Antiestrogens modulate MT1 melatonin receptor expression in breast and ovarian cancer cell lines

An interaction between cellular estrogen response and melatonin signaling mediated by G-protein coupled receptors is present in breast cancer cells. In this study, the effect of antiestrogens on basal and melatonin-modulated expression of MT1 melatonin receptor in breast and ovarian cancer cells was examined. For this purpose, the effects of the selective estrogen receptor modulator tamoxifen and pure antiestrogen ICI 182,780 on MT1 expression in estrogen receptor (ER) alpha-positive and -negative breast and ovarian cancer cell lines cultured in medium supplemented with 1 nM 17-beta estradiol were assessed by Western blot analysis. We were able to detect expression of the MT1 receptor in SK-OV-3 and OVCAR-3 cells and report its up-regulation by melatonin in both ovarian cancer cell lines. MT1 expression was observed to be significantly weaker in ERalpha-positive MCF-7 and OVCAR-3 cells than in ERalpha-negative MDA-MB-231 and SK-OV-3 cells. Treatment with the pure antiestrogen ICI 182,780 increased MT1 receptor expression in OVCAR-3 ovarian cancer cells, but decreased MT1 expression in MCF-7 breast cancer cells. No effect of ICI 182,780 on MT1 expression was observed in the ERalpha-negative cell lines SK-OV-3 and MDA-MB-231. After treatment with 4-OH tamoxifen, down-regulation of basal MT1 receptor expression in ERalpha-positive MCF-7 cells and inhibition of melatonin-induced up-regulation of MT1 in OVCAR-3 ovarian cancer cells were observed. In contrast, treatment with 4-OH tamoxifen increased the MT1 receptor level in ERalpha-negative SK-OV-3 ovarian cancer cells. Our findings support the existence of close interaction between estrogen and melatonin signaling. Moreover, our data suggest that melatonin signaling is modulated by antiestrogens in breast and ovarian cancer cells.     

雌激素及其拮抗剂对乳腺癌血管内皮生长因子的转录调节

背景与目的：肿瘤血管生成在乳腺癌的生长和转移中占有重要地位，内分泌治疗是否影响乳腺癌的肿瘤血管生成是临床关注的重要课题。本研究探讨雌激素及其拈抗剂对人乳腺癌细胞中血管内皮生长因子（VEGF）的转录调节作用及其机制。方法：应用半定量RT—PCR法检测不同浓度和作用时间下雌二醇（E2）对MCF-7乳腺癌细胞VEGF mRNA表达影响，并检测他莫昔芬（三苯氧胺，TAM）和ICI182780是否可抑制雌激素对VEGF转录的影响。结果：剂量依赖性实验显示E2浓度为1～10nmol／L时，VEGFmRNA表达水平最高，为0．125±0．006-0．112±0．014；时间依赖性实验中E2培养2h内VEGF转录水平明显升高（0．105±0．009），6h达到最大值（0．140±0．024），较未治疗组高1．5倍（P〈0．05）。TAM在浓度为1nmol／L时可轻微促进VEGFmRNA表达（0．061±0．010），但该浓度下与E2共同培养时可抑制E2诱导VEGF产生（0．070±0．001）；ICI182780同样可抑制E2诱导VEGF产生（0．068±0．001）。结论：雌激素可促进VEGFmRNA生成，其生成量依赖于雌激素的浓度和作用时间；TAM和ICI182780对雌激素诱导VEGFmRNA生成具有抑制作用，提示雌激素及其拮抗剂在转录水平调节VEOF表达，TAM通过阻遏雌激素诱导VEGF表达抑制乳腺癌肿瘤血管生成。     

Effects of a new clinically relevant antiestrogen (GW5638) related to tamoxifen on breast and endometrial cancer growth in vivo.

