EBP50通过Wnt3a/β-catenin信号通路抑制乳腺癌细胞的侵袭和转移

EBP50通过抑制LIMK/cofilin信号通路和PI3K/Akt/m TOR/MMP信号通路抑制乳腺癌细胞的侵袭和转移。然而,EBP50是否可以通过其他机制抑制乳腺癌细胞的侵袭和转移尚未可知。本研究发现,EBP50能通过Wnt3a/β-catenin信号通路影响乳腺癌细胞的侵袭和转移。Western印迹结果显示,EBP50在低转移细胞株MCF-7和正常乳腺细胞株MCF-10A中高表达,而在高转移细胞株MDA-MB-231低表达。采用RNAi技术将小RNA质粒瞬时转染乳腺癌细胞株MCF-7,同时将质粒pc DNA3.1-EBP50转入乳腺癌细胞株MDA-MB-231。Western印迹结果显示,Si EBP50/MCF-7细胞组的EBP50表达水平明显下调,MDA231/EBP50细胞组的EBP50表达水平明显上调。Transwell侵袭实验和划痕实验结果显示,用Wnt3a刺激后,Si EBP50/MCF-7细胞组体外迁移和侵袭能力明显增强,MDA231/EBP50细胞组体外迁移和侵袭能力明显减弱。Western印迹结果显示,与未用Wnt3a或同时用Wnt3a和抑制剂Dkk1刺激的相比,Si EBP50/MCF-7细胞组上皮-钙粘蛋白(Ecadherin)下调,波形蛋白(vimentin)上调,细胞核中β-联蛋白(β-catenin)的表达水平升高,而MDA231/EBP50细胞组上皮-钙粘蛋白上调,波形蛋白下调,细胞核中β-联蛋白表达下降。上述结果表明,EBP50通过Wnt3a/β-catenin信号通路影响β-联蛋白的转核,抑制EMT的发生,进而抑制乳腺癌细胞的侵袭和转移。     

高转移倾向乳腺癌细胞中p-ERK促进增殖和迁移作用的信号转导途径

在建立乳腺癌细胞MCF-7高转移倾向亚克隆LM-MCF-7细胞株的基础上,为阐明LM-MCF-7细胞具有更强增殖和迁移能力的分子机制,对其相关分子及其信号转导途径进行了探讨.免疫印迹结果显示,与MCF-7细胞相比,LM-MCF-7细胞中p-ERK1/2水平显著升高.流式细胞术和“伤口愈合”实验结果表明,ERK1/2的特异性抑制剂PD98059可明显抑制LM-MCF-7细胞的高增殖和高迁移能力.免疫印迹检测发现,与MCF-7细胞相比,LM-MCF-7细胞中与增殖和迁移相关的因子,如β-catenin、细胞周期蛋白D1、磷酸化肌球蛋白轻链（p-MLC）和肌球蛋白轻链激酶（MLCK）的水平呈明显增高,PD98059对这些因子水平的增高具有抑制作用.免疫荧光染色显示,LM-MCF-7细胞中β-catenin分布在细胞核中,应用PD98059处理后,β-catenin主要分布在胞浆中.上述研究结果表明,在LM-MCF-7细胞中活化的ERK1/2水平升高,是导致该细胞增殖和迁移能力增强的重要原因之一,与ERK1/2-MLCK-p-MLC和ERK1/2-β-catenin 细胞周期蛋白D1等信号转导途径有密切的关系.     

间充质干细胞释放 Dkk-1 抑制乳腺癌细胞 Wnt/β-catenin 途径的实验研究

研究表明,间充质干细胞具有向肿瘤细胞定向迁移并且抑制肿瘤细胞的特性,然而其分子机理目前尚不清楚.为了探讨间充质干细胞抑制肿瘤细胞作用的分子机制,应用BMMS-03人间充质干细胞的条件培养液作用于MCF-7乳腺癌细胞,通过软琼脂克隆形成实验、MTT实验、免疫印迹和免疫荧光染色等技术观察细胞克隆形成、增殖和基因表达的变化.结果显示:在BMMS-03细胞条件培养液作用下,MCF-7细胞的克隆形成和增殖受到了明显的抑制,β-catenin及其下游靶蛋白c-Myc、Bcl-2、PCNA和survivin的表达被明显下调,MCF-7细胞浆和细胞核内β-catenin的表达被明显抑制.BMMS-03细胞中Dkk-1的表达水平与MCF-7细胞相比较高.利用抗Dkk-1的抗体中和BMMS-03细胞条件培养液中的Dkk-1后,可明显拮抗BMMS-03细胞条件培养液对MCF-7细胞中β-catenin及c-Myc表达的抑制作用,基因转染使MCF-7细胞过表达Dkk-1后,MCF-7细胞的β-catenin及c-Myc的表达明显下调.同样经基因转染使BMMS-03细胞过表达Dkk-1后,其条件培养液可进一步下调MCF-7细胞β-catenin及c-Myc的表达.上述结果表明,间充质干细胞BMMS-03对乳腺癌MCF-7细胞的恶性表型具有明显抑制作用,其分子机制与间充质干细胞释放Dkk-1抑制乳腺癌细胞Wnt/β-catenin信号途径有关.     

