by the formation, retraction and/or stabilization of new synaptic contacts. While some changes in the odour environment are rapid, the synaptogenesis of adult-born neurons occurs over a longer time scale. It remains unknown how the bulbar network functions when rapid and persistent changes in environmental conditions occur but when new synapses have not been formed. Here we reveal a new form of structural remodelling where mature spines of adult-born but not early-born neurons relocate in an activity-dependent manner. Principal cell activity induces directional growth of spine head filopodia (SHF) followed by spine relocation. Principal cell-derived glutamate and BDNF regulate SHF motility and directional spine relocation, respectively; and spines with SHF are selectively preserved following sensory deprivation. Our three-dimensional model suggests that spine relocation allows fast reorganization of OB network with functional consequences for odour information processing. PMID:27578235

  16. Principal cell activity induces spine relocation of adult-born interneurons in the olfactory bulb.

    PubMed

    Breton-Provencher, Vincent; Bakhshetyan, Karen; Hardy, Delphine; Bammann, Rodrigo Roberto; Cavarretta, Francesco; Snapyan, Marina; Côté, Daniel; Migliore, Michele; Saghatelyan, Armen

    2016-01-01

    Adult-born neurons adjust olfactory bulb (OB) network functioning in response to changing environmental conditions by the formation, retraction and/or stabilization of new synaptic contacts. While some changes in the odour environment are rapid, the synaptogenesis of adult-born neurons occurs over a longer time scale. It remains unknown how the bulbar network functions when rapid and persistent changes in environmental conditions occur but when new synapses have not been formed. Here we reveal a new form of structural remodelling where mature spines of adult-born but not early-born neurons relocate in an activity-dependent manner. Principal cell activity induces directional growth of spine head filopodia (SHF) followed by spine relocation. Principal cell-derived glutamate and BDNF regulate SHF motility and directional spine relocation, respectively; and spines with SHF are selectively preserved following sensory deprivation. Our three-dimensional model suggests that spine relocation allows fast reorganization of OB network with functional consequences for odour information processing. PMID:27578235

  17. Lactoferrin protects against chemical-induced rat liver fibrosis by inhibiting stellate cell activation.

    PubMed

    Tung, Yu-Tang; Tang, Ting-Yu; Chen, Hsiao-Ling; Yang, Shang-Hsun; Chong, Kowit-Yu; Cheng, Winston T K; Chen, Chuan-Mu

    2014-01-01

    Liver diseases, which can be caused by alcohol abuse, chemical intoxication, viral hepatitis infection, and autoimmune disorders, are a significant health issue because they can develop into liver fibrosis and cirrhosis. Lactoferrin (LF), a siderophilic protein with 2 iron-binding sites, has been demonstrated to possess a multitude of biological functions, including antiinflammation, anticancer, and antimicrobial effects, as well as immunomodulatory-enhancing functions. In the current study, we induced hepatotoxicity in rats with dimethylnitrosamine (DMN) to establish a situation that would enable us to evaluate the hepatoprotective effects of LF against hepatic injury. Our results showed that DMN-induced hepatic pathological damage significantly decreased the body weight and liver index, increased the mRNA and protein levels of collagen α-1(I) (ColIα-1) and α-smooth muscle actin, and increased the hydroxyproline content. However, treatment with LF significantly increased body weight and liver index, decreased the mRNA and protein levels of ColIα-1 and α-smooth muscle actin, and suppressed the hydroxyproline content when compared with the DMN-treated group. Liver histopathology also showed that low-dose LF (100mg/kg of body weight) or high-dose LF (300 mg/kg of body weight) could significantly reduce the incidences of liver lesions induced by DMN. These results suggest that the LF exhibits potent hepatoprotection against DMN-induced liver damage in rats and that the hepatoprotective effects of LF may be due to the inhibition of collagen production and to stellate cell activation. PMID:24731632

  18. In vascular smooth muscle cells paricalcitol prevents phosphate-induced Wnt/β-catenin activation.

    PubMed

    Martínez-Moreno, Julio M; Muñoz-Castañeda, Juan R; Herencia, Carmen; Oca, Addy Montes de; Estepa, Jose C; Canalejo, Rocio; Rodríguez-Ortiz, Maria E; Perez-Martinez, Pablo; Aguilera-Tejero, Escolástico; Canalejo, Antonio; Rodríguez, Mariano; Almadén, Yolanda

    2012-10-15

    The present study investigates the differential effect of two vitamin D receptor agonists, calcitriol and paricalcitol, on human aortic smooth muscle cells calcification in vitro. Human vascular smooth muscle cells were incubated in a high phosphate (HP) medium alone or supplemented with either calcitriol 10(-8)M (HP + CTR) or paricalcitol 3·10(-8) M (HP + PC). HP medium induced calcification, which was associated with the upregulation of mRNA expression of osteogenic factors such as bone morphogenetic protein 2 (BMP2), Runx2/Cbfa1, Msx2, and osteocalcin. In these cells, activation of Wnt/β-catenin signaling was evidenced by the translocation of β-catenin into the nucleus and the increase in the expression of direct target genes as cyclin D1, axin 2, and VCAN/versican. Addition of calcitriol to HP medium (HP + CTR) further increased calcification and also enhanced the expression of osteogenic factors together with a significant elevation of nuclear β-catenin levels and the expression of cyclin D1, axin 2, and VCAN. By contrast, the addition of paricalcitol (HP + PC) not only reduced calcification but also downregulated the expression of BMP2 and other osteoblastic phenotype markers as well as the levels of nuclear β-catenin and the expression of its target genes. The role of Wnt/β-catenin on phosphate- and calcitriol-induced calcification was further demonstrated by the inhibition of calcification after addition of Dickkopf-related protein 1 (DKK-1), a specific natural antagonist of the Wnt/β-catenin signaling pathway. In conclusion, the differential effect of calcitriol and paricalcitol on vascular calcification appears to be mediated by a distinct regulation of the BMP and Wnt/β-catenin signaling pathways.

  19. Neutrophil extracellular traps downregulate lipopolysaccharide-induced activation of monocyte-derived dendritic cells.

    PubMed

    Barrientos, Lorena; Bignon, Alexandre; Gueguen, Claire; de Chaisemartin, Luc; Gorges, Roseline; Sandré, Catherine; Mascarell, Laurent; Balabanian, Karl; Kerdine-Römer, Saadia; Pallardy, Marc; Marin-Esteban, Viviana; Chollet-Martin, Sylvie

    2014-12-01

    Polymorphonuclear neutrophils (PMN) play a central role in inflammation and participate in its control, notably by modulating dendritic cell (DC) functions via soluble mediators or cell-cell contacts. Neutrophil extracellular traps (NETs) released by PMN could play a role in this context. To evaluate NET effects on DC maturation, we developed a model based on monocyte-derived DC (moDC) and calibrated NETs isolated from fresh human PMN. We found that isolated NETs alone had no discernable effect on moDC. In contrast, they downregulated LPS-induced moDC maturation, as shown by decreased surface expression of HLA-DR, CD80, CD83, and CD86, and by downregulated cytokine production (TNF-α, IL-6, IL-12, IL-23), with no increase in the expression of tolerogenic DC genes. Moreover, the presence of NETs during moDC maturation diminished the capacity of these moDC to induce T lymphocyte proliferation in both autologous and allogeneic conditions, and modulated CD4(+) T lymphocyte polarization by promoting the production of Th2 cytokines (IL-5 and IL-13) and reducing that of Th1 and Th17 cytokines (IFN-γ and IL-17). Interestingly, the expression and activities of the lymphoid chemokine receptors CCR7 and CXCR4 on moDC were not altered when moDC matured in the presence of NETs. Together, these findings reveal a new role for NETs in adaptive immune responses, modulating some moDC functions and thereby participating in the control of inflammation.

  20. STS-1 promotes IFN-α induced autophagy by activating the JAK1-STAT1 signaling pathway in B cells.

    PubMed

    Dong, Guanjun; You, Ming; Fan, Hongye; Ding, Liang; Sun, Lingyun; Hou, Yayi

    2015-08-01

    Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by the overexpression of IFN-α. IFN-α induces autophagy via the JAK1-STAT1 signaling pathway, contributing to the pathogenesis of SLE. Recent studies reported that B cells from patients with SLE and NZB/W F1 mice had enhanced autophagy activity; however, the mechanism still remains unknown. Here, we show that the protein tyrosine phosphatase STS-1 (suppressor of T-cell receptor signaling 1) was significantly overexpressed in B cells from patients with SLE and MRL/lpr mice. Notably, STS-1 promoted IFN-α-induced autophagy in B cells by enhancing the JAK1-STAT1 signaling activation. STS-1 inhibited the phosphorylation of the E3 ubiquitin protein ligase c-cbl, and subsequently promoted IFN-α-induced phosphorylation of tyrosine kinase 2, leading to JAK1-STAT1 signaling activation. Furthermore, STAT1 and JAK1 inhibitors blocked the IFN-α-induced autophagy promoted by STS-1, indicating that STS-1 promotes IFN-α-induced autophagy via the JAK1-STAT1 signaling. Our results demonstrate the importance of STS-1 in regulating IFN-α-induced autophagy in B cells, and this could be used as a therapeutic approach to treat SLE.

  1. Ozone induces a proinflammatory response in primary human bronchial epithelial cells through mitogen-activated protein kinase activation without nuclear factor-κB activation.

    PubMed

    McCullough, Shaun D; Duncan, Kelly E; Swanton, Samantha M; Dailey, Lisa A; Diaz-Sanchez, David; Devlin, Robert B

    2014-09-01

    Ground-level ozone (O3) is a ubiquitous environmental air pollutant that is a potent inducer of airway inflammation and has been linked with respiratory and cardiovascular morbidity and mortality. Some studies using transformed or immortalized cells have attributed O3-mediated expression of inflammatory cytokines with activation of the canonical NF-κB pathway. In this study, we sought to characterize the O3-mediated activation of cellular signaling pathways using primary human bronchial epithelial cells obtained from a panel of donors. We demonstrate that the O3-induced expression of proinflammatory cytokines requires the activation of the epidermal growth factor receptor/MEK/ERK and MKK4/p38 mitogen-activated signaling pathways but does not appear to involve activation of canonical NF-κB signaling. In addition to providing a novel mechanistic model for the O3-mediated induction of proinflammatory cytokines, these findings highlight the importance of using primary cells over cell lines in mechanistic studies.

  2. Activation of PI3K signaling prevents aminoglycoside-induced hair cell death in the murine cochlea.

    PubMed

    Jadali, Azadeh; Kwan, Kelvin Y

    2016-01-01

    Loss of sensory hair cells of the inner ear due to aminoglycoside exposure is a major cause of hearing loss. Using an immortalized multipotent otic progenitor (iMOP) cell line, specific signaling pathways that promote otic cell survival were identified. Of the signaling pathways identified, the PI3K pathway emerged as a strong candidate for promoting hair cell survival. In aging animals, components for active PI3K signaling are present but decrease in hair cells. In this study, we determined whether activated PI3K signaling in hair cells promotes survival. To activate PI3K signaling in hair cells, we used a small molecule inhibitor of PTEN or genetically ablated PTEN using a conditional knockout animal. Hair cell survival was challenged by addition of gentamicin to cochlear cultures. Hair cells with activated PI3K signaling were more resistant to aminoglycoside-induced hair cell death. These results indicate that increased PI3K signaling in hair cells promote survival and the PI3K signaling pathway is a target for preventing aminoglycoside-induced hearing loss.

  3. Activation of PI3K signaling prevents aminoglycoside-induced hair cell death in the murine cochlea

    PubMed Central

    Jadali, Azadeh

    2016-01-01

    ABSTRACT Loss of sensory hair cells of the inner ear due to aminoglycoside exposure is a major cause of hearing loss. Using an immortalized multipotent otic progenitor (iMOP) cell line, specific signaling pathways that promote otic cell survival were identified. Of the signaling pathways identified, the PI3K pathway emerged as a strong candidate for promoting hair cell survival. In aging animals, components for active PI3K signaling are present but decrease in hair cells. In this study, we determined whether activated PI3K signaling in hair cells promotes survival. To activate PI3K signaling in hair cells, we used a small molecule inhibitor of PTEN or genetically ablated PTEN using a conditional knockout animal. Hair cell survival was challenged by addition of gentamicin to cochlear cultures. Hair cells with activated PI3K signaling were more resistant to aminoglycoside-induced hair cell death. These results indicate that increased PI3K signaling in hair cells promote survival and the PI3K signaling pathway is a target for preventing aminoglycoside-induced hearing loss. PMID:27142333

  4. HMG-CoA reductase regulates CCL17-induced colon cancer cell migration via geranylgeranylation and RhoA activation

    SciTech Connect

    Al-Haidari, Amr A.; Syk, Ingvar; Thorlacius, Henrik

    2014-03-28

    Highlights: • Simvastatin blocked CCL17-induced and CCR4-dependent RhoA activation in HT29 cells. • CCL17/CCR4-mediated migration of colon cancer cells was antagonised by simvastatin. • Cell migration recovered by adding Mevalonate and geranylgeranyl pyrophosphate. • Targeting HMG-CoA reductase might be useful to inhibit colon cancer metastasis. - Abstract: Background: Simvastatin is widely used to lower cholesterol levels in patients with cardiovascular diseases, although accumulating evidence suggests that statins, such as simvastatin, also exert numerous anti-tumoral effects. Aim: The aim of this study was to examine the effect of simvastatin on colon cancer cell migration. Methods: Migration assays were performed to evaluate CCL17-induced colon cancer cell (HT-29) chemotaxis. In vitro tumor growth and apoptosis were assessed using a proliferation assay and annexin V assay, respectively. Active RhoA protein levels in CCL17-stimulated colon cancer cells were quantified using a G-LISA assay. Results: We found that simvastatin dose-dependently decreased CCL17-induced colon cancer cell migration. Simvastatin had no effect on colon cancer cell proliferation or apoptosis. Inhibition of beta chemokine receptor 4, CCR4, reduced CCL17-evoked activation of RhoA in colon cancer cells. Moreover, administration of mevalonate reversed the inhibitory effect of simvastatin on CCL17-induced colon cancer cell migration. Interestingly, co-incubation with geranylgeranyl pyrophosphate (GGPP) antagonized the inhibitory impact of simvastatin on colon cancer cell migration triggered by CCL17. Moreover, we observed that simvastatin decreased CCL17-induced activation of RhoA in colon cancer cells. Administration of mevalonate and GGPP reversed the inhibitory effect of simvastatin on CCL17-provoked RhoA activation in colon cancer cells. Conclusions: Taken together, our findings show for the first time that HMG-CoA reductase regulates CCL17-induced colon cancer cell migration via

  5. Notch Signaling Activation in Cervical Cancer Cells Induces Cell Growth Arrest with the Involvement of the Nuclear Receptor NR4A2

    PubMed Central

    Sun, Lichun; Liu, Mingqiu; Sun, Guang-Chun; Yang, Xu; Qian, Qingqing; Feng, Shuyu; Mackey, L. Vienna; Coy, David H.

    2016-01-01

    Cervical cancer is a second leading cancer death in women world-wide, with most cases in less developed countries. Notch signaling is highly conserved with its involvement in many cancers. In the present study, we established stable cervical cell lines with Notch activation and inactivation and found that Notch activation played a suppressive role in cervical cancer cells. Meanwhile, the transient overexpression of the active intracellular domain of all four Notch receptors (ICN1, 2, 3, and 4) also induced the suppression of cervical cancer Hela cell growth. ICN1 also induced cell cycle arrest at phase G1. Notch1 signaling activation affected the expression of serial genes, especially the genes associated with cAMP signaling, with an increase of genes like THBS1, VCL, p63, c-Myc and SCG2, a decrease of genes like NR4A2, PCK2 and BCL-2. Particularly, The nuclear receptor NR4A2 was observed to induce cell proliferation via MTT assay and reduce cell apoptosis via FACS assay. Furthermore, NR4A2's activation could reverse ICN1-induced suppression of cell growth while erasing ICN1-induced increase of tumor suppressor p63. These findings support that Notch signaling mediates cervical cancer cell growth suppression with the involvement of nuclear receptor NR4A2. Notably, Notch/NR4A2/p63 signaling cascade possibly is a new signling pathway undisclosed. PMID:27471554

  6. Notch Signaling Activation in Cervical Cancer Cells Induces Cell Growth Arrest with the Involvement of the Nuclear Receptor NR4A2.

