Influence of dehydroepiandrosterone and 5-en-androstene-3 beta, 17 beta-diol on the growth of MCF-7 human breast cancer cells induced by 17 beta-estradiol.

The effects of dehydroepiandrosterone (DHEA) and 5-en-androstene-3 beta, 17 beta-diol (ADIOL) on the proliferation of MCF-7 cells were studied both in steroid - free and estradiol (E2) supplemented media. Growth was evaluated by counting the cells after six days of culture. The results show that DHEA 500 nM and ADIOL 2 nM stimulate MCF-7 cell growth in steroid-free medium, while in medium supplemented with E2 1 nM they partly antagonize the stimulatory effect of the estrogen. The latter action is also shown by lower DHEA concentrations (20 nM, 100 nM), which have no effect when added to the steroid-free medium. Incubations carried out in the presence of labeled DHEA show its conversion to ADIOL. Moreover, tamoxifen counteracts in a dose-depended manner the stimulatory effect of DHEA and ADIOL, suggesting that it is mediated by an interaction with estrogen receptors.     

Effect of breast cyst fluid on oestrogen 17-oxidoreductase activity in MCF-7 breast cancer cells

Breast cyst fluid (BCF) was found to stimulate oestrogen 17-oxidoreductase activity in the reductive direction, i.e., conversion of oestrone (E1) to oestradiol (E2), in MCF-7 breast cancer cells. Dialysis of BCF revealed that this property of BCF was present in both dialysed BCF and dialysate, implying that both high and low mol. wt. substances were responsible for stimulating E1 to E2 conversion. Gel filtration of dialysed BCF revealed that the high mol. wt. substances responsible for the stimulation of E1 to E2 conversion had mol. wts. of approximately 11 kD and 68 kD. This property of BCF would serve to increase the concentration of E2, a steroid which may play a role in mammary carcinogenesis.     

Bile acids influence the growth, oestrogen receptor and oestrogen-regulated proteins of MCF-7 human breast cancer cells.

The effects of the major human serum bile acid, glycochenodeoxycholic acid (GCDC), as well as unconjugated chenodeoxycholic acid (CDC), on the MCF-7 human breast cancer cell line have been studied in vitro under oestrogen and bile acid deprived culture conditions. GCDC increased the growth of the breast cancer cells over the range 10-300 microM. At concentrations in excess of the bile acid binding capacity of the medium cell growth was prevented. In contrast 10 microM CDC tended to reduce cell growth. Oestrogen (ER) and progesterone (PgR) receptors, pS2 and total cathepsin D were quantified by monoclonal antibody based immunoassays. Ten to 100 microM GCDC and 10 microM CDC down-regulated ER protein and this was accompanied by induction of the oestrogen-regulated proteins PgR, pS2 and possibly cathepsin D, including increased secretion of the latter two proteins into the culture medium. All these changes were quantitatively similar to those observed with 10 nM oestradiol. The bile acid effects on ER and PgR were not due to interference with the assay procedures. Cells incubated with 50 microM GCDC or 10 microM CDC had higher pmolar concentrations of the bile acids than controls. This study suggests that naturally occurring bile acids influence the growth and steroid receptor function of human breast cancer cells.     

The androgen derivative 5alpha-androstane-3beta,17beta-diol inhibits prostate cancer cell migration through activation of the estrogen receptor beta subtype

Prostate cancer growth depends, in its earlier stages, on androgens and is usually pharmacologically modulated with androgen blockade. However, androgen-ablation therapy may generate androgen-independent prostate cancer, often characterized by an increased invasiveness. We have found that the 5alpha-reduced testosterone derivative, dihydrotestosterone (the most potent natural androgen) inhibits cell migration with an androgen receptor-independent mechanism. We have shown that the dihydrotestosterone metabolite 5alpha-androstane-3beta,17beta-diol (3beta-Adiol), a steroid which does not bind androgen receptors, but efficiently binds the estrogen receptor beta (ERbeta), exerts a potent inhibition of prostate cancer cell migration through the activation of the ERbeta signaling. Very surprisingly, estradiol is not active, suggesting the existence of different pathways for ERbeta activation in prostate cancer cells. Moreover, 3beta-Adiol, through ERbeta, induces the expression of E-cadherin, a protein known to be capable of blocking metastasis formation in breast and prostate cancer cells. The inhibitory effects of 3beta-Adiol on prostate cancer cell migration is counteracted by short interfering RNA against E-cadherin. Altogether, the data showed that (a) circulating testosterone may act with estrogenic effects downstream in the catabolic process present in the prostate, and (b) that the estrogenic effect of testosterone derivatives (ERbeta-dependent) results in the inhibition of cell migration, although it is apparently different from that linked to estradiol on the same receptor and may be protective against prostate cancer invasion and metastasis. These results also shed some light on clinical observations suggesting that alterations in genes coding for 3beta-hydroxysteroid dehydrogenases (the enzymes responsible for 3beta-Adiol formation) are strongly correlated with hereditary prostate cancer.     

