Etoposide-induced Apoptosis Through Caspase-3 And JNK Activation In HL-60 Cells

Objective:The present study aimed at investigating etoposide-induced apoptosis and the underlying mechanisms involving activation of c-Jun NH2-terminal kinase (JNK) and caspase-3 using the human leukemia cell line HL-60 cells.Methods:HL-60 cells were treated with etoposide. The cells survival rate was determined by trypan blue staining and etoposide-induced apoptosis was analyzed by AO/EB double fluorescent staining and DNA gel electrophoresis. Effects of etoposide and the caspase inhibitor, z-VAD-FMK, on JNK and caspase-3 activity were determined, respectively, by using western blotting and a caspase-3 Fluorometric Assay Kit.Results:1. Etoposide decreased HL-60 cells survival rate in a dose-dependent manner at concentration ranging from 0.2 μmol/L to 200.0 μmol/L and in a time-dependent manner at a concentration of 20.0 μmol/L.2. AO/EB double fluorescent staining showed typical apoptotic morphological changes induced by etoposide, such as a decrease in cell volume, chromatin condensation, nuclear disintegration, and apoptotic bodies, etc.; The "DNA ladder" analysis detected DNA fragmentation in the treated cells. 3. Etoposide activated JNK in a time and dose dependent manner, the activation occurred 5 min after the treatment and sustained to the end of the treatment. Z-VAD-FMK, a caspase inhibitor, had no effects on JNK.4. Etoposide induced capcase-3 activation. With the range of doses used in this study, there was a relationship between etoposide concentration and caspase-3 activity. Z-VAD-FMK obviously inhibited caspase-3.Conclusions:1. Etoposide induces the apoptosis in HL-60 cells. 2. Etoposide-induced apoptosis in HL-60 cells is at least partially mediated by JNK and caspase-3.