应用基因表达谱芯片从不同转移能力的 乳腺癌细胞中筛选转移相关基因

人乳腺癌细胞系 MCF-7 及其转移亚克隆 LM-MCF-7 为肿瘤转移分子机制的研究提供了细胞模型 . 应用基因芯片技术比较两种具有不同转移能力细胞系基因表达谱的差异,寻找乳腺癌转移相关基因 . 提取两种细胞总 RNA ,分别用 Cy5-dCTP 、 Cy3-dCTP 标记 LM-MCF-7 和 MCF-7 的 cDNA ,并与含有 21 329 个基因的芯片进行杂交并扫描,利用 GenePix Pro 4.0 图像分析软件处理数据判断基因是否在两个细胞中存在表达差异 . 经互换荧光标记物重复两次实验,共筛选出差异表达基因 67 个,其中 41 个在 LM-MCF-7 细胞中表达上调, 26 个在 LM-MCF-7 细胞中表达下调 . 应用实时定量 RT-PCR 对 7 个表达差异明显的基因进行了验证 . 生物信息学分析结果提示,上述发现的差异基因编码产物与细胞内信号转导、转录调节、应激反应、新陈代谢、发育、细胞运动、细胞凋亡和细胞粘连等功能有关 . 据文献报道,这些差异表达的基因中有 35 个与肿瘤有关,其中 9 个与乳腺癌转移有关, 6 个可能参与肿瘤浸润和转移过程 . 根据基因芯片检测的结果,从功能上对 LM-MCF-7 细胞和 MCF-7 细胞与细胞凋亡的关系进行了研究,发现具有高转移倾向的 LM-MCF-7 细胞与 MCF-7 细胞相比,抗凋亡能力较强 . 上述与肿瘤转移相关基因在肿瘤转移中的作用及其分子机理有待深入研究 .     

GALNT14对人乳腺癌MCF-7细胞迁移的影响

构建真核表达载体pcDNA 3.1-Flag-T14,重组质粒经酶切分析及测序鉴定后,利用脂质体将重组质粒转染人乳腺癌细胞系MCF-7细胞,经G418筛选并建立稳定转染GALNT14细胞株.应用半定量RT-PCR、Western blot检测稳定细胞株GALNT14 mRNA及蛋白表达水平,细胞划痕修复及穿膜试验检测GALNT14基因对MCF-7迁移能力的影响,同时RT-PCR检测GALNT14对MMP-2,MMP-9,TGF-β1及VEGF等肿瘤浸润转移相关因子表达的影响.结果显示成功构建了真核重组表达载体pcDNA 3.1-Flag-T14,经RT-PCR和Western blot检测显示成功获得了稳定表达GALNT14的MCF-7细胞株;GALNT14能够提高MCF-7细胞株的迁移能力,且能增加侵袭转移相关因子MMP-2,MMP-9,TGF-β1及VEGF的表达.结论:GALNT14可明显促进MCF-7细胞的迁移,可能在肿瘤侵袭转移中起重要作用.     

