二氢青蒿素对胃癌细胞株SGC7901增殖的影响

Effects of dihydroartemisinin on the proliferation of gastric cancer cell line SGC7901

目的观察二氢青蒿素（DHA）对胃癌细胞株SGC7901增殖的影响及作用机制。方法SGC7901细胞分为DHA组和对照组，DHA组分别加入浓度为6．25、12．50、25．00、50．00、100．00μmol／LDHA的培养基，对照组加入等体积含0．1％DMSO的培养基。不同浓度DHA处理SGC7901细胞24、48和72h后，MTT法检测细胞的增殖情况。不同浓度DHA处理SGC7901细胞24h后，流式细胞仪测定细胞周期的分布；100μmol／LDHA处理细胞24h后，采用Westernblot法测定细胞周期蛋白A（CyclinA）、CyclinD1、CyclinE、细胞周期蛋白依赖性激酶4（CDK4）和P16蛋白的表达水平；免疫共沉淀检查CDK4与CyclinD1和P16之间的相互作用。采用单因素方差分析和t检验进行统计学分析。结果6．25、12．50、25．00、50．00、100．00pomol／LDHA分别处理SGC7901细胞24、48和72h，均明显抑制细胞增殖（F=78．66，235．37，93．75，P〈0．05）；与对照组比较，不同浓度DHA组G0／G，期细胞比例均明显上升（F=18．42，P〈0．05）；100．00tzmol／LDHA处理细胞24h，CyclinD1和CDK4蛋白的相对表达量分别为0．17±O．05、0．24±0．06，较对照组的0．67±0．15、0．64±0．18明显下降（t=7．746，5．164，P〈0．05）；DHA组CyclinE蛋白的相对表达量为0．35±0．06，较对照组的0．42±0．06也有下降，但差异无统计学意义（t=2．02l，P〉0．05）；DHA组和对照组CyclinA蛋白的相对表达量分别为0．38±0．08和0．35±0．09，两组比较，差异无统计学意义（t=1．266，P〉0．05）；与对照组P16蛋白相对表达量（0．29±0．07）比较，DHA组P16蛋白相对表达量（0．54±0．12）显著升高（f=4．408，P〈0．05）。免疫共沉淀结果示DHA处理SGC7901细胞后，CDK4与CyclinD1结合减少，与P16结合增加。结论DHA将SGC7901细胞周期阻滞于Go／G．期，其主要机制可能与下调CyclinD1和CDK4表达、上调P16表达，抑制CyclinD1与CDK4的结合，促进P16与CDK4的结合有关。

Objective To investigate the effects of dihydroartemisinin （DHA） on the proliferation of gastric cancer cell line SGC7901 and its mechanism. Methods SGC7901 cells were divided into the DHA group and the control group. SGC7901 cells in the DHA group were treated with DHA of different concentrations （6.25, 12.50, 25.00, 50.00, 100.00 μmol/L）, SGC7901 cells in the control group were cultured in the 0.1% DMSO medium. The proliferation of SGC7901 cells was detected by the MTT method at different time points （24, 48, 72 hours）. Cell cycles of SGC7901 in the DHA group were observed by flow eytometry at 24 hours after treatment. The expressions of Cyclin A, Cyelin D1, Cyclin E, Cyelin-dependent kinase 4 （CDK4） and P16 were detected by Western blot after treating SGC7901 with DHA at concentration of 1001xmol/L for 24 hours. The interaction between CDK4 with Cyelin D1 or P16 was examined using the co-immunoprecipitation assay. All data were analyzed using the one-way analysis of variance or the t test. Results The proliferation of SGC7901 cells was significantly inhibited after the treatment with DHA at different concentrations （6.25, 12.50, 25.00, 50.00, 100.00 I^moL/L） for 24, 48 and 72 hours （F = 78.66, 235. 37, 93. 75, P 〈 0.05）. Compared with control group, the number of SCG7901 cells in the G0/GZ phase in the DHA group was significantly increased （F = 18.42, P 〈 0.05）. After treating SGC7901 cells with DHA for 24 hours, the protein expressions of Cyclin D1 and CDK4 were 0.67 ±0.15 and 0.64 ±0.18 in the control group, which were significantly higher than 0.17 ±0.05 and 0.24 ±0.06 in the DHA group （t =7. 746, 5. 164, P 〈0.05）. The protein expressions of Cyelin E were 0.42 ± 0.06 in the control group and 0.35 ± 0.06 in the DHA group, with no significant difference （ t = 2. 021, P 〉 0.05 ）. The protein expressions of Cyclin A were 0.35 ± 0.09 in the control group and 0.38 ± 0.08 in the DHA group, with no significant difference between the 2 groups （ t = 1. 266, P 〉 0.05 ）. The protein expressions of P16 were 0.29 ± 0.07 in the control group and 0.54 ± 0.12 in the DHA group, with significant difference between the 2 groups （ t = 4. 408, P 〈 0.05 ）. The results of co-immunoprecipitation assay showed that DHA decreased the interaction between CDK4 and Cyclin D1, and increased the interaction between CDK4 and P16. Conclusion DHA induces SGC7901 cells arrested in Go/G1 phase, and the effect may be related with its down- regulation of Cyclin D1 and CDK4, up-regulation of PI6, decreasing the interaction between CDK4 and Cyclin D1, and increasing the interaction between CDK4 and P16.