In this study, we evaluated the individual and combined effects of equol and 4-OHT, the bioactive metabolite of tamoxifen, in the ER positive MCF-7 breast cancer cells. Our findings show for the first time that equol not only does not abolish the anti-tumor effects of tamoxifen, but instead it induces apoptosis and significantly enhances tamoxifen’s pro-apoptotic effects in these cells (Figure
1A-C and Figure
2A-C). Moreover, the pan-caspase inhibitor Z-VAD-FMK significantly inhibited equol- and tamoxifen- induced apoptosis (Figure
3), suggesting that these compounds activate the caspase-mediated apoptotic pathway. However, the inhibition was not complete, suggesting that caspase-independent mechanisms may also be involved in equol and tamoxifen induced apoptosis. Previous studies support our findings showing that equol inhibits MCF-7 proliferation and induces caspase-mediated apoptosis in ER negative breast cancer cells and rat mammary tumors [
8,
48,
49]. With respect to tamoxifen, previous studies provide evidence that tamoxifen induces caspase-dependent apoptosis in MCF-7 and other types of cancer cells [
30,
32,
50‐
53]. Even though high concentrations of equol (100 μM) were required to activate MCF-7 apoptosis, which are not physiologically achievable in human plasma due to metabolic conversion of the active aglycone equol to the inactive conjugated form [
54], our results may find applications in targeted immunotherapies, which may enable maximal delivery of equol into the cancer cells. This strategy was previously used successfully for genistein, which was immunoconjugated with a monoclonal antibody and targeted to a B cell-specific receptor for treatment of an animal model of B-cell precursor leukemia [
55].
To fully explore the apoptotic pathway activated by equol and tamoxifen, we investigated their effects on key proteins involved in apoptosis, such PARP, α-fodrin and caspases −6, -7, -8 and −9. Caspase-9 is part of the intrinsic (mitochondrial) apoptotic pathway and is activated by cytochrome-c release from the mitochondria, whereas caspase-8 is part of the extrinsic apoptotic pathway activated by external signals through the death receptors [
45]. Active caspase −9 and caspase-8 in turn induce cleavage and activation of the effector caspases −3, -6 and −7 [
43,
45,
56,
57], which subsequently cleave nuclear and cytosolic targets, such as PARP and α-fodrin, resulting in cell destruction [
43,
44]. Since MCF-7 cells are deficient of functional caspase-3, the effector caspase-7 is responsible for apoptosis in these cells [
58‐
60]. Our experiments show that equol and 4-OHT induce PARP and α-fodrin proteolysis, which was significantly enhanced by their combination and partially inhibited by the pan-caspase inhibitor Z-VAD-FMK (Figure
4A), suggesting that additional proteases besides caspases may be involved in equol- and tamoxifen-induced apoptosis. Furthermore, the combination of equol and tamoxifen induced a pronounced caspase-9 and caspase-7 cleavage accompanied with cytochrome-c release into the cytosol, without affecting caspases-6 and −8 (Figure
4B and Figure
5). Treatment with either equol or tamoxifen, on the other hand, had a lesser effect on caspase-9 and caspase-7 cleavage associated with a trivial effect on cytochrome-c release from the mitochondria into the cytosol. Consequently, the combination of equol and tamoxifen is significantly more potent in inducing MCF-7 cell apoptosis than each compound alone. Therefore, our data suggest that equol and tamoxifen activate the intrinsic apoptotic pathway. Previous studies support our findings as they have shown activation of the intrinsic apoptotic pathway in MCF-7 cells by tamoxifen and daidzein [
2,
29‐
31,
51,
61,
62]. Moreover, equol and tamoxifen induced a time-dependent reduction in blc-2 expression and hence the bcl-2:bax ratio, which was further reduced by the combination of the two compounds (Figure
6). Decreased bcl-2 expression was observed in several cancer cell types treated with tamoxifen and daidzein [
14,
63,
64] and in equol-induced apoptosis in mammary carcinomas [
14,
48].