三氧化二砷诱导脑胶质瘤 U87 细胞凋亡的机制。

Department of Neurosurgery, Yantaishan Hospital, Yantai, China.

Eur Rev Med Pharmacol Sci. 2017 Nov;21(21):4875-4881.

OBJECTIVE

To investigate the mechanism of arsenic trioxide (As2O3)-induced apoptosis of glioma cells.

MATERIALS AND METHODS

U87 cells were treated by different concentrations of As2O3 (8 μmol/L, 6 μmol/L, 4 μmol/L, 2 μmol/L, 1 μmol/L and 0.5 μmol/L) for 24 h, 48 h and 72 h, respectively. Cell viability was detected by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, and the appropriate dosage and time were screened. Transferase-mediated deoxyuridine triphosphate-biotin nick end labeling (TUNEL) was used to stain cells, followed by an investigation on the apoptosis of cells. In the study of molecular mechanism, the expression of p53 in the cells was determined by immunofluorescence, and then apoptosis-related factors, Fas, FasL and Bax, were tested using Real-time polymerase chain reaction (RT-PCR). Finally, the effect of As2O3 on apoptosis-related proteins, caspase-3 and caspase-9, was investigated by Western blotting.

RESULTS

As2O3 could significantly inhibit proliferation of U87 cells, and the result of TUNEL staining displayed As2O3 had the function of inducing apoptosis. Immunofluorescence results demonstrated that p53 was highly expressed in glioma cells, which was reduced after drug administration. The results of detection of apoptosis factors using RT-PCR revealed that mRNA expressions of Fas, FasL and Bax in the glioma cells were distinctly higher than those in the As2O3 group. The result of Western blotting indicated that caspase-3 and caspase-9 proteins were highly expressed in glioma cells. Analysis of variance showed that the difference between the control group and the As2O3 group was statistically significant (p<0.01).

CONCLUSIONS

As2O3 can inhibit proliferation of glioma cells and induce its apoptosis, which may be correlated with down-regulation of expressions of apoptosis-related factors, Fas, FasL and Bax, and apoptosis-related proteins, p53, caspase-3 and caspase-9.

目的

研究三氧化二砷（As2O3）诱导神经胶质瘤细胞凋亡的机制。

材料和方法

采用不同浓度的 As2O3（8μmol/L、6μmol/L、4μmol/L、2μmol/L、1μmol/L 和 0.5μmol/L）处理 U87 细胞 24h、48h 和 72h，筛选合适的剂量和时间。采用 3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四氮唑溴盐（MTT）法检测细胞活力，并进行细胞凋亡检测。在分子机制研究中，采用免疫荧光法检测细胞中 p53 的表达，然后采用实时聚合酶链反应（RT-PCR）检测 Fas、FasL 和 Bax 等凋亡相关因子。最后，采用 Western blot 法检测 As2O3 对凋亡相关蛋白 caspase-3 和 caspase-9 的影响。

结果

As2O3 可明显抑制 U87 细胞的增殖，TUNEL 染色结果显示 As2O3 具有诱导细胞凋亡的作用。免疫荧光结果显示，p53 在神经胶质瘤细胞中高表达，药物处理后表达降低。采用 RT-PCR 检测凋亡相关因子的结果显示，Fas、FasL 和 Bax 在神经胶质瘤细胞中的 mRNA 表达明显高于 As2O3 组。Western blot 结果显示，caspase-3 和 caspase-9 蛋白在神经胶质瘤细胞中高表达。方差分析显示，对照组和 As2O3 组之间的差异有统计学意义（p<0.01）。