Induction of Apoptosis and Upregulation of Fas Ligand and Fas-Associated Death Domain by Doxorubicin in Human Jurkat Leukemia T-Cells

Objective To investigate the dose and time kinetics of induction of apoptosis induced by doxorubicin in Jurkat leukemia cells,and to explore its pertinent molecular mechanisms.Methods Human Jurkat leukemia T-cells were treated with doxorubicin at the concentration of 0.1 mg/L,0.2 mg/L,0.5 mg/L and 1.0 mg/L for 6,12,24 and 36 hours,respectively,of which one sample was pretreated with zVAD-fmk(benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone) prior to addition of doxorubicin 0.2 mg/L.Apoptosis was detected with both annexin V-FITC and propidium iodide(PI) staining and annexin V-FITC and PI double positive cells were analyzed by flow cytometry.Western blot was used to evaluate the level of Fas ligand(FasL) and FADD(Fas-associated death domain) expression.Results The differences of apoptotic cells induced by all dose of doxorubicin were not significant(P0.05) at 6 hour;at 12 hour,only the highest dose,1 mg/L,significantly induced cell apoptosis;while the lowest dose,0.1 mg/L,did not significantly caused cell apoptosis for all time points.After exposure to the doses of 0.2 and 0.5 mg/L for 24 or 36 hours,a significant increase in percentage of apoptotic cells was observed(P0.001).Apoptosis induced by doxorubicin was completely inhibited when the cells were incubated with doxorubicin in the presence of zVAD-fmk(P0.001).The level of FasL and FADD expression correspondingly increased with exposure time to doxorubicin.Conclusions Doxorubicin induces apoptosis in a dose-and time-dependent manner;upregulated FasL may initiate the activation of the Fas signaling pathway and caspases are the ultimate executioner in the induction of leukemia cell apoptosis by doxorubicin.