Changes in Radiation Sensitivity and Steroid Receptor Content Induced by Hormonal Agents and Ionizing Radiation in Breast Cancer Cells in Vitro

Possible influences of tamoxifen and estradiol on in vitro radiation sensitivity and cellular receptor content after irradiation and/or tamoxifen treatment were studied in breast cancer cell lines; estrogen receptor (ER) and progesterone receptor (PgR) positive cell lines MCF-7 and MCF-7/TAM^R-1 and the ER and PgR negative cell line MDA-MB-231. The tamoxifen resistant MCF-7/TAM^R-1 cells were more resistant to ionizing radiation than the MCF-7 and MDA-MB-231 cells. Exposure to tamoxifen made the MCF-7 cells more radiation resistant, while estradiol made the MDA-MB-231 cells more radiation sensitive. A radiation dose of 6 Gy reduced the ER content in cytosol in both MCF-7 and MCF-7/TAM^R-1 cells, but brought no alterations to the PgR content. In MCF-7/TAM^R-1 cells tamoxifen exposure significantly increased the ER and reduced the PgR content, an effect not observed in the MCF-7 cells. To conclude, the present study indicates that irradiation and tamoxifen may modify the ER and PgR content in cytosol in breast cancer cells. Hormonal treatment may alter the radiation sensitivity, even in ER negative cells, suggesting that hormonal agents may act both via receptor and non-receptor binding mechanisms.     

Antiestrogenic effects of Z-1, 1-dichloro-2,3 diphenyl-2-(4-methoxyphenyl)cyclopropane(5a) on human breast cancer cells in culture.

Compound 5a ([Z]-1, 1-Dichloro-2,3 diphenyl-2-(4-methoxyphenyl)cyclopropane) is a novel cyclopropyl compound which was shown to be a pure antiestrogen. In the present study, the antiproliferative activity of 5a was examined on estrogen receptor (ER)-positive MCF-7 and ER-negative MDA-MB-231 human breast cancer cells and A-549 human lung cancer cells using the hemocytometric trypan blue exclusion method. Compound 5a inhibited the growth of MCF-7 cells in a dose-related manner over a concentration range of 10(-9) to 10(-5) M, but did not alter the growth of MDA-MB-231 or A-549 cells. Co-administration of estradiol (10(-8) M) reversed the antiproliferative activity of 5a (10(-7) M) on MCF-7 cells. Further, an ER-dependent mechanism of action is supported by the specific ER binding of 5a in MCF-7 cells observed in this study. The influence of 5a on the cell surface morphology of MCF-7 and MDA-MB-231 cells was studied using scanning electron microscopy (SEM). Compound 5a at 10(-6) M reduced the length and density of microvilli (MV) on MCF-7 cells, which was reversed by co-administration of estradiol (10(-8) M). This compound did not alter the cell surface morphology of ER-negative MDA-MB-231 cells. In conclusion, 5a and tamoxifen inhibited the growth of ER-prositive MCF-7 cells in an estradiol-reversible manner, and had no effect on ER-negative MDA-MB-231 cells. The results of this study with human breast cancer cells suggest that 5a may be highly effective in the treatment of estrogen-dependent breast cancer and/or in the prophylactic treatment of women with a high risk of breast cancer development.     

Oestrogen sulphatase activity in hormone-dependent and hormone-independent breast-cancer cells: modulation by steroidal and non-steroidal therapeutic agents.

