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PARP Cleavage (parp + cleavage)
Selected AbstractsRole of GSK-3, activity in motor neuronal cell death induced by G93A or A4V mutant hSOD1 geneEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 2 2005Seong-Ho Koh Abstract Point mutations such as G93A and A4V in the human Cu/Zn-superoxide dismutase gene (hSOD1) cause familial amyotrophic lateral sclerosis (fALS). In spite of several theories to explain the pathogenic mechanisms, the mechanism remains largely unclear. Increased activity of glycogen synthase kinase-3 (GSK-3) has recently been emphasized as an important pathogenic mechanism of neurodegenerative diseases, including Alzheimer's disease and ALS. To investigate the effects of G93A or A4V mutations on the phosphatidylinositol-3-kinase (PI3-K)/Akt and GSK-3 pathway as well as the caspase-3 pathway, VSC4.1 motoneuron cells were transfected with G93A- or A4V-mutant types of hSOD1 (G93A and A4V cells, respectively) and, 24 h after neuronal differentiation, their viability and intracellular signals, including PI3-K/Akt, GSK-3, heat shock transcription factor-1 (HSTF-1), cytochrome c, caspase-3 and poly(ADP-ribose) polymerase (PARP), were compared with those of wild type (wild cells). Furthermore, to elucidate the role of the GSK-3,-mediated cell death mechanism, alterations of viability and intracellular signals in those mutant motoneurons were investigated after treating the cells with GSK-3, inhibitor. Compared with wild cells, viability was greatly reduced in the G93A and A4V cells. However, the treatment of G93A and A4V cells with GSK-3, inhibitor increased their viability by activating HSTF-1 and by reducing cytochrome c release, caspase-3 activation and PARP cleavage. However, the treatment did not affect the expression of PI3-K/Akt and GSK-3,. These results suggest that the G93A or A4V mutations inhibit PI3-K/Akt and activate GSK-3, and caspase-3, thus becoming vulnerable to oxidative stress, and that the GSK-3,-mediated cell death mechanism is important in G93A and A4V cell death. [source] Cyclooxygenase-2 inhibitor celecoxib augments chemotherapeutic drug-induced apoptosis by enhancing activation of caspase-3 and -9 in prostate cancer cellsINTERNATIONAL JOURNAL OF CANCER, Issue 3 2005Devendra S. Dandekar Abstract Many tumors constitutively express high levels of the inducible form of proinflammatory enzyme, cyclooxygenase-2 (COX-2). Increased COX-2 expression is associated with tumor cell resistance to many cytotoxic chemotherapy drugs. Furthermore, increased resistance to cytotoxic antitumor drugs is also known to be dependent on associated stromal cells in many tumors. We investigated whether prostate tumor-associated stromal cells, marrow-derived osteoblasts, affect cytotoxicity of 2 antitumor drugs, COL-3 and docetaxel (TXTR), and whether it is dependent on COX-2 activity. We further examined whether inhibiting the activity of COX-2 negate the stroma-induced decrease in drug sensitivity in tumor cells. COX-2-specific inhibitor celecoxib (CXB) was used to inhibit COX-2 activity and associated alteration in cell death signaling was investigated. Coculturing PC-3ML cells with osteoblasts decreased the cytotoxicity of the tested antitumor drugs and was associated with increased COX-2 activity in PC-3ML cells. A significant decrease in drug-induced PGE2 increase and an increase in cytotoxicity were observed when cells were treated with COL-3 or TXTR combined with CXB. Cytotoxicity of single or combination treatment increased apoptosis, which was associated with caspase-3 and -9 activation, PARP cleavage, increased BAD protein, but decreased protein levels of XIAP and BCL- xL. Oral administration of CXB (40 mg/kg) to mice with PC-3ML tumors for 42 days increased tumor latency, decreased tumor growth and enhanced tumor control with COL-3 or TXTR. Overall, a synergistic enhancement of antitumor activity in combination treatment was observed in vitro and an additive effect in vivo. These observations suggest a potential clinical use of combined dosing of COX-2 inhibitors and cytotoxic drugs at lower, nontoxic dose than currently used to treat advanced prostate cancer. © 2005 Wiley-Liss, Inc. [source] Lipophilic but not hydrophilic statins selectively