粉防己碱对膀胱癌T24细胞的凋亡诱导和自噬抑制作用

Tetrandrine induces apoptosis and blocks autophagic l ux of bladder cancer T24 cells

目的 :研究粉防己碱(tetrandrine,Tet)对人膀胱癌细胞增殖、凋亡以及自噬的影响,并初步探讨其作用机制。方法 :应用Tet作用几种膀胱癌细胞后,CCK-8法检测细胞增殖活性及Tet的半数抑制浓度(half maximal inhibitory concentration,IC50)。Tet处理膀胱癌T24细胞后,FCM法检测细胞凋亡状态,蛋白质印迹法检测凋亡相关蛋白caspase-3的剪切情况;同时,用Z-VAD-FMK抑制caspase活性后,采用CCK-8法检测细胞增殖活性。应用Tet处理转染了重组质粒EGFP-LC3的T24细胞,激光共聚焦显微镜下观察细胞自噬体形成情况。蛋白质印迹法检测Tet处理前后T24细胞中自噬相关标志蛋白LC3和P62的表达量。应用LC3反转实验验证Tet对T24细胞自噬流的影响。同时,使用3-MA抑制自噬体形成后,再采用CCK-8法检测膀胱癌T24细胞的增殖活性变化。结果 :Tet可以显著抑制膀胱癌细胞增殖(P<0.01),Tet作用5637、T24、EJ和J82细胞48 h的IC50值分别为(8.03±1.2)、(6.71±0.99)、(4.93±0.72)和(2.72±0.24)μmol/L。Tet能够激活caspase-3,显著诱导T24细胞凋亡(P<0.01);而Z-VAD-FMK能够部分抑制Tet的凋亡诱导作用(P<0.05)。Tet处理重组质粒EGFP-LC3转染的T24细胞后,EGFP-LC3荧光点数量较药物未处理组明显增加(P<0.01)。Tet作用后,T24细胞中LC3-Ⅱ蛋白和P62蛋白的表达水平明显上调(P<0.05)。LC3反转实验证明,Tet处理后膀胱癌细胞自噬流下游发生阻断;而3-MA抑制自噬上游信号后,Tet的抗增殖作用不能被逆转(P>0.05)。结论 :Tet可显著抑制T24细胞的增殖,诱导细胞凋亡,阻断细胞自噬。

Objective: To investigate the effects of tetrandrine(Tet) on proliferation, apoptosis and autophagy of human bladder cancer cell lines, and explore its mechamism. Methods: Cell count kit-8(CCK-8) assay was performed to analyze the effect of Tet on the proliferation of some bladder cancer cell lines and the half maximal inhibitory concentration(IC50) of Tet. After Tet treatment, the apoptosis rate of bladder cancer T24 cells was determined by flow cytometry, and the cleavage of apoptosis-related caspase-3 protein was detected by Western blotting. The activity of caspase was inhibited by Z-VAD-FMK, and then the viability of T24 cells treated by Tet was determined by CCK-8 assay. After the transfection of EGFP-LC3 into T24 cells, the cells were treated with Tet, and then the autophagosome formation was observed by confocal laser scanning microscopy assay. The expressions of autophagy-associated proteins LC3 and P62 in T24 cells after Tet treatment were detected by Western blotting. The effect of Tet on autophagic flux of T24 cells was determined by LC3 turnover assay, while the proliferative activity of T24 cells was detected by CCK-8 assay after treatment with 3-MA to inhibit autophagy. Results: Tet could obviously inhibit the proliferation of bladder cancer cells(P < 0.01). The values of IC50 of Tet(48 h) for cell lines 5637, T24, EJ and J82 were 8.03±1.2, 6.71±0.99, 4.93±0.72 and 2.72±0.24 μmol/L, respectively. Tet significantly induced apoptosis of T24 cells by caspase-3 activation(P < 0.01), and this effect could be partially antagonized by Z-VADFMK(P < 0.05). The number of EGFP-LC3 puncta was significantly increased after Tet treatment in T24 cells transfected with EGFP-LC3(P < 0.01). The expressions of LC3-Ⅱ and P62 proteins were up-regulated in T24 cells after Tet treatment(P < 0.05). The LC3 turnover assay confirmed that Tet blocked the autophagic flux in T24 cells, but 3-MA failed to rescue Tet-induced cell death(P > 0.05). Conclusion: Tet can inhibit the proliferation of bladder cancer cells, induce the apoptosis, and block the autophagy.