Integrin beta1 upregulation in MCF-7 breast cancer cells by angiotensin II.

AIMS: Integrins are a major family of cell adhesion molecules whose function is perturbed in tumour invasion and metastasis. Angiotensin II (A II) is well-known in the systemic control of water and electrolyte homeostasis and haemodynamics, but recent evidence points to an additional local renin-angiotensin system (RAS) with possible long-term trophic effects including carcinogenesis. METHODS: The effect of angiotensin II on MCF-7 human breast cancer cell line integrin expression was evaluated with immunocytochemistry (ICC) and immunoprecipitation (IP). RESULTS: The experiments demonstrated a 1.40 +/- 0.14-fold increase in beta, integrin expression on MCF-7 cells following treatment with A II. CONCLUSIONS: These findings report the first evidence of an association between integrins and the RAS in human breast cancer cells and suggest a novel research avenue for future anti-metastatic strategies, through the manipulation of cell adhesion mechanics, in the management of invasive human breast cancer.     

Caveolin-1 inhibits anchorage-independent growth, anoikis and invasiveness in MCF-7 human breast cancer cells

Caveolin-1 is an essential structural constituent of caveolae that has been implicated in mitogenic signaling and oncogenesis. Caveolin-1 is down-regulated in oncogene-transformed and tumor-derived cells. Antisense suppression of caveolin-1 or expression of a dominant negative form are sufficient for inducing cellular transformation. Expression of recombinant caveolin-1 inhibits anchorage-independent growth in cancer cells. The present study was designed to determine whether this is caused by inhibition of cancer cell survival or cell proliferation, and to test if another important property of cancer cells, i.e. matrix invasion, is modulated by expression of caveolin. Utilizing MCF-7 human breast adenocarcinoma cells stably transfected with caveolin-1 (MCF-7/Cav1), we demonstrate that caveolin-1 expression decreases MCF-7 cell proliferation rate and markedly reduces their capacity to form colonies in soft agar. The loss of anchorage-independent growth is not associated with stimulation of anoikis; in fact, MCF-7/Cav1 cells exhibit increased survival after detachment as compared with MCF-7 cells, indicating that in these cells caveolin-1 inhibits anoikis. Analysis of matrix metalloprotease release and matrix invasion revealed that expression of caveolin-1 inhibits also these important metastasis-related phenomena. Plating MCF-7 cells on a laminin matrix resulted in activation of ERK1/2, which was dramatically inhibited in MCF-7/Cav1 cells. We conclude that high expression level of caveolin-1 in human breast cancer cells exerts a negative modulatory effect on anchorage-independent growth by inhibiting cell proliferation even though matrix-independent cell survival is enhanced. Caveolin-1 expression inhibits also matrix invasion and blocks laminin-dependent activation of ERK1/2. The inhibitory effect of caveolin-1 on these transformation-dependent processes supports the hypothesis that caveolin-1 acts as a tumor suppressor protein which may impose major phenotypic changes when expressed in human cancer cells.     

紫花牡荆素抑制乳腺癌MCF-7细胞增殖与侵袭能力的研究

目的观察紫花牡荆素对乳腺癌MCF-7细胞株增殖与侵袭能力的影响并探讨其分子机制。方法应用四甲基偶氮唑蓝（MTT）比色法与侵袭实验检测紫花牡荆素对MCF-7细胞增殖与侵袭能力的影响,应用反转录PCR、Western blot法、Tunel法检测紫花牡荆素对基因表达、蛋白表达、细胞凋亡的影响。结果不同浓度紫花牡荆素均抑制MCF-7细胞增殖水平,同未加药对照组比较差异均有统计学意义（P〈0．05）,5、10、20μmol／L紫花牡荆素作用后,MCF-7细胞迁移数与未处理组相比分别降低20.3％、44．4％和50．3％（P〈0．05）。以10μmol／L紫花牡荆素处理后,凋亡细胞数增多,可以上调Bax与Caspase-3蛋白的表达水平,下调基质金属蛋白酶（MMP）-2与MMP-9的mRNA和蛋白表达水平。结论紫花牡荆素对于乳腺癌细胞恶性增殖与侵袭能力具有显著抑制作用。     

Heterologous regulation of inositol lipid hydrolysis in human breast cancer cells by oestradiol 17 beta, bombesin and fluoroaluminate.

