Dose-dependent changes of the ratio of apoptosis to proliferation by norethisterone and medroxyprogesterone acetate in human breast epithelial cells.

OBJECTIVE: There is increasing evidence that adding progestogens to estrogen replacement therapy may do more harm than good; however, whether all progestogens act equally on breast cells is debatable. Apart from estrogens, mitogenic growth factors from stromal breast tissue are important in growth-regulation of breast cells, and may modify the response to progestogens. We investigated the effects of medroxyprogesterone acetate (MPA) as well as norethisterone (NET) in the presence of a growth factor mixture and/or estradiol in normal and cancerous human epithelial breast cells. METHODS: MCF10A cells (human epithelial, estrogen- and progesterone-receptor negative, normal breast cells), HCC1500 (human estrogen and progesterone receptor-positive primary breast cancer cells) and MCF-7 cells (human estrogen and progesterone receptor-positive metastatic breast cancer cell line) were used in the experiments. The cells were incubated with progestogens at concentrations of 10(-10) to 10(-6) M for 7 days and growth factors (GFs), estradiol (E2) alone and a combination of GFs + E2. Cell proliferation rate was measured by ATP assay. Apoptosis was measured by cell death assay. Ratios of cell death : proliferation were calculated from these results. RESULTS: In MCF10A cells growth factors elicited a decrease in the ratio of apoptosis to proliferation. This effect was further stimulated by the addition of MPA, whereas NET had no effect. In HCC cells growth factors and estradiol alone and in combination led to a reduction in the ratio. This effect could be partly reversed dose-dependently by the addition of MPA and NET, being more pronounced for MPA. Similar results were found for MCF-7 cells stimulated by estradiol. CONCLUSION: The results of our investigations demonstrate that there are differences between the two progestogens NET and MPA investigated with respect to their effects on normal and cancerous cells. By increasing the mitotic rate of normal epithelial cells, MPA may increase breast cancer risk in women when used in long-term treatment. In this respect NET reacts neutral. The mitosis of pre-existing cancerous cells may be partly inhibited by the addition of both progestogens. Thus, our results indicate that it is necessary to differentiate between normal and malignant breast cells concerning the assessment of progestogens as a risk factor for breast carcinogenesis.     

Comparison of the proliferative effects of ethinylestradiol on human breast cancer cells in an intermittent and a continuous dosing regime.

Many women would prefer fewer bleeding episodes while taking oral contraceptives. For this reason and with the intention of reducing menstruation-associated symptoms, an extended-cycle contraceptive is considered in the present paper. However, it remains unknown whether this long-term treatment is associated with a different breast cancer risk from that of the usual treatment. Therefore, in the present in vitro work we intend to compare the effect of these different treatment regimens on breast cancer risk. MCF-7 cells (human estrogen- and progesterone-receptor-positive metastatic breast cancer cells) and HCC1500 cells (human estrogen- and progesterone-receptor-positive primary breast cancer cells) were incubated with physiological concentrations of ethinylestradiol (EE). Usual and extended cycles were mimicked by incubation periods of 18 hours with EE followed by 6 hours without EE and 24 hours with EE for 3 days, respectively. In both cell lines, EE elicited a significant increase in the proliferation rate. No significant difference was found between the two incubation periods. Our results indicate that continuously administered ethinylestradiol may not increase breast cancer risk in comparison to intermittent application. However, clinical studies are necessary to prove these in vitro results.     

The effects of postmenopausal hormone replacement therapy and oral contraceptives on the endogenous estradiol metabolism.

