The Ovarian Cancer Induced By Tumor Necrosis Factor Ov - 90, Apoptosis Mechanism Research

Apoptosis is the major mechanism by which multicellular organisms clear superfluous, infected, damaged or potentially dangerous cells. For many years the role of proteolytic enzymes in the regulation of apoptosis was a major research topic. Most of the work were focused on the role and regulation of Caspases, which may be indispensable for the typical apoptotic pathway. The activated Caspases acted as both the initiator of apoptosis and the executioner to cleave the key regulatory and structural molecules in cells, culminating in cell demise. However, in recent years there has been growing evidence to suggest that, in addition to Caspase-mediated proteolysis, other proteases such as Cathepsins may also be involved in the regulation of apoptosis. Cathepsins, in particular Cathespin B(CTSB), can activate Caspase-dependent or Caspase-independent pathways of cell death. The mechanism of Cathepsins apoptosis pathways may be cell type-specific, and relations between Caspases and CTSB are incompletely understood.Tumor necrosis factor a (TNFa) is a multifunctional cytokine signaling such complex and diverse cellular processes as inflammatory cytokine production, cell survival, cell proliferation, and paradoxically, cell death. TNFa-mediated cytotoxicity is of considerable importance and is mediated, in part, by its ability to induce apoptosis. TNFa can either activate Caspase-8 and trigger mitochondrial apoptosis pathway, or induce the release of Cathepsins from lysosomes into cytoplasm, bringing about the death of the cell. Despite the fact that CTSB had been identified to be involved in many cells’apoptosis, the detailed mechanism of CTSB-mediated apoptosis is still undefined. In this study, the role of CTSB and Caspases and linkage of them in the process of apoptosis in ovarian cancer OV-90 cells exposed to TNFa were investigated. The principal findings of this study were as follows:1) CTSB and Caspases are both involved in the OV-90 cells apoptosis induced by TNFa, and Caspases activation precedes lysosomal destabilization.2) Such classical TNFa signaling pathway mediated by Caspases-8 as TNFｐ�'Caspase-8�'Bid�'mitochondria�'Cytochrome c�'Caspase-9�'Caspase-3,-7,-6�'apoptosis exists in OV-90 cells. The pathway was significantly blocked by the Caspase-8 specific inhibitor z-IETD-fmk.3) In OV-90 cells, CTSB participates in the Caspase-dependent TNFa signaling pathway. The release of Cytochrome c from mitochondrial and the activation of Caspase-3 triggered by TNFa could be blocked by CTSB specific inhibitor CA074Me (CA), and Caspase-8 inhibitor z-IETD-fmk could also suppress CTSB activity, suggesting that CTSB can not only acts as an effector protease downstream to Caspase-8, but also contributes to apoptosis by induction of mitochondrial outer membrane permeabilization (MOMP), possibly via release of Cytochrome C. The inhibition of CTSB also decreased Caspase-8 activity, suggesting that CTSB can feedback influence on Caspase-8.4) Inhibition of Caspase-8 by z-IETD-fmk in OV-90 cells significantly reduced, but did not completely abolish TNFa-induced lysosomal membrane permeabilization (LMP). At the same time, CTSB inhibitor CA suppressed activity of Caspase-8 and LMP in TNFa-induced OV-90 cell death, suggesting that Caspase-8 and CTSB are responsible for TNFa-triggered LMP of OV-90 cells. Inhibition of CTSB or Caspases partially protected OV-90 cells against TNFa-induced apoptotic cell death, while inhibition of both CTSB and Caspases resulted in almost complete protection, demonstrating that TNFa induced OV-90 cell death is initiated by at least two pathways mediated by both Caspases and CTSB.5) In our experiments, we found that mitochondria permeability transition pore (MPTP) inhibitor cyclosporin A (CsA) enhanced the effect of TNFa-induced apoptosis. CsA has been described by many authors as a very specific inhibitor of mitochondrial membrane potential and permeability transition in isolated mitochondrial fractions and in isolated hepatocytes. Our data shows that CsA could intensify the apoptotic effect of TNFa in OV-90 cells, though it was not toxic to OV-90 cells when the cells were treated with CsA alone. Interestingly, CsA plus Caspase-8 inhibitor z-IETD-fmk almost completely prevented cell death induced by TNFa, which worked more efficiently than z-IETD-fmk did alone. In contrast, the inhibition effect of CsA plus CTSB inhibitor CA was less than that of CA. These observations suggest that the mechanisms of Caspase-8 and CTSB resulting in membrane organelle permeabilization are not the same, and CTSB may participate in a Caspase-8-independent pathway during TNFa induced OV-90 cell death.This study provides foundations for the understanding of CTSB-mediated apoptosis pathway in TNFa induced OV-90 cell death.