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Aminoflavone (5-amino-2-(4-amino-3-fluorophenyl)-6,8-difluoro-7-methylchromen-4-one; AF; NSC 686288), a novel anticancer candidate agent, is undergoing clinical evaluation. AF induces DNA-protein cross-links (DPCs), γ-H2AX phosphorylation, aryl hydrocarbon receptor (AhR) signaling, apoptosis and its own metabolismviacytochromeP4501A1and1A2(CYP1A1/1A2) activation in sensitive estrogen receptor positive (ER+) MCF7 breast cancer cells. Estrogen receptor negative (ER−) breast cancer is typically more aggressive with a poorer prognosis. In this investigation, we evaluated the ability of AF to induce reactive oxygen species (ROS) formation, oxidative DNA damage and apoptosis in ER− MDA-MB-468 breast cancer cells. The antioxidant,N-acetyl-l-cysteine (NAC), attenuated the cytotoxic effects of AF in MDA-MB-468 cells; an effect is also observed in ER+ T47D breast cancer cells. Nonmalignant MCF10A breast epithelial cells were resistant to the cytotoxic effects of AF. AF increased intracellular ROS, an effect blocked by NAC and the CYP1A1/1A2 inhibitor, α-Naphthoflavone (α-NF). AF induced oxidative DNA damage as evidenced by increased 8-oxo-7,8-dihydroguanine (8-oxodG) levels and DPC formation in these cells. AF caused S-phase arrest corresponding to an increase in p21(waf1/cip1) protein expression. AF induced caspase 3, 8 and 9 activation, caspase-dependent apoptotic body formation and poly [ADP-ribose] polymerase (PARP) cleavage. Pretreatment with the pan-caspase inhibitor, benzyloxycarbonyl-Val-Ala-DL-Asp(OMe)-fluoromethylketone inhibited apoptosis and partially inhibited ROS formation and oxidative DNA damage. Pretreatment with NAC attenuated AF-induced apoptotic body formation and caspase 3 activation. These studies suggest AF inhibits the growth of breast cancer cells in part, by inducing ROS production, oxidative DNA damage and apoptosis and has the potential to treat hormone-independent breast cancer.