Estrogen-dependent Human Breast Cancer Research Articles

Abstract 754: Development of death receptor agonists for breast cancer therapy

Abstract TRAIL and death receptor (TRAIL-R1 and TRAIL-R2) agonistic antibodies have been shown to induce apoptosis in cancer cells and their clinical trials have produced positive results. However, there are no small organic molecular weight compounds that bind to TRAIL-death receptors and induce apoptosis specifically in cancer cells. The purpose of this study was to identify and characterize death receptor agonists (DRAs)/ small molecular weight organic compounds that induce apoptosis in breast cancer cells. The pharmacokinetics of the DRA with a single oral dose was characterized in nude mice. Blood was collected over 72 hour period after drug administration, and analyzed by HPLC-MS/MS for plasma drug concentration. The Cmax, tmax, and AUC of DRA was measured. DRAs inhibited cell viability and colony formation, and induced apoptosis in estrogen-dependent (MCF-7) and independent (MDA-MB-231 and MDA-MB-468) human breast cancer cell lines, but had no effect on human normal mammary epithelial cells and hepatocytes. The ability of DRAs to induce apoptosis was further enhanced in the presence of TRAIL. DRAs formed active TRAIL-DISC (death-inducing signaling complex) similar to that of TRAIL. DRAs inhibited the growth of orthotopically implanted MDA-MB-231 and MDA-MB-468 tumors in nude mice. Histological examination of tumor tissues derived from DRAs-treated nude mice revealed massive caspase-3 activation and apoptosis. Pharmacokinetic data demonstrate that DRA possesses a long terminal half-life, which is acceptable for further clinical development. In conclusion, we have identified, for the first time, DRAs that can be used in the clinic for the treatment of human breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 754.

Effects of Non-Digestible Carbohydrates on the Growth of Estrogen-Dependent Human Breast Cancer (MCF-7) Tumors Implanted in Ovariectomized Athymic Mice

Non-digestible carbohydrates (NDC 4 ) have been used as a low-calorie sweetener and prebiotics that stimulate the growth of certain intestinal bacteria that support healthy colon conditions. In this study, we examined the dietary effect of commercially available NDCs on estrogen receptor positive (ER+) human breast cancer. We conducted a feeding study of fructooligosaccharides (FOSs), Fibersol 2 (F2; digestion resistant maltodextrin), Hi-Maize (HM; high amylose cornstarch), and Frutafit (FF; a range of powdered inulins) (5% in diet, w/w) to evaluate their effects on the growth of ER(+) human breast cancer (MCF-7) tumors in the presence of 17β-estradiol (E2) using an athymic xenograft model. F2, HM, and FOSs supplementation significantly reduced E2-stimulated MCF-7 tumor growth by inhibiting cellular proliferation (Ki-67) and increasing apoptosis (M30) in tumors. F2, HM, and FOSs treatments also lowered serum E2 level and reduced uterine weight compared to the control diet. NDCs treatments downregulated relative mRNA expression of the E2-responsive gene markers pS2, bcl2, bcl-xL, and cyclin D1 in MCF-7 tumors. In conclusion, the NDC intake may have a protective effect against ER(+) tumors by inhibiting cellular proliferation and increasing apoptosis.

Combination of a Novel Growth Hormone Releasing Hormone Antagonist JMR 132 and Tamoxifen Enhances the Inhibitory Effects on Growth of ZR 75 and T47D Estrogen Dependent Human Breast Cancer Cells In Vitro and In Vivo.