PURPOSE: Cross-resistance is an important issue for the evaluation of new antiestrogens to treat advanced breast cancer patients who have failed tamoxifen therapy. In addition, postmenopausal patients treated with long-term adjuvant tamoxifen show a 3-4-fold increase in the risk of developing endometrial cancer. Consequently, a new second line agent should be more antiestrogenic and less estrogen-like on the uterus, and be effective at controlling the growth of breast cancer after exposure to tamoxifen. The purpose was to evaluate the effects of the new tamoxifen analogue GW5638 on breast and endometrial cancer growth. EXPERIMENTAL DESIGN: Athymic mice were transplanted with an endometrial tumor model (ECC-1 E2) that is responsive to estrogen and has never been exposed to antiestrogen. In addition, we used three breast tumor models: a tamoxifen-na?ve tumor (T47D-E2) and two tamoxifen-stimulated tumors (MT2 TAM and MCF-7 TAM LT). The antiestrogen GW5638 (1.5 mg daily), tamoxifen (0.5 mg or 1.5 mg daily), and raloxifene (1.5 mg daily) were given p.o. The pure antiestrogen ICI182,780 (5 mg once a week) was given s.c. Western blots from MCF-7 TAM breast tumors were performed to demonstrate the regulation of estrogen receptor alpha expression by different ligands. RESULTS: Estradiol and GW5638 down-regulated the receptor compared with control. ICI182,780 completely degraded the receptor but tamoxifen had no effect. GW5638 did not promote tumor growth, and was effective in blocking the effects of postmenopausal estradiol on the growth of tamoxifen-na?ve breast and endometrial tumors. However, raloxifene did not completely block the effects of postmenopausal estradiol on the growth of tamoxifen-na?ve endometrial tumor after 14 weeks. GW5638 and ICI182,780 but not raloxifene were also effective in blocking the tamoxifen-stimulated breast tumor growth in athymic mice. CONCLUSIONS: GW5638 is more effective than raloxifene in blocking the effect of estrogen on tamoxifen-na?ve endometrial cancer. More importantly, GW5638, like the pure antiestrogen ICI182,780, is able to block the growth of breast cancer stimulated by tamoxifen differently from raloxifene. GW5638 down-regulates estrogen receptor but does not completely destroy the receptor. Therefore, based on our findings, GW5638 could be developed as a second line agent for advanced breast cancer patients and an important first line agent to evaluate as an adjuvant treatment or chemopreventive.     

Insulin receptor substrate 1 is a target for the pure antiestrogen ICI 182,780 in breast cancer cells

The pure antiestrogen ICI 182,780 inhibits insulin-like growth factor (IGF)-dependent proliferation in hormone-responsive breast cancer cells. However, the interactions of ICI 182,780 with IGF-I receptor (IGF-IR) intracellular signaling have not been characterized. Here, we studied the effects of ICI 182,780 on IGF-IR signal transduction in MCF-7 breast cancer cells and in MCF-7-derived clones overexpressing either the IGF-IR or its 2 major substrates, insulin receptor substrate 1 (IRS-1) or src/collagen homology proteins (SHC). ICI 182,780 blocked the basal and IGF-I-induced growth in all studied cells in a dose-dependent manner; however, the clones with the greatest IRS-1 overexpression were clearly least sensitive to the drug. Pursuing ICI 182,780 interaction with IRS-1, we found that the antiestrogen reduced IRS-1 expression and tyrosine phosphorylation in several cell lines in the presence or absence of IGF-I. Moreover, in IRS-1-overexpressing cells, ICI 182,780 decreased IRS-1/p85 and IRS-1/GRB2 binding. The effects of ICI 182,780 on IGF-IR protein expression were not significant; however, the drug suppressed IGF-I-induced (but not basal) IGF-IR tyrosine phosphorylation. The expression and tyrosine phosphorylation of SHC as well as SHC/GRB binding were not influenced by ICI 182,780. In summary, downregulation of IRS-1 may represent one of the mechanisms by which ICI 182,780 inhibits the growth of breast cancer cells. Thus, overexpression of IRS-1 in breast tumors could contribute to the development of antiestrogen resistance.     

