Eileen Brantley

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

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 metabolism via cytochrome P4501A1 and 1A2 (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.

Aryl Hydrocarbon Receptor Ligand 5F 203 Induces Oxidative Stress That Triggers DNA Damage in Human Breast Cancer Cells.

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.

One-pot synthesis of cinnamylideneacetophenones and their in vitro cytotoxicity in breast cancer cells

A series of cinnamylideneacetophenones were synthesized via a modified Claisen-Schmidt condensation reaction and evaluated for cytotoxicity against breast cancer cells using the Alamar Blue™ assay. Derivatives 17 and 18 bearing a 2-nitro group on the B ring, exhibited sub-micromolar cytotoxicity in MCF-7 cells (IC50=71 and 1.9 nM), respectively. Derivative 17 also displayed sub-micromolar (IC50=780 nM) cytotoxicity in MDA-MB-468 cells. Additionally, 17 and 18 displayed significantly less cytotoxicity than the chemotherapeutic doxorubicin in non-tumorigenic MCF-10A cells. This study provides evidence supporting the continued development of nitro-substituted cinnamylideneacetophenones as small molecules to treat breast cancer.

AhR ligand aminoflavone suppresses α6-integrin-Src-Akt signaling to attenuate tamoxifen resistance in breast cancer cells: CAMPBELL et al.

More than 40% of patients with luminal breast cancer treated with endocrine therapy agent tamoxifen demonstrate resistance. Emerging evidence suggests tumor initiating cells (TICs) and aberrant activation of Src and Akt signaling drive tamoxifen resistance and relapse. We previously demonstrated that aryl hydrocarbon receptor ligand aminoflavone (AF) inhibits the expression of TIC gene α6‐integrin and disrupts mammospheres derived from tamoxifen‐sensitive breast cancer cells. In the current study, we hypothesize that tamoxifen‐resistant (TamR) cells exhibit higher levels of α6‐integrin than tamoxifen‐sensitive cells and that AF inhibits the growth of TamR cells by suppressing α6‐integrin–Src–Akt signaling. In support of our hypothesis, TamR cells and associated mammospheres were found to exhibit elevated α6‐integrin expression compared with their tamoxifen‐sensitive counterparts. Furthermore, tumor sections from patients who relapsed on tamoxifen showed enhanced α6‐integrin expression. Gene expression profiling from the TCGA database further revealed that basal‐like breast cancer samples, known to be largely unresponsive to tamoxifen, demonstrated higher α6‐integrin levels than luminal breast cancer samples. Importantly, AF reduced TamR cell viability and disrupted TamR mammospheres while concomitantly reducing α6‐integrin messenger RNA and protein levels. In addition, AF and small interfering RNA against α6‐integrin blocked tamoxifen‐stimulated proliferation of TamR MCF‐7 cells and further sensitized these cells to tamoxifen. Moreover, AF reduced Src and Akt signaling activation in TamR MCF‐7 cells. Our findings suggest elevated α6‐integrin expression is associated with tamoxifen resistance and AF suppresses α6‐integrin–Src–Akt signaling activation to confer activity against TamR breast cancer.

Abstract 4177: Anticancer agent Aminoflavone restores the expression of tumor suppressor miRNA 26a and inhibits putative stemness biomarker α6-integrin in Tamoxifen resistant cells

Despite the efficacy of anti-estrogen agent Tamoxifen, commonly used to treat patients with estrogen receptor positive (ER + ) tumors, up to 40% of patients experience recurrence. Breast tumor-initiating cells (TICs), or breast cancer stem cells, exhibit Tamoxifen resistance and contribute substantially to recurrence. We recently demonstrated that investigational anticancer agent Aminoflavone (AF) disrupts mammospheres (in vitro clusters of cells enriched with TICs) by thwarting the expression of α6-integrin. In the current study, we found AF potently inhibited Tamoxifen resistant (TamR) cell growth and blocked Tamoxifen-mediated stimulation of TamR cell proliferation using the Alamar Blue assay. qPCR analyses revealed α6-integrin expression was significantly elevated in ER + breast cancer cell models of acquired and de novo Tamoxifen resistance relative to Tamoxifen sensitive cells. In particular, AF decreased the expression of both A and B variants of α6-integrin, the B variant being essential for TIC function. Western blotting revealed AF reduced total α6-integrin expression in TamR cells. Furthermore, we found that an anti-α6-integrin blocking antibody sensitized TamR cells to the active Tamoxifen metabolite, 4-hydroxy-Tamoxifen, and enhanced the efficacy of AF in these cells. AF also reduced the protein expression of p-Src, a downstream target of α6-integrin that is linked to Tamoxifen resistance and decreased breast cancer survival. In addition, miRNA sequencing of Tamoxifen sensitive and TamR mammospheres revealed differential expression of several miRNAs. Notably, miR-26a expression was down-regulated 2-fold in TamR mammospheres compared to Tamoxifen sensitive mammospheres and AF restored miR-26a expression 5-fold in TamR mammospheres. Using miRNA target prediction algorithms TargetScan and PicTar, we found miR-26a binding sites on the α6-integrin promoter. Taken together, our data suggest that AF re-expresses tumor suppressor miR-26a and inhibits the α6-integrin/Src signaling axis to reduce TIC capacity and counteract Tamoxifen resistance. Citation Format: Petreena Campbell, Leah Rowland, Anna Opoku-Agyeman, Nichole Mavingire, Ubaldo Soto, Gayathri Nagaraj, Yonghong Zhang, Sean (Xin) Chen, Charles Wang, Eileen Brantley. Anticancer agent Aminoflavone restores the expression of tumor suppressor miRNA 26a and inhibits putative stemness biomarker α6-integrin in Tamoxifen resistant cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4177. doi:10.1158/1538-7445.AM2017-4177

