Cellular FLICE/Caspase-8–Inhibitory Protein as a Principal Regulator of Cell Death and Survival in Human Hepatocellular Carcinoma

Abstract

SUMMARY:Human hepatocellular carcinomas (HCCs) show resistance to apoptosis mediated by several death receptors.Because cellular FLICE/caspase-8 -inhibitory protein (cFLIP) is a recently identified intracellular inhibitor of caspase-8 activation that potently inhibits death signaling mediated by all known death receptors, including Fas, TNF-receptor (TNF-R), and TNF-related apoptosis-inducing ligand receptors (TRAIL-Rs), we investigated the expression and function of cFLIP in human HCCs. We found that cFLIP is constitutively expressed in all human HCC cell lines and is expressed more in human HCC tissues than in nontumor liver tissues. Metabolic inhibitors, actinomycin D (ActD) or cycloheximide (CHX), dramatically rendered HCC cells sensitive to Fas-mediated apoptosis. Neither caspase-8 nor caspase-3 was activated by agonistic anti-Fas antibody alone, but both caspases were activated by Fas stimulation in the presence of ActD or CHX, indicating the importance of caspase-8 inhibitors that are sensitive to metabolic inhibitors. Actually, cFLIP expression was decreased in ActD or CHX treatment. cFLIP down-regulation induced by cFLIP antisense oligodeoxynucleotides sensitized HLE cells to Fas, TNF-R, and TRAIL-R-mediated apoptosis. Furthermore, cFLIP over-expression activated nuclear factor (NF)-B and cFLIP down-regulation attenuated NF-B activation induced by TNF-␣ or TRAIL. Pretreatment with pan-caspase-inhibitor, benzyloxycarbonyl-Val-Ala-Asp (OMe) fluoromethyl ketone (Z-VAD-fmk), restored NF-B activity attenuated by cFLIP down-regulation. cFLIP expression was increased by TNF-␣, TRAIL, or vascular endothelial growth factor but decreased by wortmannin, indicating that cFLIP expression is regulated by both the NF-B and phosphatidylinostiol-3 kinase (PI-3)/Akt pathways. These results suggest that cFLIP plays an important role in cell survival not simply by inhibiting death-receptor-mediated apoptosis but also by regulating NF-B activation in human

Differential susceptibility to TRAIL of normal versus malignant human urothelial cells

“…35 Intracellular factor(s), such as FLIP and IAPs, have previously been implicated in TRAIL resistance in some bladder 16 and hepatocellular carcinoma cells. 42 Furthermore, a previous study has correlated levels of FLIP mRNA with TRAIL resistance in malignant urothelial cells. 43 However, our study is the first to correlate apoptotic susceptibility in malignant and normal urothelial cells with expression of FLIP at the protein level.…”

Down-regulation of cFLIP following reovirus infection sensitizes human ovarian cancer cells to TRAIL-induced apoptosis

“…An increasing number of reports have indicated a role for cFLIP expression in determining TRAIL sensitivity in a variety of human cancer cells [29,51,[59][60][61], including ovarian cancer cell lines [29,34,35] and epithelial ovarian cancer tissues [36]. Decreased cFLIP expression following RNA interference has also been shown to significantly enhance cell death in TRAILtreated cancer cells [30,62], including ovarian cancer cells [33].…”

“…Inhibition of PI3 kinase has been shown to down-regulate cFLIP expression in some cancer cells [51,57,58]. After finding that inhibition of PI3 kinase using wortmannin decreased cFLIP expression in OVCAR3 cells (not shown) we investigated whether inhibition of PI3 kinase could also enhance TRAIL-induced apoptosis in OVCAR3 cells.…”

“…Phosphothionate bonds present between nucleotides were included to enhance cell permeability. Antisense cFLIP:5′-gatttcagcagacatcctac-3′and sense cFLIP:5′-catcctacagacgacttcag-3′sequences were used [51,52]. Oligonucleotides were added directly to the media and cells were incubated for 18-24 h prior to TRAIL treatment.…”

TRAIL resistance results in cancer progression: a TRAIL to perdition?

“…Even in cells resistant to traditional chemotherapy with 5 FU, a decrease in cFLIP and sensitization to TRAIL-induced apoptosis was observed. Similarly in hepatocellular carcinoma cells, sensitivity to TRAIL-induced apoptosis was cFLIP dependent (Okano et al, 2003). Interestingly, the use of peroxisome proliferators-activated receptor-g (PPARg) agonists, also lead to a reduction in cFLIP levels and sensitization to TRAIL in several solid tumor-derived cell lines (Kim et al, 2002).…”