| Object: To study the effect of As2O3 combined with γ-secretase inhibitor on drug resistance of hepatocellular carcinoma Hep G2 cells.In order to investigate the relationship between reversal of drug resistance of Hep G2 cells by As2O3 and Notch signaling pathway, and to reveal the possible mechanisms of reversal of drug resistance of Hep G2 cells by As2O3.To provide experimental basis and reference for the treatment of liver cancer.Methods: 1.Hep G2, and Hep G2/ADM cells were cultured in vitro; 2.Hep G2, and Hep G2/ADM cells received interventions respectively with As2O3(0.25,0.5,1.0,2.0,4.0,8.0 mg/ml), MW167(10,20,40umol/L), ADM(0.25,0.5, 1.0,2.0,4.0,8.0 mg/ml) for 24 h, 48 h, and 72 h. MTT method was used to detect cell proliferation and to select the optimal time and dose of As2O3 and MW167. IC50 and resistance multiples were calculatede by SPSS; 3.Effect of different drugs on cell resistance.(1)Cells were divided into four groups: control group(without intervention), the combination group(received intervention with both MW167 and As2O3), MW167group(received intervention with MW167),As2O3 group(received intervention with As2O3).(2)Cell apoptosis of each group was detected by flow cytometry.(3)Reversal multiple was computed by MTT assay.(4)RT-PCR was used to detect the gene expression of Notch-1, Bcl-2, Caspase-3, MDR-1 in each group.(5)Western blot was used to detect the protein expression of Notch-1, Bcl-2 and Caspase-3, MDR-1 in each group.Results: 1.Under the light microscope, the cultured Hep G2, Hep G2/ADM cells were found with a spindle shape,a large nucleus and growing clumped together on the wall.By comparison,cultured cells were similar with ATCC cell library pictures and were considered as human hepatoma Hep G2 cell strain; 2.To determine the doses of As2O3 and MW167 and the responding time, that results in a proliferation inhibition rate of less than 20% was set as the optimal doses and time(As2O3 0.25mg/L,MW167 10umol/L for 48h) and to intervene cells on the doses and time.IC50 values of the cells treated with As2O3, MW167, ADM decreased over time(P<0.05). Resistance multiples of different responding times were 24h(1.48 times), 48h(1.71 times), 72h(16.57 times), with a significance of P<0.05;3.Study on the drug resistance:(1)Effects of As2O3, MW167, ADM on the proliferation: the same dose with the prolonging of time, the proliferation inhibition rates increased(P<0.05); the same time along with the increase of dose, the proliferation inhibition rates increased(P<0.05).the same time the same drug between different cells proliferation inhibitory rate increased(P<0.05);the proliferation inhibition rates of different cells were increased with the same time and dose(P<0.05).(2)Reversal multiples of combined group,MW167 group and As2O3 group were 2.79 times,2.16 times and 1.79 times respectively,and the difference was significant with P<0.05.(3)Apoptosis rates of Hep G2 cells in combined group,MW167 group,As2O3 group and control group were(42.63±0.73),(28.73±0.32),(23.37±0.40) and(6.77±0.15),the differences between groups were statistically significant(P<0.05).Apoptosis rates of Hep G2/ADM cells in combined group,MW167 group,As2O3 group,control group were(52.066±0.321),(42.600±0.700),(36.166 ±0.650),(4.433±0.208),the differences between groups were statistically significant(P<0.05).(4)Compared with the blank group, gene expression levels of Bcl-2,Notch-1 in drug groups decreased(P<0.05), caspase-3 in drug groups increased(P<0.05), the combination group was more obvious, and the trend of increasing and decreasing was that the combination group>MW167group>As2O3 group(P<0.05).Gene expression of MDR-1 in Hep G2 cell was higher than in Hep G2/ADM cell(P<0.05) and was decreased in Hep G2/ADM cells of drug groups(P<0.05) with a trend that the combination group>MW167 group>As2O3 group(P<0.05).While Gene expression of MDR-1 in Hep G2 cells without difference between blank group and drug groups(P﹥0.05).Compared with the blank group, protein expression levels of Bcl-2,Notch-1 in drug groups were decreased(P<0.05), Caspase-3 in drug groups were increased(P<0.05), the combination group was more obvious with a trend that the combination group>MW167 group>As2O3 group(P<0.05).Protein expression level of MDR-1 in Hep G2/ADM cell was higher than in Hep G2 cell(P<0.05) and was decreased in Hep G2/ADM cells of drug groups(P<0.05) with a trend that the combination group>MW167 group>As2O3 group(P<0.05).While protein expression of MDR-1 in Hep G2 cell without difference between blank group and drug groups(P﹥0.05).Conclusion:1. As2O3 can inhibit Notch-1 expression, and promote Hep G2, Hep G2/ADM apoptosis and inhibit cell proliferation, reverse the cell resistance, and has a stronger effect on the Hep G2 / ADM cell.2. Combination of As2O3 and γ-secretase inhibitor has a stronger effect in reversing multidrug resistance of Hep G2/ADM cells, which may as a result of the consistency in the down-regulated expression of Bcl-2, Notch-1, MDR-1 and up-regulated of caspase-3 gene and protein expression. |
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