induce cell death in gynaecological cancers expressing high levels of HMGCoA reductaseJOURNAL OF CELLULAR AND MOLECULAR MEDICINE, Issue 5 2010S. Kato Abstract Recent reports have suggested that statins induce cell death in certain epithelial cancers and that patients taking statins to reduce cholesterol levels possess lower cancer incidence. However, little is known about the mechanisms of action of different statins or the effects of these statins in gynaecological malignancies. The apoptotic potential of two lipophilic statins (lovastatin and simvastatin) and one hydrophilic statin (pravastatin) was assessed in cancer cell lines (ovarian, endometrial and cervical) and primary cultured cancerous and normal tissues. Cell viability was studied by MTS assays and apoptosis was confirmed by Western blotting of PARP and flow cytometry. The expressions of key apoptotic cascade proteins were analysed. Our results demonstrate that both lovastatin and simvastatin, but not pravastatin, selectively induced cell death in dose- and time-dependent manner in ovarian, endometrial and cervical cancers. Little or no toxicity was observed with any statin on normal cells. Lipophilic statins induced activation of caspase-8 and -9; BID cleavage, cytochrome C release and PARP cleavage. Statin-sensitive cancers expressed high levels of HMG-CoA reductase compared with resistant cultures. The effect of lipophilic statins was dependent on inhibition of enzymatic activity of HMG-CoA reductase since mevalonate pre-incubation almost completely abrogated the apoptotic effect. Moreover, the apoptotic effect involved the inhibition of synthesis of geranylgeranyl pyrophosphate rather than farnesyl pyrophosphate. In conclusion, lipophilic but not hydrophilic statins induce cell death through activation of extrinsic and intrinsic apoptotic cascades in cancerous cells from the human female genital tract, which express high levels of HMG-CoA reductase. These results promote further investigation in the use of lipophilic statins as anticancer agents in gynaecological malignancies. [source] Differential apoptotic response of J774 macrophages to alumina and ultra-high-molecular-weight polyethylene particlesJOURNAL OF ORTHOPAEDIC RESEARCH, Issue 1 2002Alain Petit We recently identified apoptosis in in vitro wear particle-stimulated macrophages. The recent explosion of interest in apoptosis lies in the fact that it is under positive and negative regulation through evolutionary conserved biochemical pathways. It may also be possible to modulate macrophage apoptosis in the treatment of periprosthetic osteolysis. The purpose of this study was to compare the macrophage response to identically sized particles of alumina ceramic (Al2O3) and ultra-high-molecular-weight polyethylene (UHMWPE) in terms of TNF-, release and induction of apoptosis. J774 mouse macrophages were incubated for 0,24 h in the presence of Al2O3 and UHMWPE particles. TNF-, release was measured by ELISA; Poly(ADP-ribose)polymerase (PARP) and caspase-3 expression was analyzed by Western blot; DNA fragmentation (DNA laddering) was visualized on agarose gel containing ethidium bromide. Al2O3 particles induced TNF-, release after 4 h incubation with concentrations reaching 483 and 800 pg/ml after 24 h with 125 and 250 particles/macrophage, respectively (control = 161 pg/ml) (P < 0.05 vs. control). The same concentrations of UHMWPE particles induced a much larger and significant TNF-, release after only 1 h incubation, increasing up to 6250 pg/ml after 24 h (P < 0.05 vs. control). Western blot analysis demonstrated that the active caspase-3 fragment (17 kDa) and the proteolytic PARP fragment (85 kDa) were expressed after 2 h incubation with 125 and 250 Al2O3 particles/macrophage. The active caspase-3 and the PARP fragment had lower expression and appeared after a longer incubation time (8 h) with 125 and 250 UHMWPE particles/macrophage. Finally, DNA fragmentation (DNA laddering) was observed after 16 h with 125 and 250 particles of Al2O3 per macrophage whereas no laddering was induced by UHMWPE particles even after 24 h incubation. This study shows that although both Al2O3 and UHMWPE particles induce TNF-, release, this stimulation was much greater (8,10 times higher) with UHMWPE than A12O3 (P < 0.05 