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.
Conclusions: 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.
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。
Keywords: apoptosis; autophagy; brazilin; tongue squamous cell carcinoma.