Combined effects of buserelin, estradiol and tamoxifen on the growth of MCF-7 human breast cancer cells in vitro

The growth-stimulation of MCF-7 human breast cancer cells in vitro induced by 30 pM estradiol was inhibited both by the LHRH-agonist Buserelin and the anti-estrogen Tamoxifen used as single agents. Combined administration of both drugs was less effective in this respect. In the presence of estradiol Buserelin had no effect on the pattern of [35S]methionine labelled secretory proteins when examined with one-dimensional gel electrophoresis, whereas the estrogen-induced progesterone receptor synthesis was inhibited. Thus with estradiol concentrations comparable to plasma values in medically castrated patients, the LHRH-agonist Buserelin can directly inhibit breast cancer cell growth in vitro.     

Insulin-like growth factors modulate the growth inhibitory effects of retinoic acid on MCF-7 breast cancer cells

Retinoids are currently being tested for the treatment and prevention of several human cancers, including breast cancer. However, the anti-cancer and growth inhibitory mechanisms of retinoids are not well understood. All-trans retinoic acid (RA) inhibits the growth of the estrogen receptor-positive (ER+) breast cancer cell line, MCF-7, in a reversible and dose-dependent manner. In contrast, insulin-like growth factors (IGF-I,IGF-II) and insulin are potent stimulators of the proliferation of MCF-7 and several other breast cancer cell lines. Pharmacologic doses of RA (≤10^?6M) completely inhibit IGF-I-stimulated MCF-7 cell growth. Published data suggest that the growth inhibitory action of RA on IGF-stimulated cell growth is linear and dose-dependent, similar to RA inhibition of unstimulated or estradiol-stimulated MCF-7 cell growth. Surprisingly, we have found that IGF-I or insulin-stimulated cell growth is increased to a maximum of 132% and 127%, respectively, by cotreatment with 10^?7 M RA, and that 10^?9–10^?7 M RA increase cell proliferation compared to IGF-I or insulin alone. MCF-7 cells that stably overexpress IGF-II are also resistant to the growth inhibitory effects of 10^?9–10^?7 M RA. Treatment with the IGF-I receptor blocking antibody, αIR-3, restores RA-induced growth inhibition of IGF-I-treated or IGF-II-overexpressing MCF-7 cells, indicating that the IGF-I receptor is mediating these effects. IGFs cannot reverse all RA effects since the altered cell culture morphology of RA-treated cells is similar in growth-inhibited cultures and in IGF-II expressing clones that are resistant to RA-induced growth inhibition. These results indicate that RA action on MCF-7 cells is biphasic in the presence of IGF-I or insulin with 10^?9–10^?7 M RA enhancing cell proliferation and ≥ 10^?6M RA causing growth inhibition. As IGF-I and IGF-II ligands are frequently detectable in breast tumor tissues, their potential for modulation of RA effects should be considered when evaluating retinoids for use in in vivo experimental studies and for clinical purposes. Additionally, the therapeutic use of inhibitors of IGF action in combination with RA is suggested by these studies. © 1995 Wiley-Liss Inc.     

Estrogenic effects of two derivatives of icariin on human breast cancer MCF-7 cells

The aims of the present study were to determine the estrogenic activities of icariin (ICA) and its derivatives and their structure–estrogenic activity relationship. Therefore, icaritin (ICT) and desmethylicaritin (DICT) were derived from ICA. The estrogenic activities of ICA, ICT and DICT were examined by cell proliferation and progestogen receptor mRNA expression of estrogen-receptor-positive MCF-7 cells. Current studies exhibited that ICT and DICT both markedly enhanced the proliferation of MCF-7 cells; as compared to estradiol (100%), their relative proliferative effects (RPE) were 90% and 94%, respectively. Cell proliferation induced by ICT and DICT was completely antagonized by ICI182,780. ICT and DICT increased progestogen receptor (PR) at mRNA levels at 48 h after treatment, although the effects were not as prominent as 17β-estradiol (E₂). These phenomena were not observed with ICA. Results demonstrate that ICT and DICT (nonconjugated forms) possess estrogen-like activity; however, ICA appears to have no estrogenicity in the MCF-7 cell line model in vitro.     

