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目的蟾蜍灵(Bufalin)是中医抗癌药物蟾酥的有效成分之一,但其抗肿瘤的机制并不十分清楚。本研究通过分析不同浓度Bufalin对食管癌TE13细胞株迁移距离、MEK1/2及其活化形式P-MEK1/2和基质金属蛋白酶(matrix metalloproteinase,MMP)表达的影响,初步探讨Bufalin抑制食管癌TE13细胞迁移能力的作用机制。方法采用四甲基偶氮唑蓝[3-(4,5-dimethylthiazol-yl)-2,5-diphenyl tetrazolium bromide,MTT]法测定细胞药物毒性,细胞划痕实验分别测量不同浓度(0、10、25、50和100nmol/L)Bufalin处理组食管癌TE13细胞的迁移距离。蛋白质印迹法及免疫细胞化学法检测MEK1/2和P-MEK1/2蛋白的表达。逆转录-聚合酶链反应(reverse transcription-polymerase chain reaction,RT-PCR)检测MMP-2、MMP-9mRNA的表达情况。结果 MTT法结果显示,Bufalin药物最大无毒浓度(TC0)为100nmol/L。细胞划痕实验结果显示,不同浓度Bufalin处理组在划痕36h后与对照组相比迁移的距离不同,随着Bufalin浓度的增加,细胞迁移距离下降,100nmol/L迁移距离最小(F=243.163,P<0.01),Bufalin能有效抑制食管癌细胞迁移距离。蛋白质印迹法及免疫细胞化学检测结果显示,Bufalin对MEK1/2蛋白的表达无影响,但能显著抑制其活化形式P-MEK1/2的表达,并呈剂量依赖性,随Bufalin浓度的升高(0、10、25、50和100nmol/L),P-MEK1/2相对表达量逐渐下降,分别为0.710±0.006、0.676±0.010、0.656±0.010、0.599±0.020和0.521±0.021,F=76.369,P<0.01。P-MEK1/2阳性细胞数随着Bufalin浓度的升高而减少,分别为(80.330±1.905)%、(68.407±2.219)%、(67.297±1.797)%、(52.617±2.368)%和(26.97±2.912)%,F=243.348,P<0.01。RT-PCR结果显示,随着Bufalin药物浓度的升高,MMP-2mRNA相对表达量分别为0.772±0.010、0.725±0.019、0.663±0.015、0.582±0.019和0.519±0.019,F=112.990,P<0.01;MMP-9mRNA相对表达量分别为0.783±0.013、0.740±0.010、0.703±0.006、0.601±0.017和0.531±0.010,均低于对照组,F=179.417,P<0.01。结论 Bufalin可能通过下调MEK1/2的活性,从而抑制食管癌TE13细胞的迁移能力。
OBJECTIVE Bufalin is one of the active ingredients of Chinese toad drug, but the anti-tumor mechanism is not very clear. This study was designed to investigate the effect of Bufalin on esophageal cancer TE13 cell line migration distance, MEK1 / 2 and its activated form of P-MEK1 / 2 and matrix metalloproteinase (MMP) Migration mechanism of action. Methods MTT assay was used to determine cytotoxicity. Cell scratch assay was used to measure cell viability at different concentrations (0, 10, , 25, 50 and 100 nmol / L) Bufalin treatment group esophageal cancer TE13 cell migration distance. The expression of MEK1 / 2 and P-MEK1 / 2 proteins was detected by Western blotting and immunocytochemistry. Reverse transcription-polymerase chain reaction (RT-PCR) was used to detect the expression of MMP-2 and MMP-9 mRNA. Results MTT assay showed that the maximum toxic concentration of Bufalin (TC0) was 100 nmol / L. The results of cell scratch assay showed that the migration distance of Bufalin treated group was different from that of control group at 36h after scratching, with the increase of Bufalin concentration, the migration distance decreased and the migration distance of 100nmol / L was the smallest (F = 243.163, P <0.01), Bufalin can effectively inhibit esophageal cancer cell migration distance. Western blotting and immunocytochemistry showed that Bufalin had no effect on the expression of MEK1 / 2 protein, but significantly inhibited the expression of P-MEK1 / 2 in a dose-dependent manner. With the increase of Bufalin concentration The relative expression of P-MEK1 / 2 decreased gradually to 0.710 ± 0.006, 0.676 ± 0.010, 0.656 ± 0.010, 0.599 ± 0.020 and 0.521 ± 0.021, respectively, F = 76.369, P <0.01. The number of P-MEK1 / 2 positive cells decreased with the increase of Bufalin concentration, which were (80.330 ± 1.905)%, (68.407 ± 2.219)%, (67.297 ± 1.797)%, (52.617 ± 2.368)% and (26.97 ± 2.912)%, F = 243.348, P <0.01. RT-PCR results showed that with the increase of Bufalin concentration, the relative expression of MMP-2 mRNA were 0.772 ± 0.010, 0.725 ± 0.019, 0.663 ± 0.015, 0.582 ± 0.019 and 0.519 ± 0.019, respectively, F = 112.990, P <0.01 ; The relative expression levels of MMP-9 mRNA were 0.783 ± 0.013,0.740 ± 0.010,0.703 ± 0.006,0.601 ± 0.017 and 0.531 ± 0.010, respectively, which were lower than the control group, F = 179.417, P <0.01. Conclusion Bufalin may inhibit the migration of esophageal cancer TE13 cells by down-regulating the activity of MEK1 / 2.