Caveolin-白对乳腺癌耐药细胞株Hs578T／Dox生长凋亡的影响

背景与目的：作为信号转导枢纽的Caveolae与恶性肿瘤发生和耐药密切相关．Caveolin-1为其标志蛋白。本研究探讨Caveolin-1对肿瘤耐药细胞体外生长凋亡的影响及其作用机制。方法：选用乳腺癌Hs578T阿霉素耐药细胞株Hs578T／Dox．通过基因转染技术培育Caveolin-1蛋白过表达细胞株Hs578T／Dox—cav-1作为实验组．空载体转染细胞株Hs578T／Dox-vector作为对照组,Westernblot检测细胞Caveolin-1蛋白表达。研究细胞生长凋亡变化：MTF检测细胞生长曲线,流式细胞技术细胞周期分析,检测软琼脂集落形成能力。细胞培养48h,流式细胞技术检测细胞自然凋亡指数;加入凋亡诱导剂-星形孢菌素细胞培养8h,检测星形孢菌素诱导细胞凋亡指数。结果：Hs578T／Dox-cav-1较Hs578T／Dox—vector的Caveolin-1蛋白稳定过表达。Hs578T／Dox—cav-1与Hs578T／Dox-vector细胞生长曲线比较,生长明显增快（P〈0．01）,软琼脂培养集落形成体积增大、数目增加[（983．6±75．0）vs（700．8±78．9）,P〈0．01]。细胞周期分析．Hs578T／Dox—cav-1细胞S期和G2／M期细胞比率增大,增殖指数升高[（76．6±4．0）％V8．（58．0±4．1）％]。细胞自然凋亡指数[（5．7±0．5）％VS．（11．3±0．8）％]和星形孢菌素诱导凋亡指数[（13．8±1．2）％VS．（21．4±1．89）％]下降。结论：高表达Caveolin-1蛋白使乳腺癌阿霉素耐药细胞Hs578T／Dox具有更强的生长能力和抗凋亡能力。     

Overexpression of caveolin-1 induces alteration of multidrug resistance in Hs578T breast adenocarcinoma cells

Caveolin-1 is a major caveolae-coat protein involved in a variety of cell signaling processes. Some studies have suggested that the level of caveolin-1 expression positively correlates with multi-drug resistance in cancer cells. We demonstrated for the first time that Hs578T doxorubicin resistant cells (Hs578T/Doxo), which contain low levels of endogenous caveolin-1 and high levels of P-glycoprotein, are rendered drug-sensitive by overexpression of exogenous caveolin-1. MTT assays showed that after overexpressing caveolin-1, the drug resistance of Hs578T/Doxo cells to doxorubicin and cisplatin was reduced from 25.4 +/- 1.5 and 65.3 +/- 2.5 microg/ml to 0.8 +/- 0.15 and 23.2 +/- 2.1 microg/ml, respectively (i.e. reduced by 97% and 64%, respectively). Furthermore, using rhodamine-123 efflux assays, we observed a significant decrease in P-glycoprotein activity in caveolin-1 overexpressing cells, similar to that observed with 5 microM cyclosporine A or 10 microM verapamil, 2 inhibitors of P-glycoprotein activity. Using confocal microscopy, subcellular fractionation and co-immunoprecipitation assays, a possible physical interaction between caveolin-1 and P-glycoprotein in the caveolae membrane was observed in Hs578T/Doxo cells overexpressing caveolin-1. These results suggest that overexpression of caveolin-1 changes the state of the cells from drug-resistant to drug-sensitive by inhibiting P-glycoprotein transport activity.     

Suppression of staurosporine-mediated apoptosis in Hs578T breast cells through inhibition of neutral-sphingomyelinase by caveolin-1

Caveolin-1, a 21-24kDa integral membrane protein, is a principal structural component of caveolae in vivo. To investigate the roles of caveolin-1, we established stable transfectants in Hs578T breast adenocarcinoma cells that had up- and down-regulated caveolin-1 expression. In the paper, we demonstrated that caveolin-1 overexpression in Hs578T cells significantly reduced staurosporine-induced apoptosis and the levels of caveolin-1 expression positively correlated with the number of colonies and colony size in soft agar. Our findings indicate for the first time in Hs578T cells that caveolin-1 might play a pivotal role in regulating apoptosis as a suppressor rather than a facilitator through inhibition of neutral-sphingomyelinase, decrease of ceramide, furthermore, activation of Akt signaling pathway.     