乳腺癌转移中的磷脂酰胆碱和溶血磷脂酰胆碱分析（英文）

我们以前曾报道花生四烯酸(arachidonic acid,AA)代谢产物可以促进乳腺癌细胞增殖和迁移.为了进一步寻找维持高转移乳腺癌细胞中AA高水平代谢的内源机制,深入探求AA代谢促进乳腺癌细胞转移的分子机理,我们应用HPLC/ESI/MSn技术检测和分析了乳腺癌MCF-7和高转移乳腺癌LM-MCF-7细胞中溶血磷脂酰胆碱(lysophosphatidylcholines,Lyso PCs)和磷脂酰胆碱(phosphatidylcholines,PCs)的成分和含量.发现了10种Lyso PC的含量在LM-MCF-7细胞中显著高于MCF-7细胞,有6种PC可水解产生AA,它们在LM-MCF-7细胞中的含量显著低于MCF-7细胞,提示这些溶血磷脂含量的升高和磷脂含量的降低可能与乳腺癌转移相关.在LM-MCF-7细胞中,COX-2抑制剂吲哚美辛(indomethacin,Indo)和LOX抑制剂(nordihydroguaiaretic acid,NDGA)共同作用可明显下调c PLA2的活性,应用HPLC-ESI-MSn技术比较c PLA2活性下调前后LM-MCF-7细胞中Lyso PC和PC含量的变化,发现其中4种PC可被c PLA2水解产生AA.还发现,细胞内Lyso PC与PC的比值可以反映c PLA2的活性.通过以上研究进一步证实了由c PLA2活性调节的AA释放及代谢对乳腺癌转移具有重要作用.     

Janus激酶3通过影响钙池操纵性钙通道促进乳腺癌细胞的迁移北大核心CSCD

本研旨在探讨Janus激酶3 (JAK3)对乳腺癌细胞迁移能力的影响以及其作用机制。利用si RNA (si JAK3)沉默乳腺癌MCF-7细胞JAK3的表达,用划痕实验检测细胞迁移能力,用荧光钙成像技术检测钙池操纵性钙通道(store-operatedcalcium channel,SOCC)活性,用Westernblot和RT-PCR检测钙池操纵性钙内流(store-operatedcalciumentry,SOCE)过程中的重要分子Orai1和STIM1表达水平。结果显示,SOCC抑制剂2-APB对MCF-7细胞的迁移能力有抑制作用。si JAK3转染可显著抑制MCF-7细胞的迁移能力,降低SOCC活性,下调Orai1和STIM1的m RNA和蛋白表达水平。在JAK3沉默细胞中过表达Orai1或STIM1,可使MCF-7细胞迁移力恢复。上述结果提示,JAK3通过影响SOCC促进乳腺癌细胞的迁移。     

Janus激酶3通过影响钙池操纵性钙通道促进乳腺癌细胞的迁移

本研旨在探讨Janus激酶3 (JAK3)对乳腺癌细胞迁移能力的影响以及其作用机制。利用si RNA (si JAK3)沉默乳腺癌MCF-7细胞JAK3的表达,用划痕实验检测细胞迁移能力,用荧光钙成像技术检测钙池操纵性钙通道(store-operatedcalcium channel,SOCC)活性,用Westernblot和RT-PCR检测钙池操纵性钙内流(store-operatedcalciumentry,SOCE)过程中的重要分子Orai1和STIM1表达水平。结果显示,SOCC抑制剂2-APB对MCF-7细胞的迁移能力有抑制作用。si JAK3转染可显著抑制MCF-7细胞的迁移能力,降低SOCC活性,下调Orai1和STIM1的m RNA和蛋白表达水平。在JAK3沉默细胞中过表达Orai1或STIM1,可使MCF-7细胞迁移力恢复。上述结果提示,JAK3通过影响SOCC促进乳腺癌细胞的迁移。     

乳腺癌转移中的磷脂酰胆碱和溶血磷脂酰胆碱分析

我们以前曾报道花生四烯酸（arachidonic acid,AA)代谢产物可以促进乳腺癌细胞增殖和迁移。为了进一步寻找维持高转移乳腺癌细胞中AA高水平代谢的内源机制,深入探求AA代谢促进乳腺癌细胞转移的分子机理,我们应用HPLC/ESI/MSⁿ技术检测和分析了乳腺癌MCF-7和高转移乳腺癌LM-MCF-7细胞中溶血磷脂酰胆碱（lysophosphatidylcholines,LysoPCs）和磷脂酰胆碱（phosphatidylcholines,PCs）的成分和含量。我们发现了10种LysoPC的含量在LM-MCF-7细胞中显著高于MCF-7细胞,有6种PC可水解产生AA,它们在LM-MCF-7细胞中的含量显著低于MCF-7细胞,提示这些溶血磷脂含量的升高和磷脂含量的降低可能与乳腺癌转移相关。在LM-MCF-7细胞中,COX-2抑制剂吲哚美辛（indomethacin,Indo）和LOX抑制剂（nordihydroguaiaretic acid,NDGA）共同作用可明显下调cPLA2的活性,应用HPLC-ESI-MSⁿ技术比较cPLA2活性下调前后LM-MCF-7细胞中LysoPC和PC含量的变化,发现其中4种PC可被cPLA2水解产生AA。我们还发现,细胞内LysoPC与PC的比值可以反映cPLA2的活性。通过以上研究我们进一步证实了由cPLA2活性调节的AA释放及代谢对乳腺癌转移具有重要作用。     

