华蟾素抑制胃癌细胞靶点PIM3的实验研究

目的评价华蟾素的有效成分丁硫磷对胃腺癌的抑制作用和可能的作用靶点。方法以胃腺癌AGS细胞为模型,用不同浓度(0、20、50μg/mL)的丁硫磷加以处理。采用非放射性细胞增殖法检测细胞活力,采用乳酸脱氢酶(LDH)活性试剂盒测定LDH活性。Hoechst/碘化丙啶(PI)染色和膜联蛋白V-异硫氰酸荧光素(Annexin V-FITC)/PI染色检测细胞凋亡,并用流式细胞仪进行分析。蛋白质印迹法检测Bcl-2、Bax蛋白的表达水平。通过i TRAQ分析,确定丁硫磷处理后AGS胃腺癌细胞的PIM3表达基因。结果经20、50μg/mL丁硫磷处理胃腺癌AGS细胞24、48 h后细胞活性分别为(107.21±4.35)%、(96.12±3.87)%和(56.29±2.98)%、(64.27±3.91)%,显著低于0μg/mL丁硫磷(120.31±2.35)%、(119.83±3.24)%](P <0.05)。20、50μg/mL丁硫磷处理胃腺癌AGS细胞后LDH水平分别为(297.83±13.24)、(412.32±27.98) U/mg,显著高于0μg/mL丁硫磷(150.27±2.35) U/mg](P <0.05)。20、50μg/mL丁硫磷细胞凋亡率分别为(27.69±3.25)%、(43.78±6.58)%,显著高于0μg/mL丁硫磷(1.08±0.29)%](P <0.05)。20、50μg/mL丁硫磷处理胃腺癌AGS细胞的Bcl-2蛋白表达水平(32.18±3.89、14.12±2.70)显著低于0μg/mL丁硫磷(85.46±5.12),而Bax蛋白表达水平(169.84±16.63、238.91±20.12)显著高于0μg/mL丁硫磷(96.32±6.57)(P<0.05)。20、50μg/mL丁硫磷处理胃腺癌AGS细胞后PIM3 mRNA表达(51.36±6.89、15.26±3.15)和PIM3蛋白(60.28±4.52、18.92±3.42)表达低于0μg/mL丁硫磷(120.31±17.28、120.07±13.54)(P <0.05);且50μg/mL丁硫磷的以上作用较20μg/mL丁硫磷更显著(P <0.05)。结论华蟾素对胃腺癌AGS细胞具有抗癌活性,且呈剂量依赖性,其可能的作用靶点为PIM3。

Cinobufacini bufothionine inhibition against gastric adenocarcinoma cells by targeting PIM3

Objective To evaluate the inhibitory effect and possible target of bufothionine on gastric adenocarcinoma.Methods AGS gastric cell model was used to study,and treated with different concentrations of buthion(0,20,50μg/mL).Cell viability was measured by non radioactive cell proliferation assay,LDH activity was determined by LDH activity Kit.Apoptosis was detected by Hoechst/PI staining and Annexin V-FITC/PI staining,and flow cytometry was used for analysis.The expression level of Bcl-2,Bax proteins was detected using the Western blotting.PIM3 differentially expressed genes in AGS gastric adenocarcinoma cells treated with bufothionine were tested with the iTRAQ analysis.Results Cell viability of gastric adenocarcinoma AGS cells treated with 20 and 50μg/mL buthion for 24 h and 48 h were(107.21±4.35)%,(96.12±3.87)%and(56.29±2.98)%,(64.27±3.91)%,which were significantly lower than 0μg/mL buthion(120.31±2.35)%,(119.83±3.24)%](P<0.05).The LDH levels of gastric adenocarcinoma AGS cells treated with 20 and 50μg/mL buthion were(297.83±13.24)and(412.32±27.98)U/mg,respectively,which were significantly higher than 0μg/mL buthion(150.27±2.35)U/mg](P<0.05).The apoptotic rates of gastric adenocarcinoma AGS cells treated with 20 and 50μg/mL buthion were(27.69±3.25)%and(43.78±6.58)%,respectively,which were higher than the 0μg/mL buthion(1.08±0.29)%](P<0.05).The Bcl-2 protein expression level of gastric adenocarcinoma AGS cells treated with 20 and 50μg/mL buthion(32.18±3.89,14.12±2.70)was significantly lower than 0μg/mL buthion(85.46±5.12),while the Bax protein expression level(169.84±16.63,238.91±20.12)was significantly higher than 0μg/mL buthion(96.32±6.57)(P<0.05).The PIM3 mRNA(51.36±6.89,15.26±3.15)and PIM3 protein(60.28±4.52,18.92±3.42)expression of gastric adenocarcinoma AGS cells treated with 20 and 50μg/mL buthion were lower than 0μg/mL buthion(120.31±17.28,120.07±13.54)(P<0.05);and the above effect of 50μg/mL buthion were more significant than those of 20μg/mL buthion(P<0.05).Conclusion Bufothionine has antitumor activity against gastric adenocarcinoma AGS cells,it was dose-dependent,with the possible target of PIM3.