Caveolin-1对乳腺癌细胞Hs578T耐药株生长、增殖的影响

目的：探讨caveolin-1基因对乳腺癌细胞系Hs578T耐药株（Hs578T／Dox）生长、增殖的影响。方法：在乳腺癌细胞系Hs578T耐药株中转染caveolin-1基因，构建高表达caveolin-1蛋白的细胞系（Hs578T／Dox—cav），Western Blot方法证实转染成功。MTT法绘制转染前后细胞生长曲线，比较生长速度的差别；将两种细胞接种于软琼脂中，比较转染前后集落形成的区别，接种于裸鼠体内，比较成瘤情况。结果：转染caveolin-1后Hs578T耐药株的生长速度明显加快（P〈0．01），集落形成明显增多（P〈0．01），裸鼠成瘤率明显增加（P〈0．01）。x结论：caveolin-1可促进乳腺癌细胞系Hs578T耐药株的生长和增殖。     

Differential expression of CYP1A1 and CYP1B1 in human breast epithelial cells and breast tumor cells

Human cytochromes P450 1A1 (CYP1A1) and P450 1B1 (CYP1B1) catalyze the metabolic activation of a number of procarcinogens and the hydroxylation of 17beta-estradiol (E2) at the C-2 and C-4 positions, respectively. The aromatic hydrocarbon receptor (AhR) agonist 2,3,7,8- tetrachlorodibenzo-p-dioxin (TCDD) has a marked effect on estrogen metabolism in MCF-7 breast-tumor cells by induction of these two enzymes. To investigate whether induction of CYP1A1 and CYP1B1 by AhR agonists and the associated increase in E2 metabolism are common to all breast epithelial cells and breast-tumor cells, we determined the effects of TCDD on E2 metabolism, and CYP1A1 and CYP1B1 mRNA levels in a series of non-tumor-derived breast epithelial (184A1 and MCF-10A) and breast-tumor (MCF-7, T-47D, ZR-75-1, BT-20, MDA-MB-157, MDA-MB-231 and MDA-MB-436) cell lines. In 184A1 cells, which did not express detectable estrogen receptor (ER) alpha mRNA, CYP1A1 mRNA and activity were induced by TCDD, and enhanced E2 metabolism in TCDD-treated cells was predominantly E2 2-hydroxylation. In MCF-10A, MCF-7, T-47D, ZR-75-1 and BT-20 cells, which expressed varying levels of ER alpha mRNA, both CYP1A1 and CYP1B1 mRNA levels and rates of both E2 2- and 4- hydroxylation were highly elevated following exposure to TCDD. In MDA- MB-157, MDA-MB-231 and MDA-MB-436 cells, which did not express detectable ER alpha mRNA and generally displayed fibroblastic or mesenchymal rather than epithelial morphology, CYP1B1 induction was favored, and the rate of E2 4-hydroxylation exceeded that of 2- hydroxylation in TCDD-treated cells. These results show that breast epithelial cells and tumor cells vary widely with regard to AhR- mediated CYP1A1 and CYP1B1 induction, suggesting that factors in addition to the AhR regulate CYP1A1 and CYP1B1 gene expression. In these cell lines, significant CYP1A1 inducibility was restricted to cultures displaying epithelial morphology, whereas CYP1B1 inducibility was observed in cells of both epithelial and mesenchymal morphology.      

