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

Abstract

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 cell cycle at G2/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 activation of at least caspase-8, -9 and -3. The involvement of death receptor pathway was indicated by the up-regulation of both tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and its death receptor (DR5). We also showed that 2´-nitroflavone increased 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 (MAPKs) pathways were studied, it was found that p38 and c-Jun NH2-terminal kinase (JNK) pathways were activated by 2´-nitroflavone in HL-60 cells, whereas phosphorylation levels of extracellular signal-regulated kinases (ERK) 1/2 significantly decreased. 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, opposite results were obtained when ERK 1/2 pathway was inhibited, and no effect was observed in the presence of a specific inhibitor of p38 MAPK. These findings demonstrate for the first time the antitumor action of 2´-nitroflavone in haematological cancer cell lines and suggest that both JNK and ERK 1/2 cascades are involved in the apoptotic response induced by 2´-nitroflavone in HL-60 cells.