Oestrogen sulphatase may play an important role in providing intracellular oestrogens from E1S for the growth and maintenance of breast tumours. In this study, we characterized oestrogen sulphatase in the hormone-dependent (ER/PR+) MCF-7 and in the hormone-independent (ER/PR-) MDA-MB-231 breast-cancer cells and, furthermore, examined its modulation by MPA, 4-OH-A4, tamoxifen, danazol, ethinyloestradiol and DHAS in both these cell types. Our detailed study of oestrogen sulphatase activity as a function of incubation time, E1S concentration and numbers of MCF-7 and MDA-MB-231 cells showed that more E1S was hydrolysed by MDA-MB-231 cells than by MCF-7 cells at all time points and all substrate concentrations. Additionally, although the Km values of E1S for oestrogen sulphatase in both MCF-7 and MDA-MB-231 cells were similar, the Vmax values, and therefore the activity, differed greatly. The effect of various steroidal and non-steroidal compounds also suggested differences in these 2 cell lines with respect to oestrogen sulphatase inhibition or stimulation. MPA significantly increased the hydrolysis of [3H]E1S in both cell lines, possibly through its effect on membrane fluidity. Tamoxifen increased E1S hydrolysis in MDA-MB-231 cells but not in MCF-7 cells, whereas 4-OH-A4 inhibited E1S in MCF-7 cells but not in MDA-MB-231 cells. Danazol (an isoxazol derivative of 17 alpha-ethinyltestosterone), 17 alpha-ethinyloestradiol and DHAS all significantly inhibited oestrogen sulphatase activity in both cell lines. Furthermore, danazol had a growth-inhibitory effect on both MCF-7 and MDA-MB-231 cells, although MCF-7 cells appeared to be more sensitive to growth inhibition by danazol.     

Nitrosourea and nitrosocarbamate derivatives of the antiestrogen tamoxifen as potential estrogen receptor-mediated cytotoxic agents in human breast cancer cells

Summary We have prepared two analogs of the antiestrogen tamoxifen that incorporate known DNA-crosslinking functions, a chloroethyl nitrosourea and a nitrosocarbamate moiety, and we have tested their bioactivities in cultures of human breast cancer cells. Both compounds bind to the estrogen receptor from MCF-7 cells, with relative binding affinities of 0.18% for the nitrosocarbamate derivative and 0.35% for the nitrosourea derivative, while the affinity of tamoxifen is 1.8%, and that of estradiol is set at 100%. The tamoxifen-nitrosocarbamate compound demonstrated a dose-related cytotoxicity by the colony formation and cell proliferation assays that was not blocked by estradiol in either estrogen receptor-positive MCF-7 cells or estrogen receptor-negative MDA-MB-231 cells, and thus, was not studied further. Tamoxifen-nitrosourea (TAM-NU) showed dose-related cytotoxicity in MCF-7 cells that was blocked by estradiol, whereas its activity in MDA-MB-231 cells was unaffected by estradiol. N-2-(4-t-butylphenoxy)ethyl-N-chloroethyl-N-nitrosourea (BPE-NU), a control compound which contains the nitrosourea moiety but does not bind to the estrogen receptor, had no effect on cell proliferation or colony formation in MCF-7 cells, but was very inhibitory in the receptor-negative MDA-MB-231 cells. In contrast, TAM-NU was more active in the receptor-positive MCF-7 cells than in the MDA-MB-231 line. Thus, because TAM-NU appears to be active selectively against the receptor-positive cell line, and because this activity is suppressible by estradiol, its cytotoxic effect seems to be mediated via the estrogen receptor. However, since TAM-NU is active only in prolonged treatment protocols, it appears likely that its cytotoxic activity results from the hormone antagonistic effect of the hydrolysis product of TAM-NU (bis-desmethyltamoxifen), rather than from a direct receptor-mediated, DNA-directed cytotoxic action of TAM-NU itself. This study stresses the need for the use of appropriate control compounds and cell systems in order to assess whether the toxic activity displayed by hormone-cytotoxic conjugates is mediated by receptor interactions and whether it operates through the intended toxic mechanism.     

Antiestrogens modulate MT1 melatonin receptor expression in breast and ovarian cancer cell lines

