非甾体抗炎药对胃癌细胞凋亡的诱导及相关基因表达的调控

Regulation of non steroidal anti inflammatory drugs on apoptosis and apoptosis related genes of gastric cancer cell line

目的明确非甾体抗炎药(NSAID)对胃癌细胞凋亡的影响及凋亡相关基因表达的调控。方法应用MTT比色法检测NSAID对胃癌细胞生长活力的影响;吖啶橙/溴化乙锭(AO/EB)双染色、AnnexinV/PI双染色、共聚焦显微镜、流式细胞术检测细胞凋亡;RTPCR、Westernblot法检测凋亡相关基因bcl2、bax表达水平的改变。结果吲哚美辛(Indo)和阿司匹林(Asp)对胃癌细胞株AGS均有生长抑制作用,且呈时间/浓度依赖性;在药物作用6～24h生长抑制率改变最为明显;NSAID使AGS细胞发生细胞凋亡的形态学改变,Indo800μmol/L作用24h凋亡率为9.34%±1.99%,48h为38.97%±3.36%;Asp8mmol/L48h凋亡率为17.60%±3.30%,Indo的凋亡诱导作用更为明显;随着药物作用时间的延长,bcl2mRNA表达逐渐减弱,bax基因及蛋白表达逐渐增强,在药物作用6～24h改变最为明显,Bcl2蛋白未检测到。结论NSAID可诱导胃癌细胞凋亡,为NSAID的抗肿瘤应用提供了理论依据;NSAID可能通过调控bcl2、bax的基因及蛋白水平而诱导凋亡。

Objective To investigate whether non steroidal anti inflammatory drugs(NSAID) can induce apoptosis of gastric cancer cell line and elucidate the regulation of NSAID on bcl 2 family genes. Methods The anti proliferative effect of NSAID was measured by MTT assay. Apoptosis was determined by acridine orange/ethidium bromide(AO/EB) double staining, Annexin V/PI double staining, laser scanning confocul cytometry (LSC), and flow cytometry (FCM). Alteration of mRNA of bcl 2 and bax genes was detected by reverse transcription polymerase chain reaction (RT PCR). Results Both indomethacin (Indo) and aspirin (Asp) could inhibit the growth of AGS gastric cancer cells in a time/dose dependent manner. The anti proliferative effect was more obvious during 6 24 h. AGS cells appeared notable apoptosis after incubated with Indo 800 μmol/L or Asp 8 mmol/L for 24 h, the apoptosis rate was 9.34%±1.99% in 24 h and 38.97% ±3.36% in 48 h in Indo group, and 17.60%±3.30% in 48 h in Asp group. The apoptosis inducing effect of Indo was stronger than that of Asp. The bax mRNA kept increasing, while the bcl 2 gene kept decreasing during NSAID treatment. The Bax protein increased after both Indo and Asp treatment, while the Bcl 2 protein was undetectable, and the tendency was more obvious during 6 24 h. Conclusions Both Indo and Asp could induce apoptosis of AGS gastric cancer cells, which supply a further theoretical foundation to the anti cancer use of NSAID. One of the major pathways that mediated the anti tumour response of Indo and Asp in gastric cancer cells was through up regulation of bax and down regulation of bcl 2 genes expression.