Intrinsic mechanism of estradiol-induced apoptosis in breast cancer cells resistant to estrogen deprivation

Background: We previously developed an estrogen receptor (ER)-positive breast cancer cell line (MCF-7:5C) that is resistant to long-term estrogen deprivation and undergoes rapid and complete apoptosis in the presence of physiologic concentrations of 17β-estradiol. Here, we investigated the role of the mitochondrial apoptotic pathway in this process. Methods: Apoptosis in MCF-7:5C cells treated with estradiol, fulvestrant, or vehicle (control) was investigated by annexin V-propidium iodide double staining and 4′,6-diamidino-2-phenylindole (DAPI) staining. Apoptosis was also analyzed in MCF-7:5C cells transiently transfected with small interfering RNAs (siRNAs) to apoptotic pathway components. Expression of apoptotic pathway intermediates was measured by western blot analysis. Mitochondrial transmembrane potential (ψ_m) was determined by rhodamine-123 retention assay. Mitochondrial pathway activity was determined by cytochrome c release and cleavage of poly(ADP-ribose) polymerase (PARP) protein. Tumorigenesis was studied in ovariectomized athymic mice that were injected with MCF-7:5C cells. Differences between the treatment groups and control group were determined by two-sample t test or one-factor analysis of variance. All statistical tests were two-sided. Results: MCF-7:5C cells treated with estradiol underwent apoptosis and showed increased expression of proapoptotic proteins, decreased ψ_m, enhanced cytochrome c release, and PARP cleavage compared with cells treated with fulvestrant or vehicle. Blockade of Bax, Bim, and p53 mRNA expression by siRNA reduced estradiol-induced apoptosis relative to control by 76% [95% confidence interval (CI) = 73% to 79%, P<.001], 85% [95% CI = 90% to 80%, P<.001], and 40% [95% CI = 45% to 35%, P<.001], respectively, whereas blockade of FasL by siRNA had no effect. Estradiol caused complete regression of MCF-7:5C tumors in vivo. Conclusion: The mitochondrial pathway of apoptosis plays a critical role in estradiol-induced apoptosis in long-term estrogen-deprived breast cancer cells. Physiologic concentrations of estradiol could potentially be used to induce apoptosis and tumor regression in tumors that have developed resistance to aromatase inhibitors.