南方医科大学学报

南方医科大学学报 ›› 2019, Vol. 39 ›› Issue (03): 351-.doi: 10.12122/j.issn.1673-4254.2019.03.14

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巴西苏木素对舌癌Tca8113 细胞凋亡和自噬的影响及分子机制

贾亚萌，佟晓哲，范敬炎   

  • 出版日期:2019-03-20 发布日期:2019-03-20

Effect of brazilin on apoptosis and autophagy of tongue cancer Tca8113 cells and its molecular mechanism

  • Online:2019-03-20 Published:2019-03-20

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摘要： 目的探讨巴西苏木素对人舌鳞状细胞癌Tca8113细胞增殖、凋亡和自噬的影响及其分子机制，为临床用药提供理论依 据。方法用MTT法检测巴西苏木素对Tca8113细胞的增殖作用；Hoechst33342染色法观察细胞形态变化；Annexin V/PI双染 色检测巴西苏木素对Tca8113细胞凋亡程度的影响；Western blot法检测凋亡相关蛋白bax、bcl-2、cleaved caspase3及自噬相关 蛋白p-AMPK、p-mTOR、LC3B、p62 的表达；使用AMPK通路抑制剂与巴西苏木素共同作用于Tca8113 细胞，并检测通路相 关蛋白p-AMPK、p-mTOR、LC3B的表达。结果MTT结果表明，巴西苏木素能明显抑制Tca8113 细胞的增殖，24 h 时IC50为 31.17 μmol/L；Hoechst33342染色结果显示，巴西苏木素能使Tca8113细胞发生凋亡形态学改变，且与浓度正相关；Annexin V/PI 染色结果显示，不同浓度巴西苏木素作用24 h后，细胞凋亡数和凋亡率均高于空白对照组；Western blot检测相关蛋白表达结果 表明，巴西苏木素可抑制抗凋亡蛋白bcl-2，促进凋亡关键蛋白bax和cleaved-caspase3的表达，同时增加LC3B和p-AMPK的表 达，降低p62和p-mTOR表达。在与抑制剂合用后，p-AMPK与LC3B表达量虽仍略高于空白对照组，但与单纯使用巴西苏木素 相比均明显降低，而p-mTOR的表达较巴西苏木素组则明显升高（P<0.05）。结论巴西苏木素能抑制Tca8113细胞增殖并促进 其凋亡，同时可通过AMPK/mTOR通路诱导细胞发生自噬。

Abstract: Objective To investigate the effects of brazilin on the proliferation, apoptosis and autophagy of human tongue squamous cell carcinoma Tca8113 cells in vitro and explore its molecular mechanism. Methods The changes in the proliferation, morphology and apoptosis of Tca8113 cells in response to brazilin treatment were detected using MTT assay, Hoechst33342 staining, and Annexin V/PI double staining, respectively. The expressions of apoptosis-related protein Bax, Bcl-2, cleaved caspase-3 and autophagy-related proteins p-AMPK, p-mTOR, LC3B, and p62 in the treated cells were detected using Western blotting. The effect of treatment with both the AMPK pathway inhibitor and brazilin on the expressions of the pathway-related proteins p-AMPK, p-mTOR, and LC3B was assessed. Results MTT assay showed that brazilin significantly inhibited the proliferation of Tca8113 cells with an IC50 of 31.17 μmol/L at 24 h. Hoechst33342 staining showed that brazilin induced apoptotic morphological changes in Tca8113 cells in a concentration-dependent manner. Treatment with different concentrations of brazilin resulted in increased apoptosis in the cells. Brazilin obviously inhibited the expression of Bcl-2, p62 and p-mTOR and enhanced the expressions of Bax, cleaved caspase-3, LC3B and p-AMPK. The AMPK pathway inhibitor significantly inhibited the increase in p-AMPK and LC3B expressions and the decrease in p-mTOR expression induced by brazilin. Conclusion Brazilin can inhibit the proliferation and promote apoptosis in Tca8113 cells and at the same time induces autophagy in the cells through the AMPK/mTOR pathway.