    PubMed

    Sun, Lichun; Liu, Mingqiu; Sun, Guang-Chun; Yang, Xu; Qian, Qingqing; Feng, Shuyu; Mackey, L Vienna; Coy, David H

    2016-01-01

    Cervical cancer is a second leading cancer death in women world-wide, with most cases in less developed countries. Notch signaling is highly conserved with its involvement in many cancers. In the present study, we established stable cervical cell lines with Notch activation and inactivation and found that Notch activation played a suppressive role in cervical cancer cells. Meanwhile, the transient overexpression of the active intracellular domain of all four Notch receptors (ICN1, 2, 3, and 4) also induced the suppression of cervical cancer Hela cell growth. ICN1 also induced cell cycle arrest at phase G1. Notch1 signaling activation affected the expression of serial genes, especially the genes associated with cAMP signaling, with an increase of genes like THBS1, VCL, p63, c-Myc and SCG2, a decrease of genes like NR4A2, PCK2 and BCL-2. Particularly, The nuclear receptor NR4A2 was observed to induce cell proliferation via MTT assay and reduce cell apoptosis via FACS assay. Furthermore, NR4A2's activation could reverse ICN1-induced suppression of cell growth while erasing ICN1-induced increase of tumor suppressor p63. These findings support that Notch signaling mediates cervical cancer cell growth suppression with the involvement of nuclear receptor NR4A2. Notably, Notch/NR4A2/p63 signaling cascade possibly is a new signling pathway undisclosed. PMID:27471554

  7. The role of p38 MAPK activation in auranofin-induced apoptosis of human promyelocytic leukaemia HL-60 cells

    PubMed Central

    Park, Seon-Joo; Kim, In-Sook

    2005-01-01

    In a previous study, we reported an antileukaemic activity of auranofin (AF), demonstrating its dual effects: on the induction of apoptotic cell death and its synergistic action with retinoic acid on cell differentiation. In this study, we investigated the downstream signalling events of AF-induced apoptosis to determine the molecular mechanisms of AF activity. Treatment of HL-60 cells with AF induced apoptosis in a concentration- and time-dependent manner. Western blot analysis showed that AF-induced apoptosis was accompanied by the activation of caspase-8, caspase-9, and caspase-3, and the release of cytochrome c from the mitochondria. The phosphorylation and kinase activities of p38 mitogen-activated protein kinase (p38 MAPK) increased gradually until 12 h after AF (2 μM) treatment, and p38 MAPK was also activated concentration-dependently. Pretreatment with SB203580, a specific inhibitor of p38 MAPK, significantly blocked DNA fragmentation and the cleavage of procaspase-8, procaspase-3, and poly-ADP-ribose polymerase (PARP), whereas SB203580 alone had no effect. Reactive oxygen species (ROS) were also detected within 1 h after AF treatment, and the antioxidant N-acetyl-L-cysteine (NAC) effectively protected the cells from apoptosis by inhibiting the phosphorylation of p38 MAPK and the activation of caspases. These results suggest that ROS generation and the subsequent activation of p38 MAPK are essential for the proapoptotic effects of AF in human promyelocytic leukaemia HL-60 cells. PMID:16086031

  8. Methylprednisolone pulse therapy induced fall in natural killer cell activity in rheumatoid arthritis.

    PubMed

    Pedersen, B K; Beyer, J M; Rasmussen, A; Klarlund, K; Pedersen, B N; Helin, P

    1984-10-01

    Natural killer (NK) cell activity was studied in 8 patients with classic or definite rheumatoid arthritis (RA) by investigating the killing of K 562 cells by peripheral blood lymphocytes before, during, and after intravenous methylprednisolone pulse therapy (MPPT). MPPT produced a considerable fall in NK activity and after 3 months NK activity was less than half that before MPPT. PMID:6516854

  9. Acetaldehyde Induces Cytotoxicity of SH-SY5Y Cells via Inhibition of Akt Activation and Induction of Oxidative Stress

    PubMed Central

    Yan, Tingting; Zhao, Yan; Zhang, Xia

    2016-01-01

    Excessive alcohol consumption can lead to brain tissue damage and cognitive dysfunction. It has been shown that heavy drinking is associated with an earlier onset of neurodegenerative diseases such as Alzheimer's disease. Acetaldehyde, the most toxic metabolite of ethanol, is speculated to mediate the brain tissue damage and cognitive dysfunction induced by the chronic excessive consumption of alcohol. However, the exact mechanisms by which acetaldehyde induces neurotoxicity are not totally understood. In this study, we investigated the cytotoxic effects of acetaldehyde in SH-SY5Y cells and found that acetaldehyde induced apoptosis of SH-SY5Y cells by downregulating the expression of antiapoptotic Bcl-2 and Bcl-xL and upregulating the expression of proapoptotic Bax. Acetaldehyde treatment led to a significant decrease in the levels of activated Akt and cyclic AMP-responsive element binding protein (CREB). In addition, acetaldehyde induced the activation of p38 mitogen-activated protein kinase (MAPK) while inhibiting the activation of extracellular signal-regulated kinases (ERKs, p44/p42MAPK). Meanwhile, acetaldehyde treatment caused an increase in the production of reactive oxygen species and elevated the oxidative stress in SH-SY5Y cells. Therefore, acetaldehyde induces cytotoxicity of SH-SY5Y cells via promotion of apoptotic signaling, inhibition of cell survival pathway, and induction of oxidative stress. PMID:26649137

  10. Acetaldehyde Induces Cytotoxicity of SH-SY5Y Cells via Inhibition of Akt Activation and Induction of Oxidative Stress.

    PubMed

    Yan, Tingting; Zhao, Yan; Zhang, Xia

    2016-01-01

    Excessive alcohol consumption can lead to brain tissue damage and cognitive dysfunction. It has been shown that heavy drinking is associated with an earlier onset of neurodegenerative diseases such as Alzheimer's disease. Acetaldehyde, the most toxic metabolite of ethanol, is speculated to mediate the brain tissue damage and cognitive dysfunction induced by the chronic excessive consumption of alcohol. However, the exact mechanisms by which acetaldehyde induces neurotoxicity are not totally understood. In this study, we investigated the cytotoxic effects of acetaldehyde in SH-SY5Y cells and found that acetaldehyde induced apoptosis of SH-SY5Y cells by downregulating the expression of antiapoptotic Bcl-2 and Bcl-xL and upregulating the expression of proapoptotic Bax. Acetaldehyde treatment led to a significant decrease in the levels of activated Akt and cyclic AMP-responsive element binding protein (CREB). In addition, acetaldehyde induced the activation of p38 mitogen-activated protein kinase (MAPK) while inhibiting the activation of extracellular signal-regulated kinases (ERKs, p44/p42MAPK). Meanwhile, acetaldehyde treatment caused an increase in the production of reactive oxygen species and elevated the oxidative stress in SH-SY5Y cells. Therefore, acetaldehyde induces cytotoxicity of SH-SY5Y cells via promotion of apoptotic signaling, inhibition of cell survival pathway, and induction of oxidative stress.

  11. Triptolide induces protective autophagy through activation of the CaMKKβ-AMPK signaling pathway in prostate cancer cells

    PubMed Central

    Zhang, Zhe; Mao, Lin; Han, Yangyang; Yan, Jun; Lei, Ming

    2016-01-01

    Triptolide, an active compound extracted from the Chinese herb thunder god vine (Tripterygium wilfordii Hook F.), has potent anti-tumor activity. Recently, triptolide was found to induce autophagy in cancer cells. However, the effects of triptolide on autophagy in human prostate cancer (PCa) cells have not yet been clearly elucidated. In this study, we demonstrated that triptolide induces autophagy in three PCa cell lines, PC-3, LNCaP and C4–2. Furthermore, we found that triptolide mediates intracellular accumulation of free calcium by stimulating the endoplasmic reticulum (ER) stress response. This activates the CaMKKβ-AMPK signaling pathway, which in turn inhibits mTOR and activates both ULK1 and Beclin 1, finally resulting in autophagy. Moreover, we found that treatment with autophagy inhibitors 3-methyladenine (3-MA) and chloroquine (CQ) enhances triptolide-induced PCa cell death and growth inhibition. Using a PC-3-xenografted mouse model, we showed that blocking autophagy with CQ significantly promoted triptolide-induced tumor growth inhibition in vivo. Overall, our results show that triptolide induces protective autophagy through the CaMKKβ-AMPK pathway in PCa cells, implying that a combination of triptolide with autophagy inhibitors may potentially be an effective therapeutic strategy for PCa. PMID:26734992

  12. Safrole oxide induces apoptosis by activating caspase-3, -8, and -9 in A549 human lung cancer cells.

    PubMed

    Du, Aiying; Zhao, Baoxiang; Yin, Deling; Zhang, Shangli; Miao, Junying

    2006-01-01

    Previously we found that 3,4-(methylenedioxy)-1-(2',3'-epoxypropyl)-benzene (safrole oxide) induced a typical apoptosis in A549 human lung cancer cells. In this study, we further investigated which caspases were activated by safrole oxide during the apoptosis. The data showed that the activity of caspase-3, -8, and -9 was significantly enhanced by the compound, which suggested that safrole oxide might be used as a caspase promoter to initiate lung cancer cell apoptosis.

  13. The AQP-3 water channel is a pivotal modulator of glycerol-induced chloride channel activation in nasopharyngeal carcinoma cells.

    PubMed

    Zhang, Haifeng; Deng, Zhiqin; Yang, Lili; Luo, Hai; Liu, Shanwen; Li, Yuan; Wei, Yan; Peng, Shuang; Zhu, Linyan; Wang, Liwei; Chen, Lixin

    2016-03-01

    Aquaporin (AQP) and chloride channels are ubiquitous in virtually all living cells, playing pivotal roles in cell proliferation, migration and apoptosis. We previously reported that AQP-3 aquaglyceroporin and ClC-3 chloride channels could form complexes to regulate cell volume in nasopharyngeal carcinoma cells. In this study, the roles of AQP-3 in their hetero-complexes were further investigated. Glycerol entered the cells via AQP-3 and induced two different Cl(-) currents through cell swelling-dependent or -independent pathways. The swelling-dependent Cl(-) current was significantly inhibited by pretreatment with CuCl2 and AQP-3-siRNA. After siRNA-induced AQP-3 knock-down, the 140 mM glycerol isoosmotic solution swelled cells by 22% (45% in AQP-3-intact cells) and induced a smaller Cl(-) current; this current was smaller than that activated by 8% cell volume swelling, which induced by the 140 mM glycerol hyperosmotic solution in AQP-3-intact cells. This suggests that the interaction between AQP-3 and ClC-3 plays an important role in cell volume regulation and that AQP-3 may be a modulator that opens volume-regulated chloride channels. The swelling-independent Cl(-) current, which was activated by extracellular glycerol, was reduced by CuCl2 and AQP-3-siRNA pretreatment. Dialyzing glycerol into cells via the pipette directly induced the swelling-independent Cl(-) current; however this current was blocked by AQP-3 down-regulation, suggesting AQP-3 is essential for the opening of chloride channels. In conclusion, AQP-3 is the pathway for water, glycerol and other small solutes to enter cells, and it may be an essential modulator for the gating of chloride channels. PMID:26794461

  14. Amplification activation loop between caspase-8 and -9 dominates artemisinin-induced apoptosis of ASTC-a-1 cells.

    PubMed

    Xiao, Fenglian; Gao, Weijie; Wang, Xiaoping; Chen, Tongsheng

    2012-06-01

    Although caspases have been demonstrated to be involved in artemisinin (ARTE)-induced apoptosis, their exact functions are not well understood. The aim of this report is to explore the roles of caspase-8, -9 and -3 during ARTE-induced apoptosis in human lung adenocarcinoma (ASTC-a-1) cells. ARTE treatment induces a rapid generation of reactive oxygen species (ROS), and ROS-dependent apoptosis as well as the activation of caspase-8, -9 and -3 via time- and dose-dependent fashion. Of upmost importance, inhibition of caspase-8 or -9, but not caspase-3, almost completely blocks the ARTE-induced not only activation of the caspase-8, -9 and -3 but also apoptosis. In addition, the apoptotic process triggered by ARTE does not involve the Bid cleavage, tBid translocation, significant loss of mitochondrial membrane potential and cytochrome c release from mitochondria. Moreover, silencing Bax/Bak does not prevent the ATRE-induced cell death as well as the activation of caspase-8, -9 and -3. Collectively, our data firstly demonstrate that ARTE triggers a ROS-mediated positive feedback amplification activation loop between caspase-8 and -9 independent of mitochondria, which dominantly mediated the ARTE-induced apoptosis via a caspase-3-independent apoptotic pathway in ASTC-a-1 cells. Our findings imply a potential to develop new derivatives from artemisinin to effectively initiate the amplification activation loop of caspases. PMID:22434375

  15. Amplification activation loop between caspase-8 and -9 dominates artemisinin-induced apoptosis of ASTC-a-1 cells.

    PubMed

    Xiao, Fenglian; Gao, Weijie; Wang, Xiaoping; Chen, Tongsheng

    2012-06-01

    Although caspases have been demonstrated to be involved in artemisinin (ARTE)-induced apoptosis, their exact functions are not well understood. The aim of this report is to explore the roles of caspase-8, -9 and -3 during ARTE-induced apoptosis in human lung adenocarcinoma (ASTC-a-1) cells. ARTE treatment induces a rapid generation of reactive oxygen species (ROS), and ROS-dependent apoptosis as well as the activation of caspase-8, -9 and -3 via time- and dose-dependent fashion. Of upmost importance, inhibition of caspase-8 or -9, but not caspase-3, almost completely blocks the ARTE-induced not only activation of the caspase-8, -9 and -3 but also apoptosis. In addition, the apoptotic process triggered by ARTE does not involve the Bid cleavage, tBid translocation, significant loss of mitochondrial membrane potential and cytochrome c release from mitochondria. Moreover, silencing Bax/Bak does not prevent the ATRE-induced cell death as well as the activation of caspase-8, -9 and -3. Collectively, our data firstly demonstrate that ARTE triggers a ROS-mediated positive feedback amplification activation loop between caspase-8 and -9 independent of mitochondria, which dominantly mediated the ARTE-induced apoptosis via a caspase-3-independent apoptotic pathway in ASTC-a-1 cells. Our findings imply a potential to develop new derivatives from artemisinin to effectively initiate the amplification activation loop of caspases.

  16. Imaging of activated caspase-3 in living cell by fluorescence resonance energy transfer during photosensitization-induced apoptosis

    NASA Astrophysics Data System (ADS)

    Wu, Yunxia; Xing, Da; Chen, Qun; Tang, Yonghong

    2005-01-01

    Photodynamic therapy (PDT) is a novel and promising cancer treatment that employs a combination of a photosensitizing chemical and visible light, induces apoptosis in cell, and activation of caspase-3 is considered to be the final step in many apoptosis pathways. The changes of caspase-3 activation in cell during TNFα- and photodynamic therapy-induced apoptosis was measured by fluorescence resonance energy transfer (FRET) analysis. FRET probe consisting of fusions of an enhanced cyan fluorescent protein (ECFP), Venus and a linker peptide containing the caspase-3 cleavage sequence DEVD was utilized. Therefore, activated caspase-3 cleaved the linker peptide of FRET probe and disrupted the FRET signal. Human lung adenocarcinoma cell line (ASTC-a-1) were stably transfected with the plasmid (ECFP-DEVD-Venus) and then were treated by TNF-α and PDT, respectively. Experimental results indicated that caspase-3 activation resulted in cleavage of linker peptide and subsequent disruption of the FRET signal during TNFα- and photodynamic therapy-induced apoptosis, and that the activation of caspase-3 induced by photodynamic therapy was faster than that induce by TNF-α. The study supports that using FRET technique and different recombinant substrates as FRET probes could be used to detect the process of PDT-induced apoptosis and provide a new means to investigate apoptotic mechanism of PDT.

  17. Activation of Rac1-dependent redox signaling is critically involved in staurosporine-induced neurite outgrowth in PC12 cells.

    PubMed

    Kim, Du Sik; An, Jeong Mi; Lee, Han Gil; Seo, Su Ryeon; Kim, Seon Sook; Kim, Ju Yeon; Kang, Jeong Wan; Bae, Yun Soo; Seo, Jeong Taeg

    2013-02-01

    Staurosporine, a non-specific protein kinase inhibitor, has been shown to induce neurite outgrowth in PC12 cells, but the mechanism by which staurosporine induces neurite outgrowth is still obscure. In the present study, we investigated whether the activation of Rac1 was responsible for the neurite outgrowth triggered by staurosporine. Staurosporine caused rapid neurite outgrowth independent of the ERK signaling pathways. In contrast, neurite outgrowth in response to staurosporine was accompanied by activation of Rac1, and the Rac1 inhibitor NSC23766 attenuated the staurosporine-induced neurite outgrowth in a concentration-dependent manner. In addition, suppression of Rac1 activity by expression of the dominant negative mutant Rac1N17 also blocked the staurosporine-induced morphological differentiation of PC12 cells. Staurosporine caused an activation of NADPH oxidase and increased the production of reactive oxygen species (ROS), which was prevented by NSC23766 and diphenyleneiodonium (DPI), an NADPH oxidase inhibitor. Staurosporine-induced neurite outgrowth was attenuated by pretreatment with DPI and exogenous addition of sublethal concentration of H2O2 accelerated neurite outgrowth triggered by staurosporine. These results indicate that activation of Rac1, which leads to ROS generation, is required for neurite outgrowth induced by staurosporine in PC12 cells.

  18. Mercury specifically induces LINE-1 activity in a human neuroblastoma cell line.

    PubMed

    Habibi, Laleh; Shokrgozar, Mohammad Ali; Tabrizi, Mina; Modarressi, Mohammad Hossein; Akrami, Seyed Mohammad

    2014-01-01

    L1 retro-elements comprise 17% of the human genome. Approximately 100 copies of these autonomous mobile elements are active in our DNA and can cause mutations, gene disruptions, and genomic instability. Therefore, human cells control the activities of L1 elements, in order to prevent their deleterious effects through different mechanisms. However, some toxic agents increase the retrotransposition activity of L1 elements in somatic cells. In order to identify specific effects of neurotoxic metals on L1 activity in neuronal cells, we studied the effects of mercury and cobalt on L1-retroelement activity by measuring levels of cellular transcription, protein expression, and genomic retrotransposition in a neuroblastoma cell line compared with the effects in three non-neuronal cell lines. Our results show that mercury increased the expression of L1 RNA, the activity of the L1 5'UTR, and L1 retrotransposition exclusively in the neuroblastoma cell line but not in non-neuronal cell lines. However, cobalt increased the expression of L1 RNA in neuroblastoma cells, HeLa cells, and wild-type human fibroblasts, and also increased the activity of the L1 5'UTR as well as the SV40 promoter in HeLa cells but not in neuroblastoma cells. Exposure to cobalt did not result in increased retrotransposition activity in HeLa cells or neuroblastoma cells. We conclude that non-toxic levels of the neurotoxic agent mercury could influence DNA by increasing L1 activities, specifically in neuronal cells, and may make these cells susceptible to neurodegeneration over time.