Abstract Background: Tamoxifen is the oldest selective estrogen receptor modulator (SERM) developed to antagonize the estrogen receptor function on human breast cancer cells. JMR 132 is a novel GHRH Antagonist proven to be potent inhibitor of tumor growth in estrogen receptor negative human breast cancer cell lines. A combination of both substances could induce a more potent growth inhibition, as compared to the single drugs.Methods: To determine inhibitory effects of the GHRH antagonist JMR 132 and Tamoxifen alone and in combination, estrogen receptor positive cell lines ZR 75 and T 47D were incubated with substances at concentrations of 2.5µM, 5µM and 10µM. The cell viability after a period of 72 h was measured by MTT assay. To assess if the treatment arrested cell growth at a specific phase of the cell cycle or simply caused cell death, we performed a flow cytometric DNA cell cycle analysis on ZR 75 cells exposed to JMR 132, Tamoxifen and their combination. In an in vivo model, the cell line ZR 75 was xenografted subcutaneously into nude mice. Treatment groups were control, JMR 132 at a dose of 10µM, Tamoxifen at a dose of 100µgr per mouse q3 per week and the combination of both substances. All given for a period of 4 weeks.Results: In vitro 1o µM JMR 132 and Tamoxifen significantly decreased the proliferation of the ZR 75 and T47D cell lines, as compared to controls (p< 0.05). The combination of 1o µM inhibited the growth of ZR 75 was significantly by 14%, as compared to JMR 132 alone. The combination also significantly suppressed the growth of T47D by 14% and 22% respectively, as compared to Tamoxifen and JMR 132. In in vivo treatment with the combination of JMR 132 and Tamoxifen induced a significant inhibition of tumor growth of ZR 75 xenografts after 14 days compared to the single agents groups and inhibition remained significant until the end of the study on day 28. The flow cytometric DNA cell cycle analysis revealed a cell cycle arrest in the S Phase which led to apoptosis of the cells.Conclusion: Treatment of ZR 75 and T47D estrogen receptor positive human breast cancer lines with GHRH antagonist JMR 132 and its combination with Tamoxifen induced potent inhibitory effects in vitro and in vivo and led to subsequent cell death. Further studies are being conducted to elucidate the mechanisms of action of these effects. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 5073.

Selective recruitment of breast cancer anti-estrogen resistance genes and relevance for breast cancer progression and tamoxifen therapy response

Although endocrine treatment of breast cancer is effective and common practice, in advanced disease the development of resistance is nearly inevitable. To get more insight into individual genes that account for resistance against hormonal agents, we have executed functional genetic screens and subsequently evaluated the clinical relevance of several identified genes with respect to tumor aggressiveness and tamoxifen resistance in estrogen receptor-positive patients. Estrogen-dependent human breast cancer cells were transduced with different retroviral cDNA expression libraries and subjected to selective cultures with various anti-estrogens. From a total of 264 resistant cell clones, 132 different genes were recovered by PCR. By applying stringent selection criteria, we identified 15 breast cancer anti-estrogen resistance (BCAR) genes individually yielding resistance. BCAR genes were recovered with differential frequencies for the diverse culture conditions and anti-estrogen drugs. Analysis of the relation of BCAR genes (EIF1, FBXL10, HRAS, NRG1, PDGFRA, PDGFRB, RAD21, and RAF1) with tamoxifen treatment in patients with advanced disease showed significant association with clinical benefit and progression-free survival for EIF1 and PDGFRA mRNA levels. Furthermore, PDGFRA and HRAS mRNA levels were significantly associated with tumor aggressiveness in lymph node-negative patients who had not received adjuvant systemic therapy. In conclusion, our functional genetic screens showed that BCAR genes differ in their ability to confer resistance towards distinct anti-estrogens. Based on the clinical relevance of several BCAR genes, further studies are warranted to characterize the underlying mechanisms, which may ultimately lead to the development of novel treatments and more individualized management of breast cancer patients.

Trigonelline Is a Novel Phytoestrogen in Coffee Beans ,

Drinking coffee has been associated with the development of several endocrine-related cancers. The interpretation of these data has often been limited to the role that caffeine plays. Trigonelline (Trig), a niacin-related compound, is a natural constituent of coffee accounting for ∼1% dry matter in roasted beans. Studies exploring the effects of this bioactive compound on mammalian cells are limited. The initial purpose of our studies was to determine whether Trig alters the actions of estradiol (E2), using proliferation of estrogen-dependent human breast cancer (MCF-7) cells as a model system. When cells were cotreated with suboptimal doses of E2 (10 pmol/L) and Trig (100 pmol/L), an additive enhancement of MCF-7 growth was observed. In the absence of E2, Trig stimulated MCF-7 cell proliferation in a dose-responsive manner and significantly enhanced cell growth at concentrations as low as 100 pmol/L. Cotreatment of MCF-7 cells with Trig and ICI 182,780, an estrogen receptor (ER) antagonist, inhibited Trig-induced cell proliferation. Trig treatment also induced activation of estrogen response element reporter assays in MCF-7 cells and increased expression of ER target genes (pS2, progesterone receptor, and cyclin D1) similar to E2. While our data demonstrate that Trig activates the ER, competitive binding assays showed that Trig does not compete E2 off of the ER at any concentration. This suggests that Trig is activating the ER through a separate mechanism. Collectively, these data demonstrate that Trig even at low concentrations stimulates MCF-7 cell growth and that this effect is mediated through ER, clearly identifying Trig as a novel phytoestrogen.