An interaction between cellular estrogen response and melatonin signaling mediated by G-protein coupled receptors is present in breast cancer cells. In this study, the effect of antiestrogens on basal and melatonin-modulated expression of MT1 melatonin receptor in breast and ovarian cancer cells was examined. For this purpose, the effects of the selective estrogen receptor modulator tamoxifen and pure antiestrogen ICI 182,780 on MT1 expression in estrogen receptor (ER) alpha-positive and -negative breast and ovarian cancer cell lines cultured in medium supplemented with 1 nM 17-beta estradiol were assessed by Western blot analysis. We were able to detect expression of the MT1 receptor in SK-OV-3 and OVCAR-3 cells and report its up-regulation by melatonin in both ovarian cancer cell lines. MT1 expression was observed to be significantly weaker in ERalpha-positive MCF-7 and OVCAR-3 cells than in ERalpha-negative MDA-MB-231 and SK-OV-3 cells. Treatment with the pure antiestrogen ICI 182,780 increased MT1 receptor expression in OVCAR-3 ovarian cancer cells, but decreased MT1 expression in MCF-7 breast cancer cells. No effect of ICI 182,780 on MT1 expression was observed in the ERalpha-negative cell lines SK-OV-3 and MDA-MB-231. After treatment with 4-OH tamoxifen, down-regulation of basal MT1 receptor expression in ERalpha-positive MCF-7 cells and inhibition of melatonin-induced up-regulation of MT1 in OVCAR-3 ovarian cancer cells were observed. In contrast, treatment with 4-OH tamoxifen increased the MT1 receptor level in ERalpha-negative SK-OV-3 ovarian cancer cells. Our findings support the existence of close interaction between estrogen and melatonin signaling. Moreover, our data suggest that melatonin signaling is modulated by antiestrogens in breast and ovarian cancer cells.     

Differential regulation of growth and invasiveness of MCF-7 breast cancer cells by antiestrogens

Estrogen increases the ability of the estrogen-dependent MCF-7 human breast cancer cell line to both proliferate and invade through an artificial basement membrane. In studying the response of MCF-7 cells to various antiestrogens, we found that 4-hydroxytamoxifen and tamoxifen inhibited cell proliferation but increased their invasiveness. In contrast, the structurally unrelated benzothiophene antiestrogens, LY117018 and LY156758, were potent antiproliferative agents which did not stimulate invasiveness. The differential effects of these antiestrogenic agents on invasion correlated with changes in production of collagenase IV, while no significant change was seen in the chemotactic activity of the cells. Invasiveness was increased by 17 beta-estradiol or 4-hydroxytamoxifen after a few hours of treatment and was rapidly lost when 17 beta-estradiol was withdrawn. Stimulation of invasiveness with 17 beta-estradiol was blocked by the antiestrogen, LY117018. Cells from the MDA-MB-231 line which lacks estrogen receptors were not affected by estrogen or antiestrogen in terms of proliferation or invasion. These studies indicate that the invasiveness of MCF-7 cells is regulated by antiestrogens through the estrogen receptor and may be mediated by collagenase IV activity. Antiestrogens which reduce both the proliferation and invasiveness of these cells may be interesting new candidates for clinical application.     

Inhibition of colon and breast carcinoma cell growth by interleukin-4.

Within human carcinomas, there is often an infiltration of lymphocytes and other cells of the immune system. A variety of cytokines are produced by such cells that could have a paracrine influence on the growth of tumor epithelium. The effect of one of these cytokines, interleukin-4 (IL-4), on human breast and colon cancer cell lines was therefore examined. IL-4 inhibited the growth of human colon (HT 29) and breast [MCF-7 wild type (MCF-7 WT), MCF-7 Adriamycin-resistant (MCF-7r), MDA-MB-231, and MDA-MB-468] carcinoma cells in culture. Competitive binding of 125I-IL-4 demonstrated the presence of 2000 high affinity IL-4-binding sites on HT 29 cells. The Kd for specific binding of 125I-IL-4 to HT 29 cells was 77 pM. Further studies were conducted on the estrogen-dependent MCF-7 WT and estrogen-independent MDA-MB-231 breast carcinoma lines. Concentrations of IL-4 of 10-100 nM were required to significantly inhibit growth of these carcinoma cell lines; e.g., with MCF-7 WT cells, half-maximal inhibition of growth occurred at 20 nM IL-4. Specific binding of 125I-IL-4 was detected to MCF-7 WT and MDA-MB-231 cells, but the low level of binding precluded Scatchard analysis. IL-4 inhibited 90% of the 17 beta-estradiol-stimulated growth of MCF-7 WT cells in a dose-dependent manner but without a change in estrogen receptor expression. Inhibition of growth by IL-4 was less in the absence of estrogens. Combined treatment with IL-4 and other known inhibitors of breast carcinoma cell growth [transforming growth factor-beta 1 (TGF-beta 1) and the antiestrogen tamoxifen] showed additive inhibition. The hormone-independent cell lines MCF-7r and MDA-MB-231 were additively inhibited by IL-4 and TGF-beta 1. This was not the case with MDA-MB-468 cells in which inhibition by IL-4 and TGF-beta 1 was of similar magnitude but no significantly greater effect was observed on combined treatment. No secretion of IL-4 was detected from these cell lines either basally or on treatment with TGF-beta 1 or tamoxifen, and we conclude that IL-4 is a nonautocrine inhibitor of breast carcinoma cell growth.     

