2'-Nitroflavone induces apoptosis and modulates mitogen-activated protein kinase pathways in human leukaemia cells

The cytotoxic activity of 2'-nitroflavone was evaluated in different haematological cancer cell lines and its mechanism of action was further studied in HL-60 cells. 2'-Nitroflavone arrested the cell cycle at the G(2)/M phase and induced an apoptotic response characterized by an increase in the sub-G1 fraction of cells, a typical DNA ladder fragmentation, chromatin condensation and the detection of cells stained with Annexin V. Apoptosis was dependent on the activation of at least caspase-8, caspase-9 and caspase-3. The involvement of the death receptor pathway was indicated by the upregulation of both the tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) and its death receptor (DR5). We also showed that 2'-nitroflavone increased the expression levels of Bax and induced the release of cytochrome C to cytosol, suggesting the participation of the mitochondria-dependent pathway. When mitogen-activated protein kinases pathways were studied, it was found that p38 and c-Jun NH(2)-terminal kinase (JNK) pathways were activated by 2'-nitroflavone in HL-60 cells, whereas the phosphorylation levels of extracellular signal-regulated kinases (ERK) 1/2 decreased significantly. In addition, whereas both pharmacological inhibition of JNK and downregulation of JNK expression by RNA interference reduced the nitroflavone growth-inhibitory activity and the apoptotic effect, contrasting results were obtained when the ERK1/2 pathway was inhibited, and no effect was observed in the presence of a specific inhibitor of p38 mitogen-activated protein kinase. These findings show for the first time the antitumour action of 2'-nitroflavone in haematological cancer cell lines and suggest that both JNK and ERK1/2 cascades are involved in the apoptotic response induced by 2'-nitroflavone in HL-60 cells.