【摘要】 目的:以细胞周期作为抗癌药物新靶点的研究,可能是很有前途的。笔者的前期工作发现,二烯丙基二硫化物（diallyl disulfide,DADS）可抑制人胃癌BGC823细胞增殖,其增殖抑制与细胞周期G₂/M期阻滞有关;DADS可能是通过抑制细胞分裂周期蛋白25C（Cell division cycle protein 25C,Cdc25C）、cyclinB1表达使部分BGC823细胞停滞在G₂/M期,但G₂/M期阻滞的机制还未完全阐明。本研究进一步探讨DADS诱导人胃癌BGC823细胞周期G₂/M期阻滞的可能机制。方法:RT-PCR检测Chk1和Chk2在mRNA水平的改变;Western blot检测DADS处理BGC823细胞前后细胞周期相关蛋白ATM-RAD3相关基因（ATM-RAD3-related gene,ATR）、细胞周期检查点蛋白激酶1（checkpoint kinase1,Chk1）、细胞周期检查点蛋白激酶2（checkpoint kinase2,Chk2）表达和ATR、Chk1、Chk2的磷酸化程度:免疫共沉淀检测Chk1、Chk2与Cdc25C结合情况。结果:RT-PCR检测显示,Chk1和Chk2的mRNA水平在处理组与未处理组之间无显著性差异（P>0.05）。Western blot检测显示,总Chk1和Chk2蛋白表达在细胞处理前后均无明显改变,但15mg/L DADS刺激BGC823细胞2h后,处理组细胞Chk1磷酸化程度明显增加,并呈时间依赖性（P<0.05）,而Chk2磷酸化程度在处理组与未处理组之间无显著性差异（P>0.05）。15mg/L DADS作用15～120min,ATR磷酸化程度明显增加,呈时间依赖性（P<0.05）,而ATR表达无改变。免疫共沉淀分析表明,DADS能促进BGC823细胞Chk1与Cdc25C结合,而对Chk2与Cdc25C结合无影响。结论:DAD诱导人胃癌BGC823细胞G₂/M期阻滞与Chk1的活化有关,DADS可能是通过激活ATR、Chk1,调节Cdc25C的表达引起人胃癌BGC823细胞G₂/M期阻滞。更多还原

【Abstract】 Objective: Cell cycle has recently become more appealing as a new target of anti-carcinogenic agent. Diallyl disulfide （DADS） inhibits growth and induces cell cycle G₂/M arrest in human gastric cancer BGC823 cells. Cell division cycle protein 25C （Cdc25C） and CyclinB1 expression are involved in G₂/M arrest. However, mechanisms of G₂/M arrest are not yet fully understood. The aim of this study was to elucidate the mechanism of cell cycle G₂/M arrest in human gastric cancer BGC823 cells induced by DADS. Methods: The expression of Chk1 and Chk2 mRNA associated with cell cycle arrest of BGC823 cells after the induction with DADS for 1 or 2 days was detected by RT-PCR. The protein expression of cycle-related proteins ATM-RAD3-related gene （ATR）, checkpoint kinasel （Chk1）, checkpoint kinase 2 （Chk2）, P-ATR, P-Chkl and P-Chk2 was measured by Western blot. Interaction between Chk1/2 and Cdc25C was analyzed by immunoprecipitation. Results: After the cells were treated with 15 mg/L DADS for 1 or 2 days, the expression of Chk1 and Chk2 mRNA was not significantly different from that in untreated cells （P>0.05）. Western blot analysis showed that the expression of total Chkl and Chk2 treated with 15 mg/L DADS was not significantly different from that in untreated cells. But phospho-Chkl showed a significant increase after stimulation with 15 mg/L DADS for 2h to 12h and continued to increase gradually as time went on （P<0.05）. Phospho-Chk2 showed a weak expression and a weaker expression after stimulation with DADS, but the changes were not statistically significant （P>0.05）. Addition of 15 mg/L DADS to BGC823 cells for 15 min to 120 min resulted in an increase in phospho-ATR expression, whereas no changes were found in ATR expression （P<0.05）. The Chkl Ab increasingly precipitated Cdc25C in BGC823 cells treated with DADS （P<0.05）. In contrast, Chk2 Ab failed to change precipitation with Cdc25C by DADS （P>0.05）. Conclusion: Activation of Chkl was involved in cell cycle G₂/M arrest in BGC823 cells treated with DADS. Cell cycle G₂/M arrest by DADS is associated with phosphorylation of several cell cycle regulatory proteins including ATR and Chkl which regulate expression of Cdc25C.更多还原