Differential suppression of proliferation in MCF-7 and MDA-MB-231 breast cancer cells exposed to alpha-, gamma- and delta-tocotrienols is accompanied by altered expression of oxidative stress modulatory enzymes

Tocotrienols belong to the vitamin E family of chemicals known to have potent anti-proliferative and apoptotic activities against a variety of cancer cells with little to no comparable influence on the normal cells. Whether tocotrienols control the expression of phase II antioxidant enzymes in the context of their anti-carcinogenic mechanisms has not been investigated. The present studies were performed to test whether the differential growth inhibition resulting from exposure to α-, γ- and δ-tocotrienols in estrogen receptor-positive human MCF-7 and estrogen receptor-negative MDA-MB-231 breast cancer cells might be accompanied by changes in phase II antioxidant enzymes. Cell proliferation and clonogenicity in both cell lines were significantly inhibited by γ- and δ-tocotrienols with little affect when cells were similarly exposed to α-tocotrienol, at doses up to 10 μM. The expression and activity of several antioxidant enzymes in 10 μM tocotrienol-treated cells were determined by Western blot and biochemical assays. In MDA-MB-231 cells, δ- was more active than α- or γ-tocotrienols in up-regulating glutathione peroxidase; however, the three tocotrienols had comparable activity in inducing thioredoxin. In MCF-7 cells, expression of quinone reductase 2 and thioredoxin was increased by γ- and δ-tocotrienols, whereas quinone reductase 1 was unaffected by exposure to the tocotrienols. The tocotrienols also did not affect the expression and activity of superoxide dismutase in both MCF-7 and MDA-MB-231 cells, but increased catalase activity concomitant with slight reduction in the catalase expression. In MDA-MB-231 cells, treatment by tocotrienols led to several fold increase of NRF2 expression marked by corresponding decrease in KEAP1 levels. By contrast, no significant change in NRF2 and KEAP1 levels was observed in MCF-7 cells. These studies demonstrate that different tocotrienols show distinct and selective activity in regulating the NRF2-KEAP1, in coordination with the induced expression of cytoprotective oxidative stress modulatory genes and regulation of proliferation in breast cancer cells.     

Comparative studies on the polyamine metabolism and DFMO treatment of MCF-7 and MDA-MB-231 breast cancer cell lines and xenografts.

In order to characterize the estrogen receptor (ER)-positive MCF-7 and ER-negative MDA-MB-231 human breast cancer cells and xenografts, their growth kinetic parameters and some biochemical characteristics concerning the receptor status and polyamine metabolism were determined and compared. The doubling times calculated from the growth curves showed higher proliferation rate of MDA-MB-231 cells, both in culture (21 hours) and in xenograft (9.7 days), in comparison to the MCF-7 cells which had values of 32 hours and 11.6 days, respectively. Growth-dependent changes observed in the intracellular putrescine, spermidine and spermine concentrations indicated a higher activity of polyamine metabolism in the MDA-MB-231 cells and xenograft as well. However, biosynthetic key-enzyme ornithine decarboxylase activity (ODC, EC 4.1.1.17) showed neither characteristic differences between the two types of breast cancer, nor consistent relationship with their proliferation rate. Metabolic alterations of the MCF-7 and MDA-MB-231 cell lines grown in vitro were also reflected in the polyamine composition of their culture medium. Independently of their receptor status, both types of breast cancer were responsive to difluoromethylornithine (DFMO) treatment. DFMO inhibited the ODC activity totally and depleted the cellular polyamine levels. MCF-7 cells in culture were more sensitive to the antitumoral effect of DFMO than the MDA-MB-231 line, while the rate of growth inhibition did not differ significantly in the xenografts. The present results provided further evidence on the different polyamine metabolism of ER-positive MCF-7 and ER-negative MDA-MB-231 human breast cancer cells in vitro and in vivo, suggesting a correlation of hormonal modulation with polyamines as a determinant group of biological response modifiers.     

