Chemotherapy and TRAIL-mediated colon cancer cell death: the roles of p53, TRAIL receptors, and c-FLIP

Abstract

Tumor necrosis factor -related apoptosis-inducing ligand (TRAIL) has recently attracted attention as a potential therapeutic agent in the treatment of cancer. We assessed the roles of p53, TRAIL receptors, and cellular Fas-associated death domain -like interleukin-1B-converting enzyme inhibitory protein (c-FLIP) in regulating the cytotoxic effects of recombinant TRAIL (rTRAIL) alone and in combination with chemotherapy [5-fluorouracil (5-FU), oxaliplatin, and irinotecan] in a panel of colon cancer cell lines. Using clonogenic survival and flow cytometric analyses, we showed that chemotherapy sensitized p53 wild-type, mutant, and null cell lines to TRAIL-mediated apoptosis. Although chemotherapy treatment did not modulate mRNA or cell surface expression of the TRAIL receptors death receptor 4, death receptor 5, decoy receptor 1, or decoy receptor 2, it was found to down-regulate expression of the caspase-8 inhibitor, c-FLIP. Stable overexpression of the long c-FLIP splice form but not the short form was found to inhibit chemotherapy/rTRAIL -induced apoptosis. Furthermore, siRNA-mediated down-regulation of c-FLIP, particularly the long form, was found to sensitize colon cancer cells to rTRAIL-induced apoptosis. In addition, treatment of a 5-FU-resistant cell line with 5-FU down-regulated c-FLIP expression and sensitized the chemotherapy-resistant cell line to rTRAIL. We conclude that TRAIL-targeted therapies may be used to enhance conventional chemotherapy regimens in colon cancer regardless of tumor p53 status. Furthermore, inhibition of c-FLIP may be a vital accessory strategy for the optimal use of TRAIL-targeted therapies.

p53-Mediated upregulation of DcR1 impairs oxaliplatin/TRAIL-induced synergistic anti-tumour potential in colon cancer cells

“…One study reported TRAIL-enhanced apoptosis in response to chemotherapy in a large panel of colon cancer cell lines, including in the p53 þ / þ or p53 À/À HCT116 cells (Galligan et al, 2005). In contrast, our results are in favour of an antagonism between the two molecules in p53 wild-type cells such as HCT116, LS513 and LS174T.…”

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

“…In fact, this is in agreement with recent reports showing that chemotherapy-induced downregulation of cFLIP splice forms occurs in cell lines irrespectively of their p53 status and, more specifically, that ATM kinase activity is required to trigger 5-FU-and neocarzinostatin-induced and p53-independent cFLIP downregulation, which in turn sensitized hepatocellular carcinoma cell lines to TRAIL. 41,42 Identification of the factor regulated by the Ca 2 þ -CaM-p53 axis described in this report conducting DR5-DISC activity thereby remains.…”

Histone deacetylase inhibitors synergistically potentiate death receptor 4-mediated apoptotic cell death of human T-cell acute lymphoblastic leukemia cells

“…The mRNA sequences targeted by c-FLIP L siRNA was 5 0 -AAGGAA CAGCTTGGCGCTCAA-3 0 , which was used in a previous study [33]. An unrelated siRNA with a sequence of 5 0 -AATTCTCCGAACGTGTCACGT-3 0 was also employed as a control [33].…”

“…The mRNA sequences targeted by c-FLIP L siRNA was 5 0 -AAGGAA CAGCTTGGCGCTCAA-3 0 , which was used in a previous study [33]. An unrelated siRNA with a sequence of 5 0 -AATTCTCCGAACGTGTCACGT-3 0 was also employed as a control [33]. CCRF-CEM cells were transfected with 2 lM of siRNA by electroporation using MicroPorator MP-100 according to manufacturer's instruction (NanoEn Tek Inc., Korea) [31], and downregulation of c-FLIP L expression was examined by Western blotting after incubation for 48 h. After 24 h of siRNA transfection, the cells (2 9 10 4 cells/well in 96-well plate) were treated with AY4 (10 lg/ml) or TRAIL (0.5 lg/ml) alone or in combination with SAHA (1 lM) or VPA (1 mM) for 24 h prior to MTT assay.…”