Background

c-FLIP is an anti-apoptotic protein that blocks death receptor-mediated apoptosis by inhibiting caspase 8 activation. c-FLIP is frequently over-expressed in colorectal cells, and it has previously been shown that high c-FLIP expression limits the effectiveness of chemotherapy treatment. We have previously shown that reduction of c-FLIP expression using siRNA synergistically enhances the response of CRC cells to chemotherapy or TRAIL. Pan-HDAC inhibitors (HDACi) such as SAHA have been shown to be effective anti-cancer agents. Our aims were to assess the effect of HDACi on c-FLIP expression in colorectal cancer

Method

Each inhibitor was used in a panel of CRC cell lines (HCT116, HT29, H630 and RKO). Protein expression was assessed by Western blotting. Apoptosis was assessed by PARP cleavage and Flow Cytometry using PI and Annexin V/PI staining. mRNA expression was determined using RT-qPCR. Proteasomal inhibiton was achieved using MG132. Ubiquitination assays were obtained from Pierce, and precipitate visualised using Western blot.

Results

Treatment with HDAC inhibitors was found to induce apoptosis in a panel of colorectal cancer cell lines in a manner that was dependent on caspase 8, TRAIL receptor DR5 and down-regulation of c-FLIP protein expression.  Similar results were obtained using in vivo models of colorectal cancer. Further analysis revealed that HDAC inhibitors down-regulate c-FLIP expression post-tranlationally by triggering its ubiquitination and proteasomal degradation.  Using more selective HDAC inhibitors, we identified HDAC6 as the key HDAC involved in regulating c-FLIP expression as specific HDAC6 inhibition recapitulated the effects of SAHA on c-FLIP expression and apoptosis induction in colorectal cancer cell models

Conclusion

HDAC inhibitors induce apoptosis in colorectal cancer cells by down-regulating c-FLIP and thereby activating DR4/5-dependent, caspase 8-mediated apoptosis. Thus, HDAC inhibitors act as effective post-translational suppressors of c-FLIP expression. Moreover, we have identified HDAC6 as the key HDAC involved in regulating c-FLIP expression in colorectal cancer cells