Estrogen and rapamycin effects on cell cycle progression in T47D breast cancer cells

The contribution of estrogen (and progesterone) in driving cell cycle progression of hormone dependent breast cancer cells is well documented, however, the roles of the various relevant signal transduction pathways remain unclear. The immunosuppressant rapamycin is a potent inhibitor of cell cycle progression and has been used to define signal transduction pathways. In this study we have determined rapamycin's effects on cell cycle progression in estrogen dependent breast cancer cells using a novel method of inducing S-phase. In this method estradiol-17- alone induced S-phase without mitogen support. In our studies the T47D cells were quite sensitive to estradiol-17-, with half-maximal induction in the picomolar range, indicating that the estrogen can induce S-phase in the absence of mitogens such as insulin. The estrogen response does not seem to be particularly specific because estriol estrone and estradiol-17--BSA were about as effective as estradiol-17-. R5020, a progestin also induced S-Phase, while rapamycin blocked steroid driven transition of cells from G₁ to S-phase.     

Inhibition of hormone and growth factor responsive and resistant human breast cancer cells by CeReS-18, a cell regulatory sialoglycopeptide

We have previously documented that CeReS-18, a cellregulatory sialoglycopeptide, inhibits the cellular proliferation of normaland transformed cell types from a diverse rangeof species. Most cell types studies exhibit asimilar sensitivity to the reversible but growth inhibitoryeffects of CeReS-18 at 7 × 10^-8 Mconcentration, while at higher concentrations CeReS-18 can elicitcytotoxicity. The present study was conducted to examinethe effect of CeReS-18 on the proliferation ofhuman mammary epithelial carcinoma cells. MCF-7 cells, whichare estrogen receptor positive (ER⁺), and BT-20 cells,which are estrogen receptor negative (ER^-), were utilized.Both cell lines show equal sensitivity to growthinhibition elicited by CeReS-18. Complete cessation of cellcycling was achieved with 7 × 10^-8 MCeReS-18, and the arrest was shown to becompletely reversible. Flow cytometric analysis, performed on CeReS-18treated cells from both cell types, revealed thatthe majority of these cells were arrested inthe G1 phase of the cell cycle. Whencells were treated simultaneously with inhibitor and stimulatoryconcentrations of mitogens such as epidermal growth factor(EGF), basic fibroblast growth factor (b-FGF), estrogen, insulin-likegrowth factors I and II (IGFI and IGFII),no alteration of the inhibitory activity of CeReS-18was observed. CeReS-18 clearly abrogated the mitogenic activitythat these growth factors elicited with human mammarycarcinoma cells.     

Epidermal growth factor receptors in human breast cancer

Summary The capacity for specific binding of¹²⁵I-epidermal growth factor (EGF) was studied in crude membrane fractions from 95 human breast carcinomas. About 42% of the samples showed saturable, high affinity, specific binding of EGF. In 21% of the tumors we were able to demostrate high (above 10 fmoles/mg protein) binding capacity. Moreover, high EGF receptor values were associated with a low content of estradiol receptor. These studies are related to the definition of new biochemical markers in human breast cancer.     

Relationship of growth stimulated by lithium,estradiol, and EGF to phospholipase C activity in MCF-7 human breast cancer cells

Summary Lithium-stimulated MCF-7 cell proliferation was compared to proliferation stimulated by other mitogens for this cell line - estradiol (E₂) and epidermal growth factor (EGF) - and lithium was found to be effective within a narrow concentration range. Mitogenic effects of lithium on proliferation stimulated by E₂ and EGF were additive below maximum, but were not synergistic. The phosphoinositide pathway is a cell signaling system involved in cell proliferation, within which phospholipase C (PLC)-mediated hydrolysis of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P₂] leads to the production of the second messengers inositol-1,4,5-trisphosphate [Ins(1,4,5)P₃] and diacylglycerol (DAG), as well as to calcium mobilization. At mitogen concentrations which maximally stimulated cell growth, estradiol stimulated both growth and PLC activity, while EGF and lithium stimulated cell growth but had little effect on the activity of the enzyme. Dose-responses with EGF revealed that a low concentration (0.1 ng/ml, 0.017 nM) of EGF appeared to stimulate both PLC activity and cell growth, but that higher concentrations of EGF which stimulated greater proliferation inhibited PLC activity. Steady-state levels of inositol phosphates including inositol trisphosphate were increased by all three mitogens. In growth assays, the phorbol ester phorbol 12-myristate-13-acetate (PMA), which mimics the actions of DAG, stimulated some cell growth, but dioctanoylglycerol, an additional DAG analog, and the calcium ionophore A23187, alone or with the DAG analogs, had no effect. These results suggest that PLC-mediated PtdIns(4,5)P₂ hydrolysis is not primarily associated with signaling proliferation by lithium or EGF in MCF-7 breast cancer cells.     

