Estrogen inhibits paclitaxel-induced apoptosis via the phosphorylation of apoptosis signal-regulating kinase 1 in human ovarian cancer cell lines

The influence of postoperative estrogen replacement therapy on the sensitivity of ovarian cancer to paclitaxel remains elusive. We examined whether estrogen affects paclitaxel-induced apoptosis in the Caov-3 human ovarian cancer cell line, which expresses estrogen receptor. 17beta-Estradiol (E2) significantly reversed the paclitaxel-induced apoptosis and reduction of cell viability, and a highly selective estrogen receptor antagonist, ICI182,780, and a phosphatidylinositol 3-kinase inhibitor, LY294002, attenuated the reversal effect of E2 on paclitaxel-induced apoptosis and reduction of cell viability. E2 significantly induced the phosphorylation of Akt. Akt and apoptosis signal-regulating kinase 1 (ASK1) were physically associated, and E2 induced the phosphorylation of ASK1 at serine-83, which is a consensus Akt phosphorylation site. We confirmed a previous report showing that paclitaxel induces cell damage via the ASK1-c-Jun N-terminal protein kinase (JNK) cascade. E2 inhibited the paclitaxel-induced JNK activation, and the E2-induced inhibition of the paclitaxel-induced JNK activation was attenuated in cells treated with either ICI182,780 or LY294002 or transfected with ASK1S83A, in which a consensus Akt phosphorylation site at serine-83 was converted to alanine. The inhibitory effect of E2 on the paclitaxel-induced reduction of cell viability and apoptosis was diminished in cells transfected with ASK1S83A. These results indicate that E2 inhibits paclitaxel-induced cell damage by inhibiting JNK activity via phosphorylation of Akt-ASK1. Thus, treatment of ovarian cancer with paclitaxel might be less effective in the setting of postoperative estrogen replacement therapy.     

Suppression of apoptosis signal-regulating kinase 1-induced cell death by 14-3-3 proteins

Apoptosis signal-regulating kinase 1 (ASK1) is a pivotal component of a signaling pathway induced by many death stimuli, including tumor necrosis factor alpha, Fas, and the anticancer drugs cisplatin and paclitaxel. Here we report that ASK1 proapoptotic activity is antagonized by association with 14-3-3 proteins. We found that ASK1 specifically bound 14-3-3 proteins via a site involving Ser-967 of ASK1. Interestingly, overexpression of 14-3-3 in HeLa cells blocked ASK1-induced apoptosis whereas disruption of the ASK1/14-3-3 interaction dramatically accelerated ASK1-induced cell death. Targeting of ASK1 by a 14-3-3-mediated survival pathway may provide a novel mechanism for the suppression of apoptosis.     

Targeted deletion of apoptosis signal-regulating kinase 1 attenuates left ventricular remodeling

Left ventricular remodeling that occurs after myocardial infarction (MI) and pressure overload is generally accepted as a determinant of the clinical course of heart failure. The molecular mechanism of this process, however, remains to be elucidated. Apoptosis signal-regulating kinase 1 (ASK1) is a mitogen-activated protein kinase kinase kinase that plays an important role in stress-induced apoptosis. We used ASK1 knockout mice (ASK-/-) to test the hypothesis that ASK1 is involved in development of left ventricular remodeling. ASK-/- hearts showed no morphological or histological defects. Echocardiography and cardiac catheterization revealed normal global structure and function. Left ventricular structural and functional remodeling were determined 4 weeks after coronary artery ligation or thoracic transverse aortic constriction (TAC). ASK-/- had significantly smaller increases in left ventricular end-diastolic and end-systolic ventricular dimensions and smaller decreases in fractional shortening in both experimental models compared with WT mice. The number of terminal deoxynucleotidyl transferase biotin-dUDP nick end-labeling-positive myocytes after MI or TAC was decreased in ASK-/- compared with that in WT mice. Overexpression of a constitutively active mutant of ASK1 induced apoptosis in isolated rat neonatal cardiomyocytes, whereas neonatal ASK-/- cardiomyocytes were resistant to H2O2-induced apoptosis. An in vitro kinase assay showed increased ASK1 activity in heart after MI or TAC in WT mice. Thus, ASK1 plays an important role in regulating left ventricular remodeling by promoting apoptosis.     