AFPep, a novel drug for the prevention and treatment of breast cancer, does not disrupt the estrous cycle or fertility in rats

Pregnancy lowers the risk of breast cancer, largely attributable to alpha-fetoprotein (AFP). A small AFP-derived peptide (AFPep) which mimics the active site of AFP has been developed and may be useful for decreasing the risk of breast cancer for women. AFPep has been shown previously to stop the growth of estrogen-dependent human breast cancer xenografts in mice and prevent carcinogen-induced breast cancer in a rat model. Since AFPep disrupts an estrogen-responsive pathway, it is essential to assess its effects on the female reproductive cycle and fertility. Ten cycling female Sprague-Dawley rats (age 81 days) were given 100 microg AFPep in saline s.c. daily for 20 days. A second group of ten rats was given 50 microg tamoxifen s.c. daily and a third group received saline only. Vaginal smears were obtained twice per day and stained to assess estrous cycle phase. After completion of estrous cycle assessment (five cycles, 21 days), rats were maintained on drug and allowed to mate. Effects on birth of offspring and maternal body weights were assessed. AFPep had no significant effect on the incidence or duration of any estrous cycle phase, and no effect on reproductive potential or maternal body mass. Tamoxifen significantly increased the length of diestrus, locking the cycle in this phase for most animals. Only half of the tamoxifen-treated rats mated, and none became pregnant. Tamoxifen significantly slowed the rate of body mass increase. In rats, AFPep has no toxicity and no effect on female reproduction. This molecule may be developed into an attractive modality for prevention of breast cancer in women.

Combination of a novel growth hormone releasing hormone Antagonist JMR 132 and Tamoxifen enhances the inhibitory effects on growth of ZR 75 and T47D estrogen dependent human breast cancer cells in vitro and in vivo

Background: Tamoxifen is the oldest selective estrogen receptor modulator (SERM) developed to antagonize the estrogen receptor function on human breast cancer cells. JMR 132 is a novel GHRH Antagonist proven to be potent inhibitor of tumor growth in estrogen receptor negative human breast cancer cell lines. A combination of both substances could induce a more potent growth inhibition, as compared to the single drugs. Methods: To determine inhibitory effects of the GHRH antagonist JMR 132 and Tamoxifen alone and in combination, estrogen receptor positive cell lines ZR 75 and T 47D were incubated with substances at concentrations of 2,5µM, 5µM and 10µM. The cell viability after a period of 72h was measured by MTT assay. To assess if the treatment arrested cell growth at a specific phase of the cell cycle or simply caused cell death, we performed a flow cytometric DNA cell cycle analysis on ZR 75 cells exposed to JMR 132, Tamoxifen and their combination. In an in vivo model, the cell line ZR 75 was xenografted subcutaneously into nude mice. Treatment groups were control, JMR 132at a dose of 10µM, Tamoxifen at a dose of 100µgr per mouse q3 per week and the combination of both substances. All given for a period of 4 weeks. Results: In vitro 1o µM JMR 132 and Tamoxifen significantly decreased the proliferation of the ZR 75 and T47D cell lines, as compared to controls (p<0,05). The combination of 1o µM inhibited the growth of ZR 75 was significantly by 14%, as compared to JMR 132 alone. The combination also significantly suppressed the growth of T47D by 14% and 22% respectively, as compared to Tamoxifen and JMR 132. In in vivo treatment with the combination of JMR 132 and Tamoxifen induced a significant inhibition of tumor growth of ZR 75 xenografts after 14 days compared to the single agents groups and inhibition remained significant until the end of the study on day 28. The flow cytometric DNA cell cycle analysis revealed a cell cycle arrest in the S Phase which led to apoptosis of the cells. Conclusion: Treatment of ZR 75 and T47D estrogen receptor positive human breast cancer lines with GHRH antagonist JMR 132 and its combination with Tamoxifen induced potent inhibitory effects in vitro and in vivo and led to subsequent cell death. Further studies are being conducted to elucidate the mechanisms of action of these effects

Effects of soy isoflavone extracts on the growth of estrogen-dependent human breast cancer (MCF-7) tumors implanted in ovariectomized nude mice

In this study, we explored the effects of soy isoflavone extracts on the growth of estrogen-dependent human breast cancer (MCF-7) tumors implanted in ovariectomized athymic mice. The ovariectomized athymic mice were implanted with MCF-7 cells. They were fed with low, moderate and high doses of soy isoflavone extracts, at dietary concentrations of 6.25, 12.5 and 25 g/kg, respectively. The expression of ki-67 was detected by immunohistochemistry. The pS2 expression in tumors was analyzed by real-time PCR. Estrogen level in the serum was measured by chemiluminescence enzyme immunoassay. Compared with the control group, dietary soy isoflavone extracts had a significant stimulatory effect on MCF-7 tumor growth in mice (P 0.05). In conclusion, in the tested dietary concentration range soy isoflavone extracts had a stimulatory effect on tumor growth. 6.25 and 12.5 g/kg doses of soy isoflavone extracts can increase the cell proliferation in tumors and induce estrogen-responsive pS2 expression.