胰岛素对人乳腺癌细胞影响的体外实验研究

目的:探讨胰岛素能否促进人乳腺癌细胞增殖。方法:将胰岛素直接作用于体外培养的人乳腺癌细胞株(MDA-MB-543),运用四氮唑盐,直接细胞计数及流式细胞仪测定等方法观察胰岛素对MDA-MB-543细胞株活力,细胞总数,细胞周期等的影响。结果:胰岛素(0.5~16mu/ml,8~18小时)使MDA-MB-543细胞株活力,细胞总数增加,呈明显的量效关系,流式检测证实胰岛素(7.5mu/ml)使S期细胞增多。结论:在体外,胰岛素可诱导人乳腺癌细胞株MDA-MB-543增殖。     

Adiposity and estrogen receptor-positive,postmenopausal breast cancer risk: Quantification of the mediating effects of fasting insulin and free estradiol

Adiposity increases estrogen receptor (ER)-positive postmenopausal breast cancer risk. While mechanisms underlying this relationship are uncertain, dysregulated sex-steroid hormone production and insulin signaling are likely pathways. Our aim was to quantify mediating effects of fasting insulin and free estradiol in the adiposity and ER-positive postmenopausal breast cancer association. We used data from a case–cohort study of sex hormones and insulin signaling nested within the Melbourne Collaborative Cohort Study. Eligible women, at baseline, were not diagnosed with cancer, were postmenopausal, did not use hormone therapy and had no history of diabetes or diabetes medication use. Women with ER-negative disease or breast cancer diagnosis within the first follow-up year were excluded. We analyzed the study as a cumulative sampling case–control study with 149 cases and 1,029 controls. Missing values for insulin and free estradiol were multiply imputed with chained equations. Interventional direct (IDE) and indirect (IIE) effects were estimated using regression-based multiple-mediator approach. For women with body mass index (BMI) >30 kg/m² compared to women with BMI 18.5–25 kg/m², the risk ratio (RR) of breast cancer was 1.75 (95% confidence interval [CI] 1.05–2.91). The estimated IDE (RR) not through the mediators was 1.03 (95% CI 0.43–2.48). Percentage mediated effect through free estradiol was 72% (IIE-RR 1.56; 95% CI 1.11–2.19). There was no evidence for an indirect effect through insulin (IIE-RR 1.12; 95% CI 0.68–1.84; 28% mediated). Our results suggest that circulating free estradiol plays an important mediating role in the adiposity–breast cancer relationship but does not explain all of the association.     

Inhibition of human breast cancer cell growth by blockade of the mevalonate-protein prenylation pathway is not prevented by overexpression of cyclin D1

Overexpression of the cyclin D1 (CCND1) gene, encoding a downstream effector of mitogenic signals that plays a central role in G₁ phase progression, is often found in cancerous cells. In sporadic breast cancer (BC), this is one of the most frequent and early genetic lesions identified so far, found in more than 50% of the tumors. Inhibitors of the mevalonate/protein prenylation pathway belong to a new family of cancer therapeutic agents that act by blocking intracellular mitogenic signal transduction pathways, thereby preventing expansion of pre-cancerous foci and inhibiting growth of transformed cells. It is not known at present whether constitutively high intracellular levels of cyclin D1 might interfere with the cytostatic actions of mevalonate/protein prenylation inhibitors. This possibility was investigated here by assessing the cell cycle effects of Simvastatin, a non-toxic upstream inhibitor of the mevalonate pathway, on human BC MCF-7 cells expressing either normal or enhanced levels of cyclin D1 from of a stably transfected, tet-inducible expression vector. Results show that constitutive overexpression of this protein, such as that found in sporadic BCs, does not influence the growth inhibitory effects of Simvastatin in vitro. In addition, D1-overexpressing embryo fibroblasts were also found to be responsive to the cell cycle effects of mevalonate/protein prenylation pathway blockade, further suggesting that high intracellular levels of cyclin D1 do not prevent the cytostatic actions of compounds targeting this metabolic pathway.     