Protective effect of apoptosis signal-regulating kinase1 inhibitor against mice liver injury

Objective:To explore the protective effect and its molecular mechanism of apoptosis signalregulating kinase 1(ASK1) inhibitor(GS-459679) on acetaminophen-induced liver injury in mice.Methods:The model of liver injury was established by administration of acetaminophen(APAP)(300 mg/kg,i.p.) on C57BL/6 mice.Forty-eight male C57BL/6 mice were randomly divided into four groups,consisting of control group,GS group(GS-459679,30 mg/kg,i.p.),APAPinduced group,and GS combined with APAP-induced group.For GS combined with APAPinduced group,mice were treated with GS 30 min prior to administration of APAP.After mice were euthanized at 6 h or 12 h.respectively,serum levels of alanine aminotransferase(ALT) and aspartate aminotransferase(AST) were analyzed,and mRNA levels of TNF- α,IL-6 and IL-1βwere tested.The activity of glutathione(GSH),oxidized GSH(GSSG) and malondialdehyde were quantified.In addition,ASK1,P-ASK1,JNK and P-JNK protein levels were tested in all groups.Results:The ASK1 and P-ASK1 levels were up-regulated in APAP-induced group.Compared to the control group,serum levels of ALT and AST.and mRNA levels of TNF- a,IL-6 and IL-1(3were increased in APAP-induced group.Meanwhile,the levels of MAD and GSSG.and the ratio of GSSG/GSH were higher and the JNK was activatedin APAP-induced group compared with that in control group.However,compared to APAP-induced group,GS combined with APAP-induced group displayed a decrease of protein expression levels of ASK 1,P-ASKI and P-JNK,a reduction of serum levels of ALT and AST,a decrease in TNF- a.IL-6 and IL-1(3 mRNA levels,and a low ration of GSSG/GSH.Conclusions:GS-459679 treatment effectively down-regulates ASK1 and P-ASK 1 expression.Addition of GS-459679 decreases the generation of liver metabolites and inflammatory factors,reduces oxidative stress reaction,inhibits JNK activation,and then protects the responsiveness to APAP-induced liver injury.     

Apoptosis signal-regulating kinase 1 inhibits hepatocarcinogenesis by controlling the tumor-suppressing function of stress-activated mitogen-activated protein kinase

The stress-activated mitogen-activated protein kinases (MAPKs), c-Jun NH2-terminal kinase (JNK), and p38 have been implicated in hepatocarcinogenesis. Although the many interrelated functions of JNK and p38 are precisely regulated by upstream signaling molecules, little is known about upstream regulators. We investigated the role of apoptosis signal-regulating kinase 1 (ASK1), a major player in the regulation of JNK and p38 activities, in hepatocarcinogenesis using a mouse hepatocellular carcinoma (HCC) model. ASK1-deficient (ASK1(-/-) ) and wildtype (WT) mice were treated with diethylnitrosamine on postnatal day 14. Strikingly, after 7 months, approximately three times as many tumors developed in ASK1(-/-) mice as in WT mice. Although JNK and p38 activation were attenuated in ASK1(-/-) HCCs relative to WT HCCs, cell proliferation was comparable in HCCs from both types of mice. On the other hand, both cancer cell apoptosis and hyperphosphorylation of BimEL, a proapoptotic Bcl-2 family member, were suppressed in the ASK1(-/-) HCCs. ASK1(-/-) mice showed remarkable resistance to Fas-induced hepatocyte apoptosis in vivo, probably because of attenuated JNK-mediated BimEL phosphorylation and mitochondrial apoptotic pathway activation. The reintroduction of ASK1 to ASK1(-/-) mouse liver using an adenoviral vector restored Fas-induced hepatocyte death and phosphorylation of JNK and BimEL. Similar findings were obtained in tumor necrosis factor alpha-induced hepatocyte apoptosis. Furthermore, ASK1 was involved in DNA damage-induced p21 up-regulation through a p38 pathway. CONCLUSION: ASK1 is involved in death receptor-mediated apoptosis and DNA-damage response by way of stress-activated MAPK in the liver, and thus acts as a tumor suppressor in hepatocarcinogenesis. This study provides new insight into the regulation of stress- activated MAPK signaling in hepatocarcinogenesis.     