Inositol lipid turnover has been implicated in the action of oestradiol 17 beta and bombesin-related peptides on the human breast cancer cell line MCF-7. In the present study, in addition to measuring inositol lipid turnover as indicated by inositol monophosphate (IP) accumulation, we have also monitored the effect of oestradiol on the incorporation of both 3H-inositol and 14C-glycerol into MCF-7 cell phospholipids. Pre-treatment of MCF-7 cells with oestradiol (10 nM) for 48 hr stimulated a 4.3-fold increase in IP production. This was similarly accompanied by a 3.4-fold increase in the incorporation of 3H-inositol into total phosphoinositides and a 40% increase in cell growth. The oestrogen antagonist LYI 17018 completely attenuated these effects. Oestradiol also stimulated 14C-glycerol incorporation into phosphatidyl inositol, -choline and -ethanolamine by 97%, 82% and 99%, respectively. IP production in response to bombesin was potentiated by oestradiol in a dose-dependent fashion. Fluoroaluminate (AlF4-) stimulated a dose-dependent increase in IP production and oestradiol pre-treatment increased the sensitivity of this IP response to AlF4-. Medroxyprogesterone acetate inhibited bombesin-stimulated IP production but had no effect on the response to AlF4-. Our data suggest that the oestrogenic action on basal IP production in MCF-7 cells may reflect an effect on inositol lipid synthesis rather than turnover. However, the potentiation by oestradiol of both bombesin- and AlF4(-)-stimulated inositol lipid hydrolysis suggests the operation of a post-receptor regulatory mechanism(s) which is independent of the inositol lipid pool size.     

Estrogens are not required for prolactin induced growth of MCF-7 human breast cancer cells

MCF-7 human breast cancer cells in continuous culture respond to the growth promoting activity of prolactin. Within 3 days of addition to culture medium in the presence of charcoal stripped fetal bovine serum which is depleted of bovine lactogens and estrogens, prolactin at 250 ng/ml promotes a 2- to 3-fold increase in cell number. When phenol red, which is a weak estrogen agonist, is also eliminated from the medium, the cells respond to prolactin to the same extent. When cells are grown for two generations in the presence of lactogen free, phenol red free, charcoal stripped serum, the prolactin induced response is even greater. Human and ovine prolactins are equipotent. The ability of the cells to bind lactogenic hormones remains unaltered by elimination of phenol red from the growth medium. These data indicate that prolactin alone is a mitogen for human breast cancer cells in long-term culture.     

靶向VEGF的不对称siRNA抑制乳腺癌MCF-7细胞的增殖

目的:探讨靶向血管内皮生长因子(vascular endothelial growth factor,VEGF)基因的siRNA对乳腺癌MCF-7细胞的抑制效果。方法:设计靶向VEGF的4种小干扰RNA(small interfering RNA,siRNA),包括对称siRNA(siRNA21/21,siRNA23/23)与不对称siRNA(asymmetric siRNA,aiRNA;aiRNA21/23,aiRNA19/21)。siRNA转染入MCF-7细胞后,MTT及流式细胞术检测MCF-7细胞增殖及凋亡情况,RT-PCR、ELISA法检测MCF-7细胞中VEGF基因和蛋白的表达。结果:与对照组相比,aiRNA21/23、siRNA23/23、aiRNA19/21、siRNA21/21这4种siRNA都能有效抑制VEGF mRNA的表达[(71.4±5.01)%、(40.0±3.11)%、(37.2±2.79)%、(11.1±0.99)%vs(2.4±0.11)%,P<0.01],且抑制MCF-7细胞的增殖[(44.7±5.38)%、(38.5±5.67)%、(33.6±2.18)%、(33.1±3.18)%vs(2.2±0.28)%,P<0.01],其中以不对称aiRNA21/23的抑制效果最明显。与对照组比较,aiRNA21/23显著促进MCF-7细胞的凋亡[(49.9±4.02)%vs(4.7±0.91)%,P<0.01]。结论:靶向VEGF基因的siRNA可抑制MCF-7细胞的增殖、促进细胞凋亡,尤以不对称siRNA的效果最明显。     

In vitro modulation of MCF-7 breast cancer cell growth by myoepithelial cells

Growth and differentiation of MCF-7 breast adenocarcinoma cells were studied in mixed cultures of MCF-7 cells and PA 16/23 myoepithelial cells and in isolated cultures of MCF-7 cells grown in the absence and presence of conditioned medium of PA 16/23 cells. In the cocultures, the MCF-7 cells grew in smaller aggregates and showed a more differentiated phenotype than in the isolated cultures. The conditioned medium of PA 16/23 cells enhanced growth and reduced differentiation of the MCF-7 cells. Hence, a direct relationship between MCF-7 cells and PA 16/23 cells seems to play a leading role in influencing the behaviour of the former cells, thus overriding the opposite effects of soluble factors secreted by the PA 16/23 cells.     