LRP16通过调控E-钙粘合素的表达促进MCF-7细胞的侵袭生长

LRP16在原代乳腺癌组织中表达水平与雌激素受体α（ERα）表达状态以及腋窝淋巴结侵袭数目密切相关.为研究LRP16基因对乳腺癌MCF-7细胞侵袭生长的影响,并探讨涉及的分子机制,采用基质胶黏附实验与Transwell方法,检测内源性LRP16表达抑制MCF-7细胞的体外黏附、侵袭生长与迁移特征.结果表明,抑制LRP16在MCF-7细胞中的表达,降低了细胞的体外黏附、侵袭与迁移能力;采用FVB小鼠进行的实验性转移试验结果显示,抑制LRP16显著降低了MCF-7细胞的肺转移结节数目;为探索可能的分子机制,采用Western印迹方法,检测了LRP16对乳腺癌转移相关分子MMP-2, MMP-9, CD44和E-钙黏着蛋白表达的影响,结果在LRP16抑制的MCF-7细胞中只有E-钙黏着蛋白蛋白表达上调.进一步的Northern印迹与免疫组化实验结果表明,抑制LRP16可上调MCF-7细胞中E 钙黏着蛋白基因的mRNA与蛋白表达水平;共转染与双荧光素酶方法检测LRP16对E-钙黏着蛋白基因启动子的表达调控效应,结果显示,LRP16抑制E 钙黏着蛋白基因基因5′-近端启动子的转录激活,该抑制效应选择性存在于内源性ERα阳性细胞,并且依赖于雌激素的存在;染色质免疫共沉淀方法（ChIP）检测ERα与E-钙黏着蛋白基因启动子的相互作用,结果显示,在LRP16基因表达缺陷的MCF-7细胞中,ERα抗体沉淀到E-钙黏着蛋白基因启动子的DNA序列;上述研究结果表明,抑制LRP16基因表达,削弱了激素依赖型乳腺癌细胞的侵袭生长能力,其分子机制涉及了LRP16通过ERα介导对E-钙黏着蛋白基因基因转录激活的调控.     

Oleuropein inhibits migration ability through suppression of epithelial-mesenchymal transition and synergistically enhances doxorubicin-mediated apoptosis in MCF-7 cells

Distinct metastasis is one of the main causes of breast cancer (BC)-related mortality and epithelial-mesenchymal transition (EMT) is a primary step in metastasis dissemination. On the other hand, doxorubicin (DOX) is an effective chemotherapeutic agent against BC; unfortunately, its clinical use is limited by dose-dependent side effects. Therefore, extensive efforts have been dedicated to suppressing metastasis of BC and also to overcome DOX side effects together with keeping its antitumor efficacy. Studies supported the role of oleuropein (OLEU) in reducing DOX-induced side effects besides its antitumor actions. In this study, the antimigratory effect of OLEU was assessed and real-time PCR (RT-PCR) was used to detect OLEU effect on the expression level of EMT markers, in MCF-7 cells. The cytotoxic effect of OLEU and DOX was assessed by MTT assay, whereas the ratio of apoptosis was investigated by flow cytometry. The results showed that migration ability of MCF-7 cells remarkably decreased in OLEU treated group and RT-PCR results showed that OLEU may exert its antimigratory action by suppressing EMT through downregulation of sirtuin1 (SIRT1). Also, the results indicated that both OLEU and DOX were cytotoxic to MCF-7 cells, whereas DOX-OLEU cotreatment led to additive cytotoxicity and apoptosis rate. This study provides evidence regarding the suppressive role of OLEU on MCF-7 cells migration ability through suppression of EMT, and for the first time, it was proposed that SIRT1 downregulation can be involved in the OLEU antimigratory effect. Also, the findings demonstrated that OLEU can reduce DOX-induced side effects by reducing its effective dose.     

