Intrinsic Mechanism of Estradiol-Induced Apoptosis in Breast Cancer Cells Resistant to Estrogen Deprivation

• Published in: JNCI : Journal of the National Cancer Institute 2005-12, Vol.97 (23), p.1746-1759
• Main Authors: Lewis, Joan S., Meeke, Kathleen, Osipo, Clodia, Ross, Eric A., Kidawi, Noman, Li, Tianyu, Bell, Eric, Chandel, Navdeep S., Jordan, V. Craig
• Format: Article
• Language: English
• Subjects: Annexin A5 - metabolism; Antineoplastic Agents, Hormonal - pharmacology; Apoptosis - drug effects; Apoptosis Regulatory Proteins - metabolism; Biological and medical sciences; Blotting, Western; Breast cancer; Breast Neoplasms - drug therapy; Breast Neoplasms - metabolism; Breast Neoplasms - pathology; Caspase 8; Caspases - metabolism; Cell Line, Tumor; Cell Proliferation - drug effects; Cytochromes c - metabolism; Estradiol - analogs & derivatives; Estradiol - metabolism; Estradiol - pharmacology; Estrogen Antagonists - pharmacology; Estrogens; Fas Ligand Protein; fas Receptor - metabolism; Female; Flow Cytometry; Gene expression; Gene Expression Regulation, Neoplastic; Gynecology. Andrology. Obstetrics; Humans; In Situ Nick-End Labeling; Mammary gland diseases; Medical sciences; Membrane Glycoproteins - metabolism; Microscopy, Electron; Mitochondria - drug effects; Mitochondrial DNA; Poly (ADP-Ribose) Polymerase-1; Poly(ADP-ribose) Polymerases - metabolism; Polymerase Chain Reaction; Proto-Oncogene Proteins - metabolism; Proto-Oncogene Proteins c-bcl-2 - metabolism; RNA, Small Interfering - metabolism; Signal Transduction - drug effects; Transfection; Tumor Necrosis Factors - metabolism; Tumor Suppressor Protein p53 - metabolism; Tumors; Up-Regulation
• ISSN: 0027-8874; 1460-2105
• DOI: 10.1093/jnci/dji400

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