虫草素对人舌癌细胞凋亡、自噬的影响及其分子机制

目的研究虫草素对人舌癌TCA-8113细胞的细胞周期、凋亡和自噬的影响并探讨虫草素抑制舌癌发生的作用机制。方 法用CCK-8法检测虫草素对TCA-8113细胞增殖的作用；用流式细胞术检测不同浓度药物对细胞周期、细胞凋亡的影响；用荧 光定量PCR和Western blot 的方法检测凋亡相关基因Caspase-3，Caspase-9，Bcl-2、Bax；免疫组化方法检测自噬相关蛋白LC- 3β，P62，p-mTOR，AMPK。结果CCK-8 细胞增殖检测结果表明，虫草素能明显抑制TCA-8113 细胞的增殖，24 h 时IC50为 3.548 mg/mL；48 h时IC50为1.185 mg/mL；流式细胞结果显示，诱导细胞周期阻滞与S期，虫草素能显著的诱导TCA-8113细胞 凋亡，并且呈浓度依赖性。荧光定量PCR和Westen blot结果表明，虫草素可诱导促凋亡基因Bax、Caspase-3、Caspase-9的表达， 抑制抗凋亡基因Bcl-2的表达（P<0.05）；免疫组化的结果表明，虫草素可促进LC-3β和AMPK的表达，抑制P62和p-mTOR的表 达（P<0.05）。结论虫草素对TCA-8113细胞的抑制并促进细胞凋亡，同时通过AMPK/mTOR通路诱导细胞自噬。

Effect of cordycepin on apoptosis and autophagy of tongue cancer cells in vitro and the molecular mechanism

Objective To study the effect of cordycepin on cell cycle, apoptosis and autophagy of human tongue cancer TCA- 8113 cells and explore the mechanism of cordycepin for inhibiting the occurrence of tongue cancer. Methods CCK-8 method was used to assess the inhibitory effect of cordycepin on TCA-8113 cell proliferation in vitro. The cell cycle and cell apoptosis of TCA-8113 cells treated with different concentrations of cordycepin were analyzed using flow cytometry. The expressions of apoptosis-related genes caspase-3, caspase-9, Bcl-2, and Bax were examined using quantitative real-time PCR and Western blotting, and immunohistochemistry was used to detect the expressions of autophagy-related proteins LC-3β, P62, p-mTOR, and AMPK. Results CCK-8 assay showed that cordycepin significantly inhibited the proliferation of TCA-8113 cells in a concentration-dependent manner with an IC50 of 3.548 mg/mL at 24 h and an IC50 of 1.185 mg/mL at 48 h. Flow cytometric analysis showed that cordycepin caused cell cycle arrest at S phase and dose-dependently increased the apoptotic rate of TCA- 8113 cells. Treatment of the cells with cordycepin enhanced the expressions of Bax, caspase-3 and caspase-9 at both the mRNA and protein levels and inhibited the expression of the antiapoptotic gene Bcl-2. Immunohistochemistry demonstrated that cordycepin promoted the expression of LC-3β and AMPK and inhibited the expression of P62 and p-mTOR. Conclusion Cordycepin inhibits the proliferation and induces apoptosis of HCT-116 cells through the mitochondrial pathway and induces autophagy via the AMPK/mTOR pathway.