65 and 47 kDa forms of estrogen receptor in human breast cancer: relation with estrogen responsiveness

Summary In breast cancer nearly 40% of estrogen receptor (ER) positive patients do not respond to hormone therapy. As several species of ER have been described, we examined 41 breast cancers for: (1) the presence of ER and progesterone receptor (PR); (2) the molecular weight (Mr) of ER; (3) estrogen responsiveness, appreciated by the ability of a piece of tumor transplanted in nude mice to show an estrogen-induced protein synthesis (PR synthesis). We found that there are: two species of ER with different Mr (65 and 47 kDa), and three species of tumors (36% containing the highest form of ER alone, 49% bearing the two components in variable amounts, and 15% bearing only the minor species). Eleven of these 41 tumors could be assayed for PR synthesis induction, showing that estrogen responsiveness is correlated with the major component. Due to the limited number of samples (11) the data are preliminary, but they strongly suggest that the different forms of ER could exist in the living cell with different functional abilities.     

Apoptotic mechanisms in T47D and MCF-7 human breast cancer cells

To investigate the mechanisms underlying apoptosis in breast cancer cells, staurosporine was used as an apoptotic stimulus in the human breast cancer cell lines MCF-7 and T47D. Staurosporine induced dose and time dependent increases in DNA fragmentation which was abrogated by z-VAD-fmk. MCF-7 cells did not express caspase-3, suggesting that DNA fragmentation occurred in the absence of caspase-3 and that other caspases may be involved. Staurosporine induced DEVDase activity in T47D cells suggesting the involvement of caspase-3 and/or caspase-7, yet there was no DEVDase activity in MCF-7 cells, probably ruling out the involvement caspase-7. However, staurosporine induced the cleavage of pro-caspase-6 in MCF-7 cells, but not in T47D cells. Caspase dependent PARP cleavage was detected in MCF-7 cells at 3 h, whereas only partial PARP cleavage was detected in T47D cells and then only after 24 h. Moreover, staurosporine led to cytochrome c release at 2 h in MCF-7 cells and 6 h in T47D cells. In addition, a time dependent and caspase-independent reduction of the mitochondrial transmembrane potential was observed; which appeared to occur after the release of cytochrome c. Translocation of Bax from the cytosol to mitochondria was observed in both cell types, and this preceded cytochrome c release in both T47D and MCF-7 cells. Apoptotic events in both cell types differ temporally, involving activation of different caspases and mitochondrial changes.     

Autocrine and paracrine growth regulation of breast cancer: Clinical implications

Summary Research using experimental models of human breast cancer has broadened our understanding of the possible biochemical pathways regulating breast cancer growth. Breast cancer cells express receptors for and respond to a variety of steroid and polypeptide hormones and growth factors. Specific oncogenes are also expressed in breast cancer cells, and levels of expression may relate to tumor growth and aggressiveness. Recent studies have shown that breast cancer cells can even synthesize and secrete various growth factors that could stimulate tumor growth through autocrine and/or paracrine mechanisms. Secretion of some of these growth factors is regulated by estrogen, providing a possible mechanism for estrogen induced growth. Knowledge of these growth regulatory pathways has potentially important clinical implications. Blockade of these pathways offers new possible treatment strategies, much as antiestrogens have been used to inhibit tumor growth. Quantification of the expression of certain oncogenes, growth factor receptors, or the growth factors themselves, may provide prognostic information for the individual patient. Finally, it is plausible that measurement of these tumor products in body fluids might provide tumor markers that are useful in the diagnosis and treatment of breast cancer.     

