华蟾素注射液对人肝癌HepG-2细胞DNA拓扑异构酶Ⅰ的影响

The effect of cinobufacini injection on DNA topoisomerase Ⅰ of human hepatocellular carcinoma HepG-2 cells

背景与目的:华蟾素注射液是传统抗肿瘤中药制剂,目前药效机制尚不明确。本研究旨在探讨华蟾素注射液对DNA拓扑异构酶Ⅰ(topoisomerase Ⅰ,TOPOⅠ)的影响。方法:采用噻唑蓝还原法(MTT)观察华蟾素注射液对人肝癌HepG-2细胞增殖的影响;采用流式细胞术(FCM)检测对HepG-2细胞周期的影响;采用RT-PCR法检测对HepG-2细胞TOPOⅠmRNA表达的影响;采用TOPOⅠ介导的负超螺旋PBR322 DNA解旋反应检测华蟾素对TOPOⅠ酶活性的影响;采用负超螺旋PBR322 DNA检测华蟾素对DNA的直接抑制作用。结果:华蟾素注射液对HepG-2细胞增殖具有抑制作用,其抑制效应具有时间和剂量依赖性;并将HepG-2细胞阻滞于S期;RT-PCR检测表明,华蟾素注射液可下调TOPOⅠmRNA表达,表现浓度依赖效应;对TOPOⅠ介导的负超螺旋PBR322 DNA解旋反应有抑制作用;对负超螺旋PBR322 DNA没有直接抑制作用。结论:华蟾素注射液能够抑制HepG-2细胞增殖,影响DNA拓扑异构酶Ⅰ酶的mRNA表达及活性可能为机制之一。

Background and purpose：The cinobufacini injection is a traditional antitumor drug.However,its mechanism is still unclear.The purpose of this study was to observe the effect of cinobufacini injections in DNA TOPOⅠ of human hepatocellular carcinoma HepG-2 cells.Methods：The cells that were proliferated were assessed by MTT assay.Cell cycles were shown through FCM.TOPOⅠmRNA expression was analyzed through RT-PCR.The activity of TOPOⅠ was measured by TOPOⅠ mediated super coiled PBR322 relaxation.Supercoiled PBR322 was also used to determine the direct DNA breakages.Results：Cinobufacini injections significantly inhibited HepG-2 cells proliferation in ways that were dependent on dosages and time.Induced tumor cells arrest at the S-phase.TOPOⅠ mRNA expression decreased in a manner that was dependent on dosages which inhibited the TOPOⅠ mediated DNA relaxations.However,the cinobufacini injections could not directly induce DNA breakage at any concentration.Conclusion：Cinobufacini injections can inhibit human hepatocellular carcinoma HepG-2 cells proliferation.The regulation of topoisomerase Ⅰ activity and mRNA expression may be one of the mechanisms that causes the cinobufacini injection to contribute against tumor.