Molecular Mechanisms Of Action Of Isoflavones

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14.4.1 Regulation of the Expression of Genes Related to Cell Cycle and Apoptosis

To explore the molecular mechanisms by which genistein induces cell cycle arrest, we have examined the expression of cell cycle-related genes including cyclins, CDC2, and cyclin dependent kinase inhibitors (CDKIs). Our results showed that the treatment of cells with different concentrations of genistein caused a dose-dependent decrease in the expression of cyclin Bj,50,66 corresponding with the G2/M phase cell cycle arrest as observed by flow cytometry. Isoflavone-induced G2/M arrest has been associated with the inhibition of CDC2 kinase activity.77,79 We and other investigators have also found significant up-regulation of p21WAFJ expression in genistein-treated cancer cells compared to control cells.31,32,38,50,51,66,73,79 Moreover, our microarray data showed that genistein inhibited cell growth through downregulation of cell proliferation and cell cycle-related genes (cyclin B, CDC25A, TGF-P, ki67).80 These results suggest that downregulation of cyclin B1, CDC2, CDC25A, TGF-P, and ki67, and upregulation of p21WAF1 could be one of the mechanisms by which genistein arrests cancer cells in G2/M phase and inhibits cancer cell growth.

To explore the molecular mechanisms by which genistein induces apoptosis, our laboratory has examined the expression of genes that are critically involved in the apoptotic pathways after genistein treatment. The results showed that genistein treatment reduced Bcl-2 protein expression and significantly increased expression of Bax in all cancer cells tested.31,32,50,66,73,81 Other investigators also reported that soy isoflavones could induce apoptosis in human hepatoma cells and breast cancer cells through caspase-3 activation and downregulation of Bcl-2, Bcl-XL, and HER-2/neu.39,77,82 Kazi et al.83 showed that genistein induced apoptosis by inhibition of proteasome and induction of p27KIP1, iKB-a, and Bax.83 These results suggest that caspase activation, inhibition of proteasome, upregulation of

Bax, and downregulation of Bcl-2, Bcl-XL, and HER-2/neu may be additional molecular mechanisms by which isoflavones induce apoptosis.

14.4.2 Regulation of Cell Signaling Pathways 14.4.2.1 NF-kB Pathway

Nuclear factor-KB (NF-kB) pathway plays important roles in the control of cell growth, differentiation, apoptosis, inflammation, stress response, and many other physiological processes in cellular signaling. Because of its importance in cancer development and progression, NF-kB has been described as a major culprit and a therapeutic target in cancer.84-88 To investigate whether genistein regulates cell growth and apoptosis through NF-kB pathway, our laboratory examined NF-kB DNA-binding activity in genistein treated PC3 and LNCaP prostate cancer cells by electrophoretic mobility shift assay (EMSA).89 The results showed that genistein significantly inhibited NF-kB DNA-binding activity in both cell lines and abrogated the induction of NF-kB DNA-binding activity stimulated by either H2O2 or TNF-a. These results demonstrated that genistein inhibits NF-kB DNA-binding activity in both nonstimulated and stimulated conditions.89 Similar results have been reported by other investigators, showing that NF-kB DNA binding and COX-2 promoter activity were enhanced by TNF-a, and these effects were inhibited by genistein in human lung epithelial cells.90

It has been known that NF-kB DNA-binding activity could be activated by IkB phosphorylation, IkB could be phosphorylated by activated IkB kinase (IKK), and IKK could be phosphorylated and activated by an upstream kinase, mitogen-activated kinase kinase 1 (MEKK1).91-94 The results from our laboratory showed that genistein treatment inhibited MEKK1 kinase activity and reduced the amount of phosphorylated IKB in prostate cancer cells. Cells treated with TNF-a or H2O2 showed increased MEKK1 kinase activity and genistein pretreat-ment blocked MEKK1 kinase activation stimulated by TNF-a or H2O2. These results suggest that genistein may inhibit MEKK1 kinase activity and subsequent phosphorylation of IkB, thereby resulting in the inactivation of NF-kB.