线粒体融合蛋白-2基因增强人乳腺癌T47D细胞对小白菊内酯的敏感性

目的:探讨线粒体融合蛋白-2(mitofusin-2,Mfn-2)基因表达对人乳腺癌T47D细胞对小白菊内酯敏感性的影响。方法:Real-time PCR检测不同乳腺癌细胞系(T47D、MDA-MB-231、MCF-7、MDA-MB-435及HCC38)中Mfn-2 mRNA的表达。LipofectamineTM2000体外转染pEGFP和pEGFP-Mfn-2质粒至人乳腺癌T47D细胞,real-time PCR和Western blotting检测T47D细胞中Mfn-2 mRNA和蛋白的表达,MTT法检测T47D细胞的增殖,流式细胞术检测T47D细胞凋亡率及线粒体膜电位。结果:与正常乳腺细胞相比,Mfn-2 mRNA在乳腺癌HCC38细胞系中高表达,在T47D等其他细胞系中均低表达。pEGFP-Mfn-2转染48 h后,T47D细胞中Mfn-2 mRNA和蛋白的表达均明显上调。与pEGFP转染组相比,pEGFP-Mfn-2转染组T47D细胞在小白菊内酯(0.05 mol/L)作用下的存活率明显降低[(47.93±2.21)%vs(56.93±2.05)%,P<0.05]。流式细胞术检测结果显示:50 mmol/L小白菊内酯作用下,pEGFP-Mfn-2转染组与pEGFP转染组相比,T47D细胞凋亡率升高[(71.2±2.1)%vs(38.8±2.6)%,P<0.05],而线粒体膜电位明显降低[(1.6±0.1)%vs(5.0±0.5)%,P<0.05]。结论:pEGFP-Mfn-2转染可增强T47D细胞对小白菊内酯的敏感性。     

三苯氧胺与来曲唑治疗ER阳性乳腺癌的临床疗效及对血清雌二醇水平和远期预后的影响

目的探讨三苯氧胺与来曲唑治疗雌激素受体(ER)阳性乳腺癌的临床疗效及对血清雌二醇(E2)水平和远期预后的影响。方法采用简单随机抽样法将110例ER阳性乳腺癌患者分为对照组(n=53)和研究组(n=57)。两组患者均接受长春瑞滨联合顺铂治疗,对照组患者在此基础上接受三苯氧胺治疗,研究组患者在此基础上接受来曲唑治疗,随访3年。比较两组患者的临床疗效、血清肿瘤标志物和E2水平、子宫内膜情况、不良反应发生率及生存情况。结果研究组患者的疾病控制率为92.98%,高于对照组的79.25%,差异有统计学意义(P<0.05)。治疗前,两组患者的血清糖类抗原153(CA153)、癌胚抗原(CEA)及E2水平比较,差异均无统计学意义(P>0.05)。治疗后3个月,两组患者的血清CEA和CA153水平均较本组治疗前降低,且研究组患者的血清CEA和CA153水平均低于对照组,差异均有统计学意义(P<0.05)。治疗后3、6、12个月,研究组患者的血清E2水平均低于治疗前,对照组患者的血清E2水平均高于治疗前,差异均有统计学意义(P<0.05)。病理结果提示,治疗后对照组中子宫内膜息肉2例,内膜不典型增生1例。两组患者恶心呕吐、便秘、肝肾功能不全、血小板减少、粒细胞减少及白细胞降低的发生率比较,差异均无统计学意义(P> 0.05)。研究组患者的3年生存情况优于对照组(P<0.05)。结论来曲唑治疗ER阳性乳腺癌的临床疗效优于三苯氧胺,且可降低血清肿瘤标志物和E2水平,改善远期预后。     