Anti-proliferative and apoptotic effects of oleuropein and hydroxytyrosol on human breast cancer MCF-7 cells

Olive oil intake has been shown to induce significant levels of apoptosis in various cancer cells. These anti-cancer properties are thought to be mediated by phenolic compounds present in olive. These beneficial health effects of olive have been attributed, at least in part, to the presence of oleuropein and hydroxytyrosol. In this study, oleuropein and hydroxytyrosol, major phenolic compound of olive oil, was studied for its effects on growth in MCF-7 human breast cancer cells using assays for proliferation (MTT assay), cell viability (Guava ViaCount assay), cell apoptosis, cellcycle (flow cytometry). Oleuropein or hydroxytyrosol decreased cell viability, inhibited cell proliferation, and induced cell apoptosis in MCF-7 cells. Result of MTT assay showed that 200 μg/mL of oleuropein or 50 μg/mL of hydroxytyrosol remarkably reduced cell viability of MCF-7 cells. Oleuropein or hydroxytyrosol decrease of the number of MCF-7 cells by inhibiting the rate of cell proliferation and inducing cell apoptosis. Also hydroxytyrosol and oleuropein exhibited statistically significant block of G₁ to S phase transition manifested by the increase of cell number in G₀/G₁ phase.     

Multihormonal regulation of insulin-like growth factor-I-related protein in MCF-7 human breast cancer cells

MCF-7 human breast cancer cells have been studied for hormonal regulation of secretion of an insulin growth factor-I (IGF-I)-related growth factor. 17 beta-Estradiol, which is required for tumorigenesis of the cell line in the nude mouse and which stimulates proliferation in vitro, was able to significantly induce IGF-I secretion at 10(-13) M, with maximal induction at 10(-11) M. Under optimal conditions IGF-I could be induced 4-fold after 4 days. Demonstration of estrogenic stimulations required removal of phenol red, a weak estrogen, from the cell culture medium. In addition to estrogen, insulin, epidermal growth factor, and transforming growth factor alpha induce both cellular proliferation and IGF-I secretion, while growth inhibitory antiestrogens, transforming growth factor beta, and glucocorticoids have the opposite effect. In each case, modulations in IGF-I secretion preceeded effects on cellular proliferation. IGF-I was not regulated by human GH, basic fibroblast growth factor, platelet-derived growth factor, or PRL, none of which affected proliferation rate. Thus, regulation of IGF-I secretion in human breast cancer is controlled by different hormones from those previously reported in human fibroblasts. Regulation of IGF-I by neither estrogen nor antiestrogen was associated with changes in steady-state mRNA levels; thus regulation may occur at a step beyond mRNA. We conclude that IGF-I production is tightly coupled to growth regulation by estrogens, antiestrogens, and other hormones and may contribute to autocrine and/or paracrine growth regulation by these agents in breast cancer.     

Structure-function relationships of the raloxifene-estrogen receptor-alpha complex for regulating transforming growth factor-alpha expression in breast cancer cells.

Amino acid Asp-351 in the ligand binding domain of estrogen receptor alpha (ERalpha) plays an important role in regulating the estrogen-like activity of selective estrogen receptor modulator-ERalpha complexes. 4-Hydroxytamoxifen is a full agonist at a transforming growth factor alpha target gene in situ in MDA-MB-231 human breast cancer cells stably transfected with the wild-type ERalpha. In contrast, raloxifene (Ral), which is also a selective estrogen receptor modulator, is a complete antiestrogen in this system. Because D351G ERalpha allosterically silences activation function-1 activity in the 4-hydroxytamoxifen-ERalpha complex with the complete loss of estrogen-like activity, we examined the converse interaction of amino acid 351 and the piperidine ring of the antiestrogen side chain of raloxifene to enhance estrogen-like action. MDA-MB-231 cells were either transiently or stably transfected with Asp-351 (the wild type), D351E, D351Y, or D351F ERalpha expression vectors. Profound differences in the agonist and antagonist actions of Ralcenter dotERalpha complexes were noted only in stable transfectants. The agonist activity of the Ralcenter dotERalpha complex was enhanced with D351E and D351Y ERalpha, but raloxifene lost its agonist activity with D351F ERalpha. The distance between the piperidine nitrogen of raloxifene and the negative charge of amino acid 351 was critical for estrogen-like actions. The role of the piperidine ring in neutralizing Asp-351 was addressed using compound R1h, a raloxifene derivative replacing the nitrogen on its piperidine ring with a carbon to form cyclohexane. The derivative was a potent agonist with wild type ERalpha. These results support the concept that the side chain of raloxifene shields and neutralizes the Asp-351 to produce an antiestrogenic ERalpha complex. Alteration of either the side chain or its relationship with the negative charge at amino acid 351 controls the estrogen-like action at activating function 2b of the selective estrogen receptor modulator ERalpha complex.     

