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.

Targeted pharmacotherapeutic agents have been successful in treating women with breast cancer that either over-expresses human epidermal growth factor receptor 2 (Her2/neu) or is dependent upon estrogen receptor signaling. However, patients with Her2/neu-negative or hormone-independent breast cancer do not benefit from these targeted agents. A candidate anticancer agent 2-(4-amino-3-methylphenyl)-5-benzothiazole (5F 203) targets the aryl hydrocarbon receptor signaling pathway and potently inhibits the growth of breast cancer cells irrespective of either estrogen receptor or Her2/neu status. 5F 203 may represent a promising agent for women who lack targeted therapeutic options. We have previously assessed the role reactive oxygen species (ROS) play in mediating the anticancer activity of 5F 203. In our current studies, we pretreated 5F 203-sensitive breast cancer cells with rotenone prior to treatment with 5F 203. This pretreatment diminished 5F 203-mediated ROS formation as measured by DCF-DA staining followed by flow cytometry. This suggests 5F 203-induced ROS production is partially derived from the mitochondria. Using the comet assay and a JC1 dye-based fluorometry assay respectively, we found 5F 203 induced single-strand break formation and mitochondrial membrane potential loss in sensitive breast cancer cells. Pretreatment with the antioxidant N-acetyl-L-cysteine partially diminished apoptosis as assessed by relief contrast microscopy and the Annexin V/PI assay. This implies that 5F 203-induced apoptosis depends at least in part on ROS derived from mitochondria. We also analyzed 84 oxidative stress-responsive genes using PCR-array and determined that 5F 203 significantly increased the expression of cytoglobin, a novel tumor suppressor, in sensitive breast cancer cells. These data imply ROS and cytoglobin contribute to the anticancer activity of 5F 203. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3658.

Breakthroughs are needed in breast cancer therapy to improve clinical outcomes. Emerging evidence suggests that tumorigenesis stems, in part, from epigenetically silenced tumor suppressor genes (TSGs) and restoring TSGs may represent a viable strategy to treat breast cancer. We previously found that aryl hydrocarbon receptor (AhR) agonist 2-(4-amino-3-methylphenyl)-5-fluorobenzothiazole (5F 203) exhibits potent cytotoxicity, increases reactive oxygen species (ROS), induces DNA damage and up-regulates the expression of putative tumor suppressor gene cytoglobin in breast cancer cells. In the current study, we seek to delineate the mechanism by which 5F 203 induces DNA damage and cytoglobin expression in susceptible breast cancer cells. We found that 5F 203 activated p38 mitogen activated protein kinase (p38) and c-Jun-N terminal kinase (JNK) signaling in breast cancer cells. Pretreatment with antioxidant N-acetyl-L-cysteine or AhR inhibitor α-naphthoflavone diminished 5F 203-mediated p38 or JNK activation in a cell context-dependent fashion. Pretreatment with pharmacological inhibitors of p38 or JNK suppressed 5F 203-mediated increases in intracellular ROS to suggest the presence of a positive feedback loop. 5F 203 induced oxidative DNA damage in breast cancer cells but not breast epithelial MCF-10A cells unlike AhR agonist benzo[a]pyrene which induced oxidative DNA damage more indiscriminately. Pretreatment with p38 or JNK inhibitors suppressed 5F 203-induced single strand break formation and cytoglobin mRNA expression in breast cancer cells. Our data show 5F 203 confers anticancer activity in breast cancer cells in part by increasing ROS via a positive feedback loop to sustain p38 and JNK activation resulting in DNA damage and cytoglobin restoration. Citation Format: Leah K. Rowland, Lancelot S. McLean, Petreena Campbell, Cheri N. Watkins, Dain Zylstra, Louisa H. Amis, Maheswari Senthil, Eileen Brantley. Aryl hydrocarbon receptor agonist 5F 203 induces oxidative stress triggering DNA damage and cytoglobin up-regulation in human breast cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2565. doi:10.1158/1538-7445.AM2015-2565