Zoledronic acid induces apoptosis and changes the TRAIL/OPG ratio in breast cancer cells

Breast cancer has a propensity to metastasize to bone, thus causing pathological fractures. Bisphosphonates are established drugs in the treatment of bone metastasis that inhibit osteoclast activity and interrupt the vicious cycle of osteoclast–tumor cell interactions. We evaluated the direct effects of zoledronic acid on estrogen receptor (ER)-negative MDA-MB-231 and ER-positive MCF-7 breast cancer cells. While zoledronic acid (100 μM) inhibited MDA-MB-231 cell proliferation after 72 h, and induced apoptosis via activation of caspase-3 and -7, it had only minor effects on MCF-7 cells. In addition, zoledronic acid induced apoptosis by up-regulating TNF-related apoptosis-inducing ligand (TRAIL) in MDA-MB-231 cells (p < 0.01), but had no effect on the expression of its decoy receptor osteoprotegerin (OPG). In MCF-7 cells, both cytokines were suppressed by zoledronic acid. In conclusion, zoledronic acid enhanced the TRAIL-to-OPG ratio in TRAIL-sensitive MDA-MB-231 cells, indicating that the TRAIL/OPG cytokine system is a bisphosphonate-responsive target in breast cancer.     

Essiac® and Flor-Essence® herbal tonics stimulate the in vitro growth of human breast cancer cells

SummaryBackground People diagnosed with cancer often self-administer complementary and alternative medicines (CAMs) to supplement their conventional treatments, improve health, or prevent recurrence. Flor-Essence^? and Essiac^? Herbal Tonics are commercially available complex mixtures of herbal extracts sold as dietary supplements and used by cancer patients based on anecdotal evidence that they can treat or prevent disease. In this study, we evaluated Flor-Essence^? and Essiac^? for their effects on the growth of human tumor cells in culture.Methods The effect of Flor-Essence^? and Essiac^? herbal tonics on cell proliferation was tested in MCF-7, MDA-MB-436, MDA-MB-231, and T47D cancer cells isolated from human breast tumors. Estrogen receptor (ER) dependent activation of a luciferase reporter construct was tested in MCF-7 cells. Specific binding to the ER was tested using an ICI 182,780 competition assay.Results Flor-Essence^? and Essiac^? herbal tonics at 1%, 2%, 4% and 8% stimulated cell proliferation relative to untreated controls in both estrogen receptor positive (MCF-7 and T47D) and estrogen receptor negative (MDA-MB-231 and MDA-MB-436) cell lines. Exposure to the tonics also produced a dose-dependent increase in ER dependent luciferase activity in MCF-7 cells. A 10⁻⁷ M concentration of ICI 182,780 inhibited the induction of ER dependent luciferase activity by Flor-Essence^? and Essiac^?, but did not affect cell proliferation.Conclusion Flor-Essence^? and Essiac^? Herbal Tonics can stimulate the growth of human breast cancer cells through ER mediated as well as ER independent mechanisms of action.     