MiR-487a Promotes TGF-β1-induced EMT,the Migration and Invasion of Breast Cancer Cells by Directly Targeting MAGI2

Tumor metastasis is a complex and multistep process and its exact molecular mechanisms remain unclear. We attempted to find novel microRNAs (miRNAs) contributing to the migration and invasion of breast cancer cells. In this study, we found that the expression of miR-487a was higher in MDA-MB-231breast cancer cells with high metastasis ability than MCF-7 breast cancer cells with low metastasis ability and the treatment with transforming growth factor β1 (TGF-β1) significantly increased the expression of miR-487a in MCF-7 and MDA-MB-231 breast cancer cells. Subsequently, we found that the transfection of miR-487a inhibitor significantly decreased the expression of vimentin, a mesenchymal marker, while increased the expression of E-cadherin, an epithelial marker, in both MCF-7 cells and MDA-MB-231 cells. Also, the inactivation of miR-487a inhibited the migration and invasion of breast cancer cells. Furthermore, our findings demonstrated that miR-487a directly targeted the MAGI2 involved in the stability of PTEN. The down-regulation of miR-487a increased the expression of p-PTEN and PTEN, and reduced the expression of p-AKT in both cell lines. In addition, the results showed that NF-kappaB (p65) significantly increased the miR-487a promoter activity and expression, and TGF-β1 induced the increased miR-487a promoter activity via p65 in MCF-7 cells and MDA-MB-231 cells. Moreover, we further confirmed the expression of miR-487a was positively correlated with the lymph nodes metastasis and negatively correlated with the expression of MAGI2 in human breast cancer tissues. Overall, our results suggested that miR-487a could promote the TGF-β1-induced EMT, the migration and invasion of breast cancer cells by directly targeting MAGI2.     

Cathepsin G-induced malignant progression of MCF-7 cells involves suppression of PAF signaling through induced expression of PAFAH1B2

Breast cancer is primarily classified into ductal and lobular types, as well as into noninvasive and invasive cancer. Invasive cancer involves lymphatic and hematogenous metastasis. In breast cancer patients with distant metastases, a neutrophil-derived serine protease; cathepsin G (Cat G), is highly expressed in breast cancer cells. Cat G induces cell migration and multicellular aggregation of MCF-7 human breast cancer cells; however, the mechanism is not clear. Recently, platelet-activating factor (PAF)-acetylhydrolase (PAF-AH), the enzyme responsible for PAF degradation, was reported to be overexpressed in some tumor types, including pancreatic and breast cancers. In this study, we investigated whether PAF-AH is involved in Cat G-induced aggregation and migration of MCF-7 cells. We first showed that Cat G increased PAF-AH activity and elevated PAFAH1B2 expression in MCF-7 cells. The elevated expression of PAFAH1B2 was also observed in human breast cancer tissue specimens by immunohistochemical analysis. Furthermore, knockdown of PAFAH1B2 in MCF-7 cells suppressed the cell migration and aggregation induced by low concentrations, but not high concentrations, of Cat G. Carbamoyl PAF (cPAF), a nonhydrolyzable PAF analog, completely suppressed Cat G-induced migration of MCF-7 cells. In addition, PAF receptor (PAFR) inhibition induced cell migration of MCF-7 cells even in the absence of Cat G, suggesting that Cat G suppresses the activation of PAFR through enhanced PAF degradation due to elevated expression of PAFAH1B2 and thereby induces malignant phenotypes in MCF-7 cells. Our findings may lead to a novel therapeutic modality for treating breast cancer by modulating the activity of Cat G/PAF signaling.     

Pyruvate Carboxylase Is Up-Regulated in Breast Cancer and Essential to Support Growth and Invasion of MDA-MB-231 Cells

Pyruvate carboxylase (PC) is an anaplerotic enzyme that catalyzes the carboxylation of pyruvate to oxaloacetate, which is crucial for replenishing tricarboxylic acid cycle intermediates when they are used for biosynthetic purposes. We examined the expression of PC by immunohistochemistry of paraffin-embedded breast tissue sections of 57 breast cancer patients with different stages of cancer progression. PC was expressed in the cancerous areas of breast tissue at higher levels than in the non-cancerous areas. We also found statistical association between the levels of PC expression and tumor size and tumor stage (P < 0.05). The involvement of PC with these two parameters was further studied in four breast cancer cell lines with different metastatic potentials; i.e., MCF-7, SKBR3 (low metastasis), MDA-MB-435 (moderate metastasis) and MDA-MB-231 (high metastasis). The abundance of both PC mRNA and protein in MDA-MB-231 and MDA-MB-435 cells was 2-3-fold higher than that in MCF-7 and SKBR3 cells. siRNA-mediated knockdown of PC expression in MDA-MB-231 and MDA-MB-435 cells resulted in a 50% reduction of cell proliferation, migration and in vitro invasion ability, under both glutamine-dependent and glutamine-depleted conditions. Overexpression of PC in MCF-7 cells resulted in a 2-fold increase in their proliferation rate, migration and invasion abilities. Taken together the above results suggest that anaplerosis via PC is important for breast cancer cells to support their growth and motility.