Enhanced TNF-alpha-induced apoptosis in Fanconi anemia type C-deficient cells is dependent on apoptosis signal-regulating kinase 1

Fanconi anemia (FA) is a chromosomal instability disorder characterized by progressive bone marrow failure. Experimental evidence suggests that enhanced oxidant and myelosuppressive cytokine-mediated apoptosis of hematopoietic stem and progenitor cells contributes to the pathogenesis of marrow failure in FA. However, the molecular mechanisms responsible for the apoptotic phenotype in hematopoietic cells are incompletely understood. Recent data in Fancc-/- murine embryonic fibroblasts (MEFs) implicate increased oxidant-induced apoptotic signaling through the redox-dependent protein, apoptosis signal-regulating kinase 1 (Ask1). Here, we examined whether altered Ask1 signaling participated in the proapoptotic phenotype of primary Fancc-/- MEFs and hematopoietic progenitors treated with the myelosuppressive cytokine tumor necrosis factor-alpha (TNF-alpha). Our data indicate that TNF-alpha induces hyperactivation of Ask1 and the downstream effector p38 in Fancc-/- MEFs. In addition,Ask1 inactivation in Fancc-/- MEFs and hematopoietic progenitors restored survival to wild-type (WT) levels in the presence of TNF-alpha. Furthermore, targeting the Ask1 pathway by using either antioxidants or a p38 inhibitor protected Fancc-/- MEFs and c-kit+ cells from TNF-alpha-induced apoptosis. Collectively, these data argue that the predisposition of Fancc-/- hematopoietic progenitors to apoptosis is mediated in part through altered redox regulation and Ask1 hyperactivation.     

The effect of apoptosis signal-regulating kinase 1 gene transfer on rat collagen induced arthritis

OBJECTIVE: To examine the apoptosis-inducing effect of apoptosis signal-regulating kinase 1 (ASK1) gene transfer into synovial cells in vitro and in vivo. METHODS: An adenovirus vector was constructed so that a constitutively active form of ASK1 gene (ASK1DeltaN) was expressed in the presence of the Cre recombinase. The ASK1DeltaN and Cre adenovirus vectors were cotransduced into cultured synoviocytes derived from patients with rheumatoid arthritis (RA), and apoptosis was evaluated by TUNEL and Hoechst staining. Collagen induced arthritis (CIA) was induced in 8-week-old male DA rats, and 10 days later the 2 adenovirus vectors were coadministered into the ankle joints of the animals. As indicators of severity of arthritis, swelling of the ankle and articular index (AI) scores were evaluated, while histopathological observation of articular tissue was also performed. RESULTS: In the cultured human RA synoviocytes, overexpression of the ASK1DeltaN significantly reduced cell viability and induced apoptosis. In the CIA rats transduced with the ASK1DeltaN gene, arthritis was significantly promoted in terms of the swelling of the ankle joints and elevation of the AI scores. Histopathological observation also revealed that the constitutively active ASK1 induced massive infiltration of inflammatory cells into the synovial membrane as well as proliferation of synovial fibroblasts. Degeneration of the synovial membrane was not evident. CONCLUSION: Adenoviral transduction of ASK1DeltaN induced apoptosis in RA synoviocytes in vitro, but not in CIA synovium in vivo.     

Small heat-shock protein Hsp20 attenuates beta-agonist-mediated cardiac remodeling through apoptosis signal-regulating kinase 1

Chronic stimulation of the beta-adrenergic neurohormonal axis contributes to the progression of heart failure and mortality in animal models and human patients. In cardiomyocytes, activation of the beta-adrenergic pathway has been shown to result in transiently increased expression of a cardiac small heat-shock protein Hsp20. The present study shows that cardiac overexpression (10-fold) of Hsp20 may protect the heart against beta-agonist-induced cardiac remodeling, associated with isoproterenol (50 mug/g per day) infusion for 14 days. Hsp20 attenuated the cardiac hypertrophic response, markedly reduced interstitial fibrosis, and decreased apoptosis. Contractility was also preserved in hearts with increased Hsp20 levels. These beneficial effects were associated with attenuation of the ASK1-JNK/p38 (apoptosis signal-regulating kinase 1/c-Jun NH(2)-terminal kinase/p38) signaling cascade triggered by isoproterenol, whereas there was no difference in either extracellular signal-related kinase 1/2 or Akt activation. Parallel in vitro experiments supported the inhibitory role of Hsp20 on enforced ASK1-JNK/p38 activation in both H9c2 cells and adult rat cardiomyocytes. Immunostaining studies also demonstrated that Hsp20 colocalizes with ASK1 in cardiomyocytes. Taken together, our findings indicate that (1) beta-agonist-induced cardiac injury is associated with activation of the ASK1-JNK/p38 cascade; (2) increased expression of Hsp20 attenuates the induction of remodeling, dysfunction, and apoptosis in response to sustained beta-adrenergic stimulation; and (3) the beneficial effects of Hsp20 are at least partially attributable to inhibition of the ASK1-signaling cascade.     

