广藿香醇下调PD-L1的表达对人胃癌细胞抗肿瘤作用的研究

Effect of Patchouli alcohol on anti-tumor effect of gastric cancer cells by down-regulation of PD-L1 expression

目的研究广藿香醇能否通过下调人胃癌细胞中关键促癌因子PD-L1的表达进而抑制人胃癌细胞的恶性行为以及下调PD-L1表达的机制。方法使用不同剂量的广藿香醇作用于常规培养的人胃癌细胞SGC-7901和BGC-823,用CCK-8实验、细胞划痕实验和流式细胞技术分别检测不同浓度的广藿香醇对SGC-7901和BGC-823细胞的增殖能力、迁移能力和凋亡能力的影响,用荧光定量PCR法检测SGC-7901和BGC-823细胞中PD-L1、NF-κB的mRNA表达水平的变化,用Western-blot法检测SGC-7901和BGC-823细胞中PD-L1、NF-κB的蛋白表达水平的变化。结果经实时荧光定量PCR法检测人胃正常上皮细胞GSY和胃癌细胞系细胞BGC-823、MGC-803、SGC-7901、MKN-74中mRNAPD-L1、NF-κB mRNA表达水平,最终筛选出BGC823、SGC7901进行后续实验。与对照组比较,0.08、0.1、0.3、0.5 mmol/L药物组中BGC-823、SGC-7901细胞的增殖能力显著降低,且降低幅度随加药浓度升高而增大。经计算SGC-7901的IC_(50)浓度为0.37 mmol/L,BGC-823的IC_(50)浓度为0.27 mmol/L,因此选择浓度为0.3 mmol/L、0.5 mmol/L的广藿香醇进行后续实验。与人胃癌细胞SGC-7901、BGC-823的对照组比较,广藿香醇各加药组细胞的划痕愈合率均有明显下降,差异有统计学意义(P<0.05),并且0.3、0.5 mmol/L的广藿香醇对BGC823细胞的划痕愈合率的影响更为显著。经AnnexinV-FITC/PI双染后,0.3、0.5 mmol/L广藿香醇作用后能明显诱导SGC-7901、BGC-823细胞发生凋亡。加药组(广藿香醇浓度分别为0.3,0.5 mmol/L)作用于SGC-7901、BGC-823细胞24 h后,细胞凋亡率显著高于对照组(P<0.05),并且广藿香醇对SGC-7901、BGC-823细胞的凋亡作用呈剂量依赖,与对照组比较,0.3、0.5 mmol/L加药组的两种胃癌细胞的凋亡率均显著提高(P<0.05)。在SGC-7901中,与对照组比较,广藿香醇各加药组细胞中PD-L1、NF-κB的mRNA均明显下降(P<0.05);在BGC-823中,与对照组比较,广藿香醇各加药组细胞中PD-L1、NF-κB的mRNA均明显下降(P<0.05)。在SGC-7901细胞中,与对照组比较,广藿香醇各加药组细胞中PD-L1、NF-κB的蛋白表达含量均明显下降(P<0.05);在BGC-823细胞中,与对照组比较,广藿香醇各加药组细胞中PD-L1、NF-κB的蛋白表达含量均明显下降(P<0.05)。结论广藿香醇可以通过下调PD-L1的表达抑制胃癌细胞的恶性行为,并且这种作用机制与广藿香醇抑制胃癌细胞中NF-κB的表达有关。

Objective To investigate the inhibitory effect of Patchouli alcohol on the malignant behaviors of gastric cancer cells by down-regulation of programmed cell death-ligand 1(PD-L1)expression,and its mechanism.Methods In vitro cultured BGC-823 and SGC-7901 gastric cancer cells were treated with different concentrations of patchouli alcohol.The effects of different concentrations of patchouli alcohol on the proliferation,migration and apoptosis of SGC-7901 and BGC-823 cells were detected by the cell counting kit-8(CCK-8)assays,cell scratch test and flow cytometry.The quantitative real time polymerase chain reaction(qRT-PCR)was conducted to detect the level of mRNA expression level of PD-L1 and nuclear factor-kappaB(NF-κB)in SGC-7901 and BGC-823 cells;Western blot was performed to detect the protein expressions of PD-L1 and NF-κB.Results The expression levels of mRNA of PD L1 and NF-κB in normal human gastric epithelial cells GSY and gastric cancer cell lines involving BGC-823,MGC-803,SGC-7901 and MKN-74 were detected by qRT-PCR,BGC-823 and SGC-7901 were finally screened for subsequent experiments.Compared with the blank control group,the proliferative capacity of BGC-823 and SGC-7901 cells in the 0.08mmol/L,0.1mmol/L,0.3mmol/L and 0.5 mmol/L drug groups were significantly decreased,and the degree of decrease rose with increase in the drug concentration.The inhibitory concentration 50%(IC_(50))of SGC-7901 upon calculation was 0.37mmol/L,and that of BGC-823 was 0.27mmol/L.Consequently,Patchouli alcohol with the concentration of 0.3mmol/L or 0.5mmol/L was selected for subsequent experiments.Patchouli alcohol in 0.3mmol/L and 0.5mmol/L after double staining of the Annexin V-Fluorescein Isothiocyanate(FITC)/Propidium Iodide(PI)could significantly induce SGC-7901 and BGC-823 cells to have apoptosis.After 24h,the apoptosis rate of SGC-7901 and BGC-823 cells in the treatment group(the concentration of Patchouli alcohol was 0.3mmol/L and 0.5mmol/L,respectively)were significantly higher relative to the corresponding control group(P<0.05),the effect of Patchouli alcohol on the apoptosis of SGC-7901 and BGC-823 cells was dose-dependent,and the apoptosis rate of SGC-7901 and BGC-823 cells in the treatment group in 0.3mmol/L and 0.5mmol/L was significantly increased compared with the blank group(P<0.05).In the SGC-7901,mRNA of both PD-L1 and NF-κB in the cells of Patchouli alcohol dosing groups decreased significantly compared with the blank group(P<0.05).In BGC-823,mRNA of both PD-L1 and NF-κB in cells of Patchouli alcohol dosing groups decreased significantly compared with the blank group(P<0.05).In SGC-7901 cells,the protein expression content of both PD-L1 and NF-κB in cells of Patchouli alcohol dosing groups decreased significantly compared with the blank group(P<0.05);In BGC-823 cells,protein expression content of both PD-L1 and NF-κB in cells of Patchouli alcohol dosing group decreased significantly compared with the blank group(P<0.05),which was related to the suppression of NF-κB expression in gastric cancer cells by Patchouli alcohol.Conclusion Patchouli alcohol is capable of inhibiting the malignant behavior of gastric cancer cells by down-regulating the expression of PD-L1,and this mechanism is possibly related to the inhibitory effect of patchouli alcohol on NF-κB in gastric cancer cells.