Antiestrogen, trans-tamoxifen modulation of human breast cancer cell growth

To gain further insight into how estrogens modulate cell function, the effects of estrogen on cell proliferation were studied in human breast cancer cells. We examined the effects of estrogen on the proliferation of three human breast cancer cell lines that differed in their estrogen receptor contents. Ten nM estradiol markedly stimulated the proliferation of MCF-7 human breast cancer cells that contained high levels of estrogen receptor (1.15±0.03 pmole/mg protein) over that of control. In T47D cells that contained low levels of estrogen receptor (0.23±0.05 pmole/mg protein), Ten nM estrogen slightly stimulated the proliferation over that of control. MDA-MB-231 cells, that contained no detectable levels of estrogen receptors, had their growth unaffected by estrogen. These results showed their sensitivity to growth stimulation by estrogen correlated well with their estrogen receptor content. Also we examined the effect of estrogen on cellular progesterone receptor level as well as plasminogen activator activity in MCF-7 cells. Ten nM estradiol showed maximal stimulation of progesterone receptor level as well as plasminogen activator activity in MCF-7 cells. It is not clear whether these stimulations of progesterone receptor and plasminogen activator activity by estrogen are related to the estrogen stimulation of cell proliferation of MCF-7 cells. Studies with estrogen in human breast cancer cells in culture indicate that sensitivity to growth stimulation by estrogen correlates well with estrogen receptor contents.     

Assaying the estrogenicity of phytoestrogens in cells of different estrogen sensitive tissues.

There is currently much concern that a wide range of both synthetic and naturally occurring environmental chemicals may act as endocrine disruptors (ED), and may adversely affect humans and wildlife. We examined the estrogenic effects of the phytoestrogens daidzein (DAI), equol (EQU) and O-desmethylangolensin (O-DMA), two metabolites of DAI, in three different assays. Binding affinity to the estrogen receptor alpha was 1000-10,000-fold lower compared with the endogenous estrogen estradiol. In the receptor positive cell line MCF-7 the phytoestrogens induced the expression of a reporter gene. The E-SCREEN is based on the estrogen-receptor binding induced proliferation of the human breast cancer cell line MCF-7. We also adapted the E-SCREEN for the estrogen-receptor positive human ovarian cancer cell line BG-1. The tested phytoestrogens induced cell proliferation in both cell lines, but not in the receptor negative human breast cancer cell line MDA-MB-231. The phytoestrogen-induced cell proliferation could be blocked by addition of the receptor antagonist 4-hydroxytamoxifen (OHT). Combination treatments with the endogenous estrogen estradiol showed competitive effects in MCF-7 cells. These studies demonstrated that the tested phytoestrogens exerted estrogenic responses in cells derived from two different tissues, breast and ovary. Furthermore, we demonstrated that BG-1 cells are a suitable additional cell system to investigate estrogenicity of test compounds.     

Growth medium for the evaluation of antiestrogenic compounds in MCF-7 cell culture.

The primary aim of the present study was to define a set of cell culture conditions that would be optimal for the evaluation of antiestrogens using estrogen dependent MCF-7 human breast cancer cells in culture. Therefore, the present study examined for the influence of media supplements which are known to alter the growth rate and estrogen receptor content of MCF-7 cells in culture. In this study, MCF-7 cell proliferation was evaluated using the hemocytometric trypan blue exclusion method. The results of this study clearly indicate that 1) charcoal-dextran stripped serum offers no advantage over low estradiol (less than 100 fM) calf serum which is available commercially, and 2) calf serum is as effective as fetal calf serum, in the presence or absence of insulin, in time-dependent growth of MCF-7 cells. Thus, growth media containing 5% non-stripped low estradiol (less than 100 fM) calf serum without insulin supplementation was found to be optimal for the evaluation of the antitumor activity of antiestrogenic compounds using MCF-7 cells in culture.     

