双酚A诱导人乳腺癌MCF-7细胞上皮间质化的研究

双酚A(BPA)是一类典型的环境雌激素,能干扰机体正常内分泌系统.作为一种激素相关疾病,乳腺癌的发生发展与BPA暴露的关系已引起人们的关注.本研究旨在探讨BPA对人乳腺癌MCF-7细胞上皮细胞间质化(EMT)的诱导作用.经BPA处理的MCF-7细胞,采用MTT试验检测BPA对细胞活力的影响,Transwell试验检测BPA对细胞迁移能力的影响,Real-time RT-PCR、Western Blot检测BPA对上皮型蛋白标志物E-cadherin、间质型蛋白标志物Vimentin及EMT相关转录因子Snail表达的影响.结果发现,低浓度BPA能够促进MCF-7细胞的增殖,显著增强细胞的迁移能力.BPA处理能抑制E-cadherin的表达,促进Vimentin及Snail的表达.研究结果提示,BPA处理可能通过促进Snail的表达而调控EMT相关蛋白标志物的表达,进而增强乳腺癌MCF-7细胞的迁移能力.     

Expression alterations of cytochromes P4501A1, 2E1, and 3A,and their receptors AhR and PXR caused by 1-octyl-3-methylimidazolium chloride in mouse mammary carcinoma cells

Cytochrome P450s (CYPs) play a key role in the metabolism of a wide range of environmental xenobiotics and endogenous compounds. The expression and activity levels of CYPs can be elevated by a process of induction involving the activation of nuclear receptors. The effects of the ionic liquid 1-octyl-3-methylimidazolium chloride ([C₈mim][Cl]) on the expression of cytochrome P450 members, including CYP1A1, CYP2E1, and CYP3A, as well as on aryl hydrocarbon receptor (AhR) and pregnane X receptor (PXR) in mouse mammary carcinoma cells (EMT6) were investigated by using quantitative real-time PCR in the present study. The results reveal that [C₈mim][Cl]-exposure up-regulates the expressions of CYP1A1, CYP2E1, and CYP3A at mRNA level, suggesting that imidazolium-based ionic liquids can activate CYPs. Our results also suggest that [C₈mim][Cl]-mediated CYP3A induction be PXR-dependent. This result may be beneficial to evaluating the environmental toxicity of imidazolium-based ionic liquids and investigating the metabolism of imidazolium-derivative drugs.     

低浓度TBBPA暴露促进子宫内膜癌细胞侵袭和迁移的毒性效应与机理

为了研究低浓度四溴双酚A(TBBPA)暴露对人子宫内膜癌恶化的影响,本实验采用人子宫内膜癌细胞Ishikawa暴露于不同浓度的TBBPA,探讨其毒性效应及可能存在的分子机理.实验采用MTT法检测Ishikawa细胞的存活率,用倒置显微镜观察TBBPA对Ishikawa细胞形态的影响,用细胞划痕与Transwell实验检测细胞迁移侵袭情况,用实时荧光定量PCR与Western blotting分别检测上皮间充质转化(Epithelial Mesenchymal Transition,EMT)标志物及相关转录因子mRNA与蛋白水平表达量.结果显示,Ishikawa细胞暴露于不同浓度TBBPA(1.00×10-9、1.00×10-8、1.00×10-7mol·L-1)后,细胞存活率明显升高,细胞形态由卵圆形逐渐变为长梭形,细胞侵袭率、细胞划痕愈合率、细胞迁移率与对照组相比显著升高,差异具有统计学意义.在mRNA和蛋白水平上,EMT标志物E-cadherin表达量显著下降,Vimentin和N-cadherin表达量明显上升;EMT上游转录因子Twist和Snail-1的含量也显著增加.结果表明,TBBPA暴露是通过改变细胞内E-cadherin、Vimentin和N-cadherin的含量进而激活Ishikawa细胞上皮间充质转化,导致人子宫内膜癌的恶化;而激活上皮间充质转化的过程可能与TBBPA诱导Twist和Snail-1的表达有关.     

Cardiotoxin III suppresses the invasiveness of MDA-MB-231 cells by targeting proto-oncogene tyrosine-protein kinase Src and reversing mesenchymal-to-epithelial transition

The epithelial-to-mesenchymal transition is the first step required for breast cancer to initiate metastasis. The aberrant activation of proto-oncogene tyrosine-protein kinase Src regulates multiple functions during tumor progression. Cardiotoxin III, a basic polypeptide isolated from Naja naja atra venom, has been shown to exhibit anticancer activity; however, the effect of cardiotoxin III on the epithelial-to-mesenchymal transition of cancer cells remains elusive. Exposure of MDA-MB-231 cells to cardiotoxin III resulted in morphological changes and upregulation of E-cadherin with a concomitant decrease in N-cadherin and vimentin protein levels, resulting in the inhibition of cell migration and invasion. Cardiotoxin III induced downregulation of snail and slug expression. Simultaneously, cardiotoxin III suppressed Src phosphorylation and downstream activation of focal adhesion kinase, of the docking protein p130^cas, and of paxillin. In addition, cardiotoxin III inhibited the phosphorylation of extracellular signal-regulated kinase and phosphatidylinositol 3-kinase. The Src specific inhibitor 4-amino-5-(4-chlorophenyl)-7-(dimethylethyl)pyrazolo[3,4-d]pyrimidine decreased in the phosphorylation and the expression changes of epithelial-to-mesenchymal transition markers in a similar way. Thus, cardiotoxin III has a novel anticancer effect by suppressing proto-oncogene tyrosine-protein kinase activity, reversing epithelial-to-mesenchymal transition.