vs. control). As well, the induction of apoptosis, as measured by activation of caspase-3, PARP cleavage and DNA laddering, is different for these two particles, being faster and more important with Al2O3 than UHMWPE. We hypothesize that the ability of Al2O3 to induce macrophage apoptosis may explain the lower TNF-, release observed with these particles and explain the differences seen in osteolysis patterns of ceramic,ceramic (CC) vs. metal,polyethylene (Mpe) articulations. In conclusion, apoptosis may be a major internal mechanism to decrease macrophage activity and may be a desired therapeutic endpoint. The identification of an apoptosis-related pathway in the macrophage response to ceramic particles provides crucial data for a rational approach in the treatment and/or prevention of periprosthetic osteolysis. © 2002 Orthopaedic Research Society. Published by Elsevier Science Ltd. All rights reserved. [source] Participation of various kinases in staurosporine-induced apoptosis of RAW 264.7 cellsJOURNAL OF PHARMACY AND PHARMACOLOGY: AN INTERNATI ONAL JOURNAL OF PHARMACEUTICAL SCIENCE, Issue 11 2002Kouya Yamaki Staurosporine induced apoptosis of RAW 264.7 cells, a mouse macrophage-like cell line, as determined by DNA fragmentation, the increase of annexin V-stained cells, and the cleavage of poly(ADP- ribose)polymerase (PARP), a substrate of caspase. Analysis of the increase in the percentage of sub-G1 cells revealed that the DNA fragmentation occurred in a time- and concentration-dependent manner at 0.021,2.1 ,m of staurosporine. Staurosporine induced phosphorylation of p38 mitogen-activated protein kinase (MAPK) but suppressed spontaneous phosphorylation of p44/42 MAPK. The p38 MAPK inhibitor SB203580, the MAPK/extracellular signal-regulated kinase kinase inhibitor PD98059 and the phosphatidylinositol 3-kinase (P13K) inhibitor LY294002 potentiated the staurosporine-induced PARP cleavage and DNA fragmentation. The protein kinase A (PKA) inhibitor H-89 potentiated the staurosporine-induced DNA fragmentation without potentiating the PARP cleavage. In contrast, the protein kinase C (PKC) inhibitor Ro-31,8425 suppressed the PARP cleavage and DNA fragmentation. These findings suggested that staurosporine induces apoptosis via the caspase cascade in RAW 264.7 cells. The staurosporine-induced apoptosis is positively regulated by PKC, negatively regulated by p38 MAPK, p44/42 MAPK and P13K via the caspase cascade, and negatively regulated by PKA without regulation of caspase activation. [source] Betulin induces mitochondrial cytochrome c release associated apoptosis in human cancer cellsMOLECULAR CARCINOGENESIS, Issue 7 2010Yang Li Abstract We examined whether betulin, a naturally abundant compound, has anticancer functions in human cancer cells. The results showed that betulin significantly inhibited cell viability in cervix carcinoma HeLa cells, hepatoma HepG2 cells, lung adenocarcinoma A549 cells, and breast cancer MCF-7 cells with IC50 values ranging from 10 to 15,µg/mL. While betulin exhibited only moderate anticancer activity in other human cancer cells such as hepatoma SK-HEP-1 cells, prostate carcinoma PC-3, and lung carcinoma NCI-H460, with IC50 values ranging from 20 to 60,µg/mL, it showed minor growth inhibition in human erythroleukemia K562 cells (IC50,>,100,µg/mL). We further investigated the mechanism of anticancer activity by betulin, using HeLa cells as an experimental model. Betulin (10,µg/mL) induces apoptotic cell death, as evidenced by morphological characteristics such as membrane phosphatidylserine translocation, nuclear condensation/fragmentation, and apoptotic body formation. A kinetics analysis showed that the depolarization of mitochondrial membrane potential and the release of mitochondrial cytochrome c occurred as early as 30,min after treatment with betulin. Betulin, unlike its chemical derivative betulinic acid, did not directly trigger mitochondrial cytochrome c release in isolated mitochondria. Importantly, Bax and Bak were rapidly translocated to the mitochondria 30,min after betulin treatment. The sequential activation of caspase-9 and caspase-3/-7 and the cleavage of poly(ADP-ribose) polymerase (PARP) were observed behind those mitochondrial events. Furthermore, specific downregulation of