RUNX3基因真核表达载体的构建及其在乳腺癌T47D细胞中的表达

目的:构建真核表达载体pcDNA3.1(+)-RUNX3,并在乳腺癌T47D细胞株中表达。方法:应用基因重组技术和限制性内切酶EcoRI和XhoI酶切,构建并鉴定pcDNA3.1(+)-RUNX3真核表达载体,经脂质体Lipofectamine2000介导质粒转染T47D细胞后应用逆转录-聚合酶链反应(RT-PCR)和Western blot实验,检测RUNX3在T47D细胞中的表达。结果:重组真核表达载体pcDNA3.1(+)-RUNX3经限制性内切酶EcoRI和XhoI酶切,电泳后显示1.3kb的RUNX3目的片段和5.4kb的pcDNA3.1(+)载体片段。测序证实酶切片段与gene bank中登记的RUNX3序列相同,证实pcDNA3.1(+)-RUNX3真核表达载体构建成功。经RT-PCR和Western blot检测,表明转染pcDNA3.1(+)-RUNX3的T47D细胞RUNX3阳性表达。结论:重组真核表达载体构建正确,并建立稳定表达RUNX3的T47D细胞系,从而为后续研究提供有用的细胞研究模型。     

Secreted growth factors from estrogen receptor-negative human breast cancer do not support growth of estrogen receptor-positive breast cancer in the nude mouse model

Estrogen receptor (ER)-negative MDA-231 human breast cancer cells have been shown to secrete high concentrations of several growth factors including transforming growth factor-alpha and insulin-like growth factor I, which could have important autocrine or paracrine growth regulatory functions and, additionally, could explain the rapid autonomous growth of these cells. In contrast, the hormone-responsive, ER-positive MCF-7 cells secrete low levels of these factors constitutively. Since estrogen treatment increases secretion of these growth factors in MCF-7 cells, it has been postulated that these growth factors mediate estrogen's growth effects through an autocrine mechanism. To test this hypothesis we reasoned that growth factors supplied by MDA-231 cells should support growth of MCF-7 cells in an estrogen-depleted environment. Inoculation of castrated female athymic nude mice with MDA-231 cells resulted in rapid tumor growth. However, MDA-231 tumors did not support growth of MCF-7 cells inoculated on the opposite flank by an endocrine mechanism; MCF-7 tumors required estrogen supplementation for growth. To determine if MDA-231 cells could support MCF-7 growth by a paracrine mechanism, various mixtures of the two cell lines were coinoculated at the same site in castrated or in estrogen-supplemented mice. ER was not detectable in tumors derived from a mixed inoculum, indicating the absence of MCF-7 cell growth. Furthermore, DNA flow cytometry of these tumors revealed only a single G₁ peak representative of MDA-231 cells in estrogen-deprived mice. On the other hand, two distinct G₁ peaks representing both MDA-231 and MCF-7 cells were detected in tumors grown in estrogen-supplemented mice. These data demonstrate that growth factors from estrogen-independent MDA-231 cells are not capable of replacing estrogen for growth stimulation of MCF-7 cells. Either estrogen-stimulated growth of MCF-7 cells requires other secreted factors not supplied by MDA-231 cells, or it involves a different mechanism.     

Estrogen receptors,progesterone receptors,and cell proliferation in human breast cancer

Summary The breast is a target organ for estrogens and progesterone. These hormones control several functions of the normal and abnormal mammary epithelium including cell proliferation. Most of the actions of estrogens and progesterone are mediated via specific steroid receptors, and one would expect that proliferating cells should contain estrogen receptors (ER) and/or progesterone receptors (PR). However, the correlation between receptor expression and cell proliferation is still controversial. In the present study we have examined 29 human breast cancer samples; in 17 of them we evaluated the simultaneous ER and PR localization with that of proliferating cell nuclear antigen (PCNA) and silver-stained nucleolar organizer regions (AgNORs) in a cell-by-cell study. We found that in almost 50% of the tumor biopsies examined, the cells expressing ER were significantly associated with elevated cell proliferation. In another group (38%) there were not significant differences between ER expression and cell proliferation. In only one of the samples (6%) the cells expressing ER showed lower cell proliferation. The study also revealed that in 44% of the tumors the PR expressing cells were associated with elevated cell proliferation. In a second group the PR expression was not significantly associated with cell proliferation (33% of the cases). Finally, in 22% of the samples the cells carrying PR showed lower cell proliferation. We also detected lower ER immunoreactivity in 30% of the breast cancer biopsies with one of the monoclonal antibodies against ER (antibody 1D5 directed against the A/B domain). This group of tumors was PR-negative (or very weakly positive) and had high proliferation. The presence of tumors with abnormal ER proteins and displaying ER/PR significantly associated with elevated cell proliferation could have implications in human breast cancer treatment.     