Dietary genistein negates the inhibitory effect of letrozole on the growth of aromatase-expressing estrogen-dependent human breast cancer cells (MCF-7Ca) in vivo

Genistein (GEN), a soy isoflavone, stimulates growth of estrogen-dependent human tumor cells (MCF-7) in a preclinical mouse model for postmenopausal breast cancer. Antiestrogens and aromatase inhibitors are frontline therapies for estrogen-dependent breast cancer. We have demonstrated that dietary GEN can negate the inhibitory effect of tamoxifen. In this study, we evaluated the interaction of dietary GEN (at 250-1000 p.p.m. in the American Institute of Nutrition 93 growth diet) and an aromatase inhibitor, letrozole (LET), on the growth of tumors in an aromatase-expressing breast cancer xenograft model (MCF-7Ca) in the presence and absence of the substrate androstenedione (AD). Dietary GEN (250 and 500 p.p.m.) or implanted AD stimulated MCF-7Ca tumor growth. Implanted LET inhibited AD-stimulated MCF-7Ca tumor growth. In the presence of AD and LET, dietary GEN (250, 500 and 1000 p.p.m.) reversed the inhibitory effect of LET in a dose-dependent manner. Uterine wet weight, plasma estradiol (E(2)) levels (enzyme-linked immunosorbent assay) and total plasma GEN and LET levels (liquid chromatography-electrospray/tandem mass spectrometry) were measured. Ki-67 (cellular proliferation), aromatase and pS2 protein expression in tumors were evaluated using immunohistochemical (IHC) analysis. In conclusion, dietary GEN increased the growth of MCF-7Ca tumors implanted in ovariectomized mice and could also negate the inhibitory effect of LET on MCF-7Ca tumor growth. These findings are significant because tumors, which express aromatase and synthesize estrogen, are good candidates for aromatase therapy dietary and GEN can reverse the inhibitory effect of LET on tumor growth and adversely impact breast cancer therapy. Caution is warranted for consumption of dietary GEN by postmenopausal women with estrogen-dependent breast cancer taking LET treatment.

New steroidal aromatase inhibitors: suppression of estrogen-dependent breast cancer cell proliferation and induction of cell death.

BackgroundAromatase, the cytochrome P-450 enzyme (CYP19) responsible for estrogen biosynthesis, is an important target for the treatment of estrogen-dependent breast cancer. In fact, the use of synthetic aromatase inhibitors (AI), which induce suppression of estrogen synthesis, has shown to be an effective alternative to the classical tamoxifen for the treatment of postmenopausal patients with ER-positive breast cancer. New AIs obtained, in our laboratory, by modification of the A and D-rings of the natural substrate of aromatase, compounds 3a and 4a, showed previously to efficiently suppress aromatase activity in placental microsomes. In the present study we have investigated the effects of these compounds on cell proliferation, cell cycle progression and induction of cell death using the estrogen-dependent human breast cancer cell line stably transfected with the aromatase gene, MCF-7 aro cells.ResultsThe new steroids inhibit hormone-dependent proliferation of MCF-7aro cells in a time and dose-dependent manner, causing cell cycle arrest in G0/G1 phase and inducing cell death with features of apoptosis and autophagic cell death.ConclusionOur in vitro studies showed that the two steroidal AIs, 3a and 4a, are potent inhibitors of breast cancer cell proliferation. Moreover, it was also shown that the antiproliferative effects of these two steroids on MCF-7aro cells are mediated by disrupting cell cycle progression, through cell cycle arrest in G0/G1 phase and induction of cell death, being the dominant mechanism autophagic cell death. Our results are important for the elucidation of the cellular effects of steroidal AIs on breast cancer.