Estrogen Responsiveness of IBEP-2, A New Human Cell Line Derived from Breast Carcinoma

IBEP-2, an established cell line recently derived from breast carcinoma, was characterized with regard to estrogen receptor (ER) expression, cell mitogenic response to estrogenic stimulation and sensitivity to antiestrogens. In addition, we examined ER modulation following binding of agonist and antagonists, and the ER-mediated induction of progesterone receptor (PgR). ER level in IBEP-2 cells, determined by enzyme-linked immunoassay (EIA), was slightly higher than that measured in MCF-7 cells (662 v.s. 595 fmol/mg protein). When tested on IBEP-2 and MCF-7, various agonists stimulated cell growth with EC50's reflecting different estrogenic potencies (E(2) approximately diethylstilbestrol > E(1) > genistein). IBEP-2 appeared slightly more sensitive than MCF-7, especially to E(2) (at least 4-fold difference between EC50 values). By contrast, IBEP-2 and MCF-7 were equally sensitive to the growth inhibitory effect of antiestrogens 4-hydroxy-tamoxifen (OH-Tam) and ICI 182,780. As revealed by immunoblotting and immunofluorescence using anti-ER alpha antibodies, ER expression in IBEP-2 cells was modulated by E(2) and estrogen antagonists like it has been shown in other ER-positive cell lines, that is, E(2) and ICI 182,780 caused ER downregulation, whereas OH-Tam induced ER accumulation. Ligand-induced downregulation of ER involved degradation in proteasomes, since it was suppressed by the proteasome inhibitor MG-132. Exposure of IBEP-2 cells to E(2) resulted in a marked (at least 25-fold) induction of PgR, documented by EIA, immunoblotting and immunofluorescence. PgR induction due to E(2) was not modified by MG-132. Interestingly, MG-132 alone produced an ER-independent increase of PgR expression. IBEP-2 might prove to be valuable to study ER-mediated induction of PgR.     

Cross-resistance of triphenylethylene-type antiestrogens but not ICI 182,780 in tamoxifen-stimulated breast tumors grown in athymic mice.

The triphenylethylene antiestrogens, idoxifene (Idox) and toremifene (Tor), are structurally related analogues of tamoxifen (Tam) and were developed to improve the therapeutic index for advanced breast cancer patients. However, the issue of cross-resistance with Tam for these new agents is critical for clinical testing because the majority of breast cancer patients have already received or failed adjuvant Tam. The goal of this study was to determine the effectiveness of Idox as an antitumor agent in three models of Tam-stimulated breast cancer in athymic mice and compare the results with the actions of Tor and ICI 182,780 in a Tam-stimulated MCF-7 tumor model. We first compared the activities of Tam and Idox in the 17beta-estradiol (E2)-stimulated MCF-7 tumor line MT2:E2. Tam and Idox reduced E2-stimulated growth by 65-70% (week 9: P = 0.0009 for Tam, P = 0.0005 for Idox versus E2 alone). However, Tam (1.5 mg daily) and Idox (1.0 mg daily) both produced T47D breast tumors in athymic mice during 23 weeks of treatment (12 tumors/22 sites and 15 tumors/18 sites, respectively). Tam and Idox stimulated tumor growth equally in two different Tam-stimulated MCF-7 models and in a T47D model. Tor was completely cross-resistant with Tam in the MCF-7 tumor model, which implied that neither Idox nor Tor would be effective as a second-line endocrine therapy after Tam failure and may offer no therapeutic advantages over Tam as adjuvant therapies. In contrast, ICI 182,780, a pure antiestrogen currently being tested as a treatment for breast cancer after Tam failure, had no growth-stimulatory effect on the MCF-7 Tam-stimulated breast tumor line. This agent may provide an advantage as an adjuvant therapy and increase the time to treatment failure.     

A potent specific pure antiestrogen with clinical potential.

