三白草酮调节PD-1/PD-L1信号通路对食管癌细胞恶性生物学行为及免疫抑制的影响

Effects of Sauchinone on the Malignant Biological Behaviors and Immune Suppression of Esophageal Cancer Cells by Regulating the PD-1/PD-L1 Signaling Pathway

该文旨在探究三白草酮(Sau)对食管癌细胞恶性生物学行为、免疫抑制的影响以及对PD-1/PD-L1信号通路的调控机制。培养人食管癌细胞KYSE-510和正常食管上皮细胞系Het-1A,用不同浓度Sau处理细胞,以CCK-8法检测细胞存活率。将KYSE-510细胞随机分为KYSE-510组、Sau低浓度(Sau-L)组、Sau中浓度(Sau-M)组、Sau高浓度(Sau-H)组和Sau-H+pcDNA-PD-1组。采用CCK-8法检测各组KYSE-510细胞存活率;采用平板克隆实验检测各组KYSE-510细胞的克隆形成情况;采用Transwell实验检测各组KYSE-510细胞的迁移及侵袭情况;采用流式细胞术检测各组KYSE-510细胞的凋亡情况;采用免疫印迹法(Western blot)检测各组细胞中PD-1/PD-L1信号通路相关蛋白以及血管内皮生长因子(VEGF)、转化生长因子β1(TGF-β1)的相对表达水平。结果显示,Sau对KYSE-510细胞增殖的抑制作用比对Het-1A更显著。与KYSE-510组相比,Sau-L组、Sau-M组和Sau-H组细胞存活率、克隆形成数量、迁移和侵袭细胞数以及PD-1、PD-L1、VEGF、TGF-β1蛋白表达水平均降低(P<0.05),细胞凋亡率升高(P<0.05);而在高浓度Sau处理的KYSE-510细胞中过表达PD-1则逆转了以上指标的变化趋势(P<0.05)。总之,Sau能够抑制食管癌细胞的恶性生物学行为,解除免疫抑制,其机制可能与PD-1/PD-L1信号通路被抑制有关。

The objective of this study was to investigate the effects of Sau(sauchinone)on the malignant biological behaviors and immune suppression of esophageal cancer cells,and its regulatory mechanism on the PD-1/PD-L1 signaling pathway.Human esophageal cancer cells KYSE-510 and normal esophageal epithelial cell line Het-1A were cultured and treated with different concentrations of Sau.The cell survival rate was detected by CCK-8 method.KYSE-510 cells were randomly separated into KYSE-510 group,Sau low-concentration(Sau-L)group,Sau medium-concentration(Sau-M)group,Sau high-concentration(Sau-H)group,and Sau-H+pcDNA-PD-1 group.CCK-8 method was applied to detect the survival rate of KYSE-510 cells.Plate cloning experiment was applied to detect the clonogenesis ability of KYSE-510 cells.Transwell experiment was applied to detect the migration and invasion abilities of KYSE-510 cells.Flow cytometry was applied to detect the apoptosis of KYSE-510 cells.Western blot was applied to detect the relative expression levels of PD-1/PD-L1 signaling pathway-related proteins,VEGF(vascular endothelial growth factor),and TGF-β1(transforming growth factor-β1).The results showed that the inhibitory effect of Sau on the proliferation of KYSE-510 cells was more significant than that of Het-1A.Compared with the KYSE-510 group,the cell survival rate,clone formation quantity,migration and invasion cell numbers,the protein expression levels of PD-1,PD-L1,VEGF,and TGF-β1 in the Sau-L,Sau-M,and Sau-H groups all reduced(P<0.05),while the cell apoptosis rate increased(P<0.05).However,overexpression of PD-1 in KYSE-510 cells treated with high concentration of Sau reversed the trend of changes of the above indica-tors(P<0.05).In conclusion,Sau can inhibit the malignant biological behaviors of esophageal cancer cells,relieve immune suppression,and its mechanism may be related to the inhibition of the PD-1/PD-L1 signaling pathway.