The estradiol metabolism may be of clinical relevance in the pathophysiology of various diseases; the increase in D-ring metabolites over A-ring metabolites in breast cancer patients is of special interest. Since estrogen therapy has been blamed for increasing the risk of breast cancer, the effects of hormonal replacement therapy (HRT) and oral contraception were investigated on the ratio of the main D-ring metabolite, 16alpha-hydroxyestrone (16-OHE1), to the main A-ring metabolite, 2-hydroxyestrone (2-OHE1). In our study, hormone replacement therapy (HRT) in postmenopausal women consisted of administration of estradiol valerate either with or without addition of the progestin dienogest. In the second part of the study, women of reproductive age received ethinylestradiol plus dienogest or ethinylestradiol plus norethisterone acetate as oral contraceptives (OC). 2-OHE1 and 16-OHE1 were measured by enzyme immunoassay in 8 h night-urine collected before and after 3 months of hormone administration. With HRT, that is, estradiol valerate or estradiol valerate plus dienogest, the ratios before treatment were 0.47 and 0.60; after 3 months, they were 0.54 and 0.52, respectively. There were no significant differences. With oral contraception, that is, ethinylestradiol plus dienogest or norethisterone acetate, the ratios before administration were 0.62 and 0.68, vs. 0.31 and 0.54 after 3 months, respectively. The ratio after ethinylestradiol and dienogest was significantly lower after treatment. HRT and OC in the estrogen-progestin combinations tested did not impose any negative effects on estradiol metabolism--they did not elicit a higher D-ring metabolism, which is considered to increase breast cancer risk.     

Inhibition of human breast cancer cell proliferation with estradiol metabolites is as effective as with tamoxifen.

The anti-estrogenic substance tamoxifen is effective in the adjuvant therapy applied in human breast cancer. Since it partly exhibits estrogenic activity and has serious side-effects, however, pure anti-estrogenic compounds are being sought. In our experimental study, we compared the anti-proliferative effect of estradiol and 13 endogenous estradiol metabolites on human breast cancer cells with the effect of tamoxifen. We used MCF-7 and MDA-MB 231, the well-established estrogen receptor-positive and -negative cell lines. 4-hydroxytamoxifen, the active metabolite of tamoxifen, estradiol and 13 estradiol metabolites were tested in concentrations ranging from 3.1 to 100 microM. Incubation time was 4 days and cell proliferation was measured by means of the ATP chemosensitivity test. 4-hydroxytamoxifen showed an IC50 value of 27 microM and 18 microM in MCF-7 and MDA-MB 231 cells, respectively. Estradiol and its metabolites were anti-proliferative in both cell lines. A few A-ring metabolites were more effective in inhibiting cell proliferation than D-ring metabolites and the parent substance 17beta-estradiol. 4-OHE1, 2-MeOE1 and 2-MeOE2 were as effective in both cell lines as tamoxifen. For the first time it has been demonstrated that endogenous estradiol metabolites are equally anti-proliferative as tamoxifen in the context of human breast cancer cells. Since some of these metabolites exhibit no estrogenic activity, they are likely to be valuable in clinical studies of chemoprevention and adjuvant therapy of breast cancer.     

Effects of Combination of Estradiol with Selective Progesterone Receptor Modulators (SPRMs) on Human Breast Cancer Cells In Vitro and In Vivo

Use of estrogen or estrogen / progestin combination was an approved regimen for menopausal hormonal therapy (MHT). However, more recent patient-centered studies revealed an increase in the incidence of breast cancer in women receiving menopausal hormone therapy with estrogen plus progestin rather than estrogen alone. Tissue selective estrogen complex (TSEC) has been proposed to eliminate the progesterone component of MHT with supporting evidences. Based on our previous studies it is evident that SPRMs have a safer profile on endometrium in preventing unopposed estrogenicity. We hypothesized that a combination of estradiol (E2) with selective progesterone receptor modulator (SPRM) to exert a safer profile on endometrium will also reduce mammary gland proliferation and could be used to prevent breast cancer when used in MHT. In order to test our hypothesis, we compared the estradiol alone or in combination with our novel SPRMs, EC312 and EC313. The compounds were effectively controlled E2 mediated cell proliferation and induced apoptosis in T47D breast cancer cells. The observed effects were found comparable that of BZD in vitro. The effects of SPRMs were confirmed by receptor binding studies as well as gene and protein expression studies. Proliferation markers were found downregulated with EC312/313 treatment in vitro and reduced E2 induced mammary gland proliferation, evidenced as reduced ductal branching and terminal end bud growth in vivo. These data supporting our hypothesis that E2+EC312/EC313 blocked the estrogen action may provide basic rationale to further test the clinical efficacy of SPRMs to prevent breast cancer incidence in postmenopausal women undergoing MHT.     