New calmodulin antagonists inhibit in vitro growth of human breast cancer cell lines independent of their estrogen receptor status

Calmodulin plays a key role in the regulation of cell proliferation and calmodulin antagonists may offer a new therapeutic approach in the treatment of breast cancer. Three new specific calmodulin antagonists with improved potency were synthesized and screened on human breast cancer cell lines known to be estrogen receptor (ER)-positive or -negative. These calmodulin antagonists significantly inhibited cell growth as measured by the MTT proliferation assay (p<0.001). Their IC50 values were in the low micromolar range against both ER-positive and -negative variants of the MCF-7 cell line. Two other breast cancer cell lines (ER-positive T-47D and ER-negative MDA-MB-231) were also inhibited by these calmodulin antagonists with IC50 values in a similar range. The level of inhibition was independent of any stimulation of cell growth by estradiol. Calmodulin antagonists effectively reduced cell growth of both ER-positive and -negative human breast cancer cell lines in vitro. Calmodulin antagonists represent a novel therapeutic approach requiring further investigation.     

Stilbene-based inhibitors of estrone sulfatase with a dual mode of action in human breast cancer cells

Estrone sulfate (E1S) is an endogenous prodrug that delivers estrone and, subsequently, estradiol to target cells, after hydrolysis by the enzyme estrone sulfatase, which is active in various tissues including hormone-dependent breast cancer. Blockade of this enzyme should reduce the estrogen level in breast cancer cells and prevent hormonal growth stimulation. In this study, a number of sulfamoyloxy-substituted stilbenes with side chains that guarantee antiestrogenic activity were synthesized and evaluated as inhibitors of estrone sulfatase. They inhibited this enzyme in human MDA-MB 231 breast cancer cells, with IC(50) values in the submicromolar range. The effects of both the free hydroxy derivatives and the sulfamates on gene activation were determined in transfected MCF-7/2a breast cancer cells stimulated either with estradiol or with estrone sulfate. The analysis of data revealed a dual mode of action of the majority of compounds. They blocked gene expression by inhibition of estrone sulfatase and by antiestrogenic action. This pharmacological profile was also observed in assays on antiproliferative activity. The most potent derivative 8 g inhibited the growth of wild-type human MCF-7 cells with an IC(50) value of 13 nM.     

Estrogen receptor enhances the antiproliferative effects of trichostatin A and HC-toxin in human breast cancer cells

Trichostatin A, an antifungal antibiotics, and HC-toxin are potent and specific inhibitors of histone deacetylase activity. Histone deacetylase inhibitors are new class of chemotherapeutic drugs able to induce tumor cell apoptosis and/ or cell cycle arrest. In this study, the antiproliferative activities of trichostatin A and HC-toxin were compared between estrogen receptor positive human breast cancer cell MCF-7 and estrogen receptor negative human breast cancer cell MDA-MB-468. Trichostatin A and HC-toxin showed potent antiproliferative activity in both MCF-7 and MDA-MB-468 cells. In MCF-7 cells that contain high level estrogen receptor, trichostatin A and HC-toxin brought about three-times more potent cell growth inhibitory effect than estrogen receptor negative MDA-MB-468 cells. Both trichostatin A and HC-toxin showed cell cycle arrest at G₂/M phases of MCF-7 and MDA-MB-468 cells in a dose- and time- dependent manner. Trichostatin A and HC-toxin also induced apoptosis from MCF-7 and MDA-MB-468 cells in a dose- and time-dependent manner. Results of this study suggested that antiproliferative effects of trichostatin A and HC-toxin might be involved in estrogen receptor signaling pathway, but cell cycle arrest and apoptosis of trichostatin A and HC-toxin might not be involved in estrogen receptor system of human breast cancer cells.     

Insulin and estrogen receptor ligand influence the FGF-2 activities in MCF-7 breast cancer cells

From the MCF-7 cell line we have developed, a human mammary cancer cell subline with the same karyotype as the mother strain and named MCF-7(SF), able to grow in serum-free chemically defined medium. This cell subline was firstly used to analyze the effect of basic fibroblast growth factor (FGF-2) in estrogen-receptor-positive human breast cancer cells. FGF-2 like estradiol is able to increase cell proliferation and pS2 expression but was also found to inhibit progesterone receptor (PR) expression. The anti-estrogen tamoxifen partly counteracts the effects of FGF-2 and to discriminate between its two main mediators (estrogen receptor vs. anti-estrogen binding site, AEBS) we compare the efficacies of pure anti-estrogen (ICI 182,780) and AEBS ligand (PBPE). It appears that pure anti-estrogen counteracts cell growth and pS2 effects of FGF-2 since AEBS ligand inhibits the cell growth but has no activity on pS2 expression. Secondly, adding insulin (10(-6)M) in the culture medium induces a strong increase in cell proliferation, which then elicits an inhibitory effect of FGF-2 and addition of anti-estrogens, are less efficient to further decrease growth, since the effects of FGF-2 and anti-estrogens on pS2 expression are conserved.     

