The Role Of Bcl-2 And Bax In Nitric Oxide Induced Apoptosis Of Human Hepatocellular Carcinoma

ObjectiveTo investigate the role of bcl-2 and bax in nitric oxide induced apoptosis of human hepatocellular carcinoma cells.MethodsThe expression of bcl-2 and bax mRNA in hepatocellular carcinoma cell lines HepG2 and SMMC-7721 was detected by RT-PCR. The recombinant plasmids were constructed by inserting antisense bcl-2 cDNA sequence or antisense bax cDNA sequence into the plasmid pcDNA3.1/Hygro(-). HepG2 cells were transfected with pcDNA3.1/Hygro(-)bcl-2 and pcDNA3.1/Hygro(-)bax by Lipofectamine and postive clones were identified by RT-PCR and immuno-blot. The cloned cells were further investigated for the effects of SNP on proliferation by MTT assay, fluorescent imaging and flow cytometry.1. The levels of bcl-2 and bax mRNA expression were reduced and the ratio ofbax/cl-2 mRNA was elevated in both SMMC-7721 and HepG2 cell lines treated with 1. 0mmol/L SNP. The decreased degree of bcl-2 and bax mRNA expression was smaller in HepG2 cell than that in SMMC-7721 cell.The alteration time of bcl-2 and bax mRNA expression was later in HepG2 cell than that in SMMC-7721.2. The bax and bcl-2 fragment were specifically amplified by RT-PCR and DNAsegment joining technique,then the recombinant plasmids containing antisense bcl-2 or bax cDNA fragment was constructed. The positive recombinant plasmid was screened and digested by two different endonucleases. The DNA sequence of recombinant plasmids were identical with that published by GeneBank .3. HepG2 cells were transfected with the recombinant plasmids. The Hygromycin Bresistence gene fragment in transfected cells was amplified by RT-PCR and the level of bcl-2 and bax expression was lower than that of HepG2 by immuno-blot analysis.4. In four cell lines,the growth and the proliferation of HepG2-bax cells was the fastest, HepG2-bcl-2 were the lowest, HepG2 and HepG2-pcDNA3.1 cells were similar.5. SNP can not only inhibit the growth and proliferation but also induce apoptosis of human hepatocellular carcinoma cells HepG2 > HepG2-bcl-2, HepG2-bax and HepG2-pcDNA3.1. However, the sensitivity of four cell lines to NO was different: HepG2-bcl-2 was the highest, HepG2-bax was the lowest, both HepG2 and HepG2-pcDNA3.1 was similar.Conclusion1. SNP induced apoptosis in human hepatocellular carcinoma cells may be correlated with the change of bcl-2 and bax mRNA expression and increase of the ratio of bax/bcl-2 mRNA.2. The recombinant plasmids containing antisense bcl-2 and bax cDNA fragement were successfully construted.3. Both HepG2-bcl-2 and HepG2-bax were sucessfully cloned, in which the level of bcl-2 and bax expression was lower.4. bcl-2 can inhibit but bax can promote the apoptosis of hepatocellular carcinoma cells induced by NO.In conclusion, these results indicate that bcl-2 and bax may play an important role in NO induced apoptosis.