二烯丙基二硫化物诱导胃癌BGC823 细胞周期G2/M期阻滞的机制*

Mechanism of Cell Cycle G2/M Arrest in Human Gastric Cancer BGC 823 Cells Induced by Diallyl Disulfide

    摘要
  • 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BGC 823 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）、cyclinB 1 表达使部分BGC 823 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BGC 823 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BGC 823 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/LDADS刺激BGC 823 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BGC 823 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BGC 823 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BGC 823 细胞G2/M期阻滞。

     

    Abstract: Objective:Cell cycle has recently become more appealing as a new target of anti-carcinogen -ic 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 CyclinB 1 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 G 2/M arrest in human gastric cancer BGC 823 cells induced by DADS. Methods:The expression of Chkl 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 kinase 1 (Chk1), checkpoint kinase 2 (Chk2), P-ATR, P-Chk1 and P-Chk 2 was measured by Western blot. Interaction between Chk1/2 and Cdc 25C was analyzed by immuno-precipitation. Results: After the cells were treated with15mg/L DADS for 1 or 2 days, the expression of Chkland Chk 2 mRNA was not significantly different from that in untreated cells (P>0.05). Western blot analysis showed that the expression of total Chk 1 and Chk 2 treated with 15mg/L DADS was not significantly different from that in untreated cells. But phospho-Chk 1 showed a significant increase after stimulation with 15mg/L DADS for2h 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 15mg/L DADS to BGC 823 cells for 15min to 120 min resulted in an increase in phospho-ATR expression, whereas no changes were found in ATR expression ( P<0.05). The Chk 1 Ab in-creasingly 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 Chk1 was involved in cell cy-cle G2/M arrest in BGC 823 cells treated with DADS. Cell cycle G 2/M arrest by DADS is associated with phos-phorylation of several cell cycle regulatory proteins including ATR and Chk 1 which regulate expression of Cdc 25C.

     

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