Genistein, a predominant component of soy products, has been shown to have anti-cancer properties [2
]. This study revealed a novel mechanism that genistein utilizes to inhibit proliferation. Proliferation of EGF treated colon cancer cells is mediated by loss of FOXO3 activity [19
], and here we showed this pathway to be inhibited by genistein. Upstream, genistein inhibits EGF induced loss of FOXO3 activity by targeting the PI3K/Akt pathway. Downstream, genistein inhibits EGF-induced FOXO3 disassociation from p53(mut), which further promotes FOXO3 activity and leads to increased expression of the p27kip1 cell cycle inhibitor, which inhibits proliferation in colon cancer cells.
We demonstrated that one of the anti-proliferative mechanisms of genistein in colon cancer cells is to promote FOXO3 activity by inhibiting EGF-induced FOXO3 phosphorylation (inactivation) via the PI3K/Akt pathway. Active FOXO3 negatively regulates proliferation of colon cancer cells [36
], and we showed that its inactivation is an essential step in EGF-mediated proliferation [19
]. Although some studies demonstrated that high concentrations of genistein can downregulate EGFR in prostate cells [37
], we showed that the concentration of genistein used for this study did not affect EGFR expression in colon cancer cells and had modest effects on activation of EGFR that are most likely non-specific. It has been shown that genistein inhibits EGF-stimulated serine, threonine, and tyrosine phosphorylation [38
]. Also, genistein affects estrogen receptors [39
], which are critical in colon cancer progression [14
]. Therefore, we speculate, that genistein inhibit Akt independently of EGFR, by attenuating either kinase activity downstream of EGFR or blocking estrogen receptor. It has been previously demonstrated that genistein inhibits proliferation in colon cancer cells via PI3K/Akt [40
], a pathway known to be critical to colon cancer progression [10
], however downstream mechanisms were not well understood. This study demonstrated that genistein inhibits PI3K/Akt activation that leads to prevention of FOXO3 phosphorylation (inactivation) in colon cancer cells and revealed a new mechanism whereby genistein attenuates proliferation of colon cancer cells.
Active FOXO3 attenuates proliferation by upregulation of the cell cycle inhibitor p27kip1 [36
], and we showed that EGF-induced FOXO3 disassociation from the p27kip1 promoter [19
] is inhibited by genistein in colon cancer cells. In prostate and breast cancer cells, the anti-proliferative effects of genistein occur by increasing levels of the cell cycle inhibitor p27kip1 [43
], but upstream mechanisms were not understood. Here we showed that genistein increases p27kip1 expression in colon cancer cells by promoting FOXO3 binding to the p27kip1 promoter. It is important to take into account that increased p27kip1 by genistein is most likely one of the mechanisms of inhibition of proliferation and that the other targeted molecules also play a role. Also, this study demonstrated that for increased p27kip1 expression, interaction between FOXO3 and mutated tumor suppressor p53 is required. In contrast to human lung cancer cells where genistein increased wild type but not mutated p53 [45
], in colon cancer HT-29 cells we showed that genistein increased expression of mutated p53. Although, wild type p53 interacts with FOXO3 thereby decreasing its activity in the FOXO3-53 complex [29
], this study demonstrated that mutated p53 increased FOXO3 activity in HT-29 cells. Additionally EGF treatment did not affect interactions between wild type p53 and FOXO3 further supporting that a mutation of p53 is most likely accountable for the genistein effect. Since a mutation of p53 is critical to colon cancer development [30
], the anti-proliferative properties of genistein may relate to targeting mutated p53 and thus promoting FOXO3 activity and cell cycle arrest.
This study showed that genistein inhibits proliferation of colon cancer cells by attenuating a negative effect of EGF on tumor suppressor FOXO3 activity, thereby promoting FOXO3 interaction with mutated p53, which leads to expression of p27kip1 and cell cycle arrest. These findings support a potential role of genistein in combination with other chemopreventive agents [3
] for the treatment of colon cancer.