Leptin and insulin growth factor I in relation to breast cancer (Greece)

Objectives: Because both breast cancer and the hormone leptin are associated with obesity and reproductive phenomena in women, we have examined whether there is a relationship between leptin and breast cancer among premenopausal and postmenopausal women. We have also evaluated in this dataset the association of IGF-I with breast cancer. Methods: Seventy-five cases, diagnosed during mammographic screening, with incident breast cancer were matched for age and type of permanent residence with seventy-five controls from those screened negative in the same study base. Results: There was no evidence for an association between IGF-I and either premenopausal or postmenopausal breast cancer risk or between leptin and postmenopausal breast cancer. Among premenopausal women, however, there was a strong and statistically significant inverse association of leptin with breast cancer. Conclusion: We did not confirm the positive association, reported from other investigations, of IGF-I with premenopausal breast cancer risk. We have found evidence, however, that leptin may be inversely related to breast cancer risk among premenopausal women. The latter finding is not biologically implausible and deserves to be examined in additional datasets.     

人表皮生长因子受体-2及雌激素受体与乳腺癌的关系

乳腺癌与人表皮生长因子受体-2及雌激素受体关系密切,这两种受体也是乳腺癌的分类标准和治疗靶点。在大多数乳腺癌患者中,人表皮生长因子受体-2信号途径和雌激素受体信号途径参与了细胞的增生存活过程。而且在乳腺癌病例中,人表皮生长因子受体-2和雌激素受体呈现出一定程度的负相关。这说明这两种受体活化后有一些联系。本文简要综述了人表皮生长因子受体-2和雌激素受体的联系以及这种联系在乳腺癌治疗中的意义。     

Control of breast cancer cell growth by steroids and growth factors: Interactions and mechanisms

Summary Over the past two decades, the simple model for control of breast cancer growth involving one or two factors acting directly or indirectly via endocrine pathways has turned into a complex model implicating numerous interacting factors and the diverse cell populations constituting breast tumors. Current approaches to breast cancer therapy now require integration of these multiple parameters and enhanced understanding of the different levels of their intricate interactions.     

Expression of insulin-like growth factor binding proteins by T-47D human breast cancer cells: regulation by progestins and antiestrogens

Summary We have used ligand blotting and Northern blotting techniques to examine the effects of progestins and antiestrogens on expression of insulin-like growth factor binding proteins (IGFBPs) by T-47D human breast cancer cells under conditions where these agents are growth inhibitory. Under basal conditions, conditioned medium from T-47D cells was found to contain IGFBPs of 39, 33, and 27 kDa. Northern blot and/or Western blot analysis have identified these as IGFBP 2, 5, and 4, respectively. Medroxyprogesterone acetate (MPA) treatment resulted in a time- and dose-dependent decrease in IGFBP 4 and 5 mRNA abundance and secretion of these proteins, while little if any effect was observed on IGFBP 2 expression. A decrease in the steady state mRNA levels for IGFBP 4 and 5 was observed with as little as 0.1 nM MPA. Using 10 nM MPA a maximum decrease in IGFBP 4 and 5 mRNA levels was observed between 12 and 24 hours. While RU 486 alone had little or no effect on IGFBP 4 expression, it inhibited the effect of MPA. However, in the same samples, IGFBP 5 expression was inhibited by RU 486, and RU 486 was unable to reverse the effects of progestins on the expression of IGFBP 5. Furthermore, another synthetic progestin, Org 2058, but not dexamethasone, inhibited IGFBP 4 and IGFBP 5 expression. The antiestrogen ICI 164384 also transiently decreased the steady state mRNA levels of both IGFBP 4 and IGFBP 5. Regulation of expression of the IGFBPs by these agents suggests a potential role for the IGFBPs in the growth response of T-47D cells to these agents.