The influence of a novel cyclopropyl antiestrogen (compound 7a) on human breast cancer cells in culture

Compound 7a ([Z]-1,1,-dichloro-2,3-diphenyl-2-(4-(2-dimethylamino)ethoxy)phenyl) cyclopropane, dihydrogen citrate salt) is a novel cyclopropyl antiestrogen which was shown to be an estrogen antagonist without estrogen agonist activity. The antiproliferative activity of 7a was examined on estrogen receptor (ER)positive MCF-7 and ER-negative MDA-MB-231 human breast cancer cells and A-549 human lung cancer cells. Compound 7a inhibited the growth of MCF-7 cells in a dose-related manner over a concentration range of 10^–9 to 10^–5M, but did not alter the growth of MDA-MB-231 or A-549 cells. The antiproliferative activity of 7a (10^–7M) on MCF-7 cells was reversed by co-administration of estradiol (10^–8M). An ER-dependent mechanism of action is also supported by the specific ER binding of 7a in MCF-7 cells observed in this study. A study of cell surface morphology using scanning electron microscopy (SEM) revealed that compound 7a at 10^–6M reduced the length and density of microvilli (MV) on MCF-7 cells, which was reversed by co-administration of estradiol (10^–8M). Compound 7a did not alter the cell surface morphology of ER-negative MDA-MB-231 cells. In conclusion, 7a inhibited the growth of ER-positive MCF-7 cells in an estradiol-reversible manner, and had no effect on ER-negative MDA-MB-231 cells or A-549 lung cancer cells. The results of this study support the antiestrogenic action of 7a previously observedin vivo and suggest that 7a may be highly effective in the treatment of estrogen-dependent breast cancer and/or in the prophylactic treatment of women with a high risk of breast cancer development.     

Associations of retinoids, tamoxifen and alpha-interferon 2a in human breast cancer

We evaluated in vitro the potentiating and/or synergistic antitumor effects among retinoids (all-trans-retinoic acid, tRA, and 13-cis-retinoic acid, 13cRA), alpha-interferon 2a (alpha-IFN 2a) and tamoxifen (TAM) on both estrogen receptor positive (ER(+)) and negative (ER(-)) human breast cancer cell lines. In our experimental model, the three studied agents showed antiproliferative activity in ER(+) cell lines MCF-7 and ZR-75.1, while alpha-IFN 2a was the most effective drug in the ER(-) cell line MDA-MB-231. Retinoids and TAM exerted a strong apoptotic effect in MCF-7 cells, while such an effect was obtained in MDA-MB-231 cells by alpha-IFN 2a. The tested combinations displayed different effects in the different evaluated cell lines: i) in MCF-7 cells tRA + TAM showed additive activity, both tRA + alpha-IFN 2a and TAM + alpha-IFN 2a association displayed a synergistic effect, and a further potentiation of the antiproliferative action was detected with the triple combination; ii) in ZR-75.1 cell line an additive activity was showed by tRA + TAM and TAM + alpha-IFN 2a, while tRA + alpha-IFN 2a produced synergistic action; iii) in MDA-MB-231 cell line only alpha-IFN 2a displayed a strong antiproliferative effect, and no significant potentiation was exerted by any drug association. The feasibility and activity of such combinations have been tested in two pilot clinical trials on patients with metastatic breast cancer: both the tested associations were tolerable, with good treatment compliance and low toxicity. The different antiproliferative and apoptotic effects observed in vitro on apparently similar breast cancer cell lines prompted us to a further investigation of the mutual biological modulations of these drug combinations, in view of a better selection of patients who might potentially benefit from these treatments.     

Antitumor mechanism of action of a cyclopropyl antiestrogen (compound 7b) on human breast cancer cells in culture