  19. Salicylic acid induced cysteine protease activity during programmed cell death in tomato plants.

    PubMed

    Kovács, Judit; Poór, Péter; Szepesi, Ágnes; Tari, Irma

    2016-06-01

    The hypersensitive response (HR), a type of programmed cell death (PCD) during biotic stress is mediated by salicylic acid (SA). The aim of this work was to reveal the role of proteolysis and cysteine proteases in the execution of PCD in response of SA. Tomato plants were treated with sublethal (0.1 mM) and lethal (1 mM) SA concentrations through the root system. Treatment with 1 mM SA increased the electrolyte leakage and proteolytic activity and reduced the total protein content of roots after 6 h, while the proteolytic activity did not change in the leaves and in plants exposed to 0.1 mM SA. The expression of the papain-type cysteine protease SlCYP1, the vacuolar processing enzyme SlVPE1 and the tomato metacaspase SlMCA1 was induced within the first three hours in the leaves and after 0.5 h in the roots in the presence of 1 mM SA but the transcript levels did not increase significantly at sublethal SA. The Bax inhibitor-1 (SlBI-1), an antiapoptotic gene was over-expressed in the roots after SA treatments and it proved to be transient in the presence of sublethal SA. Protease inhibitors, SlPI2 and SlLTC were upregulated in the roots by sublethal SA but their expression remained low at 1 mM SA concentration. It is concluded that in contrast to leaves the SA-induced PCD is associated with increased proteolytic activity in the root tissues resulting from a fast up-regulation of specific cysteine proteases and down-regulation of protease inhibitors. PMID:27165526

  20. Salicylic acid induced cysteine protease activity during programmed cell death in tomato plants.

    PubMed

    Kovács, Judit; Poór, Péter; Szepesi, Ágnes; Tari, Irma

    2016-06-01

    The hypersensitive response (HR), a type of programmed cell death (PCD) during biotic stress is mediated by salicylic acid (SA). The aim of this work was to reveal the role of proteolysis and cysteine proteases in the execution of PCD in response of SA. Tomato plants were treated with sublethal (0.1 mM) and lethal (1 mM) SA concentrations through the root system. Treatment with 1 mM SA increased the electrolyte leakage and proteolytic activity and reduced the total protein content of roots after 6 h, while the proteolytic activity did not change in the leaves and in plants exposed to 0.1 mM SA. The expression of the papain-type cysteine protease SlCYP1, the vacuolar processing enzyme SlVPE1 and the tomato metacaspase SlMCA1 was induced within the first three hours in the leaves and after 0.5 h in the roots in the presence of 1 mM SA but the transcript levels did not increase significantly at sublethal SA. The Bax inhibitor-1 (SlBI-1), an antiapoptotic gene was over-expressed in the roots after SA treatments and it proved to be transient in the presence of sublethal SA. Protease inhibitors, SlPI2 and SlLTC were upregulated in the roots by sublethal SA but their expression remained low at 1 mM SA concentration. It is concluded that in contrast to leaves the SA-induced PCD is associated with increased proteolytic activity in the root tissues resulting from a fast up-regulation of specific cysteine proteases and down-regulation of protease inhibitors.

  1. An activated Notch1 signaling pathway inhibits cell proliferation and induces apoptosis in human esophageal squamous cell carcinoma cell line EC9706.

    PubMed

    Lu, Zhaoming; Liu, Hongtao; Xue, Lexun; Xu, Peirong; Gong, Tianxiao; Hou, Guiqin

    2008-03-01

    Previous studies have demonstrated that Notch1 signaling pathway plays a major role in maintaining the balance of cell proliferation, differentiation and apoptosis, and is closely associated with tumorigenesis. However, roles of Notch1 signaling pathway in esophageal squamous cell carcinoma (ESCC), which is a common cause of mortality in China, remain poorly understood. Therefore, a novel strategy for seeking a rational molecular therapeutic target for ESCC is urgently needed. The purpose of this study is to examine the effect of the active Notch1 signaling pathway on the proliferation and apoptosis of ESCC cells and to investigate the underlying molecular mechanisms in carcinogenesis of the esophagus. The results revealed that a constitutively activated Notch1 signaling pathway was observed in ESCC cell line EC9706, through a pcNICD vector mediated expression system. Clearly, the activated Notch1 signaling pathway gave rise to proliferation suppression of the cells, accompanied with a cell cycle inhibition at the G0/G1 phase and apoptosis. In contrast to the expression of CDK2, cyclin D1 and cyclin E observed in EC9706 cells untreated and transfected with pcDNA3.1, there was a markedly decrease in the cells stably expressing Notch1 NICD. Up- and down-regulations of GSK3 beta and beta-catenin, respectively, indicated that Notch1 inhibited proliferation and induced apoptosis of EC9706 cells through Wnt-mediated signaling pathway. These findings suggest that Notch1 signaling pathway may participate in carcinogenesis of the esophagus.

  2. Lipoxin A4 protects against lipopolysaccharide-induced sepsis by promoting innate response activator B cells generation.

    PubMed

    Cheng, Qiong; Wang, Zheng; Ma, Ruihua; Chen, Yongtao; Yan, Yan; Miao, Shuo; Jiao, Jingyu; Cheng, Xue; Kong, Lingfei; Ye, Duyun

    2016-10-01

    Sepsis is a serious disease that leads to severe inflammation, dysregulation of immune system, multi-organ failure and death. Innate response activator (IRA) B cells, which produce granulocyte-macrophage colony-stimulating factor (GM-CSF), protect against microbial sepsis. Lipid mediator lipoxin A4 (LXA4) exerts anti-inflammatory and immunoregulatory effects, and it has been reported that LXA4 receptor ALX/FPR2 is expressed on B cells. Here, we investigated the potential role of LXA4 on IRA B cells in lipopolysaccharide (LPS)-induced sepsis. We found that LXA4 significantly promoted the expansion of splenic IRA B cells and increased GM-CSF expression in splenic B cells with LPS stimulation. After splenectomy, LXA4 treatment did not change the serum or peritoneal IL-1β, IL-6 and TNF-α levels in LPS-induced sepsis. LXA4 accelerated the migration of peritoneal B cells to spleen for their differentiation into IRA B cells, whereas this effect was independent of peritoneal macrophage. Furthermore, LXA4 enhanced the phosphorylation level of signal transducer and activator of transcription 5 (STAT5) in splenic B cells. These results suggest that LXA4 protects against LPS-induced sepsis by promoting the generation and migration of splenic IRA B cells, and the underlying molecular mechanism may be related to STAT5 activation. It might provide new insights and therapeutic approaches for treating sepsis.

  3. Lipoxin A4 protects against lipopolysaccharide-induced sepsis by promoting innate response activator B cells generation.

    PubMed

    Cheng, Qiong; Wang, Zheng; Ma, Ruihua; Chen, Yongtao; Yan, Yan; Miao, Shuo; Jiao, Jingyu; Cheng, Xue; Kong, Lingfei; Ye, Duyun

    2016-10-01

    Sepsis is a serious disease that leads to severe inflammation, dysregulation of immune system, multi-organ failure and death. Innate response activator (IRA) B cells, which produce granulocyte-macrophage colony-stimulating factor (GM-CSF), protect against microbial sepsis. Lipid mediator lipoxin A4 (LXA4) exerts anti-inflammatory and immunoregulatory effects, and it has been reported that LXA4 receptor ALX/FPR2 is expressed on B cells. Here, we investigated the potential role of LXA4 on IRA B cells in lipopolysaccharide (LPS)-induced sepsis. We found that LXA4 significantly promoted the expansion of splenic IRA B cells and increased GM-CSF expression in splenic B cells with LPS stimulation. After splenectomy, LXA4 treatment did not change the serum or peritoneal IL-1β, IL-6 and TNF-α levels in LPS-induced sepsis. LXA4 accelerated the migration of peritoneal B cells to spleen for their differentiation into IRA B cells, whereas this effect was independent of peritoneal macrophage. Furthermore, LXA4 enhanced the phosphorylation level of signal transducer and activator of transcription 5 (STAT5) in splenic B cells. These results suggest that LXA4 protects against LPS-induced sepsis by promoting the generation and migration of splenic IRA B cells, and the underlying molecular mechanism may be related to STAT5 activation. It might provide new insights and therapeutic approaches for treating sepsis. PMID:27494686

  4. 8-Farnesyloxycoumarin induces apoptosis in PC-3 prostate cancer cells by inhibition of 15-lipoxygenase-1 enzymatic activity.

    PubMed

    Hosseinymehr, Minoo; Matin, Maryam M; Sadeghian, Hamid; Bahrami, Ahmad Reza; Kaseb-Mojaver, Nasrin

    2016-10-01

    Prostate cancer is the second most common cancer in men worldwide. Overexpression of 15-lipoxygenase-1 (15-LOX-1) has been reported in prostate cancer patients. This study aimed to investigate the cytotoxic and anticancer effects of 8-farnesyloxycoumarin (8f), a prenylated coumarin, by inhibition of 15-LOX-1 activity, in prostate cancer cells. The activity of 15-LOX-1 and the inhibitory effects of 8f on this enzyme were first assessed in PC-3 and DU145 prostate cancer cells. The MTT assay was used to examine the cytotoxicity effects of 8f on PC-3 cells following 15-LOX-1 inhibition. To determine the type of cell death, chromatin condensation and DNA damage were examined by DAPI staining and comet assay, respectively. Furthermore, the effects of 8f on the cell cycle were evaluated by PI staining and flow cytometry. The activity of 15-LOX-1 was determined to be higher in PC-3 compared with DU145 cells; thus, this cell line was selected for further experiments. 8f induced cell death in PC-3 cells in a dose-dependent and time-dependent manner, with IC50 values similar to cisplatin, which was used as a control. However, 8f did not significantly affect the viability of HFF3, human foreskin fibroblast cells, under identical conditions. The appearance of apoptotic cells after 8f treatment was confirmed by the presence of PC-3 cells containing condensed chromatin as shown by DAPI staining. The comet assay indicated the induction of DNA damage in cancerous cells compared with normal cells. In addition, 8f induced a potent G1 cell-cycle arrest in PC-3 cells. Our results showed that the antitumor effects of 8f on PC-3 cells were promoted by apoptosis induction, probably via inhibition of 15-LOX-1 activity, thus suggesting that 8f may have therapeutic value in prostate cancer treatment. PMID:27362790

  5. A novel copper complex induces paraptosis in colon cancer cells via the activation of ER stress signalling.

    PubMed

    Gandin, Valentina; Pellei, Maura; Tisato, Francesco; Porchia, Marina; Santini, Carlo; Marzano, Cristina

    2012-01-01

    Platinum anticancer drugs have been used for three decades despite their serious side effects and the emerging of resistance phenomena. Recently, a phosphine copper(I) complex, [Cu(thp)(4)][PF(6)] (CP), gained special attention because of its strong antiproliferative effects. CP killed human colon cancer cells more efficiently than cisplatin and oxaliplatin and it overcame platinum drug resistance. CP preferentially reduced cancer cell viability whereas non-tumour cells were poorly affected. Colon cancer cells died via a programmed cell death whose transduction pathways were characterized by the absence of hallmarks of apoptosis. The inhibition of 26S proteasome activities induced by CP caused intracellular accumulation of polyubiquitinated proteins and the functional suppression of the ubiquitin-proteasome pathway thus triggering endoplasmic reticulum stress. These data, providing a mechanistic characterization of CP-induced cancer cell death, shed light on the signaling pathways involved in paraptosis thus offering a new tool to overcome apoptosis-resistance in colon cancer cells.

  6. Infection-induced type I interferons activate CD11b on B-1 cells for subsequent lymph node accumulation

    PubMed Central

    Waffarn, Elizabeth E.; Hastey, Christine J.; Dixit, Neha; Choi, Youn Soo; Cherry, Simon; Kalinke, Ulrich; Simon, Scott I.; Baumgarth, Nicole

    2016-01-01

    Innate-like B-1a lymphocytes rapidly redistribute to regional mediastinal lymph nodes (MedLN) during influenza infection to generate protective IgM. Here we demonstrate that influenza infection-induced type I interferons directly stimulate body cavity B-1 cells and are a necessary signal required for B-1 cell accumulation in MedLN. Vascular mimetic flow chamber studies show that type I interferons increase ligand-mediated B-1 cell adhesion under shear stress by inducing high-affinity conformation shifts of surface-expressed integrins. In vivo trafficking experiments identify CD11b as the non-redundant, interferon-activated integrin required for B-1 cell accumulation in MedLN. Thus CD11b on B-1 cells senses infection-induced innate signals and facilitates their rapid sequester into secondary lymphoid tissues, thereby regulating the accumulation of polyreactive IgM producers at sites of infection. PMID:26612263

  7. 1800MHz Microwave Induces p53 and p53-Mediated Caspase-3 Activation Leading to Cell Apoptosis In Vitro

    PubMed Central

    Xing, Fuqiang; Zhan, Qiuqiang; He, Yiduo; Cui, Jiesheng; He, Sailing; Wang, Guanyu

    2016-01-01

    Recent studies have reported that exposure of mammalian cells to microwave radiation may have adverse effects such as induction of cell apoptosis. However, the molecular mechanisms underlying microwave induced mammalian cell apoptosis are not fully understood. Here, we report a novel mechanism: exposure to 1800MHz microwave radiation induces p53-dependent cell apoptosis through cytochrome c-mediated caspase-3 activation pathway. We first measured intensity of microwave radiation from several electronic devices with an irradiation detector. Mouse NIH/3T3 and human U-87 MG cells were then used as receivers of 1800MHz electromagnetic radiation (EMR) at a power density of 1209 mW/m2. Following EMR exposure, cells were analyzed for viability, intracellular reactive oxygen species (ROS) generation, DNA damage, p53 expression, and caspase-3 activity. Our analysis revealed that EMR exposure significantly decreased viability of NIH/3T3 and U-87 MG cells, and increased caspase-3 activity. ROS burst was observed at 6 h and 48 h in NIH/3T3 cells, while at 3 h in U-87 MG cells. Hoechst 33258 staining and in situ TUNEL assay detected that EMR exposure increased DNA damage, which was significantly restrained in the presence of N-acetyl-L-cysteine (NAC, an antioxidant). Moreover, EMR exposure increased the levels of p53 protein and p53 target gene expression, promoted cytochrome c release from mitochondrion, and increased caspase-3 activity. These events were inhibited by pretreatment with NAC, pifithrin-α (a p53 inhibitor) and caspase inhibitor. Collectively, our findings demonstrate, for the first time, that 1800MHz EMR induces apoptosis-related events such as ROS burst and more oxidative DNA damage, which in turn promote p53-dependent caspase-3 activation through release of cytochrome c from mitochondrion. These findings thus provide new insights into physiological mechanisms underlying microwave-induced cell apoptosis. PMID:27689798

  8. Activation of p53 contributes to pseudolaric acid B-induced senescence in human lung cancer cells in vitro

    PubMed Central

    Yao, Guo-dong; Yang, Jing; Li, Qiang; Zhang, Ye; Qi, Min; Fan, Si-miao; Hayashi, Toshihiko; Tashiro, Shin-ichi; Onodera, Satoshi; Ikejima, Takashi

    2016-01-01

    Aim: Pseudolaric acid B (PAB), a diterpene acid isolated from the root bark of Pseudolarix kaempferi Gordon, has shown to exert anti-tumor effects via inducing cell cycle arrest followed by apoptosis in several cancer cell lines. Here we reported that PAB induced a mitotic catastrophe in human lung cancer A549 cells, which resulted in senescence without apoptosis or necrosis. Methods: Three human lung cancer cell lines (A549, H460 and H1299 cells) were examined. Cell growth inhibition was assessed with MTT assay. Cell cycle distribution was determined using a flow cytometer. Cell nuclear morphology was observed under a fluorescence microscope. Senescent cells were detected using SA-β-Gal staining. Apoptotic and senescent protein expression was examined using Western blot analysis. The expression of p53 and p21 in the cells was downregulated by siRNAs. Results: Treatment with PAB (5–80 μmol/L) inhibited the growth of A549 cells in dose- and time-dependent manners. Prolonged treatment with PAB (20 μmol/L) caused G2/M arrest at day 1 followed by mitotic catastrophe from day 2, which eventually resulted in cell senescence between days 3 and 4 without cell death (apoptosis or necrosis). Knockdown of p53 expression with siRNA significantly suppressed PAB-induced senescence in A549 cells (p53 wild). Furthermore, PAB-induced senescence was also observed in human lung cancer H460 cells (p53 wild), but not in human lung cancer H1299 cells (p53 null). Conclusion: The anti-tumor action of PAB against human lung cancer A549 cells in vitro involves the induction of senescence through activation of the p53 pathway. PMID:27041461

  9. Prostaglandin E2 Prevents Hyperosmolar-Induced Human Mast Cell Activation through Prostanoid Receptors EP2 and EP4

    PubMed Central

    Torres-Atencio, Ivonne; Ainsua-Enrich, Erola; de Mora, Fernando; Picado, César; Martín, Margarita

    2014-01-01

    Background Mast cells play a critical role in allergic and inflammatory diseases, including exercise-induced bronchoconstriction (EIB) in asthma. The mechanism underlying EIB is probably related to increased airway fluid osmolarity that activates mast cells to the release inflammatory mediators. These mediators then act on bronchial smooth muscle to cause bronchoconstriction. In parallel, protective substances such as prostaglandin E2 (PGE2) are probably also released and could explain the refractory period observed in patients with EIB. Objective This study aimed to evaluate the protective effect of PGE2 on osmotically activated mast cells, as a model of exercise-induced bronchoconstriction. Methods We used LAD2, HMC-1, CD34-positive, and human lung mast cell lines. Cells underwent a mannitol challenge, and the effects of PGE2 and prostanoid receptor (EP) antagonists for EP1–4 were assayed on the activated mast cells. Beta-hexosaminidase release, protein phosphorylation, and calcium mobilization were assessed. Results Mannitol both induced mast cell degranulation and activated phosphatidyl inositide 3-kinase and mitogen-activated protein kinase (MAPK) pathways, thereby causing de novo eicosanoid and cytokine synthesis. The addition of PGE2 significantly reduced mannitol-induced degranulation through EP2 and EP4 receptors, as measured by beta-hexosaminidase release, and consequently calcium influx. Extracellular-signal-regulated kinase 1/2, c-Jun N-terminal kinase, and p38 phosphorylation were diminished when compared with mannitol activation alone. Conclusions Our data show a protective role for the PGE2 receptors EP2 and EP4 following osmotic changes, through the reduction of human mast cell activity caused by calcium influx impairment and MAP kinase inhibition. PMID:25329458