either caspase-9, Bax, or Bak by siRNA effectively reduced PARP cleavage and caspase-3 activation. Taken together, the lines of evidence demonstrate that betulin triggers apoptosis of human cancer cells through the intrinsic apoptotic pathway. © 2010 Wiley-Liss, Inc. [source] Inositol hexaphosphate downregulates both constitutive and ligand-induced mitogenic and cell survival signaling, and causes caspase-mediated apoptotic death of human prostate carcinoma PC-3 cells,MOLECULAR CARCINOGENESIS, Issue 1 2010Mallikarjuna Gu Abstract Constitutively active mitogenic and prosurvival signaling cascades due to aberrant expression and interaction of growth factors and their receptors are well documented in human prostate cancer (PCa). Epidermal growth factor (EGF) and insulin-like growth factor-1 (IGF-1) are potent mitogens that regulate proliferation and survival of PCa cells via autocrine and paracrine loops involving both mitogen-activated protein kinase (MAPK)- and Akt-mediated signaling. Accordingly, here we assessed the effect of inositol hexaphosphate (IP6) on constitutive and ligand (EGF and IGF-1)-induced biological responses and associated signaling cascades in advanced and androgen-independent human PCa PC-3 cells. Treatment of PC-3 cells with 2,mM IP6 strongly inhibited both growth and proliferation and decreased cell viability; similar effects were also observed in other human PCa DU145 and LNCaP cells. IP6 also caused a strong apoptotic death of PC-3 cells together with caspase 3 and PARP cleavage. Mechanistic studies showed that biological effects of IP6 were associated with inhibition of both constitutive and ligand-induced Akt phosphorylation together with a decrease in total Akt levels, but a differential inhibitory effect on MAPKs extra cellular signal-regulated kinase 1/2 (ERK1/2), c- Jun N-terminal protein kinase (JNK1/2), and p38 under constitutive and ligand-activated conditions. Under similar condition, IP6 also inhibited AP-1 DNA-binding activity and decreased nuclear levels of both phospho and total c-Fos and c- Jun. Together, these findings for the first time establish IP6 efficacy in inhibiting aberrant EGF receptor (EGFR) or IGF-1 receptor (IGF-1R) pathway-mediated sustained growth promoting and survival signaling cascades in advanced and androgen-independent human PCa PC-3 cells, which might have translational implications in advanced human PCa control and management. © 2009 Wiley-Liss, Inc. [source] Cooperative inhibitory effect of ZD1839 (Iressa) in combination with 17-AAG on glioma cell growth,MOLECULAR CARCINOGENESIS, Issue 5 2006Daniel R. Premkumar Abstract ZD1839 ("Iressa") is an orally active, selective epidermal growth factor (EGF) receptor-tyrosine kinase inhibitor. We evaluated the antitumor activity of ZD1839 in combination with HSP90 antagonist, 17-AAG in malignant human glioma cell lines. ZD1839 independently produced a dose-dependent inhibition of cellular proliferation in glioma cells grown in culture with time- and dose-dependent accumulation of cells in G1 phase of the cell cycle on flow cytometric analysis, although the concentrations required for optimal efficacy were at or above the limits of clinically achievable levels. Because the heat shock protein (HSP) is involved in the conformational maturation of a number of signaling proteins critical to the proliferation of malignant glioma cells, we hypothesized that the HSP90 inhibitor 17-AAG would potentiate ZD 1839-mediated glioma cytotoxicity by decreasing the activation status of EGF receptor, as well as downregulating the levels of other relevant signaling effectors. We, therefore, examined the effects of ZD1839 and 17-AAG, alone and in combination, on signal transduction and apoptosis in a series of malignant glioma cell lines. Simultaneous exposure to these inhibitors significantly induced cell death and quantitative analysis revealed that interaction between ZD1839 and 17-AAG-induced cytotoxicity was synergistic, leading to a pronounced increase in active caspase-3 and PARP cleavage. No significant growth inhibition or caspase activation was seen in control cells. The enhanced cytotoxicity of this combination was associated with diminished Akt activation and a significant downregulation of EGFR receptor, Raf-1 and mitogen