Abstract 577: Equol enhances tamoxifen's antitumor effect by induction of caspase-mediated apoptosis in MCF-7 breast cancer cells

Abstract

Abstract Soy phytoestrogens, such as daidzein and its metabolite equol, have been proposed to be responsible for the low breast cancer rate in Asian women. However, since these compounds possess both estrogenic and anti-estrogenic properties, it remains unknown whether they provide protection against tumor development and progression. Since the majority of estrogen receptor positive (ER+) breast cancer patients are treated with the selective estrogen receptor modulator tamoxifen, the basic objective of this project is to determine whether equol enhances or inhibits tamoxifen's anti-tumor effect and to identify the molecular mechanisms involved. For this purpose, we examined the individual and combined effects of equol and tamoxifen on the ER+ breast cancer cell line MCF-7 (ERα+/−Rα+). We found that equol at concentrations higher than 10 μM significantly reduced the viability of MCF-7 cells. Furthermore, the combination of equol (100 μM) and 4-hydroxytamoxifen (4-OHT; 10 μM) reduced MCF-7 cell viability and induced apoptosis more effectively than each compound alone. Subsequent treatment of MCF-7 cells with the pan-caspase inhibitor Z-VAD-FMK and the calpain/cathepsin inhibitor ALLN demonstrated that equol and 4-OHT induce apoptosis via both caspase-depended and -independed mechanisms. In order to further investigate the apoptotic pathways employed by equol, 4-OHT and their combination, we evaluated the expression of key proteins involved in apoptosis such as PARP, α-fodrin, AIF, bcl-2, bax, p21 and p53 using Western blot analysis. The combination of 4-OHT and equol induced PARP and α-fodrin cleavage which was inhibited by Z-VAD-FMK indicating that their combination induces caspase-depended apoptosis. This was accompanied by reduced bcl-2:bax ratio confirming a pro-apoptotic effect. The combination of equol and 4-OHT induced caspase-7 cleavage indicating a significant role for caspase-7 in equol- and tamoxifen-induced apoptosis. Finally, upon determination of the effect of equol and tamoxifen on estrogen receptor expression in MCF-7 cells, it was found that equol reduces the ERα:ERα ratio, while 4-OHT increases it. When cells were treated with both compounds we observed that the effect of equol prevailed, providing an additional explanation for the beneficial combined effects of equol and tamoxifen. In conclusion, the effects of equol in potentiating tamoxifen's anti-tumor effect may be explained by the induction of the caspase-mediated apoptotic pathway and the reduction of the ERα:ERα ratio. These results can find applications in clinical or pre-clinical cancer chemoprevention studies against breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 577. doi:1538-7445.AM2012-577