Abstract 2565: Aryl hydrocarbon receptor agonist 5F 203 induces oxidative stress triggering DNA damage and cytoglobin up-regulation in human breast cancer cells

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

Abstract 215: Aminoflavone exhibits in vivo efficacy on cancer stem cells in a spontaneous estrogen-dependent breast cancer murine model

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Stem-cell populations have been identified in a range of solid tumors and possess the capacity to self renew. It is thought that these cancer stem cells (CSCs) play an important role in cancer establishment, progression, and resistance to current treatments. CSCs have been identified in primary breast cancer tissues and cell lines derived from metastatic tumors. Traditional cancer therapies are effective at debulking some tumors but often fail to produce long-term clinical remissions, presumably due to their inability to eradicate the cancer stem cell population. Therefore, novel treatments aimed at targeting this population can potentially enhance treatment of both primary and metastatic tumors. Aminoflavone (AF; NSC 686288) is a novel anticancer agent. Previous work from our research group demonstrated that AF is a ligand of the aryl hydrocarbon receptor (AhR) and displays cytotoxicity in the MCF-7 breast cancer cell line. A prodrug formulation of AF (AFP464, NSC710464), recently entered phase II clinical trials. Moreover, we previously found that AF disrupts MCF-7 mammosphere formation via AhR signaling activation to inhibit alpha 6-integrin expression. To investigate the efficacy of AF in vivo, we used an estrogen dependent, Tamoxifen-sensitive spontaneous M05 model system involving 1-year-old virgin female BALB/c mice developed in our animal facility. The M05 tumor is a semi-differentiated adenocarcinoma that expresses estrogen and progesterone receptors. After AF treatment (12 mg/kg, i.p., QD × 5 days) tumor size and growth rate decreased substantially compared to control animals. Cytometric analyses of cells obtained from AF-treated and control animals revealed that Lin (-)/ CD29 (Hi)/CD24 (+) cancer stem cells were lower in animals treated with AF than in the vehicle control group. We also observed a diminished capacity for mammosphere formation in cells obtained from tumors treated with AF. These data suggest AF diminishes the quantity of breast cancer stem cells as well as their integrity and capacity to form mammospheres in vitro and in vivo. These findings shed additional insight into the molecular mechanism of anticancer action for this novel anticancer agent to strengthen the rationale for its inclusion in breast cancer treatment regimens. Citation Format: Mariana Callero, Damian Berardi, Laura Todaro, Marina Simian, Eileen J. Brantley, Ubaldo Soto, Andrea Loaiza-Perez. Aminoflavone exhibits in vivo efficacy on cancer stem cells in a spontaneous estrogen-dependent breast cancer murine model. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 215. doi:10.1158/1538-7445.AM2014-215

Abstract LB-198: Ryl hydrocarbon receptor agonist Aminoflavone disrupts mammosphere formation via alpha 6 integrin inhibition

Introduction: Emerging evidence has suggested that cancer stem cells (CSCs) play a prominent role in the initiation and dissemination of a myriad of malignancies including breast cancer. CSCs have the capacity to self-renew within heterogeneous tumor cells. Currently available therapeutic options for breast cancer are incapable of suppressing CSC growth. Alpha 6 integrin contributes to CSC growth while aryl hydrocarbon receptor (AhR) activation impedes CSC proliferation. AhR agonist Aminoflavone, a synthetic flavonoid, is currently undergoing clinical evaluation for the treatment of breast cancer. We hypothesize that Aminoflavone alters the integrity and formation of cell suspension clusters initiated by breast cancer stem cells (mammospheres) by modulating alpha 6 integrin expression in an AhR-dependent manner Experimental Procedures: Mammospheres derived from MCF-7 breast cancer cells were exposed to Aminoflavone at varying times. We also treated mammospheres with Tamoxifen an anticancer agent frequently used in the treatment of breast cancer. Following Aminoflavone treatment, cells were analyzed microscopically to evaluate alterations in mammosphere structure. In addition, the expression of molecular targets associated with CSC growth in addition to alpha 6 integrin were evaluated by Western blotting and quantitative RT-PCR analysis. Results: We observed that Aminoflavone, but not Tamoxifen, disaggregates MCF-7 mammospheres. In contrast, Aminoflavone did not affect mammospheres produced from AhR defective MCF-7 (AHR100) cells. Aminoflavone abrogated the ability of secondary mammospheres to form. In addition, alpha-6-integrin expression was abolished following Aminoflavone treatment in MCF-7 but not AHR100 cells. The expression of several genes whose products regulate alpha-6 integrin expression were also modulated following Aminoflavone treatment in MCF-7 cells. Conclusion: These data suggest that Aminoflavone disrupts mammosphere formation via AhR signaling activation that results in alpha 6 integrin inhibition. These findings support continued efforts to develop novel AhR ligands with efficacy against breast CSCs as a viable therapeutic strategy for breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-198. doi:1538-7445.AM2012-LB-198

Abstract 3658: 5F 203 modulates oxidative stress, induces single-strand break formation and promotes mitochondrial membrane potential loss in sensitive breast cancer cells

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.