Thrombospondin-1 induces endothelial cell apoptosis and inhibits angiogenesis by activating the caspase death pathway

Thrombospondin-1 (TSP1) is a potent natural inhibitor of angiogenesis. Although TSP1 has been reported to induce endothelial cell apoptosis in vitro and to downregulate neovascularization in vivo, the molecular mechanisms that link these two processes have yet to be established. Here we report that TSP1 mediates endothelial cell apoptosis and inhibits angiogenesis in association with increased expression of Bax, decreased expression of Bcl-2, and processing of caspase-3 into smaller proapoptotic forms. The ability of TSP1 to induce both endothelial cell apoptosis in vitro and to suppress angiogenesis in vivo was blocked by the caspase-3 inhibitor z-DEVD-FMK. TSP1 also attenuated VEGF-mediated Bcl-2 expression in endothelial cells in vitro and angiogenesis in vivo. Furthermore, TSP1 induced endothelial cell apoptosis and inhibited neovascularization in sponge implants in SCID mice. We conclude that TSP1 induces endothelial cell apoptosis and inhibits neovascularization by altering the profile of survival gene expression and activating caspase-3.     

Interaction between integrin alpha9beta1 and vascular cell adhesion molecule-1 (VCAM-1) inhibits neutrophil apoptosis

According to the prevailing paradigm, neutrophils are short-lived cells that undergo spontaneous apoptosis within 24 hours of their release from the bone marrow. However, neutrophil survival can be significantly prolonged within inflamed tissue by cytokines, inflammatory mediators, and hypoxia. During screening experiments aimed at identifying the effect of the adhesive microenvironment on neutrophil survival, we found that VCAM-1 (CD106) was able to delay both spontaneous and Fas-induced apoptosis. VCAM-1-mediated survival was as efficient as that induced by the cytokine IFN-beta and provided an additive, increased delay in apoptosis when given in combination with IFN-beta. VCAM-1 delivered its antiapoptotic effect through binding the integrin alpha9beta1. The alpha9beta1 signaling pathway shares significant features with the IFN-beta survival signaling pathway, requiring PI3 kinase, NF-kappaB activation, as well as de novo protein synthesis, but the kinetics of NF-kappaB activation by VCAM-1 were slower and more sustained compared with IFN-beta. This study demonstrates a novel functional role for alpha9beta1 in neutrophil biology and suggests that adhesive signaling pathways provide an important extrinsic checkpoint for the resolution of inflammatory responses in tissues.     

Progression of heart failure was suppressed by inhibition of apoptosis signal-regulating kinase 1 via transcoronary gene transfer.

OBJECTIVES: We examined whether the inhibition of apoptosis signal-regulating kinase 1 (ASK1) would attenuate the progression of heart failure in TO-2 hamsters with hereditary dilated cardiomyopathy. BACKGROUND: Heart failure remains the leading cause of mortality and requires novel therapies targeting the biologically relevant processes within cardiomyocytes that lead to cell death. Apoptosis signal-regulating kinase 1 is a key signaling molecule for cardiomyocyte death. METHODS: We generated recombinant adeno-associated virus (rAAV) expressing an N-terminal truncated form of the dominant-negative mutant of ASK1 (ASKdeltaN(KR)). TO-2 hamsters were subjected to an in vivo rAAV transcoronary transfer. RESULTS: ASKdeltaN(KR) retained its dominant-negative activity in vitro. The rAAV expressing ASKdeltaN(KR) treatment inhibited ASK1 activation in the hamster hearts and suppressed progression of ventricular remodeling such as chamber dilation, impairment of contractile and relaxation functions, and fibrosis. Inhibition of ASK1 reduced the number of apoptotic cells and selectively attenuated c-Jun NH2-terminal kinase activation. Although the deficiency of delta-sarcoglycan, a genetic defect in the hamster, leads to the degradation of dystrophin, the treatment significantly protected hearts from this degradation, probably by inhibiting calpain activation. CONCLUSIONS: Apoptosis signal-regulating kinase 1 is involved in the pathogenesis of heart failure progression, mediated through c-Jun NH2-terminal kinase-mediated apoptosis and calpain-dependent dystrophin cleavage, and may be a therapeutic target to treat patients with heart failure.     

