The Effect Of Ascorbic Acid On The Apoptosis Induced By Arsenic Trioxide In Human Hepatocarcinoma Cells And Normal Hepatic Cells

Part one Cell culture and Morphology testObjective: Normal hepatic L-02 cells and hepatocarcinoma HepG2 cells were cultured in vitro, to investigate the effect of ascorbic acid on the growing inhibition induced by arsenic trioxide in human hepatocarcinoma cells.Methods: Normal hepatic L-02 cells and hepatocarcinoma HepG2 cells were cultured in vitro. The viability of the test cells was assessed with both the Trypan blue exclusion and MTT tetrazolium (3- [4, 5-dimethyl-thiazol -2 -yl] -2, 5 -diphenyltetralzolium bromide, 5.0mg/mL) assays.Results: In comparison with the use of As₂O₃ ( 2.0μmol/L ) separately for various interval, the inhibition ability of HepG2 cells were significantly increased by the combination of As₂O₃ (2.0μmol/L)and AA(50μmol/L) : the growth inhibition ratio(MTT assays) was increased from (23.36±0.92) % to (43.95±1.56) %, even greater than the effect of high concentration (As₂O₃ 4.0μmol/L) (41.45±1.10)%(P<0.05).Conclusion: AA could enhance the ability of As₂O₃'s growing and inhibition in human hepatocarcinoma cells.Part two Apoptosis assessment and Cell cycle analysisObjective: Normal hepatic L-02 cells and hepatocarcinoma HepG2 cells were cultured in vitro, to investigate the effect of ascorbic acid on the apoptosis induced by arsenic trioxide in human hepatocarcinoma cells, and analyze the changes of cell cycle. Methods: the apotosis induced by AA, As₂O₃ and the combination of AA and As₂O₃ and changes of cell cycle were identified and quantified by flow cytometry (FCM) with annexin-V staining and PI-staining.Results: In comparison with the use of As₂O₃ (2.0μmol/L )separately for various interval, the inhibition ability and the apoptosis ratio of HepG2 cells were significantly increased by the combination of As₂O₃ (2.0μmol/L)and AA(50μmol/L) : the the apoptosis ratio was increased from (11.37±1.20) % to (16.06±0.86) % (P<0.01). Cell cycle analysis showed that the combination of AA and As₂O₃ effectively enhanced cell cycle's arresting in S phase, and enhanced the apoptosis.Conclusion: Combined AA with As₂O₃ in HepG2 cells could enhance apoptosis induced by As₂O₃.Part three The safety of the combination of AA and As₂O₃Objective: To test the the safety of the combination of AA and As₂O₃Methods: Normal hepatic L-02 cells and hepatocarcinoma HepG2 cells were cultured in vitro. The viability of the test cells was assessed with both the Trypan blue exclusion and MTT tetrazolium ( 3- [ 4, 5– dimethyl– thiazol– 2 - yl]– 2 , 5 - diphenyltetralzolium bromide, 5.0 mg/mL) assays.Results: the growth inhibition ratio(MTT assays) was increased from (36.54±0.82) % to (51.98±1.52) %, even greater than the effect of high concentration (As₂O₃ 4.0μmol/L)( 45.27±1.96) (P<0.01); however in normal hepatic L-02 cells, it was less than the highest concentration (4.0μmol/L) of As₂O₃.Conclusion: Combined AA with As₂O₃ in HepG2 cells could enhance growth inhibition induced by As₂O₃, but the enhancement ability was not parallel in normal hepatic L-02 cells by the combination.