  10. Long-term climbing fibre activity induces transcription of microRNAs in cerebellar Purkinje cells.

    PubMed

    Barmack, Neal H; Qian, Zuyuan; Yakhnitsa, Vadim

    2014-09-26

    Synaptic activation of central neurons is often evoked by electrical stimulation leading to post-tetanic potentiation, long-term potentiation or long-term depression. Even a brief electrical tetanus can induce changes in as many as 100 proteins. Since climbing fibre activity is often associated with cerebellar behavioural plasticity, we used horizontal optokinetic stimulation (HOKS) to naturally increase synaptic input to floccular Purkinje cells in mice for hours, not minutes, and investigated how this activity influenced the transcription of microRNAs, small non-coding nucleotides that reduce transcripts of multiple, complementary mRNAs. A single microRNA can reduce the translation of as many as 30 proteins. HOKS evoked increases in 12 microRNA transcripts in floccular Purkinje cells. One of these microRNAs, miR335, increased 18-fold after 24 h of HOKS. After HOKS stopped, miR335 transcripts decayed with a time constant of approximately 2.5 h. HOKS evoked a 28-fold increase in pri-miR335 transcripts compared with an 18-fold increase in mature miR335 transcripts, confirming that climbing fibre-evoked increases in miR335 could be attributed to increases in transcription. We used three screens to identify potential mRNA targets for miR335 transcripts: (i) nucleotide complementarity, (ii) detection of increased mRNAs following microinjection of miR335 inhibitors into the cerebellum, and (iii) detection of decreased mRNAs following HOKS. Two genes, calbindin and 14-3-3-θ, passed these screens. Transfection of N2a cells with miR335 inhibitors or precursors inversely regulated 14-3-3-θ transcripts. Immunoprecipitation of 14-3-3-θ co-immunoprecipitated PKC-γ and GABAAγ2. Knockdown of either 14-3-3-θ or PKC-γ decreased the serine phosphorylation of GABAAγ2, suggesting that 14-3-3-θ and PKC-γ under the control of miR335 homeostatically regulate the phosphorylation and insertion of GABAAγ2 into the Purkinje cell post-synaptic membrane. PMID:25135969

  11. Quercetin induces bladder cancer cells apoptosis by activation of AMPK signaling pathway

    PubMed Central

    Su, Qiongli; Peng, Mei; Zhang, Yuqing; Xu, Wanjun; Darko, Kwame Oteng; Tao, Ting; Huang, Yanjun; Tao, Xiaojun; Yang, Xiaoping

    2016-01-01

    Quercetin, a natural existing polyphenol compound, has shown anticancer capacity for liver, breast, nasopharyngeal and prostate carcinoma but has not been clinically approved yet. This might be due to lack of clear mechanistic picture. Bladder cancer is one of the most common cancers of the urinary tract in the world. In China, bladder cancer has the highest rate of incidence out of all malignancies of the urinary system. The anticancer application of quercetin on bladder cancer has not been investigated either. This study was aimed to examine the mechanisms of quercetin on inhibition of bladder cancer. First, two human and one murine bladder cancer cell lines were tested in vitro for inhibitory sensitivity by MTT and cologenic assays. Second, AMPK pathway including 4E-BP1 and S6K were examined by western blot. Quercetin induces apoptosis and inhibits migration. We are the first to show that quercetin displays potent inhibition on bladder cancer cells via activation of AMPK pathway. PMID:27186419

  12. Ganoderma lucidum Polysaccharides Induce Macrophage-Like Differentiation in Human Leukemia THP-1 Cells via Caspase and p53 Activation.

    PubMed

    Hsu, Jia-Wei; Huang, Hsuan-Cheng; Chen, Shui-Tein; Wong, Chi-Huey; Juan, Hsueh-Fen

    2011-01-01

    Differentiation therapy by induction of tumor cells is an important method in the treatment of hematological cancers such as leukemia. Tumor cell differentiation ends cancer cells' immortality, thus stopping cell growth and proliferation. In our previous study, we found that fucose-containing polysaccharide fraction F3 extracted from Ganoderma lucidum can bring about cytokine secretion and cell death in human leukemia THP-1 cells. This prompted us to further investigate on how F3 induces the differentiation in human leukemia cells. We integrated time-course microarray analysis and network modeling to study the F3-induced effects on THP-1 cells. In addition, we determined the differentiation effect using Liu's staining, nitroblue tetrazolium (NBT) reduction assay, flow cytometer, western blotting and Q-PCR. We also examined the modulation and regulation by F3 during the differentiation process. Dynamic gene expression profiles showed that cell differentiation was induced in F3-treated THP-1 cells. Furthermore, F3-treated THP-1 cells exhibited enhanced macrophage differentiation, as demonstrated by changes in cell adherence, cell cycle arrest, NBT reduction and expression of differentiation markers including CD11b, CD14, CD68, matrix metalloproteinase-9 and myeloperoxidase. In addition, caspase cleavage and p53 activation were found to be significantly enhanced in F3-treated THP-1 cells. We unraveled the role of caspases and p53 in F3-induced THP-1 cells differentiation into macrophages. Our results provide a molecular explanation for the differentiation effect of F3 on human leukemia THP-1 cells and offer a prospect for a potential leukemia differentiation therapy.

  13. Matriptase is required for the active form of hepatocyte growth factor induced Met, focal adhesion kinase and protein kinase B activation on neural stem/progenitor cell motility.

    PubMed

    Fang, Jung-Da; Lee, Sheau-Ling

    2014-07-01

    Hepatocyte growth factor (HGF) is a chemoattractant and inducer for neural stem/progenitor (NS/P) cell migration. Although the type II transmembrane serine protease, matriptase (MTP) is an activator of the latent HGF, MTP is indispensable on NS/P cell motility induced by the active form of HGF. This suggests that MTP's action on NS/P cell motility involves mechanisms other than proteolytic activation of HGF. In the present study, we investigate the role of MTP in HGF-stimulated signaling events. Using specific inhibitors of phosphatidylinositol-3-kinase (PI3K), protein kinase B (Akt) or focal adhesion kinase (FAK), we demonstrated that in NS/P cells HGF-activated c-Met induces PI3k-Akt signaling which then leads to FAK activation. This signaling pathway ultimately induces MMP2 expression and NS/P cell motility. Knocking down of MTP in NS/P cells with specific siRNA impaired HGF-stimulation of c-Met, Akt and FAK activation, blocked HGF-induced production of MMP2 and inhibited HGF-stimulated NS/P cell motility. MTP-knockdown NS/P cells cultured in the presence of recombinant protein of MTP protease domain or transfected with the full-length wild-type but not the protease-defected MTP restored HGF-responsive events in NS/P cells. In addition to functioning as HGF activator, our data revealed novel function of MTP on HGF-stimulated c-Met signaling activation.

  14. Telomerase activation as a repair response to radiation-induced DNA damage in Y79 retinoblastoma cells.

    PubMed

    Akiyama, Masaharu; Ozaki, Kohji; Kawano, Takeshi; Yamada, Osamu; Kawauchi, Kiyotaka; Ida, Hiroyuki; Yamada, Hisashi

    2013-10-28

    The molecular mechanism of telomerase activation induced by ionizing radiation (IR) remains poorly understood. We demonstrate that DNA damage induced by IR at doses of 2-5 Gy triggers activation of Akt, predominant to that of protein phosphatase 2A (PP2A), resulting in human telomerase reverse transcriptase (hTERT) phosphorylation and increased telomerase activity in Y79 cells. DNA damage induced by IR at doses greater than 10 Gy might trigger PP2A activation, predominant to that of Akt, resulting in hTERT dephosphorylation and decreased telomerase activity. Our results suggest that differential activation of Akt and PP2A may be responsible for telomerase regulation. PMID:23850566

  15. Dendritic morphology, synaptic transmission, and activity of mature granule cells born following pilocarpine-induced status epilepticus in the rat

    PubMed Central

    Gao, Fei; Song, Xueying; Zhu, Dexiao; Wang, Xiaochen; Hao, Aijun; Nadler, J. Victor; Zhan, Ren-Zhi

    2015-01-01

    To understand the potential role of enhanced hippocampal neurogenesis after pilocarpine-induced status epilepticus (SE) in the development of epilepsy, we quantitatively analyzed the geometry of apical dendrites, synaptic transmission, and activation levels of normotopically distributed mature newborn granule cells in the rat. SE in male Sprague-Dawley rats (between 6 and 7 weeks old) lasting for more than 2 h was induced by an intraperitoneal injection of pilocarpine. The complexity, spine density, miniature post-synaptic currents, and activity-regulated cytoskeleton-associated protein (Arc) expression of granule cells born 5 days after SE were studied between 10 and 17 weeks after CAG-GFP retroviral vector-mediated labeling. Mature granule cells born after SE had dendritic complexity similar to that of granule cells born naturally, but with denser mushroom-like spines in dendritic segments located in the outer molecular layer. Miniature inhibitory post-synaptic currents (mIPSCs) were similar between the controls and rats subjected to SE; however, smaller miniature excitatory post-synaptic current (mEPSC) amplitude with a trend toward less frequent was found in mature granule cells born after SE. After maturation, granule cells born after SE did not show denser Arc expression in the resting condition or 2 h after being activated by pentylenetetrazol-induced transient seizure activity than vicinal GFP-unlabeled granule cells. Thus our results suggest that normotopic granule cells born after pilocarpine-induced SE are no more active when mature than age-matched, naturally born granule cells. PMID:26500490

  16. Requirement of ERα and basal activities of EGFR and Src kinase in Cd-induced activation of MAPK/ERK pathway in human breast cancer MCF-7 cells

    SciTech Connect

    Song, Xiulong Wei, Zhengxi; Shaikh, Zahir A.

    2015-08-15

    Cadmium (Cd) is a common environmental toxicant and an established carcinogen. Epidemiological studies implicate Cd with human breast cancer. Low micromolar concentrations of Cd promote proliferation of human breast cancer cells in vitro. The growth promotion of breast cancer cells is associated with the activation of MAPK/ERK pathway. This study explores the mechanism of Cd-induced activation of MAPK/ERK pathway. Specifically, the role of cell surface receptors ERα, EGFR, and Src kinase was evaluated in human breast cancer MCF-7 cells treated with 1–3 μM Cd. The activation of ERK was studied using a serum response element (SRE) luciferase reporter assay. Receptor phosphorylation was detected by Western blot analyses. Cd treatment increased both the SRE reporter activity and ERK1/2 phosphorylation in a concentration-dependent manner. Cd treatment had no effect on reactive oxygen species (ROS) generation. Also, blocking the entry of Cd into the cells with manganese did not diminish Cd-induced activation of MAPK/ERK. These results suggest that the effect of Cd was likely not caused by intracellular ROS generation, but through interaction with the membrane receptors. While Cd did not appear to activate either EGFR or Src kinase, their inhibition completely blocked the Cd-induced activation of ERK as well as cell proliferation. Similarly, silencing ERα with siRNA or use of ERα antagonist blocked the effects of Cd. Based on these results, it is concluded that not only ERα, but also basal activities of EGFR and Src kinase are essential for Cd-induced signal transduction and activation of MAPK/ERK pathway for breast cancer cell proliferation. - Highlights: • Low micromolar concentrations of Cd rapidly activate ERK1/2 in MCF-7 cells. • Signal transduction and resulting cell proliferation require EGFR, ERα, and Src. • These findings implicate Cd in promotion of breast cancer.

  17. Aspergillus oryzae lectin induces anaphylactoid oedema and mast cell activation through its interaction with fucose of mast cell-bound non-specific IgE.

    PubMed

    Yamaki, K; Yoshino, S

    2011-11-01

    We investigated whether Aspergillus oryzae lectin (AOL), a fucose-specific lectin, induces anaphylactoid reactions and mast cell activation. The injection of AOL into footpads of mice produced a dose-related acute paw oedema. The AOL-induced oedema was attenuated by predose of histamine H1 receptor blocker or pretreatment of the lectin with fucose before injection and was not observed in SCID and mast cell-deficient WBB6F1-W/Wv mice. These results suggested that the AOL-induced anaphylactoid reaction was mediated by histamine released from mast cells. In addition, the activation of mast cells was seemed to be induced by the crosslinking of IgE on the cell surface following the binding of AOL to fucose residues in IgE. Consistent with the in vivo results, AOL induced the degranulation of the rat mast cell line RBL2H3 sensitized with monoclonal IgE. As AOL induced the increase in intracellular Ca(2+) concentration of IgE-sensitized RBL2H3 cells as well as antigen stimulation, AOL could input signals from FcεRI. The degranulation of IgE-sensitized RBL2H3 cells by AOL was diminished by pretreatment of AOL with fucose. Defucosylated IgE did not induce degranulation of RBL2H3 cells in response to AOL stimulation, in spite of its ability to induce degranulation by antigen stimulation as intact IgE. These results indicated that AOL bound to fucose residue of IgE causing antigen-independent IgE-mediated mast cell activation and anaphylactoid reactions in vitro and in vivo, respectively. AOL bound to human IgE as well as to mouse IgE, suggesting the possible implication of AOL in the allergic response to Aspergillus oryzae in humans.

  18. Mitochondrial reactive oxygen species-mediated NLRP3 inflammasome activation contributes to aldosterone-induced renal tubular cells injury

    PubMed Central

    Ding, Wei; Guo, Honglei; Xu, Chengyan; Wang, Bin; Zhang, Minmin; Ding, Feng

    2016-01-01

    Aldosterone (Aldo) is an independent risk factor for chronic kidney disease (CKD), and although Aldo directly induces renal tubular cell injury, the underlying mechanisms remain unclear. NLRP3 inflammasome and mitochondrial reactive oxygen species (ROS) have recently been implicated in various kinds of CKD. The present study hypothesized that mitochondrial ROS and NLRP3 inflammasome mediated Aldo–induced tubular cell injury. The NLRP3 inflammasome is induced by Aldo in a dose- and time-dependent manner, as evidenced by increased NLRP3, ASC, caspase-1, and downstream cytokines, such as interleukin (IL)-1β and IL-18. The activation of the NLRP3 inflammasome was significantly prevented by the selective mineralocorticoid receptor (MR) antagonist eplerenone (EPL) (P < 0.01). Mice harboring genetic knock-out of NLRP3 (NLRP3−/−) showed decreased maturation of renal IL-1β and IL-18, reduced renal tubular apoptosis, and improved renal epithelial cell phenotypic alternation, and attenuated renal function in response to Aldo-infusion. In addition, mitochondrial ROS was also increased in Aldo-stimulated HK-2 cells, as assessed by MitoSOXTM red reagent. Mito-Tempo, the mitochondria-targeted antioxidant, significantly decreased HK-2 cell apoptosis, oxidative stress, and the activation of NLRP3 inflammasome. We conclude that Aldo induces renal tubular cell injury via MR dependent, mitochondrial ROS-mediated NLRP3 inflammasome activation. PMID:27014913

  19. Platelet-leukocyte interaction in adhesion to endothelial cells induced by platelet-activating factor in vitro.

    PubMed Central

    Hirafuji, M.; Shinoda, H.

    1991-01-01

    1. Platelet-activating factor (PAF, 10 nM) did not induce platelet adhesion to endothelial cells cultured in monolayer but it induced their adhesion to protein-coated plastic. However, PAF induced a marked platelet adhesion to endothelial cells when polymorphonuclear leukocytes (PMNs) were present. Lyso-PAF had no effect. 2. Phase-contrast microscopic examination showed that single platelets rather than their aggregates adhered to the endothelial cell surface around aggregating and adhering PMNs. 3. Significant platelet adhesion was induced by PAF at concentrations higher that 0.01 nM with the maximal response at 10 nM. Platelet adhesion occurred within minutes after PAF addition, reaching a maximum approximately after 30 min. Platelet adhesion also occurred significantly at a PMN:platelet ratio of 1:800, and linearly up to 1:50. 4. The PAF-induced platelet adhesion was suppressed by three structurally unrelated PAF antagonists, WEB 2086, ONO 6240 and BN 52021, in a concentration-dependent manner. 5. PAF also increased PMN adhesion to endothelial cell monolayers, which was further augmented by the presence of platelets. 6. The present study demonstrates that PAF induces platelet adhesion to endothelial cells in vitro when PMNs are present and that there is a close interaction between platelets and PMNs in their adhesion to endothelial cells. The present study further suggests that PMNs could play a central role in platelet adhesion to vascular endothlium in certain pathological conditions. Images Figure 2 PMID:1884095

  20. TIPE2 Inhibits Hypoxia-Induced Wnt/β-Catenin Pathway Activation and EMT in Glioma Cells.

    PubMed

    Liu, Zhi-Jun; Liu, Hong-Lin; Zhou, Hai-Cun; Wang, Gui-Cong

    2016-01-01

    Hypoxia-induced epithelial-to-mesenchymal transition (EMT) could facilitate tumor progression. TIPE2, the tumor necrosis factor-α (TNF-α)-induced protein 8-like 2 (also known as TNFAIP8L2), is a member of the TNF-α-induced protein 8 (TNFAIP8, TIPE) family and has been involved in the development and progression of several tumors. However, the effects of TIPE2 on the EMT process in glioma cells and the underlying mechanisms of these effects have not been previously reported. In our study, we assessed the roles of TIPE2 in the EMT process in glioma cells in response to hypoxia. Our results indicated that TIPE2 expression was significantly decreased in human glioma cell lines. TIPE2 overexpression significantly inhibited hypoxia-induced migration and invasion, as well as suppressed the EMT process in glioma cells. Furthermore, TIPE2 overexpression prevented hypoxia-induced expression of β-catenin, cyclin D1, and c-myc in human glioma cells. In summary, these data suggest that TIPE2 overexpression inhibited hypoxia-induced Wnt/β-catenin pathway activation and EMT in glioma cells. PMID:27656836

  1. Crocin, the main active saffron constituent, mitigates dichlorvos-induced oxidative stress and apoptosis in HCT-116 cells.