Proliferation-stimulating effects of icaritin and desmethylicaritin in MCF-7 cells

Icariin, icaritin and desmethylicaritin are constituents of Epimedium with a similar structure to genistein and daidzein. Using the modified MCF-7 cell proliferation assay (E-SCREEN assessment system), these compounds were tested for their estrogen-like activities. Icaritin and desmethylicaritin, but not icariin, strongly stimulated the proliferation of MCF-7/BUS cells. Cell cycle analysis revealed that the proliferation stimulatory effect was associated with a marked increase in the number of MCF-7/BUS cells in S phase and a significant increase in the G2/M population, with effects similar to those of estradiol. These actions were dose dependent (range from 1 nM to 10 microM) and could be significantly inhibited by the specific estrogen receptor antagonist ICI 182,780 [7 alpha-[9(4,4,5,5,5-pentafluoropentyl)sulfinyl]nonyl)-estra-1,3,5(10)-triene-3,17beta-diol)]. The estrogen receptor-regulated progesterone receptor and PS2 mRNA levels were increased by treatment with icaritin or desmethylicaritin within 24 h and the effects were also reversed by ICI 182,780. It was concluded that icaritin and desmethylicaritin are novel phytoestrogens and that the estrogenic effects of icaritin and desmethylicaritin are mediated by the estrogen receptor.     

(Anti)estrogenic effects of phytochemicals on human primary mammary fibroblasts, MCF-7 cells and their co-culture

In the public opinion, phytochemicals (PCs) present in the human diet are often considered beneficial (e.g. by preventing breast cancer). Two possible mechanisms that could modulate tumor growth are via interaction with the estrogen receptor (ER) and inhibition of aromatase (CYP19). Multiple in vitro studies confirmed that these compounds act estrogenic, thus potentially induce tumor growth, as well as aromatase inhibitory, thus potentially reduce tumor growth. It is thought that in the in vivo situation breast epithelial (tumor) cells communicate with surrounding connective tissue by means of cytokines, prostaglandins and estradiol forming a complex feedback mechanism. Recently our laboratory developed an in vitro co-culture model of healthy mammary fibroblasts and MCF-7 cells that (at least partly) simulated this feedback mechanism (M. Heneweer et al., TAAP vol. 202(1): 50-58, 2005). In the present study biochanin A, chrysin, naringenin, apigenin, genistein and quercetin were studied for their estrogenic properties (cell proliferation, pS2 mRNA) and aromatase inhibition in MCF-7 breast tumor cells, healthy mammary fibroblasts and their co-culture. The proliferative potency of these compounds in the MCF-7 cells derived from their EC(50)s decreased in the following order: estadiol (4*10(-3) nM)>biochanin A (9 nM)>genistein (32 nM)>testosterone (46 nM)>naringenin (287 nM)>apigenin (440 nM)>chrysin (4 microM). The potency to inhibit aromatase derived from their IC(50)s decreased in the following order: chrysin (1.5 microM)>naringenin (2.2 microM)>genistein (3.6 microM)>apigenin (4.1 microM)>biochanin A (25 microM)>quercetin (30 microM). The results of these studies show that these PCs can induce cell proliferation or inhibit aromatase in the same concentration range (1-10 microM). Results from co-cultures did not elucidate the dominant effect of these compounds. MCF-7 cell proliferation occurs at concentrations that are not uncommon in blood of individuals using food supplements. Results also indicate that estrogenicity of these PCs is quantitatively more sensitive than aromatase inhibition. It is suggested that perhaps a more cautionary approach should be taken for these PCs before taken as food supplements.     

Keratinocyte growth factor stimulates the migration and proliferation of breast cancer cells in a culture wounding model.