The effect of progesterone and synthetic progestins on serum- and estradiol-stimulated proliferation of human breast cancer cells.

The results from the Women's Health Initiative study on enhanced breast cancer risk in postmenopausal women using an estrogen/progestin combination clearly indicate the need for a comparison of different progestins with regard to cancer risk. To shed some light on this issue, we have investigated the influence of progesterone and various synthetic C19- and C21-progestins on cell proliferation of a human breast cancer cell line in vitro. Of special interested was the comparison of two different regimens commonly used in HRT, sequential and continuous combination with estradiol. We used the human breast cancer cell line MCF-7 as a model. Progesterone (P), chlormadinone acetate (CMA), dienogest (DNG), gestodene (GSD), 3-ketodesogestrel (KDG), levonorgestrel (LNG), medroxyprogesterone acetate (MPA), and norethisterone (NET) were investigated in the range of 0.01nm to 10 micro M alone and in combination with 10 nM estradiol. Cell proliferation was measured after 7 days using the ATP chemosensitivity test. Tested alone, CMA, DNG, GSD, KDG, MPA and NET significantly stimulated cell proliferation, but only at high dosages. Sequentially combined with estradiol, only CMA inhibited cell proliferation over the whole concentration range. Slight effects were found for DNG, GSD and KDG, whereas P and MPA only showed an effect at the highest concentration. NET had no significant effect on cell proliferation. Continuously combined, all progestins exhibited an inhibitory effect over the whole concentration range. The most prominent effects were found for P, CMA, GSD, and KDG. Only slight effects were found for DNG, MPA and NET. Our in vitro results indicate that the influence on breast cancer risk using HRT in postmenopausal women might depend on the type of progestin used as well as on the regimen applied. However, the net inhibitory in vitro effect of the progestins at clinically relevant dosages is rather minimal, and whether progestins in general can reduce breast cancer risk in long-term treatment remains uncertain. Further clinical trials are urgently needed to clarify this issue.     

Sex hormone-binding globulin selectively modulates estradiol-regulated genes in MCF-7 cells.

Human sex hormone-binding globulin inhibits the effects of estradiol on proliferation and apoptosis of breast cancer cells. We report here the effect of sex hormone-binding globulin on estradiol regulation of gene expression in MCF-7 breast cancer cells using a selected set of genes. Estradiol upregulates genes that are positive regulators of proliferation (e.g., bcl-2, c-fos, c-myc, cyclin D) or/and related to more aggressive form of breast cancer (e.g. BRCA-1, EGF-R) and downregulates two genes (c-jun and ERalpha). Sex hormone-binding globulin modulates only a selected group of estradiol-controlled genes (inhibiting upregulation of bcl-2, c-myc, EGF-R, PR, and downregulation of ERalpha), starting 48 hours after treatment. Our study demonstrates that in breast cancer cells, sex hormone-binding globulin is effective on few selected genes which are involved in cell growth and apoptosis or related to cell estrogen-dependence and that the protein regulation of estradiol effect is selected and specific. Sex hormone-binding globulin action in estrogen breast cancer cells is strongly associated to cell growth and estrogen-sensitivity.     

