异长春花碱抑制人乳腺癌细胞转移侵袭的作用及其机制

Inhibition of vinorelbine on invasion and metastasis of breast cancer cell and its mechanism

目的探讨异长春花碱抑制人乳腺癌细胞转移侵袭的作用及其机制。方法人乳腺癌MCF-7和MDA-MB-435细胞分别用异长春花碱1、10μmol/L处理后,MTS法检测细胞黏附能力,Transwell法检测细胞侵袭能力,ELISA法检测细胞转移生长因子-β(TGF-β)分泌的变化,明胶酶谱法检测基质金属蛋白酶(MMP)-2和MMP-9活性,Western blotting法检测肿瘤细胞E-钙黏素、N-钙黏素、MMP-2、MMP-9、p-JNK及p-Akt蛋白的表达,RT-PCR法检测肿瘤细胞E-钙黏素、N-钙黏素及MMP-2、MMP-9基因的表达,双荧光素酶报告基因实验考察AP-1和核因子-κB(NF-κB)活性的变化。结果异长春花碱1、10μmol/L给药后,细胞黏附能力:MCF-7细胞分别下降34.8%、66.8%,MDA-MB-435细胞分别下降42.6%、72.3%;侵袭能力:MCF-7细胞分别下降44.4%、72.2%,MDA-MB-435细胞分别下降47.7%、86.4%;TGF-β分泌:MCF-7细胞分别下降28.2%、52.1%,MDA-MB-435细胞分别下降39.0%、55.1%;E-钙黏素基因表达:MCF-7细胞分别上调86.5%、181.6%,MDA-MB-435细胞分别上调116.6%、160.7%;N-钙黏素基因表达:MCF-7细胞分别下降33.7%、74.1%,MDA-MB-435细胞分别下降57.6%、76.9%;MMP-2基因表达:MCF-7细胞分别下降71.6%、88.4%,MDA-MB-435细胞分别下降57.4%、69.4%;MMP-9基因表达:MCF-7细胞分别下降15.0%、84.0%,MDA-MB-435细胞分别下调22.1%、73.0%;E-钙黏素蛋白表达显著上调,N-钙黏素蛋白表达明显下调,MMP-2和MMP-9及p-JNK、p-Akt蛋白表达显著下降;AP-1活性:MCF-7细胞分别下降27.7%、68.2%,MDA-MB-435细胞分别下降34.8%、71.4%;NF-κB活性:MCF-7细胞分别下降18.4%、44.8%,MDA-MB-435细胞分别下降24.4%、51.9%。结论异长春花碱可抑制人乳腺癌MCF-7和MDA-MB-435细胞体外侵袭能力,其机制可能与下调肿瘤转移相关信号通路及上皮间质转化表达有关。

Objective To investigate the inhibition of vinorelbine （VRB） on the invasion and metastasis of breast cancer cell and its mechanism. Methods The MCF-7 and MDA-MB-435 cells were treated with 1 and 10 μmol/L VRB. The cell adhesion was tested by MTS, the invasion was detected by Transwell, secretion of TGF-β was detected using ELISA, the activities of MMP-2 and MMP-9 were detected by zymography, the expression of proteins, including E-cadherin （E-CAD）, N-cadherin （N-CAD）, MMP-2, and MMP-9, p-JNK and, p-Akt was evaluated by Western blotting, RT-PCR was used to detect E-CAD, N-CAD, MMP-2, and MMP-9 genes, and dual-luciferase reporter assay was used to validate the activities of AP-1 and NF-κB. Results After being treated with 1 and 10 μmol/L VRB, for MCF-7 and MDA-MB-435, the adhesion ability was decreased by 34.8% and 66.8%, 42.6% and 72.3%; The metastatic ability was decreased by 44.4% and 72.2%, 47.7% and 86.4%; The secretion of TGF-]3 was reduced by 28.2% and 52.1%, 39.0% and 55.1%, significantly; The mRNA expression levels of E-CAD were increased by 86.5% and 181.6%, 116.6% and 160.7%; while the levels of N-CAD were decreased by 33.7% and 74.1%, 57.6% and 76.9%; The gene expression of MMP-2 was decreased by 71.6% and 88.4%, 57.4% and 69.4%; The gene expression of MMP-9 was decreased by 15.0% and 84.0%, 22.1% and 73.0%, respectively. The protein expression of E-CAD was up-regulated while the protein expression of N-CAD, MMP-2, MMP-9, p-JNK, and p-Akt were down-regulated significantly; And dual-luciferase reporter assay validated VRB could inhibit the transcriptional activity of AP-1 and NF-κB by 27.7% and 68.2%, 34.8% and 71.4%, 18.4% and 44.8%, 24.4% and 51.9%, respectively. Conclusion VRB could inhibit the metastasis of breast cancer MCF-7 and MDA-MB-435 cells via down-regulation of inhibition and blocking of signaling pathway correlated with metastasis and epithelial-mesenchymal transition.