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为了探讨S8联合小分子化合物Z18的抗肿瘤效应,本研究通过联合用药方式处理HeLa细胞后,采用台盼蓝染色和流式细胞仪对细胞死亡率和死亡方式进行检测。电镜实验证实Z18诱导细胞自噬,并利用Western Blotting检测联合用药后LC3-II的变化和使用转染了GFP-LC3或mRFP-EGFP-LC3的HeLa细胞检测联合用药对细胞自噬和自噬流的影响。最后通过ATG5 siRNA和CQ抑制自噬和自噬流,观察联合用药对细胞的自噬和死亡的变化,进一步研究S8对Z18的抗肿瘤作用的影响。结果显示,S8联合用药后,细胞死亡率显著升高(P<0.05),死亡方式主要以坏死为主。S8明显地促进了Z18对HeLa细胞的毒性,其促进作用具有剂量依赖性。但联合用药并未降低Z18引起的自噬空泡集聚,LC3-II蛋白表达也未发生变化。抑制自噬后,并未影响联合用药促进细胞死亡的结果。因此,Z18在联合使用S8的情况下,细胞自噬水平没有发生明显变化,而细胞死亡比例明显升高,自噬并不影响联合用药诱导的细胞死亡。S8可能通过调节细胞坏死的其他途径进而促进细胞对Z18的敏感性,并进一步使细胞发生坏死。
In order to investigate the antitumor effect of Z18 combined with small molecule compound Z8, we studied the cell death rate and death pattern of HeLa cells by the combination of drug treatment and trypan blue staining and flow cytometry. Electron microscopy confirmed that Z18 induced autophagy. Western Blotting was used to detect the changes of LC3-II after combined treatment. HeLa cells transfected with GFP-LC3 or mRFP-EGFP-LC3 were used to test the effect of the combination therapy on autophagy and autophagy Impact. At last, ATG5 siRNA and CQ were used to inhibit the autophagy and autophagy, to observe the changes of autophagy and death caused by the combined drugs, and to further study the effect of S8 on the anti-tumor effect of Z18. The results showed that after combined treatment with S8, cell death rate was significantly increased (P <0.05), and the main death mode was necrosis. S8 significantly promoted the toxicity of Z18 to HeLa cells in a dose-dependent manner. However, combination therapy did not reduce the concentration of autophagic vacuoles induced by Z18, and the expression of LC3-II protein did not change. Inhibition of autophagy did not affect the combination of drugs to promote cell death results. Therefore, Z18 in the combined use of S8, the level of autophagy did not change significantly, while the proportion of cell death was significantly increased, autophagy does not affect the combination of drug-induced cell death. S8 may further promote cell sensitivity to Z18 by regulating other pathways of cell necrosis and further necrosis of cells.