Assessment of estrogenic recruitment before chemotherapy in advanced breast cancer: Preliminary results of a double-blind randomized study of the eortic breast cancer cooperative group

We investigated whether estrogenic recruitment could enhance the antitumor effect of chemotherapy in 165 patients with advanced breast cancer, presumably sensitive to hormonal treatments (ER + and/or PgR + lesions). The therapeutic regimen consisted of: (a) estrogenic suppression by aminoglutethimide 1 g/day + hydrocortisone 40 mg/day; surgical castration in premenopausal patients only; (b) FAC (5FU 500 mg/m²; ADM 50 mg/m²; CPA 500 mg/m²) for 3 weeks; (c) following randomization, exactly 24h prior to chemotherapy, patients had to take 1 tablet of either placebo (PL) or 50 μg ethinylestradiol (EE2). Tolerance, responses, time to progression and median survival were identical in both groups. Thus, EE2 before chemotherapy did not contribute to the efficacy of this particular therapeutic regimen, which yielded an overall response rate of 64%. We conclude that the validity of the hormonal recruitment concept has not yet been established in clinical practice, so that this approach remains experimental.     

Estrogen receptor-beta agonist diarylpropionitrile counteracts the estrogenic activity of estrogen receptor-alpha agonist propylpyrazole-triol in the mammary gland of ovariectomized Sprague Dawley rats

Although estrogen can bind both types of estrogen receptors, estrogen receptor-alpha (ERα) is dominant in mediating estrogenic activity in the mammary gland and uterus. Excessive estrogenic activity such as estrogen-based postmenopausal hormone replacement therapy increases the risk for breast and endometrial cancers. The adverse effect of estrogen on uterine endometrium can be opposed by progestins; however, estrogen-plus-progestin regimen imposes substantially greater risk for breast cancer than estrogen alone. In this study, we used ERα-selective agonist propylpyrazole-triol (PPT) and ERβ-selective agonist diarylpropionitrile (DPN) to activate ERα and estrogen receptor-beta (ERβ) separately in an ovariectomized rat model and determined whether PPT-activated ERα function in the mammary gland can be suppressed by DPN activated ERβ. Ovariectomized rats were randomly divided into six groups and treated with DMSO (control), DPN, PPT, PPT/DPN, PPT/Progesterone, and PPT/Progesterone/DPN, respectively. In the mammary gland, PPT but not DPN increased cell proliferation and amphiregulin gene expression; importantly, the stimulatory effect of PPT on mammary cell proliferation and amphiregulin gene expression can be suppressed by DPN. In the uterus, the effect of PPT on uterine weight and endometrial cell proliferation was not inhibited by DPN but can be inhibited by progesterone. These data provide in vivo evidence that PPT activated ERα activity in the mammary gland can be opposed by ERβ-selective agonist DPN, which may be explored for the development of better hormone replacement therapy regimen with less risk for breast cancer.     

Prenatal and pubertal exposure to 17α-ethinylestradiol cause morphological changes in the prostate of old gerbils

This study evaluated such as exposure to ethinylestradiol during the prenatal (18th–22nd day) and pubertal (42nd–49th day) periods acts on the male ventral prostate and female prostate of 12-month old gerbils. We performed the analysis to serum hormone levels for estradiol and testosterone. The prostates were submitted to morphometric and immunohistochemical analyses. Exposure to ethinylestradiol during these developmental periods decreased the testosterone serum levels in males and increased the estradiol serum levels in females. Morphologically, prostate intraepithelial neoplasia and disorders in the arrangement of the fibrous components were observed in the prostate glands of both sexes of gerbil exposed to ethinylestradiol during development periods. In the male prostate, the ethinylestradiol promoted decreased in the frequency of positive epithelial cell for androgen receptor (AR) and increased the frequency of positive stromal cell for estrogen receptor α. However, in the female prostate, this synthetic estrogen caused AR upregulation and increased cell proliferation. This study shows that the exposure to ethinylestradiol during development phases alters the morphology and the hormonal signaling in the male and female prostates of old gerbils, confirming the action of ethinylestradiol as endocrine disruptor.     