Modulation by oestrogen and progestins/antiprogestins of alpha interferon receptor expression in human breast cancer cells

Human breast cancer ZR-75-1 cells expressed 1516 (105) (mean [S.D.]) interferon (IFN) receptors (IFNR) per cell with K_d of 0.61 (0.15) nmol/l. Oestrogen independent ZR-PR-LT and tamoxifen resistant ZR-75-9a1 8 μmol/l cells expressed similar numbers of IFNR. ZR-75-9a1 cells, which had been maintained in the absence of tamoxifen or known oestrogenic activity for 46 weeks, expressed a significantly higher number of IFNR (3170 [315]). Exposure of ZR-75-1 cells to 10⁻⁹ mol/l 17β-oestradiol (E2) led to a consistent reduction in IFNR numbers whilst 10⁻⁶ mol/l tamoxifen slightly increased IFNR expression. Since IFN increases oestrogen receptors in this cell line, IFN and E2 appear to have opposite effects on expression of each others' receptor. 10⁻⁹ mol/l medroxy progesterone acetate and mifepristone significantly increased IFNR numbers whilst ORG 2058 decreased IFNR expression and ZK 98.299 had no effect. Progestin/antiprogestin induced IFNR increase in this cell line correlated with down-regulation of progesterone receptor (PR). Thus an IFN/ER/PR axis may exist in ZR-75-1 cells and variants.     

Association of genetic polymorphisms in CYP19 and CYP1A1 with the oestrogen receptor-positive breast cancer risk

Since tamoxifen has been shown to reduce the risk of oestrogen receptor (ER)-positive, but not ER-negative, breast cancers in a chemoprevention trial (P-1), it is important to develop assays to assess risk factors for ER-positive breast cancer in order to appropriately select candidates for chemoprevention with tamoxifen. Thus, the significance of genetic polymorphisms of genes involved in oestrogen biosynthesis (CYP19) and metabolism (CYP1A1) as a risk factor for ER-positive breast cancers was evaluated. A case-control study was conducted with 257 breast cancer patients and 191 healthy female controls. Two polymorphisms, CYP19 (TTTA repeats) in intron 4 and CYP1A1 6235C/T in the 3' non-coding region, and their association with the breast cancer risk after adjustment for the other epidemiological risk factors were examined. CYP19 (TTTA)7(-3bp) allele carriers showed a significantly (P<0.05) increased risk of ER-positive breast cancers (Odds Ratio (OR)=1.72, 95% Confidence Interval (CI) 1.10-2.69), but not ER-negative breast cancers. CYP1A1 6235C allele carriers showed a non-significant (P=0.06) trend towards a decreased risk of ER-positive breast cancers (OR=0.65, 95% CI 0.42-1.02), but not ER-negative breast cancers. The combination of these two polymorphisms was found to be more useful in the assessment of the ER-positive breast cancer risk (OR=3.00, 95% CI=1.56-5.74) than the CYP19 (TTTA)7(-3bp) polymorphism alone. The combination of CYP19 (TTTA)7(-3bp) and CYP1A1 6235C/T polymorphisms is associated with an ER-positive, but not ER-negative, breast cancer risk, and, thus, would be useful in the selection of candidates for chemoprevention with tamoxifen.     

Molecular classification of selective oestrogen receptor modulators on the basis of gene expression profiles of breast cancer cells expressing oestrogen receptor alpha

The purpose of this study was to classify selective oestrogen receptor modulators based on gene expression profiles produced in breast cancer cells expressing either wtERalpha or mutant(351)ERalpha. In total, 54 microarray experiments were carried out by using a commercially available Atlas cDNA Expression Arrays (Clontech), containing 588 cancer-related genes. Nine sets of data were generated for each cell line following 24 h of treatment: expression data were obtained for cells treated with vehicle EtOH (Control); with 10(-9) or 10(-8) M oestradiol; with 10(-6) M 4-hydroxytamoxifen; with 10(-6) M raloxifene; with 10(-6) M idoxifene, with 10(-6) M EM 652, with 10(-6) M GW 7604; with 5 x 10(-5) M resveratrol and with 10(-6) M ICI 182,780. We developed a new algorithm 'Expression Signatures' to classify compounds on the basis of differential gene expression profiles. We created dendrograms for each cell line, in which branches represent relationships between compounds. Additionally, clustering analysis was performed using different subsets of genes to assess the robustness of the analysis. In general, only small differences between gene expression profiles treated with compounds were observed with correlation coefficients ranged from 0.83 to 0.98. This observation may be explained by the use of the same cell context for treatments with compounds that essentially belong to the same class of drugs with oestrogen receptors related mechanisms. The most surprising observation was that ICI 182,780 clustered together with oestrodiol and raloxifene for cells expressing wtERalpha and clustered together with EM 652 for cells expressing mutant(351)ERalpha. These data provide a rationale for a more precise and elaborate study in which custom made oligonucleotide arrays can be used with comprehensive sets of genes known to have consensus and putative oestrogen response elements in their promoter regions.     

