Diallyl Trisulfide Induces HL-60 Cell Apoptosis By Activating Mixed Lineage Kinase 3/c-Jun N-terminal Kinase Mediated Via Reactive Oxygen Species

Objective: To explore the mechanism of HL-60 cell apoptosis induced by diallyl trisulfid and the role of reactive oxygen species in which process.Method: HL-60 cells were either treated with various doses of DATS alone, or DATS combination with antioxidant N-acetyl-L-cysteine (NAC) for 0.5, 1, 3, 6, 12h, respectively. The intracellular ROS level was detected by flow cytometry. The morphological change of apoptosis was observed by Hoechst staining. DNA fragmentation in HL-60 cells was detected by DNA Ladder test. HL-60 cells were either treated with100μM DATS or DATS combination with CEP-1347(a MLK special inhibitor) for 3h, The Phosphorylation of Mixed Lineage Kinase 3 and JNK was analyzed by Western blot.Results: The results from flow cytometry indicated that 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 during the time from 0.5 to 3h.and reached at maximuam(89.7±3.67) when HL-60 cells were incubated with DATS of 150μM for 3 hours, and then maintain a high level. The results from Hoechst staining and DNA Ladder test showed that Karyopyknosis, caryorrhexis and limpid DNA Ladder was observed after treatment of HL-60 cells with DATS from 6 to 24 hours. NAC, a special blocking agent of ROS, attenuates the HL-60 cell apoptotic effect induced by DATS.Our results also indicated that DATS induced phosphorylation of Mixed Lineage Kinase 3 and JNK. HL-60 cells were treated with 100μM DATS for 12h,The protein expression of JNK, and that pretreatment of NAC and CEP-1347 decreased the phosphorylation of MLK3 and JNK induced by 100μM DATS for 3h.Conclusion: DATS induces the phosphorylation of MLK3 and JNK, which mediated by reactive oxygen species in HL-60 cells. MLK3/JNK signaling by DATS is involved HL-60 cell apoptosis.