Role of manganese superoxide dismutase on growth and invasive properties of human estrogen-independent breast cancer cells

Manganese superoxide dismutase (MnSOD) is known to play a role in cancer. MnSOD exerts a tumor suppressive effect in estrogen-dependent human breast cancer cells. In the present study we investigated the in vitro role of MnSOD in the growth of some aggressive and highly metastatic estrogen-independent breast cancer cells, i.e., MDA-MB231 and SKBR3 cells. We show that estrogen-independent cells expressed a significantly higher basal MnSOD level compared to estrogen-dependent human breast cancer cell lines (MCF-7 and T47D). For MDA-MB231 cells, the high-MnSOD level was accompanied by an overproduction of intracellular hydrogen peroxide (H2O2) and by a low expression of the major H2O2-detoxifying enzymes, catalase, and peroxiredoxin 3, compared to MCF-7 cells. Suppression of MnSOD expression by antisense RNA was associated with a decrease of H2O2 content and caused a stimulation of growth with a reduced cell doubling time but induced a decrease of colony formation. Furthermore, treatment of MDA-MB231 cells with H2O2 scavengers markedly reduced tumor cell growth and colony formation. In addition, MnSOD suppression or treatment with H2O2 scavengers reduced the invasive properties of MDA-MB231 cells up to 43%, with a concomitant decrease of metalloproteinase-9 activity. We conclude that MnSOD plays a role in regulating tumor cell growth and invasive properties of estrogen-independent metastatic breast cancer cells. These action are mediated by MnSOD-dependent H2O2 production. In addition, these results suggest that MnSOD up-regulation may be one mechanism that contributes to the development of metastatic breast cancers.

Genistein stimulates growth of human breast cancer cells in a novel, postmenopausal animal model, with low plasma estradiol concentrations

We have demonstrated that genistein (GEN) stimulates growth of estrogen-dependent breast tumors in vivo. In this study, we evaluated whether dietary GEN can act in an additive manner with low circulating levels of 17beta-estradiol (E2). We developed an E2 delivery system using silastic implants that yield low circulating plasma E2 levels similar to those observed in postmenopausal women. We inserted various concentrations of E2 silastic implants (1:127, 1:63, 1:31, 1:15 and 1:7 = E2:cholesterol) and injected estrogen-dependent human breast cancer (MCF-7) cells into ovariectomized athymic mice. The E2 implants tested (1:127-1:7) generated 30.1-101.6 pM E2 in plasma, which is comparable to the E2 levels observed in postmenopausal women. The E2 implants stimulated MCF-7 tumor growth in a dose-dependent manner. We selected the 1:31 ratio of E2 implant to evaluate if dietary GEN acts in an additive manner with low E2 levels to influence the growth of MCF-7 tumors. Ovariectomized mice were divided into four groups: MCF-7 control, 500 ppm GEN, 1:31 E2, and 1:31 E2 + 500 ppm GEN. At week 17, the average tumor sizes were 7.6, 32.1, 67.4 and 106.8 mm2 for these groups, respectively (P < 0.05), demonstrating that 500 ppm GEN additively stimulated MCF-7 tumor growth in the presence of low levels of E2. In summary, we established a preclinical mouse model that results in E2 blood concentrations similar to those found in postmenopausal women. Further, we observed that these concentrations regulate the growth rate of MCF-7 breast tumors. Using this model, we demonstrated that dietary GEN in the presence of low levels of circulating E2 act in an additive manner to stimulate estrogen-dependent tumor growth in vivo. Results from this study suggest that consumption of products containing GEN may not be safe for postmenopausal women with estrogen-dependent breast cancer.

Effects of dietary daidzein and its metabolite, equol, at physiological concentrations on the growth of estrogen-dependent human breast cancer (MCF-7) tumors implanted in ovariectomized athymic mice

Genistein and daidzein are the main isoflavones in legumes. Equol is an intestinal bacterial metabolite of daidzein. In this study, we evaluated the estrogenic potential of daidzein and synthetic (+/-)-equol to stimulate growth of estrogen-dependent breast cancer (MCF-7) in vitro and in vivo. We hypothesize that estrogenic effects of daidzein and (+/-)-equol could modulate the growth of MCF-7 cells both in vitro and also once implanted into ovariectomized athymic mice. At concentrations between 0.001 and 50 microM, daidzein and (+/-)-equol stimulated the growth of MCF-7 cells with maximal stimulation at 1 muM in vitro. To evaluate their effects on the growth of MCF-7 cells implanted in ovariectomized athymic mice, two dietary dose-response studies [daidzein (125, 250, 500 and 1000 p.p.m.) and (+/-)-equol (250, 500 and 1000 p.p.m.)] were conducted. Tumor size and body weight were monitored weekly during the study. At completion of the study, we analyzed cellular proliferation of tumors using immunohistochemical staining (ki-67), pS2 expression in tumors using a real time quantitative reverse transcription-polymerase chain reaction (qRT-PCR), and total daidzein and (+/-)-equol levels in plasma using liquid chromatography-electrospray tandem mass spectrometry (LC-ES/MS/MS). Dietary daidzein had a slight but significant stimulatory effect on MCF-7 tumor growth in mice. No significant induction of pS2 mRNA (an estrogen-responsive marker) in tumors by dietary daidzein was observed. Total plasma daidzein concentrations in plasma were between 0.25 and 1.52 microM. Dietary equol treatment (for 37 weeks) did not stimulate MCF-7 tumor growth. There were no statistical differences in tumor size, proliferation and pS2 expression among any treatment groups. Total equol concentrations in plasma were 2.10-3.21 microM. In conclusion, daidzein and (+/-)-equol have proliferative effects on MCF-7 cell growth in vitro within the concentration range tested. Dietary daidzein had a slight but significant stimulatory effect on tumor growth, whereas (+/-)-equol did not stimulate the growth of estrogen-dependent breast tumor growth in athymic mice, increase the cell proliferation in tumors, or induce an estrogen-responsive pS2 expression. Total daidzein or (+/-)-equol plasma levels in mice fed the isoflavones were in the range that stimulated MCF-7 cell growth in vitro. These results suggest that pharmacokinetic and/or metabolic factors attenuate the estrogenic effects of daidzein and equol in vivo.

