Objective To examine the effects of the MEK inhibitor on human pancreatic cancer cells, and to explore the molecular mechanisms. Methods Human pancreatic cancer cell lines CFPANC1, PANC1 and MiaPaCa2 were treated with MEK inhibitor PD98059 or DMSO, the sensitivity was analyzed by an MTT assay, and cell cycle distribution was evaluated by flow cytometry( FCM), The transcriptional level and protein expression of tumor suppressor genes were detected by real-time RT-PCR and western blot respectively. DNA methyltransferase (Dnmt)1, 3a and 3b were also assayed by western blot, The methylation status of the promoter of the p16INK4A gene was assayed by methylation-specific PCR (MSP). Results PD98059 inhibited to various degrees the growth of three pancreatic cancer cell lines, accompanied by G0-G1 cell cycle arrest. PD98059 up-regulated the expression of p16INK4a, p21WAF1, p27KIP1 mRNA, demethylated the hypermethylation status of p16INK4a gene promoter, and decreased Dnmtl and Dnmt3b in CFPANC1 and PANC1 cell lines. PD98059 only increased the expression of p27KIP1, while the changes of p16INK4a, p21WAF1 and Dnmt were less marked in MiaPaCa2 cell line. Conclusions MEK inhibitor PD98059 down-regulate the activation of Dnmt and up-regulate tumor supress genes, along with the inhibition of cell proliferation and cell cycle progression.%目的 观察细胞外信号调节激酶信号通路(MEK)抑制剂对人胰腺癌细胞生长及细胞周期相关的抑癌基因表达的影响。方法 培养人胰腺癌细胞系CFPAC1、PANC1和MiaPaCa2，应用50μmol/L的MEK抑制剂PD98059处理细胞24h，四甲基偶氮唑蓝(MTT)法检测细胞增殖，流式细胞仪分析细胞周期，实时定量PCR法检测p16INK4a、p21 WAF1和p27KIP1 mRNA的表达，蛋白质印迹法检测DNA甲基化酶(Dnmt)1、3a和3b表达，甲基化特异性PCR(MSP)分析p16INK4a基因启动子甲基化状况。结果 PD98059处理24h后，CFPAC1、PANC1和MiaPaCa2细胞的增殖抑制率分别为69％、78％和45％；G0/G1期细胞比例分别从(68.21±0.73)％、(56.54±0.68)％、(54.89±0.79)％增加到(80.37±0.65)％、(72.05±0.52)％、(79.21±0.93)％(P值均＜0.05)；S期和G2/M期细胞比例相应减少。PD98059处理后，CFPAC1、PANC1细胞p27KIP1、p21WAF1和p16INK4a mRNA表达增加，Dnmt1和Dnmt3b蛋白表达减少；p16INK4a启动子甲基化状态被去除。而MiaPaCa2细胞仅p27KIP1 mRNA表达增加；p21WAF1、p16INK4a mRNA和Dnmt表达均无明显变化。结论 MEK通路抑制剂可能通过下调DNA甲基化酶、上调细胞周期相关抑癌基因表达而抑制胰腺癌细胞周期进展和细胞增殖。
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