Nongenomic oestrogen signalling in oestrogen receptor negative breast cancer cells: a role for the angiotensin II receptor AT1

Introduction  

Oestrogens can mediate some of their cell survival properties through a nongenomic mechanism that involves the mitogen-activated protein kinase (MAPK) pathway. The mechanism of this rapid signalling and its dependence on a membrane bound oestrogen receptor (ER), however, remains controversial. The role of G-protein-coupled receptor and epidermal growth factor (EGF) receptor in an ER-independent signalling pathway modulated by oestrogen was investigated.     

Steroid hormone receptor gene expression in human breast cancer cells: inverse relationship between oestrogen and glucocorticoid receptor messenger RNA levels

The relative expression in human breast cancer cells of messenger ribonucleic acids (mRNA) encoding different steroid hormone receptors is unknown. Accordingly, mRNA levels in total RNA extracted from 13 human breast cancer cell lines were measured by Northern analysis employing complementary DNA probes for the human oestrogen (ER), progesterone (PR), androgen (AR), vitamin D3 (VDR) and glucocorticoid receptors (GR). The 7 ER+ lines expressed a single 6.4 kilobases (kb) ER mRNA. Interestingly, low concentrations of ER mRNA were detected in the ER- cell lines, MDA-MB-330 and BT 20. PR mRNA, predominantly a 13.5 kb species, was expressed in the 6 lines known to be ER+, PR+ by radioligand binding; however, one ER+ cell line, MDA-MB-134, failed to express PR mRNA. A 10.5 kb AR mRNA was expressed at significantly higher levels in ER+ than ER- cell lines. All cell lines expressed a single 4.6 kb mRNA for VDR and a single 7.4 kb mRNA for GR. ER and PR mRNA levels were positively correlated (p = 0.011) and each was positively correlated with androgen receptor (AR) mRNA levels (p less than or equal to 0.009). ER, PR and AR mRNAs were negatively associated with GR levels (p less than or equal to 0.012), while ER and AR mRNA levels were negatively correlated with mRNA for the epidermal growth factor receptor. In contrast, levels of VDR mRNA were unrelated to the concentration of any other steroid receptor mRNA. Our data demonstrate the coordinate expression of ER, PR and AR genes, and an inverse relationship between sex steroid hormone receptor and GR gene expression in human breast cancer cell lines.     

Interactions between MAP kinase and oestrogen receptor in human breast cancer

PurposeThe oestrogen receptor (ERα) may be activated in a ligand-dependent manner, via oestrogen, or in a ligand-independent manner, via signal transduction pathways. Mitogen Activated Protein Kinase (MAPK) is known to directly phosphorylate ERα at serine 118 in a ligand-independent manner. This study investigated the interaction between MAPK and ERα in breast cancer.Materials & methodsImmunohistochemical experiments were undertaken to determine the expression of MAPK, pMAPK and pER(ser118) in breast tumours to determine their clinical relevance. Immunofluorescent experiments were performed, on MCF-7 breast cancer cells, to monitor the phosphorylation and localisation of MAPK and ERα in response to oestrogen, heregulin and a MAPK inhibitor.ResultsOestrogen and Heregulin stimulated phosphorylation of ERα and its nuclear translocation, but heregulin induced this at levels much lower than those observed with oestrogen. Following stimulation with heregulin, but not oestrogen, treatment with MAPK inhibitor reduced the levels of nuclear pER(ser118). In cells treated with both oestrogen and heregulin, nuclear pER(ser118) was visible; but at levels comparable with heregulin treatment alone.ConclusionThis study confirms that ligand-mediated phosphorylation is associated with rapid nuclear localisation of ERα, due to oestrogen binding. ERα is phosphorylated at serine 118 in a ligand-independent manner. Preventing nuclear translocation of pMAPK reduced the levels of ligand-independent, but not ligand-dependent phosphorylation of ERα. Co-stimulation with both oestrogen and heregulin suggested that heregulin mediated signalling determines the subcellular localisation of ERα. Activation of ERα by direct phosphorylation may result in its rapid deactivation due to degradation or nuclear export.     

