DADS诱导人胃癌细胞周期阻滞相关基因的差异表达

目的研究二烯丙基二硫(diallyl disulfide,DADS)诱导人胃癌MGC803细胞G2/M周期阻滞相关基因表达的变化。方法MTT法、流式细胞术分别分析DADS对MGC803细胞的抑制作用与细胞周期分布的影响;功能基因芯片检测周期相关差异表达基因;Western blot、RT-PCR验证周期相关基因的表达。结果MTT法、流式细胞术证明DADS可明显抑制MGC803细胞增殖并诱导G2/M期阻滞。基因芯片结果显示,30 mg.L-1DADS作用MGC803细胞4 h后,表达上调的基因有CDC45L、CDK5R1、p21、CUL4A、GADD45α、RAD17、TP53,下调的基因是ANAPC5、ATR、BRCA1、CCNA2、CCNB2、CCNE1、CCNE2、CDC16、CDC6、CDK5RAP1、GTF2H1、MCM5、RAD9A、RGC32。RT-PCR显示30 mg.L-1DADS处理MGC803细胞4、8、12 h后,p21、GADD45α时间依赖性的表达增强(P<0.05),CyclinB1呈时间依赖性的表达降低(P<0.05)。TP53在4、8、12 h后与对照组比较表达明显增强(P<0.05)。Western blot结果表明,30 mg.L-1DADS作用MGC803细胞6、12、24 h后,p53、GADD45α、p21蛋白表达上调,CyclinB1蛋白表达下调(P<0.01)。结论DADS诱导人胃癌MGC803细胞G2/M周期阻滞是由多基因协同作用的结果,可能通过两种途径共同调节完成的。

The differential expressions of cell cycle arrest-associated genes in gastric cancer cells induced by diallyl disulfide

Aim To investigate the changes in cycle-related genes expressions in human gastric cancer MGC803 cells induced by diallyl disulfide(DADS).Methods MGC803 cells growth inhibition was measured by MTT assay.Flow cytometry was used to determine the change of cell cycle.Differential genes expressions of DADS group and control group were analyzed by cell cycle microarray.Some cycle-related genes expressions were detected by RT-PCR and Western blot.Results MTT assay and flow cytometry showed DADS could inhibit MGC803 cell proliferation and induce cell cycle arrest in G2/M phase.After treatment with 30 mg·L-1 DADS for 4 h,CDC45L,CDK5R1,p21,CUL4A,GADD45α,RAD17,TP53 genes were up-regulated(P<0.05),and ANAPC5,ATR,BRCA1,CCNA2,CCNB2,CCNE1,CCNE2,CDC16,CDC6,CDK5RAP1,GTF2H1,MCM5,RAD9A,RGC32 genes down-regulated(P<0.05).After treatment with 30 mg·L-1 DADS for different times,RT-PCR showed TP53,GADD45α and p21 were up-regulated while CyclinB1 was down-regulated(P<0.05).Western blot showed TP53,GADD45α and p21 were up-regulated whereas CyclinB1 was down-regulated(P<0.05).Conclusion Cell cycle arrest of MGC803 cells induced by DADS relates with various genes perhaps through two signal transduction pathways.