Radiosensitization Of Breast Cancer Cells By Vitamin D Or Chloroquine Through Modulation Of The Dual Cytoprotective And Cytotoxic Functions Of Autophagy

Introduction: Previous studies in our laboratory have demonstrated that the active form of vitamin D, 1,25-di hydroxy vitamin D3 (1,25-D3), or its analogs such as EB 1089, can confer enhanced sensitivity to irradiation. Studies using MCF-7 breast tumor cells indicated that radiosensitization was likely to be occurring through the promotion of autophagic cell death. The current work was designed to extend these findings to another (p53 wild type estrogen receptor positive) breast tumor cell line, ZR-75-1, which expresses functional apoptosis executioner caspase, caspase 3. Experimental Procedure: In vitro studies assessed the effects of 100nM 1,25-D3 alone, fractionated doses (4 × 2 Gy) of radiation (IR) delivered over a period of 2 days, and the combination treatment of 100nM 1,25-D3 concurrently with radiation. Radiation sensitivity was assessed by clonogenic survival and by monitoring cell viability over a period of 6 days by trypan blue exclusion. Apoptosis was evaluated based on nuclear fragmentation and western blotting for cleavage of caspase-3 and PARP; autophagy was monitored by acridine orange staining for formation of autophagolysosomes and autophagic flux based on degradation of p62 by western blotting. Pharmacologic inhibition of autophagy utilized 200nM Bafilomycin A1 and 5µM Chloroquine while genetic inhibition involved knockdown of Atg5. Results: 1,25-D3 enhanced sensitivity to ionizing radiation in the ZR-75-1 breast tumor line based on reduced clonogenic survival (50-70% at all radiation doses) and markedly greater suppression of cell viability (60% for 1,25-D3 + IR vs 25% for IR alone). There was no evidence for apoptosis with either radiation alone or 1,25-D3 + IR (lack of nuclear fragmentation or condensation by DAPI/TUNEL staining, lack of caspase-3 or PARP cleavage). Acridine orange staining/flow cytometry and p62 degradation were indicative of autophagy with either radiation alone or 1,25-D3 + IR. Autophagy blockade with chloroquine, bafilomycin or silencing of Atg5 increased sensitivity to radiation alone while attenuating the impact of 1,25-D3 + radiation. Conclusion: Treatment with 1,25-D3 has the capacity to radiosensitize ZR-75-1 breast cancer cells by increasing the rate and extent of cell killing through autophagy. In contrast, inhibition of radiation induced autophagy also confers sensitivity to radiation. We conclude that autophagy appears to be cytoprotective in response to radiation alone and cytotoxic in response to 1,25-D3 + radiation treatment. Consequently, modulation of the dual functions of autophagy may provide a strategy for radiosensitization of breast tumor cells. Supported by Award Number F31CA144812 from the National Cancer Institute and by American Institute for Cancer Research Grant # 06A058-REV. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2675. doi:10.1158/1538-7445.AM2011-2675