Effect Of The G₂/M Arrest Induced By Diallyl Disulfide In Chk-transfected Human Gastric Cancer MGC803 Cell Line

Objective: Diallyl disulfide (DADS) has G2/M arrest effects in various cancer cell lines in vitro, but its mechanisms remain unclear. This study was designed to construct the eukaryotic expression vector of human Chk1and Chk2 gene, observe its expression in gastric cancer MGC803 cells in vitro, and explore the association of Chk1 and Chk2 expression in G2/M arrest induced by diallyl disulfide in human gastric cancer MGC803 cells.Methods: MGC803 cells growth inhibition was measured by HE staining. Stably transfect the eukaryotic expression vector of human Chk1 and Chk2 genes into huaman gastric cancer cell line MGC803 to get the cell strain that can express the genes stably. Collect the cells and detect the expression of Chk1and Chk2 genes in MGC803 cell by RT-PCR and western blot. Flow cytomery was used to determine the change of cell cycle. The expression profile of cell cycle-associated genes in MGC803 cells and in Chk1- and Chk2-transfected MGC803 cells exposed to DADS were indentified by SuperArray Human Cell cycle Gene Array. RT-PCR and Western blot analysis the expression of cell cycle-related genes and protein.Results: As exposed to inversion microscope and optics microscope, after 30mg·L-1 DADS was added to culture medium 12h, MGC803 cells showed malignance declined as nuclear-to-cytoplasmic ratio was reliable, the number of nucleoli in the cell nuclear decreased, size of nucleus shortened, and coloring of nucleus turned weak.After treated with 30mg·L-1DADS for different times, Northern blot analysis showed the level of Chkl and Chk2 mRNA had no significant difference between the DADS treatment and control in MGC803 cells (P>0.05). Which showed that the level of Chkl and Chk2 mRNA had no relationship with cell cycle arrest.Eukaryotic expression vector of recombined human Chk1 and Chk2 genes were successfully transfected MGC803 cells stably by liposome method. The result of RT-PCR and western blot showed that the cells strain can high express Chk1 and Chk2 genes stably. Flow cytometry analysis revealed that treated MGC803 cells, Chk1- or Chk2- transfected MGC803 cells with 30mg·L-1DADS accumulated cells in the G2/M phase, which exhibited a time-dependent model, respectively (P<0.05). The proportion of cells in the G2/M phase after treatment with 30mg·L-1DADS for 12h was comparable (26.4%,41.3%,24.4%), and more than three times that occurring in untreated cells (6.9%,9.4%,8.6%). DADS treatment for 24h resulted in accumulation of 51.5%, 57.5% and 45.5% of cells in G2/M phase respectively.It was revealed that the expression of Chk1 and Chk2 proteins had no change after treated with 30mg·L-1 DADS for 24h by immuocytochemistry (P>0.05).Expression of Chk1 and Chk2 had no change after treated with 30mg·L-1 DADS for 12h, 24h, 36h and 48h by RT-PCR, respectively, (P>0.05). Which indicated that the level of Chkl and Chk2 mRNA had not related with cell cycle arrest in Chk1- and Chk2-transfected MGC803 cells.Western blot analysis showed that phosphorylation of Chk1 was increased in Chk1-transfected MGC803 cells treated with 30mg·L-1 DADS for 2h, 4h, 8h and 12h in a time-dependent model (P<0.05). At the same time, the total Chk2 abundance did not change, and the expression of Phospho-Chk2 was weak in Chk2-transfected MGC803 cells, but its expression had no significant difference with control group by DADS (P>0.05).Conclusion:1. Eukaryotic expression vector of recombined human Chk1 and Chk2 genes were successfully transfected MGC803 cells stably by liposome method.2. Chk1 was related with DADS arrestted human gastric cancer cells at G2/M by increaseing the expression of p-Chk1.