Previous studies from this laboratory have described a series of 7 alpha-alkylamide analogues of estradiol with pure antiestrogenic activity, exemplified by ICI 164,384. A new compound, 7 alpha-[9-(4,4,5,5,5-pentafluoropentylsulfinyl)nonyl]estra-1,3,5(10 )- triene-3,17 beta-diol (ICI 182,780) has now been identified which has significantly increased antiestrogenic potency and retains pure estrogen antagonist activity. The antiuterotrophic potency of ICI 182,780 in the immature rat was more than 10-fold greater than that of ICI 164,384 (50% effective doses of 0.06 and 0.9 mg/kg, respectively). This order of magnitude increase of in vivo potency was also reflected, in part, by intrinsic activity at the estrogen receptor. The relative binding affinities of ICI 182,780 and ICI 164,384 were 0.89 and 0.19, respectively, compared with that of estradiol (1.0). Similarly, the in vitro growth-inhibitory potency of ICI 182,780 exceeded that of ICI 164,384 in MCF-7 human breast cancer cells, where 50% inhibitory concentrations of 0.29 and 1.3 nM, respectively, were recorded. ICI 182,780 was a more effective inhibitor of MCF-7 growth than 4'-hydroxytamoxifen, producing an 80% reduction of cell number under conditions where 4'-hydroxytamoxifen achieved a maximum of 50% inhibition. This increased efficacy was reflected by a greater reduction of the proportion of cells engaged in DNA synthesis in ICI 182,780-treated cell cultures compared with tamoxifen-treated cells. Sustained antiestrogenic effects, following a single parenteral dose of ICI 182,780 in oil suspension, were apparent in both rats and pigtail monkeys. In vivo, antitumor activity of ICI 182,780 was demonstrated with xenografts of MCF-7 and Br10 human breast cancers in nude mice. A single injection of ICI 182,780 provided antitumor efficacy equivalent to that of daily tamoxifen treatment for at least 4 weeks. The properties of ICI 182,780 identify this pure antiestrogen as a prime candidate with which to evaluate the potential therapeutic benefits of complete estrogen withdrawal in endocrine-responsive human breast cancer.     

Effects of 4-hydroxytamoxifen and a novel pure antioestrogen (ICI 182780) on the clonogenic growth of human breast cancer cells in vitro.

We have investigated the effects on breast cancer cell growth of 4-hydroxytamoxifen (4OHT), a conventional antioestrogen with agonist activity, and 7 alpha-[9-(4,4,5,5,5-pentafluoropentylsulphinyl)nonyl]oestra- 1,3,5,(10)- triene-3,17 beta-diol (ICI 182780), a novel, pure antioestrogen, using established human breast cancer cell lines and cancer cells obtained directly from breast cancer patients with malignant pleural effusions who had relapsed on tamoxifen. The effects of the two agents were assessed using the Courtenay-Mills clonogenic assay, which measures the growth of single cancer cells as colonies suspended in soft agar. The standard assay was modified by the use of defined serum- and phenol red-free growth medium. The growth of oestrogen receptor (ER)-positive MCF-7 cells in the assay was oestrogen responsive. Both antioestrogens inhibited the stimulatory effects of 1 nM oestradiol, but ICI 182780 caused significantly greater inhibition than 4OHT at 0.1-1.0 microM concentrations. In the absence of oestradiol, 4OHT but not ICI 182780 caused significant stimulation of colony formation at low (0.01-1.00 nM) concentrations. Neither antioestrogen had any effects on colony formation by the ER-negative Hs578T cell line. Successful colony formation was obtained in primary cultures from six out of eight malignant effusions. Colony formation was significantly stimulated by 0.1 nM oestradiol in four cases and by 10 nM 40HT in two cases. In contrast, ICI 182780 exhibited no intrinsic stimulatory activity and significantly inhibited both oestradiol- and 4OHT-stimulated cell growth. We conclude that the agonist activity of 4OHT and other conventional antioestrogens may cause treatment failure in some patients by stimulating breast cancer cell growth. The new, pure antioestrogen ICI 182780 is a more potent oestrogen antagonist than 4OHT and exhibits no growth-stimulatory activity. This agent may therefore offer therapeutic advantages over conventional antioestrogens in patients with advanced breast cancer and may be effective after conventional agents have failed.     

Effect of estrogen and antiestrogens on cell proliferation and synthesis of secreted proteins in the human breast cancer cell line MCF-7 and a tamoxifen resistant variant subline, AL-1.

