The Effects Of Tripeptide Tyroserleutide On The Cell Cycle Of Human Hepatorcllular Carcinma And Its Preliminary Mechanisms

Objective:To explore the inhibition effects of YSL on the cell cycle of human hepatocellular carcinoma BEL-7402 cells, and to approach its preliminary mechanisms by affecting cell cycle control factors in tumor cells.Methods:1. The effect of YSL on cell cycle of G0/G1 phase and S phase of human hepatocellular carcinoma BEL-7402 cells was assayed by FCM method.2. H2A.X phosphorylation flow cytometry assay and comet assay were used to estimate the double-stranded DNA breaks of BEL-7402 cells induced by YSL.3. The activity of CDK4/cyclin D kinase of G0/G1 phase was determined by colorimetric methods; Western blot was applied for CDK4 and cyclin D protein expression of human hepatocellular carcinoma BEL-7402 cells by YSL in vitro; and Real-time PCR was applied for CDK4 and cyclin D mRNA level of human hepatocellular carcinoma BEL-7402 cells by YSL in vitro.4. The activity of CDK2/cyclin E kinase of G0/G1 phase was determined by colorimetric methods; Western blot was applied for CDK2 and cyclin E protein expression of human hepatocellular carcinoma BEL-7402 cells by YSL in vitro; and Real-time PCR was applied for CDK2 and cyclin E mRNA level of human hepatocellular carcinoma BEL-7402 cells by YSL in vitro.5. The activity of CDK2/cyclin A kinase of S phase was determined by colorimetric methods; Western blot was applied for CDK2 and cyclin A protein expression of human hepatocellular carcinoma BEL-7402 cells by YSL in vitro; and Real-time PCR was applied for CDK2 and cyclin A mRNA level of human hepatocellular carcinoma BEL-7402 cells by YSL in vitro.Results: 1. Cells were synchronized on G0/G1 phase by serum starvation method, YSL (6.4mg/ml,3.2mg/ml) could increase the percentage of Go/G1 phase cells, decreasing the percentage of S phase cells. Cells were synchronized on G1/S phase by double thymidine blocking method, YSL could increase the percentage of S phase cells, decreasing the percentage of G2/M phase cells.2. YSL(6.4mg/ml,3.2mg/ml) could increase the percentage of positive cells of phosphorylation histone H2A.X. By comet assay YSL also could induce DNA damage in human hepatocellular carcinoma BEL-7402 cells.3. YSL (6.4mg/ml,3.2mg/ml) could remarkably inhibit the activity of CDK4/cyclin D kinase. YSL could significantly inhibited CDK4 and cyclin D protein expression, but could not affect mRNA level of CDK4 and cyclin D.4. YSL (6.4mg/ml,3.2mg/ml) could remarkably inhibit the activity of CDK2/cyclin E kinase. YSL could significantly inhibited CDK2 and cyclin E protein expression, but could not affect mRNA level of CDK2 and cyclin E.5. YSL (6.4mg/ml,3.2mg/ml) could remarkably inhibit the activity of CDK2/cyclin A kinase. YSL could significantly inhibited CDK2 and cyclin A protein expression, but could not affect mRNA level of CDK2 and cyclin A.Conclusion:YSL could arrest the cell cycle of human hepatocellular carcinoma BEL-7402 cells on G0/G1 phase and S phase. The mechanisms maybe by DNA damage caused by YSL, CDK/cyclin kinase complexes firstly experienced the signal of DNA damage, then the activity of CDK/cyclin kinase complexes were decreased, which induce the cell cycle arrested. And at last, YSL induces tumor cells apoptosis.