Intrinsic and extrinsic factors in estrogen action in human breast cancer: role of polyamines and pituitary factors

Although polyamines are important in regulating proliferation of mammalian cells, their role in hormone induction of cell growth has not been delineated. In the estradiol-responsive human breast cancer cell line, T-47D clone 11, estradiol (10(-10) M) was able to stimulate cell proliferation and the activity of ornithine decarboxylase (ODC), the first and rate-limiting enzyme in the biosynthesis of polyamines. alpha-Difluoromethylornithine (DFMO), a specific inhibitor of ODC, blocked the estradiol-induced cell proliferation and ODC activity. Exogenous addition of putrescine, the natural product of ODC, rescued the inhibitory effect of DFMO. In addition, DFMO abolished the estradiol-induced growth of several other estrogen-responsive human breast cancer cell lines but did not affect the growth of hormone-independent cell lines. Further, a serum factor was found to be required for estradiol to exert its effect. To gain insight into the nature of this and possibly other extrinsic factors involved, the effect of estradiol on the proliferation of T-47D cells transplanted into athymic nude mouse was evaluated. In this in vivo system, estradiol alone produced only moderate growth of the human breast tumor. The simultaneous transplantation of a prolactin (PRL)- and growth hormone (GH)-secreting rat pituitary tumor or normal rat pituitary glands at a different site dramatically potentiated the effect of estradiol on the growth of the breast tumor xenograft. Purified PRL or GH were without effect, indicating that the active pituitary factor is neither PRL nor GH. Further, conditioned medium from rat pituitary tumor cells potentiated the mitogenic effect of estradiol on T-47D and several other estrogen receptor-positive human breast cancer cell lines in vitro under serum-free condition. In conclusion, we have identified both intrinsic (polyamines) and extrinsic (pituitary/serum) factors that are importance for estrogen to exert its mitogenic action. The next goal will be to elucidate the mechanisms of action of these molecules in the modulation of estrogen action.     

Regulation of epidermal growth factor-receptor by estrogen and antiestrogen in the human breast cancer cell line MCF-7

Regulation of breast tumor proliferation depends in a large part on a variety of hormones and growth factors. In this report we show that estrogen and antiestrogen modulate epidermal growth factor-receptor (EGF-R) level in the human breast cancer MCF-7 cells with opposite mechanisms. Although a short-term treatment (24h to 48h) with estradiol leads to a decrease in EGF-R number, the addition of hormone in cell culture for 5 days increases EGF-R level with a maximal effect observed at 10(-10) M estradiol. In contrast, when cells are treated with the antiestrogen hydroxytamoxifen, a dose-dependent decrease in EGF-R level occurs. We also report that EGF is able to induce estrogen receptors and, to a lesser extent, progesterone receptors when added to MCF-7 cell cultures. These results demonstrate an interaction between both estrogen receptor and EGF receptor growth promoting systems in target cells. The implications of such an interaction in the understanding of human breast cancer hormone responsiveness and, in the development of therapies, are discussed.     

Effect of antiestrogens and aromatase inhibitor on basal growth of the human breast cancer cell line MCF-7 in serum-free medium

Antiestrogens are efficient inhibitors of estrogen-mediated growth of human breast cancer. Besides inhibiting estradiol-stimulated growth, antiestrogens may have a direct growth-inhibitory effect on estrogen receptor (ER) positive cells and thus be more efficient than aromatase inhibitors, which will only abrogate estrogen-dependent tumor growth. To address this issue, we have used the human breast cancer cell line MCF-7/S9 as a model system which is maintained in a chemically defined medium without serum and estrogen. The addition of estradiol results in an increase in cell growth rate. Thus, the MCF-7/S9 cell line is estrogen-responsive but not estrogen-dependent. Three different types of antiestrogens, namely tamoxifen, ICI 182,780 and EM-652 were found to exert a significant and dose-dependent inhibition of basal growth of MCF-7/S9 cells. The growth-inhibitory effect of the three antiestrogens was prevented by simultaneous estradiol treatment. Antiestrogen treatment also reduced the basal pS2 mRNA expression level, thus indicating spontaneous estrogenic activity in the cells. However, treatment with the aromatase inhibitor had no effect on basal cell growth, excluding that endogenous estrogen synthesis is involved in basal growth. These data demonstrate that in addition to their estrogen antagonistic effect, antiestrogens have a direct growth-inhibitory effect which is ER-mediated. Consequently, in the subset of ER positive breast cancer patients with estrogen-independent tumor growth, antiestrogen therapy may be superior to treatment with aromatase inhibitors which only inhibit estrogen formation but do not affect cancer cell growth in the absence of estrogens.     

