PKCδ-mediated regulation of FLIP expression in human colon cancer cells

FLICE-like inhibitory protein (FLIP), a naturally occurring caspase-inhibitory protein that lacks the critical cysteine domain necessary for catalytic activity, is a negative regulator of Fas-induced apoptosis. Decreased FLIP levels sensitize tumor cells to Fas- and TRAIL-mediated apoptosis; however, the cellular mechanisms regulating FLIP expression have not been defined. Here, we examined the roles of the PKC and NF-κB pathway in the regulation of FLIP in human colon cancers. FLIP mRNA levels were increased in Caco-2 cells by treatment with PMA; actinomycin D completely inhibited the induction of FLIP by PMA, indicating transcriptional regulation. PKC inhibitors Gö6983 and Ro-31-8220 blocked PMA-stimulated FLIP expression. Pretreatment with the PKCδ-selective inhibitor rottlerin or transfection with PKCδ siRNA inhibited PMA-induced FLIP expression, which identifies a role for PKCδ in FLIP induction. Treatment with the proteasome inhibitor, MG132, or the NF-κB inhibitor (e.g., PDTC and gliotoxin), or overexpression of the superrepressor of IκB-α inhibited PMA-induced upregulation of FLIP. Moreover, PMA-induced NF-κB transactivation was blocked by GF109203x. In conclusion, our results demonstrate a critical role for PKCδ/NF-κB in the regulation of FLIP in human colon cancer cells.