Induction of apoptosis by TLK199 in human leukemia cells

2270 TLK199 (ezatiostat hydrochloride) is a novel glutathione analog inhibitor of the enzyme glutathione S-transferase P1-1 (GST P1-1) that is currently under investigation for the treatment of cytopenias due to myelodysplastic syndrome or chemotherapy. GST P1-1 has been shown to negatively regulate JNK activity and decrease cellular reactive oxygen species (ROS) levels. Since both JNK activity and ROS levels have a role in cell proliferation, differentiation and apoptosis, a GST P1-1 inhibitor may modulate these cellular processes. Previously we have reported that TLK199 stimulates colony formation from normal peripheral blood and bone marrow cells at non-cytotoxic doses. In this study, we investigated additional effects of TLK199 on the growth of TF-1 erythroleukemia and HL-60 promyelocytic cells. Treatment with TLK199 in these leukemia cell lines resulted in apoptosis and increase in ROS levels. JNK activation was observed in TF-1 but not HL-60 cells. Consistent with the induction of apoptosis, treatment with TLK199 resulted in cleavage of PARP protein in a dose- and time-dependent manner, indicating the activation of caspase 3. In HL-60 cells, caspase 3 was maximally induced by 40 μM TLK199 in 5.5 hours, and its activation resulted in degradation of anti-apoptotic Mcl-1 protein. Treatment with TLK199 led to a loss of mitochondrial membrane potential and activation of caspase 9, but not caspase 8, suggesting that TLK199 induced the intrinsic apoptotic pathway. In support of the caspase cascade leading to loss of cell viability by TLK199, the pan-caspase inhibitor z-VAD-FMK protected HL-60 cells from undergoing apoptosis in the presence of TLK199 and increased the cytotoxic concentration of TLK199 by 2.5-fold. Microarray analysis of gene expression in TF-1 and HL-60 cells was performed. In both cell lines, TLK199 treatment resulted in the upregulation of several genes involved in the cellular response to ER stress. Consistent with the activation of JNK in TF-1 cells, TLK199 treatment upregulated genes for AP-1 transcription factors such as c- jun . These data indicate that TLK199 induces apoptosis in human leukemia cell lines TF-1 and HL-60 by activation of the caspase cascade and that caspase activation mediates the cytotoxic effects of TLK199.