The human breast cancer cell line MCF-7 contains estrogen receptors and responds to estrogens with an increase in growth rate and to antiestrogens with a decrease in growth rate. Estrogen stimulation of cell proliferation is concomitant with an increase in the synthesis and secretion of three proteins with mol. wt 52 kDa, 61 kDa and 66 kDa and a decrease in the synthesis and secretion of a 42 kDa protein. The antiestrogen ICI 164,384 has a complete estrogen antagonistic effect on the synthesis of these secreted proteins, whereas the antiestrogen tamoxifen has an agonistic effect on the synthesis and secretion of the 52 kDa protein. We believe that the above mentioned estrogen regulated secreted proteins are either directly or indirectly involved in control of cell proliferation, and the less pronounced inhibitory effect of tamoxifen on cell proliferation compared to ICI 164,384 may be due to agonistic effects of tamoxifen. A tamoxifen resistant variant of the MCF-7 cell line, the AL-1 subline, can be growth inhibited by ICI 164,384, although a higher concentration is needed to inhibit the AL-1 cells compared to the parent MCF-7 cells. Tamoxifen has no effect on secreted proteins from the AL-1 cells, whereas ICI 164,384 has a complete estrogen antagonistic effect on secreted proteins, indicating that the mechanisms by which estrogens and antiestrogens influence cell proliferation may be via up and down regulation of secreted proteins with growth regulatory functions.     

Estrogen and antiestrogens alter breast cancer invasiveness by modulating the transforming growth factor-β signaling pathway

In the later stages of breast cancer, estrogen receptor (ER)α-negative cancers typically have higher histological grades than ERα-positive cancers, and transforming growth factor (TGF)-β promotes invasion and metastasis. Our previous study indicated that ERα inhibited TGF-β signaling by inducing the degradation of Smad in an estrogen-dependent manner. In the present study, we report that the suppressive effects of ERα and estrogen on tumor progression are mediated by inhibiting TGF-β signaling. Furthermore, we investigated the effects of antiestrogens such as ICI182,780 (ICI) or tamoxifen (TAM) on TGF-β signaling and breast cancer invasiveness. The levels of total Smad and pSmad were reduced by estrogen, whereas ICI slightly increased them, and TAM had no effect. To investigate the effect of antiestrogens on breast cancer invasiveness, we generated highly migratory and invasive MCF-7-M5 cells. The migration and invasion of these cells were suppressed by the inhibitor of TGF-β receptor kinase, SB-505124, and estrogen. However, antiestrogens did not suppress the migration and invasion of these cells. In addition, we screened TGF-β target genes whose expression was reduced by estrogen treatment and identified four genes associated with breast cancer invasiveness and poor prognosis. The expression of these genes was not decreased by antiestrogens. These observations provide a new insight into estrogen function and the mechanisms underlying estrogen-mediated suppression of tumor progression.     

Anti-proliferative and anti-estrogenic effects of ICI 164,384 and ICI 182,780 in 4-OH-tamoxifen-resistant human breast-cancer cells

The effects of the anti-estrogens 4-hydroxytamoxifen (OHTam), ICI 164,384 and ICI 182,780 were tested on the MCF-7/LCC2 breast-carcinoma cell line, which grows significantly in the presence of OHTam and serves as a model for studying anti-estrogen resistance of estrogen-receptor-positive breast cancer. Cell proliferation and cathepsin-D secretion were strongly inhibited by either ICI 182,780 or ICI 164,384 alone or ICI 164,384 in combination with 17-p-estradiol (E2) or OHTam. ICI 164,384 alone did not affect the cathepsin-D and pS2 mRNA levels, but antagonized the stimulatory effects of E2 or OHTam on these 2 mRNAs. OHTam was more effective than E2 in increasing cathepsin-D mRNA levels, supporting the idea that anti-estrogen-resistant breast cancer continues to over-express cathepsin-D. These data show that the steroidal anti-estrogens ICI 164,384 and ICI 182,780 retain their ability to inhibit cell proliferation and the estrogen-responsiveness of cathepsin-D and pS2 genes in the OHTam-resistant MCF-7/ LCC2 cell line. These pure anti-estrogens may thus be efficient second-line treatments of some Tamoxifen-resistant tumors.     