Effects of the environmental estrogens bisphenol A, o,p'-DDT, p-tert-octylphenol and coumestrol on apoptosis induction, cell proliferation and the expression of estrogen sensitive molecular parameters in the human breast cancer cell line MCF-7

In the presented study, we have analysed effects of the environmental estrogens bisphenol A (BPA), p-tert-octylphenol (OCT), o,p'-DDT (DDT) and coumestrol (COU) on cell proliferation, apoptosis induction, progesterone receptor (PR) and androgen receptor (AR) mRNA expression and ER alpha protein expression in comparison to estradiol (E2) and the selective ER modulator (SERM) raloxifene (RAL) and the pure antiestrogen faslodex (ICI 182780) in the human breast cancer cell line MCF-7. A dose dependent analysis of the cell cycle distribution of MCF-7 cells after administration of OCT, DDT and COU revealed a significant induction of cell proliferation and reduced rate of apoptosis. Maximum induction of cell proliferation and the lowest rate of apoptosis could be observed at a dose of 10(-6)M. Interestingly, administration of BPA reduces the rate of apoptosis, but does not enhance proliferation at any dose analysed. PR mRNA expression in MCF-7 cells was up regulated after administration of COU and DDT, whereas treatment with BPA and OCT did not effect PR mRNA expression. AR mRNA expression was down regulated by COU, but not effected by BPA, DDT and OCT. The expression of ER alpha protein in the breast cancer cells was slightly down regulated by COU and DDT, but unaffected by BPA and OCT. In summary and in comparison to the effects observed after administration of E2, RAL and ICI our data indicate that none of the analysed compounds exhibit properties comparable to RAL and ICI. COU and DDT exhibit properties which are very similar to E2. Administration of BPA and OCT did not effect any of the estrogen sensitive molecular parameters analysed. Nevertheless OCT is a very potent stimulator of cell proliferation in MCF-7 cells. Surprisingly, BPA is not able to induce the proliferation of MCF-7 breast cancer cells, but turns out to be a very potent inhibitor of apoptosis. For this reason and in agreement to the effects of BPA on the molecular parameters analysed, we conclude that BPA does not act in a classical estrogen like manner in MCF-7 breast cancer cells.     

Review of <Emphasis Type="Italic">Molecular Biology of Human Cancers</Emphasis> (<Emphasis Type="Italic">An Advanced Student’s Text</Emphasis>, by Wolfgang Schulz (Department of Urology and Center for Biological and Medical Research,Heinrich Heine University,Düsseldorf) ISBN 1-4020-3185-8

Postmenopausal women with estrogen receptor positive (ER⁺) breast cancer frequently respond paradoxically to estrogen administration with tumor regression. Using both LTED and E8CASS cells derived from MCF-7 breast cancer cells by long-term estrogen-deprivation, we previously reported that 17 -estradiol (estradiol) is a powerful, pro-apoptotic hormone which kills the cancer cells through activation of the Fas/FasL death receptor pathway. We postulated that the mitochondrial interactive protein Bcl-2 might play a role in the regulation of estradiol-induced apoptosis in both LTED and E8CASS cells. In this study, we assessed estradiol effects on cell growth, proliferation and apoptosis. Additionally we investigated the effect of estradiol on caspase activation, NF-KB and Bcl-2 expression. The functional role of Bcl-2 in estradiol-induced apoptosis was further studied by knockdown or decrease of Bcl-2 with siRNA. Our results show that estradiol significantly inhibited cell growth primarily through a pro-apoptotic action involving caspase-7 and 9 activations (p < 0.01). Basal Bcl-2 and NF-KB levels were greatly elevated and estradiol decreased NF-KB, but not Bcl-2 expression. Knockdown of Bcl-2 expression with siRNA decreased the levels of this protein by 9 fold (p < 0.01). This reduction markedly sensitized both LTED and E8CASS cells to the pro-apoptotic action of estradiol, leading to a synergistic induction of apoptosis and a concomitant reduction in cell number (p < 0.01). Therefore, down-regulation of Bcl-2 synergistically enhanced estradiol-induced apoptosis in ER⁺ postmenopausal breast cancer cells.     