Growth factors are known to influence the progression, motility and invasiveness of tumor cells. In a previous study, we reported that conditioned media from NIH 3T3 cells (mouse fibroblast), which contains KGF, increased the motile morphology of estrogen receptor (ER)-positive breast cancer cells and produced no effect on ER-negative cells. The present study examined the influence of human KGF on two estrogen receptor (ER)-positive human breast cancer cell lines (MCF-7 and T-47D) using a culture wounding model to evaluate cell proliferation and migration over a period of 48h. In the present study we observed that KGF enhanced the migration and proliferation of both MCF-7 and T-47D breast cancer cells. In both cell lines the response to KGF was found to be both dose- and time-dependent. However, the total migration and proliferation response of the MCF-7 cells to KGF was much greater than that observed in the T-47D cells. The results of this study demonstrate that human KGF enhances the migration and proliferation of human breast cancer cells. Further, these results support the concept that KGF may be an early signal in the progression of breast cancer to a more motile and metastatic phenotype.     

Possible aryl hydrocarbon receptor-independent pathway of 2,3,7,8-tetrachlorodibenzo-p-dioxin-induced antiproliferative response in human breast cancer cells

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a ligand with high affinity for the aryl hydrocarbon receptor (AhR). It suppresses 17β-estradiol (E2)-induced cell proliferation in human breast cancer cells. Although it has been theorized that the AhR is involved in TCDD-induced antiestrogenic activity and antiproliferation in human breast cancer cells, some evidence suggests that these activities of chlorinated aromatic compounds also occur by AhR-independent pathways. Here, we investigated the possibility of TCDD-induced antiproliferative responses in human breast cancer cells through AhR-independent pathways. Compared with that in vehicle-treated controls, DNA synthesis was significantly suppressed in MCF-7 cells and ZR75-1 cells treated with TCDD at a very low concentration (0.01 nM), whereas that in human ovarian carcinoma OVCAR3 cells, human cervical carcinoma HeLa cells and human choriocarcinoma JEG-3 cells was unaffected, even by exposure to 10 nM TCDD. The suppression induced by TCDD was not associated with the estrogen receptor α-signaling pathway. Another AhR agonist, 3,3',4,4',5-pentachlorobiphenyl, had no effect on DNA synthesis in MCF-7 cells at concentrations high enough to induce the transactivation function of the AhR. Furthermore, in MCF-7 cells, knockdown of the AhR by RNA interference had no effect on TCDD-induced antiproliferation. These findings suggest that the principal pathways of TCDD-induced antiproliferation in breast cancer cells are not AhR dependent.     

雌激素受体对乳腺癌细胞截短型神经激肽受体-1 的调控作用

摘要： 目的 探讨雌激素受体 α （ERα） 阳性的乳腺癌细胞中 ERα 对神经激肽受体-1 截短型变异体 （NK1R-Tr）的调控作用, 以及 ERα 是否通过调控 NK1R-Tr 的表达, 间接调控细胞的增殖能力。方法 染色质免疫共沉淀（CHIP） 实验验证 ERα 是否可以结合到 NK1R-Tr 启动子上游的 ERα 反应元件, 直接调控 NK1R-Tr 的表达; 荧光素酶报告基因实验验证 ERα 是否对 NK1R-Tr 的表达起正性调控作用。Western blot 实验和 RT-PCR 实验检测乳腺癌细胞系 MCF-7 和 T47D 的 ERα 和 NK1R-Tr 在蛋白水平和 mRNA 水平的表达情况; 以及在 ERα 激动剂雌二醇 （E2）刺激的条件下, 小干扰 RNA 敲除 ERα 后, NK1R-Tr 在不同水平的表达情况; 小干扰 RNA 敲除 NK1R-Tr 后, CCK-8 和克隆形成实验检测敲除 NK1R-Tr 的乳腺癌细胞的增殖能力。结果 在 NK1R-Tr 基因启动子上游存在 ERα 的反应元件, ERα 在 E2 存在条件下作用于该反应元件, 对 NK1R-Tr 的表达起正性调控作用。同样在 E2 刺激的条件下,敲除乳腺癌细胞 MCF-7 内源性 ERα 后, NK1R-Tr 在蛋白水平和 mRNA 水平的表达均下降; 且敲除 NK1R-Tr 的 MCF-7 细胞增殖能力较未敲除组明显降低。结论 在 ERα 阳性的乳腺癌细胞中, ERα 正性调控 NK1R-Tr 的表达,从而增强细胞的增殖能力。     

Growth inhibitory and antimitogenic activity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in T47D human breast cancer cells.

