南方医科大学学报

南方医科大学学报 ›› 2021, Vol. 41 ›› Issue (6): 847-853.doi: 10.12122/j.issn.1673-4254.2021.06.06

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三硫二苄基抑制头颈部鳞状癌细胞HN30的增殖并诱导其凋亡

徐 璐，钱 峰，孙 磊   

  • 出版日期:2021-06-20 发布日期:2021-07-05

Dibenzyl trisulfide inhibits proliferation and induces apoptosis of HN30 cells via Akt/p53 signaling pathway

  • Online:2021-06-20 Published:2021-07-05

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摘要： 目的 探究三硫二苄基（DTS）抑制头颈癌细胞HN30增殖和促进其凋亡的作用机制。方法 通过克隆形成实验检测DTS对不同头颈癌细胞HN30、HN12、SCC25增殖能力的影响，并用MTT实验检测不同浓度DTS对HN30细胞活力的影响；DTS（3、10、30 μmol/L）刺激HN30细胞24 h，经Annexin Ⅴ-FITC/PI双染及JC-1荧光探针染色，采用流式细胞仪检测DTS对HN30 细胞凋亡及线粒体膜电位的影响，并通过免疫印迹实验检测不同浓度 DTS 作用对凋亡相关蛋白 caspase 3、cleavedcaspase-3和Bcl-2表达的影响；通过免疫印迹实验检测HN30细胞在DTS（10 μmol/L）不同作用时间（0、0.5、1、2、4、8、16 h） 下，Akt/p53磷酸化水平的变化。结果 克隆形成实验发现1 μmol/L DTS能够明显抑制头颈癌细胞HN30、HN12及SCC25的增殖。MTT实验发现，与溶剂对照组相比，HN30细胞的细胞活力在DTS作用下呈剂量依赖性降低，在100 μmol/L DTS作用时效果显著（P<0.001）。采用Annexin Ⅴ-FITC/PI双染及JC-1荧光探针染色后，流式细胞术检测发现随着DTS作用浓度增高，HN30细胞的凋亡细胞比例逐渐增高（30 μmol/L DTS作用下，P<0.01），线粒体膜电位逐渐降低（30 μmol/L DTS作用下， P<0.001）。与溶剂对照组相比，DTS 刺激 24 h 后，HN30 细胞中 cleaved caspase-3 的表达升高（30 μmol/L DTS 作用下，P< 0.01），Bcl-2的表达降低（30 μmol/L DTS作用下，P<0.001）。与溶剂对照组相比，10 μmol/L DTS刺激HN30细胞16 h后，Akt磷酸化水平被显著抑制（P<0.001），而p53的磷酸化水平升高（P<0.01）。结论 DTS抑制HN30细胞的增殖，并诱导凋亡，其作用机制可能与Akt/p53信号通路有关。

关键词: 三硫二苄基；头颈部鳞状癌细胞；Akt；p53

Abstract: Objective To explore the effect of dibenzyl trisulfide (DTS) on cell proliferation and apoptosis in human head and neck squamous cell carcinoma (HNSCC) HN30 cells. Methods The effects of DTS on proliferation of HNSCC cell lines HN30, HN12, and SCC25 were examined by assessing colony formation ability of the treated cells. The effect of different concentrations of DTS on viability of HN30 cells was assessed using MTT assay. HN30 cells were treated with 3, 10, or 30 μmol/L DTS for 24 h, and the cell apoptosis and mitochondrial membrane potential (MMP) were detected using flow cytometry with annexin Ⅴ-FITC/PI double staining and JC-1 fluorescent probe staining. Western blotting was performed to determine the protein expressions of caspase-3, cleaved caspase-3 and Bcl-2 in the treated cells. The phosphorylation levels of Akt and p53 in HN30 cells were detected using Western blotting after treatment with 10 μmol/L DTS for 0.5, 1, 2, 4, 8, or 16 h. Results DTS at 1 μmol/L significantly inhibited the proliferation of HN30, HN12 and SCC25 cells as shown by colony formation assay. MTT assay showed that DTS dose-dependently decreased HN30 cell viability as compared with the solvent control group, and 100 μmol/L DTS produced the strongest inhibitory effect (P<0.0001). Treatment with DTS below 30 μmol/L concentration-dependently promoted apoptosis (P<0.01) and lowered the MMP (P<0.01) of HN30 cells, and after treatment for 24 h, the cells showed significantly increased cleaved caspase-3 (P<0.01) and decreased Bcl-2 expression (P<0.01). Treatment with 10 μmol/L DTS for 16 h significantly inhibited Akt phosphorylation (P<0.001) and enhanced p53 phosphorylation (P<0.01) in HN30 cells. Conclusion DTS inhibits proliferation and induces apoptosis of HN30 cells possibly through mechanisms involving the inhibition of Akt and the activation of p53.

Key words: dibenzyl trisulfide; head and neck squamous carcinoma cells; Akt; p53