大黄素对胰腺癌裸鼠皮下移植瘤抑癌基因去甲基化作用

摘要

目的 通过建立胰腺癌Panc1细胞株皮下移植瘤模型,探讨大黄素对胰腺癌皮下移植瘤抑癌基因p16、RASSF1A、ppENK启动子区CpG岛的去甲基化作用的影响.方法 首先建立人胰腺癌细胞裸鼠模型,后给予不同药物浓度的大黄素(0、30、50、70mg/kg)处理裸鼠,观察大黄素对裸鼠移植瘤生长的影响,采用MSP、RT-PCR以及Western blot法分别检测不同组别移植瘤的3个抑癌基因甲基化状态以及mRNA、蛋白的改变.结果 大黄素能够抑制胰腺癌裸鼠皮下移植瘤的生长,随着大黄素浓度的升高,其对胰腺癌裸鼠移植瘤的生长抑制率逐渐升高,特别50、70mg/kg浓度组与对照组比较,差异有统计学意义(P＜0.05).MSP结果显示大黄素可使裸鼠移植瘤组织的甲基化条带减弱,非甲基化条带增强,同时可以使经RT-PCR和Western blot法结果证实的低表达或无表达的mRNA和蛋白表达增强或者重新表达,与对照组比较,差异有统计学意义(P＜0.05).结论 通过体内实验笔者证实大黄素可以对胰腺癌裸鼠移植瘤抑癌基因p16、RASSF1A、ppENK发挥不同程度的去甲基化作用,使因启动子区甲基化而失表达的抑癌基因重新表达,大黄素抑制胰腺癌裸鼠移植瘤生长可能和其具有对抑癌基因去甲基化作用有关.%Objective To study the demethylation effect of emodin on tumor suppressor gene p16,RASSF1A,ppENKpromoter CpG Island in pancreatic cancer xenografthrough the establishment of pancreatic cancer cell line Pancl subcutaneous xenograft model.Methods After pancreatic cancer xenograft in nude mice model beening established,All the mice were randomly divided into 4,groups and treated intraperitoneally (IP) with saline and various concentrations (30,50,70mg/kg) of emodin.To observe the effect of emodin on tumor growth.MSP was used to detect methylation status changes of tumor suppressor gene p16,RASSF1A,ppENK gene.While mRNA and protein production of the three tumor suppressor genes were evaluated by RT-PCR and Western blotting.Results The emodin can inhibit the growth of the tumor xenografts in nude mice.With the increase of emodin concentration,the growth inhibition rate of pancreatic cancer xenografts in nude mice gradually increased,50mg/kg and 70mg/kg concentrations were compared with control group (P ＜ 0.05).MSP confirmed that the methylated status of the promoter regions of pI6,RASSF1 A,ppENK has decreased,at the same time,the unmethylated status has enhanced.The RT-PCR and Western blotting results confirmed enhanced expression or re-expression of mRNA and proteinin pancreatic cancer xenograf.(compared with control group,P ＜ 0.05).Conclusion Emodin can exert different degrees of demethylation effect on tumor suppressor gene p16,RASSF1 A,ppENK gene in pancreatic cancer xenografts,making the none expressed tumor suppressor gene to re-express.Emodin inhibites pancreatic cancer cells growth,which may be related to its demethylation of tumor suppressor genes.