The Study On The Effects And Molecular Mechanisms Of The Apoptosis Induced By ω-3 Polyunsaturated Fatty Acid In Hepatocarcinoma Hepg2 Cell Line

Objective: This study was to investigate the effect of theω-3 polyunsaturated fatty acids, the main component of eicosapentaenoic acid(eicosapentaenoic acid ,EPA), Docosahexaenoic acid(Docosahexaenoic acid,DHA),on the proliferation,apoptosis and the levels of SOD and MDA of human hepatocarcinoma HepG2 cells,and to explore its molecular mechanism.This study could provide a new way of liver cancer therapy.Methods: The human hepatocarcinoma cell line HepG2 was used in vitro. (1)MTT assay was used to examine the effects of DHA or EPA on HepG2 cell, and the cytotoxicity effects of DHA or EPA alone and combined with 5-FU on HepG2 human hepatoma cells. (2) Hoechst33258 staining was performed to examine apoptosis morphous. (3) Flow cytometry assay was used to investigate the apoptosis rate. (4) Xanthine oxidase assay was used to determine the activity of total SOD, TBA assay was used to determine the levels of MDA.Results: 1.Cytotoxicity of DHA, EPA in HepG2 cells. DHA could inhibit the proliferation of HepG2 cells in concentration-dependent way, the inhibitary rate increased with the prolonged time.The IC50 value of DHA at 24, 48, and72h was (160.87±2.13)μg/ml, (82.39±1.76)μg/ml, (70.71±1.52)μg/ml respectively. Similarly, EPA could inhibit the proliferation of HepG2 cells in concentration-dependent way,the inhibitary rate increased with the prolonged time.The IC50 value of EPA at 24,48,and72h was are (89.09±1.56)μg/ml, (72.68±2.05)μg/ml, (56.4±1.38)μg/ml.2. Cytotoxicity of DHA or EPA combined 5-FU on HepG2 cells.The tumor inhibitory rates of administration of DHA(20,40,60μg/ml)or EPA (20,40,60μg/ml) combined with 5-FU(5μg/ml) were higher than that of 5-FU administration alone. Therefore,ω-3 polyunsaturated fatty acids is expected to become Chemotherapy helping drugs. 3. Effects of EPA or DHA on apoptosis of HepG2 cells.Hochest33258 staining showed that contronl groups nucleus were Large, smooth surface, more parks, cell nuclear Blu-ray. After the EPA and DHA (80μg/ml) treatment intervention for 48h, Density of nuclear staining increased that its shape was half-moon and to unite around the nuclear membrane and nucleolus cracking.4. Apoptosis rate of HepG2 cells by flow cytometryTreated with 60,80μg/ml DHA for 48h, the apoptosis rates of DHA were 6.52%(60μg/ml),14.67%(80μg/ml), respectively. Treated with 60,80μg/ml EPA for 48h, the apoptosis rate of EPA groups were 12.31%(60μg/ml),32.12%(80μg/ml) . With the concentration increasing, apoptosis rate increased.5. Effect of EPA or DHA on total SOD activity and MDA content in HepG2 cells.After treatment with 60, 80 or 100μg/ml of EPA or DHA, Total SOD in the treated cells decreased (P <0.05). After treatment with 80 or 100μg/ml of EPA or DHA, the total cellular SOD activity has a more significant reduction than that in cells treated with 60μg/ml of EPA or DHA(P <0.05). After treatment with 80 or 100μg/ml of EPA or DHA, MDA levels in the cells were significantly higher (P <0.05).Conclusions:1. DHA, EPA may inhibit human hepatoma cell line HepG2 proliferation. 2. DHA or EPA has synergism effects on 5-FU. 3. DHA, EPA may induce HepG2 cell apoptosis. 4. DHA,EPA can inhibit the growth and induce apoptosis of HepG2 cells possibly through the mechanism involved in lipid peroxidation .