Effects Of Diallyl Disulfide On Expression Of Apoptosis And Cell Cycle-associated Genes In Human Leukemia HL-60 Cells

Part OneEffects of Diallyl Disulfide on Cell Proliferation Inhibition and Apoptosis Induction in HL-60 CellsObjective: To investigate the effects of diallyl disulfide (DADS) on cell proliferation, apoptosis and cell cycle in human leukemia cell line HL-60.Methods: Cell proliferation inhibition was estimated by growth curve and average doubling time. Cell cycle was detected by flow cytometry. Cell apoptosis was verified by cell morphology observed under light microscope, flow cytometry with PI and AnnexinⅤ/PI staining, and DNA agarose gel electrophoresis. Results: (1) Growth curve showed that cell proliferation could be significantly inhibited by 60μmol·L-1 and 120μmol·L-1 DADS(P<0.05). (2) Average doubling time of HL-60 was delayed from 18.45h in the control group to 30.95h in the 60μmol·L-1 group and 60.70h in the 120μmol·L-1 group(P<0.05). (3) The cell proliferation inhibition was associated with cell cycle arrest. S and G2/M phase arrest could be induced by 15μmol·L-1 and 30μmol·L-1 DADS and G1 phase arrest could be induced by 60μmol·L-1 and 120μmol·L-1 DADS(P<0.05). (4) Flow cytometry analysis showed that the apoptotic rate of HL-60 cells treated with 15, 30, 60μmol·L-1 DADS for 24h respectively, was (3.45±0.35)%,(10.90±0.85)%,(33.25±1.63)%,increased in a concentration-dependent manner(P<0.05). (5) AnnexinⅤ/PI staining showed that the apoptotic rate of HL-60 cells treated with 60μmol·L-1 DADS for 4, 8, 12h respectively, was (4.60±0.45)%,(8.51±0.45)%,(16.92±0.77)%, increased in a time-dependent manner(P<0.05). (6) After treated with 60μmol·L-1 DADS for 24h, DNA extracted from HL-60 cells displayed a characteristic ladder pattern on agarose gel electrophoresis, and typical morphologic changes were observed under microscope, including cell shrinkage, nuclear condensation, and formation of apoptotic bodies.Conclusion:1. DADS could significantly inhibit the proliferation of HL-60 cells, which was associated with cell cycle arrest.2. 60μmol·L-1 DADS could effectively induce apoptosis of HL-60 cells.Part TwoThe Expression Profile Analysis of Related Genes Induced by DADS in HL-60 CellsObjective: To explore the expression profile of related genes induced by DADS in HL-60 cells with gene array and its molecular mechanisms. Methods: Total RNA was isolated from cells, untreated and treated with 60μmol·L-1 DADS for 24h, and reverse-transcribed into a cDNA probe labeled with Bio-16-dUTP. cDNA was hybridized to the Human Apoptosis and Cell Cycle Gene Array of Superarray Bioscience, containing 288 key genes involved in apoptosis, cell cycle and oxidative stress. The gene expression profile was analysed by GEArray Analyzer software. The microarray results were comfirmed by RT-PCR and Western blot.Results: (1) 6 genes up-regulated, including Fas-L,TRAF1,TRAF5,E2F6,IL8,CXCL10, and 22 genes down-regulated, including Bag-1,BNIP3,MCL-1,TNFRSF10C,TNFRSF8,Cyclin B,Cyclin C,Cks1p9,CUL4A,E2F-4,Hus1, MCM2,MKI67,MRE11B,NEDD8,PCNA,SKP1A,GPX1,CAT,DNAJA1,HSPA4,HSPA9B, were found with Gene Array. (2) The results of Fas-L and Bag-1 selected for RT-PCR and Western blot were consistent with those from Gene Array.Conclusion:1. DADS could inhibt cell proliferation of HL-60 cells via down-regulate MCM2,MKI67 and PCNA.2. DADS could induce cell cycle arrest of HL-60 cells via down-regulate CyclinB1,CKS1B,CUL4A,NEDD8 and SKP1A.3. DADS could induce apoptosis of HL-60 cells via up-regulate Fas-L and down-regulate Bag-1,MCL1,TNFRSF8,TNFRSF10C, which might be mediated by mutiple signal transduction pathways.