氧化苦参碱对人乳腺癌 MCF -7 细胞的生长抑制作用及其机制研究

目的：了解中药苦参的有效成分氧化苦参碱对人乳腺癌细胞系MCF-7肿瘤细胞的增殖及其相关的调控机制的干预作用.方法：以乳腺癌细胞株（MCF-7）为研究对象,利用MTT法检测氧化苦参碱的体外杀伤活性;RT-PCR法、Western blot法、流式细胞技术、细胞免疫荧光计数检测基因、蛋白表达水平,对其相关调控基因的表达水平进行检测.结果：氧化苦参碱对体外培养的MCF-7细胞的生长有明显的抑制作用;可以诱导乳腺癌 MCF-7细胞凋亡,而且这种生长抑制及促进凋亡的作用可能是通过抑制Wnt/β-catenin信号转导通路的活性来实现.结论：氧化苦参碱可以通过抑制Wnt/β-catenin信号转导通路的活性来抑制人乳腺癌MCF-7细胞系的增殖,促进其凋亡.     

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.     

Paradoxical regulation of Bcl-2 family proteins by 17beta-oestradiol in human breast cancer cells MCF-7.

Tumorigenesis is related to the dysregulation of cell growth or cell death pathways. Hence, elucidation of the mechanisms involved in the modulation of pro- or anti-apoptotic proteins is important in furthering understanding of breast cancer aetiology and may aid in designing prevention and treatment strategies. In the present study, we examined the role of 17beta-oestradiol on the regulation of apoptosis in the breast cancer cell line MCF-7. Using multi-probe RNAase protection assays, we found changes in the mRNA levels of several Bcl-2 family proteins upon treatment of MCF-7 cells with 17beta-oestradiol. Unexpectedly, we found a paradoxical effects of 17beta-oestradiol on two anti-apoptotic proteins Bcl-2 and Bcl-x. Treatment with 17beta-oestradiol resulted in up-regulation of Bcl-2 mRNA and protein, but down-regulated Bcl-x(L) mRNA and protein. The effect of 17beta-oestradiol on Bcl-x(L) occurred at concentration-dependent fashion. The effect was specific to 17beta-oestradiol since other steroid hormones exert no effect on Bcl-x(L). Tamoxifen, an anti-oestrogen, blocked the down-regulation of Bcl-x(L) by 17beta-oestradiol demonstrating this effect is oestrogen receptor-dependent. We speculate that different members of the Bcl-2 family proteins may be regulated through different pathway and these pathways may be modulated by 17beta-oestradiol.     

siＲNA 介导的eIF3b基因沉默抑制乳腺癌细胞MCF-7的侵袭和迁移

目的:探讨siＲNA介导的eIF3b基因沉默对乳腺癌细胞MCF-7侵袭和迁移能力的影响。方法:设计eIF3b基因的小分子干扰RNA(siRNA)片段,脂质体介导转染入乳腺癌细胞株MCF-7,转染分3个组:I组(空白对照组)、II组(转染非特异性对照组)和III组（转染eIF3b-siRNA组）。应用qＲT-PCＲ和Western blotting免疫印迹法检测转染前后eIF3b基因mＲNA和蛋白表达水平;运用Matrigel 胶模型细胞迁移和侵袭实验检测MCF-7细胞迁移和侵袭能力的变化。结果:与I组（1.09±0.19）、II组（1.05±0.17）相比,III组eIF3b 蛋白表达水平（0.45±0.06）明显下降,差异具有统计学意义(P＜0.05),I组和II组eIF3b蛋白表达水平比较,差异无统计学意义(P＞0.05)。III组eIF3b mRNA相对表达水平为0.54±0.08,明显低于I组(1.12±0.16)和II组(1.08±0.18),差异有统计学意义（P＜0.05）,I组和II组eIF3b mRNA表达比较,差异无统计学意义(P＞0.05)。细胞迁移实验中,III组细胞明显少于I组和II组(P＜0.05);细胞侵袭实验中,III组穿过人工基底膜的细胞明显少于I组和II组(P＜0.05)。结论:下调eIF3b表达可明显抑制乳腺癌细胞侵袭和迁移。