Effect And Mechanism Of Target For Induction Of Differentiation And Cycle Arrest In Human Gastric Cancer Cells By Diallyl Disulfide

Gastric cancer is the most common type of malignancy in China, and its morbidity and mortality rank first now. There are 400,000 people or so with gastric cancer, and 300,000 people succumb to it, which is much higher than that in the west developed countries. At present, therapeutic effect of orthodox surgical therapy, radiation therapy and chemotherapy is comparatively weak, easy to palindromia, furthermore , their side effect is serious and the 5-year survival rate of gastric cancer remains only about 30%. Therefore, it is an urgently solved topic to raise the 5-year survival rate, explore new method to treat gastric cancer, and look for new effective anticancer drugs with low toxicity and inexpensive price. Now human gastric cancer reversion research has become leading edge field in life science research. DADS inhibits the in vitro growth of colon, lung, breast and gastric cancer cell lines, and leukemia cell lines and it becomes a very potential candicate anticancer drug.【DADS inhibited growth of human gastric cancer MGC803 cells and induced them differentiation】 MTT assay, electron microscope,Con A-mediated cell agglutination, ALP specific activity detection and optics microscope, cytochemical staining, scrape-loading and dye transfer technology were employed to confirm the DADS-induced cell growth inhibition and differentiation in MGC803 cell line. Exposure of human gastric cancer MGC803 cells to DADS from 25 to 35mg·L^-1 for 96 h had less absorbance value than control,which exhibited a dose-dependent model (P<0.05),whereas DADS induced-growth inhibition rate enhanced from 11.3%,35.2%,50.7% to 60.6% as assessed by the MTT test(P<0.05).As exposed to electron microscope, MGC803 cells took on malignance declining as cellular heteromorphism diminished, nucleocytoplasmic proportion was reliable to reasonableness, the number of microvilli on cell surface decreased, cellular apparatus were abundant in plasm and intercellular conjunctional structure appeared after it being exposed to 30mg·L^-1 DADS; Con A-mediated cell agglutination ratio was 26.98% versus controls 79.10% (p<0.05) ; cell ALP specific activity decreased by 66.12% from 0.120 U/g to 0.040 U/g (p<0.05). Cellular skeletin synthesized and reconstructured and recruitment of intercellular communication of gap junction were also observed with cytochemical technology; Gastric cancer cells showed negative of dye transfer,while positive transfer of LY dye from the load cells at the scraped-line to neighboring cells in gastric cancer cells induced by DADS. Immunihistichemical stain indicated that expression of p21WAF1 enhanced and that mutant p53,p21ras,c-myc downregulation expression and ther was no change in pRb. All these showed above suggested that human gastric cancer cells were induced differentiation into normal cells by DADS. Flow cytometry analysis revealed that treatment MGC803 cell with increasing quantities of DADS increased in the percentage of cells in the G₂/M phase. The proportion of cells in the G₂/M phase after treatment with 30mg·L^-1 DADS for 36 h was comparable (58.7%), and more than four times that occurring in untreated cells (13.8%). Exposure of human gastric cancer BGC823 cells to DADS from 5 to 20 mg·L^-1 for 96 h had less absorbance value than control, which exhibited a dose-dependent model (P<0.05), whereas DADS induced-growth inhibition rate enhanced from 34.2,39.2,55.7,69.6% as assessed by the MTT test (P<0.05).Which suggest that DADS inhibited growth of human gastric cancer BGC823 cells. After BGC823 cells were treated with 15mg·L^-1 DADS, flow cytometry analysis displayed that cell percentage in S phase of the DADS treated MGC803 cells Was decreased,whereas cell percentage in G₂ phase Was significantly increased(P<0.05),and little change in Gl phase was found. Which is the same as that of MGC803 cells. Much attention has been focused on the ERK pathway as a possible target for newly designed anti-neoplastic drugs. Therefore, it is essential to establish the role that the ERK pathway plays in the process that leads to differentiation within a specific tissue or cell type. In this study, ERK activity is observed in the DADS-induced differentiation effect in human gastric cancer cell line MGC803. Western blot analysis revealed that although DADS did not influence the quantity of ERK1/2 protein expressed, it did decrease its phosphorylation in a concentration-dependent manner, compared with the controls (P<0.05). At 30 mg·L^-1, DADS inhibited the activation of ERK1/2 in 15-30 min (P<0.05). These results suggested that the DADS-induced differentiation of MGC803 cells involved an alteration of the ERK1/2 signaling pathway .Conclusion: Differentiation and growth inhibition was induced by DADS. Cell Cycle G₂/M Arrest was involved in growth inhibition and differentiation induced by DADS in the Human Gastric Cancer Cells. ERK is involved in the differentiation and growth inhibition induced by diallyl disulfide in the human gastric cancer cells.