    PubMed

    Ben Salem, Intidhar; Boussabbeh, Manel; Kantaoui, Hiba; Bacha, Hassen; Abid-Essefi, Salwa

    2016-08-01

    The protective effects of Crocin (CRO), a carotenoid with wide spectrum of pharmacological effects, against the cytotoxicity and the apoptosis produced by exposure to Dichlorvos (DDVP) in HCT116 cells were investigated in this work. The cytotoxicity was monitored by cell viability, ROS generation, antioxidant enzymes activities, malondialdehyde (MDA) production and DNA fragmentation. The apoptosis was assessed through the measurement of the mitochondrial transmembrane potential (ΔΨm) and caspases activation. The results indicated that pretreatment of HCT116 cells with CRO, 2h prior to DDVP exposure, significantly increased the survival of cells, inhibited the ROS generation, modulated the activities of catalase (CAT) and superoxide dismutase (SOD) and reduced the MDA level. The reduction in mitochondrial membrane potential, DNA fragmentation and caspases activation were also inhibited by CRO. These findings suggest that CRO can protect HCT116 cells from DDVP-induced oxidative stress and apoptosis. PMID:27470340

  2. Dengue-induced autophagy, virus replication and protection from cell death require ER stress (PERK) pathway activation

    PubMed Central

    Datan, E; Roy, S G; Germain, G; Zali, N; McLean, J E; Golshan, G; Harbajan, S; Lockshin, R A; Zakeri, Z

    2016-01-01

    A virus that reproduces in a host without killing cells can easily establish a successful infection. Previously, we showed that dengue-2, a virus that threatens 40% of the world, induces autophagy, enabling dengue to reproduce in cells without triggering cell death. Autophagy further protects the virus-laden cells from further insults. In this study, we evaluate how it does so; we show that dengue upregulates host pathways that increase autophagy, namely endoplasmic reticulum (ER) stress and ataxia telangiectasia mutated (ATM) signaling followed by production of reactive oxygen species (ROS). Inhibition of ER stress or ATM signaling abrogates the dengue-conferred protection against other cell stressors. Direct inhibition of ER stress response in infected cells decreases autophagosome turnover, reduces ROS production and limits reproduction of dengue virus. Blocking ATM activation, which is an early response to infection, decreases transcription of ER stress response proteins, but ATM has limited impact on production of ROS and virus titers. Production of ROS determines only late-onset autophagy in infected cells and is not necessary for dengue-induced protection from stressors. Collectively, these results demonstrate that among the multiple autophagy-inducing pathways during infection, ER stress signaling is more important to viral replication and protection of cells than either ATM or ROS-mediated signaling. To limit virus production and survival of dengue-infected cells, one must address the earliest phase of autophagy, induced by ER stress. PMID:26938301

  3. Immunomodulation of airway epithelium cell activation by mesenchymal stromal cells ameliorates house dust mite-induced airway inflammation in mice.

    PubMed

    Duong, Khang M; Arikkatt, Jaisy; Ullah, M Ashik; Lynch, Jason P; Zhang, Vivian; Atkinson, Kerry; Sly, Peter D; Phipps, Simon

    2015-11-01

    Allergic asthma is underpinned by T helper 2 (Th2) inflammation. Redundancy in Th2 cytokine function and production by innate and adaptive immune cells suggests that strategies aimed at immunomodulation may prove more beneficial. Hence, we sought to determine whether administration of mesenchymal stromal cells (MSCs) to house dust mite (HDM) (Dermatophagoides pteronyssinus)-sensitized mice would suppress the development of Th2 inflammation and airway hyperresponsiveness (AHR) after HDM challenge. We report that the intravenous administration of allogeneic donor MSCs 1 hour before allergen challenge significantly attenuated the features of allergic asthma, including tissue eosinophilia, Th2 cytokine (IL-5 and IL-13) levels in bronchoalveolar lavage fluid, and AHR. The number of infiltrating type 2 innate lymphoid cells was not affected by MSC transfer, suggesting that MSCs may modulate the adaptive arm of Th2 immunity. The effect of MSC administration was long lasting; all features of allergic airway disease were significantly suppressed in response to a second round of HDM challenge 4 weeks after MSC administration. Further, we observed that MSCs decreased the release of epithelial cell-derived alarmins IL-1α and high mobility group box-1 in an IL-1 receptor antagonist-dependent manner. This significantly decreased the expression of the pro-Th2 cytokine IL-25 and reduced the number of activated and antigen-acquiring CD11c(+)CD11b(+) dendritic cells in the lung and mediastinal lymph nodes. Our findings suggest that MSC administration can ameliorate allergic airway inflammation by blunting the amplification of epithelial-derived inflammatory cytokines induced by HDM exposure and may offer long-term protection against Th2-mediated allergic airway inflammation and AHR.

  4. Zeranol induces COX-2 expression through TRPC-3 activation in the placental cells JEG-3.

    PubMed

    Zhu, Yun; Yao, Xiaoqiang; Leung, Lai K

    2016-09-01

    Transient Receptor Potential Channels (TRPs) are commonly expressed in the reproductive tissues in human. Many female reproductive processes have been associated with these TRPs. The mycotoxin zeranol or α-zearalanol is derived from fungi in the Fusarium family. Limited exposure to zeranol appears to be safe. In North America, farmers are using synthetic zeranol to promote growth in livestock. As the health risks of exposure to residual zeranol have not been determined, this practice is disallowed in the European Community. In the present study the cellular calcium levels were elevated in JEG-3 cells treated with zeranol at or above 10nM. Subsequent study indicated that expressions of TRP channels were induced. In response to the calcium flow, ERK, P38 and PKCβ were activated and COX-2 expression was increased. Specific TRP inhibitors were employed to establish the connection between the ion channel activity and COX-2 expression, and TRPC-3 appeared to be the triggering mechanism. Since the involvement of COX-2 is implicated in placental development and parturition, exposure to this mycotoxin poses a potential threat to pregnant women. PMID:27224899

  5. Diethyldithiocarbamate induces apoptosis in neuroblastoma cells by raising the intracellular copper level, triggering cytochrome c release and caspase activation.

    PubMed

    Matias, Andreza C; Manieri, Tânia M; Cipriano, Samantha S; Carioni, Vivian M O; Nomura, Cassiana S; Machado, Camila M L; Cerchiaro, Giselle

    2013-02-01

    Dithiocarbamates are nitrogen- and sulfur-containing compounds commonly used in pharmacology, medicine and agriculture. The molecular effects of dithiocarbamates on neuronal cell systems are not fully understood, especially in terms of their ability to accumulate copper ions inside the cell. In this work, the molecular effects of N,N-diethyldithiocarbamate (DEDTC) were studied in human SH-SY5Y neuroblastoma cells to determine the role of copper in the DEDTC toxicity and the pathway trigged in cell by the complex Cu-DEDTC. From concentration-dependent studies, we found that 5 μM of this compound induced a drastic decrease in viable cells with a concomitant accumulation in intracellular copper resulted from complexation with DEDTC, measured by atomic absorption spectroscopy. The mechanism of DEDTC-induced apoptosis in neuronal model cells is thought to occur through the death receptor signaling triggered by DEDTC-copper complex in low concentration that is associated with the activation of caspase 8. Our results indicated that the mechanism of cell death involves cytochrome c release forming the apoptosome together with Apaf-1 and caspase 9, converting the caspase 9 into its active form, allowing it to activate caspase 3 as observed by immunofluorescence. This pathway is induced by the cytotoxic effects that occur when DEDTC forms a complex with the copper ions present in the culture medium and transports them into the cell, suggesting that the DEDTC by itself was not able to cause cell death and the major effect is from its copper-complex in neuroblastoma cells. The present study suggests a role for the influence of copper by low concentrations of DEDTC in the extracellular media, the absorption and accumulation of copper in the cell and apoptotic events, induced by the cytotoxic effects that occur when DEDTC forms a complex with the copper ions. PMID:22951949

  6. Uric acid induces oxidative stress and growth inhibition by activating adenosine monophosphate-activated protein kinase and extracellular signal-regulated kinase signal pathways in pancreatic β cells.

    PubMed

    Zhang, Yongneng; Yamamoto, Tetsuya; Hisatome, Ichiro; Li, Youfeng; Cheng, Weijie; Sun, Ning; Cai, Bozhi; Huang, Tianliang; Zhu, Yuzhang; Li, Zhi; Jing, Xubin; Zhou, Rui; Cheng, Jidong

    2013-08-15

    Hyperuricaemia is a disorder of purine metabolism, and is strongly associated with insulin resistance and abnormal glucose metabolism. As the producer of insulin, pancreatic β cells might be affected by elevated serum uric acid levels and contribute to the disregulated glucose metabolism. In this study, we investigated the effect of high uric acid on rat pancreatic β cell function. Under high uric acid condition, proliferation of pancreatic β cells was inhibited, production of reactive oxygen species increased, and glucose stimulated insulin secretion was also compromised. Further examination on signal transduction pathways revealed that uric acid-induced ROS is involved in the activation of adenosine monophosphate-activated protein kinase (AMPK), and extracellular signal-regulated kinase (ERK). Pharmacological inhibition of ERK activation rescued β cells from growth inhibition. More importantly, activation of ERK induced by uric acid is significantly diminished by AMPK inhibitor, indicating ERK as a downstream target of AMPK in response to high uric acid condition. We also investigated the transportation channel for uric acid into pancreatic β cells. While major urate transporter URAT1 is not expressed in β cells, organic anion transporter (OAT) inhibitor successfully blocked the activation of ERK by uric acid. Our data indicate that high uric acid levels induce oxidative damage and inhibit growth of rat pancreatic β cells by activating the AMPK and ERK signal pathways. Hyperuricemia may contribute to abnormal glucose metabolism by causing oxidative damage and function inhibition of pancreatic β cells.

  7. Measurement of Ligand-Induced Activation in Single Viable T Cells Using the lacZ Reporter Gene

    NASA Astrophysics Data System (ADS)

    Karttunen, Jaana; Shastri, Nilabh

    1991-05-01

    We have used the bacterial β-galactosidase gene (lacZ) as a reporter gene for the rapid measurement of T-cell antigen receptor (TCR)-mediated activation of individual T cells. The reporter construct contained the lacZ gene under the control of the nuclear factor of activated T cells (NF-AT) element of the human interleukin 2 enhancer [Fiering, S., Northrop, J. P., Nolan, G. P., Matilla, P., Crabtree, G. R. & Herzenberg, L. A. (1990) Genes Dev. 4, 1823-1834]. The activity of the intracellular lacZ enzyme was analyzed by flow cytometric measurement of fluorescein accumulation in cells loaded with the fluorogenic β-galactosidase substrate fluorescein di-β-D-galactopyranoside. As a model system, the T-cell hybridoma BO4H9.1, which is specific for the lysozyme peptide (amino acids 74-88)/A^b complex, was transfected with the NF-AT-lacZ construct. lacZ activity was induced in 50-100% of the transfectant cells following exposure to pharmacological agents, to the physiological peptide/major histocompatibility complex ligand, or to other TCR-specific stimuli. Interestingly, increasing concentrations of the stimulus increased the fraction of lacZ^+ cells, but not the level of lacZ activity per cell. Even under widely varying levels of stimulus, the level of lacZ activity in individual lacZ^+ cells remained within a remarkably narrow range. These results demonstrate that TCR-mediated activation can be readily measured in single T cells and strongly suggest that, once committed to activation, the level of NF-AT transcriptional activity in individual T cells is independent of the form or concentration of stimulus. This assay is likely to prove useful for the study of early activation events in individual T cells and of TCR ligands.

  8. Activation of reactive oxygen species/AMP activated protein kinase signaling mediates fisetin-induced apoptosis in multiple myeloma U266 cells.

    PubMed

    Jang, Ki Young; Jeong, Soo-Jin; Kim, Sun-Hee; Jung, Ji Hoon; Kim, Ji-Hyun; Koh, Wonil; Chen, Chang-Yan; Kim, Sung-Hoon

    2012-06-28

    We investigated the molecular mechanisms responsible for fisetin-induced apoptosis in U266 cells. Fisetin elicited the cytotoxicity in U266 cells, manifested as an increased fraction of the cells with sub-G1 content or stained positively with TUNEL labeling. Fisetin enhanced caspase-3 activation, downregulation of Bcl-2 and Mcl-1(L), and upregulation of Bax, Bim and Bad. Fisetin activated AMPK as well as its substrate acetyl-CoA carboxylase (ACC), along with a decreased phosphorylation of AKT and mTOR. Fisetin also stimulated generation of ROS in U266 cells. Conversely, compound C or N-acetyl-L-cystein blocked fisetin-induced apoptosis. Our data suggest that fisetin-induced apoptosis in U266 cells is through ROS and AMPK pathways.

  9. Imaging of Ras/Raf activity induced by low energy laser irradiation in living cell using FRET

    NASA Astrophysics Data System (ADS)

    Wang, Fang; Chen, Tong-Sheng; Xing, Da

    2005-01-01

    Ras/Raf signaling pathway is an important signaling pathway that governs cell proliferation, differential and apoptosis. Low-energy laser irradiation (LELI) was found to modulate various processes. Generally, cell proliferation is induced by low doses LELI and apoptosis is induced by high doses LELI. Mechanism of biological effect of LELI has not been clear. Recently, activation of MEK (mitogen-activated protein kinase) and ERK (extracellular-signal-regulated kinase), which are downstream protein kinases of Ras/Raf, are observed during LELI-induced cell proliferation by immunoprecipitation and western blot analysis. RaichuRas reporter consisting of fusions of H-ras, the Ras-binding domain of Raf (RafRBD), a cyan fluorescent protein (CFP) and a yellow fluorescent protein (YFP). Therefore, intramolecular binding of GTP-Ras to RafRBD brings CFP close to YFP and increases FRET between CFP and YFP. Human lung adenocarcinoma cell line (ASTC-a-1) was transfected with the plasmid (pRaichuRas) and then treated with LELI at dose of 60J/cm2. Effect of LELI on Ras/Raf in physiological condition of living cells was observed by fluorescence resonance energy transfer (FRET) technique during lung adenocarcinoma cell apoptosis induced by high dose (60J/cm2) LELI. Experimental results showed that after high dose LELI treatment, the binding of Ras and Raf decreases obviously, Ras/Raf signaling pathway deregulates and cell apoptosis occurs.

  10. Analysis of caspase3 activation in ChanSu-induced apoptosis of ASTC-a-1 cells by fluorescence techniques

    NASA Astrophysics Data System (ADS)

    Sun, Lei; Chen, Tongsheng; Wang, Longxiang; Wang, Huiying

    2008-02-01

    ChanSu(CS), a traditional Chinese medicine, is composed of many chemical compoments. It is isolated from the dried white secretion of the auricular and skin glands of toads, and it has been widely used for treating the heart diseases and other systemic illnesses. However, it is difficult to judge antitumor effect of agents derived from ChanSu and the underlying mechanism of ChanSu inducing cell apoptosis is still unclear. This report was performed to explore the inhibitory effect and mechanism of ChanSu on human lung adenocarcinoma cells (ASTC-a-1). Fluorescence emission spectra and fluorescence resonance energy transfer (FRET) were used to study the caspase-3 activation during the ChanSu-induced human lung adenocarcinoma (ASTC-a-1) cell apoptosis. CCK-8 was used to assay the inhibition of ChanSu on the cell viability. The cells expressing stably with SCAT3 was used to examine if caspase-3 was activated by ChanSu using acceptor photobleaching technique. Our data showed that treatment of ASTC-a-1 cell with ChanSu resulted in the inhibition of viability and induction of apoptosis in a dose-dependent manner and the SCAT3 was almost cleaved 24 h after ChanSu treatment, implying that ChanSu induced cell apoptosis via a caspase-3-dependent death pathway. Our findings extend the knowledge about the cellular signaling mechanisms mediating ChanSu-induced apoptosis.

  11. ERK1/2 activation by angiotensin II inhibits insulin-induced glucose uptake in vascular smooth muscle cells.

    PubMed

    Izawa, Yuki; Yoshizumi, Masanori; Fujita, Yoshiko; Ali, Nermin; Kanematsu, Yasuhisa; Ishizawa, Keisuke; Tsuchiya, Koichiro; Obata, Toshiyuki; Ebina, Yousuke; Tomita, Shuhei; Tamaki, Toshiaki

    2005-08-15

    Clinical evidence suggests a relationship between hypertension and insulin resistance, and cross-talk between angiotensin II (Ang II) and insulin signaling pathways may take place. We now report the effect of Ang II on insulin-induced glucose uptake and its intracellular mechanisms in vascular smooth muscle cells (VSMC). We examined the translocation of glucose transporter-4 (GLUT-4) and glucose uptake in rat aortic smooth muscle cells (RASMC). Mitogen-activated protein (MAP) kinases and Akt activities, and phosphorylation of insulin receptor substrate-1 (IRS-1) at the serine and tyrosine residues were measured by immunoprecipitation and immunoblotting. As a result, Ang II inhibited insulin-induced GLUT-4 translocation from cytoplasm to the plasma membrane in RASMC. Ang II induced extracellular signal-regulated kinase (ERK) 1/2 and c-Jun N-terminal kinase (JNK) activation and IRS-1 phosphorylation at Ser307 and Ser616. Ang II-induced Ser307 and Ser616 phophorylation of IRS-1 was inhibited by a MEK inhibitor, PD98059, and a JNK inhibitor, SP600125. Ang II inhibition of insulin-stimulated IRS-1 tyrosyl phophorylation and Akt activation were reversed by PD98059 but not by SP600125. Ang II inhibited insulin-induced glucose uptake, which was also reversed by PD98059 but not by SP600125. It is shown that Ang II-induced ERK1/2 activation inhibits insulin-dependent glucose uptake through serine phophorylation of IRS-1 in RASMC.

