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)

Abstract

Aminoflavone (AF, NSC 686288) is beginning clinical trials. It induces replication-mediated histone H2AX phosphorylation, DNA-protein crosslinks and activates p53. Here, we studied p21 CIP1/WAF1 and Mdm2 responses to AF. Although p53 stabilization and phosphorylation at serine 15 increased with dose and time of exposure, Mdm2 and p21 CIP1/WAF1 protein levels displayed a biphasic response, as they accumulated at submicromolar doses and then decreased with increasing AF. As both Mdm2 and p21 CIP1/WAF1 mRNA levels increased with AF concentration without reduction at higher concentrations, we measured the half-lives of Mdm2 and p21proteins. Mdm2 and p21 CIP1/WAF1 half-lives were shortened with increasing AF concentrations. Proteasomal degradation appears responsible for the decrease of both Mdm2 and p21 CIP1/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 p21 CIP1/WAF1 and with enhanced apoptosis. These results suggest that the cellular responses to AF are determined at least in part by Mdm2 and p21 CIP1/WAF1 protein levels, as well as by Akt activity, leading either to cell cycle arrest when Mdm2 and p21 CIP1/WAF1 are elevated, or to apoptosis when Mdm2 and p21 CIP1/WAF1 are degraded by the proteasome and Akt insufficiently activated to protect against apoptosis.

Aminoflavone induces oxidative DNA damage and reactive oxidative species‐mediated apoptosis in breast cancer cells

“…This is consistent with what was determined in a recent publication reporting apoptosis induction by AF in other sensitive cancer cells. 12 The biphasic apoptotic response correlates with the biphasic response seen for DNA damage induced by AF in MDA-MB-468 cells.…”

“…36 Previous studies demonstrate the ability of AF to induce p21 expression in MCF-7 cells. 8,12 To determine whether p21 expression levels are altered in MDA-MB-468 cells following AF treatment, we exposed these cells at varying time points (2-18 hr) to AF (1 lM) and measured p21 protein expression using Western blotting. Data demonstrate that AF induces an increase in p21 expression within 2 hr and sustains this effect for the duration of treatment (Fig.…”

“…Sensitive cells possess constitutively high levels of sulfotransferases which can convert AF into active species and depend upon AhR signaling pathway activation resulting in CYP1A1 or CYP1A2 induction in response to the apoptotic and DNA damaging effects of AF. 10,12 Certain ER2 breast cancer cells (i.e., MDA-MB-435) demonstrate resistance to AF 6,7 since they contain low sulfotranferase expression levels and are unresponsive to AhR signaling activation.…”

“…Besides its estrogen receptor status, MDA-MB-468 cells differ from MCF-7 cells in possessing a mutated form of p53 while MCF-7 cells possess wild type p53. [14][15][16] We anticipated that differences may occur in the ability of AF to induce p21 (waf1/cip1) expression, 12 a protein that TM dye reagent was added and absorbance or fluorescence was measured to determine the anticancer activity of AF as determined in Materials and Methods. Data represent the mean of at least 3 independent experiments performed in quadruplicate; bars, SEM.…”

Association of Human Cytochrome P450 1A1 (CYP1A1) and Sulfotransferase 1A1 (SULT1A1) Polymorphisms with Differential Metabolism and Cytotoxicity of Aminoflavone

“…It is entering phase II trials for the treatment of breast cancer. Mechanistic studies on the cytotoxicity of AF have revealed that AF induces DNA damage associated with DNA-protein cross-link, DNA singlestrand breaks, and DNA replication-dependent DNA double-strand breaks (3,5). AF also inhibits DNA synthesis and induces S-phase arrest (3).…”