Effect of the Selective Estrogen Receptor Modulator Arzoxifene on Repopulation of Hormone-Responsive Breast Cancer Xenografts between Courses of Chemotherapy

Selective inhibition of repopulation of clonogenic tumor cells between courses of chemotherapy has potential to improve the effectiveness of treatment. Here we study arzoxifene, a short-acting selective estrogen receptor modulator, for its potential to inhibit repopulation in estrogen-dependent human breast cancer MCF-7 xenografts between courses of chemotherapy. Proliferation of tumor cells was evaluated by cyclin D1 expression and uptake of 5-bromo-2'-deoxyuridine. Arzoxifene decreased cell proliferation in xenografts. To model adjuvant treatment of human breast cancer, MCF-7 cells were injected s.c. into nude mice and four groups of mice received the following treatments beginning after implantation: (a) control (vehicle solution); (b) arzoxifene alone, 5 days per week by oral gavage for 3 weeks; (c) 5-fluorouracil (5-FU) or paclitaxel i.p. weekly, for 3 doses; and (d) arzoxifene following each cycle of chemotherapy. The incidence of tumors with volume > or =50 mm(3) was determined as a function of time. MCF-7 xenografts developed in 100% of control mice by 4 weeks after implantation. Paclitaxel or 5-FU alone had minor effects to delay the appearance of xenografts whereas arzoxifene alone caused longer delay. Combined treatment with arzoxifene given between cycles of 5-FU or paclitaxel had substantial effects, with approximately 50% tumor incidence by 5 weeks. Our results indicate that arzoxifene can inhibit repopulation of hormone-responsive MCF-7 breast cancer xenografts when given between courses of chemotherapy. The scheduling of short-acting hormonal agents between courses of adjuvant chemotherapy for human breast cancer has potential to improve the outcome of treatment.

Effects of zearalenone on proliferation and apoptosis in MCF-7 cells

To explore the effects of zearalenone (ZEA) on proliferation and apoptosis in estrogen-dependent human breast cancer MCF-7 cells and the likely underlying molecular mechanisms. Cell viability was determined by MTT assay and cell cycle distribution by cytometry. Apoptosis was detected by Cell Death Detection ELISA and cytometry, respectively. The expressions of bax and bcl-2 were examined using multiple RT-PCR and Western-blot both at mRNA and protein level, respectively. The current study confirmed the previous studies that ZEA could stimulate proliferation in MCF-7 cells with inducing a profound increase in S phase and a modest increase in G(2)/M phase that was accompanied by a decrease in G(0)/G(1) phase. ZEA could inhibit apoptosis in MCF-7 cells following estrogen ablation at a range of concentrations of 2 nmol/L -96 nmol/L. Western blot and RT-PCR analysis revealed that the anti-apoptotic bcl-2 was upregulated at both protein and mRNA level, together with the downregulation of pro-apoptotic bax. ZEA should have possessed comparative estrogenic activity and could promote the progression of MCF-7 cells through the cell cycle by a decreasing in the G(0)/G(1) phase and by a significant increasing in S-phase. The pro-proliferative activity of ZEA was due to inhibition of apoptosis through regulation of bax/bcl-2 expression.

