华蟾素通过调控基质金属蛋白酶抑制人胃腺癌细胞MGC-803侵袭与迁移

Effect of Cinobufacini on Invasion and Migration of Human Gastric Cancer MGC-803 Cells by Regulating Matrix Metalloproteinases

目的：探讨华蟾素对人胃腺癌MGC-803细胞的侵袭与迁移的影响及其分子机制。方法：噻唑蓝（MTT）比色法检测华蟾素（10,20,40,80,160,320 mg·L-1）对人胃腺癌MGC-803细胞增殖的抑制影响;transwell小室法检测华蟾素对MGC-803细胞侵袭与迁移能力的影响;蛋白免疫印迹法（Western blot）检测经华蟾素作用后的MGC-803细胞中基质金属蛋白酶-2（matrix metalloproteinases-2,MMP-2）,基质金属蛋白酶-9（matrix metalloproteinases-9,MMP-9）和基质金属蛋白酶抑制因子-1（tissue inhibitors of metalloproteinases-1,TIMP-1）,基质金属蛋白酶抑制因子-2（tissue inhibitors of metalloproteinases-2,TIMP-2）的蛋白表达水平;实时荧光定量PCR（Real-time PCR）检测经华蟾素处理后的MGC-803细胞中MMP-2,MMP-9,TIMP-1,TIMP-2的mRNA表达情况。结果：（1）MTT比色法结果显示,与空白组比较,华蟾素（10,20,40,80,160,320 mg·L-1）对胃腺癌MGC-803细胞的增殖具有显著抑制作用（P〈0.01）,并且华蟾素对MGC-803细胞的抑制作用呈浓度和时间依赖。（2）transwell实验结果显示,与空白组比较,华蟾素（10,20,40 mg·L-1）对胃腺癌MGC-803细胞的侵袭与迁移能力具有不同程度的抑制作用（P〈0.05）（3）Real-time PCR和Western blot实验结果显示,与空白组比较,华蟾素（10,20,40 mg·L-1）能够下调MMP-2,MMP-9蛋白和mRNA的表达,上调TIMP-1,TIMP-2蛋白和mRNA的表达。结论：华蟾素能明显抑制胃腺癌MGC-803细胞的侵袭与迁移,其机制可能与上调TIMP-1,TIMP-2的转录水平,进而下调MMP-2,MMP-9蛋白水平有关。

Objective： To investigate the effect of cinobufacini on the invasion and migration of human gastric adenocarcinoma MGC-803 cells and its molecular mechanisms. Method： The inhibitory effect of cinobufacini on the proliferation of human gastric cancer MGC-803 cells was detected by methye thiazolye telrazlium（ MTT） assay. The effect of cinobufacini on the invasion and migration of MGC-803 cells was determined by transwell assay. The protein expressions of matrix metalloproteinases-2（ MMP-2）,matrix metalloproteinases-9（ MMP-9）,tissue inhibitors of metalloproteinases-1（ TIMP-1） and tissue inhibitors of metalloproteinases-2（ TIMP-2） were examined by Western blot. After treatment with different concentrations of cinobufacini,the mRNA expressions of MMP-2,MMP-9,TIMP-1 and TIMP-2 in MGC-803 cells were detected by Real-time PCR. Result：（1） MTT assay showed that cinobufacini（ 10,20,40,80,160,320 mg·L-1） significantly inhibited the proliferation of gastric cancer MGC-803 cells（ P〈 0. 01） in a dose and time-dependent manner.（2） Transwell assay results showed that cinobufacini（ 10, 20, 40 mg ·L-1） inhibited the invasion and migration of gastric adenocarcinoma MGC-803 cells to varying degrees（ P 〈0. 05）.（3） Real-time PCR and Western blot results showed that cinobufacini（ 10,20,40 mg·L-1） could down-regulate the protein and mRNA expressions of MMP-2,MMP-9 and up-regulate protein and mRNA expressions of TIMP-1,TIMP-2. Conclusion： Cinobufacini can obviously inhibit the invasion and migration of gastric cancer MGC-803 cells. The underlying mechanism may be related to the up-regulation of the transcription of TIMP-1 and TIMP-2 and the down-regulation of the protein levels of MMP-2 and MMP-9.