S8联合小分子化合物Z18的抗肿瘤效应研究

 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。

Synergistic Anticancer Effect of Z18 Combined with S8 in vitro

To study the synergistic anticancer effect of Z18 combined with S8 in vitro against HeLa cell, we firstly determined the cell death rate using the trypan blue exclusion method and the necrotic cell death was identified by flow cytometry. After being confirmed the autophagy induction in Z18-treated cells, we next analyzed LC3-II conversion by the western blotting and used GFP-LC3 or mRFP-EGFP-LC3 expressing cells to check the autophagic activity and the autophagic flux after the combination treatment. Finally, we inhibited the autophagic activity or the autophagic flux by using ATG5 siRNA or CQ and determined the cell death rate after the combination treatment. It is shown that the pharmacologic inhibitor of Fatty Acid Synthase (FAS) S8 would promote the Z18-mediated cell death in HeLa cells (P<0.05) with the increase of the concentration of S8 and the cell would die mainly as a result of necrotic processes. However, when Z18 is combined with S8, S8 has no effect on the autophagic activity induced by Z18, such as LC3-II conversion. Inhibited autophagic activity has no effect on the cell death after the combination treatment. It is suggested that S8 does not change the autophagic activity but increase the cell death rate in HeLa cells. The enhancement of Z18 anti-tumor activity by S8 may be through another pathway of cell necrosis, and is not related to the autophagy pathway.