【Checkpoint Kinase Signaling is Involved in Cell Cycle G₂/M Arrest Induced by Diallyl Disulfide in the Human Gastric Cancer Cell Line MGC803 and BGC823】 Anticancer insights derived from cell cycle research has given birth to the idea of cell cycle G₂ checkpoint abrogation as a cancer cell specific therapy, based on the discovery that many cancer cells have a defective G1 checkpoint resulting in a dependence on the G₂ checkpoint during cell replication. The most promising target to date seems to be Chk1 and there will be a growing number of selective inhibitors of Chk1 available in the future with a variety of activities that promise to have potential G₂ checkpoint abrogation qualities. Cell cycle has recently become more appealing as a new target of anti-carcinogenic agent.It is a new mechanism that the role of Chk was reduced by anti-carcinogenic drug in tumor cells. Furthermore, G₂/M cell cycle arrest is mainly related with the Chk2-Cdc25C-Cdc2/cyclin B1 pathway in cancer cells.It was found that Cdc25C and CyclinB1 expression are critical events in G₂/M arrest of MGC803 cells by DADS. DADS increased the expression of cyclinB1 by 20% after 12 h. DADS inhibited the expression of the cell cycle-associated phosphatase Cdc25C by 80% after 48 h. Western blot analysis showed that DADS treatment of BGC823 cells decreased the level of Cdc25C in a time-dependent model (P<0.05). DADS increased the expression of cyclinB1 after 12h and then its expression decreased after 24h (P<0.05), which tendency is similar to MGC803 cells by DADS. Cdc25C was thought to be the major effector of the G₂/M DNA damage checkpoint response. Cdc25C which triggers cyclinB1/CDK1 complex is controlled by Chk1 or/and Chk2. So Chk1 and Chk2 activity was observed by DADS on human gastric cancer cells. Western blot analysis showed that phosphorylation of Chk1 was decreased following treatment of MGC803 and BGC823 cells with DADS after 1hrs in a time-dependent model. At the same time,the total Chk1 abundance did not change.Expression of Phospho-Chk2 was weak in these two untreated cell lines,furthermore, its expression wasn't changed by DADS (P>0.05). Expression of Chk1 and Chk2 had no change after treated with DADS. The level of Chkl and Chk2 mRNA had no significant difference between the DADS treatment and contro1 by RT-PCR (P>0.05). Which showed that the level of Chkl and Chk2 mRNA had no relationship with cell cycle arrest.ATR is capable of specifically phosphorylating Chkl. Western blot analysis showed that Phospho-ATR was inactivated by DADS from 15 or 30 to 90 min in MGC803cells or BGC823 cells respectively(P<0.05). Expression of ATR had no change after treated with DADS. Coimmunoprecipitated Chk1 or Chk2 was detected by anti-Chk1 or anti-Chk2 immunoprecipitation (IP) followed by anti-Cdc25C immunoblotting (IB), DADS enhanced binding activity of Chk1 kinase with Cdc25C in MGC803 and BGC823 cells,but didn't influence binding activity of Chk2 kinase with Cdc25C. Which verified that DADS induced G₂/M arrest by Chk1- Cdc25C on MGC803cells and BGC823 cells.Conclusion: Checkpoint kinase Chk1 may be the target to arrest cell cycle by DADS. ATR/ Chk1/Cdc25C/ cyclin B1 was regulated by DADS to arrest Human Gastric Cancer Cells at G₂/M.【Checkpoint Kinase Signaling is regulated in Cell Cycle G₂/M Arrest Induced by Chk1 and Chk2 RNAi】To investigate the effect of Chk1 or Chk2 gene silence on the cell viability of human gastric cancer cell line MGC803 and BGC823, and explore the role of Chk1 and Chk2 in cell cycle arrest. The siRNA targeting at Chk1 or Chk2 gene was transfected into MGC803 and BGC823 cells. The mRNA and protein expression of Chk1 was detected by RT-PCR and Western-blot respectively. The Chk1 or Chk2 expression at mRNA and protein levels in Chk1 or Chk2 siRNA-transfected group was reduced markedly respectively as compared with that in negative control and empty vector-transfected group (P<0.05).Cell viability was determined via MTT assay. Treated with DADS, cells-transfected viability by Chk1 SiRNA was decreased by 18.3% or 20.3% in MGC803 and BGC823 cells respectively (P<0.05). Inhibition of the Chk1 expression in Chk1 siRNA transfected group in BGC823 cells significantly abrogated G₂/M arrest induced by DADS, and the proportion of the cells in G₂/M phase was more than two double lower than that in other control groups (P<0.05).While inhibition of the Chk2 expression in Chk2 siRNA transfected group had little effect on G₂/M arrest.Chk1 gene silence increased expression of Cdc25C and cyclinB1,and inhibition of expression of Cdc25C and cyclinB1 by DADS was blocked (P<0.05). On the contrary,Chk2 gene silence can not do so.Conclusion: Chk1 gene silence can inhibited and growth of Human Gastric Cancer Cells, and take synergistic effect with DADS. DADS arrestted Human Gastric Cancer Cells at G₂/M by Chk1/Cdc25C /CyclinB1, and its main target was Chk1. Chk2 didn't participate G₂/M arrest by DADS in Human Gastric Cancer Cells.