Manipulation of cell cycle kinetics: Influence on the cytotoxicity of doxorubicin in human breast cancer cells

In vitro exposure of estrogen receptor-negative (ER-) EVSA-T human breast cancer cells to insulin and/or estradiol had no effect on cell cycle distribution, in contrast to a 3–5-fold increase in the percentages of cells in the S-phase of the cell cycle in the ER+ MCF-7 cell line. Estrogen pretreatment of MCF-7 cells followed by incubation with doxorubicin resulted in an augmented inhibition of cell growth compared to unstimulated controls. This delay in growth was accompanied by a decrease in the percentages of cells actively synthesizing DNA, and by an augmented percentage of cells exhibiting a G₂M-amount of DNA at the end of a 6–9 day period of culture in complete growth medium.     

Purification and characterization of an alpha 1-antichymotrypsin-like 66 kDa protein from the human breast cancer cell line, MCF-7.

The synthesis of a 66 kDa protein immunoreactive with antibodies to human alpha 1-antichymotrypsin (alpha 1-ACT) is induced by estradiol (E2) in the human breast cancer cell line MCF-7. We have purified this alpha 1-ACT-like 66 kDa protein from medium conditioned by MCF-7 cells, performed a comparative physico-chemical characterization with serum alpha 1-ACT, and analysed its presumed positive regulatory effect on growth of MCF-7 cells. The 66 kDa protein is a functional antiproteinase which is antigenically identical to serum alpha 1-ACT. The 66 kDa protein does however deviate from serum alpha 1-ACT with respect to mol. wt. and pattern of microheterogeneity, the molecular mechanism for this is probably an incomplete glycoprotein processing in the MCF-7 cells. The results of our growth experiments suggest that the 66 kDa protein is a minor positive growth regulatory factor, which may contribute to breast carcinoma cell proliferation in a cooperative manner.     

Interaction of estradiol and high density lipoproteins on proliferation of the human breast cancer cell line MCF-7 adapted to grow in serum free conditions

The responsiveness of the human mammary carcinoma cell line MCF-7 to estradiol and tamoxifen treatment has been studied in different culture conditions. Cells from exponentially growing cultures were compared with cells in their initial cycles after replating from confluent cultures ("confluent-log" cells). It has been observed that estradiol stimulation of tritiated thymidine incorporation decreases with cell density and that "confluent-log" cells are estrogen unresponsive for a period of four cell cycles in serum-free medium conditions. On the other hand, growth of cells replated from exponentially growing, as well as from confluent cultures, can be inhibited by tamoxifen or a combined treatment with tamoxifen and the progestin levonorgestrel. This growth inhibitory effect can be rescued by estradiol when cells are replated from exponentially growing cultures. The growth inhibitory effect cannot be rescued by estradiol alone (10(-10) to 10(-8) M) when cells are replated from confluent cultures. In this condition, the addition of steroid depleted serum is necessary to reverse the state of estradiol unresponsiveness. Serum can be replaced by high density lipoproteins but not by low density lipoproteins or lipoprotein deficient serum. The present data show that estradiol and HDL interact in the control of MCF-7 cell proliferation.     

芫菁斑蝥素对喉癌细胞和胃癌细胞的抑制作用

【目的】 研究提取自眼斑芫菁Mylabris cichorii (Linnaeus)体内的斑蝥素对人喉癌HEP-2细胞和人胃癌BGC-823细胞的抑制、以及对细胞周期分布的影响。【方法】 将斑蝥素作用于经体外培养的人喉癌HEP-2细胞和人胃癌BGC-823细胞, 采用MTT法进行体外细胞抑制实验, 测定斑蝥素对这2种癌细胞生长的抑制率与剂量效应;采用流式细胞术测定斑蝥素处理的人喉癌HEP-2细胞的细胞周期;并通过光学显微镜观察其细胞形态学改变。【结果】 斑蝥素浓度为1.28 μmol/L时, 对HEP-2细胞有显著抑制作用, 且随药物浓度升高其抑制作用增强, 呈剂量效应关系, 抑制中浓度为2.88 μmol/L;斑蝥素浓度为20.4 μmol/L时, 对BGC-823细胞有显著抑制作用, 且随药物浓度升高其抑制作用增强, 呈剂量效应关系, 抑制中浓度为54.85 μmol/L。用浓度1.44和2.88 μmol/L的斑蝥素处理HEP-2细胞24 h后, G2-M期分布从8.21%增加到22.29%, S期细胞分布从14.33%增加到21.61%, 且随药物浓度升高其阻滞作用增加, 呈剂量效应关系。G0-G1期细胞分布都有所降低, 从77.45%降低到56.10%, G0-G1期峰前无显著的亚二倍体峰出现, 说明斑蝥素未能够诱导HEP-2细胞发生凋亡。光镜检查显示：HEP-2细胞可出现细胞收缩、胞膜突出、核碎裂等现象。【结论】 斑蝥素对治疗喉癌的效果可能较为理想, 而对胃癌的作用则不明显。     

