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Calcitriol or 1,25-dihydroxyvitamin D3, the hormonally active form of vitamin D, as well as vitamin D analogs, has been shown to increase sensitivity to ionizing radiation in breast tumor cells. The current studies indicate that the combination of 1,25-dihydroxyvitamin D3 with radiation appears to kill p53 wild-type, estrogen receptor-positive ZR-75-1 breast tumor cells through autophagy. Minimal apoptosis was observed based on cell morphology by DAPI and TUNEL staining, annexin/PI analysis, caspase-3, and PARP cleavage as well as cell cycle analysis. Induction of autophagy was indicated by increased acridine orange staining, RFP-LC3 redistribution, and detection of autophagic vesicles by electron microscopy, while autophagic flux was monitored based on p62 degradation. The autophagy inhibitors, chloroquine and bafilomycin A1, as well as genetic suppression of the autophagic signaling proteins Atg5 or Atg 7 attenuated the impact of the combination treatment of 1,25 D3 with radiation. In contrast to autophagy mediating the effects of the combination treatment, the autophagy induced by radiation alone was apparently cytoprotective in that either pharmacological or genetic inhibition increased sensitivity to radiation. These studies support the potential utility of vitamin D for improving the impact of radiation for breast cancer therapy, support the feasibility of combining chloroquine with radiation for the treatment of breast cancer, and demonstrate the existence of an "autophagic switch" from cytoprotective autophagy with radiation alone to cytotoxic autophagy with the 1,25 D3-radiation combination.

A Switch Between Cytoprotective and Cytotoxic Autophagy in the Radiosensitization of Breast Tumor Cells by Chloroquine and Vitamin D