Dose–response transition from cell cycle arrest to apoptosis with selective degradation of Mdm2 and p21WAF1/CIP1 in response to the novel anticancer agent, aminoflavone (NSC 686288)

Aminoflavone (AF, NSC 686288) is beginning clinical trials. It induces replication-mediated histone H2AX phosphorylation, DNA–protein crosslinks and activates p53. Here, we studied p21CIP1/WAF1 and Mdm2 responses to AF. Although p53 stabilization and phosphorylation at serine 15 increased with dose and time of exposure, Mdm2 and p21CIP1/WAF1 protein levels displayed a biphasic response, as they accumulated at submicromolar doses and then decreased with increasing AF. As both Mdm2 and p21CIP1/WAF1 mRNA levels increased with AF concentration without reduction at higher concentrations, we measured the half-lives of Mdm2 and p21CIP1/WAF1 proteins. Mdm2 and p21CIP1/WAF1 half-lives were shortened with increasing AF concentrations. Proteasomal degradation appears responsible for the decrease of both Mdm2 and p21CIP1/WAF1, as MG-132 prevented their degradation and revealed AF-induced Mdm2 polyubiquitylation. AF also induced protein kinase B (Akt) activation, which was reduced with increasing AF concentrations. Suppression of Akt by small interfering RNA was associated with downregulation of Mdm2 and p21CIP1/WAF1 and with enhanced apoptosis. These results suggest that the cellular responses to AF are determined at least in part by Mdm2 and p21CIP1/WAF1 protein levels, as well as by Akt activity, leading either to cell cycle arrest when Mdm2 and p21CIP1/WAF1 are elevated, or to apoptosis when Mdm2 and p21CIP1/WAF1 are degraded by the proteasome and Akt insufficiently activated to protect against apoptosis.