It has been reported that some chemotherapeutic agents such as cisplatin and docetaxel induce the activation of NF-kB in cancer cells, and this may be responsible for drug resistance in cancer cells. Therefore, inactivation of NF-kB in combination with chemotherapeutic agents may lead to better killing of cancer cells by existing chemotherapeutic agents. Indeed, we have found that the combination treatment of genistein with lower doses of docetaxel or cisplatin elicited significantly greater inhibition of cell growth compared to either agent alone.95 The combination treatment induced more apoptosis compared to single agents. By EMSA, we found that NF-kB activity was significantly increased by docetaxel or cisplatin treatment, and the NF-KB-inducing activity of these agents was completely abrogated in cells pretreated with genistein. These results clearly suggest that genistein pretreatment, which inactivates NF-kB activity, together with other cellular effects of genistein, may contribute to increased cell growth inhibition and apoptosis with nontoxic doses of docetaxel or cisplatin, which could be a novel strategy for the treatment of cancer.

14.4.2.2 Akt Pathway

Akt pathway is an important cell signal transduction pathway. It has been known that Akt is activated by phospholipid binding and phosphorylation at Thr308 by PDK1 or at Ser473 by PDK2.96 Activated Akt functions to promote cell survival by inhibiting apoptosis through inactivation of pro-apoptotic factors.97-99 Akt also regulates NF-kB pathway via phosphorylation and activation of molecules in NF-kB pathway. Because of its importance in promoting cell survival, Akt has received much attention and has been believed to be a therapeutic target in cancer.100,101 We have previously investigated the effects of genistein on Akt pathway in PC3 prostate cancer cells.55 We found that genistein reduced the level of phosphorylated Akt protein and the Akt kinase activity under nonstimulated condition. Genistein also abrogated Akt activation stimulated by EGF, suggesting the inactivation of Akt kinase under both nonstimulated and stimulated conditions after genistein treatment.

We have further investigated the inhibitory mechanisms of genistein on Akt and NF-kB pathways by transfection experiments.55 Akt expression construct (pLNCX-Akt) was transiently co-transfected with NF-kB-Luc reporter construct into PC3 prostate cancer cells. Luciferase assay showed an induced luciferase activity in PC3 cells co-transfected with pLNCX-Akt and NF-kB-Luc. However, genistein inhibited the luciferase activity in PC3 cells co-transfected with pLNCX-Akt and NF-kB-Luc. Furthermore, genistein abrogated the activation of Akt in transfected cells stimulated by EGF. EMSA testing for NF-kB DNA-binding activity in transfected cells also showed similar results. These results demonstrate that genistein exerts its inhibitory effects on NF-kB pathway through Akt pathway. Downregulation of NF-kB and Akt signaling pathways by genistein may be one of the molecular mechanisms by which genistein inhibits cancer cell growth and induces apoptosis. Another study by other investigators also demonstrated that genistein could inhibit Akt activation induced by estradiol in MCF-7 cells.102 These studies provide strong molecular evidence showing the anticancer effects of isoflavone genistein through Akt and NF-kB regulation.

14.4.2.3 AR and ER Pathways

It has been known that androgen receptor (AR) signaling pathway is involved in the development and progression of prostate cancer through regulation of transcription of prostate specific antigen (PSA).103 104 We have investigated the effects of genistein on the expression of PSA through androgen regulation105 and found that genistein at low concentration (<10 |imol/L) transcriptionally downregulated AR, decreased nuclear protein binding to ARE, and, thereby, inhibited the transcription and protein expression of PSA in androgen-sensitive LNCaP cells. However, higher concentrations (10 to 50 | mol/L) of genistein was needed to significantly inhibit PSA secretion in VeCaP cells, which are androgen-insensitive, and no alternation in the AR expression or ARE binding activity was observed. By transfection experiments, we found that genistein inhibited PSA synthesis in prostate cancer cells through both androgen-dependent and androgen-independent pathways. These results demonstrate the inhibitory effects of genistein on AR and PSA.

Because of its structural similarity to estrogen, isoflavones have been expected to exert their effects through ER signaling pathway. However, an experimental study has found that isoflavones at different concentrations may exhibit different effects. Genistein might either induce breast cancer cell proliferation by estrogenic agonistic properties (at concentrations <1 |imol/L) or prevent hormone-dependent growth of breast cancer cells by potential estrogen-antagonistic activity (at concentrations >5 |imol/L) dependent on its concentrations.106 Moreover, experimental studies show that isoflavones also have inhibitory effects on hormone-independent cancers. These results suggest that isoflavones may be potent chemopreventive and/or therapeutic agents for cancers, regardless of hormone responsiveness.

