乳腺癌细胞表面MHCⅡ类抗原和共刺激分子表达的研究

目的研究乳腺癌细胞表面MHCⅡ类抗原与共刺激分子CD40、CD80(B7-1)和CD86(B7-2)的表达.方法采用流式细胞技术检测5株乳腺癌细胞MCF-7、SK-BR-3、T47D、MDA-MB-435s和ZR-75 -30表面MHCⅡ类抗原与共刺激分子CD40、CD80(B7-1)和CD86(B7-2)的表达,并与正常乳腺细胞HBL-100作比较. 结果 5株乳腺癌细胞MHCⅡ类分子表达均与正常乳腺细胞HBL-100差异有显著性(P<0.05), MCF-7细胞表达水平最低,约为HBL-100的1/5.MDA-MB-435s与ZR-75-30细胞表达水平为HBL-100的2倍,MDA-MB-435s荧光强度也比HBL-100及其他乳腺癌细胞高,但MDA-MB -435s细胞表面CD40分子表达水平最低,约为MCF-7与HBL-100 CD40分子表达水平的10%. MDA-MB-435s细胞的CD80和CD86分子表达水平与HBL-100相当(P>0.05),另4株乳腺癌细胞的CD80和CD86分子表达均比HBL-100低(P<0.05). 结论乳腺癌细胞表面MHCⅡ与共刺激分子表达异常,不同细胞表面分子的表达有所差异.乳腺癌细胞可通过这些分子表达异常而发生免疫逃逸.     

miR-21通过靶基因ZNF326调控乳腺癌细胞侵袭、迁移的分子机制研究

目的:探讨微小RNA-21（miR-21）靶向锌指蛋白326(ZNF326)对乳腺癌细胞侵袭、迁移的影响及其分子机制。方法:采用qRT-PCR与Western blot法检测乳腺癌细胞株HCC70、MDA-MB-231、BT549、MDA-MB-468与正常细胞株HBL-100中miR-21与ZNF326的表达。以miR-21表达量最高的乳腺癌HCC70细胞为研究对象,分为NC组、anti-miR-con组、anti-miR-21组、pcDNA-control组、pcDNA-ZNF326组;共转染分组为anti-miR-21+si-con组、anti-miR-21+si-ZNF326组。MTT法检测细胞增殖能力,Transwell迁移及侵袭实验检测细胞迁移及侵袭能力。双荧光素酶报告基因系统验证miR-21与ZNF326的靶向调控关系。Western blot法检测CDK1、MMP-2蛋白的表达。结果:与正常细胞株HBL-100相比,乳腺癌细胞株HCC70、MDA-MB-231、BT549、MDA-MB-468中miR-21高表达,ZNF326 mRNA及蛋白表达水平均显著下降;分别与anti-miR-con组、pcDNA-control组比较,anti-miR-21组与pcDNA-ZNF326组HCC70细胞增殖能力显著下降,细胞迁移及侵袭能力明显降低,CDK1、MMP-2的表达水平明显降低;双荧光素酶实验结果表明miR-21能靶向结合ZNF326的3' UTR并调控其表达;与anti-miR-21+si-con组相比,anti-miR-21+si-ZNF326组HCC70细胞增殖、迁移及侵袭能力均明显增强,促进CDK1、MMP-2表达。结论:miR-21可靶向抑制ZNF326基因的表达,进而促进乳腺癌细胞增殖、迁移及侵袭。     

Expression of Ets-related transcription factors and matrix metalloproteinase genes in human breast cancer cells

The expression levels of ets and MMP genes was examined in two breast cancer cell lines of differing invasive potential. The more invasive MDA-MB-231 cell line had higher levels of Ets-1, Ets-2, PEA3, ERM, Tel, Net, MMP-13 and -14 mRNA than MCF-7 cells. MMP-1, -3 and -16 mRNAs were expressed equally. TPA stimulated MMP-1, -9 and TIMP-1 mRNA expression in both cell lines. MMP-2 and MMP-7 mRNAs were not detected in either cell line. The Ets-1 protein was only detected in MDA-MB-231 cells and its level increased following TPA stimulation. TPA induced MMP-9 activity in MCF-7 cells and increased its activity in MDA-MB-231 cells, however, MMP-2 activity was not detected.     