  12. Mechano-growth factor induces migration of rat mesenchymal stem cells by altering its mechanical properties and activating ERK pathway

    SciTech Connect

    Wu, Jiamin; Wu, Kewen; Lin, Feng; Luo, Qing; Yang, Li; Shi, Yisong; Song, Guanbin; Sung, Kuo-Li Paul

    2013-11-08

    Highlights: •MGF induced the migration of rat MSC in a concentration-dependent manner. •MGF enhanced the mechanical properties of rMSC in inducing its migration. •MGF activated the ERK 1/2 signaling pathway of rMSC in inducing its migration. •rMSC mechanics may synergy with ERK 1/2 pathway in MGF-induced rMSC migration. -- Abstract: Mechano-growth factor (MGF) generated by cells in response to mechanical stimulation has been identified as a mechano effector molecule, playing a key role in regulating mesenchymal stem cell (MSC) function, including proliferation and migration. However, the mechanism(s) underlying how MGF-induced MSC migration occurs is still unclear. In the present study, MGF motivated migration of rat MSCs (rMSCs) in a concentration-dependent manner and optimal concentration of MGF at 50 ng/mL (defined as MGF treatment in this paper) was demonstrated. Notably, enhancement of mechanical properties that is pertinent to cell migration, such as cell traction force and cell stiffness were found to respond to MGF treatment. Furthermore, MGF increased phosphorylation of extracellular signal-regulated kinase (ERK), ERK inhibitor (i.e., PD98059) suppressed ERK phosphorylation, and abolished MGF-induced rMSC migration were found, demonstrating that ERK is involved molecule for MGF-induced rMSC migration. These in vitro evidences of MGF-induced rMSC migration and its direct link to altering rMSC mechanics and activating the ERK pathway, uncover the underlying biomechanical and biological mechanisms of MGF-induced rMSC migration, which may help find MGF-based application of MSC in clinical therapeutics.

  13. Microglia Induce Neurotoxic IL-17+ γδ T Cells Dependent on TLR2, TLR4, and TLR9 Activation

    PubMed Central

    Derkow, Katja; Krüger, Christina; Dembny, Paul; Lehnardt, Seija

    2015-01-01

    Background Interleukin-17 (IL-17) acts as a key regulator in central nervous system (CNS) inflammation. γδ T cells are an important innate source of IL-17. Both IL-17+ γδ T cells and microglia, the major resident immune cells of the brain, are involved in various CNS disorders such as multiple sclerosis and stroke. Also, activation of Toll-like receptor (TLR) signaling pathways contributes to CNS damage. However, the mechanisms underlying the regulation and interaction of these cellular and molecular components remain unclear. Objective In this study, we investigated the crosstalk between γδ T cells and microglia activated by TLRs in the context of neuronal damage. To this end, co-cultures of IL-17+ γδ T cells, neurons, and microglia were analyzed by immunocytochemistry, flow cytometry, ELISA and multiplex immunoassays. Results We report here that IL-17+ γδ T cells but not naïve γδ T cells induce a dose- and time-dependent decrease of neuronal viability in vitro. While direct stimulation of γδ T cells with various TLR ligands did not result in up-regulation of CD69, CD25, or in IL-17 secretion, supernatants of microglia stimulated by ligands specific for TLR2, TLR4, TLR7, or TLR9 induced activation of γδ T cells through IL-1β and IL-23, as indicated by up-regulation of CD69 and CD25 and by secretion of vast amounts of IL-17. This effect was dependent on the TLR adaptor myeloid differentiation primary response gene 88 (MyD88) expressed by both γδ T cells and microglia, but did not require the expression of TLRs by γδ T cells. Similarly to cytokine-primed IL-17+ γδ T cells, IL-17+ γδ T cells induced by supernatants derived from TLR-activated microglia also caused neurotoxicity in vitro. While these neurotoxic effects required stimulation of TLR2, TLR4, or TLR9 in microglia, neuronal injury mediated by bone marrow-derived macrophages did not require TLR signaling. Neurotoxicity mediated by IL-17+ γδ T cells required a direct cell-cell

  14. Matrix fibronectin disruption and altered endothelial cell adhesion induced by activated leukocytes

    SciTech Connect

    Vincent, P.; Richards, P.; Saba, T.; DelVecchio, P.

    1986-03-01

    Sequestration of activated leukocytes (PMN) within the lung may contribute to pulmonary vascular injury following trauma, sepsis, or intravascular coagulation. Monolayers of cultured rat endothelial cells were utilized to evaluate the effect of activated PMNs on endothelial cell attachment and the extracellular fibronectin matrix over a 4 hr incubation interval. Rat endothelial cells were identified by immunofluorescent staining of Factor VIII R:Ag. Endothelial cells were labeled with /sup 51/Cr in order to establish a cell injury assay in which the release of pelletable (cell associated) or non-pelletable activity was measured in the media. PMN activation was verified by chemiluminescence activity. Following phorbol myristate acetate (PMA) the leukocytes aggregated, chemiluminesced, and caused detachment of /sup 51/Cr endothelial cells. Endothelial detachment increased as a function of time with a plateau by 3 hrs. Immunofluorescent analysis of extracellular fibronectin in endothelial cell cultures revealed disruption of the fibrillar matrix fibronectin in association with endothelial cell disadhesion. Matrix fibronectin disruption was not seen with PMNs or PMA alone. Thus, disruption of the fibronectin matrix by released proteases may contribute to endothelial cell detachment.

  15. Silymarin prevents palmitate-induced lipotoxicity in HepG2 cells: involvement of maintenance of Akt kinase activation.

    PubMed

    Song, Zhenyuan; Song, Ming; Lee, David Y W; Liu, Yanze; Deaciuc, Ion V; McClain, Craig J

    2007-10-01

    Whereas adipocytes have a unique capacity to store excess free fatty acids in the form of triglyceride in lipid droplets, non-adipose tissues, such as liver, have a limited capacity for storage of lipids. Saturated long-chain fatty acids, such as palmitate, are the major contributors to lipotoxicity. Silymarin is a mixture of flavonolignans, extracted from the milk thistle (Silibum marianum). Its hepatoprotective properties have been studied both in vitro and in vivo; however, its effect on palmitate-induced lipotoxicity has not been investigated. The objective of this study was to investigate (i) whether silymarin could protect HepG2 cells from palmitate-induced cell death in an in vitro model, and (ii) possible mechanisms involved in this hepatoprotective role of silymarin. HepG2 cells were treated with palmitate in the absence or presence of silymarin and supernatants or cell lysates were collected at varying time-points. Cell death was assayed by measuring DNA fragmentation, caspase-3 activity and lactate dehydrogenase release. Lipid peroxidation was assessed by measuring malondialdehyde and 4-hydroxyalkenals. Akt kinase activity was also measured. Incubation with palmitate caused significant death in HepG2 cells. Palmitate incubation did not cause significant changes in reactive oxygen species production or intracellular glutathione content, but markedly inhibited Akt kinase activity. Pre-treatment of HepG2 cells with silymarin prevented palmitate-induced inhibition of Akt kinase activity and attenuated cell death. Our results suggest that silymarin may be an effective agent in protecting hepatocytes from saturated fatty acids-induced cell death. These data also provide a further rationale for exploration of the use of silymarin in the treatment of non-alcoholic steatohepatitis.

  16. Inhibition of fatty acid amide hydrolase activates Nrf2 signalling and induces heme oxygenase 1 transcription in breast cancer cells

    PubMed Central

    Li, H; Wood, J T; Whitten, K M; Vadivel, S K; Seng, S; Makriyannis, A; Avraham, H K

    2013-01-01

    BACKGROUND AND PURPOSE Endocannabinoids such as anandamide (AEA) are important lipid ligands regulating cell proliferation, differentiation and apoptosis. Their levels are regulated by hydrolase enzymes, the fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MGL). Here, we investigated whether FAAH or AEA are involved in NF (erythroid-derived 2)-like 2 (Nrf2)/antioxidant responsive element (ARE) pathway. EXPERIMENTAL APPROACH The aim of this study was to analyse the effects of AEA or FAAH inhibition by the URB597 inhibitor or FAAH/siRNA on the activation of Nrf2-ARE signalling pathway and heme oxygenase-1 (HO-1) induction and transcription. KEY RESULTS Endogenous AEA was detected in the immortalized human mammary epithelial MCF-10A cells (0.034 ng per 106 cells) but not in MCF-7 or MDA-MB-231 breast cancer cells. Because breast tumour cells express FAAH abundantly, we examined the effects of FAAH on Nrf2/antioxidant pathway. We found that inhibition of FAAH by the URB597 inhibitor induced antioxidant HO-1 in breast cancer cells and MCF-10A cells. RNAi-mediated knockdown of FAAH or treatment with AEA-activated ARE-containing reporter induced HO-1 mRNA and protein expression, independent of the cannabinoid receptors, CB1, CB2 or TRPV1. Furthermore, URB597, AEA and siRNA-FAAH treatments induced the nuclear translocation of Nrf2, while siRNA-Nrf2 treatment and Keap1 expression blocked AEA, URB597 and si-FAAH from activation of ARE reporter and HO-1 induction. siRNA-HO-1 treatment decreased the viability of breast cancer cells and MCF-10A cells. CONCLUSIONS AND IMPLICATIONS These data uncovered a novel mechanism by which inhibition of FAAH or exposure to AEA induced HO-1 transcripts and implicating AEA and FAAH as direct modifiers in signalling mediated activation of Nrf2-HO-1 pathway, independent of cannabinoid receptors. PMID:23347118

  17. Lysophosphatidic acid induces reactive oxygen species generation by activating protein kinase C in PC-3 human prostate cancer cells

    SciTech Connect

    Lin, Chu-Cheng; Lin, Chuan-En; Lin, Yueh-Chien; Ju, Tsai-Kai; Huang, Yuan-Li; Lee, Ming-Shyue; Chen, Jiun-Hong; Lee, Hsinyu

    2013-11-01

    Highlights: •LPA induces ROS generation through LPA{sub 1} and LPA{sub 3}. •LPA induces ROS generation by activating PLC. •PKCζ mediates LPA-induced ROS generation. -- Abstract: Prostate cancer is one of the most frequently diagnosed cancers in males, and PC-3 is a cell model popularly used for investigating the behavior of late stage prostate cancer. Lysophosphatidic acid (LPA) is a lysophospholipid that mediates multiple behaviors in cancer cells, such as proliferation, migration and adhesion. We have previously demonstrated that LPA enhances vascular endothelial growth factor (VEGF)-C expression in PC-3 cells by activating the generation of reactive oxygen species (ROS), which is known to be an important mediator in cancer progression. Using flow cytometry, we showed that LPA triggers ROS generation within 10 min and that the generated ROS can be suppressed by pretreatment with the NADPH oxidase (Nox) inhibitor diphenylene iodonium. In addition, transfection with LPA{sub 1} and LPA{sub 3} siRNA efficiently blocked LPA-induced ROS production, suggesting that both receptors are involved in this pathway. Using specific inhibitors and siRNA, phospholipase C (PLC) and protein kinase C (PKC) were also suggested to participate in LPA-induced ROS generation. Overall, we demonstrated that LPA induces ROS generation in PC-3 prostate cancer cells and this is mediated through the PLC/PKC/Nox pathway.

  18. Augmentation by transferrin of IL-2-inducible killer activity and perforin production of human CD8+ T cells.

    PubMed Central

    Nakamura, A; Sone, S; Nabioullin, R; Sugihara, K; Munekata, M; Nishioka, Y; Nii, A; Ogura, T

    1993-01-01

    The effects of human transferrin (Tf) on lymphokine (IL-2)-activated killer (LAK) induction from blood lymphocytes of healthy donors was examined. LAK cells were induced by 6-day incubation in medium with recombinant human IL-2 of lymphocytes, and their cytotoxic activity was assessed by measuring 51Cr release from NK-resistant Daudi cells. Tf alone did not induce any LAK activity, but in combination with IL-2, it augmented LAK induction dose- and time-dependently. This augmenting effect was completely abolished by pretreatment with anti-Tf antiserum. Tf augmented the proliferative response of lymphocytes to IL-2 and their expressions of receptors for IL-2 and Tf. CD8+ T cells were isolated from purified blood lymphocytes using antibody-bound magnetic beads. Addition of Tf to cultures of CD8+ cells resulted in significant augmentation of killer cell induction and perforin (PFP) production after 4 days stimulation with IL-2. These results indicate that Tf is important in generation of IL-2-inducible killer properties and PFP activity of human CD8+ T cells. PMID:8467561

  19. Activation of Nrf2 by cadmium and its role in protection against cadmium-induced apoptosis in rat kidney cells

    SciTech Connect

    Chen Jun; Shaikh, Zahir A.

    2009-11-15

    Kidney is the primary target organ in chronic cadmium (Cd) toxicity, and oxidative stress plays an important role in this process. The nuclear transcription factor Nrf2 binds to antioxidant response elements (AREs) and regulates genes involved in protecting cells from oxidative damage. Whether kidney cells respond to Cd by activating Nrf2 is unknown. This study was designed to examine the Cd-induced activation of Nrf2 transcriptional activity in a stable rat kidney cell line, NRK-52E, and to investigate the protection this might offer against apoptosis. The cells were treated with 5-20 muM CdCl{sub 2} for 5 h, followed by a recovery period of up to 24 h. A concentration-dependent increase (up to 2.9-fold) in the level of reactive oxygen species was noted upon termination of 5-h Cd treatment. The Nrf2-ARE binding activity also increased and peaked (6.1-fold) at 10 muM Cd concentration. Time-course study revealed that the binding activity increased at 1 h of Cd treatment and peaked 2 h post Cd treatment. Apoptosis was detected 6 h post treatment with Cd and a concentration- and time-dependent increase in the apoptotic cell population occurred during the next 18 h. Over-expression of Nrf2 by transient transfection conferred resistance against Cd-induced apoptosis. Conversely, suppression of Nrf2 expression by specific siRNA resulted in greater sensitivity of the cells to Cd by decreasing the levels of two antioxidant enzymes, hemeoxygenase-1 and glutamate-cysteine ligase. Taken together, these results suggest that in kidney cells the activation of Nrf2 is an adaptive intracellular response to Cd-induced oxidative stress, and that Nrf2 is protective against Cd-induced apoptosis.

  20. Anti-Inflammatory Effects of Levalbuterol-Induced 11β-Hydroxysteroid Dehydrogenase Type 1 Activity in Airway Epithelial Cells

    PubMed Central

    Randall, Matthew J.; Kostin, Shannon F.; Burgess, Edward J.; Hoyt, Laura R.; Ather, Jennifer L.; Lundblad, Lennart K.; Poynter, Matthew E.

    2015-01-01

    Airway epithelial NF-κB activation is observed in asthmatic subjects and is a cause of airway inflammation in mouse models of allergic asthma. Combination therapy with inhaled short-acting β2-agonists and corticosteroids significantly improves lung function and reduces inflammation in asthmatic subjects. Corticosteroids operate through a number of mechanisms to potently inhibit NF-κB activity. Since β2-agonists can induce expression of 11β-HSD1, which converts inactive 11-keto corticosteroids into active 11-hydroxy corticosteroids, thereby potentiating the effects of endogenous glucocorticoids, we examined whether this mechanism is involved in the inhibition of NF-κB activation induced by the β-agonist albuterol in airway epithelial cells. Treatment of transformed murine Club cells (MTCC) with (R)-albuterol (levalbuterol), but not with (S)- or a mixture of (R + S)- (racemic) albuterol, augmented mRNA expression of 11β-HSD1. MTCC were stably transfected with luciferase (luc) reporter constructs under transcriptional regulation by NF-κB (NF-κB/luc) or glucocorticoid response element (GRE/luc) consensus motifs. Stimulation of NF-κB/luc MTCC with lipopolysaccharide (LPS) or tumor necrosis factor-α (TNFα) induced luc activity, which was inhibited by pretreatment with (R)-, but not (S)- or racemic albuterol. Furthermore, pretreatment of GRE/luc MTCC with (R)-, but not with (S)- or racemic albuterol, augmented 11-keto corticosteroid (cortisone) induced luc activity, which was diminished by the 11β-HSD inhibitor glycyrrhetinic acid (18β-GA), indicating that there was a conversion of inactive 11-keto to active 11-hydroxy corticosteroids. LPS- and TNFα-induced NF-κB/luc activity was diminished in MTCC cells treated with a combination of cortisone and (R)-albuterol, an effect that was inhibited by 18β-GA. Finally, pretreatment of MTCC cells with the combination of cortisone and (R)-albuterol diminished LPS- and TNFα-induced pro-inflammatory cytokine

  1. Role of Snail Activation in Alcohol-induced iNOS-mediated Disruption of Intestinal Epithelial Cell Permeability

    PubMed Central

    Forsyth, Christopher B.; Tang, Yueming; Shaikh, Maliha; Zhang, Lijuan; Keshavarzian, Ali

    2013-01-01

    Background Chronic alcohol use results in many pathological effects including alcoholic liver disease (ALD). ALD pathogenesis requires endotoxemia. Our previous studies showed that increased intestinal permeability is the major cause of endotoxemia and that this gut leakiness is dependent on alcohol stimulation of inducible nitric oxide synthase (iNOS) in both alcoholic subjects and rodent models of alcoholic steatohepatitis (ASH). The mechanism of the alcohol-induced, iNOS-mediated disruption of the intestinal barrier function is not known. We have recently shown that alcohol stimulates activation of the transcription factor Snail and biomarkers of epithelial mesenchymal transition. Since activated Snail disrupts tight junctional proteins , we hypothesized that activation of Snail by iNOS might be one of the key signaling pathways mediating alcohol stimulated intestinal epithelial cell hyperpermeability. Methods We measured intestinal permeability in alcohol-fed C57BL/6 control and iNOS KO mice and measured Snail protein expression in the intestines of these mice. We then examined intestinal epithelial permeability using the Caco-2 cell model of the intestinal barrier ± siRNA inhibition of Snail. We assessed Snail activation by alcohol in Caco-2 cells ± inhibition of iNOS with L-NIL or siRNA. Finally, we assessed Snail activation by alcohol ± inhibition with siRNA for p21-activated kinase (PAK1). Results Our data show that chronic alcohol feeding promotes intestinal hyperpermeability in wild type BL/6 but not in iNOS KO mice. Snail protein expression was increased in the intestines of alcohol-treated wild type mice but not in iNOS KO mice. SiRNA inhibition of Snail significantly inhibited alcohol-induced hyperpermeability in Caco-2 cell monolayers. Alcohol stimulation of SnailpS246 activation was blocked by inhibition of iNOS with L-NIL or with siRNA. SiRNA inhibition of PAK1 significantly inhibited alcohol-mediated activation of Snail in Caco-2 cells

  2. 1,10-Phenanthroline promotes copper complexes into tumor cells and induces apoptosis by inhibiting the proteasome activity.

