Calycosin Inhibits ER-Negative Breast Cancer Cells Through Downregulating ER-α30-Mediated PI3K/AKT Signaling Pathway

Objectives: To investigate the role of the novel estrogen receptor(ER)splice variant: ER-30 in isoflavone phytoestrogen calycosin-induced anticancer effects in ER-negative breast cancer cells,as well as the underlying molecular mechanism in it.We aim to provide a promising therapeutic option for breast cancer patients and disclose a better understanding of ER-α30 in ER-negative breast cancer development and progression.Methods: Two ER-negative breast cancer cell lines(MDA-MB-231 and SK-BR-3)were cultured with different concentrations of calycosin(0,20,40 and 80 μmol/L)for 48 h.CCK-8 and colony formation assays were performed to assess the proliferation of ER-negative breast cancer cells.Hoechst33258,flow cytometry and western blot assays were performed to assess cell apoptosis and the expression levels of characteristic apoptotic biomarkers Bcl-2,Bax,Caspase3 and Cleaved-Caspase3.At the same time,the expression levels of ER-α30 in human normal mammary epithelial MCF-10 A cells,ER-negative breast cancer MDA-MB-231 and SK-BR-3 cells,as well as the effect of calycosin on ER-α30 expression were assessed by western blot and q RT-PCR assays,respectively.Subsequently,the gain-of-function and loss-of-function models were constructed by plasmid against ER-α30.The efficiency of overexpression and knockdown was verified by western blot.CCK-8,colony formation,Hoechst33258,flow cytometry and western blot assays were performed to assess cell proliferation,apoptosis,and apoptotic biomarkers in ER-negative breast cancer cells after overexpression and knockdown of ER-α30.Then,the activation of PI3K/AKT signaling pathway was detected by western blot in transfected cells with or without calycosin treatment.Results: CCK-8,colony formation,Hoechst33258 and flow cytometry assays showed that calycosin significantly inhibited cell proliferation and induced apoptosis in a concentration-dependent manner in ER-negative breast cancer MDA-MB-231 and SK-BR-3 cells.The expression levels of pro-apoptotic protein Bax,Cleaved-Caspase3 and the ratio of Cleaved-Caspase3/Caspase3 were increased,while anti-apoptotic protein Bcl-2and the ratio of Bcl-2/Bax were decreased after calycosin treatment.It was verified by western blot and q RT-PCR assays that ER-α30 was highly expressed in ER-negative breast cancer MDA-MB-231 and SK-BR-3 cells compared with human normal mammary epithelial MCF-10 A cells.More importantly,calycosin suppressed the expression of ER-α30 in a dose-dependent manner.Knockdown of ER-α30 inhibited proliferation and induced apoptosis in ER-negative breast cancer cells,and promoted the anticancer effects of calycosin,while overexpression of ER-α30 showed the opposite effects.Further investigations in mechanism proved that calycosin inactivated p-PI3 K and p-AKT expressions by suppressing ER-α30 expression,thereby inhibiting tumor growth of ER-negative breast cancer cells.Conclusions: Calycosin,a typical isoflavone phytoestrogen,inhibits proliferation and induces apoptosis through down-regulation of ER-α30expression and inactivation of downstream PI3K/AKT signaling pathway in ER-negative breast cancer cells.