activated protein kinase (MAPK). Cells exposed to 17-AAG and ZD1839 displayed a significant reduction in cell cycle regulatory proteins, such as CDK4 and CDK6. Taken together, these findings suggest that ZD1839, an EGF receptor tyrosine kinase inhibitor, plays a critical role in regulating the apoptotic response to 17-AAG and that multi-site targeting of growth signaling and cell survival pathways could provide a potent strategy to treat patients with malignant gliomas. © 2006 Wiley-Liss, Inc. [source] Adverse effects associated with persistent stimulation of Leydig cells with hCG in vitroMOLECULAR REPRODUCTION & DEVELOPMENT, Issue 11 2009Archana Aggarwal The detrimental effects of persistent stimulation with hCG were investigated in rat Leydig cells in vitro. Significant rise in lipid peroxidation and reactive oxygen species (ROS) with concomitant attenuation in the activities of antioxidant enzymes: superoxide dismutase, catalase, and glutathione- S -transferase was observed. Transcripts for catalase and superoxide dismutase were also depleted. Subsequent to each hCG challenge, the total antioxidant capacity in the target cells also declined significantly (P,<,0.05). There was an increase in cell apoptosis (23%), which was associated with a rise in caspase-3 activity, PARP cleavage, and Fas, FasL, caspase-8 expression. While Bax and Caspase-9 expression remained unchanged, Bcl-2 demonstrated a marked decline. Taken together, the above data indicate that persistent hCG stimulation of Leydig cells induced adverse effects leading to oxidative stress and apoptosis which was channeled primarily through the extrinsic pathway. Mol. Reprod. Dev. 76: 1076,1083, 2009. © 2009 Wiley-Liss, Inc. [source] Downmodulation of Bcl-2 sensitizes metastatic LNCaP-LN3 cells to undergo apoptosis via the intrinsic pathwayTHE PROSTATE, Issue 6 2010Renduo Song Abstract BACKGROUND We explored the mechanisms of apoptosis after Bcl-2 protein downmodulation in metastatic LNCaP-LN3 cells (LN3). METHODS LNCaP, LNCaP-Pro5 (Pro5) and LN3 cells were cultured in 5% charcoal-stripped serum (CSS) or in R1881 (synthetic androgen) and bicalutamide (synthetic anti-androgen) and growth inhibition was assessed. Expression levels of androgen receptor (AR) and Bcl-2 were determined. LN3 cells were transfected with small interfering RNA Bcl-2 (siRNA Bcl-2) or control siRNA oligonucleotides. Rates of apoptosis and proliferation were obtained. Cytochrome c localization in treated and control cells was assessed,±,cyclosporine A (CsA). Caspases 9, 3, and poly (ADP-ribose) polymerase cleavage (PARP) were measured upon downmodulation of Bcl-2; and cell growth inhibition in vitro after Bcl-2 modulation combined with docetaxel chemotherapy was determined. RESULTS LN3 cells maintained growth under castrate conditions in vitro. AR protein amplification did not explain castrate-resistant LN3 cell growth. Bcl-2 protein levels in LN3 cells were significantly higher than in Pro5 cells, and were effectively downmodulated by siRNA Bcl-2. Subsequently increased apoptosis and decreased proliferation mediated by cytochrome c was noted and this was reversed by CsA. siRNA Bcl-2-transfected LN3 cells exhibited elevated levels of caspases 9, 3, and PARP cleavage. Exposure of LN3 cells to docetaxel led to increased apoptosis, and simultaneous downmodulation of Bcl-2 substantially enhanced this effect. CONCLUSIONS Downmodulation of Bcl-2 in metastatic castrate-resistant LNCaP-LN3 cells led to apoptosis via a cytochrome c -dependent pathway that was enhanced with docetaxel treatment. Prostate 70: 571,583, 2010. © 2009 Wiley-Liss, Inc. [source] p53 is dispensable for the induction of apoptosis after inhibition of protein kinase CK2THE PROSTATE, Issue 2 2010Carolin C. Schneider Abstract BACKGROUND Protein kinase CK2 is a ubiquitously expressed heterotetramer consisting of two catalytic ,/,, and two regulatory , subunits. Expression of CK2 is highly elevated in tumor cells where it protects cells from apoptosis. A variety of different compounds were tested as inhibitors of protein kinase CK2 in order to find new therapy strategies. To analyze the role of p53 in the response to CK2 inhibition we used one of the most specific CK2 inhibitors available, TBB, in different prostate cancer