巴弗洛霉素A1对舌鳞状细胞癌Tca8113细胞增殖及自噬的抑制作用

Inhibitory effect of bafilomycin A1 on proliferation and autophagy of touge squamous cell carcinoma Tca8113 cells

目的探讨巴弗洛霉素A1(Baf-A1)对人舌鳞状细胞癌Tca8113细胞增殖、凋亡及自噬的影响。方法采用终浓度为0.1、0.5μmol/L的Baf-A1处理Tca8113细胞,并设不加药的对照组。MTT比色法测定对照组及不同浓度Baf-A1处理0、24、48、72 h细胞的增殖情况,流式细胞仪检测0.5μmol/L Baf-A1处理72 h的细胞周期,Western blotting、细胞免疫荧光检测自噬标记蛋白LC3B的表达,电子透射显微镜观察细胞自噬体形成情况。结果 Baf-A1呈剂量依赖性抑制Tca8113细胞增殖,0.5μmol/L Baf-A1处理72 h时细胞增殖抑制作用最明显。流式细胞仪检测显示,与对照组比较,Baf-A1组sub-G1期、G1期细胞增多(P<0.05),S期和G2/M期细胞减少(P<0.05)。Baf-A1组LC3B-II蛋白的相对表达量为1.83±0.23,高于对照组的0.79±0.18(P<0.01);细胞免疫荧光检测显示,对照组中可见少量LC3B表达,而Baf-A1组72 h后可见大量LC3B点状聚集物;电镜观察显示,与对照组比较,0.5μmol/L Baf-A1处理后可见大量自噬体形成,自噬溶酶体形成增加。结论 Baf-A1对舌鳞癌Tca8113细胞有增殖抑制作用,可能与其阻滞细胞周期和抑制细胞自噬有关。

Objective To explore the inhibitory effect of bafilomycin A1 （ Bar-A1 ） on cell proliferation and autophagy of touge squamous cell carcinoma Tca8113 cells. Methods Tca8113 cells were cultured with Baf-A1（0. 1,0. 5 μmoL/L） , and cells without treatment were set as control group. The cell proliferation was determined at 0,24,48 and 72 h by MTr assay in control group and Bar- A1 group. Flow cytometry was applied to measure cell cycle treated with 0. 5 μmol/L Baf-A1 for 72 h. Western blotting and immunoflu- oreseence were used to analyze autophagy marker LC3B protein expression. Autophagosome formation was observed by transmission e- lectron microscope. Results Bar-A1 could dose-dependently inhibit the proliferation of Tca8113 cells. The highest proliferative inhibi- tion was observed at 72 h after cells treated with 0. 5 μmol/L Baf-A1. Compared with control group, Tca8113 cells in Baf-A1 group in- creased in sub-G1 and G1 phase（P〈0. 05） , and decreased in S and GE/M phase（P〈0. 05）. LC3B-II expression of Bar-A1 group was 1.83±0. 23, which was significantly higher than 0. 79±0. 18 of control group（P〈0. 05）. Immunofluorescence showed that little LC3B could be seen in control group, while large amount of LC3B puncta was observed in Baf-A1 group after 72 h. Compared with control group, large amount of autophagosomes were observed and the formation of autolysosomes increased in Baf-A1 group. Conclusion Baf-A1 has a toxic effect on the proliferation of Tca8113 ceils. It may be related to cell cycle arrest and autophagy inhibition.