Background: New cancer therapeutic strategies must be investigated that enhance prostate cancer treatment while minimizing associated toxicities. We have previously shown that genistein, the major isoflavone found in soy, enhanced prostate cancer radiotherapy in vitro and in vivo. In this study, we investigated the cellular and molecular interaction between genistein and radiation using PC-3 human prostate cancer cells.

We previously showed that genistein, the major bioactive component of soy isoflavones, acts as a radiosensitizer and potentiates prostate tumor cell killing by radiation in vitro and in animal tumor models in vivo. However, when given alone in vivo, pure genistein promoted increased lymph node metastasis, which was not observed with a soy isoflavone mixture consisting of genistein, daidzein, and glycitein. In this study, we show that soy inhibits tumor cell growth and potentiates radiation-induced cell killing in vitro like pure genistein. In an orthotopic model, combining soy isoflavones with tumor irradiation inhibited prostate tumor growth. To determine the molecular mechanisms by which soy isoflavones potentiate radiotherapy, we investigated apurinic/apyrimidinic endonuclease 1/redox factor-1 (APE1/Ref-1) and nuclear factor KB (NF-KB), two signaling molecules involved in survival pathways. Soy isoflavones decreased APE1/Ref-1 expression in vitro, whereas radiation up-regulated it. Pretreatment with soy isoflavones followed by radiation inhibited APE1/Ref-1 expression. APE1/Ref-1 decrease correlated with decreased DNA-binding activity of NF-KB mediated by soy isoflavones and radiation, thus promoting cell killing. In vivo treatment of prostate tumors with soy isoflavones and radiation downregulated APE1/Ref-1 protein expression and NF-KB activity, confirming the molecular alterations observed in vitro. The down-regulation of APE1/Ref-1 and NF-KB by isoflavones, in vitro and in vivo, supports our hypothesis that these markers represent biological targets of isoflavones. Indeed, a 2-fold increase in APE1/Ref-1 expression, obtained by cDNA transfection, resulted in a 2-fold increase in NF-KB DNA-binding activity, and both of which were downregulated by soy isoflavones, confirming the cross-talk between these molecules and, in turn, causing radiosensitization. [Cancer Res 2007;67(5):2141-9]

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...

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Dietary intake of foods rich in antioxidant properties is suggested to be cancer protective. Foods rich in antioxidant properties include grape (Vitis vinifera), one of the world's largest fruit crops and most commonly consumed fruits in the world. The composition and cancer-protective effects of major phenolic antioxidants in grape skin and seed extracts are discussed in this review. Grape skin and seed extracts exert strong free radical scavenging and chelating activities and inhibit lipid oxidation in various food and cell models in vitro. The use of grape antioxidants are promising against a broad range of cancer cells by targeting epidermal growth factor receptor (EGFR) and its downstream pathways, inhibiting over-expression of COX-2 and prostaglandin E2 receptors, or modifying estrogen receptor pathways, resulting in cell cycle arrest and apoptosis. Interestingly, some of these activities were also demonstrated in animal models. However, in vivo studies have demonstrated inconsistent antioxidant efficacy. Nonetheless, a growing body of evidence from human clinical trials has demonstrated that consumption of grape, wine and grape juice exerts many health-promoting and possible anti-cancer effects. Thus, grape skin and seed extracts have great potential in cancer prevention and further investigation into this exciting field is warranted.

The biological mechanisms responsible for aging remain poorly understood. We propose that increases in DNA damage and mutations that occur with age result from a reduced ability to repair DNA damage. To test this hypothesis, we have measured the ability to repair DNA damage in vitro by the base excision repair (BER) pathway in tissues of young (4-month-old) and old (24-month-old) C57BL/6 mice. We find in all tissues tested (brain, liver, spleen and testes), the ability to repair damage is significantly reduced (50-75%; P<0.01) with age, and that the reduction in repair capacity seen with age correlates with decreased levels of DNA polymerase beta (beta-pol) enzymatic activity, protein and mRNA. To determine the biological relevance of this age-related decline in BER, we measured spontaneous and chemically induced lacI mutation frequency in young and old animals. In line with previous findings, we observed a three-fold increase in spontaneous mutation frequency in aged animals. Interestingly, lacI mutation frequency in response to dimethyl sulfate (DMS) does not significantly increase in young animals whereas identical exposure in aged animals results in a five-fold increase in mutation frequency. Because DMS induces DNA damage processed by the BER pathway, it is suggested that the increased mutagenicity of DMS with age is related to the decline in BER capacity that occurs with age. The inability of the BER pathway to repair damages that accumulate with age may provide a mechanistic explanation for the well-established phenotype of DNA damage accumulation with age.

We previously showed that treatment of prostate cancer cells with soy isoflavones and radiation resulted in greater cell killing in vitro, and caused downregulation of NF-jB and APE1/Ref-1. APE1/Ref-1 functions as a redox activator of transcription factors, including NF-jB and HIF-1a. These molecules are upregulated by radiation and implicated in radioresistance of cancer cells. We extended our studies to investigate the role of HIF-1a survival pathway and its upstream Src and STAT3 molecules in isoflavones and radiation interaction. Radiation induced phosphorylation of Src and STAT3 leading to induction of HIF-1a. Genistein, daidzein or a mixture of soy isoflavones did not activate this pathway. These data were observed both in PC-3 (AR-) and C4-2B (ARþ) androgen-independent cell lines. Pretreatment with isoflavones inhibited Src/STAT3/HIF-1a activation by radiation and nuclear translocation of HIF-1a. These findings correlated with decreased expression of APE1/Ref-1 and DNA binding activity of HIF-1a and NF-jB. In APE1/Ref-1 cDNA transfected cells, radiation caused a greater increase in HIF-1a and NF-jB activities but this effect was inhibited by pretreatment with soy prior to radiation. Transfection experiments indicate that APE1/Ref-1 inhibition by isoflavones impairs the radiation-induced transcription activity of NF-jB and HIF-1a. This mechanism could result in the inhibition of genes essential for tumor growth and angiogenesis, as demonstrated by inhibition of VEGF production and HUVECs tube formation. Our novel findings suggest that the increased responsiveness to radiation mediated by soy isoflavones could be due to pleiotropic effects of isoflavones blocking cell survival pathways induced by radiation including Src/STAT3/HIF-1a, APE1/Ref-1 and NF-jB.

Pretreatment with genistein, a bioactive component of soy isoflavones, potentiated cell killing induced by radiation in human PC-3 prostate cancer cells in vitro. Using an orthotopic xenograft in nude mice, we demonstrated that genistein combined with prostate tumor irradiation caused greater inhibition of primary tumor growth and increased control of spontaneous metastasis to para-aortic lymph nodes, increasing mouse survival. Paradoxically, treatment with genistein alone increased metastasis to lymph nodes. This observation is of concern in relation to soy-based clinical trials for cancer patients. To address whether this observation is because nude mice have an impaired immune system, these studies were repeated in orthotopic RM-9 prostate tumors in syngeneic C57BL/6 mice. The combination of genistein with radiation in this model also caused a greater inhibition of primary tumor growth and spontaneous metastasis to regional para-aortic lymph nodes, whereas treatment with genistein alone showed a trend to increased lymph node metastasis. Data from the syngeneic and xenograft models are comparable and indicate that the combination of genistein with radiotherapy is more effective and safer for prostate cancer treatment than genistein alone, which promotes metastatic spread to regional lymph nodes.