Study On The Effects And Mechanisms Of Dihydroartemisinin On The Biological Behavior Of Hepatocellular Carcinoma

Part1.Molecular mechanism of Dihydroartemisinin to induce apoptosis and inhibit migration in human liver cancer cell line Huh-7 with mutant TP53Objective:To investigate the effect and molecular mechanism of DHA on the proliferation, apoptosis, and migration of human liver cancer cell (Huh-7) with mutant TP53 in vitro.Methods:Huh-7 with mutant TP53 cell line were cultured in Dulbecco’s modified Eagle’s Medium(DMEM)/high-glucose medium and Roswell Park Momorial Insitute-1640 medium supplemented with 10% fetal bovine serum, 100mg/ml penicillin and 100mg/ml stretomycin at 5% CO2 and 37℃.Huh-7 with mutant TP53 cells were exposed to DHA,EPO for 48h, and the MTT assay was used to observe the effect of DHA, EPO on the proliferation of Huh-7 with mutant TP53 cells. the flow cytometry was used to verify their effect on the apoptosis of Huh-7 with mutant TP53 cells. Scratch method was used to observe the cell migration ability.Immunofluorescence assay were used to observe the effect on the DNA damage.Then the phosphorylation levels of H2AX and STAT3 of Huh-7 with mutant TP53 cells was detected by Western blot.Results:DHA significantly inhibited the growth of Huh-7 cell and the inhibition was dependent on the drug concentration(P<0.05), The IC50 of Huh-7 cell lines by DHA for 48h were 10.6±1.2μmol/L; the results of the flow cytometry showed that DHA can promote the apoptosis of Huh-7 cell. DHA (3.15±0.39) and EPO (3.15±0.39) increases the apoptosis respectively(P<0.05), compared with the control group (1.03±0.29); The results of the Scratch method showed that DHA can inhibited the migration rate of cells crawl toward the scratches, compared with the control group and EPO group; DHA increased the phosphorylated H2AX levels and decrased the phosphorylated STAT3 levels,but the expression of STAT31evels were not effected by DHA.Conclusions:For Huh-7 with mutant TP53 cells,DHA can inhibit their growth ability, and the inhibition might be related to up-regulation the phosphorylation level of H2AX; DHA can promote their apoptosis ability, and the pro-apoptosis was associated with down-regulation the phosphorylation level of STAT3.Part2.Molecular mechanism of Dihydroartemisinin combined with Cisplatin to inhibit proliferation and induce apoptosis in human liver cancer cell line H uh-7 with mutant TP53Objective:To investigate the effect and molecular mechanism of DHA combined with Cisplatin on the proliferation, apoptosis of human liver cancer cell (Huh-7) with mutant TP53.Methods:Huh-7 with mutant TP53 cell line were cultured in Dulbecco’s modifi ed Eagle’s Medium(DMEM)/high-glucose medium and Roswell Park Momorial In situte-1640 medium supplemented with 10% fetal bovine serum,100mg/ml penicill in and 100mg/ml stretomycin at 5% CO2 and 37℃. the MTT assay was used to observe the effect of DHA, DDP and DHA+DDP on the proliferation of Huh-7 with mutant TP53 cells; the median-effect principle were used to analysis the Inf luence curve of single drug and combination therapy on the growth of tumor cel 1s; the expression levels of bax, bcl-2 and the phosphorylation levels of STAT3 AKT, ERK1/2 of Huh-7 with mutant TP53 cells was detected by Western blot.Results:DHA significantly inhibited the growth of Huh-7 cells and the inhibition was dependent on the drug concentration (p<0.05); the results of the median-ef fect principle showed that DHA had a synergistic effect on the cytotoxicity of D DP in Huh-7 cells, the combination index is 0.667; the results of the West blot showed that DHA and DDP can both down-regulate the expression of bax,, and down-regulate the phosphorylated AKT、ERK、STAT3 levels, The combined effe ct of two drugs is more significant (p<0.05);Conclusions:DHA had a synergistic effect on the cytotoxicity of DDP in Huh-7 cells and Inhibit of cell cycle progression, the pro-apoptosis was associated with down-regulation the expression of baxand down-regulation the phosphorylation lev els of AKT、ERK、STAT3.Part3. Association of hOGG1 Ser326Cys polymorphism with susceptibility to hepatocellular carcinomaObjective:Since, the relationship between hOGG1 Ser326Cys polymorphism and HCC was inconsistent in the recent literatures. The present meta-analysis based on previous studies was to obtain precise estimation on the issue.Methods:A computer search was carried out from PubMed, CBM and EMBASE databases. A total of nine case-control publications with 2583 HCC patients and 2271 controls were included in the meta-analysis. Pooled odds ratios (ORs) and 95% confidence intervals (CIs) were calculated to evaluate the relationship of Ser326Cys polymorphism and HCC susceptibility. Z test was used to assess the significance of pooled OR. The fixed-effect model or random-effect model was employed according to heterogeneity.Results:Overall, hOGG1 Ser326Cys polymorphism was in relation with increased risk for HCC under the following genetic models:GG versus CC (OR=2.51,95% CI=1.67-3.78); GG versus CG + CC (OR=2.27,95% CI=1.57-3.30); GG + CG versus CC (OR=1.13,95% CI=1.03-1.24). The subgroup analysis by ethnicity suggested that high risk for HCC was observed in Asians with GG and GG + CG genotype (GG versus CC:OR=2.17,95% CI=1.49-3.17; GG versus CG + CC:OR=1.96,95% CI=1.41-2.73; GG+ CG versus CC:OR=1.13,95% CI=1.03-1.25). For subgroup analysis based on source of control, GG genotype of Ser326Cys was significantly associated with HCC risk in hospital-based (HB) controls (GG versus CC:OR=2.31,95% CI=1.50-3.56; GG versus CG + CC:OR=2.17,95% CI= 1.44-3.28), as well as in population-based (PB) models (GG vs. CC:OR=2.80,95% CI=1.16-6.77; GG versus CG+CC:OR=2.39, 95% CI=1.08-5.30).Conclusions:According to the results, The hOGG1 Ser326Cys polymorphism of chemosensitivity was associated with increased risk of HCC.