ShRNA-mediated BMI-1 gene silencing inhibits gastrointestinal stromal tumor cell telomerase activity and enhances apoptosis

Gastrointestinal stromal tumors (GISTs) are the most frequently occurring mesenchymal tumors of the gastrointestinal tract. Telomerase activity is well acknowledged as a critical factor in oncogenesis. The objective of the present study is to evaluate the effect of BMI gene silencing on proliferation, apoptosis and telomerase activity in human GIST882 cells. GIST882 cells were transfected with a eukaryotic expression vector of an shRNA fragment. The silencing efficiency in the GIST882 cells was determined by RT-qPCR and a western blot analysis. After the shRNA-BMI-1 plasmid was transfected into the GIST882 cells and nude mice, a cell counting kit-8 (CCK-8) assay and flow cytometry were utilized to detect the GIST882 cell proliferation, the apoptosis rate and the cell cycle. Tumor growth was observed by tumor xenograft in nude mice. Telomerase activity and telomere length were detected by a Southern blot and a target region amplified polymorphism. The shRNA-BMI-1 recombinant plasmid was successfully constructed. The mRNA and protein expression of the BMI-1 gene in GIST882 cells was suppressed by the shRNA-BMI-1 recombinant plasmid. Meanwhile, BMI-1 gene silencing inhibited the cell proliferation, tumor growth, and cell cycle in the GIST882 cells. However, cell apoptosis was increased and telomerase activity was decreased with the silencing of the BMI-1 gene. Collectively, the results of this study suggest that silencing the BMI-1 gene may provide a new target for the treatment of GISTs.     

Hyperglycemia inhibits vascular smooth muscle cell apoptosis through a protein kinase C-dependent pathway

We hypothesized that the pathogenesis of diabetic vasculopathy involves the abnormal regulation of vascular smooth muscle cell (VSMC) apoptosis. In nondiabetic mice, a reduction in carotid artery blood flow resulted in a significant loss of medial VSMCs via apoptosis (normal flow 84+/-1 viable VSMCs, reduced flow 70+/-5 viable VSMCs; n=12, P:<0.01). In contrast, flow-induced VSMC apoptosis was markedly attenuated in streptozotocin-induced diabetic mice (normal flow 85+/-2 viable VSMC, reduced flow 82+/-4 viable VSMC; n=13, NS). In accord with our in vivo findings, the exposure of cultured rat and human VSMCs to high glucose (17.5 mmol/L) significantly attenuated the induction of apoptosis in response to serum withdrawal (rat VSMCs in normal [5.5 mmol/L] glucose 28+/-1%, high D-glucose 19+/-2%; P:<0.0001). High glucose also inhibited apoptosis induced by Fas ligand (100 ng/mL) (normal 23+/-2%, high D-glucose 13+/-2%; P:<0.006). Supplementation with the nonmetabolized enantiomer L-glucose had no effect. We confirmed reports that high glucose activates protein kinase C (PKC) and demonstrated that PKC blockade with long-term phorbol ester treatment or calphostin C prevented the antiapoptotic effect (P:<0. 001). Moreover, the upregulation of either PKCalpha or PKCbetaII expression was sufficient to inhibit serum withdrawal-induced apoptosis (control 25+/-2%, PKCalpha 11+/-2%, PKCbetaII 8+/-2%; P:<0. 0001), whereas the upregulation of PKCdelta had no significant effect. Taken together, these findings demonstrate that hyperglycemia inhibits VSMC apoptosis via a PKC-dependent pathway.