aminoflavone and Breast-Neoplasms

Breast cancer is the most common cancer in women, with more than 1.7 million diagnoses worldwide per annum. Metastatic breast cancer remains incurable, and the presence of triple-negative phenotypes makes targeted treatment impossible. The aryl hydrocarbon receptor (AhR), most commonly associated with the metabolism of xenobiotic ligands, has emerged as a promising biological target for the treatment of this deadly disease. Ligands for the AhR can be classed as exogenous or endogenous and may have agonistic or antagonistic activity. It has been well reported that agonistic ligands may have potent and selective growth inhibition activity in a number of oncogenic cell lines, and one (aminoflavone) has progressed to phase I clinical trials for breast cancer sufferers. In this study, we examine the current state of the literature in this area and elucidate the promising advances that are being made in hijacking the cytosolic-to-nuclear pathway of the AhR for the possible future treatment of breast cancer.

Topics: Animals; Apoptosis; Basic Helix-Loop-Helix Transcription Factors; Breast Neoplasms; Cell Line, Tumor; Cell Nucleus; Cell Proliferation; Crystallography, X-Ray; Cytosol; Female; Flavonoids; Gene Expression Regulation, Neoplastic; Humans; Ligands; Molecular Conformation; Oncogenes; Pharmaceutical Preparations; Protein Domains; Receptors, Aryl Hydrocarbon

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.

Topics: Breast Neoplasms; Drug Resistance, Neoplasm; Female; Flavonoids; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; Integrin alpha6; Ligands; MCF-7 Cells; Neoplastic Stem Cells; Oncogene Protein v-akt; Receptors, Aryl Hydrocarbon; Signal Transduction; src-Family Kinases; Tamoxifen

Traditional chemotherapies debulk tumors but fail to produce long-term clinical remissions due to their inability to eradicate tumor-initiating cells (TICs). This necessitates therapy with activity against the TIC niche. Αlpha6-integrin (α6-integrin) promotes TIC growth. In contrast, aryl hydrocarbon receptor (AhR) signaling activation impedes the formation of mammospheres (clusters of cells enriched for TICs). We investigated the ability of AhR agonist Aminoflavone (AF) and AF pro-drug (AFP464) to disrupt mammospheres derived from breast cancer cells and a M05 mammary mouse model of breast cancer respectively. We further examined the capacity of AF and AFP464 to exhibit anticancer activity and modulate the expression of 'stemness' genes including α6-integrin using immunofluorescence, flow cytometry and qRT-PCR analysis. AF disrupted mammospheres and prevented secondary mammosphere formation. In contrast, AF did not disrupt mammospheres derived from AhR ligand-unresponsive MCF-7 cells. AFP464 treatment suppressed M05 tumor growth and disrupted corresponding mammospheres. AF and AFP464 reduced the expression and percentage of cells that stained for 'stemness' markers including α6-integrin in vitro and in vivo respectively. These data suggest AFP464 thwarts bulk breast tumor and TIC growth via AhR agonist-mediated α6-integrin inhibition.

Topics: Active Transport, Cell Nucleus; Animals; Antineoplastic Agents; Basic Helix-Loop-Helix Transcription Factors; Breast Neoplasms; Cell Proliferation; Dose-Response Relationship, Drug; Female; Flavonoids; Gene Expression Regulation, Neoplastic; Humans; Integrin alpha6; Ligands; Mammary Neoplasms, Experimental; MCF-7 Cells; Mice, Inbred BALB C; Neoplastic Stem Cells; Phenotype; Prodrugs; Receptors, Aryl Hydrocarbon; Signal Transduction; Spheroids, Cellular; Time Factors

Numerous studies have implicated the aryl hydrocarbon receptor (AhR) as a potential therapeutic target for several human diseases, including estrogen receptor alpha (ERα) positive breast cancer. Aminoflavone (AF), an activator of AhR signaling, is currently undergoing clinical evaluation for the treatment of solid tumors. Of particular interest is the potential treatment of triple negative breast cancers (TNBC), which are typically more aggressive and characterized by poorer outcomes. Here, we examined AF's effects on two TNBC cell lines and the role of AhR signaling in AF sensitivity in these model cell lines.. AF sensitivity in MDA-MB-468 and Cal51 was examined using cell counting assays to determine growth inhibition (GI50) values. Luciferase assays and qPCR of AhR target genes cytochrome P450 (CYP) 1A1 and 1B1 were used to confirm AF-mediated AhR signaling. The requirement of endogenous levels of AhR and AhR signaling for AF sensitivity was examined in MDA-MB-468 and Cal51 cells stably harboring inducible shRNA for AhR. The mechanism of AF-mediated growth inhibition was explored using flow cytometry for markers of DNA damage and apoptosis, cell cycle analysis, and β-galactosidase staining for senescence. Luciferase data was analyzed using Student's T test. Three-parameter nonlinear regression was performed for cell counting assays.. Here, we report that ERα-negative TNBC cell lines MDA-MB-468 and Cal51 are sensitive to AF. Further, we presented evidence suggesting that neither endogenous AhR expression levels nor downstream induction of AhR target genes CYP1A1 and CYP1B1 is required for AF-mediated growth inhibition in these cells. Between these two ERα negative cell lines, we showed that the mechanism of AF action differs slightly. Low dose AF mediated DNA damage, S-phase arrest and apoptosis in MDA-MB-468 cells, while it resulted in DNA damage, S-phase arrest and cellular senescence in Cal51 cells.. Overall, this work provides evidence against the simplified view of AF sensitivity, and suggests that AF could mediate growth inhibitory effects in ERα-positive and negative breast cancer cells, as well as cells with impaired AhR expression and signaling. While AF could have therapeutic effects on broader subtypes of breast cancer, the mechanism of cytotoxicity is complex, and likely, cell line- and tumor-specific.

