Role of Inhibition of DNA Double-Strand Break Repair and Autophagy in Pancreatic Cancer Cytotoxicity and Radiation-Induced Cell Killing

Abstract

Low survival rates of pancreatic ductal adenocarcinoma (PDAC) are attributed to its detection at advanced stage and high chemotherapy and radiation resistance. Previous work in the lab has shown that resistance to radiation therapy can be decreased through simultaneous inhibition of the anti-apoptotic NF-kB cell survival signaling pathway by dimethyl-amino-parthenolide (DMAPT) and inhibition of Warburg cancer metabolism by dichloroacetate (DCA). We sought to determine the mechanism by which dual treatment with DMAPT and DCA increases cytotoxicity and radiation-induced cell killing in pancreatic cancer. Established pancreatic cancer cell lines, MIA-PaCa-2, Panc-1, and AsPc-1 cells were plated in T-25 flasks. For single fraction experiments, cells were treated with no drugs or both DMAPT and DCA at 24 and 48 hours after plating and irradiated with 0-6Gy with 160 kVp X-rays. To assess split dose repair, experimental groups were 5Gy, 2 x 2.5Gy, 5Gy with DMAPT and DCA, and 2x2.5Gy with DMAPT and DCA. Colonies were counted after 10 days of growth, and plating efficiency and survival fractions were calculated. Western blots of autophagy specific ATGs, LC-3a/b, and P62- SQSTM1 proteins were also determined. To assess radiation DNA damage response (DDR), cells were grown on chambered slides, treated with no drugs or dual DMAPT and DCA, irradiated at 2Gy, and fixed at 0hr, 1hr, and 24hrs after irradiation. After labeling with Gamma-H2Ax antibody, 53BP1 antibody, and DAPI, cells were imaged on a Leica Sp8 confocal microscope. Foci quantification was performed using ImageJ software. Forty-eight hours of DMAPT and DCA dual treatment significantly reduced plating efficiency of MIA-PaCa-2 cells from 0.406±0.092 (40.6%) to 0.253±.0.10 (25.3%), of Panc-1 cells from 0.528±0.11 (52.8%) to 0.0755±0.044 (7.55%), and of AsPc-1 cells from 0.294±0.16 (29.4%) to 0.0300±0.031 (3.0%). Cell death involves alterations of autophagy regulatory proteins. Treatment of cells with two 2.5Gy fractions given four hours apart with DMAPT and DCA dual treatment significantly reduced cell survival fraction compared to split-dose fractionated cells without drug treatment in MIA-PaCA-2 (0.224±0.098 to 0.0716±0.064), Panc-1(0.260±0.040 to 0.0130±0.012), and AsPc-1 cells (0.121±0.011 to 0.0370±0.026). Furthermore, for all three cell lines, persistence of gamma-H2Ax foci was seen at 24 hours post 2Gy irradiation in DMAPT and DCA dual treated cells compared to 2 Gy irradiated only cells, where foci levels returned to baseline levels. Dual DMAPT and DCA treatment of pancreatic cancer decreases plating efficiency, inhibits autophagy, inhibits split dose repair, and results in persistence of gamma-H2Ax foci at 24 hours post irradiation. We conclude that dual DMAPT and DCA treatment of pancreatic cancer increases radiation-induced cell killing through inhibition of DNA double-strand break repair.