Mechanism Investigation Of The Apoptosis Induced By Novel Naphthoquinone Derivatives In HRas^G12V Transgenic NIH3T3 Cell Line

The ras gene,a firstly discovered proto-oncogene,mediates cell proliferation,apoptosis and differentiation.The findings show that ras gene mutation has played a catalytic role in the occurrence of cancer,development and metastasis during nearly forty years.It is of great importance of ras gene in biological function to draw scientists’ attentions.Of all the cancer types known to humans,cancer contained ras gene mutation is about 30%.Patient suffered from primary liver cancer that contained H-ras gene mutation is about 70%,moreover,patient suffered from highly metastatic primary liver cancer that contained H-ras gene mutation is about 88%,then mostly brings out ill prognosis and easily recurrent profile.At present,there is no drug candidate for treating primary liver cancer aimed at H-ras gene mutation.Therefore,the aim at this study is to synthesize and screen compounds that specifically inhibited the Ras-AKT mutation according to the structure activity relationships of naphthoquinone derivatives,and to provide theoretical basis for the development of drugs by structure activity relationships analysis,then laying the foundation to the clinical drug development targeted patient suffered from primary liver cancer that contained H-ras gene mutation.In this study,several naphthoquinone derivatives were designed based on the analysis of structure activity relationships,and the intermediate product(5,8-dimethoxy-1,4-naphthoquinone)was synthetized applying chemical synthesis methods,then synthetizing the objective compounds by Michael Addition,further identifying the structures by hydrogen and carbon spectrum;The cytotoxicity of naphthoquinone derivatives were tested for HRasG12 V transgenic NIH3T3 cell line and NIH3T3 cell line in different concentrations by MTT assay,further screening the derivative which specifically induce the death of HRasG12 V transgenic NIH3T3 cell line;The ROS level was detected during cell death by flow cytometry,and the ratio of apoptosis and necrosis in both cell lines administrated by compound were measured though PI/Annexin V assay,then identifying the type of cell death induced by compound.The apoptosis signaling(AKT/GSK3β/β-catenin and AKT/IκBα/NFκB)of Ras downstream were checked in both cell lines administrated by compound though western blotting.The function of compound was confirmed by the comparison of the ROS scavenger(NAC)and the AKT activator(FGF2).The results showed naphthoquinone derivatives substituted for Hydroxyl alkylthiols possessed the highest cytotoxicity,naphthoquinone derivatives substituted for carboxyl possessed the certain cytotoxicity,but there was no difference between two different cells,naphthoquinone derivatives substituted for Alkyl amines possessed the lowest cytotoxicity,the cell death in HRasG12 V transgenic NIH3T3 cell line induced by BZNQ,but it had no cytotoxicity in NIH3T3 cell though MTT assay.The result indicated BZNQ lead to cell death in HRasG12 V transgenic NIH3T3 cell line though the induction of apoptosis,and the ROS level was downregulated;the apoptosis wasn’t induced by the downregulation of ROS level compared with the ROS scavenger(NAC)by flow cytometry;The result revealed BZNQ induced apoptosis through the inhibition of AKT phosphorylation,thus blocking the AKT/GSK3β/β-catenin and AKT/IκBα/NFκB signaling pathway,which further to downregulate anti-apoptotic proteins(Bcl-2 and Bcl-x L).The results illustrated FGF2 promoted the AKT phosphorylation downregulated by BZNQ,and the apoptosis was inhibited induced by BZNQ in HRasG12 V transgenic NIH3T3 cell line though co-processing experiment contained BZNQ and AKT activator(FGF2).The naphthoquinone derivative BZNQ added to benzyl mercaptans had cytotoxicity and specifically induced apoptosis in HRasG12 V transgenic NIH3T3 cell line,while it had no cytotoxicity in NIH3T3 cell line;the ROS level was decreased by BZNQ,but the apoptosis wasn’t induced though the downregulation of ROS level;BZNQ specifically induced apoptosis through the inhibition of AKT phosphorylation activated by HRasG12 V,thus blocking the AKT/GSK3β/β-catenin and AKT/IκBα/NFκB signaling pathway,which further to downregulate Bcl-2 and Bcl-x L in HRasG12 V transgenic NIH3T3 cell line.