Time dependent of epigenetic effect of disulfiram on tumor suppressor gene of RASSF1A in Hela cancer cell line

Seyedeh naghme Noorirad, M. Pourghasem, Farideh Feizi, Z. Abedian, M. Ghasemi, Z. Babazadeh, Navid Rabiee, Cellular
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引用次数: 3

Abstract

Introduction: Cervical cancer is the third most common tumor among women. Surgery, radiotherapy, and chemotherapy are common treatments, however high stage tumors have frequently poor prognosis. Nowadays, the epigenetic reversion introduced as an efficient strategy of treatment of cervical cancer. In the process, inhibitors of DNA methyltransferase (DNMT) induce re-expression of tumor suppressor genes. Among these inhibitors, disulfiram (DSF) has been suggested as non-nucleoside analogous. In this research, we evaluated the epigenetic effect of DSF on demethylation of the tumor suppressor gene, RASSF1A, in Hela cell line. Materials and methods: Hela cells were cultured and treated with different doses from 2.5 to 37.5µM during 24, 48 and 72 hours. MTT assay was carried out to find half maximal inhibitory concentration (IC50). The methylation specific PCR (MSP) assay was applied to evaluate methylation pattern. Results: The IC50 of DSF was determined at the 2.5, 12.5, and 15µM after72 hours. The MSP results showed partial demethylation at mentioned concentrations after 72h but unmethylated band was not observed after 24h. Conclusion: Our findings indicated that, IC50 of DSF exerted a biphasic effect in Hela cell line and at least 72 hours treatment is needed for the epigenetic reversion of DSF on RASSF1Ain Hela cell line.
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双硫仑对Hela癌细胞RASSF1A抑癌基因表观遗传影响的时间依赖性
宫颈癌是女性中第三大常见肿瘤。手术、放疗和化疗是常用的治疗方法,但高分期肿瘤往往预后不良。目前,表观遗传逆转作为一种有效的治疗宫颈癌的策略被引入。在此过程中,DNA甲基转移酶(DNMT)抑制剂诱导肿瘤抑制基因的重新表达。在这些抑制剂中,双硫仑(DSF)被认为是非核苷类似物。在这项研究中,我们评估了DSF对Hela细胞系肿瘤抑制基因RASSF1A去甲基化的表观遗传影响。材料和方法:以2.5 ~ 37.5µM的剂量培养Hela细胞24、48、72小时。MTT法测定半数最大抑菌浓度(IC50)。采用甲基化特异性PCR (MSP)方法评价甲基化模式。结果:测定72小时后,在2.5、12.5、15µM作用下DSF的IC50。MSP结果显示,在上述浓度下,72h后出现部分去甲基化,但24h后未观察到未甲基化条带。结论:我们的研究结果表明,DSF的IC50在Hela细胞系中具有双相作用,并且DSF对RASSF1Ain Hela细胞系的表观遗传逆转至少需要72小时的治疗。
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