Combinatorial anti-proliferative effects of tamoxifen and naringenin: The role of four estrogen receptor subtypes

Breast cancer is the most diagnosed diseases and the second-leading cause of death in females, among which the estrogen receptor positive (ER +) patients are more common of all cases. In present study, we selected MCF-7 as an in vitro model and investigated the combinatorial anti-proliferative effects of tamoxifen and naringenin on ER + breast cancer, and then explored the potential mechanisms involved in mitochondrial dysfunction and oxidative stress mediated by several estrogen receptor subtypes. Six assessment endpoints including cell viability, cell migration, cell cycle, cell apoptosis, mRNA, and protein expression were estimated. Tamoxifen and naringenin were shown to inhibit the cell growth of MCF-7 cells at higher concentrations, and co-exposure with them significantly inhibited cell proliferation in an additive manner. Results from a wound healing assay indicated that the combined treatment of tamoxifen and naringenin markedly suppressed cell migration compared with the single exposure by downregulating the expression of MMP-9 and MMP-2. Flow cytometry analysis revealed that the combined treatment of tamoxifen and naringenin blocked cell cycle in G2/M phase through suppressing the transcription of cell cycle regulation proteins. Simultaneously, co-treatment with them also induced cell apoptosis by regulating the expression of mitochondrial apoptotic proteins as well as by simulating the production of reactive oxygen species (ROS). Real-time quantitative polymerase chain reaction (RT-qPCR) and western blotting (WB) analysis results further demonstrated that the two nuclear estrogen receptors-ER alpha 66 and ER beta, as well as the two membrane estrogen receptors-ER alpha 36 and GPR30 were downregulated when cells were exposed to single tamoxifen, whereas naringenin treatment group not only downregulated the expression of ER alpha 66 and GPR30 but also upregulated ER beta and ER alpha 36. Interestingly, co-treatment group resulted in significant downregulation of ER alpha 66, ER alpha 36 and GPR30 but upregulation of ER beta. Taken together, co-treatment of tamoxifen and naringenin could inhibit cell proliferation more effectively in ER + breast cancer cells, which was associated with a modulation of the expression levels of several estrogen receptors.