Down-regulation of Bcl-2 enhances estrogen apoptotic action in long-term estradiol-depleted ER<Superscript>+</Superscript> breast cancer cells

Postmenopausal women with estrogen receptor positive (ER⁺) breast cancer frequently respond paradoxically to estrogen administration with tumor regression. Using both LTED and E8CASS cells derived from MCF-7 breast cancer cells by long-term estrogen-deprivation, we previously reported that 17 -estradiol (estradiol) is a powerful, pro-apoptotic hormone which kills the cancer cells through activation of the Fas/FasL death receptor pathway. We postulated that the mitochondrial interactive protein Bcl-2 might play a role in the regulation of estradiol-induced apoptosis in both LTED and E8CASS cells. In this study, we assessed estradiol effects on cell growth, proliferation and apoptosis. Additionally we investigated the effect of estradiol on caspase activation, NF-KB and Bcl-2 expression. The functional role of Bcl-2 in estradiol-induced apoptosis was further studied by knockdown or decrease of Bcl-2 with siRNA. Our results show that estradiol significantly inhibited cell growth primarily through a pro-apoptotic action involving caspase-7 and 9 activations (p < 0.01). Basal Bcl-2 and NF-KB levels were greatly elevated and estradiol decreased NF-KB, but not Bcl-2 expression. Knockdown of Bcl-2 expression with siRNA decreased the levels of this protein by 9 fold (p < 0.01). This reduction markedly sensitized both LTED and E8CASS cells to the pro-apoptotic action of estradiol, leading to a synergistic induction of apoptosis and a concomitant reduction in cell number (p < 0.01). Therefore, down-regulation of Bcl-2 synergistically enhanced estradiol-induced apoptosis in ER⁺ postmenopausal breast cancer cells.     

Puerariae radix isoflavones and their metabolites inhibit growth and induce apoptosis in breast cancer cells

Puerariae radix (PR) is a popular natural herb and a traditional food in Asia, which has antithrombotic and anti-allergic properties and stimulates estrogenic activity. In the present study, we investigated the effects of the PR isoflavones puerarin, daidzein, and genistein on the growth of breast cancer cells. Our data revealed that after treatment with PR isoflavones, a dose-dependent inhibition of cell growth occurred in HS578T, MDA-MB-231, and MCF-7 cell lines. Results from cell cycle distribution and apoptosis assays revealed that PR isoflavones induced cell apoptosis through a caspase-3-dependent pathway and mediated cell cycle arrest in the G2/M phase. Furthermore, we observed that the serum metabolites of PR (daidzein sulfates/glucuronides) inhibited proliferation of the breast cancer cells at a 50% cell growth inhibition (GI₅₀) concentration of 2.35 μM. These results indicate that the daidzein constituent of PR can be metabolized to daidzein sulfates or daidzein glucuronides that exhibit anticancer activities. The protein expression levels of the active forms of caspase-9 and Bax in breast cancer cells were significantly increased by treatment with PR metabolites. These metabolites also increased the protein expression levels of p53 and p21. We therefore suggest that PR may act as a chemopreventive and/or chemotherapeutic agent against breast cancer by reducing cell viability and inducing apoptosis.     

How to evaluate the risk-benefit ratio of the low-dose hormone replacement therapy?

Since the results of the women health initiative study showing an overall negative risk-benefit ratio with 0.625 mg of conjugated estrogens plus 2.5 mg of medroxyprogesterone acetate, the use of the lowest effective dose of steroids in hormone replacement therapy (HRT) is recommended.

A low-dose regimen appears to induce less side effects such as breast tenderness or leg pain than do higher dose preparations.

The decrease in hot flashes with low-dose estrogens, range 60–70%, is less than the 80–90% reduction with standard dosing. But this mean that 60–70% of menopausal women do not need higher doses.