槲皮素对人乳腺癌细胞MDA-MB-435S增殖及侵袭力的影响

 目的 探讨槲皮素对人乳腺癌细胞MDA-MB-435S 增殖及侵袭能力的影响。 方法 人乳腺癌细胞MDA-MB-435S以12.5、25、50、100、200μM 终浓度的槲皮素处理后,台盼蓝拒染法检测细胞的增殖状况、流式细胞术法检测细胞的凋亡率、Boyden小室法检测细胞的侵袭能力。 结果 人乳腺癌细胞MDA-M-435S经槲皮素处理后,其体外增殖及侵袭能力明显下降的同时凋亡率明显上升,且与药物的剂量呈正相关,当槲皮素终浓度达到200μM时,MDA-MB-435S细胞的生长及侵袭能力几乎完全受到抑制。 结论 槲皮素在体外剂量依赖性的抑制人乳腺癌细胞MDA-MB-435S的增殖及侵袭能力并能诱导其凋亡,为槲皮素用于乳腺癌的预防和治疗提供了部分依据。     

Alteration of hTERT full-length variant expression level showed different gene expression profiles and genomic copy number changes in breast cancer

We analyzed hTERT splicing patterns with respect to telomerase activity in breast cancer. Using a cDNA microarray in 22 cell lines, we observed the difference in expression profiling based on the different levels of full-length variant expression with 71 selected genes. Using 33 known genes that act with the telomerase complex, we performed unsupervised clustering with all cell lines, and found a clustering tendency related to the full-length variant expression level. Using array-based CGH, highly altered genomic copy number changes were found more often in MCF-7 (159 genes) than in MDA-MB-231 (109 genes) and MDA-MB-435 (49 genes), suggesting more genomic changes in MCF-7 cells. On comparing MCF-7 with MDA-MB231 and MDA-MB-435 cell lines, we identified 8 genes with different copy numbers, including dystroglycan, which is located in the p12-21.2 area of chromosome 3. In conclusion, alterations in the level of the full-length variant of hTERT showed different gene expression profiles and genomic copy number changes in breast cancer, which require further study into their cause-and-effect relationship.     

Syntenin is overexpressed and promotes cell migration in metastatic human breast and gastric cancer cell lines

Two human breast cancer cell lines of differing invasive and metastatic potential, MDA-MB-435 and MCF7, were examined using subtractive suppression hybridization in a search for any genes associated with metastasis. Of the 17 cDNAs identified as being differentially expressed genes, it was determined that syntenin was overexpressed in metastatic MDA-MB-435 cells. Expression analysis showed that the expression level of syntenin was well correlated with invasive and metastatic potential in various human breast and gastric cancer cell lines. Moreover, gastric tumor tissues exhibited a much higher syntenin mRNA expression than their normal counterparts. Syntenin-transfected MCF7 cells migrated more actively, and showed an increased invasion rate relative to vector-transfectants or parental MCF7 in vitro, without evidencing any effect on the adhesion to fibronectin, type I collagen and laminin. Similarly, the forced expression of syntenin to human gastric cancer cell line Az521 increased its migratory and invasive potential in vitro. Syntenin-expressing MCF7 cells were associated with the appearance of numerous cell surface extensions and with pseudopodia formation on collagen I, suggesting that syntenin may be involved in the signaling cascade to actin-reorganization. Mutation study suggested that PDZ2 domain of syntenin could be an essential role in its stimulatory effect on the cell migration. This is the first demonstration that syntenin, a PDZ motif-containing protein, can be overexpressed during the metastatic progression of human breast and gastric cancer cells and that it can function as a metastasis-inducing gene.     

Zinc and its transporter ZIP10 are involved in invasive behavior of breast cancer cells

Zinc is an essential element, necessary for sustaining all life. Zinc deficiency causes taste impairments, immune deficiency, skin problems, and growth and mental retardation. Recent reports suggest that zinc is associated with an increased risk of cancer, although it is still unclear whether zinc or its transporters are involved in cancer progression. Here we show that zinc and its transporter ZIP10 are involved in the invasive behavior of breast cancer cells. The screening of clinical samples for ZIP10 mRNA expression suggested that ZIP10 was significantly associated with the metastasis of breast cancer to the lymph node. In addition, the expression of ZIP10 mRNA was higher in the invasive and metastatic breast cancer cell lines MDA-MB-231 and MDA-MB-435S than in less metastatic breast cancer cell lines, such as MCF7, T47D, ZR75-1 and ZR75-30. In in vitro cell migration assays, the depletion of zinc transporter ZIP10 and intracellular zinc inhibited the migratory activity of MDA-MB-231 and MDA-MB-435S cells. These results showed that zinc and ZIP10 play an essential role in the migratory activity of highly metastatic breast cancer cells, and suggest ZIP10 as a possible marker for the metastatic phenotype of breast cancer and a promising target of novel treatment strategies.     