Acquired antiestrogen resistance in MCF-7 human breast cancer sublines is not accomplished by altered expression of receptors in the ErbB-family

Development of acquired resistance against antiestrogen treatment is a serious problem in human breast cancer, and knowledge of alterations resulting in resistance is important for selection of further treatment. To mimic the clinical situation we have established a series of MCF-7 human breast cancer cell lines by long term treatment with the antiestrogens tamoxifen, ICI 164,384, and ICI 182,780. Common for these cell lines is a decreased expression of the estrogen receptor (ER). In human breast cancer, lack of response to endocrine therapy is often associated with decreased expression of the estrogen receptor and increased expression of epidermal growth factor receptor (EGFR) and/or HER-2/neu (ErbB-2). Our antiestrogen resistant cell lines did not express altered levels of EGFR, HER-2/neu, ErbB-3, or ErbB-4. Estrogen and antiestrogen regulation of HER-2/neu expression was essentially similar in parent and resistant MCF-7 cells. Treatment with antibodies to HER-2/neu (Herceptin^TM) did not affect growth of MCF-7 cells or resistant cells, indicating that in this in vitro model system, acquired antiestrogen resistance does not emerge from activation of the HER-2/neu signaling pathway. In MCF-7 cells transfected with HER-2/neu and/or ErbB-3, overexpression alone did not result in resistance. However, addition of heregulinl-1 abolished the inhibitory activity of ICI 182,780 on both vector and HER-2/neu/ErbB-3 transfected MCF-7 cells, demonstrating that activation of the HER-2/neu receptor signaling pathway can override the growth inhibitory effect of ICI 182,780.     

Expression of constitutively active Akt-3 in MCF-7 breast cancer cells reverses the estrogen and tamoxifen responsivity of these cells in vivo.

PURPOSE: Prior studies had suggested that Akt activity is elevated in a subset of breast cancers. In this study, to test the effect of active Akt-3 on estrogen receptor function, we have produced MCF-7 cells, which express active Akt-3 and examined the estrogen responsiveness of these cells in vivo and in vitro. Experimental Design: MCF-7 cells expressing active Akt-3 were studied for estradiol (E2) responsiveness in vitro by both using an estrogen receptor element reporter construct as well as looking at induction of endogenous genes. These cells were also studied in vivo after injection into nude, ovariectomized mice by following tumor growth rates in the presence or absence of E2, tamoxifen, or the pure antiestrogen, ICI 182,780 (fulvestrant). RESULTS: Akt-3-expressing cells were found to produce tumors in mice in the absence of E2 that were approximately equivalent in size to control cells in mice given E2. Moreover, the formation of tumors by the Akt-3 cells was greatly suppressed by E2, stimulated by tamoxifen, and unaffected by ICI 182,780. In the in vitro assays for gene induction by E2, the Akt-3-expressing cells exhibited similar E2 and tamoxifen responsiveness as the control cells. CONCLUSIONS: These results indicate that expression of active Akt-3 in MCF-7 cells results in E2-independent tumor growth. Moreover, the growth of these tumors is inhibited by E2 and enhanced by tamoxifen. Finally, these tumors are resistant to ICI 182,780. These findings suggest that the amount of active Akt present in breast cancers may be important in the relative efficacy of different treatments.     

Human breast cancer cell lines resistant to pure anti-estrogens are sensitive to tamoxifen treatment

The pure steroidal anti-estrogens ICI 164,384 and ICI 182,780 are very potent growth inhibitors of the estrogen receptor-positive human breast cancer cell line MCF-7. However, long-term treatment of MCF-7 cells with 10^?7 M concentrations of these compounds results in selection of proliferating colonies of resistant cells. Our report describes 4 ICI 164,384- and 3 ICI 182,780-resistant MCF-7 sublines established after long-term treatment. Resistant sublines are estrogen receptor-positive, and all sublines have lost expression and estrogen inducibility of the progesterone receptor protein. Based on IC₅₀ concentrations, all tested resistant sublines had a reduced sensitivity to pure anti-estrogens on the order of 100- to 1000-fold compared with parent MCF-7 cells. All resistant cell lines have survived propagation for more than 15 subcultivations in the presence of 10^?7 M pure anti-estrogen. The MCF-7/182^R-6 subline has been tested for stability of resistance and appeared to be stably resistant after 13 weeks of propagation without the selective pressure of ICI 182,780. Cell lines resistant to the ICI 182,780 compound are cross-resistant to the ICI 164,384 compound and vice versa. However, the sublines resistant to pure anti-estrogens are sensitive to tamoxifen. Our results show that although the pure steroidal anti-estrogens are very potent growth inhibitors, they do not circumvent development of resistance. © 1995 Wiley-Liss, Inc.     