Mitogenic effect of estradiol on MCF-7 human breast cancer cells can be modulated by serum

The MCF-7 human breast cancer cell line responds to estradiol stimulation in vitro by increased proliferation only if prolonged subcultures in dextran-coated charcoal-treated fetal calf serum have been made previously. This growth stimulation is not obtained when cells are grown in medium containing 5% untreated fetal calf serum. We describe here the culture conditions under which we obtain a reproducible estradiol effect on cell growth.     

Growth inhibition of human breast cancer cells induced by calcitonin

The human breast cancer cell line (T47D) has specific, high affinity calcitonin receptors and calcitonin-responsive adenylate cyclase. Human, salmon and [Asu1,7]eel calcitonin inhibited cell growth in a dose-related manner with almost equipotency. Analogues of human calcitonin demonstrated slight cell growth inhibition. We found extreme growth inhibition with daily treatment with dibutyryl cyclic AMP (10(-4) M). In contrast to calcitonin 1,25-(OH)2D3 had a biphasic effect on cell growth. Physiological doses (5 X 10(-10) M) of 1,25-(OH)2D3 stimulated growth of T47D, whereas treatment by supraphysiological amounts (2.5 X 10(-7) M) caused significant inhibition of growth. Calcitonin and 1,25-(OH)2D3 appeared to have additive effects.     

Stimulation of growth of human breast cancer cell lines in culture by linoleic acid

Linoleic acid, an omega-6 unsaturated fatty acid, stimulated growth of the MDA-MB-231 and MCF-7 human breast cancer cell lines in culture. Responses of the estrogen-independent MDA-MB-231 cells both in serum-free medium and with 1% fetal bovine serum added were positively correlated with linoleic acid concentration over the entire range examined (5-750 ng/ml). Growth stimulation of the estrogen-responsive MCF-7 cell line was maximal at a LA concentration of 500 ng/ml when cultured in 1% fetal bovine serum-containing medium with added estradiol. Linoleic acid had no mitogenic effect on three human cancer cell lines derived from sites other than breast, or on untransformed 3T3 cells.     

Mutagenic activity of estradiol evaluated by an in vitro micronucleus assay. Short communication

The objective of the present study was to evaluate possible genetic changes in cultured human lymphocytes treated with estradiol, using the cytokinesis block micronucleus assay. Eight experimental concentrations of estradiol were used (range from 10(-10) M to 0.7 x 10(-4) M). The obtained results indicate that estradiol exhibits aneugenic and/or clastogenic effects, expressed as increased frequency of micronucleated lymphocytes at two highest experimental concentrations used in this investigation. In addition to genotoxic effects, these concentrations decreased the cytokinesis block proliferation index (CBPI) and percentage of binucleated cells, indicating the cell cycle delay and possible cytotoxic effects. In conclusion, estradiol treatment might represent a human health risk, especially if overdosed or used for a prolonged period of time.     

Selectivity of hemoregulatory peptide (HP5b) action in culture

A synthetic analog of a hemoregulatory peptide associated with mature human granulocytes (HP5b) has been investigated for inhibitory effects on various cell types in culture as compared to inhibitory action on mouse and human myelopoietic colonies (CFU-gm), which occurs from 1 X 10(-13) to 1 X 10(-6) M in vitro. This includes colony formation by lymphoid T and B cells in capillary cultures, as well as mitogen activation of T, B and NK cells. At higher concentrations, i.e., above 1 X 10(-7) M, an inhibitory effect was found on colony formation. Neither the production of interleukin (IL) 3 by mitogen-activated T cells, nor the proliferation of the IL-3-dependent L/B cell line were affected by the peptide up to 1 X 10(-5) M. A slight inhibitory effect was found above 1 X 10(-9) M on mouse 3T3 fibroblasts. A series of malignant cell lines was also tested. No effect was seen between 1 X 10(-11) and 1 X 10(-7) M on human mammary carcinoma cells in culture. On Ehrlich ascites mouse mammary carcinoma cells a 30% inhibition was seen at 10(-6) M. On a human glioblastoma cell line (GaMg) no effect was seen, and on a rat glioma cell line (BT5C) an inhibitory effect was seen at 1 X 10(-7) M and above. No significant inhibition of cell growth was seen on SC1 mouse lymphoma cells from 1 X 10(-9) to 1 X 10(-5) M during 7 days of culture. The investigated normal and malignant cell types in culture were thus not inhibited in very low concentrations which act on CFU-gm. However, a variable inhibitory effect was found at higher concentrations where the inhibition of myelopoiesis was maximal and at concentrations where the inhibition is released. The hemoregulatory peptide thus seems to be a concentration-dependent selective inhibitor of myelopoiesis. The finding that various malignant cells do not respond at lower concentrations supports the possibility of using the peptide as a protector of normal cells during cancer chemotherapy.     