 Cyclopropyl compound 7b [(Z)-1,1-dichloro2-[4-[2-(dimethylamino)ethoxy] phenyl]-2-(4-methoxy-phenyl)-3-cyclopropane] has been shown to be a pure antiestrogen in mouse uterine tissue. Antitumor activity was examined by evaluating the influence of 7b on the proliferation, estrogen receptor (ER) affinity and cell-surface morphology of ER-positive and ER-negative human breast cancer cells in culture. The antiproliferative potency of 7b was found to be equal to tamoxifen in ER-positive MCF-7 human breast cancer cells. Further, the antiproliferative activities of 7b and tamoxifen were reversed by coadministration of estradiol. Accordingly, the antiproliferative activity of compound 7b appears to be estrogen-mediated since it did not influence the growth of either ER-negative MDA-MB-231 human breast cells or A-549 human lung cancer cells in culture. An ER-dependent mechanism of action is also supported by the specific binding affinity of 7b for ER in MCF-7 cells. Further, a study of cell surface morphology using scanning electron microscopy (SEM) revealed that 7b reduced the density and distribution of microvilli (MV) on MCF-7 cells, which was reversed by coadministration of estradiol. Compound 7b did not alter the cell surface morphology of ER-negative MDA-MB-231 cells. In conclusion, 7b inhibited the growth of ER-positive MCF-7 cells in an estradiol-reversible manner, and had no effect on either ER-negative MDA-MB-231 cells or A-549 lung cancer cells. The results of this study confirm an antiestrogenic mechanism of action for 7b as previously observed in vivo and suggest that 7b would be effective in the treatment of estrogen-dependent breast cancer or as a prophylactic treatment for women with a high risk of breast cancer development. Received: 6 January 1995/Accepted: 9 October 1995     

miRNA-34a抑制乳腺癌细胞MCF-7、MDA-MB-231的增殖与侵袭

目的:研究miRNA-34a(miR-34a)对乳腺癌细胞MCF-7、MDA-MB-231的生物调控作用。方法:采用定量PCR检测人乳腺上皮细胞MCF-10A,乳腺癌细胞株MCF-7、T47D、MDA-MB-231、MDA-MB-453、Hs578T中miR-34a的表达水平。通过miR-34a mimics分别上调MCF-7、MDA-MB-231细胞中miR-34a的表达水平,MTT和Transwell检测肿瘤细胞增殖能力、侵袭力等生物学行为的变化。结果:乳腺癌细胞MCF-7、T47D、MDA-MB-231、MDA-MB-453、Hs578T中miR-34a处于低表达水平。通过miR-34a mimics上调MCF-7、MDA-MB-231细胞中miR-34a的表达后,细胞的增殖能力被miR-34a抑制（P＜0.05）,miR-34a对细胞侵袭有显著抑制作用（P＜0.05）。结论:miR－34a在乳腺癌细胞MCF-7、T47D、MDA-MB-231、MDA-MB-453及Hs578T中低表达,miR-34a抑制乳腺癌细胞MCF-7、MDA-MB-231的细胞增殖和侵袭能力。     

Estrogen receptor and hormone responsiveness of medullary thyroid carcinoma cells in continuous culture

We have examined the estrogen responsiveness and estrogen receptor in medullary thyroid carcinoma using a model of an established human cell line, TT. TT cells bind [3H]estradiol with high affinity. Scatchard analysis reveals a single class of binding site with a concentration of 173 fmol/10(6) cells and a dissociation constant of 2.1 x 10(-9) M, values which are comparable to those of a well established model cell line for estrogen responsiveness, MCF-7 human breast cancer cell line. Estradiol in physiological concentrations moderately stimulated TT cell proliferation, whereas in pharmacological concentrations it markedly inhibited cell growth. [3H]Thymidine incorporation into acid-insoluble material was also stimulated following a 5-day treatment with 5 x 10(-9) M estradiol. Tamoxifen at a concentration of 1 microM reduced cell proliferation by 43-48% after 5-7 days of treatment. The growth suppression induced by tamoxifen was reversed by addition of 10 nM estradiol. This is the first report of estrogen growth stimulation and tamoxifen growth inhibition of a tumor cell line derived from human medullary thyroid carcinoma.     

Microtubule disruption induced by estradiol in estrogen receptor-positive and -negative human breast cancer cell lines

