686288), a novel anticancer candidate agent, is undergoing clinical evaluation. AF induces DNA-protein cross-links (DPCs), c-H2AX phosphorylation, aryl hydrocarbon receptor (AhR) signaling, apoptosis and its own metabolism via cytochrome P4501A1 and 1A2 (CYP1A1/1A2) activation in sensitive estrogen receptor positive (ER1) MCF7 breast cancer cells. Estrogen receptor negative (ER2) 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 ER2 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 ER1 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, a-Naphthoflavone (a-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. ' 2007 Wiley-Liss, Inc.

Breast tumors often show profound sensitivity to exogenous oxidative stress. Investigational agent 2-(4-amino-3-methylphenyl)-5-fluorobenzothiazole (5F 203) induces aryl hydrocarbon receptor (AhR)-mediated DNA damage in certain breast cancer cells. Since AhR agonists often elevate intracellular oxidative stress, we hypothesize that 5F 203 increases reactive oxygen species (ROS) to induce DNA damage, which thwarts breast cancer cell growth. We found that 5F 203 induced single-strand break formation. 5F 203 enhanced oxidative DNA damage that was specific to breast cancer cells sensitive to its cytotoxic actions, as it did not increase oxidative DNA damage or ROS formation in nontumorigenic MCF-10A breast epithelial cells. In contrast, AhR agonist and procarcinogen benzo[a]pyrene and its metabolite, 1,6-benzo[a]pyrene quinone, induced oxidative DNA damage and ROS formation, respectively, in MCF-10A cells. In sensitive breast cancer cells, 5F 203 activated ROS-responsive kinases: c-Jun-N-terminal kinase (JNK) and p38 mitogen activated protein kinase (p38). AhR antagonists (alpha-naphthoflavone, CH223191) or antioxidants (N-acetyl-l-cysteine, EUK-134) attenuated 5F 203-mediated JNK and p38 activation, depending on the cell type. Pharmacological inhibition of AhR, JNK, or p38 attenuated 5F 203-mediated increases in intracellular ROS, apoptosis, and single-strand break formation. 5F 203 induced the expression of cytoglobin, an oxidative stress-responsive gene and a putative tumor suppressor, which was diminished with AhR, JNK, or p38 inhibition. Additionally, 5F 203-mediated increases in ROS production and cytoglobin were suppressed in AHR100 cells (AhR ligand-unresponsive MCF-7 breast cancer cells). Our data demonstrate 5F 203 induces ROS-mediated DNA damage at least in part via AhR, JNK, or p38 activation and modulates the expression of oxidative stress-responsive genes such as cytoglobin to confer its anticancer action.

ABSTRACT:Fluorinated 2-(4-amino-3-methylphenyl)benzothiazoles possess potent antiproliferative activity against certain cancer cells, similar to the unfluorinated 2-(4-amino-3-methylphenyl)benzothiazole (DF 203, NSC 674495). In "sensitive" cancer cells, DF 203 is metabolized by, can induce expression of, and binds covalently to CYP1A1. Metabolism appears to be essential for its antiproliferative activity through DNA adduct formation. However, a biphasic dose-response relationship compromises its straightforward development as a chemotherapeutic agent. We investigated whether fluorinated benzothiazoles inhibit cancer cell growth without the biphasic dose-response, and whether the fluorinated benzothiazoles are also metabolized into Benzothiazoles contain a benzene ring fused to a thiazole ring (Shi et al., 1996). These small molecules display antitumor properties that are modulated by substitutions at specific positions on the benzothiazole pharmacophore Kashiyama et al., 1999;Wells et al., 2000;Hutchinson et al., 2001Hutchinson et al., , 2002. 2-(4-Amino-3-methylphenyl)benzothiazole (DF 203) represents the lead compound for a recently explored series, and possesses potent antitumor properties in select breast, ovarian, and renal cancer cell lines of the 60-cell line, disease-oriented NCI Anticancer Drug Screen . DF 203 displays a unique biphasic dose-response relationship, with more potent activity at concentrations below 1 M but reduced antiproliferative activity at concentrations ranging from 1 to 100 M. The precise mechanism by which DF 203 and similar benzothiazoles display their antiproliferative effects has not been fully elucidated. Recent studies, however, suggest that their ability to become metabolized and covalently bind to subcellular targets contributes to their anticancer effects (Kashiyama et al., 1999;Chua et al., 2000;Hutchinson et al., 2001). Metabolism and covalent binding to subcellular This is part 25 of the series "Antitumor 2-(4-aminophenyl)

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