The T47D human breast cancer cells used in this study express relative high levels of the progesterone receptor (637 +/- 118 fmol/mg protein) and lower levels of the estrogen and aryl hydrocarbon (Ah) receptors (81 +/- 3.4 and 55 +/- 8.2 fmol/mg protein, respectively). Treatment of these cells with 0.1, 1.0 and 10 nM concentrations of 17 beta-estradiol, transforming growth factor-alpha (TGF-alpha) and epidermal growth factor (EGF) resulted in concentration-dependent increase in cell proliferation and the ratios of mitogen-treated/control cell numbers were 2.46, 2.00 and 1.90, respectively. In contrast, insulin did not significantly stimulate T47D cell proliferation and insulin-like growth factor-I (IGF-I) was active only at a concentration of 10 nM. In parallel studies, the proliferative agents also stimulated the uptake of [3H]thymidine into cellular DNA. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) did not affect T47D cell growth at concentrations of 0.1 and 1.0 nM, whereas at a concentration of 10 nM a 44% decrease in cell numbers was observed. In cells cotreated with TCDD plus 100 ng/ml insulin or 10 nM 17 beta-estradiol, EGF, TGF-alpha and IGF-I, TCDD caused a concentration-dependent decrease in cell proliferation and [3H]thymidine uptake. For example, at a 10 nM concentration of TCDD there was a 32, 45, 29, 25 and 32% decrease in the 17 beta-estradiol, TGF-alpha, EGF, IGF-I and insulin-induced cell growth, respectively. These results confirm the antiproliferative activity of TCDD in T47D cells and this was similar to results previously reported in MCF-7 human breast cancer cells for the interaction of TCDD and 17 beta-estradiol. In addition, the data also show that TCDD inhibits the growth stimulatory effects of other polypeptide growth factors in T47D cells.     

Peripheral-type benzodiazepine receptors in the regulation of proliferation of MCF-7 human breast carcinoma cell line.

Peripheral-type benzodiazepine receptors (PBR) have been implicated in cell proliferation. The aim of the present study was to test the effect of the PBR ligands PK 11195 and Ro 5-4864 and the central-type benzodiazepine receptor ligand clonazepam on breast carcinoma cell proliferation, using [3H] thymidine incorporation. We then carried out a study to identify where the PBR-specific ligands Ro 5-4864 and PK 11195 act in the cell cycle, using flow cytometric analysis. We found PBR expression in the malignant breast cancer tumors, representing various levels of estrogen and/or progesterone receptors, as well as in the MCF-7 breast carcinoma cell line. PK 11195 and Ro 5-4864 inhibited cell proliferation at concentrations of 10(-5) to 10(-4) M, while clonazepam (the central-type benzodiazepine receptor-specific ligand) had no effect. In this same concentration range, PK 11195 and Ro 5-4864, in contrast to clonazepam, induced an accumulation of MCF-7 cells in both the G0-G1 and G2-M phases of the cell cycle. The present study demonstrates that PBR ligands play a role in regulating cell proliferation in the human breast carcinoma cell line MCF-7.     

Effects of estradiol, PCB3, and their hydroxylated metabolites on proliferation, cell cycle, and apoptosis of human breast cancer cells

Certain estradiol metabolites and industrial pollutants, like polychlorinated biphenyls, may play a more important role in enhancing breast cancer risk than 17β-estradiol. The aim of this study was to compare the effects of 17β-estradiol (E2) with that of the air pollutant 4-chlorobiphenyl (PCB3) and four of their hydroxylated metabolites on cell cycle, proliferation, and apoptosis in MCF-7 human breast cancer cells at concentrations of 0.1-10nM (E2, 2-OH-E2, and 4-OH-E2) and 0.3-300nM (PCB3, 4-OH-PCB3, and 3, 4-diOH-PCB3) and 24-260h of exposure. E2 increased cell proliferation and cells in S-phase at all time points. 2-OH-E2 and 4-OH-E2 had no effect on the cell cycle, but a stimulatory action on cell proliferation from 72 to 260h of exposure to 4-OH-E2 and at 260h to 2-OH-E2 was seen. E2 and its metabolites had no effect on apoptosis. PCB3 and 4-OH-PCB3 showed no effect on proliferation, apoptosis or cell cycle distribution at any concentration and time point. Longer time exposures to 3,4-di-OH-PCB at 300nM caused a decrease of cells and an increase in G2/M and apoptotic cells. These results confirm the proliferative effect of E2 and its metabolite 4-OH-E2 in estrogen receptor positive breast cancer cells, but show no mitogenic activity for PCB3 and 4-OH-PCB3. However, the cell cycle and apoptosis effects of 3,4-diOH-PCB3 need further analysis.     

