Effect Of Raddeanin A On The Proliferation Of Human Hepatic Cancer Cell, Induce-cell Apoptosis Activity, Expressions Of VEGF And HIF-1α

In order to study the inhibition mechanism of raddeanin A on human hepatoma HepG2 cells in vitro, the two areas on the following topics for research.Part One. Study the effect of raddeanin A on the proliferation of human hepatic cancer cell, induce-cell apoptosis activity in HepG2Objective: To study the effect of raddeanin A on the proliferation of human hepatic cancer cell, induce-cell apoptosis activity in HepG2.Methods:MTT assay and colonogenic assay were used to detect the cell viability inhibitory effect of raddeanin A on HepG2 cell. The apoptosis of HepG2 cell by raddeanin A was studied by acridine orange/ethylene dibromide(AO/EB) and DNA agarose gel electrophoresis technique.Results:(1) The raddeanin A has significant inhibition of proliferation for HepG2 in vitro and the 50% inhibition concentration (IC50) was 8.94microg/mL; HepG2 were co-cultured with 5, 10, 20 and 40ug/mL raddeanin A for 72 h, against the cells proliferation with inhibitory rate of (24.81±0.55)%, (39.17±1.30)%, (62.45±7.56)%, and (79.93±5.48)% at 24h; (35.67±0.81)%, (49.73±9.18)%, (71.62±1.03)%, and (87.06±1.84)% at 48h; (42.52±1.31)%, (59.75±7.47)%, (78.85±3.15)%, and (91.36±0.84) % at 72h, respectively. Each dose group in comparison with the vehiculum group, were significant differences in various time point(P<0.001). Colonogenic assay demonstrated that the colony-forming rate were 15.73%, 7.2%, and 3.33%, respectively, via colony-forming inhibitory rate were (66.75±1.66)%, (84.25±3.11)%, and (92.55±2.33)%, respectively, compared with the control group were significantly different(P<0.001). (2) Raddeanin A can induce HepG2 cell apoptosis.Conclusion:(1) Raddeanin A inhibited the proliferation of HepG2 cell growth and at certain range the inhibitory effects were depended on the medicine concentration and treating times. (2) Raddeanin A has strong effect of anti-tumor activity and potential medicinal value. (3) Raddeanin A can induce HepG2 apoptosis. Part Two. Study the effect of raddeanin A on the expressions of vascular endothelial growth factor(VEGF) and hypoxia-inducible factor-1α(HIF-1α) in HepG2Objective:To study the effect of raddeanin A on the expressions of vascular endothelial growth factor (VEGF) and hypoxia-inducible factor-1α(HIF-1α) in HepG2.Methods:ELISA was used to determine the protein lever of VEGF secreted by HepG2 cell. RT-PCR was used to detect mRNA levels of VEGF and HIF-1αin HepG2 cell. The protein lever of VEGF and HIF-1αin HepG2 cell were detected by Western Blotting. Results:(1) Treated with Raddeanin A(5,10,20μg / mL) at 24h, the secretion of VEGF was reduced with dose-dependent, (P = 0.000); (2) Raddeanin A reduced mRNA level of VEGF and HIF-1αin HepG2 cell.①HepG2 were co-cultured with 0, 5, 10, and 20ug/mL raddeanin A for 24h , the mRNA level of HIF-1αand VEGF were decreased. The gray scale of HIF-1αratio toβ-actin were (0.65±0.07), (0.43±0.08), (0.19±0.03), and (0.08±0.02), ( P = 0.000); The gray scale of VEGF ratio toβ-actin were (0.96±0.07), (0.75±0.09), (0.55±0.10), and (0.32±0.11), (P = 0.000), respectively.②10μg/mL raddeanin A deal with HepG2 cells for 0h, 6h, 12h, 24h, the mRNA level of HIF-1αand VEGF were decreased. The gray scale of HIF-1αratio toβ-actin were (0.73±0.08) , (0.44±0.12), (0.24±0.09), and (0.07±0.03), (P = 0.000); The gray scale of VEGF ratio toβ-actin were (0.77±0.05), (0.53±0.09), (0.32±0.12), and (0.15±0.04), (P = 0.000), respectively. (3) Raddeanin A suppressed HIF-1αand VEGF protein expression.①HepG2 cells were co-cultured with different dose( 0, 5, 10, and 20ug/mL ) for 24h , the protein level of HIF-1αand VEGF were decreased. The gray scale of HIF-1 ratio toβ-actin were (0.84±0.08), (0.39±0.06), (0.28±0.04), and (0.08±0.03),(F=103.37 P=0.000);The gray scale of VEGF ratio toβ-actin were (2.08±0.28), (1.43±0.22), (0.72±0.15), and (0.17±0.10),( P=0.000), respectively.②10μg/mL raddeanin A deal with HepG2 cells at 0h, 6h, 12h, 24h, the protein level of HIF-1αand VEGF were decreased. The gray scale of HIF-1αratio toβ-actin were (2.31±0.33), (1.70±0.17), (1.30±0.96), and (0.09±0.13),( P=0.000);The gray scale of VEGF ratio toβ-actin were (2.47±0.05), (1.36±0.02), (0.30±0.02), and (0.07±0.02),(P=0.000), respectively. Conclusion:(1) Raddeanin A inhibited the expression of HIF-1αand VEGF mRNA, and at certain range the inhibitory effects were depended on the medicine concentration and treating times. (2) Raddeanin A inhibited the expression of HIF-1αand VEGF protein, and at certain range the inhibitory effects were depended on the medicine concentration and treating times. (3) Raddeanin A can inhibit tumor cells may be for inhibition the growth neovascularization.