Distinct effects of gonadotropin-releasing hormone analogs and 4-hydroxytamoxifen on pS2 mRNA expression with respect to cell proliferation in MCF-7 breast cancer cells.

Gonadotropin-releasing hormone (GnRH) analogs and antiestrogens display direct antiestrogenic effects on the proliferation of hormone-sensitive breast cancer cells. This study aimed to determine whether growth inhibition of 17 beta-estradiol-stimulated MCF-7 breast cancer cells by the agonist D-Trp6 GnRH, the GnRH antagonist BIM 21009C and 4-hydroxy-tamoxifen (OHT) respectively occurred through alterations of the estrogen receptor (ER)-mediated intracellular pathway. The pS2 mRNA expression is primarily dependent on activated ER in MCF-7 cells, and pS2 protein could act as a growth factor. Drug effect on pS2 mRNAs were qualitatively compared to those on the cell cycle. Unlike OHT, GnRH analogs did not suppress the 17 beta-estradiol-induced pS2 mRNA expression whilst the cell cycle was blocked. The pS2 mRNA expression was induced by D-Trp6 GnRH alone without effect on the cell cycle. The outcome of our study is double. Firstly, GnRH analogs are distinct from OHT as regards their effects on the ER-mediated intracellular pathway. Secondly, pS2 mRNA expression is not strictly related to MCF-7 cell proliferation, suggesting that pS2 protein has a function other than that of critical growth regulator.     

Mifepristone induces growth arrest, caspase activation, and apoptosis of estrogen receptor-expressing, antiestrogen-resistant breast cancer cells.

PURPOSE: A major clinical problem in the treatment of breast cancer is the inherent and acquired resistance to antiestrogen therapy. In this study, we sought to determine whether antiprogestin treatment, used as a monotherapy or in combination with antiestrogen therapy, induced growth arrest and active cell death in antiestrogen-resistant breast cancer cells. EXPERIMENTAL DESIGN: MCF-7 sublines were established from independent clonal isolations performed in the absence of drug selection and tested for their response to the antiestrogens 4-hydroxytamoxifen (4-OHT) and ICI 182,780 (fulvestrant), and the antiprogestin mifepristone (MIF). The cytostatic (growth arrest) effects of the hormones were assessed with proliferation assays, cell counting, flow cytometry, and a determination of the phosphorylation status of the retinoblastoma protein. The cytotoxic (apoptotic) effects were analyzed by assessing increases in caspase activity and cleavage of poly(ADP-ribose) polymerase. RESULTS: All of the clonally derived MCF-7 sublines expressed estrogen receptor and progesterone receptor but showed a wide range of antiestrogen sensitivity, including resistance to physiological levels of 4-OHT. Importantly, all of the clones were sensitive to the antiprogestin MIF, whether used as a monotherapy or in combination with 4-OHT. MIF induced retinoblastoma activation, G(1) arrest, and apoptosis preceded by caspase activation. CONCLUSIONS: We demonstrate that: (a) estrogen receptor(+)progesterone receptor(+), 4-OHT-resistant clonal variants can be isolated from an MCF-7 cell line in the absence of antiestrogen selection; and (b) MIF and MIF plus 4-OHT combination therapy induces growth arrest and active cell death of the antiestrogen-resistant breast cancer cells. These preclinical findings show potential for a combined hormonal regimen of an antiestrogen and an antiprogestin to combat the emergence of antiestrogen-resistant breast cancer cells and, ultimately, improve the therapeutic index of antiestrogen therapy.