In vitro selection of aptamer S1 against MCF-7 human breast cancer cells

Breast cancer is the most common female cancer. However, the known effective specific biomarkers for breast cancer are still scarce. Abnormal membrane proteins serve as ideal biomarkers for disease diagnoses, therapeutics and prognosis. Thus aptamers (single-stranded oligonucleotide molecules) with molecular recognition properties can be used as efficient tools to sort cells based on differences in cell surface architecture between normal and tumor cells. In this study, we aimed to screen specific aptamer against MCF-7 human breast cancer cells. Cell-SELEX process was performed to isolate aptamers from a combinatorial single-stranded nucleic acid library that selectively targeting surface proteins of MCF-7 cells in contrast with MCF-10A human mammary epithelial cells. The process was repeated until the pool was enriched for sequences that specifically recognizing MCF-7 cells in monitoring by flow cytometry. Subsequently, the enriched pool was cloned into bacteria, and positive clones were sequenced to obtain individual sequences. Representative sequences were chemically synthesized and evaluated their binding affinities to MCF-7 cells. As a result, an aptamer S1 was finally identified to have high binding affinity with equilibrium dissociation constant (K_d) value of 29.9 ± 6.0 nM. FAM-labeled aptamer S1 induced fluorescence shift in MCF-7 cells but not in MCF-10A human mammary epithelial cells, or MDA-MB-453 and MDA-MB-231 human breast cancer cells. Furthermore, result of cell imaging observed from laser confocal fluorescence microscope showed that MCF-7 cells exhibited stronger fluorescence signal resulted from Cy5-labeled aptamer S1 than MCF-10A cells. The above findings suggested that S1 may be a specificity and selectivity aptamer for MCF-7 cells and useful for the breast cancer detection and diagnosis.     

Melatonin increases p53 and p21WAF1 expression in MCF-7 human breast cancer cells in vitro.

The aim of the present work was to study whether melatonin, at physiological concentrations, exerts its antiproliferative effects on MCF-7 human breast cancer cells by inducing the expression of some of the proteins involved in the control of the cell cycle. MCF-7 cells were cultured for 48 h in DMEM media containing either melatonin (1 nM) or the diluent (0.001% ethanol). At this concentration, after 48 hours of incubation, melatonin reduced the number of viable cells in relation to controls. The decreased cell proliferation was coincident with a significant increase in the expression of p53 as well as p21WAF1 proteins. These results demonstrate that melatonin inhibits MCF-7 cell proliferation by inducing an arrest of cell cycle dependent on an increased expression of p21WAF1 protein, which is mediated by the p53 pathway.     

Selective fraction of Scutellaria baicalensis and its chemopreventive effects on MCF-7 human breast cancer cells

Based on our previous observation, the whole Scutellaria baicalensis extract (SbE) did not show significant breast cancer cell inhibitory effect. In this study, we isolated a baicalin-deprived-fraction (SbF1) of Scutellaria baicalensis, and baicalin-fraction (SbF3), and evaluated their anti-breast cancer properties using MCF-7 cells. The content of four flavonoids in extract/fractions were determined using high performance liquid chromatography. Analytical data showed that in SbF1, the major constituents are baicalein and wogonin, while SbF3 only contains baicalin. The antiproliferative effects of fractions and SbE were assayed using modified trichrome stain method. SbF1 showed significant antiproliferative effect. Treated with 100 μg/ml of SbF1 for 72 h inhibited MCF-7 cell growth by 81.6%, while in the same treatment concentration, SbF3 increased cell growth by 22.6%. SbF1 was recognized as an active fraction of SbE. The effects of four flavonoids in SbE, scutellarin, baicalin, baicalein and wogonin, were determined, and data showed that baicalein and wogonin significantly inhibited MCF-7 cell growth. In contrast, in certain concentrations, scutellarin and baicalin increased cancer cell growth. The effects of SbF1 on cell cycle and apoptosis were assayed using flow cytometry. SbF1 arrested MCF-7 cells in S- and G2/M-phases, and significantly increased induction of cell apoptosis. These combined phytochemical and biological data provide evidence for further chemopreventive studies of the baicalin-deprived SbE on breast cancer.     

Visfatin stimulates proliferation of MCF-7 human breast cancer cells

Obesity, a condition characterized by increased fat content and altered secretion of adipokines, is a risk factor for postmenopausal breast cancer. Visfatin has recently been established as a novel adipokine that is highly enriched in visceral fat. Here we report that visfatin regulated proliferation of MCF-7 human breast cancer cells. Exogenous administration of recombinant visfatin increased cell proliferation and DNA synthesis rate in MCF-7 cells. Furthermore, visfatin activated G1-S phase cell cycle progression by upregulation of cyclin D1 and cdk2 expression. Visfatin also increased the expression of matrix metalloproteinases 2, matrix metalloproteinases 9, and vascular endothelial growth factor genes, suggesting that it may function in metastasis and angiogenesis of breast cancer. Taken together, these findings suggest that visfatin plays an important role in breast cancer progression.     