Semaphorin4C 在乳腺癌子宫内膜癌及前列腺癌中的表达及意义*

目的:研究轴突导向分子Semap horin4C（Sema4C）在乳腺癌、子宫内膜癌及前列腺癌组织和细胞水平的表达情况及其与上述三种恶性肿瘤转移的关系。方法:应用免疫组化技术检测45例乳腺癌、42例子宫内膜癌及49例前列腺癌组织中Sema4C的表达情况,分别采用蛋白免疫印迹法Western Blot及细胞免疫荧光方法检测Sema4C 在三种乳腺癌细胞（MDA-MB-435S、MDA-MB-231、MCF-7）、两种子宫内膜癌细胞株（AN3CA、HEC-1-B）和两种前列腺癌细胞株（PC- 3M-1E8、PC- 3M-2B4）的蛋白表达及其亚细胞定位情况。结果:乳腺癌和子宫内膜癌中,其分别在有淋巴结转移的乳腺癌组和子宫内膜癌组的原发灶中的表达高于无淋巴结转移组,差异有统计学意义（P<0.05）。 前列腺癌中,Sema4C 的阳性表达率随着Gleason评分增加而升高（P<0.05）。 Western blot在七株肿瘤细胞均检测到Sema4C 的表达,且提示Sema4C 的表达与细胞的转移潜能呈正相关。荧光显微镜下观察到Sema4C 主要定位在细胞膜和细胞浆。结论:Sema4C 在乳腺癌、子宫内膜癌和前列腺癌组织和细胞中表达具有普遍性,且与上述三种恶性肿瘤转移关系密切,有望成为上述三种恶性肿瘤治疗的一个新的分子靶点。      

Lineage infidelity of MDA-MB-435 cells: expression of melanocyte proteins in a breast cancer cell line

The origin of cell lines is critical in defining cell type-specific biological functions. Several reports suggested that the MDA-MB-435 cell line, a cell line extensively used for studying breast cancer biology, has a gene expression pattern most compatible with melanocyte origin. However, we demonstrate that MDA-MB-435 cells express breast-specific or epithelial-specific markers. Also, MDA-MB-435 cells were induced to express breast differentiation-specific proteins and secrete milk lipids as observed in other well-established breast cancer cell lines. Notably, MDA-MB-435 cells also expressed melanocyte-specific proteins as did another highly aggressive breast cancer cell line. MDA-MB-435 xenograft tissue sections stained entirely positive for epithelium-specific markers but only partially positive for melanocyte-specific markers. Thus, MDA-MB-435 is most likely a breast epithelial cell line that has undergone lineage infidelity.     

RECK expression in pancreatic cancer: its correlation with lower invasiveness and better prognosis.

BACKGROUND: The reversion-inducing cysteine-rich protein with Kazal motifs (RECK) gene was initially isolated as a transformation suppressor gene. The RECK gene is expressed widely in normal organs but is undetectable in many tumor-derived cell lines. When artificially expressed in such cell lines, RECK negatively regulates at least matrix metalloprotease (MMP)-9, MMP-2, and MT1-MMP activation and suppresses the invasive and metastatic potentials of these cells. Clinical relevance of these observations, however, is yet to be established. The aim of this study was to examine RECK expression in pancreatic cancer, where intensive invasiveness and metastasis are frequently observed, and investigate its clinical significance. We also analyzed the correlation between RECK expression and MMP activation. METHODS: (a) RECK expression in surgically resected tissue samples of invasive ductal carcinomas of the pancreas (n = 50) was examined immunohistochemically, and its correlation with clinicopathological factors was analyzed; and (b) gelatin zymography was used for the detection of latent and activated forms of MMP-2 and MMP-9 in some of the tissue samples (n = 33). The gelatinase activity was quantified by densitometory, and the ratio of intensity of the active MMP-2 band to the total intensity of the pro- and active MMP-2 bands was evaluated as an indicator of MMP-2 activation. The MMP-9 activation was also studied. RESULTS: Among the 50 ductal carcinoma samples, 26 (52%) were stained positive for RECK. In the normal pancreas, both acinar and beta cells were stained positive, but ductal cells did not. Tumors with positive RECK staining were significantly less invasive as compared with RECK-negative tumors (P = 0.0438). Importantly, patients who had tumors with high RECK expression showed significantly better prognosis than those who had RECK-negative tumors (P = 0.0463, by Log-rank test). Zymographic analysis indicated significant inverse correlation between the level of RECK expression and extent of MMP-2 activation (P = 0.0374). CONCLUSIONS: Our findings support the hypothesis that the RECK protein has negative effects on the invasiveness of pancreatic cancer by inhibiting MMP-2 activation and suggest the potential value of RECK as a prognostic molecular marker for pancreatic cancer.     

