Calcitriol or 1,25 dihydroxy vitamin D3, the hormonally active form of vitamin D, as well as vitamin D analogs, have been shown to increase sensitivity to ionizing radiation in breast tumor cells. The current studies indicate that the combination of 1,25 dihydroxy vitamin 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.
Bioluminescent Resonance Energy Transfer is a naturally occurring phenomenon that can be exploited to explore protein-protein interactions in real-time in intact cells and cellular extracts. It detects energy transferred between a bioluminescent donor enzyme (Renilla luciferase) fusion protein and a fluorescent (GFP(2), a mutant of Green Fluorescent Protein) acceptor fusion protein. Optimal detection of BRET(2) energy transfer relies on the distance and orientation generated by the fusion proteins. This chapter describes in detail the BRET(2) assay as it is used to examine the physical interaction between the nuclear receptor ERalpha and the transcriptional coactivator SRC-1. Description of methods include selection of donor and acceptor combinations, fusion construct generation and validation, cell culture and transfection, individual fluorescence and luminescence detection, BRET(2) detection, and data analysis.
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
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