cell lines. METHODS We treated prostate cancer cells with the CK2 inhibitor TBB and determined its effect on CK2 activity by an in vitro phosphorylation assay and its effect on viability by an MTT assay. Furthermore, we analyzed changes in the expression of p53 and PARP cleavage by Western Blot analysis. RESULTS Inhibition of CK2 by TBB led to a decrease in cell viability and apoptosis in two cell lines which express wild-type p53 whereas two other cell lines expressing mutant or no p53 failed to show signs of apoptosis. Moreover, cell lines expressing wild-type p53 showed an increase of the amount of p53 and of its transactivation efficiency. However, down-regulation of p53 by RNAi showed that p53 is not necessary for the induction of apoptosis. CONCLUSIONS Wild-type p53 is not necessary for the induction of apoptosis by TBB in prostate cancer cells. Prostate 70: 126,134, 2010. ©2009 Wiley-Liss, Inc. [source] Experimental therapy of prostate cancer with novel natural product anti-cancer ginsenosides,THE PROSTATE, Issue 8 2008Wei Wang Abstract BACKGROUND Ginseng and its components exert various biological effects, including antioxidant, anti-carcinogenic, anti-mutagenic, and anti-tumor activity, and recent research has focused on their value in human cancer prevention and treatment. We recently isolated 25-hydroxyprotopanaxadiol (25-OH-PPD) and 25-hydroxyprotopanaxatriol (25-OH-PPT) from Panax ginseng and evaluated their anti-cancer activity in vitro. METHODS We compared the effects of the two compounds on human prostate cancer LNCaP and PC3 cells in vitro and in a mouse PC3 xenograft tumor model. We also accomplished a preliminary determination of the mechanisms of action of the compounds. RESULTS 25-OH-PPD, but not 25-OH-PPT, inhibited prostate cancer cell growth and proliferation, induced apoptosis, and led to arrest in the G1 phase of the cell cycle. In nude mice bearing PC3 xenograft tumors, 25-OH-PPD inhibited tumor growth in a dose-dependent manner and could be safely combined with chemotherapeutic agents (taxotere and gemcitabine) and radiation therapy to improve the anti-tumor effects. Further, in both PC3 and LNCaP cell lines, 25-OH-PPD increased expression of p21, p27, and Bax, induced PARP cleavage and activated caspases. The compound also reduced expression of MDM2, E2F1, Bcl2, cdk2/4/6, and cyclin D1, which correlated with the cell cycle arrest in G1 and the decrease in proliferation. Moreover, 25-OH-PPD demonstrated low toxicity to non-cancer cells and no observable host toxicity in animals either alone or in combination with conventional therapies. CONCLUSIONS The newly identified ginsenoside 25-OH-PPD may have potential as a novel prostate cancer therapeutic agent. Prostate 68:809,819, 2008. © 2008 Wiley-Liss, Inc. [source] Contribution of death receptor and mitochondrial pathways to Fas-mediated apoptosis in the prostatic carcinoma cell line PC3THE PROSTATE, Issue 4 2002Natalya V. Guseva Abstract BACKGROUND Two main pathways of apoptosis in mammalian cells have been described: the death receptor pathway and the mitochondrial pathway. Two different cell types have been identified for Fas-mediated apoptosis, each using almost exclusively one of two different signaling pathways. Human prostatic carcinoma cell line, PC3 is sensitive to Fas-mediated apoptosis, but relation of receptor and mitochondrial pathways is not clear. METHODS Cell viability was estimated by calcein assay. Apoptosis was determined by preparation of DNA ladder. Expression of Fas-associated death domain-dominant negative (FADD-DN) and Bcl-2, activation of caspases, PARP, DFF45, Bid cleavage, and cytochrome c release were assessed using Western blotting techniques. [35S] Methionine-labeled caspase-3 was transcribed in vitro and translated using the TNT kit (Promega). A vector containing caspase-3 was prepared by the ligation of EcoR I/BamHI flanked PCR fragment of full size caspase-3 cDNA into pBlusckript II SK(+/,) (Stratagen). RESULTS Overexpression of both FADD-DN and Bcl-2 genes prevent Fas-mediated apoptosis in PC3. As predicted, overexpression of FADD-DN prevented activation of caspase-8 and Bid cleavage and attenuated the release of cytochrome c and activation of caspases -2, -7, and -9. Bcl-2 overexpression did not affect