尿多酸肽联合丁酸钠抑制乳腺癌细胞生长的体外实验研究

In vitro study about the inhibitory effect of CDAⅡ in combination with sodium butyrate on breast cancer cells

目的:探讨尿多酸肽(uroacitide,CDAⅡ)与组蛋白去乙酰化酶抑制剂丁酸钠(sodium butyrate,SB)联合应用能否使人乳腺癌细胞MCF7维甲酸β2受体(retinoic acid receptorβ2,RARβ2)基因启动子去甲基化并诱导基因表达;以及联合用药能否协同抑制细胞生长、诱导细胞调亡。方法:培养乳腺癌细胞MCF7,分为CDAⅡ、SB两个单药组和两药联合组。分别用甲基化特异性聚合酶链反应(methylation-specific polymerase chain reaction,MSP)和反转录-聚合酶链反应(reverse transcription polymerase chain reaction,RT-PCR)检测RARβ2基因启动子甲基化状态和表达;Hoechst33342/碘化丙啶(propidiumiodide,PI)染色和DNA末端原位标记染色法(terminal deoxynucleotidyl trans-ferase mediated dUTP-biotin nick end labeling,TUNEL)检测调亡;甲基噻唑基四唑(MTT)比色法检测细胞增殖。结果:CDAⅡ或SB单药均不能使RARβ2基因启动子去甲基化,也不能诱导基因表达,联合用药使基因启动子发生部分去甲基化并诱导了基因mRNA的表达;Hoechst33342/PI后,荧光显微镜下可见联合用药组出现明显的凋亡细胞,TUNEL法显示联合用药组细胞凋亡率显著高于两个单药组(39.5%vs5.2%,8.1%,P<0.05);MTT比色法显示与单独用药相比,联合用药对肿瘤细胞的生长抑制作用更显著,两药呈现交互作用(F=15,P<0.05)。结论:CDAⅡ与组蛋白去乙酰化酶抑制剂SB具有协同抑制乳腺癌细胞增殖、诱导细胞凋亡的作用,这可能与两药联合应用能够使RARβ2基因启动子去甲基化并诱导基因表达相关。

Objective：To investigate if the combined use of CDAⅡ and sodium butyrate can induce demethylation and re-expression of retinoic acid receptorβ2（RARβ2）gene in cultured human breast cancer cells MCF7.To explore if the two drugs can inhibit cell growth and induce cell apoptosis synergetically.Methods：MCF7 cell line was treated with CDAⅡ,sodium butyrate,combination of the two drugs respectively.Methylation was assessed by methylation-specific polymerase chain reaction（MSP）for RARβ2 gene.Gene expression was evaluated by reverse transcription polymerase chain reaction（RT-PCR）.Apoptosis was detected by terminal deoxynucleotidyl transferase mediated dUTP-biotin nick end labeling（TUNEL）and Hoechst33342/propidiumiodide（PI）staining.Cell growth inhibition was measured by MTT assay.Results：Neither CDAⅡ nor sodium butyrate induced demethylation and re-expression of RARβ2 gene,Combination of the two drugs partially demethylated gene promoter accompanied by re-expression of RARβ2.The apoptotic cells in the double-drug group were obvious following Hoechst33342/PI staining.The percentage of apoptotic cells in the double-drug group was significantly higher than that of the two single-drug group（39.5% vs 5.2%,8.1%）（P〈0.05）.MTT assay showed that compared with single drug,combination of the two drugs inhibited cell growth more significantly,and the two drugs showed interaction（F=15,P〈0.05）.Conclusion：We showed that CDAⅡ,in combination with histone deacetylase inhibitor sodium butyrate synergetically inhibited the proliferation of MCF7 breast cancer cell line and induced apoptosis,in which demethylation and re-expression of RARβ2 gene induced by combination of the two drugs may be involved.