Estrogen-stimulated glucuronidation of dihydrotestosterone in MCF-7 human breast cancer cells

The non-aromatizable androgen dihydrotestosterone (DHT) has been shown to exert a potent inhibitory effect on the proliferation of some human breast cancer cell lines. DHT, however, has little or no significant inhibition on MCF-7 cell proliferation in either the presence or absence of estradiol (E₂). Since the metabolism of DHT into non-active compounds may be responsible for the observed lack of androgenic effect in this cell line, we have investigated the metabolic fate of labeled DHT in MCF-7 cells. A time course incubation was performed with 1 nM [³H]DHT and analysis of the various metabolites formed revealed a time-dependent increase in glucuronidated steroids which was stimulated more than 4-fold by 0.1 nM E₂. The major glucuronidated steroid was androstane-3, 17β-diol in both control and E₂-stimulated cells, comprising 22 ± 1.2% and 30 ± 0.6% of the total radioactivity in the medium, respectively. Other steroid glucuronides observed included DHT, androstane-3β, 17β-diol, and androsterone, all of which were elevated in the E₂-treated cells relative to control values. The present data show that E₂ exerts a stimulatory effect on the glucuronidation of androgens and their metabolites in the estrogen-dependent breast cancer celll line MCF-7. Since glucuronidation is an effective means of cellular elimination of active steroids, such a pathway may be considered as a possible site of regulation of breast cancer cell growth by hormones.     

Androgens inhibit estrogen action in MCF-7 human breast cancer cells

We have observed that the estrogen-dependent augmentation of cytoplasmic progesterone receptor (PR_c) content in MCF-7 human breast cancer cells is completely inhibited in the presence of testosterone (T) or dihydrotestosterone (DHT)¹. This effect is neither a result of altered PR_c affinity for test ligand nor a result of the direct interaction of either androgen with PR_c. Furthermore, the antiestrogenic activity of DHT is blocked in the presence of the antiandrogen, cyproterone, indicating that it is mediated through the androgen receptor. Further investigations of this cell line may provide important insights into the effects of sex steroids upon the hormonal regulation of a variety of healthy and malignant human tissues.     

Estradiol inhibits glucocorticoid receptor expression and induces glucocorticoid resistance in MCF-7 human breast cancer cells

Our study has shown that treatment of MCF-7 human breast cancer cells with 17-beta estradiol (E(2)) produced significant decreases in glucocorticoid receptor (GR) concentrations and GR mRNA levels. E(2) pre-treatment of MCF-7 cells stably transfected with the GR responsive pMTV-CAT reporter (MCF-7-MTV cells), caused significant attenuation of dexamethasone (DEX)-induced chloramphenicol acetyl transferase (CAT). In MCF-7 cells transiently transfected with [(GRE)(3)-Luc] reporter plasmid, E(2) pre-treatment significantly suppressed DEX-induced luciferase, which was abolished by the estrogen receptor antagonist ICI 182,780. We examined the effect of chronic E(2) treatment as well as E(2) withdrawal on GR function and abundance. MCF-7-MTV cells were treated with vehicle (control) or E(2) for up to 16 days. A third group received E(2) for 5 days followed by E(2) withdrawal from day 6 to 16. Chronic E(2) treatment almost totally abrogated DEX-induced CAT and reduced GR to very low levels. Interestingly, in the group subjected to E(2) withdrawal, neither the DEX response nor GR abundance recovered and reached control values suggesting that the estrogen mediated suppression is long lasting and could not be easily reversed. The E(2) induced resistance to glucocorticoid action may be of potential clinical significance in a number of settings including breast cancer, neuroendocrine response to stress and osteoporosis and could possibly contribute to the differences in glucocorticoid responsiveness among patients.     

Binding of the new morphiceptin analogs to human MCF-7 breast cancer cells and their effect on growth

In the present study, we reported on the synthesis of two new mu-opioid peptide analogs, [D-1-Nal3]morphiceptin and [D-1-Nal4]-morphiceptin [1-Nal=3-(1-naphthyl)-alanine] which expressed receptor binding affinities at least at the level of the primary opioid ligands. The new analogs also labeled mu-opioid receptors on the cells of human breast cancer MCF-7 cell line with affinity much higher than that of endomorphins and morphiceptin, the well-known mu-selective opioid peptides. However, none of the tested peptides significantly decreased cell proliferation of MCF-7 cells.