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.

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Nearly 40 000 women die annually from breast cancer in the United States. Clinically available targeted breast cancer therapy is largely ineffective in triple negative breast cancer (TNBC), characterized by tumors that lack expression of the estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (Her2). TNBC is associated with a poor prognosis. Previous reports show that aryl hydrocarbon receptor (AhR) partial agonist 2‐(4‐amino‐3‐methylphenyl)‐5‐fluorobenzothiazole (5F 203) selectively inhibits the growth of breast cancer cells, including those of the TNBC subtype. We previously demonstrated that 5F 203 induced the expression of putative tumor suppressor gene cytoglobin (CYGB) in breast cancer cells. In the current study, we determined that 5F 203 induces apoptosis and caspase‐3 activation in MDA‐MB‐468 TNBC cells and in T47D ER+ PR + Her2 − breast cancer cells. We also show that caspases and CYGB promote 5F 203–mediated apoptosis in MDA‐MB‐468 cells. 5F 203 induced lysosomal membrane permeabilization (LMP) and cathepsin B release in MDA‐MB‐468 and T47D cells. In addition, silencing CYGB attenuated the ability of 5F 203 to induce caspase‐3/‐7 activation, proapoptotic gene expression, LMP, and cathepsin B release in MDA‐MB‐468 cells. Moreover, 5F 203 induced CYGB protein expression, proapoptotic protein expression, and caspase‐3 cleavage in MDA‐MB‐468 cells and in MDA‐MB‐468 xenograft tumors grown orthotopically in athymic mice. These data provide a basis for the development of AhR ligands with the potential to restore CYGB expression as a novel strategy to treat TNBC.

Quassinoids often exhibit anti-oxidant and anti-proliferative activity. Emerging evidence suggests that these natural metabolites also display chemopreventive actions. In this study, we investigated the potential for the quassinoid glaucarubulone glucoside (Gg), isolated from the endemic Jamaican plant Castela macrophylla (Simaroubaceae), to display potent cytotoxicity and inhibit human cytochrome P450s (CYPs), particularly CYP1A enzymes, known to convert polyaromatic hydrocarbons (PAHs) into carcinogenic metabolites. Gg reduced the viability of MCF-7 breast adenocarcinoma cells (IC50 = 121 nM) to a greater extent than standard of care anticancer agents 5-fluorouracil, tamoxifen (IC50 > 10 μM) and the tamoxifen metabolite 4-hydroxytamoxifen (IC50 = 2.6 μM), yet was not cytotoxic to non-tumourigenic MCF-10A breast epithelial cells. Additionally, Gg induced MCF-7 breast cancer cell death. Gg blocked increases in reactive oxygen species in MCF-10A cells mediated by PAH benzo[a]pyrene (B[a]P) metabolite benzo[a]pyrene 1,6 quinone, yet down-regulated the expression of genes that promote antioxidant activity in MCF-7 cells. This implies that Gg exhibits anti-oxidant and cytoprotective actions in non-tumourigenic breast epithelial cells and pro-oxidant, cytotoxic actions in breast cancer cells. Furthermore, Gg inhibited the activities of human CYP1A according to non-competitive kinetics and attenuated the ability of B[a]P to induce CYP1A gene expression in MCF-7 cells. These data indicate that Gg selectively suppresses MCF-7 breast cancer cell growth without impacting non-tumourigenic breast epithelial cells and blocks B[a]P-mediated CYP1A induction. Taken together, our data provide a rationale for further investigations of Gg and similar plant isolates as potential agents to treat and prevent breast cancer.

Despite advances in targeted therapeutic agents for women with breast cancer that either express estrogen receptor (ER) alpha or over-expresses human epidermal growth factor receptor 2 (Her2), nearly 40,000 women die each year due to this malignancy. An investigational anticancer agent 2-(4-amino-3-methylphenyl)-5-benzothiazole (5F 203) potently inhibits the growth of breast cancer cells irrespective of either ER or Her2 status. In our current studies we used the Annexin V/PI assay to determine that 5F 203 induced a dose-dependent increase in the percentage of ER/Her2-positive T47D cells and ER/Her2-negative MDA-MB-468 breast cancer cells in early apoptosis. Cell death was not entirely caspase-dependent as pretreatment with pan-caspase inhibitor z-VAD-fmk only partially suppressed apoptosis. A pathway specific PCR-array revealed the ability for 5F 203 to increase the mRNA expression of pro-apoptotic genes bcl-2-antagonist/killer 1 (BAK1) and lymphotoxin alpha (LTA). Using the Acridine Orange assay, we found that 5F 203-mediated cell death was associated with lysosomal membrane permeabilization in both T47D and MDA-MB-468 cells. Additionally, we determined using the Cathepsin B assay that 5F 203 promoted the release of cathepsin B from the lysosomes in both breast cancer cell types. These data indicate 5F 203 upregulates BAK1 and LTA expression and promotes lysosomal cell death triggering the release of cathepsin B in breast cancer cells to confer its anticancer action. Citation Format: Eileen J. Brantley, Gabriell Thorne, Devin Daly, Dain Zylstra, Lancelot McLean. Investigational agent 5F 203 modulates apoptotic regulatory gene expression and induces lysosomal membrane permeabilization in breast cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2102. doi:10.1158/1538-7445.AM2013-2102