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

Joan S. Lewis, Kathleen Meeke, Clodia Osipo, Eric A. Ross, Noman Kidawi, Tianyu Li, Eric Bell, Navdeep S. Chandel, V. Craig Jordan*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

188 Scopus citations

Abstract

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 (ψm) 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 ψm, 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.

Original languageEnglish (US)
Pages (from-to)1746-1759
Number of pages14
JournalJournal of the National Cancer Institute
Volume97
Issue number23
DOIs
StatePublished - Dec 2005

ASJC Scopus subject areas

  • Oncology
  • Cancer Research

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