Hypoxia stimulates breast carcinoma cell invasion through MT1-MMP and MMP-2 activation

The process of cancer cell invasion involves degradation of the extracellular matrix (ECM) by proteases, integrin adhesion and cell motility. The role of ECM degrading proteases on the hypoxia-induced invasion of breast carcinoma cells was investigated. Hypoxia markedly increased the invasion capacity of MDA-MB-231 and MDA-MB-435 breast carcinoma cell lines. Matrix metalloproteinase (MMP) inhibitors blocked the hypoxia-induced invasion, whereas other protease inhibitors had no effect. Antibodies or siRNAs blocking either membrane type-1 MMP (MT1-MMP) or MMP-2 were effective in reducing the hypoxia-induced invasion. Serum-free reconstitution experiments confirmed the involvement of the MT1-MMP/MMP-2/tissue inhibitor of metalloproteinase-2 complex in this hypoxia-induced response. Overexpression of MT1-MMP in a poorly invasive breast cancer cell line, T47-D, promoted hypoxia-induced invasion and MMP-2 activation. Cell surface accumulation and activation of MT1-MMP without apparent regulation at the mRNA or protein levels indicated a post-translational adaptive response to hypoxia. Inhibition of the small GTPase RhoA eliminated the hypoxia-induced invasion and blocked the localization of MT1-MMP to the plasma membrane. Zymographic and molecular analysis of human breast tumors showed a strong correlation between hypoxic microenvironments and MMP-2 activation without changes in MT1-MMP expression. Our studies suggest that hypoxic tumor microenvironments promote breast cancer invasion through an MT1-MMP-dependent mechanism.     

Effects of green tea polyphenol on methylation status of RECK gene and cancer cell invasion in oral squamous cell carcinoma cells

RECK is a novel tumour suppressor gene that negatively regulates matrix metalloproteinases (MMPs) and inhibits tumour invasion, angiogenesis and metastasis. In the present study, we investigated the effects of epigallocatechin-3-gallate (EGCG), a major polyphenol in green tea, on the methylation status of the RECK gene and cancer invasion in oral squamous cell carcinoma cell lines. Our results showed that treatment of oral cancer cells with EGCG partially reversed the hypermethylation status of the RECK gene and significantly enhanced the expression level of RECK mRNA. Inhibition of MMP-2 and MMP-9 levels was also observed in these cells after treatment with EGCG. Interestingly, EGCG significantly suppressed cancer cell-invasive ability by decreasing the number of invasive foci (P<0.0001) as well as invasion depth (P<0.005) in three-dimensional collagen invasion model. Although further investigation is required to assess the extent of contribution of RECK on MMPs to the suppression of invasive behaviour, these results support the conclusion that EGCG plays a key role in suppressing cell invasion through multiple mechanisms, possibly by demethylation effect on MMP inhibitors such as RECK.     