Skin temperature rise induced by calcitonin gene-related peptide in gonadotropin-releasing hormone analogue-treated female rats and alleviation by Keishi-bukuryo-gan, a Japanese herbal medicine

The effects of Keishi-bukuryo-gan on calcitonin gene-related peptide (CGRP)-induced elevation of skin temperature were investigated in gonadotropin-releasing hormone (GnRH) analogue-treated female rats. Leupline® (1.0 mg/kg) as the GnRH analogue was subcutaneously (s.c.) injected into female rats. After Keishi-bukuryo-gan (100–1,000 mg/kg, p.o.) or 17β-estradiol (0.010 mg/kg, s.c.) was administered to GnRH analogue-treated rats for 14 days, CGRP-induced skin temperature elevation, concentration of plasma 17β-estradiol and pituitary gonadotropin (luteinizing hormone; LH, and follicle stimulating hormone; FSH) were measured. In addition, effects of 17β-estradiol and Keishi-bukuryo-gan on the proliferation of estrogen-dependent human breast cancer (MCF-7) cells were investigated under in vitro conditions. GnRH analogue significantly lowered the concentrations of plasma 17β-estradiol and pituitary gonadotropins. Tissue weights of the ovaries and uterus were also decreased by the analogue. Under the condition of estrogen deficiency, intravenous (i.v.) injection of exogenous CGRP (10 μg/kg) elevated the skin temperature of the hind paws more significantly than it did in sham-treated control rats. Estrogen supplementation inhibited this elevation of skin temperature with restoration of both the lowered plasma estrogen level and the decreased uterine weight in GnRH analogue-treated rats. On the other hand, Keishi-bukuryo-gan inhibited the elevation of skin temperature in a dose-dependent manner without restoring the plasma estrogen level and uterine weight. In addition, in an in vitro study, MCF-7 cells proliferated in a dose-dependent manner by the addition of 17β-estradiol (10 −13–10 −8M) to the medium. However, Keishi-bukuryo-gan (10 −6–10 −4 mg/ml) did not activate the MCF-7 cell proliferation. These results suggest that Keishi-bukuryo-gan, which does not exhibit estrogen activity, may be useful for the treatment of hot flashes in women who are undergoing medical ovariectomy with a GnRH analogue.

Dietary Flaxseed Enhances the Inhibitory Effect of Tamoxifen on the Growth of Estrogen-Dependent Human Breast Cancer (MCF-7) in Nude Mice

This study determined the effect of 10% dietary flaxseed (FS) and tamoxifen (TAM), alone and in combination, on the growth of estrogen-dependent human breast cancer (MCF-7) in athymic mice with or without 17beta-estradiol (E2) supplementation. Ovariectomized mice received injection with MCF-7 cells, were implanted with an E2 pellet (1.7 mg), and fed the basal diet (BD). When tumor reached approximately 40 mm2, the E2 implant was removed, and mice were randomized to the following groups and maintained at either low (E2 pellet removed) or high E2 level (new E2 pellet implanted) for 6 weeks: (a) positive control with new E2 pellet, fed BD, (b) negative control with no E2 implant, fed BD, (c) TAM group with TAM pellet (5 mg) implant, fed BD, (d) FS group fed 10% FS, (e) FS+TAM group with TAM implant, fed 10% FS. Tumor growth was monitored weekly. At low E2 level, FS regressed the pretreatment tumor size by 74%. TAM regressed tumor initially but later induced an increase so that the tumor size was finally similar to the pretreatment size. A tumor regression >53% was induced by FS+TAM than by TAM alone. At high E2 level, FS, TAM, and FS+TAM inhibited the tumor growth by 22, 41, and 50%, respectively, compared with the positive control. Decreased tumor size was attributable to reduced tumor cell proliferation and increased apoptosis. FS inhibited the growth of human estrogen-dependent breast cancer and strengthened the tumor-inhibitory effect of TAM at both low and high E2 levels.

Estrogen-like effects of ultraviolet screen 3-(4-methylbenzylidene)-camphor (Eusolex 6300) on cell proliferation and gene induction in mammalian and amphibian cells

We tested the ultraviolet screen 3-(4-methylbenzylidene)-camphor (4-MBC; Eusolex 6300), which has been implicated as a potential endocrine disruptor, for its potential to bind to and activate endogenous estrogen receptors (ER) and to mediate ER-dependent changes in gene transcription, in hepatocytes of the water-dwelling South African clawed frog Xenopus laevis. We were able to confirm previous findings that 4-MBC accelerates cell proliferation in estrogen-dependent human breast cancer cells (MCF-7). Results of competitive binding assays of [3H]17β-estradiol and 4-MBC using cytosolic protein preparations from Xenopus hepatocytes indicated that 4-MBC weakly binds to the ER. 4-MBC at a concentration of 100μmol/L is not able to completely replace estradiol from the receptor. However, when 4-MBC was tested in a gene induction assay using the relative amount of ER transcript as a marker for ER-dependent transcriptional activation, we found that micromolar concentrations of this substance produced an increase in the amount of ER mRNA that was not different from the amount of mRNA that was observed upon activation of cells with 17β-estradiol in concentrations above 1nmol/L. The results indicate that 4-MBC has the potential to change physiological and developmental processes mediated by ER signaling mechanisms. It may therefore be a potentially harmful substance for water-dwelling animals when present in the environment at micromolar concentrations.