Estrogen-dependent cyclin E-cdk2 activation through p21 redistribution.

In order to elucidate the mechanisms by which estrogens and antiestrogens modulate the growth of breast cancer cells, we have characterized the changes induced by estradiol that occur during the G1 phase of the cell cycle of MCF-7 human mammary carcinoma cells. Addition of estradiol relieves the cell cycle block created by tamoxifen treatment, leading to marked activation of cyclin E-cdk2 complexes and phosphorylation of the retinoblastoma protein within 6 h. Cyclin D1 levels increase significantly while the levels of cyclin E, cdk2, and the p21 and p27 cdk inhibitors are relatively constant. However, the p21 cdk inhibitor shifts from its association with cyclin E-cdk2 to cyclin D1-cdk4, providing an explanation for the observed activation of the cyclin E-cdk2 complexes. These results support the notion that cyclin D1 has an important role in steroid-dependent cell proliferation and that estrogen, by regulating the activities of G1 cyclin-dependent kinases, can control the proliferation of breast cancer cells.     

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.     

Bioflavonoid interaction with rat uterine type II binding sites and cell growth inhibition

Competition analysis with a number of known bioflavonoids demonstrated that these compounds (luteolin, quercetin, pelargonin) compete for [3H]estradiol binding to cytosol and nuclear type II sites in rat uterine preparations. The inhibition of [3H]estradiol binding to type II sites was specific and these bioflavonoids did not interact with the rat uterine estrogen receptor. Since estradiol stimulation of nuclear type II sites in the rat uterus is highly correlated with cellular hypertrophy and hyperplasia, we assessed the effects of these compounds on the growth of MCF-7 human breast cancer cells in culture and on estradiol stimulation of uterine growth in the immature rat. The data demonstrated that addition of quercetin (5-10 micrograms/ml) to MCF-7 cell cultures resulted in a dose-dependent inhibition of cell growth (DNA/flask). This effect was reversible by removal of quercetin from the culture medium, or by the addition of 10 nM estradiol-17 beta to these cell cultures containing this bioflavonoid. Since estradiol-17 beta (10 nM) stimulated nuclear type II sites and proliferation of MCF-7 cells, we believe bioflavonoid inhibition of MCF-7 cell growth may be mediated through an interaction with nuclear type II sites. This hypothesis was confirmed by in vivo studies which demonstrated that injection of luteolin or quercetin blocked estradiol stimulation of nuclear type II sites in the immature rat uterus and this correlated with an inhibition of uterine growth (wet and dry weight). These studies suggest bioflavonoids, through an interaction with type II sites, may be involved in cell growth regulation.     

Stimulation of PC cell-derived growth factor (epithelin/granulin precursor) expression by estradiol in human breast cancer cells

PC cell-derived growth factor (PCDGF) is an 88 kDa glycosylated protein isolated from a highly tumorigenic mouse teratoma derived cell line which is similar to the epithelin/granulin precursor. Using Northern blot and western blot analyses, we detect the expression of PCDGF mRNA and protein in MCF-7 human breast cancer cells. We show that 17-beta-estradiol stimulates PCDGF mRNA and protein expression in a time and dose-dependent manner. The stimulation of PCDGF expression by 17-beta-estradiol was observed as early as 4 hours and reached a maximum at 12 hours. Maximal stimulation of PCDGF mRNA and protein expression by 17-beta-estradiol was observed at a concentration of 10(-8) M. The stimulation of PCDGF expression by 17-beta-estradiol was completely inhibited by treatment with actinomycin D and with the antiestrogen 4-hydroxytamoxifen. The stimulation of PCDGF expression was also demonstrated in another human estrogen-responsive cell line T47D. The results presented here provide evidence of a novel estradiol responsive gene product in human breast cancer cell lines and give information about the hormonal control of epithelin/granulin (PCDGF) expression in these cells.     

