The Cell Cycle-associated Genes Holded-up By Diallyl Disulfide Was Screened By Genic Chip In Human Nasopharyngeal Carcinoma Cell Line CNE2

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 biological effects of cell arrest induced by DADS on human gastric cancer cell line CNE-2 and its molecular mechanisms.Methods:The growth inhibition effect of DADS on CNE2 cell line was measured by MTT assay and cell counting and HE staining. Flow cytomery was used to determine the change of cell cycle. The expression profile of cell cycle-associated genes in CNE-2 cells exposed to DADS was indentified by SuperArray Human Cell cycle Gene . The microarray results were comfirmed by RT-PCR and Western blot.Results: The MTT assay showed that DADS inhibited growth of CNE2 cells significantly and exhibited a dose dependent modal. Adding 50μmol/L,100μmol/L,200μmol/L and 400μmol/L DADS for 48 hours suppressed CNE2 growth by 2.78%,6.09%,22.64%,57.05%, respectively. Cell counting showed that average doubling time retarded from 24.48h in normal cultured CNE2 cells to 92.36h in 400μmol/L DADS experimental CNE2 cells (P<0.05). Light microscope examination indicated that heteromorphism and karyoplasmic ratio were decreased when treated with 50μmol/L,100μmol/L,200μmol/L and 400μmol/L DADS for 48 hours in a dose and time dependent. Flow cytometry analysis revealed that treating CNE2 cells with increasing quantities of DADS increased the percentage of cells in the G0/G1 phase. After treated with 200umol/L DADS, 20 genes were up-regulated: RECK,VEGF,PLK3,SUGT1,FLCN,TACC1,FRK,HECA,JMY,KRAS,PARD6B,PDGFB,CDKN2B,CYLD,HK2,IL1B,CCNG2,SASH1,FOSB,DUSP1 (P<0.01); 11 genes were down-regulated: GTSE1,PLK1,TFDP1,RCC1,ANAPC7,RAN,SMC4L1,BUB1B,CTCF,BRCA2,KLK10 (P<0.01). Some genes were directly related with cell cycle G2/M phase, including CDK5R1,TP53,CDKN1A,GADD45α,ANAPC5,ATR,BRCA1,CCNCA2,CCNB2,CDC6,CDC16,GTF2H1,RGC32 etc. The results of CDKN2B and RCC1 selected for RT-PCR and Western blot were consistent with those from Gene Array.Conclusion:1. DADS can inhibit CNE-2 cell proliferation and induce it cell cycle arrest in G0/G1 phase.2. Cell cycle arrest of CNE-2 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. G0/G1 arrest of CNE-2 cells by DADS may be related to enhance expression of CDKN2B and down-regulation of CyclinB1.