Hexachlorobenzene induces cell proliferation and IGF-I signaling pathway in an estrogen receptor α-dependent manner in MCF-7 breast cancer cell line

Hexachlorobenzene (HCB) is an organochlorine pesticide widely distributed in the biosphere. ERα regulates the expression of genes involved in growth and development, and plays an important role in breast cancer. The present study focuses attention on the effect of HCB (0.005, 0.05, 0.5, 5 μM) on cell proliferation in estrogen receptor α (ERα)(+) MCF-7, and ERα(−) MDA-MB-231 breast cancer cell lines. We also studied the insulin growth factor-I (IGF-I) signaling pathway in MCF-7 cells. HCB (0.005 and 0.05 μM) stimulated cell proliferation in MCF-7, but not in MDA-MB-231 cells. The pesticide increased apoptosis in MCF-7, at HCB (0.5 and 5 μM). At these doses, HCB induced the expression of the aryl hydrocarbon receptor (AhR)-regulated gene cytochrome P4501A1. MCF-7 cells exposed to HCB (0.005 and 0.05 μM) overexpressed IGF-IR and insulin receptor (IR). IRS-1-phosphotyrosine content was increased in a dose dependent manner. ICI 182,780 prevented HCB-induced cell proliferation and IGF-I signaling in MCF-7 cells incubated in phenol-red free media. In addition, HCB (0.005 μM) increased c-Src activation, Tyr537-ERα phosphorylation and ERα down-regulation. Taken together, our data indicate that HCB stimulation of cell proliferation and IGF-I signaling is ERα dependent in MCF-7 cells.     

Cytostatic and antiestrogenic effects of 2-(indol-3-ylmethyl)-3,3'-diindolylmethane, a major in vivo product of dietary indole-3-carbinol.

Under acidic conditions, indole-3-carbinol (13C) is converted to a series of oligomeric products thought to be responsible for the biological effects of dietary 13C. Chromatographic separation of the crude acid mixture of 13C, guided by cell proliferation assay in human MCF-7 cells, resulted in the isolation of 2-(indol-3-ylmethyl)-3,3'-diindolylmethane (LTr-1) as a major antiproliferative component. LTr-1 inhibited the growth of both estrogen-dependent (MCF-7) and -independent (MDA-MB-231) breast cancer cells by approximately 60% at a non-lethal concentration of 25 microM. LTr-1 had no apparent effect on the proliferation of MCF-7 cells in the absence of estrogen. LTr-1 was a weak ligand for the estrogen receptor (ER) (IC50 70 microM) and efficiently inhibited the estradiol (E2)-induced binding of the ER to its cognate DNA responsive element. The antagonist effects of LTr-1 also were exhibited in assays of endogenous pS2 gene expression and in cells transiently transfected with an estrogen-responsive reporter construct (pERE-vit-CAT). LTr-1 activated both binding of the aryl hydrocarbon (Ah) receptor to its cognate DNA responsive element and expression of the Ah receptor-responsive gene CYP1A1. LTr-1 was a competitive inhibitor of CYP1A1-dependent ethoxyresorufin-O-deethylase (EROD) activity. In summary, these results demonstrated that LTr-1, a major in vivo product of I3C, could inhibit the proliferation of both estrogen-dependent and -independent breast tumor cells and that LTr-1 is an antagonist of estrogen receptor function and a weak agonist of Ah receptor function.     