The same applies to bone preservation which is dose dependent: the number of non-respondant women will be higher than with standard doses. However, randomized double-blind, placebo controls trials have defined positive effects on bone of low doses of HRT with adequate calcium and Vitamin D in elderly women. The use of bone densitometry and of biochemical markers of bone turnover is mandatory in women using low or ultra-low-dose preparations.

In spite of the lack of trials conducted with low-dose HRT, this treatment seems to be safer:

• the plasma levels of estradiol are lower; as far as breast cancer risk is concerned, the decrease of this subrogate marker is considered as favourable;

• the increase in breast density is less pronounced;

• the nurses's health study found a dose relationship for stroke, with no increase in risk with low-dose of estrogens;

• the effects on subrogate markers of cardiovascular risk seem to be more favourable.

Beside the low-dose HRT, one must consider some other facts:

• the “critical window” theory: it is biologically plausible that HRT, if started early after the menopause can slow the progression of coronary atherosclerosis;

• the way of administration of HRT: some observational studies have shown no increase in the risk of venous thromboembolism risk among women treated with transdermal estrogens;

• the progestogen used: a French cohort study recently performed found no increase in breast cancer risk with the use of micronized progesterone meanwhile the increase in risk observed with other progestogens was similar to the findings of the WHI study.

In the future, it is conceivable that more comprehensive pharmacogenomic studies will lead to effective algorithms for individualizing the right dose of steroids to be used in HRT.     

Characterization of the stimulatory effect of medroxyprogesterone acetate and chlormadinone acetate on growth factor treated normal human breast epithelial cells

OBJECTIVE: Evidence is increasing that adding progestogens to hormone replacement therapy may be more harmful than beneficial, however it is debatable whether all progestogens act equally on breast cells. Mitogenic growth factors from stromal breast tissue are important in growth-regulation of breast cells, and may modify responses to progestogens. We investigated the effect of two C-21 derivatives, medroxyprogesterone acetate (MPA) and chlormadinone acetate (CMA) on growth-factor treated normal breast epithelial cells and tried to explore the underlying mechanisms of proliferation. METHOD: MCF10A (human epithelial, estrogen- and progesterone-receptor negative normal breast cells) were incubated with MPA or CMA at 0.1 and 1 microM for 7 days with the growth factors (GFs) EGF, bFGF and IGF-I at 1pM. The same combinations, as well as growth factors alone, were also incubated with the proliferation inhibitors PD98059 and LY294002 at 1 microM for 4 days. Cell proliferation rate was measured by the ATP-assay. RESULTS: MPA 0.1 and 1 microM, and CMA 1 microM in combination with GFs both significantly increased cell proliferation rate, with MPA having the greatest effect. MPA- and CMA-induced proliferation of GF stimulated cells was blocked by both PD98059 (selective inhibitor of MAP kinases) and LY294002 (phosphatidylinositol 3-kinase inhibitor); GF stimulated cells could not be significantly reduced by any of the inhibitors used. CONCLUSION: MPA and CMA have a stimulatory effect on benign growth factor stimulated MCF10A cells, possibly via activation of MAP kinase and subsequent substrates and activation of PI3-kinase. GF induced proliferation appear to be mediated by pathways other than those investigated here. Growth factors and progestogens therefore have an additive, synergistic effect on cell proliferation, eliciting their effects via different pathways.     

