非甾体消炎药诱导胃癌细胞凋亡及其机制的研究

NSAIDs induced apoptosis of gastric cancer cell lines and the mechanism

目的 :明确非甾体消炎药 (NSAIDs)能否诱导胃癌细胞凋亡 ;明确不同的p5 3基因表型对NSAIDs诱导的细胞凋亡是否有影响 ;明确NSAIDs对细胞凋亡相关基因Bcl 2及Bax表达的调控。方法 :通过MTT比色法检测NSAIDs对细胞生长活力的影响 ;应用丫啶橙 (AO)染色、Annexin V/PI双染色、共聚焦显微镜、流式细胞术检测细胞凋亡 ;应用RT PCR、Western blot方法检测bcl 2、bax基因及蛋白水平的改变。结果 :NSAIDs药物吲哚美辛 (Indo)和阿司匹林 (Asp)对胃癌细胞株AGS(p5 3+/ +)、MKN2 8(p5 3 / )均有显著的生长抑制作用 ,且呈时间 /浓度依赖性增强 ;在相同作用条件下 ,AGS细胞的凋亡率明显高于MKN2 8细胞 ,处理组MKN2 8细胞凋亡数量虽有所增多 ,但与正常对照组相比不具有统计学意义 ;随着药物作用时间的延长 ,Bcl 2基因mRNA表达逐渐减弱 ,Bax基因及蛋白表达逐渐增强 ,在药物作用 6～ 2 4小时改变最为明显。结论 :一定浓度的NSAIDs作用一定时间后 ,可诱导胃癌细胞凋亡 ,这为NSAIDs的抗肿瘤应用增加了理论依据 ;NSAIDs不能诱导p5 3基因突变的MKN2 8胃癌细胞株发生显著的凋亡 ,p5 3基因突变可能阻断了NSAIDs的凋亡诱导效应 ;NSAIDs可能通过调控Bcl 2。

Purpose:To investigate whether NSAIDs can induce apoptosis of gastric cancer cell lines,to observe the effect of different p53 genotype on NSAIDs induced apoptosis,to elucidate the regulation of NSAIDs on expression of apoptosis related genes. Methods:The anti-proliferative effect of NSAIDs was measured by MTT assay.Apoptosis was determined by acridine orange(AO) staining,Annexin-V/PI double staining,laser scanning cytometry(LSC) and flow cytometry (FCM).Alteration of bcl-2 and bax genes was detected by reverse transcription polymerase chain reaction (RT-PCR).Protein expression was determined by Western-blotting.Results: Indomethacin (Indo) and Aspirin (Asp) inhibited both AGS(wild-type p53)and MKN28(mutant p53) gastric cancer cell lines growth in a time/dose dependent manner.AGS cell line was more sensitive to NSAIDs,which apoptosis percentage was significantly higher than MKN28 under the same condition. The percentages of apoptosis of MKN28 were somewhat higher among NSAIDs treated groups compared with the normal control group,but these slight differences were not statistically significant. The bax mRNA kept increasing since NSAIDs treatment accompanied by a decrease of bcl-2 gene.The Bax protein increased after treatment while the Bcl-2 protein was undetectable, which tendency was more obvious during 6-24hs.Conclusion: Both Indo and Asp could induce apoptosis in gastric cancer cell lines,which adds further theoretical foundation to the anti-cancer use of NSAIDs.NSAIDs could not induce notable apoptosis of MKN28,which indicated the mutant p53 gene perhaps blocked NSAIDs induced apoptosis.One of the major pathways that mediated the anti-tumour response of NSAIDs in gastric cancer cells was through up-regulation of bax and down-regulation of bcl-2 genes and/or proteins.[