VORINOSTAT RADIOSENSITIZES PANCREATIC CANCERS BY INHIBITING DNA REPAIR AND RADIATION-INDUCED EGFR AND NF-κB SIGNALING

Abstract

Background: Histone deacetylases (HDACs) promote chromatin condensation and silence tumor suppressor genes by transcriptional repression. We hypothesized that vorinostat, an HDAC inhibitor, could radiosensitize pancreatic cancers via decondensation of DNA for greater susceptibility to radiation-induced DNA strand breaks. Methods: MiaPaCa-2, AsPC-1 and Colo357FG.cells were tested for viability using an XTT assay after vorinostat treatment for 72h. Intrinsic radiosensitivity was assessed by clonogenic survival analysis. Apoptosis was assayed by Annexin V flow-cytometry, live-and-dead assay, morphological analysis and Western blot analysis of PARP cleavage and caspase 3 activation. DNA repair was analyzed by a split-dose clonogenic assay, evaluation of γ-H2AX foci, and Western blot (WB) analysis of levels of repair proteins Ku70, Ku86, Rad50, and DNA-PKc. WB analysis of phospho- and total-EGFR and gel shift analysis of NF-κB activity were also performed. Results: Vorinostat inhibited proliferation of MiaPaCa-2, AsPC-1 and Colo357FG cells in a dose-dependent manner (IC50 2μM, 5μM and 5μM, respectively). 48hr pretreatment with vorinostat led to radiosensitization with enhancement factors (at 10% survival) of 1.1, 1.4 and 5.6 for 500nM, 1μM and 3μM vorinostat, respectively, for MiaPaCa-2 and 1.6 for 2μM vorinostat for Colo357FG. Vorinostat enhanced radiation-induced cell death/apoptosis on all assays (additive but not synergistic). Since 3μM vorinostat eliminated the shoulder of the clonogenic survival curve, inhibition of sublethal DNA damage repair was analyzed. When a single fraction of 4Gy was split into two fractions of 2Gy each, 6hrs apart, the recovery of clonogenicity was reduced to14% with vorinostat from that of 32% without vorinostat. Vorinostat prolonged the expression of γ-H2AX foci in radiated cells and decreased the expression of Rad50 and DNA-PKc. Radiation induced transient EGFR phosphorylation and NF-κB activation. Vorinostat inhibited this radiation-inducible pro-survival signaling. Conclusions: Vorinostat sensitizes pancreatic cancer to radiation by inhibiting DNA repair and suppressing radiation-induced EGFR and NF-κB pro-survival signaling pathways.