    PubMed

    Zhang, Zhen; Bi, Caifeng; Schmitt, Sara M; Fan, Yuhua; Dong, Lili; Zuo, Jian; Dou, Q Ping

    2012-12-01

    Indole-3-acetic acid and indole-3-propionic acid, two potent natural plant growth hormones, have attracted attention as promising prodrugs in cancer therapy. Copper is known to be a cofactor essential for tumor angiogenesis. We have previously reported that taurine, L-glutamine, and quinoline-2-carboxaldehyde Schiff base copper complexes inhibit cell proliferation and proteasome activity in human cancer cells. In the current study, we synthesized two types of copper complexes, dinuclear complexes and ternary complexes, to investigate whether a certain structure could easily carry copper into cancer cells and consequently inhibit tumor proteasome activity and induce apoptosis. We observed that ternary complexes binding with 1,10-phenanthroline are more potent proteasome inhibitors and apoptosis inducers than dinuclear complexes in PC-3 human prostate cancer cells. Furthermore, the ternary complexes potently inhibit proteasome activity before induction of apoptosis in MDA-MB-231 human breast cancer cells, but not in nontumorigenic MCF-10A cells. Our results suggest that copper complexes binding with 1,10-phenanthroline as the third ligand could serve as potent, selective proteasome inhibitors and apoptosis inducers in tumor cells, and that the ternary complexes may be good potential anticancer drugs.

  3. 1,10-Phenanthroline promotes copper complexes into tumor cells and induces apoptosis by inhibiting the proteasome activity.

    PubMed

    Zhang, Zhen; Bi, Caifeng; Schmitt, Sara M; Fan, Yuhua; Dong, Lili; Zuo, Jian; Dou, Q Ping

    2012-12-01

    Indole-3-acetic acid and indole-3-propionic acid, two potent natural plant growth hormones, have attracted attention as promising prodrugs in cancer therapy. Copper is known to be a cofactor essential for tumor angiogenesis. We have previously reported that taurine, L-glutamine, and quinoline-2-carboxaldehyde Schiff base copper complexes inhibit cell proliferation and proteasome activity in human cancer cells. In the current study, we synthesized two types of copper complexes, dinuclear complexes and ternary complexes, to investigate whether a certain structure could easily carry copper into cancer cells and consequently inhibit tumor proteasome activity and induce apoptosis. We observed that ternary complexes binding with 1,10-phenanthroline are more potent proteasome inhibitors and apoptosis inducers than dinuclear complexes in PC-3 human prostate cancer cells. Furthermore, the ternary complexes potently inhibit proteasome activity before induction of apoptosis in MDA-MB-231 human breast cancer cells, but not in nontumorigenic MCF-10A cells. Our results suggest that copper complexes binding with 1,10-phenanthroline as the third ligand could serve as potent, selective proteasome inhibitors and apoptosis inducers in tumor cells, and that the ternary complexes may be good potential anticancer drugs. PMID:23053530

  4. ERK1/2 activation modulates pyocyanin-induced toxicity in A549 respiratory epithelial cells.

    PubMed

    Forbes, Amanda; Davey, Andrew K; Perkins, Anthony V; Grant, Gary D; McFarland, Amelia J; McDermott, Catherine M; Anoopkumar-Dukie, Shailendra

    2014-02-01

    Pyocyanin (PCN), a virulence factor produced by Pseudomonas aeruginosa, has many damaging effects on mammalian cells. Several lines of evidence suggest that this damage is primarily mediated by its ability to generate oxidative stress. However mechanisms underlying PCN-induced oxidative injury remain unclear. Although oxidative stress and subsequent MAPK signaling has been shown to modulate cell death in other models, its role in PCN-induced cytotoxicity remains unknown. Therefore the aim of this study was to investigate the role of redox-sensitive MAPK in PCN-induced toxicity in A549 cells. Here we show that PCN (50μM) rapidly increased ERK1/2 phosphorylation after 5min. Pre-treatment of A549 cells with the MEK1/2 inhibitor U0126 (10μM) decreased PCN-induced ERK1/2 phosphorylation and protected cells against apoptosis and cell injury suggesting a role for ERK signalling. In contrast, JNK and p38 MAPK phosphorylation remained unchanged following exposure to PCN and pretreatment with either the JNK or p38 MAPK inhibitors (10μM SP600125 and 10μM SB203580, respectively) did not afford protection against PCN toxicity. This would suggest that PCN-induced cytotoxicity appears to occur independently of JNK and p38 MAPK signaling pathways. Finally, although we confirm that oxidative stress contributes to PCN-induced toxicity, our data suggest the contribution of oxidative stress is independent of ERK1/2 signaling. These findings may provide insight for novel targeted therapies to reduce PCN-mediated lung injury in patients with chronic P. aeruginosa respiratory infections.

  5. AMP Kinase Activation Alters Oxidant-Induced Stress Granule Assembly by Modulating Cell Signaling and Microtubule Organization.

    PubMed

    Mahboubi, Hicham; Koromilas, Antonis E; Stochaj, Ursula

    2016-10-01

    Eukaryotic cells assemble stress granules (SGs) when translation initiation is inhibited. Different cell signaling pathways regulate SG production. Particularly relevant to this process is 5'-AMP-activated protein kinase (AMPK), which functions as a stress sensor and is transiently activated by adverse physiologic conditions. Here, we dissected the role of AMPK for oxidant-induced SG formation. Our studies identified multiple steps of de novo SG assembly that are controlled by the kinase. Single-cell analyses demonstrated that pharmacological AMPK activation prior to stress exposure changed SG properties, because the granules became more abundant and smaller in size. These altered SG characteristics correlated with specific changes in cell survival, cell signaling, cytoskeletal organization, and the abundance of translation initiation factors. Specifically, AMPK activation increased stress-induced eukaryotic initiation factor (eIF) 2α phosphorylation and reduced the concentration of eIF4F complex subunits eIF4G and eIF4E. At the same time, the abundance of histone deacetylase 6 (HDAC6) was diminished. This loss of HDAC6 was accompanied by increased acetylation of α-tubulin on Lys40. Pharmacological studies further confirmed this novel AMPK-HDAC6 interplay and its importance for SG biology. Taken together, we provide mechanistic insights into the regulation of SG formation. We propose that AMPK activation stimulates oxidant-induced SG formation but limits their fusion into larger granules. PMID:27430620

  6. NF-kB activity-dependent P-selectin involved in ox-LDL-induced foam cell formation in U937 cell

    SciTech Connect

    Wang, Yi; Wang, Xiang; Sun, Minghui; Zhang, Zhenyu; Cao, Heng; Chen, Xiaoqing

    2011-08-05

    Highlights: {yields} Ox-LDL induced foam cell formation in the human U937 promonocytic cell line in a dose- and time-dependent manner. {yields} Ox-LDL induced expression of P-selectin through degradation of IkBa and augment of NF-kB activity and protein level during macrophage-derived foam cell formation. {yields} P-selectin and NF-kB may be identified as pivotal regulators of ox-LDL-induced foam cell formation. {yields} Therapy based on the inhibition of P-selectin and NF-kB may complement conventional treatments to prevent atherosclerosis. -- Abstract: Oxidized low-density lipoprotein (ox-LDL) plays a critical role in regulation of atherosclerosis. However, little is known about the role of Nuclear factor kB (NF-kB) activity-dependent P-selectin in ox-LDL-induced foam cell formation during atherosclerosis. In this study, we first investigated ox-LDL induced foam cell formation in the human U937 promonocytic cell line in a dose- and time-dependent manner. Treatment of U937 cells with ox-LDL increased lipid accumulation as well as intracellular cholesterol content. Next, a comparative analysis of gene expression profiling using cDNA microarray and Real-time-PCR indicated that ox-LDL exposure induced, in three treated groups, an extremely marked increase in the mRNA level of P-selectin. Protein levels of P-selectin and its upstream regulators IkBa and NF-kB showed that NF-kB pathway is involved in the ox-LDL-induced foam cell formation. Finally, overexpression of NF-kB significantly accelerated, whereas, inhibition of NF-kB with siRNA remarkably attenuated ox-LDL-induced macrophage-derived foam cell formation. It was concluded that the activity of NF-kB is augmented during macrophage-derived foam cell formation. Activation of NF-kB increased, whereas, inhibition of NF-kB decreased ox-LDL-induced P-selectin expression and lipid accumulation in macrophages, suggesting ox-LDL induced expression of P-selectin through degradation of IkBa and activation of NF-kB in the

  7. Virion encapsidated HIV-1 Vpr induces NFAT to prime non-activated T cells for productive infection.

    PubMed

    Höhne, Kristin; Businger, Ramona; van Nuffel, Anouk; Bolduan, Sebastian; Koppensteiner, Herwig; Baeyens, Ann; Vermeire, Jolien; Malatinkova, Eva; Verhasselt, Bruno; Schindler, Michael

    2016-07-01

    The majority of T cells encountered by HIV-1 are non-activated and do not readily allow productive infection. HIV-1 Vpr is highly abundant in progeny virions, and induces signalling and HIV-1 LTR transcription. We hence hypothesized that Vpr might be a determinant of non-activated T-cell infection. Virion-delivered Vpr activated nuclear factor of activated T cells (NFAT) through Ca(2+) influx and interference with the NFAT export kinase GSK3β. This leads to NFAT translocation and accumulation within the nucleus and was required for productive infection of unstimulated primary CD4(+) T cells. A mutagenesis approach revealed correlation of Vpr-mediated NFAT activation with its ability to enhance LTR transcription and mediate cell cycle arrest. Upon NFAT inhibition, Vpr did not augment resting T-cell infection, and showed reduced G2/M arrest and LTR transactivation. Altogether, Vpr renders unstimulated T cells more permissive for productive HIV-1 infection and stimulates activation of productively infected as well as virus-exposed T cells. Therefore, it could be involved in the establishment and reactivation of HIV-1 from viral reservoirs and might have an impact on the levels of immune activation, which are determinants of HIV-1 pathogenesis.

  8. Virion encapsidated HIV-1 Vpr induces NFAT to prime non-activated T cells for productive infection

    PubMed Central

    Höhne, Kristin; Businger, Ramona; van Nuffel, Anouk; Bolduan, Sebastian; Koppensteiner, Herwig; Baeyens, Ann; Vermeire, Jolien; Malatinkova, Eva; Verhasselt, Bruno; Schindler, Michael

    2016-01-01

    The majority of T cells encountered by HIV-1 are non-activated and do not readily allow productive infection. HIV-1 Vpr is highly abundant in progeny virions, and induces signalling and HIV-1 LTR transcription. We hence hypothesized that Vpr might be a determinant of non-activated T-cell infection. Virion-delivered Vpr activated nuclear factor of activated T cells (NFAT) through Ca2+ influx and interference with the NFAT export kinase GSK3β. This leads to NFAT translocation and accumulation within the nucleus and was required for productive infection of unstimulated primary CD4+ T cells. A mutagenesis approach revealed correlation of Vpr-mediated NFAT activation with its ability to enhance LTR transcription and mediate cell cycle arrest. Upon NFAT inhibition, Vpr did not augment resting T-cell infection, and showed reduced G2/M arrest and LTR transactivation. Altogether, Vpr renders unstimulated T cells more permissive for productive HIV-1 infection and stimulates activation of productively infected as well as virus-exposed T cells. Therefore, it could be involved in the establishment and reactivation of HIV-1 from viral reservoirs and might have an impact on the levels of immune activation, which are determinants of HIV-1 pathogenesis. PMID:27383627

  9. Constitutively active Notch1 induces growth arrest of HPV-positive cervical cancer cells via separate signaling pathways

    SciTech Connect

    Talora, Claudio; Cialfi, Samantha; Segatto, Oreste; Morrone, Stefania; Kim Choi, John; Frati, Luigi; Paolo Dotto, Gian; Gulino, Alberto; Screpanti, Isabella . E-mail: isabella.screpanti@uniroma1.it

    2005-05-01

    Notch signaling plays a key role in cell-fate determination and differentiation in different organisms and cell types. Several reports suggest that Notch signaling may be involved in neoplastic transformation. However, in primary keratinocytes, Notch1 can function as a tumor suppressor. Similarly, in HPV-positive cervical cancer cells, constitutively active Notch1 signaling was found to cause growth suppression. Activated Notch1 in these cells represses viral E6/E7 expression through AP-1 down-modulation, resulting in increased p53 expression and a block of pRb hyperphosphorylation. Here we show that in cervical cancer cell lines in which Notch1 ability to repress AP-1 activity is impaired, Notch1-enforced expression elicits an alternative pathway leading to growth arrest. Indeed, activated Notch1 signaling suppresses activity of the helix-loop-helix transcription factor E47, via ERK1/2 activation, resulting in inhibition of cell cycle progression. Moreover, we found that RBP-J{kappa}-dependent Notch signaling is specifically repressed in cervical cancer cells and this repression could provide one such mechanism that needs to be activated for cervical carcinogenesis. Finally, we show that inhibition of endogenous Notch1 signaling, although results in a proliferative advantage, sensitizes cervical cancer cell lines to drug-induced apoptosis. Together, our results provide novel molecular insights into Notch1-dependent growth inhibitory effects, counteracting the transforming potential of HPV.

  10. Virion encapsidated HIV-1 Vpr induces NFAT to prime non-activated T cells for productive infection.

    PubMed

    Höhne, Kristin; Businger, Ramona; van Nuffel, Anouk; Bolduan, Sebastian; Koppensteiner, Herwig; Baeyens, Ann; Vermeire, Jolien; Malatinkova, Eva; Verhasselt, Bruno; Schindler, Michael

    2016-07-01

    The majority of T cells encountered by HIV-1 are non-activated and do not readily allow productive infection. HIV-1 Vpr is highly abundant in progeny virions, and induces signalling and HIV-1 LTR transcription. We hence hypothesized that Vpr might be a determinant of non-activated T-cell infection. Virion-delivered Vpr activated nuclear factor of activated T cells (NFAT) through Ca(2+) influx and interference with the NFAT export kinase GSK3β. This leads to NFAT translocation and accumulation within the nucleus and was required for productive infection of unstimulated primary CD4(+) T cells. A mutagenesis approach revealed correlation of Vpr-mediated NFAT activation with its ability to enhance LTR transcription and mediate cell cycle arrest. Upon NFAT inhibition, Vpr did not augment resting T-cell infection, and showed reduced G2/M arrest and LTR transactivation. Altogether, Vpr renders unstimulated T cells more permissive for productive HIV-1 infection and stimulates activation of productively infected as well as virus-exposed T cells. Therefore, it could be involved in the establishment and reactivation of HIV-1 from viral reservoirs and might have an impact on the levels of immune activation, which are determinants of HIV-1 pathogenesis. PMID:27383627

  11. Facile synthesis of autophagonizer and evaluation of its activity to induce autophagic cell death in apoptosis-defective cell line.

