The role and regulation of Flice-Like Inhibitory Protein (FLIP) in cisplatin resistance in human ovarian cancer cells in vitro

Abstract

Human ovarian cancer is the most lethal gynecological malignancy. Although cisplatin (CDDP) and paclitaxel are the first-line chemotherapeutic agents for ovarian cancer, chemoresistance is a major therapeutic problem with mechanisms involved still not well understood. Dysregulation of the apoptotic cascade may be a causative factor for chemoresistance. Indeed gene products involved in the regulation of apoptosis, including the PI3K/Akt and Flice-Like Inhibitory Protein (FLIP) are frequently over-expressed and/or dysregulated in chemoresistant cells. FLIP, a caspase-8 analogue, modulates death receptor-mediated apoptosis by preventing caspase-8 activation. Since FLIP may have a critical involvement in ovarian cancer chemoresistance, it is importance to understand its role, the molecular and cellular mechanisms by which FLIP is regulated, and if and how dysregulated FLIP degradation is involved in the etiology of chemoresistance. Human ovarian cancer cell lines were used to establish the possible involvement of FLIP in the regulation of CDDP sensitivity in vitro and to assess the mechanism by which CDDP regulates FLIP. We demonstrated that CDDP down-regulates FLIP content, induces caspase-8 and -3 activation and apoptosis in chemosensitive ovarian cancer cells, but not in their resistant counterparts. Moreover, we observed that FLIP over-expression in chemosensitive cells attenuates CDDP sensitivity, while FLIP down-regulation sensitizes chemoresistant cells to CDDP. We further demonstrated that CDDP had no effects on FLIP mRNA abundance but down-regulated FLIP protein content in chemosensitive cells, a response attenuated by proteasome inhibitors. Itch is an E3 ligase protein involved in protein ubiquitination. CDDP also enhances FLIP-p53-Itch cell membrane co-localization, and FLIP ubiquitination in chemosensitive but not resistance cells. In addition, Itch silencing attenuates CDDP-induced FLIP ubiquitination. p53 siRNA also attenuates FLIP-Itch and FLIP-p53 interactions and FLIP ubiquitination. While Akt activation inhibits CDDP-induced FLIP degradation and apoptosis in chemosensitive cells, these responses are facilitated by dominant-negative Akt expression in their resistant variants. Finally, p53 siRNA attenuates CDDP-induced and dominant negative (DN)-Akt-facilitated FLIP-p53 binding and FLIP ubiquitination in resistant cells. These studies improve our understanding of FLIP involvement in chemoresistance, the mechanism by which CDDP regulates FLIP, and dysregulation of FLIP degradation in chemoresistance in ovarian cancer.