Regulation of fas ligand expression in breast cancer cells by estrogen: functional differences between estradiol and tamoxifen

During neoplastic growth and metastasis, the immune system responds to the tumor by developing both cellular and humoral immune responses. In spite of this active response, tumor cells escape immune surveillance. We previously showed that FasL expression by breast tumor plays a central role in the induction of apoptosis of infiltrating Fas-immune cells providing the mechanism for tumor immune privilege. In the present study, we showed that FasL in breast tissue is functionally active, and estrogen and tamoxifen regulate its expression. We identified an estrogen recognizing element like-motif in the promoter region of the FasL gene, suggesting direct estrogen effects on FasL expression. This was confirmed by an increase in FasL expression in both RNA and protein levels in hormone sensitive breast cancer cells treated with estradiol. This effect is receptor mediated since tamoxifen blocked the estrogenic effect. Interestingly, tamoxifen also inhibited FasL expression in estrogen-depleted conditions. Moreover, an increase in FasL in breast cancer cells induces apoptosis in Fas bearing T cells and, tamoxifen blocks the induction of apoptosis. These studies provide evidence that tamoxifen inhibits FasL expression, allowing the killing of cancer cells by activated lymphocytes. This partially explains the protective effect of tamoxifen against breast cancer.     

Synthesis and biological evaluation of 4,6-diaryl-2-pyrimidinamine derivatives as anti-breast cancer agents

Breast cancer is the most frequently diagnosed cancers and the leading causes of cancer death among females worldwide. Estrogen receptor positive has been identified as the predominant internal reasons, involving in more than 70% breast cancer patients and SERMs which competes with estradiol for the binding to ERα in breast tissue are widely used in the treatment of ER+ breast cancer, such as tamoxifen, raloxifene. However, many SERMs may cause negative side effects due to their estrogenic activity in other tissues and approximate 50% of patients with ER-positive tumors either initially do not respond or become resistant to these drugs. Here, a series of designed 4,6-diaryl-2-pyrimidinamine derivatives had been synthesized to treat estrogen receptor positive breast cancer by simultaneously antagonizing ER and inhibiting VEGFR-2. Bioactivity evaluation showed that these compounds could significantly inhibit the proliferation of MCF-7, HUVEC and Ishikawa cells. Further studies identified compound III-3A could antagonize against estrogen action and inhibit the phosphorylation of VEGFR-2 as well as inhibit angiogenesis in vivo. The results indicated designed 4,6-diaryl-2-pyrimidinamine derivatives can be used to further study as anti-breast cancer drugs.     

Estrogen replacement therapy (ERT) in high-risk cancer patients.

Menopausal estrogens are now being prescribed not only for symptom relief, but also to prevent the long-term sequelae of estrogen deficiency, namely osteoporosis and atherosclerotic disease. The well-established association between endometrial cancer and estrogen replacement therapy (ERT) has become less of a clinical concern due to the recognition of the protective effect of progestogens in this setting. A small literature has emerged suggesting that extending ERT to the woman with a history of endometrial carcinoma imposes no increased risk of recurrence and may improve survival. Candidates for ERT should be women with a better prognostic profile with reference to their cancer. The relationship between ERT and breast cancer remains a topic of intense debate and investigation. Overall, the current literature finds no significant increase in risk among healthy women without a family history of breast cancer. There are no guidelines with reference to the woman with a history of breast cancer and the use of ERT. The most prudent approach with this population is to consider alternative treatments until more is known.     

K+ Channel Expression in Human Breast Cancer Cells: Involvement in Cell Cycle Regulation and Carcinogenesis

K⁺ channels are a most diverse class of ion channels in the plasma membrane and are distributed widely throughout a variety of cells including cancer cells. Evidence has been accumulating from fundamental studies indicating that tumour cells possess various types of K⁺ channels and that these K⁺ channels play important roles in regulating tumor cell proliferation, cell cycle progression and apoptosis. Moreover, a significant increase in K⁺ channel expression has been correlated with tumorigenesis, suggesting the possibility of using these proteins as transformation markers and perhaps reducing the tumor growth rate by selectively inhibiting their functional activity. Significant progress has been made in defining the properties of breast K⁺ channels, including their biophysical and pharmacological properties and distribution throughout different phases of the cell cycle in breast cell line MCF-7. This review aims to provide a comprehensive overview of the current state of research into K⁺ channels/currents in breast cancer cells. The possible mechanisms by which K⁺ channels affect tumor cell proliferation and cell cycle progression are discussed.