The Molecular Mechanism Of Reactive Oxygen Species-mediated C-Jun N-terminal Kinase Activation Induced By Diallyl Trisulfide In HL-60 Cells

Objective : To explore the molecular mechanism of c-Jun N-terminal kinase activation and the role of reactive oxygen species in the activiation of c-Jun N-terminal kinase indured by diallyl trisulfide in HL-60 cells.Method: HL-60 cells were treated with various doses of DATS for 0.5, 1, 3, 6, 12h, intracellular ROS level was detected by flow cytometry. HL-60 cells were either treated with 150μM DATS alone, or DATS combination with antioxidant N-acetyl-L-cysteine (NAC) for 0.5, 1, 3, 6, 12h. The intracellular ROS level was detected by flow cytometry. The protein expression of JNK, GST-πand JIP, and phosphorylation of JNK, c-Jun and MLK3 was analyzed by Western blot. The GST-π-JNK complex and JIP-JNK complex was measured by coimmunoprecipitation and Western blot. The role of both MLK3 in JNK activation and JNK in c-jun phosphorylation was evaluated by the change in phosphorylation level of JNK and c-Jun after pretreatment HL-60 cells with CEP-1347, a MLK special inhibitor, or SP600125, a JNK special inhibitor.Results: DATS significantly increased the intracellular ROS level in HL-60 cells, which is a dose-and time-dependent. The fluorescence intensities of ROS increased in the dose-and time-dependent maner from 0.5 to 3h,reached at maximuam(89.7±3.67) when HL-60 cells were incubated with DATS of 150μM for 3 h, then maintained a high level. NAC pretreatment can dramatically decreased the ROS level in HL-60 cells.DATS had little effect on the protein expression of JNK and JIP, but induced phosphorylation of JNK, c-Jun and MLK3 after treated-HL-60 cells for 12h. The phosphorylation level of c-Jun increased at 3h and remained high level. The phosphorylation level of JNK reached the maximuam at 3h, and then dropped slightly, The phosphorylation of c-Jun and MLK3 increased at time-dependent maner. Antioxidant NAC attenuated the expression of p-JNK,c-Jun,p-c-Jun,p-MLK3. CEP-1347 decreased the phosphorylation level of JNK. SP600125 down-regulated the phosphorylation level of c-Jun induced by 150μM DATS for 3h in HL-60 cells. DATS had little effect on the protein expression of GST-πwithin 6h, and then decreased at 12h. NAC pretreatment did not affect the expression of GST-πand JIP. DATS induced GST-πdissociation from JNK, the minimum binding at 3h. NAC can effectively attenuated JNK dissociation from GST-πinduced by DATS. DATS accelerated the formation of JNK-JIP complex, the maximum binding at 3h. NAC had little effect on formation of JNK-JIP complex.Conclusion1. DATS induced ROS generation and phosphorylation of MLK3, JNK and c-Jun in HL-60 cells.2. ROS induced by DATS mediated MLK3/JNK/c-Jun signaling activation, and promoted GST-πdissociation from JNK.3. DATS activated JNK through activating MLK3 and blocked inhibitory effect of GST-πon JNK.