Effects of estrogens on the cytoplasmic microtubule networkwere examined by the indirect immunofluorescence method usinganti-ß-tubulin antibody. Estradiol, a naturally occurringestrogen, decreased the amount of cytoplasmic microtubule fibersduring interphase in the human breast cancer cell lines MCF-7and MDA-MB-231. Since MDA-MB-231 is an estrogen receptor-negativecell line, estradiol-induced microtubule disruption seems tobe independent of estradiol binding to receptors. The effectiveconcentration of estradiol required for induction of microtubuledisruption in 50% of the cells (EC₅₀) was 81 µM for MCF-7cells and 82 µM for MDA-MB-231 cells. A synthetic estrogen,diethylstilbestrol, also induced a decrease in microtubule fibers,with an EC₅₀ value of 48 µM for MCF-7 cells and 50 µMfor MDA-MB-231 cells. When estrogentreated and microtubule-depolymerizedcells were washed and the medium was replaced with fresh, intracellularmicrotubule networks reappeared within 3 h. When MCF-7 cellswere cultured for 4 days with estradiol (50 µM), cellgrowth was completely inhibited. However, estrone affected themicrotubule network and cell proliferation only slightly. Theseresults suggest that estradiol-induced microtubule disruptionis closely related to its inhibitory effect on cell growth.     

Characterization of estrogen and progesterone receptors and the dissociated regulation of growth and progesterone receptor stimulation by estrogen in MDA-MB-134 human breast cancer cells

We have examined the properties of the estrogen receptor and progesterone receptor in MDA-MB-134 human breast cells and have evaluated the effects of estrogen on cell proliferation and progesterone receptor levels in these cells as indices of hormonal sensitivity. These cells contain high levels of estrogen receptor (approximately 1.5 pmol/mg DNA) and low levels of progesterone receptor (0.15 pmol/mg DNA). More than 80% of the estrogen receptor is found in the nuclear fraction in the absence of estrogen, and the Kd of the receptor for estradiol is approximately 1.5 X 10(-10) M. Upon exposure to estradiol, the receptors become occupied, but there is no processing or apparent decrease in either nuclear or total cellular estrogen receptor content, as can be seen in MCF-7 human breast cancer cells. The nuclear estrogen receptor sediments as a 4.6 S species on high salt sucrose gradients, and it can be detected on sodium dodecyl sulfate-polyacrylamide gel immunoblot analysis as a species of molecular weight 65,000, identical to that of the MCF-7 estrogen receptor, using the monoclonal antibodies D75P3 gamma and H222Sp gamma prepared against the MCF-7 estrogen receptor. The estrogen receptor shows binding selectivity for estrogens and antiestrogens, and its affinity for ligands follows the order diethylstilbestrol (190%) greater than estradiol (100%) greater than estriol (13%) greater than tamoxifen (3%), as expected for estrogen receptor. Hence the receptor appears normal in many of its physicochemical properties and in terms of its binding affinity and specificity for estrogens and antiestrogens. Control cells contain low levels of progesterone receptor that display high affinity (Kd = 6 X 10(-9) M) for the synthetic progestin R5020, but exposure to estradiol (10(-11)-10(-7)M) fails to increase cellular progesterone receptor levels. In contrast, estradiol markedly stimulates the rate of cell proliferation, while tamoxifen suppresses the growth of control and of estradiol treated cells. Hence, our data show that these cells, which contain substantial levels of estrogen receptor, respond to estrogen with enhanced cell proliferation but fail to have their progesterone receptor level modulated by estradiol. These cells represent an interesting and unusual situation in which estrogenic regulation of proliferation and the stimulation of progesterone receptor are dissociated. These cells should prove useful in further evaluation of estrogenic regulation of cell proliferation and specific protein synthesis in human breast cancer.     

Correlation of estrogen receptors and charge alterations of regulatory subunits of cAMP-dependent protein kinases in human mammary tumor cells

The soluble cAMP-dependent protein kinase activities of two estrogen receptor-containing (MCF-7 and ZR-75-1) and two estrogen receptor-lacking (BT-20 and MDA-MB-231) established human mammary tumor cell lines were analyzed by DEAE-cellulose chromatography and photoaffinity labeling with 8-azido-[32P]cAMP. Predominantly, type I isoenzyme was present in MDA-MB-231 cells, type II protein kinase was the main form in ZR-75-1 and BT-20 cells; whereas MCF-7 cytosols contained equal amounts of both protein kinase types. No correlations between estrogen receptor content and cAMP-dependent protein kinase holoenzyme ratios of isoenzymes were found. A distinctly greater heterogeneity of charge isomers of cAMP-binding proteins (regulatory subunits) was observed in estrogen receptor-containing cells.