Activation of insulin-like growth factor I receptor-mediated pathway by ginsenoside Rg1

Ginsenoside Rg1, an active ingredient in ginseng, was previously shown to be a novel class of potent phytoestrogen. The present study aims at investigating the molecular mechanisms involved in mediating its actions in human breast cancer (MCF-7) cells. Rg1 (1 pM) stimulates cell proliferation (P<0.01) and estrogen-responsive pS2 mRNA expression (P<0.05) without alteration of estrogen receptor alpha (ERalpha) protein or mRNA expression in MCF-7 cells. In addition, 10(-14)-10(-4) M of Rg1 does not demonstrate specific binding to ERalpha.We hypothesize that Rg1 may exert its actions in MCF-7 cell via the activation of crosstalk between ER- and insulin growth factor I receptor (IGF-IR)-dependent pathways. The results indicate that Rg1 significantly increases IGF-IR expression and IGF-IR promoter activity in MCF-7 cells (P<0.05). Cotreatment of MCF-7 cells with 1 muM of estrogen antagonist ICI 182,780 completely abolishes the effects of Rg1 on IGF-IR expression.Furthermore, Rg1 enhances tyrosine phosphorylation of IRS-1 in MCF-7 cells upon IGF-I stimulation and the activation of IRS-1 phosphorylation is also ER-dependent. Taken together, our results suggest that Rg1 not only increases IGF-IR expression but also enhances IGF-IR-mediated signaling pathways in MCF-7 cells. The stimulation of IGF-IR expression by Rg1 in MCF-7 cells appears to require ER, and its actions might involve ligand-independent activation of ER.     

Evaluation of the estrogenic activity of the constituents in the fruits of Vitex rotundifolia L. for the potential treatment of premenstrual syndrome

The ethanol extract of the fruits of Vitex rotundifolia (VRE) and its four major compounds (casticin, luteolin, rotundifuran and agnuside) were tested for their estrogen-like activity by using the modified cell proliferation assay (E-SCREEN assessment system), as well as the estrogen receptor (ER(alpha)), estrogen receptor-regulated progesterone receptor and pS2 mRNA expression in MCF-7 cells. The results showed that only agnuside and rotundifuran could stimulate the proliferation of MCF-7 cells. These actions were dose dependent (range from 100 nM to 10 microM) and could be significantly inhibited by the specific estrogen receptor antagonist ICI 182,780. The estrogen receptor ER(alpha) and the estrogen receptor-regulated progesterone receptor and pS2 mRNA levels were increased by treatment with rotundifuran and agnuside within 24 h, and the effects could be reversed by ICI 182,780. The standardization of the extract and constituents were carried out by means of a high-performance liquid chromatography-fingerprint. It was concluded that VRE and its compounds showed estrogen-like activity and that the estrogenic effects of rotundifuran and agnuside were mediated by the estrogen inducible gene, which may be useful in regulating the hormone levels to treat related diseases. However, further studies are required to assess the physiological significance of VRE in animals and man.     

Blockade of MUC1 expression by glycerol guaiacolate inhibits proliferation of human breast cancer cells

We sought to determine whether administration of glycerol guaiacolate at an optimal biological dose inhibits human breast cancer cell growth. Human breast cancer MCF-7 and ZR-75-1 cells were treated with glycerol guaiacolate and the therapeutic efficacy and biological activity of this drug was investigated on breast cancer cell growth. MCF-7 cells were injected into the mammary fat pad of overectamized female athymic nude mice. Ten days later, animals were treated with daily intraperitoneal injections of glycerol guaiacolate for six weeks. Tumor size and volume was monitored and immunohistochemistry analysis on MUC1, p21 and ki-67 was performed. Glycerol guaiacolate decreased breast cancer cell growth in a dose-dependent manner, decreased cell migration, and caused G1 cell cycle arrest. Our results demonstrate that glycerol guaiacolate inhibits MUC1 protein and mRNA expression levels and significantly increased p21 expression in human breast cancer cells as well as induced PARP cleavage. Similarly, glycerol guaiacolate inhibited breast tumor growth in vivo as well as enhanced p21 expression and decreased breast tumor cell proliferation (ki-67 expression). Collectively, our results demonstrate that glycerol guaiacolate decreased MUC1 expression and enhanced cell growth inhibition by inducing p21 expression in breast cancer cells. These findings suggest that glycerol guaiacolate may provide a novel and effective approach for the treatment of human breast cancer.