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 17beta-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 (psim) 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 psim, 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.


CONCLUSIONS
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.
Lewis
Joan S
JS
Fox Chase Cancer Center, Philadelphia, PA 19111, USA.
Meeke
Kathleen
K
Osipo
Clodia
C
Ross
Eric A
EA
Kidawi
Noman
N
Li
Tianyu
T
Bell
Eric
E
Chandel
Navdeep S
NS
Jordan
V Craig
VC
D017304:Annexin A5 / Q000378:metabolism; D018931:Antineoplastic Agents, Hormonal / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects; D051017:Apoptosis Regulatory Proteins / Q000378:metabolism; D015153:Blotting, Western; D001943:Breast Neoplasms / Q000188:drug therapy / Q000378:metabolism* / Q000473:pathology*; D053181:Caspase 8; D020169:Caspases / Q000378:metabolism; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D045304:Cytochromes c / Q000378:metabolism; D004958:Estradiol / Q000031:analogs & derivatives / Q000378:metabolism* / Q000494:pharmacology; D004965:Estrogen Antagonists / Q000494:pharmacology; D053222:Fas Ligand Protein; D005260:Female; D005434:Flow Cytometry; D000077267:Fulvestrant; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D020287:In Situ Nick-End Labeling; D008562:Membrane Glycoproteins / Q000378:metabolism; D008854:Microscopy, Electron; D008928:Mitochondria / Q000187:drug effects; D000071137:Poly (ADP-Ribose) Polymerase-1; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D034741:RNA, Small Interfering / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D014162:Transfection; D048069:Tumor Necrosis Factors / Q000378:metabolism; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism; D015854:Up-Regulation; D019014:fas Receptor / Q000378:metabolism
D017304:Annexin A5; D018931:Antineoplastic Agents, Hormonal; D051017:Apoptosis Regulatory Proteins; C438378:BBC3 protein, human; D004965:Estrogen Antagonists; C505661:FASLG protein, human; D053222:Fas Ligand Protein; D008562:Membrane Glycoproteins; C498702:PMAIP1 protein, human; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D034741:RNA, Small Interfering; D048069:Tumor Necrosis Factors; D016159:Tumor Suppressor Protein p53; D019014:fas Receptor; D000077267:Fulvestrant; D004958:Estradiol; D045304:Cytochromes c; C499320:PARP1 protein, human; D000071137:Poly (ADP-Ribose) Polymerase-1; D011065:Poly(ADP-ribose) Polymerases; C505530:CASP8 protein, human; D053181:Caspase 8; D020169:Caspases