The Analysis Of The Differential Expression Of Cell Cycle-associated Genes Induced By Diallyl Disulfide In Human Gastric Cancer Cells

Objective: Diallyl disulfide (DADS) has cell cycle arrest effects on various cancer cell lines in vitro, but its mechanisms remain unclear. This study was designed to investigate the related genes expression of cell arrest induced by DADS on human gastric cancer cell line MGC803 and its molecular mechanisms.Methods: MGC803 cell growth inhibition was measured by MTT array and HE staining. Flow cytomery was used to determine the change of cell cycle. The expression profile of cell cycle-associated genes in MGC803 cells exposed to DADS was 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-1DADS 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.MTT assay showed that apparently growth inhibition in MGC803 cell line treated with DADS could be seen and exhibited a dose-dependent model. Adding 20, 30, 40,50mg·L-1DADS for 24 h suppressed MGC803 cells growth by 18.38%,30.67%,38.87%,43.05% (P<0.05), and 29.58%,40.13%,45.29%,53.57%(P<0.05)for 48 h, and 34.84%,48.66%,62.38%,68.07%(P<0.05)for 72h. Flow cytometry analysis revealed that treating MGC803 cells with 20, 30mg·L-1DADS accumulated cells in the G2/M phase(P<0.05), which exhibited a dose-dependent model. The proportion of cells in the G2/M phase after treatment with 30mg·L-1DADS for 12h was comparable (26.4%), and more than three times that occurring in untreated cells (6.9%).After treated with 30mg·L-1DADS, 7 genes were up-regulated: CDC45L,CDK5R1(p35),CDKN1A(p21),CUL4A,GADD45α,RAD17,TP53; 14 genes were down-regulated: ANAPC5,ATR,BRCA1,CCNA2,CCNB2,CCNE1,CCNE2,CDC16,CDC6,CDK5RAP1,GTF2H1,MCM5,RAD9A,RGC32. Some genes were directly related with cell cycle G2/M phase, including CDK5R1,TP53,p21,GADD45α,ANAPC5,ATR,BRCA1,CCNCA2,CCNB2,CDC6,CDC16,GTF2H1,RGC32 etc.After treated with 30mg·L-1DADS for different times, RT-PCR analysis showed the TP53,GADD45αand p21 were up-regulated while CyclinB1 was down-regulated.Western blot analysis showed p53,GADD45αand p21 were up-regulated whereas CyclinB1 was down-regulated.Conclusion:1. DADS can inhibit MGC803 cell proliferation and induce it cell cycle arrest in G2/M phase.2. Cell cycle arrest of MGC803 cells induced by DADS relates with various genes through coregulating of various signal transduction pathways. Among of the differential expression genes, CyclinE1 and CyclinE2 that are blong to G1 phase genes have also changed, which demonstrates DADS has some impact on whole cell cycle. 3. G2/M arrest of MGC803 cells by DADS may be related to enhance expression of p53, GADD45α, p21 and down-regulation of CyclinB1.