Effect Of Recombination Onconase With Human Serum Albumin Fusion Protein On Apoptosis Of Rat Hepatoma Cell RH-35

Onconase(ONC),which was a native protein extracted from embryos of the Northern leopard frog,and has strong cytotoxicity effect on various tumor cells and solid tumors in vitro and in vivo.and was the uniqueness ribonuclease which has enter clinical trials.ONC that has been used in clinical trial is purified from embryos of Rana pipiens by a time-consuming and laborious process,It is hard to meet the clinical need beacuse of icomplexing preparation technics and difficulty of obtaining.In order to get high expression of fusion protein,Human serum albumin(HSA)it has important biological function and extensive clinical applications,thus it has been expressed sucessfully in P.pastoris systerm,and is regarded an ideal carrier.In this paper,four linker peptides,including end to end,five amino acids of Gly4Ser1,ten amino acids of(Gly4Ser1)2 and fifteen amino acids of(Gly4Ser1)3,were inserted into the fusion geneHSA-ONC genes,all the referred to as rHSA-M(0,1,2,3)-ONC(M=Gly4Ser1).To investigate the apoptosis and cell growth inhibition in vitro effects of recombination onconase(rONC)with Human serum albumin fusion protein on rat hepatoma cell RH-35.After RH-35 cell in vitro were treated with various concentrations of rONC and rHSA-M(0,1,2,3)-ONC.The proliferation rates of the RH-35 cells in various groups were detected by SRB;the apoptotic rates of the RH-35 cells in various groups were analyzed by flow cytometry;the mRNA and protein expressions of BCL-2、Bax of the RH-35 cells in various groups were detected by RT-PCR and Western Blot;the migration activity of the RH-35 cells in various groups were detected by wound healing test.The proliferation of RH-35 cell were significantly inhibited after treated with rONC and rHSA-M(0,1,2,3)-ONC for 24 hours and this inhibitory action was positive correlated with time and dose-dependent(P<0.05).The inhibition rate of cells treated with rONC was obviously increased than that treated with rHSA-M(0,1,2,3)-ONC.The inhibition rate of cells treated with rHSA-M0-ONC was lowest among a variety of drugs treatments.The proportion of cells in G0/G1 phases remarkably rise after 24 h treatment with rONC,which is more than treatment with rHSA-M(0,1,2,3)-ONC.Cell nucleus splitting and chromatin were found in the cells.The cell apoptosis rate was also increasing.The apoptosis rate of cells treated with rONC was obviously increased than that treated with rHSA-M(0,1,2,3)-ONC The apoptosis rate of cells treated with rHSA-M0-ONC was lowest among a variety of drugs treatments.rONC and rHSA-M(0,1,2,3)-ONC could down-regulate the expression level of BCL-2 and up-regulate BAX gene levels and protein levels.The inhibition of cell migration activity treated with rONC was obviously increased than that treated with rHSA-M(0,1,2,3)-ONC.As the growth of the connecting peptide on the inhibition of cell proliferation and the promotion of cell apoptosis was more significant.So we can draw the conclusion that rONC and rHSA-M(0,1,2,3)-ONC affect the growth and apoptosis of the RH-35 cell,But the inhibition of proliferation and the promotion of apoptosis is different,rHSA-M(0,1,2,3)-ONC biological activity related to the length of connecting peptide.As the growth of the connecting peptide,the inhibition of cell proliferation and the promotion of cell apoptosis gradually strengthened.rONC and rHSA-M(0,1,2,3)-ONC could inhibit the growth and induce apoptosis of RH-35 cell in vitro due to the down-regulated expression of BCL-2 and up-regulated expression of BAX.Apoptosis mechanism may be related to the expression of BAX and BCL-2.