β-Sitosterol, β-Sitosterol Glucoside, and a Mixture of β-Sitosterol and β-Sitosterol Glucoside Modulate the Growth of Estrogen-Responsive Breast Cancer Cells In Vitro and in Ovariectomized Athymic Mice

We hypothesized that the phytosterols beta-sitosterol (BSS), beta-sitosterol glucoside (BSSG), and Moducare (MC; BSS:BSSG = 99:1) could modulate the growth of estrogen-dependent human breast cancer cells in vitro and in vivo. The present study evaluated the estrogenic and antiestrogenic effects of BSS, BSSG, and MC (0.001 to 150 micromol/L) on the proliferation of Michigan Cancer Foundation 7 (MCF-7) cells in vitro. Both BSS (>1 micromol/L) and MC (>50 micromol/L) increased MCF-7 cell proliferation. Treatment with 150 micro mol/L of BSS and MC increased cell growth by 2.4 and 1.5 times, respectively, compared to the negative control (NC) group. However, BSSG had no effect at the concentrations tested. The effects of dietary BSS, BSSG, and MC on the growth of MCF-7 cells implanted in ovariectomized athymic mice were also evaluated. Estrogenic effects of the phytosterols were evaluated in the NC, BSS, BSSG, and MC treatment groups, and antiestrogenic effects were evaluated in the 17 beta-estradiol (E(2)), E(2) + BSS, E(2) + BSSG, and E(2) + MC treatment groups. Mice were treated with dietary BSS (9.8 g/kg AIN93G diet), BSSG (0.2 g/kg diet), or MC (10.0 g/kg diet) for 11 wk. Dietary BSS, BSSG, and MC did not stimulate MCF-7 tumor growth. However, dietary BSS, BSSG, and MC reduced E(2)-induced MCF-7 tumor growth by 38.9% (P < 0.05), 31.6% (P = 0.08), and 42.13% (P < 0.05), respectively. The dietary phytosterols lowered serum E(2) levels by 35.1, 30.2, and 36.5% in the E(2) + BSS, E(2) + BSSG, and E(2) + MC groups, respectively (P < 0.05), compared to that of the E(2) treatment group. Estrogen-responsive pS2 mRNA expression in tumors did not differ among groups, but expression of the antiapoptotic marker B-cell lymphoma/leukemia-2 (bcl-2) in tumors from the E(2) + MC group was downregulated, compared to that of the E(2) treatment group. In summary, BSS and MC stimulated MCF-7 cell growth in vitro. Although BSSG comprises only 1% of MC, BSSG made MC less estrogenic than BSS alone in vitro. However, dietary BSS and MC protected against E(2)-stimulated MCF-7 tumor growth and lowered circulating E(2) levels.

The Transcriptional Repressor Sp3 Is Associated with CK2-phosphorylated Histone Deacetylase 2

Sp1 and Sp3 are ubiquitously expressed mammalian transcription factors that function as activators or repressors. Although both transcription factors share a common domain involved in forming multimers, we demonstrate that Sp1 and Sp3 form separate complexes in estrogen-dependent human breast cancer cells. Sp1 and Sp3 complexes associate with histone deacetylases (HDACs) 1 and 2. Although most HDAC2 is not phosphorylated in the breast cancer cells, HDAC2 bound to Sp1 and Sp3 and cross-linked to chromatin in situ is highly enriched in a phosphorylated form that has a reduced mobility in SDS-polyacrylamide gels. We show that protein kinase CK2 is associated with and phosphorylates HDAC2. Alkaline phosphatase treatment of HDAC2 and Sp1 and Sp3 complexes reduced the associated HDAC activity. Protein kinase CK2 is up-regulated in several cancers including breast cancer, and Sp1 and Sp3 have key roles in estrogen-induced proliferation and gene expression in estrogen-dependent breast cancer cells. CK2 phosphorylation of HDAC2 recruited by Sp1 or Sp3 could regulate HDAC activity and alter the balance of histone deacetylase and histone acetyltransferase activities and dynamic chromatin remodeling of estrogen-regulated genes.