Expression of CYP1A1 and CYP1B1 depends on cell-specific factors in human breast cancer cell lines: role of estrogen receptor status.

The impact of estrogen receptor (ER) was examined for expression and activity of cytochrome P4501B1 (CYP1B1) and cytochrome P4501A1 (CYP1A1) in two pairs of ER+/ER- human breast epithelial cell lines derived from single lineages, and representing earlier (T47D) or later (MDA-MB-231) stages of tumorigenesis. Acute loss of ER was evaluated using the anti-estrogen ICI 182,780 (ICI). In all lines, CYP1B1 was expressed constitutively and was induced by 2,3,7, 8-tetrachlorodibenzo-p-dioxin (TCDD), whereas CYP1A1 was expressed only following induction. Expression of each CYP (with or without TCDD) was greater in T47D cells than MDA cells. The ER impacted expression of these genes in opposite directions. The ER- phenotype was associated with less TCDD-induced CYP1A1 expression, but greater basal and induced CYP1B1 expression. A 48 h treatment of ER+ cells with ICI did not revert the P450 expression pattern to that of ER- cells. Based on activities of recombinant enzyme and expression levels, differences in 7,2-dimethylbenz [a]anthracene (DMBA) metabolism between the cell lines were consistent with differences in CYP1A1 and CYP1B1 expression. In T47D lines, basal microsomal DMBA metabolism was primarily due to CYP1B1, based on regioselective metabolite distribution and inhibition by anti-CYP1B1 antibodies (>80%). Metabolism in TCDD-induced microsomes was mostly due to CYP1A1 and was inhibited by anti-CYP1A1 antibody (>50%). TCDD-induced MDA+ cells demonstrated CYP1A1 activity, whereas TCDD-induced MDA- cells displayed CYP1B1 activity. Aryl hydrocarbon receptor (AhR) levels, but not AhR nuclear translocator protein (ARNT) levels were highly dependent on cell type; AhR was high and ER-independent in MDA, and low and ER-linked in T47D. AhR levels were insensitive to ICI. ER does not directly modulate the expression of CYP1A1, CYP1B1 or AhR. Indeed, factors that have replaced ER in growth regulation during clonal selection predominate in this regulation. Characteristics unique to each cell line, including ER status, determine CYP1A1 and CYP1B1 expression.     

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.     

Recombinant human interferon alpha increases oestrogen receptor expression in human breast cancer cells (ZR-75-1) and sensitizes them to the anti-proliferative effects of tamoxifen

Exposure of ZR-75-1 human breast cancer cells for 48 h to human recombinant interferon alpha (IFN alpha) resulted in increased expression of oestrogen receptors as measured in a whole cell binding assay. This effect was inversely proportional to dose being significant following treatment with 10-100 IU IFN ml-1 and was only observed at a low initial cell plating density. The extent of the increase in oestrogen receptor levels ranged from 1.2- to 7.2-fold following treatment with 10 IU IFN ml-1. No increase in progesterone receptor expression was observed under the same experimental conditions. Concentrations of IFN which increased oestrogen receptor levels had no effect on cell proliferation. IFN (500 IU ml-1) inhibited cell proliferation and the combination of this treatment with tamoxifen (2 microM) had a greater anti-proliferative effect than either drug alone although there was no evidence of synergism. However, a 5-day pretreatment of cells with IFN (10 IU ml-1) markedly sensitised them to the growth-inhibiting effect of a subsequent 6-day exposure to tamoxifen.