We previously reported that genistein, the bioactive isoflavone of soybeans, acts as a radiosensitizer for prostate cancer. Pretreatment of tumor cells with genistein potentiated radiation-induced killing in vitro and in orthotopic models in vivo. However, pure genistein promoted increased lymph node metastasis, when administered alone in vivo. We investigated in vitro and in vivo the effects of soy isoflavones (genistein, daidzein and glycitein) as soy pills of similar composition are used in human interventions but not pure genistein. Soy isoflavones inhibited cell survival and potentiated radiation cell killing in PC-3 tumor cells, in vitro. Increased cell killing correlated with inhibition of antiapoptotic molecules Bcl-xL and survivin, upregulation of proapoptotic Bax molecule and PARP cleavage, suggesting activation of apoptotic pathways. In vivo, using the PC-3 orthotopic metastatic mouse model, soy isoflavones and prostate tumor irradiation led to enhanced control of primary tumor growth and metastasis, as observed with pure genistein and radiation. Interestingly, treatment with soy isoflavones did not increase metastasis to para-aortic lymph nodes in contrast to the consistent increase caused by pure genistein. Histologically prostate tumors, treated with soy isoflavones and radiation, showed tumor destruction and in situ tissue alterations, comparable with genistein and radiation effects. However, genistein, but not soy isoflavones, caused induction of HIF1-a in prostate tumors, suggesting that induction of hypoxia by pure genistein could contribute to increased metastasis. Our studies demonstrate the safety and potential role of soy isoflavones for enhancing the therapeutic effect of radiotherapy in prostate cancer. ' 2007 Wiley-Liss, Inc.Key words: prostate cancer; soy isoflavones; genistein; radiation In the United States, prostate cancer (PCa) is the most commonly diagnosed cancer in men as well as the second leading cause of male cancer deaths. 1,2 Localized PCa is sensitive to conventional radiotherapy using megavoltage photons (X-rays), yet residual disease causes clinical relapse. 2 Thus, for improving the local control of PCa, the combination of radiation with additional antitumor agents is warranted. Epidemiological studies indicate that there is an inverse association between prostate cancer risk and consumption of phytoestrogens including isoflavones. [3][4][5] The isoflavones in soybeans mainly include genistein, daidzein and glycitein. 5 Genistein, the most bioactive isoflavone, has demonstrated inhibition of tumor cell growth in human tumor cell lines. 5 In particular, genistein inhibited the growth of human PCa cells in vitro by affecting the cell cycle and inducing apoptosis. 5 To improve the therapeutic efficacy of radiotherapy for prostate cancer, we have previously investigated the potential of genistein to act as a radiosensitizer. 6-9 Genistein significantly increased tumor cell death when given prior to radiation in several human cancer cell lines in vitro, including the PC-3 PCa...