Prostate cancer (PCa) is the most common malignant tumor in men. Epidemiologic studies have shown an inverse association between dietary intake of soy and PCa risk. We have previously shown that pre-treatment of PC-3 human PCa cell lines with genistein, the major isoflavone in soy, enhanced radiation-induced cell killing. In this study, we show that genistein combined with radiation caused 80% inhibition in PC-3 colony formation compared to 40% with genistein at 15μM and 65% with 3Gy photon radiation. This effect was also observed with human breast and renal cancer cell lines suggesting that our results are not restricted to PC-3 cells and can be applied to other cancer types as well. Importantly, the sequence of genistein followed by radiation, followed by continuous exposure of genistein was required to produce the optimal effect. The molecular mechanism behind colony formation inhibition was also investigated. Cell cycle analysis demonstrated that a more significant G2/M cell cycle arrest was induced by genistein and radiation compared to each modality alone, with treatment being dose- and time-dependent. Enhanced G2/M arrest strongly correlated with a greater decrease in cyclin B1 and a greater increase in p21 protein expression levels as determined by western blot analysis. We also found strong nuclear localization of p21 in response to genistein, with highest levels of nuclear localization found in genistein pre-treated PC-3 cells exposed to radiation. To understand the mechanism behind cell cycle inhibition, we investigated the activation of NF-κB, a well-known transcription factor critically involved in the survival of cells. Genistein was found to decrease activation of NF-κB, whereas radiation showed an increase detectable as early as 30 minutes post-radiation. Interestingly, we found that radiation-induced activation of NF-κB activity was completely abrogated by pre-treatment of cells with genistein, probably driving the cells toward apoptosis. Accordingly, analysis of the protein expression level of cleaved PARP, a known marker of apoptosis, demonstrated that PC-3 cells pre-treated with genistein followed by radiation treatment had the highest level of cleaved PARP protein expression compared with genistein or radiation alone, indicating increased cell death. These findings demonstrate that the combined treatment of PCa cells with genistein and radiation inhibits their growth in vitro by affecting cell cycle progression and inducing a cascade of molecular events that drive cells toward apoptosis, thus indicating that genistein could be effective in combination with radiation for the treatment of PCa.
[Proc Amer Assoc Cancer Res, Volume 46, 2005]