The histone deacetylase inhibitor vorinostat (SAHA) down-regulates c-FLIP and sensitizes human non-small cell lung carcinoma cell lines to ionising radiation
Session type: Poster / e-Poster / Silent Theatre session
Non-small cell lung carcinoma (NSCLC) patients have a very poor prognosis, 65% of patients are diagnosed with stage III or IV disease, which has a 5-year survival rate of 2-19%. Radiation therapy makes a significant clinical contribution to the treatment of advanced stage NSCLC. Strategies to increase the therapeutic index of radiation are sought to improve treatment outcomes. c-FLIP is an anti-apoptotic protein that blocks death receptor-mediated apoptosis by preventing caspase 8 activation at the death-inducing signalling complex (DISC). We have previously shown that c-FLIP is an inhibitor of apoptosis induced by DNA damaging drugs and that the HDAC inhibitor vorinostat down-regulates c-FLIP protein in various cancer types. The role of c-FLIP in mediating resistance to radiotherapy has not been previously determined.
c-FLIP protein was silenced using siRNA transfection. Protein levels were determined by Western blotting. To assess apoptosis PARP cleavage was detected by Western blotting and sub-G1 population analysed by flow cytometry. Clonogenic assays were used to assess long-term survival. Caspase activation was determined using caspase activity assays and Western blotting. The DNA damage response was assessed by γH2AX foci counting and flow cytometry.
Silencing c-FLIP expression enhanced cell death induced by ionising radiation, as detected by PARP cleavage, caspase activity and sub-G1 population. Similarly, pre-treatment with vorinostat sensitized cells to ionizing radiation. γH2AX is a sensitive marker of the cellular response to the presence of DNA double-strand breaks. γH2AX levels were increased and prolonged in cells in which c-FLIP protein expression was down-regulated.
Our results indicate that c-FLIP down-regulation sensitizes NSCLC cells to DNA damage induced apoptosis.Thus, pharmacological inhibition of c-FLIP, using the HDAC inhibitor vorinostat, or other methods is a potential therapeutic approach for enhancing ionising radiation induced DNA damage and cell death.