NOAD, a novel nitric oxide donor, induces G2/M phase arrest and apoptosis in human hepatocellular carcinoma Bel-7402 cells

Abstract

O2-(2,4-dinitro-5-{[2-(12-en-28-b-d-galactopyranosyl-oleanolate-3-yl)-oxy-2-oxoethyl]amino}phenyl)1-(N-hydroxyethylmethylamino)diazen-1-ium-1,2-diolate (NOAD), a novel NO-releasing derivative of oleanolic acid (OA), is an active cytotoxic component. In this study, NOAD induced a rise in intracellular NO levels and showed cytotoxic effects which were prevented by hemoglobin (NO scavenger). Meanwhile, NOAD induced G2/M phase cell cycle arrest in a concentration-dependent manner. Analysis of the cell cycle regulatory proteins demonstrated that NOAD did not change the steady-state levels of cyclin A, cyclin B, cyclin E, Cdk2 and Cdk4, but decreased the protein levels of Cdk1 and Cdc25C. Meanwhile, the levels of phosphorylation of Cdc25C and Cdk1 were significantly increased by NOAD in a concentration-dependent manner. Moreover, NOAD modulated the phosphorylation of protein kinases Chk2. During the G2/M arrest, cyclin-dependent kinase inhibitors (CDKIs), p21WAF1/CIP1 and p27kip1 were increased in a concentration-dependent manner. In addition, NOAD also caused a marked increase in the apoptotic cells, as characterized by fragmented nuclei, sub G1 formation, the level of 8-OHDG increase and poly (ADP-ribose) polymerase (PARP) cleavage, which was associated with activation of caspase-3, caspase-8 and caspase-9. Up-regulation of Bax and down-regulation of Bcl-2 were also observed in Bel-7402 cells treated with NOAD. These data suggest that NOAD produces anti-tumor effect via induction of G2/M cell cycle arrest and apoptosis.