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.

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In MCF-7 breast tumor cells, ionizing radiation promoted autophagy that was cytoprotective; pharmacological or genetic interference with autophagy induced by radiation resulted in growth suppression and/or cell killing (primarily by apoptosis). The hormonally active form of vitamin D, 1,25D 3 , also promoted autophagy in irradiated MCF-7 cells, sensitized the cells to radiation and suppressed the proliferative recovery that occurs after radiation alone. 1,25D 3 enhanced radiosensitivity and promoted autophagy in MCF-7 cells that overexpress Her-2/neu as well as in p53 mutant Hs578t breast tumor cells. In contrast, 1,25D 3 failed to alter radiosensitivity or promote autophagy in the BT474 breast tumor cell line with low-level expression of the vitamin D receptor. Enhancement of MCF-7 cell sensitivity to radiation by 1,25D 3 was not attenuated by a genetic block to autophagy due largely to the promotion of apoptosis via the collateral suppression of protective autophagy. However, MCF-7 cells were protected from the combination of 1,25D 3 with radiation using a concentration of chloroquine that produced minimal sensitization to radiation alone. The current studies are consistent with the premise that while autophagy mediates a cytoprotective function in irradiated breast tumor cells, promotion of autophagy can also confer radiosensitivity by vitamin D (1,25D 3 ). As both cytoprotective and cytotoxic autophagy can apparently be expressed in the same experimental system in response to radiation, this type of model could be utilized to distinguish biochemical, molecular and/or functional differences in these dual functions of autophagy.

Accumulating evidence indicates that the chemokine receptor CCR5 and the chemokine CCL5 may be involved in the proliferation and metastasis of prostate cancer. Consequently, chemokine receptor CCR5 antagonists could potentially act as anti prostate cancer agents. As the first natural product CCR5 antagonist, anibamine provides a novel chemical structural skeleton compared with other known antagonists identified through high-throughput screening. Our studies demonstrate that anibamine produces significant inhibition of prostate cancer cell proliferation at micromolar to submicromolar concentrations as well as suppressing adhesion and invasion of the highly metastatic M12 prostate cancer cell line. Preliminary in vivo studies indicate that anibamine also inhibits prostate tumor growth in mice. These findings indicate that anibamine may prove to be a novel lead compound for the development of prostate cancer therapeutic agents. Keywordschemokine receptor CCR5; antagonist; anibamine; prostate cancer Prostate cancer is the most common non-cutaneous solid cancer occurring amongst men in the USA, and the second most common malignant cause of male death worldwide 1 . Current therapies remain limited to surgery, radiation, and/or androgen ablation 2 . Recent investigations indicate that there is a relationship between some inflammatory processes and cancer, specifically, prostate cancer development [3][4][5][6][7][8][9][10][11][12][13] . For example, the prostate cancer cell lines PC-3, DU145, and LNCaP express the chemokine CCL5 (RANTES) and the * Corresponding author: Department of Medicinal Chemistry, Virginia Commonwealth University, 800 East Leigh Street, P.O. Box 980540, Richmond, VA 23298-0540, USA. Tel: 01-804-828-0021; Fax: 01-804-828-7625; yzhang2@vcu.edu . # These two authors contribute equally to the manuscript.Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. Anibamine (Figure 1), a novel pyridine quaternary alkaloid recently isolated from Aniba sp., was found to bind to CCR5 with an IC 50 of 1 μM in competition with 125I-gp120, an HIV viral envelop protein 14 . Thus far, anibamine is the first known natural product acting as a CCR5 antagonist. While the chemokine receptor CCR5 has mainly been targeted in HIV therapies since it was first cloned more than a decade ago 15-21 , CCR5 antagonists could provide a novel therapeutic approach for prostate cancer treatment through the inhibition of CCL5 induced cell proliferation. NIH Public AccessAnibamine has a novel structural skeleton compared to other CCR5 antagonists identified through high-throughput screening. Con...