14.4.3 Regulation of the Expression of Genes Related to Angiogenesis and Metastasis

Genistein has been shown to reduce the angiogenic and metastatic potential of cancers.76 107 We have examined the effect of genistein on the expression of MMPs in MDA-MB-435 breast cancer cells transfected with c-erbB-2,32 which has been shown to promote secretion of MMPs and subsequent metastasis in experimental models.108 We found that the expression of c-erbB-2, MMP-2, and MMP-9 in MDA-MB-435 cells stably transfected with c-erbB-2 was much higher than that in parental MDA-MB-435 cells. However, the high expression of c-erbB-2, MMP-2, and MMP-9 in 435 transfectants was significantly downregulated by genistein treatment.32 These results suggest that genistein may inhibit the expression of c-erbB-2 and subsequently decrease the secretion of MMPs in breast cancer cells.

To further explore the molecular mechanisms by which genistein exerts its anti-angiogenic and anti-metastatic effects on cancer cells, we have utilized microarray to determine the gene expression profile altered by genistein treat-ment.109 We found that genistein downregulated the expression of MMP-9, protease M, uPAR, VEGF, neuropilin, TSP, BPGF, LPA, TGF-0, TSP-1, and PAR-2, and upregulated the expression of connective tissue growth factor and connective tissue activation peptide.109 All of these genes are related to angiogenesis and metastasis. The microarray data were confirmed by RT-PCR, Western blot, and zymographic analysis at the mRNA and protein levels. We have also conducted animal experiment using SCID-human prostate cancer bone metastasis model. We found that dietary genistein significantly inhibited the growth of PC3 bone tumor and the expression of MMPs.49 Our results demonstrate that genistein regulates the transcription and translation of genes critically involved in the control of angiogenesis, tumor cell invasion and metastasis, and inhibits prostate cancer cell growth in bone metastasis model, suggesting that genistein may be a potent agent against metastatic cancers.

14.4.4 Regulation of Oxidative Stress

Isoflavones have been known to function as antioxidants. Since increased oxida-tive stress is related to carcinogenesis, it has been believed that isoflavones may inhibit carcinogenesis through antioxidative effect of isoflavones. It has been shown that isoflavone reduces hydrogen peroxide-induced DNA damage in sperm110 and that genistein inhibits tumor promoter, 12-0-tetradecanoylphorbol-13-acetate (TPA)-induced hydrogen peroxide production in human polymorphonuclear leukocytes and HL-60 cells,111 suggesting the inhibitory effect of isofla-vones on carcinogenesis. Genistein has also shown to stimulate antioxidant gene expression in colon cancer cells,112 and to inhibit ultraviolet (UV) irradiation-induced oxidative stress in epidermal carcinoma,113 suggesting its inhibitory effects on cancer cells.

Because oxidative stress activates NF-kB DNA binding activity,114 we have investigated whether the effect of isoflavone supplementation could inactivate NF-kB and reduce oxidative damage in lymphocytes in human subjects.115 The lymphocytes from healthy male subjects were harvested from peripheral blood and cultured for 24 h in the absence and presence of genistein. We found that genistein treatment inhibited basal levels of NF-kB DNA binding activity and abrogated TNF-a induced NF-kB activity.115 When human subjects received 50 mg of isoflavone supplements Novasoyâ„¢ twice daily for 3 weeks, TNF-a failed to activate NF-kB activity in lymphocytes harvested from these subjects, while lymphocytes from these subjects collected prior to isoflavone intervention showed activation of NF-kB DNA binding activity upon TNF-a treatment.115 These results suggest that isoflavone supplementation has a protective effect against TNF-a-induced NF-kB activation in humans both in vitro and in vivo. We have also investigated the effect of isoflavone supplementation on oxidative DNA damage by measuring the levels of 5-OHmdU, which represents the endogenous status of cellular oxidative stress, in the peripheral blood lymphocytes of normal human subjects before and after isoflavone supplementation. The results showed that 5-OHmdU was significantly decreased after 3 weeks of isoflavone supplementation.115 These results demonstrate that isoflavones may exert their chemopreventive effects through regulation of oxidative stress.

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