caspase-8 activation and cleavage of Bid but blocked the release of cytochrome c and activation of mitochondria localized caspases -2, -7, and,9. Overexpression of FADD-DN and Bcl-2 affected the activation of caspase-3 and PARP cleavage differently: FADD-DN attenuated the activation of caspase-3 and PARP cleavage whereas Bcl-2 overexpression prevented caspase-3 activation and completely blocked cleavage of PARP. CONCLUSIONS These data suggest that activation of caspase-8 is necessary but not sufficient to complete Fas-mediated apoptosis in PC3 cells without activation of the mitochondrial pathway. In addition, caspase-3 activation after Fas-receptor ligation involves two steps and is dependent on mitochondrial activation. Prostate 51: 231,240, 2002. © 2002 Wiley-Liss, Inc. [source] Down-regulation of myeloid cell leukemia 1 by epigallocatechin-3-gallate sensitizes rheumatoid arthritis synovial fibroblasts to tumor necrosis factor ,,induced apoptosisARTHRITIS & RHEUMATISM, Issue 5 2009Salahuddin Ahmed Objective Overexpression of the antiapoptotic protein myeloid cell leukemia 1 (Mcl-1) in rheumatoid arthritis (RA) synovial fibroblasts is a major cause of their resistance to tumor necrosis factor , (TNF,),induced apoptosis. This study was undertaken to evaluate the efficacy of epigallocatechin-3-gallate (EGCG) in down-regulating Mcl-1 expression and its mechanism of RA synovial fibroblast sensitization to TNF,-induced apoptosis. Methods EGCG effects on cultured RA synovial fibroblast cell morphology, proliferation, and viability over 72 hours were determined by microscopy and a fluorescent cell enumeration assay. Caspase 3 activity was determined by a colorimetric assay. Western blotting was used to evaluate the apoptosis mediators poly(ADP-ribose) polymerase (PARP), Mcl-1, Bcl-2, Akt, and nuclear translocation of NF-,B. Results In RA synovial fibroblasts, EGCG (5,50 ,M) inhibited constitutive and TNF,-induced Mcl-1 protein expression in a concentration- and time-dependent manner (P < 0.05). Importantly, EGCG specifically abrogated Mcl-1 expression in RA synovial fibroblasts and affected Mcl-1 expression to a lesser extent in osteoarthritis and normal synovial fibroblasts or endothelial cells. Inhibition of Mcl-1 by EGCG triggered caspase 3 activity in RA synovial fibroblasts, which was mediated via down-regulation of the TNF,-induced Akt and NF-,B pathways. Caspase 3 activation by EGCG also suppressed RA synovial fibroblast growth, and this effect was mimicked by Akt and NF-,B inhibitors. Interestingly, Mcl-1 degradation by EGCG sensitized RA synovial fibroblasts to TNF,-induced PARP cleavage and apoptotic cell death. Conclusion Our findings indicate that EGCG itself induces apoptosis and further sensitizes RA synovial fibroblasts to TNF,-induced apoptosis by specifically blocking Mcl-1 expression and, hence, may be of promising adjunct therapeutic value in regulating the invasive growth of synovial fibroblasts in RA. [source] Chlamydia trachomatis -infected host cells resist dsRNA-induced apoptosisCELLULAR MICROBIOLOGY, Issue 9 2010Linda Böhme Summary Human pathogenic Chlamydia trachomatis have evolved sophisticated mechanisms to manipulate host cell signalling pathways in order to prevent apoptosis. We show here that host cells infected with C. trachomatis resist apoptosis induced by polyI:C, a synthetic double-stranded RNA that mimics viral infections. Infected cells displayed significantly reduced levels of PARP cleavage, caspase-3 activation and a decrease in the TUNEL positive population in the presence of polyI:C. Interestingly, the chlamydial block of apoptosis was upstream of the initiator caspase-8. Processing of caspase-8 was reduced in infected cells and coincided with a decrease in Bid truncation and downstream caspase-9 cleavage. Moreover, the enzymatic activity of caspase-8, measured by a luminescent substrate, was significantly reduced in infected cells. Caspase-8 inhibition by Chlamydia was dependent on cFlip as knock-down of cFlip decreased the chlamydial block of caspase-8 activation and consequently reduced apoptosis inhibition. Our data implicate that chlamydial infection interferes with the host cell's response to viral infections and thereby influences the fate of the cell. [source] | |||||||||||||