The Mechanism Of Diallyl Trisulfide Induce NADPH Oxidase Activation By Activating Src Kinase In Human Promyelocytic HL-60 Cells

Objective: To explore the mechanism of NADPH oxidase activation by diallyl trisulfide (DATS) in the human promyelocytic leukemia HL-60 cells.Methods: HL-60 cells were treated with DATS for 15min at the concentration of 50,100,150μM. Western blot was used to detect the Src kinase activation. HL-60 cells were treated with 150μM DATS for 0min, 5min, 15min, 30min, 60min, 180min respectively, the phosphorylation level of Src, p130Cas, CrkⅡ, NADPH oxidase subunit p47phox was analyzed by Western blot. HL-60 cells were pre-incubated with Src special inhibitor PP2 for 30 min and then treated with 150μM DATS for 15min, the phosphorylation levels of Src, p130Cas, CrkⅡand NADPH oxidase subunit p47phox was observed. The mRNA expression of NADPH oxidase subunit p47phoxand Rac2 was measured by RT-PCR after treating with 150μM DATS for 0min, 5min, 15min, 30min, 60min, 180min in HL-60 cells.The expression of p47phoxand Rac2 mRNA was detected in HL-60 cells treated with 150μM DATS for 15min after pretreatment with PP2. The activity of NADPH oxidase in HL-60 cells was evaluated by Colorimetric NBT reduction test.Results: The results from Western blot showed that phosphorylation level of Src kinase was correlated with DATS concentration.After HL-60 cells treated with DATS at a various concentration for 15min.The highest phosphorylation level of Src kinase was occurred at a concentration of 150μM DATS. Src, p130Cas, CrkⅡand p47phox phosphorylation was induced by 150μM DATS at the indicated time.The highest phosphorylation level was occurred for Src at 15min, for p130Cas and p47phoxat 30min. PP2 decreased the phosphorylation level of Src, p130Cas, CrkⅡand p47phox in HL-60 cells.The results from RT-PCR indicated DATS induced mRNA expression of p47phoxand Rac2 mRNA in time-dependent manner and that PP2 reduced mRNA expression of p47phoxand Rac2 induced by DATS. The result from NBT reduction test showed that NADPH oxidase activation induced by DATS can be inhibited by PP2 pretreatment in HL-60 cells.Conclusion:1. DATS induces the phosphorylation of Src, p130Cas, CrkⅡand p47phox and increases the mRNA levels of Rac2 and p47phox in HL-60cells.2. DATS activates NADPH oxidase, associating with signaling pathways of Src-- p47phox and Src-p130Cas-CrkⅡ-Rac2 in HL-60 cells.