CD95-mediated apoptosis is impaired at receptor level by cellular FLICE-inhibitory protein (long form) in wild-type p53 human ovarian carcinoma

Purpose: Ovarian carcinoma is a highly lethal malignancy that often becomes resistant to chemotherapy. Alterations in apoptotic signals and p53 status contribute to drug resistance, and CD95-mediated apoptosis is also deficient in resistant cells. We analyzed the mechanism of resistance to CD95-mediated apoptosis in ovarian carcinoma cell lines differing in p53 status. Experimental Design: CD95-mediated apoptosis was induced by agonistic anti-CD95 antibody, and the apoptotic cascade was monitored with biochemical and functional assays. Results: CD95-mediated apoptosis was blocked in human ovarian cancer cells. In cell lines with wild-type p53, treatment with the protein synthesis inhibitor cycloheximide (CHX) together with anti-CD95 overcame the resistance, suggesting the presence of a labile inhibiting protein. Indeed, the labile protein cellular FLICE-inhibitory protein long form (c-FLIP_L) was found to block caspase-8 recruitment to the death-inducing signaling complex (DISC), and sensitization of cells by CHX was due to c-FLIP_L down-modulation at the DISC level. Down-regulation of c-FLIP_L with antisense oligonucleotides increased CD95-mediated apoptosis as in cells sensitized by CHX, demonstrating the direct involvement of c-FLIP_L in apoptosis resistance. Removal of c-FLIP_L block at DISC level allowed full activation of the mhochondrial pathway and, eventually, apoptosis in wild-type p53 cells, whereas in cells with mutated p53, c-FLIP_L involvement in CD95-mediated apoptosis resistance appeared to be irrelevant. Immunohistochemical analysis of an ovarian tumor tissue array revealed c-FLIP_L expression in samples with no p53 accumulation (P = 0.034), and a significant (P = 0.037) inverse relationship between c-FLIP_L and p53 expression levels was also observed in 27 epithelial ovarian cancer specimens with known p53 status. Conclusion: The inhibitory protein c-FLIP _L is involved in resistance to CD95-mediated apoptosis in ovarian carcinoma cells with wild-type p53.