Effects of 25-hydroxycholesterol, cholesterol, and isoprenoid derivatives on the G1 progression in Swiss 3T3 cells

The effect of inhibition of 3-Hydroxy-3-methylglutaryl Coenzyme A reductase (HMG CoA reductase) on cell cycle progression in proliferating 3T3 cells was studied. It was found that short transient exposures to the HMG CoA reductase inhibitor 25-hydroxycholesterol temporarily blocked the cell cycle traverse in the postmitotic half of G1 (G1pm), whereas cells in the subsequent cell cycle phases were unaffected. The kinetics of the cell cycle delay, induced by 25-hydroxycholesterol, resembled the kinetics of the delay induced by serum depletion, which also inhibited the activity of HMG CoA reductase. In contrast to the case of serum depletion, platelet derived growth factor (PDGF), which efficiently prevented the decrease of HMG CoA reductase in serum-free medium, was not capable of preventing the growth inhibitory effect following treatment by 25-hydroxycholesterol. However, cholesterol and two isoprenoids, dolichol and coenzyme Q, were effective in this respect. In addition, dolichol counteracted the cell cycle delay following short periods of serum starvation.     

Alteration of steroid hormone sensitivity during the cultivation of human mammary carcinoma cells

Summary During early cultivation steps of the newly derived and karyotyped human mammary carcinoma line EFM-19, the cells developed faster growth rates and became increasingly less responsive to the presence of serum in the culture medium. No drastic alterations of the morphology and of the karyotype were observed, and carcinoembryogenic antigen remained expressed during the course of the cultivation. In experimental incubations at various time intervals after the explantation, the cell proliferation was analyzed for dose-dependent effects of estradiol, cortisol, progesterone, and testosterone. After 16 wk of cultivation of the stock culture in the presence of estradiol, the cells had acquired a distinct sensitivity to estradiol resulting in permanent growth enhancement. The withdrawal of cortisol from the medium of the stock culture subsequently provoked the loss of the initially noted stimulation of the proliferation by cortisol. The stimulatory effect of progesterone on the proliferation was reversed to inhibition when the stock culture was deprived of cortisol in the growth medium. The results indicate that the choice of steroid hormones in the stock culture medium was determining the quality of the cellular growth responses. This work was supported by the Deutsche Forschungsgemeinschaft, SFB34.     

Adenosine stimulation of proliferation of breast carcinoma cell lines: evaluation of the [3H]thymidine assay system and modulatory effects of the cellular microenvironment in vitro

The purine nucleoside adenosine is produced at increased levels in the tissues of solid cancers as a result of local hypoxia. Adenosine inhibits the cell-mediated anti-tumor immune response, promotes tumor cell migration and angiogenesis, and stimulates the proliferation of tumor cells. We examined the stimulatory effect of adenosine on DNA synthesis, cell cycle progression, and cell proliferation in MCF7 and T-47D breast carcinoma cell lines in culture, and identified factors that modulate the growth response. The ability of adenosine to stimulate DNA synthesis, as measured by the incorporation of [(3)H]thymidine, was independent of the total radioactivity of the [(3)H]thymidine up to 10 microCi/ml, total thymidine concentrations up to 100 microM, and the labeling interval. It was also not affected by the presence of low-molecular-weight compounds (such as thymidine and adenosine) in the serum used to supplement the medium. Adenosine stimulated DNA synthesis and cell proliferation with an EC(50) of 4-6 microM and a maximum response at 30-100 microM, when given as a single addition. The stimulatory effect of adenosine involved progression through the cell cycle and a genuine increase in cell number, in the absence of significant apoptotic or necrotic cell death. The mitogenic effect of adenosine was dependent upon the culture cell density, with an optimum adenosine response at around 50% of confluent density. The response was also highly dependent upon the form of the serum addition to the growth medium, with the best response elicited in the presence of low concentrations of nonfetal bovine serum, although adenosine was mitogenic under standard culture conditions. The effects of serum supplementation and cell density were not due to differences in the rate of adenosine metabolism by either serum or cellular enzymes, but appeared to result from changes in the sensitivity to adenosine of the cell population in response to environmental cues. We, therefore, find that adenosine is consistently mitogenic for human breast carcinoma cells, and that the [(3)H]thymidine incorporation assay is a valid measure of this response. The data are consistent with the stimulatory effect of adenosine on cell proliferation being modulated by the local cellular environment.