c-FLIP inhibits chemotherapy-induced colorectal cancer cell death

Abstract

c-FLIP inhibits caspase 8 activation and apoptosis mediated by death receptors such as Fas and DR5. We studied the effect of c-FLIP on the apoptotic response to chemotherapies used in colorectal cancer (CRC) (5-fluorouracil, oxaliplatin and irinotecan). Simultaneous downregulation of both c-FLIP splice forms c-FLIP L and c-FLIP S with siRNA synergistically enhanced chemotherapy-induced apoptosis in p53 wild-type (HCT116p53 þ / þ , RKO), null (HCT116p53 À/À ) and mutant (H630) CRC cell lines. Furthermore, overexpression of c-FLIP L , but not c-FLIP S , potently inhibited apoptosis induced by chemotherapy in HCT116p53 þ / þ cells, suggesting that c-FLIP L was the more important splice form in mediating chemoresistance. In support of this, siRNA specifically targeted against c-FLIP L synergistically enhanced chemotherapy-induced apoptosis in a manner similar to the siRNA targeted against both splice forms. Inhibition of caspase 8 blocked the enhanced apoptosis induced by c-FLIP-targeted (FT) siRNA and chemotherapy. Furthermore, we found that downregulating cell surface DR5, but not Fas, also inhibited apoptosis induced by FT siRNA and chemotherapy. Interestingly, these effects were not dependent on activation of DR5 by its ligand TRAIL. These results indicate that c-FLIP inhibits TRAILindependent, DR5-and caspase 8-dependent apoptosis in response to chemotherapy in CRC cells. Moreover, targeting c-FLIP in combination with existing chemotherapies may have therapeutic potential for the treatment of CRC.

5-Fluorouracil signaling through a calcium–calmodulin-dependent pathway is required for p53 activation and apoptosis in colon carcinoma cells

“…Moreover, as a correspondent localization pattern was not observed in HCT116 p53 À / À cells using the same incubation time, we concluded that an efficient accumulation of DISC components DR5, FADD and caspase-8 in plasma membranes of 5-FU-treated cells is a p53-dependent or -facilitated event. Thus, in agreement with some previous reports, 11,12 these results indicate the DR5-receptor as a p53-dependent mediator of 5-FU-induced apoptosis, also suggesting that the CD95-DISC, formed for still unidentified reason(s), remains inactive with respect to caspase-8 activation, or that contribution of this complex is insignificant. 16 Analysis of potential regulatory factors upstream of DR5-DISC formation In contrast to classical extrinsic death pathways triggered by ligands of the TNF family, 5-FU-induced apoptosis most certainly emerges from either DNA or RNA damage.…”

“…However, as shown by immunostaining, in response to 5-FU, both TNF-family receptors DR5 and CD95 are accumulated in the plasma membrane (Figure 1a), indicating that either one of them or both could have a vital role for efficient apoptosis. As conflicting evidences exist in this matter, 12,13,17 we decided to assess the contribution of each individual receptor to initiator caspase-8 and effector caspase-3 activation by means of small interfering RNA (siRNA) technology (Figures 1b and c). This experimental approach clearly demonstrated that DR5 but not CD95 is the sole receptor required for caspase-8 activation and further processing of caspase-2 and effector caspase-3 upon treatment with 5-FU.…”

“…11 Similar data have been provided by using tumor cell lines, although conflicting evidences exist postulating DR4, DR5 or CD95 as the regulatory receptor for caspase-8 activation and apoptosis. [12][13][14] Interestingly, death receptor ligand does not seem to be required for DISC formation in this particular apoptosis pathway. 12 Most DR5 expression studies in clinical tumor sections do not experimentally verify whether the death receptor is induced as a consequence of chemotherapy, or if it is a response to tumorogenesis.…”

“…[12][13][14] Interestingly, death receptor ligand does not seem to be required for DISC formation in this particular apoptosis pathway. 12 Most DR5 expression studies in clinical tumor sections do not experimentally verify whether the death receptor is induced as a consequence of chemotherapy, or if it is a response to tumorogenesis. On the other hand, an examination of the response of three human colon tumors to TNF-related apoptosis-inducing ligand (TRAIL) alone and in combination with chemotherapy drugs, using SCID mice engrafted with intact patient surgical colorectal cancer specimens, revealed that tumors treated with carnitine palmitoyltransferase-11 (CPT-II) showed increased membrane expression of DR5, also suggesting that CPT-II may increase sensitivity to TRAIL by upregulation of DR5.…”

Combined inhibition of FLIP and XIAP induces Bax-independent apoptosis in type II colorectal cancer cells

“…We have shown previously that FLIP gene silencing can induce apoptosis in colorectal cancer cells and sensitize them to TRAIL-and chemotherapy-induced apoptosis (Longley et al, 2006;Wilson et al, 2007). It has been suggested that chemotherapy-induced DR5 upregulation sensitizes type II colorectal cancer cells to TRAIL when mito-chondrial-mediated apoptosis is blocked by converting them into type I cells (Wang and El-Deiry, 2003).…”