    PubMed

    Nguyen, Jennifer; Chen, Luxi; Kumar, Dhiraj; Lee, Jiyong

    2016-10-01

    Some cancer cells are resistant to apoptosis, rendering them irresponsive towards apoptosis-inducing chemotherapy drugs. Another mode of action to kill these apoptosis-defective cells is essential and autophagy, a dynamic process that degrades cytoplasmic contents for cellular maintenance, has been considered as one of the alternate routes. A small molecule inducer of autophagy, autophagonizer was reported to induce cell death through a novel process that is independent of extrinsic apoptosis and the normal signaling pathways of autophagy. Here, we describe an efficient synthetic procedure for the autophagonizer. The newly synthesized autophagonizer (DK-1-49) resulted in an accumulation of autophagy-associated LC3-II and enhanced levels of autophagosomes and acidic vacuoles. Furthermore, cell viability was inhibited by autophagic cell death in not only human cancer cells but also Bax/Bak double-knockout cells. These findings highlight that intrinsic apoptosis is not also involved in the induction of cellular death by the autophagonizer suggesting the autophagonizer is a promising candidate for anticancer therapeutics for cancer cells that are resistant to apoptosis-inducing chemotherapy. PMID:27597252

  12. T helper-1 lymphocytes induce dendritic cell tumor killing activity by an interferon-γ-dependent mechanism

    PubMed Central

    LaCasse, Collin J.; Janikashvili, Nona; Larmonier, Claire B.; Alizadeh, Darya; Hanke, Neale; Kartchner, Jessica; Situ, Elaine; Centuori, Sara; Har-Noy, Michael; Bonnotte, Bernard; Katsanis, Emmanuel; Larmonier, Nicolas

    2012-01-01

    Dendritic cells (DCs) encompass a heterogeneous population of cells capable of orchestrating innate and adaptive immune responses. The ability of DCs to act as professional antigen presenting cells has been the foundation for the development and utilization of these cells as vaccines in cancer immunotherapy. DCs are also endowed with the non-conventional property of directly killing tumor cells. The current study investigates the regulation of murine DC cytotoxic function by T lymphocytes. We provide evidence that CD4+ Th-1, but not Th-2, Th-17 cells or Treg are capable of inducing DC cytotoxic function. IFN-γ was identified as the major factor responsible for Th-1-induced DC tumoricidal activity. Tumor cell killing mediated by Th-1-activated killer DCs (Th-1 KDCs) was dependent on inducible nitric oxide synthase (iNOS) expression and nitric oxide (NO) production. Importantly, Th-1 KDCs were capable of presenting the acquired antigens from the killed tumor cells to T lymphocytes in vitro or in vivo. These observations open new possibilities for the application of KDCs in cancer immunotherapy. PMID:22075702

  13. Casticin induces ovarian cancer cell apoptosis by repressing FoxM1 through the activation of FOXO3a

    PubMed Central

    JIANG, LING; CAO, XIAO-CHENG; CAO, JIAN-GUO; LIU, FEI; QUAN, MEI-FANG; SHENG, XI-FENG; REN, KAI-QUN

    2013-01-01

    Casticin, a polymethoxyflavone, is reported to have anticancer activities. The aim of the present study was to examine the molecular mechanisms by which casticin induces apoptosis in ovarian cancer cells. The human ovarian cancer cell lines SKOV3 and A2780 were cultured in vitro. Various molecular techniques, including histone/DNA enzyme-linked immunosorbent assay (ELISA), reverse transcription polymerase chain reaction (RT-PCR), western blot analysis and gene transfection, were used to assess the expression of FOXO3a and forkhead box protein M1 (FoxM1) in casticin-treated ovarian cancer cell lines. Casticin-induced apoptotic cell death was accompanied by the activation of transcription factor FOXO3a, with a concomitant decrease in the expression levels of FoxM1 and its downstream target factors, namely survivin and polo-like kinase 1 (PLK1), and an increase in p27KIP1. A small inhibitory RNA (siRNA) knockout of FoxM1 potentiated casticin-induced apoptosis in ovarian cancer cells. Silencing FOXO3a expression using siRNA increased FoxM1 expression levels and clearly attenuated the induction of apoptosis by casticin treatment. These results show that casticin-induced apoptosis in ovarian cancer may be caused by the activation of FOXO3a, leading to FoxM1 inhibition. PMID:23761826

  14. Matrine-induced autophagy regulated by p53 through AMP-activated protein kinase in human hepatoma cells.

    PubMed

    Xie, Shan-Bu; He, Xing-Xing; Yao, Shu-Kun

    2015-08-01

    Matrine, one of the main extract components of Sophora flavescens, has been shown to exhibit inhibitory effects on some tumors through autophagy. However, the mechanism underlying the effect of matrine remains unclear. The cultured human hepatocellular carcinoma cell line HepG2 and SMMC‑7721 were treated with matrine. Signal transduction and gene expression profile were determined. Matrine stimulated autophagy in SMMC‑7721 cells in a mammalian target of rapamycin (mTOR)-dependent manner, but in an mTOR-independent manner in HepG2 cells. Next, in HepG2 cells, autophagy induced by matrine was regulated by p53 inactivation through AMP-activated protein kinase (AMPK) signaling transduction, then AMPK suppression switched autophagy to apoptosis. Furthermore, the interferon (IFN)-inducible genes, including interferon α-inducible protein 27 (IFI27) and interferon induced transmembrane protein 1 (IFITM1), which are downstream effector of p53, might be modulated by matrine-induced autophagy. In addition, we found that the p53 protein isoforms, p53β, p53γ, ∆133p53, and ∆133p53γ, due to alternative splicing of intron 9, might be regulated by the p53-mediated autophagy. These results show that matrine induces autophagy in human hepatoma cells through a novel mechanism, which is p53/AMPK signaling pathway involvement in matrine-promoted autophagy.

  15. Activation of salt-inducible kinase 2 promotes the viability of peritoneal mesothelial cells exposed to stress of peritoneal dialysis

    PubMed Central

    Wang, H-H; Lin, C-Y; Su, S-H; Chuang, C-T; Chang, Y-L; Lee, T-Y; Lee, S-C; Chang, C-J

    2016-01-01

    Maintaining mesothelial cell viability is critical to long-term successful peritoneal dialysis (PD) treatment. To clarify the viability mechanism of peritoneal mesothelial cells under PD solutions exposure, we examined the mechanisms of cellular response to this stress conditions. Here we report that the proteasome activity is inhibited when treated with PD solutions. Proteasome inhibition-mediated activation of salt-inducible kinase 2 (SIK2), an endoplasmic reticulum-resident protein, is important for mesothelial cell viability. SIK2 is mobilized to promote autophagy and protect the cells from apoptosis under PD solution or MG132 treatment. Immunofluorescence staining showed that SIK2 is colocalized with LC3B in the autophagosomes of mesothelial cells treated with PD solution or derived from patients undergoing PD treatment. SIK2 activation is likely via a two-step mechanism, upstream kinases relieving the autoinhibitory conformation of SIK2 molecule followed by autophosphorylation of Thr175 and activation of kinase activity. These results suggest that activation of SIK2 is required for the cell viability when proteasome activity is inhibited by PD solutions. Maintaining or boosting the activity of SIK2 may promote peritoneal mesothelial cell viability and evolve as a potential therapeutic target for maintaining or restoring peritoneal membrane integrity in PD therapy. PMID:27441650

  16. PROLACTIN-INDUCED TYROSINE PHOSPHORYLATION, ACTIVATION AND RECEPTOR ASSOCIATION OF FOCAL ADHESION KINASE (FAK) IN MAMMARY EPITHELIAL CELLS

    EPA Science Inventory

    Prolactin-Induced Tyrosine Phosphorylation, Activation and Receptor
    Association of Focal Adhesion Kinase (FAK) in Mammary Epithelial Cells.
    Suzanne E. Fenton1 and Lewis G. Sheffield2. 1U.S. Environmental Protection
    Agency, MD-72, Research Triangle Park, NC 27711, and

  17. Hsc70 facilitates TGF-β-induced activation of Smad2/3 in fibroblastic NRK-49F cells.

    PubMed

    Ikezaki, Midori; Higashimoto, Natsuki; Matsumura, Ko; Ihara, Yoshito

    2016-08-26

    Heat-shock cognate protein 70 (Hsc70), a molecular chaperone constitutively expressed in the cell, is involved in the regulation of several cellular signaling pathways. In this study, we found that TGF-β-induced phosphorylation and nuclear translocation of Smad2/3 were suppressed in fibroblastic NRK-49F cells treated with small interfering RNA (siRNA) for Hsc70. In the cells underexpressing Hsc70, transcriptional induction of connective tissue growth factor (CTGF), a target gene of the TGF-β signaling, was also suppressed in the early phase of TGF-β stimulation. Upon stimulation with TGF-β, Hsc70 interacted with Smad2/3, suggesting functional interactions of Hsc70 and Smad2/3 for the activation of TGF-β-induced Smad signaling. Although the expression of heat-shock protein 70 (Hsp70) was upregulated in the cells treated with Hsc70 siRNA, TGF-β-induced Smad activation was not affected in the cells overexpressing Hsp70. Collectively, these results indicate that Hsc70, but not Hsp70, supportively regulates TGF-β-induced Smad signaling in NRK-49F cells.

  18. Non-genomic immunosuppressive actions of progesterone inhibits PHA-induced alkalinization and activation in T cells.

    PubMed

    Chien, Eileen Jea; Chang, Ching-Pang; Lee, Wen-Feng; Su, Tsung-Hsien; Wu, Chia-Hsun

    2006-09-01

    Progesterone is an endogenous immunomodulator, and can suppress T-cell activation during pregnancy. When analyzed under a genome time scale, the classic steroid receptor pathway does not have any effect on ion fluxes. Therefore, the aim of this study was to investigate whether the non-genomic effects on ion fluxes by progesterone could immunosuppress phytohemagglutinin (PHA)-induced human peripheral T-cell activation. The new findings indicated that, first, only progesterone stimulated both [Ca2+]i elevation and pHi decrease; in contrast, estradiol or testosterone stimulated [Ca2+]i elevation and hydrocortisone or dexamethasone stimulated pHi decrease. Secondly, the [Ca2+]i increase by progesterone was dependent on Ca2+ influx, and the acidification was blocked by Na+/H+ exchange (NHE) inhibitor, 3-methylsulphonyl-4-piperidinobenzoyl, guanidine hydrochloride (HOE-694) but not by 5-(N,N-dimethyl)-amiloride (DMA). Thirdly, progesterone blocked phorbol 12-myristate 13-acetate (PMA) or PHA-induced alkalinization, but PHA did not prevent progesterone-induced acidification. Fourthly, progesterone did not induce T-cell proliferation; however, co-stimulation progesterone with PHA was able to suppress PHA-induced IL-2 or IL-4 secretion and proliferation. When progesterone was applied 72 h after PHA stimulation, progesterone could suppress PHA-induced T-cell proliferation. Finally, immobilization of progesterone by conjugation to a large carrier molecule (BSA) also stimulated a rapid [Ca2+]i elevation, pHi decrease, and suppressed PHA-induced proliferation. These results suggested that the non-genomic effects of progesterone, especially acidification, are exerted via plasma membrane sites and suppress the genomic responses to PHA. Progesterone might act directly through membrane specific nonclassical steroid receptors to cause immunomodulation and suppression of T-cell activation during pregnancy.

  19. Ganoderma lucidum stimulates NK cell cytotoxicity by inducing NKG2D/NCR activation and secretion of perforin and granulysin.

    PubMed

    Chang, Chih-Jung; Chen, Yi-Yuan M; Lu, Chia-Chen; Lin, Chuan-Sheng; Martel, Jan; Tsai, Sheng-Hui; Ko, Yun-Fei; Huang, Tsung-Teng; Ojcius, David M; Young, John D; Lai, Hsin-Chih

    2014-04-01

    Ganoderma lucidum (G. lucidum) is a medicinal mushroom long used in Asia as a folk remedy to promote health and longevity. Recent studies indicate that G. lucidum activates NK cells, but the molecular mechanism underlying this effect has not been studied so far. To address this question, we prepared a water extract of G. lucidum and examined its effect on NK cells. We observed that G. lucidum treatment increases NK cell cytotoxicity by stimulating secretion of perforin and granulysin. The mechanism of activation involves an increased expression of NKG2D and natural cytotoxicity receptors (NCRs), as well as increased phosphorylation of intracellular MAPKs. Our results indicate that G. lucidum induces NK cell cytotoxicity against various cancer cell lines by activating NKG2D/NCR receptors and MAPK signaling pathways, which together culminate in exocytosis of perforin and granulysin. These observations provide a cellular and molecular mechanism to account for the reported anticancer effects of G. lucidum extracts in humans.

  20. γ-Tocotrienol induces apoptosis in human T cell lymphoma through activation of both intrinsic and extrinsic pathways.

    PubMed

    Wilankar, Chandan; Khan, Nazir M; Checker, Rahul; Sharma, Deepak; Patwardhan, Raghavendra; Gota, Vikram; Sandur, Santosh Kumar; Devasagayam, T P A

    2011-01-01

    Tocotrienols are members of vitamin E family and possess broad biological activities including antioxidant, anti-inflammatory and antitumor effects. In the present study, we examine the potential of α-tocotrienol (AT) and γ-tocotrienol (GT) in inhibiting the proliferation of human T cell lymphoma Jurkat cells and elucidate the pathways involved in anti tumor effects of GT. GT but not AT inhibited proliferation and induced apoptosis in Jurkat cells in a dose dependent manner. GT treatment resulted in elevated mitochondrial ROS production, activation of JNK and suppression of ERK and p38 MAPK. GT also induced calcium release, loss of mitochondrial membrane potential and cytochrome c release from the mitochondria. These changes were accompanied by increase in Bax expression with a concomitant decrease in Bcl-xl expression suggesting activation of mitochondrial apoptotic pathway. GT induced increase in mitochondrial ROS was abrogated by catalase. Besides, GT also up-regulated surface expression of Fas and FasL on Jurkat cells. Further, caspase activation and PARP degradation were also seen in cells treated with GT. Inhibitors of caspase-8 and caspase-9 significantly abrogated GT mediated apoptosis. In contrast GT was not toxic to normal human peripheral blood mononuclear cells suggesting differential cytotoxicity towards normal lymphocytes and transformed lymphoma cells. Cellular uptake studies with tocotrienols showed higher intracellular accumulation of GT as compared to AT which may be responsible for its better antitumor activity. Our results show antitumor effects of GT in human lymphoma cells via increased mitochondrial ROS generation and activation of both intrinsic and extrinsic apoptotic pathways.

  1. Butyrate-induced proapoptotic and antiangiogenic pathways in EAT cells require activation of CAD and downregulation of VEGF

    SciTech Connect

    Belakavadi, Madesh . E-mail: belakama@umdnj.edu; Prabhakar, B.T.; Salimath, Bharathi P.

    2005-10-07

    Butyrate, a short-chain fatty acid produced in the colon, induces cell cycle arrest, differentiation, and apoptosis in transformed cell lines. In this report, we study the effects of butyrate (BuA) on the growth of Ehrlich ascites tumor (EAT) cells in vivo. BuA, when injected intraperitoneally (i.p) into mice, inhibited proliferation of EAT cells. Further, induction of apoptosis in EAT cells was monitored by nuclear condensation, annexin-V staining, DNA fragmentation, and translocation of caspase-activated DNase into nucleus upon BuA-treatment. Ac-DEVD-CHO, a caspase-3 inhibitor, completely inhibited BuA-induced apoptosis, indicating that activation of caspase-3 mediates the apoptotic pathway in EAT cells. The proapoptotic effect of BuA also reflects on the antiangiogenic pathway in EAT cells. The antiangiogenic effect of BuA in vivo was demonstrated by the downregulation of the secretion of VEGF in EAT cells. CD31 immunohistochemical staining of peritoneum sections clearly indicated a potential angioinhibitory effect of BuA in EAT cells. These results suggest that BuA, besides regulating other fundamental cellular processes, is able to modulate the expression/secretion of the key angiogenic growth factor VEGF in EAT cells.

  2. Romidepsin reduces histone deacetylase activity, induces acetylation of histones, inhibits proliferation, and activates apoptosis in immortalized epithelial endometriotic cells.

    PubMed

    Imesch, Patrick; Fink, Daniel; Fedier, André

    2010-12-01

    Romidepsin inhibited HDAC activity, produced acetylation of the histone proteins, up-regulated p21, and down-regulated cyclins B1 and D1, resulting in proliferation inhibition and apoptosis activation in 11z immortalized epithelial endometriotic cells. Our findings provide evidence that endometriotic cells are sensitive to the epigenetic effects of romidepsin and suggest that endometriosis may be therapeutically targeted by romidepsin.

  3. Substrate-induced PC12 cell differentiation without filopodial, lamellipodial activity or NGF stimulationa.

    PubMed

    Lamour, Guillaume; Souès, Sylvie; Hamraoui, Ahmed

    2015-03-01

    Nanoscale gradients in energy of adhesion are physical cues from the extracellular environment that can significantly affect cell functions and enhance the neuronal differentiation of PC12 cells. How such surface effects can trigger differentiation and initiate neurite outgrowth, remains to be elucidated. Here we used surface modification, atomic force microscopy and immunofluorescence to analyze PC12 cells. We studied the kinetics of neurites growth under cytochalasin-B treatment, known as an inhibitor of actin polymerization. We found that neither filopodia nor lamellipodia are involved in detecting the surface effects that induce the differentiation of PC12 cells. This finding suggests that the solution to this problem lies beyond identifying a precise cytoskeleton-associated cell-substrate intermediate. Thus, a more comprehensive model is probably required to identify the mechanism by which cell-substrate interactions are eventually translated into a differentiation signal. PMID:25350917

  4. SILAC-Based Mass Spectrometry Analysis Reveals That Epibrassinolide Induces Apoptosis via Activating Endoplasmic Reticulum Stress in Prostate Cancer Cells

    PubMed Central

    2015-01-01

    Epibrassinolide (EBR) is a polyhydroxylated sterol derivative and biologically active compound of the brassinosteroids. In addition to well-described roles in plant growth, EBR induces apoptosis in the LNCaP prostate cancer cells expressing functional androgen receptor (AR). Therefore, it is suggested that EBR might have an inhibitory potential on androgen receptor signaling pathway. However, the mechanism by which EBR exerts its effects on LNCaP is poorly understood. To address this gap in knowledge, we used an unbiased global proteomics approach, i.e., stable-isotope labeling by amino acids in cell culture (SILAC). In total, 964 unique proteins were identified, 160 of which were differentially expressed after 12 h of EBR treatment. The quantification of the differentially expressed proteins revealed that the expression of the unfolded protein response (UPR) chaperone protein, calreticulin (CALR), was dramatically downregulated. The decrease in CALR expression was also validated by immunoblotting. Because our data revealed the involvement of the UPR in response to EBR exposure, we evaluated the expression of the other UPR proteins. We demonstrated that EBR treatment downregulated calnexin and upregulated BiP and IRE1α expression levels and induced CHOP translocation from the cytoplasm to nucleus. The translocation of CHOP was associated with caspase-9 and caspase-3 activation after a 12 h EBR treatment. Co-treatment of EBR with rapamycin, an upstream mTOR pathway inhibitor, prevented EBR-induced cell viability loss and PARP cleavage in LNCaP prostate cancer cells, suggesting that EBR could induce ER stress in these cells. In addition, we observed similar results in DU145 cells with nonfunctional androgen receptor. When proteasomal degradation of proteins was blocked by MG132 co-treatment, EBR treatment further induced PARP cleavage relative to drug treatment alone. EBR also induced Ca2+ sequestration, which confirmed the alteration of the ER pathway due to drug