Gene regulation by phorbol 12-myristate 13-acetate in MCF-7 and MDA-MB-231, two breast cancer cell lines exhibiting highly different phenotypes

We have examined the effects of the protein kinase C (PKC)-activator phorbol 12-myristate 13-acetate (PMA) on gene expression in two breast cancer cell (BCC) lines exhibiting highly different phenotypes. These are the estrogen receptor alpha (ERalpha)-positive, weakly invasive, luminal epithelial-like MCF-7 and the ERalpha-negative, highly invasive, fibroblast-like MDA-MB-231. They express constitutively low and high PKC activities, respectively. After a 24-h exposition to 100 nM PMA, the number of genes showing an altered expression at the 2-fold change level was much higher in MCF-7 (n=435) than in MDA-MB-231 (n=18) BCC. Four of these genes, namely CDC2, CENPA, NR4A1 and MMP10, were altered in the same way in both cell lines. Two genes were regulated in an opposite way: ID1 and EVA1. Many of the genes down-regulated in MCF-7 BCC appeared to be preferentially expressed in the G1, S, and/or G2 phases of the cell cycle. The ERalpha gene, ESR1, and other genes associated to the ERalpha-positive, luminal epithelial-like BCC phenotype were down-regulated, while a series of genes related to a more aggressive, fibroblast-like BCC phenotype were up-regulated. Other altered genes were notably linked to cell architecture, supporting profound effects of PMA on cell morphology and motility, as well as on the interactions between BCC and their neighboring proteins. Of note, all the modulated genes involved in proteolysis and its control were up-regulated. In summary, PMA effects suggest that PKC activation may induce, to some extent, a more fibroblast-like phenotype in the ERalpha-positive, luminal epithelial-like MCF-7 BCC, and significantly modulate the interactions of these cells with their environment.     

Induction of apoptosis and inhibition of c-erbB-2 in MDA-MB-435 cells by genistein.

Breast cancer is the most common cancer and second leading cause of cancer related deaths in women in the United States. Genistein is a protein tyrosine kinase inhibitor and prominent isoflavonoid in soy products and has been proposed as the agent responsible for lowering the rate of breast cancer in Asian women. We have previously shown that genistein inhibits the growth of MDA-MB-231 breast cancer cells, regulates the expression of apoptosis-related genes, and induces apoptosis through a p53-independent pathway. In this study, we investigated these effects of genistein in the breast cancer cell line MDA-MB-435 and 435.eB cells that were established by transfecting c-erbB-2 cDNA into MDA-MB-435. We also investigated the effect of genistein on matrix metalloproteinase (MMP) secretion previously shown to be effected by erbB-2 transfection. Genistein was found to inhibit MDA-MB-435 and 435.eB cell growth. Induction of apoptosis was also observed in these cell lines when treated with genistein, as measured by DNA laddering, poly(ADP-ribose) polymerase (PARP) cleavage, and flow cytometric analysis. We also found an up-regulation of Bax and p21WAF1 expression and down-regulation of Bcl-2 and c-erbB-2 in genistein-treated cells. Gelatin zymography showed that genistein inhibits the secretion of MMP in the breast cancer cells. From these results, we conclude that genistein inhibits the growth of MDA-MB-435 breast cancer cells, induces apoptosis, regulates the expression of genes, and may inhibit invasion and metastasis of breast cancer cells. These findings suggest that genistein may be a potentially effective chemopreventive or therapeutic agent against breast cancer.     

高转移性人乳腺癌细胞亚克隆的筛选

目的筛选高转移潜能的人乳腺癌细胞亚克隆,为人类乳腺癌转移相关的体内外研究提供实验对象。方法通过有限稀释法分离和培养人乳腺癌细胞MDA-MB-435S的单克隆;采用细胞生物学方法体外评价亚克隆的克隆形成、增殖、运动和侵袭能力;将MDA-MB-435S亚克隆细胞通过乳腺脂肪垫接种于免疫双缺陷SCID鼠,验证体外筛选克隆的体内转移能力。定量资料采用独立样本t检验进行统计学分析。结果筛选得到MDA-MB-435S的高转移亚克隆14-E5;14-E5与亲代细胞呈梭形、伪足较长的细胞形态明显不同,体积比亲代细胞小,呈多边形,触角增多;细胞的体外克隆形成能力、运动能力、侵袭能力及体内自发转移能力均较亲代显著增强(P0.050);细胞周期S期和G2/M期比例比亲代减少,增殖能力较亲代降低(t=7.047,P=0.002);细胞异质黏附能力、体内成瘤率与亲代之间的差异无统计学意义(P0.050)。结论筛选并建立了高转移潜能人乳腺癌细胞亚克隆细胞株14-E5,可以用于乳腺癌转移基因筛选、转移机制研究、抗转移药物的研发和评价抗转移实验性治疗疗效。