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. 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.

The apoptotic action of estrogen following exhaustive antihormonal therapy: a new clinical treatment strategy.

Long-term antihormonal therapy is effective at controlling the recurrence of estrogen receptor (ER)-positive breast cancer, but there may be unanticipated consequences for the development of new forms of drug resistance. Laboratory studies of exhaustive antihormonal therapy demonstrate there are at least two phases of resistance to selective ER modulators (SERMs; tamoxifen and raloxifene) and to estrogen withdrawal (aromatase inhibitors). In Phase I drug resistance, estrogen or a SERM promote tumor growth, but in Phase II drug resistance estrogen induces apoptosis. Understanding of the new biology of estrogen action has clinical relevance. There are paradoxical interactions between fulvestrant and postmenopausal levels of estrogen that cause robust growth of Phase II tamoxifen resistance or autonomous aromatase-resistant tumors. These new data suggest a rational approach for the treatment of patients with ER-positive breast cancer that have failed exhaustive antihormonal treatment. Low-dose estrogen could be used to debulk patients followed by fulvestrant in a low estrogen environment (aromatase treatment) to maintain tumor control.

Selective estrogen receptor modulators (SERMs): mechanisms of anticarcinogenesis and drug resistance.

Despite the beneficial effects of estrogens in women's health, there is a plethora of evidence that suggest an important role for these hormones, particularly 17beta-estradiol (E(2)), in the development and progression of breast cancer. Most estrogenic responses are mediated by estrogen receptors (ERs), either ERalpha or ERbeta, which are members of the nuclear receptor superfamily of ligand-dependent transcription factors. Selective estrogen receptor modulators (SERMs) are ER ligands that in some tissues (i.e. bone and cardiovascular system) act like estrogens but block estrogen action in others. Tamoxifen is the first SERM that has been successfully tested for the prevention of breast cancer in high-risk women and is currently approved for the endocrine treatment of all stages of ER-positive breast cancer. Raloxifene, a newer SERM originally developed for osteoporosis, also appears to have preventive effect on breast cancer incidence. Numerous studies have examined the molecular mechanisms for the tissue selective action of SERMs, and collectively they indicate that different ER ligands induce distinct conformational changes in the receptor that influence its ability to interact with coregulatory proteins (i.e. coactivators and corepressors) critical for the regulation of target gene transcription. The relative expression of coactivators and corepressors, and the nature of the ER and its target gene promoter also affect SERM biocharacter. This review summarizes the therapeutic application of SERMs in medicine; particularly breast cancer, and highlights the emerging understanding of the mechanism of action of SERMs in select target tissues, and the inevitable development of resistance.

Activation of cyclin D1 by estradiol and spermine in MCF-7 breast cancer cells: a mechanism involving the p38 MAP kinase and phosphorylation of ATF-2.

Estradiol (E2) and the naturally occurring polyamines (putrescine, spermidine, and spermine) play important roles in breast cancer cell growth and differentiation. We examined the effects of E2 and spermine on the phosphorylation and DNA binding of activating transcription factor-2 (ATF-2) in MCF-7 breast cancer cells. ATF-2 is a transcription factor involved in estrogenic regulation of cyclin D1 gene, and thereby cell cycle progression. DNA affinity immunoblot assays showed a six- to eightfold increase in the binding of ATF-2 to a 74-mer ATF/CRE oligonucleotide (ODN1) from cyclin D1 promoter in the presence of 4 nM E2 and 0.5 mM spermine, compared to untreated control. Individual treatments with E2 or spermine caused a twofold or lower increase in ATF-2 binding to ODN1. Immunoblotting with phospho-ATF-2 antibody showed that increased DNA binding of ATF-2 was associated with its phosphorylation. A p38 MAP kinase inhibitor, PD169316, inhibited ATF-2 phosphorylation. In contrast, the MEK-ERK1/2 inhibitor, PD98059, or the JNK inhibitor, SP600125, had no significant effect on DNA binding of ATF-2. Cyclin D1 promoter (-1745CD1) activity increased by approximately 12-fold (above control) in the presence of E2 and spermine, compared to a sixfold increase in the presence of E2 alone and a twofold increase with spermine. Cells transfected with a dominant negative mutant of ATF-2 showed decreased transactivation of cyclin D1 promoter in response to E2 and spermine. These results indicate that spermine can enhance E2-induced cell signaling and cyclin D1 transcription by activation of the p38 MAP kinase and phosphorylation of ATF-2, contributing to breast cancer cell proliferation.

Differential effects of 16alpha-hydroxyestrone and 2-methoxyestradiol on cyclin D1 involving the transcription factor ATF-2 in MCF-7 breast cancer cells.

We studied the effects of 2-methoxyestradiol (2-ME2) and 16alpha-hydroxyestrone (16alpha-OHE1), two metabolites of estradiol (E2), on DNA synthesis, cell cycle progression and cyclin D1 protein levels in estrogen receptor-positive MCF-7 cells. E2 and 16alpha-OHE1 stimulated DNA synthesis, and 2-ME2 inhibited the stimulatory effects of these agents. E2 and 16alpha-OHE1 stimulated the progression of cells from G1 to S phase and this effect was attenuated by 2-ME2. Western blot analysis showed that E2 and 16alpha-OHE1 increased cyclin D1 protein level by about fourfold compared with control. 2-ME2 had no significant effect on cyclin D1; however, it prevented the accumulation of cyclin D1 in the presence of E2 and 16alpha-OHE1. Cells transfected with a cyclin D1 reporter gene and treated with E2 or 16alpha-OHE1 showed 7- and 9.5-fold increase respectively in promoter activity compared with control. This activity was significantly inhibited by 2-ME2. Cyclin D1 transactivation was mediated by the cAMP response element (CRE) region, which binds activating transcription factor 2 (ATF-2). DNA affinity assay showed 2.5- and 3.5-fold increases in ATF-2 binding to CRE in the presence of E2 and 16alpha-OHE1 respectively. The binding of ATF-2 was inhibited by the presence of 2-ME2. These results show that 2-ME2 can downregulate cyclin D1 and thereby cell cycle progression by a mechanism involving the disruption of ATF-2 binding to cyclin D1 promoter.