Topics: Antineoplastic Agents; Apoptosis; Basic Helix-Loop-Helix Transcription Factors; Breast Neoplasms; Cell Proliferation; Cytochrome P-450 CYP1A1; Cytochrome P-450 CYP1B1; DNA Damage; Dose-Response Relationship, Drug; Estrogen Receptor alpha; Female; Flavonoids; Genes, Reporter; Humans; MCF-7 Cells; Receptors, Aryl Hydrocarbon; RNA Interference; S Phase Cell Cycle Checkpoints; Signal Transduction; Transfection

Aminoflavone is a unique DNA damaging agent currently undergoing phase I evaluation in a prodrug form (AFP464). In anticipation of combination regimens, interactions between aminoflavone and several anticancer drugs were investigated in MCF-7 breast cancer cells to determine whether synergistic cancer cell killing effects were observed.. Colony formation assays were performed to assess the effect of combining aminoflavone with a variety of anticancer drugs. Changes in initial uptake, retention or efflux of aminoflavone and the second agent were compared to the behavior of drugs alone. Key features required for aminoflavone activity in cell culture models were also explored, focusing on the obligatory induction of CYP1A1/1A2 and binding of reactive aminoflavone metabolites to tumor cell total macromolecules and DNA.. Aminoflavone was synergistic when co-incubated with paclitaxel, camptothecin or SN38. Uptake of neither aminoflavone nor any of the other three compounds was altered in combination incubations. Paclitaxel did not inhibit DNA binding of aminoflavone metabolites, while camptothecin did. Aminoflavone-induced CYP1A1 induction was observed in the presence of camptothecin or paclitaxel.. Aminoflavone is a promising therapeutic agent for breast cancer due to its unique mechanism of action compared to commonly used drugs. Combined treatments utilizing aminoflavone in conjunction with paclitaxel or camptothecin may provide an even greater cytotoxic effect than achieved with aminoflavone alone.

Topics: Antineoplastic Combined Chemotherapy Protocols; Breast Neoplasms; Camptothecin; Cell Line, Tumor; Colony-Forming Units Assay; Cytochrome P-450 CYP1A1; Cytochrome P-450 CYP1A2; DNA; Drug Synergism; Enzyme Induction; Female; Flavonoids; Humans; Irinotecan; Paclitaxel

Aminoflavone (AF), the active component of a novel anticancer agent (AFP464) in phase I clinical trials, is a ligand of the aryl hydrocarbon receptor (AhR). AhR dimerizes with HIF-1beta/AhR, which is shared with HIF-1alpha, a transcription factor critical for the response of cells to oxygen deprivation. To address whether pharmacologic activation of the AhR pathway might be a potential mechanism for inhibition of HIF-1, we tested the effects of AF on HIF-1 expression. AF inhibited HIF-1alpha transcriptional activity and protein accumulation in MCF-7 cells. However, inhibition of HIF-1alpha by AF was independent from a functional AhR pathway. Indeed, AF inhibited HIF-1alpha expression in Ah(R100) cells, in which the AhR pathway is functionally impaired, yet did not induce cytotoxicity, providing evidence that these effects are mediated by distinct signaling pathways. Moreover, AF was inactive in MDA-MB-231 cells, yet inhibited HIF-1alpha in MDA-MB-231 cells transfected with the SULT1A1 gene. AF inhibited HIF-1alpha mRNA expression by approximately 50%. Notably, actinomycin-D completely abrogated the ability of AF to downregulate HIF-1alpha mRNA, indicating that active transcription was required for the inhibition of HIF-1alpha expression. Finally, AF inhibited HIF-1alpha protein accumulation and the expression of HIF-1 target genes in MCF-7 xenografts. These results show that AF inhibits HIF-1alpha in an AhR-independent fashion, and they unveil additional activities of AF that may be relevant for its further clinical development.

Topics: Animals; Basic Helix-Loop-Helix Transcription Factors; Breast Neoplasms; Cell Line, Tumor; DNA Damage; Female; Flavonoids; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Ligands; Mice; Mice, Nude; Receptors, Aryl Hydrocarbon; RNA, Messenger; RNA, Small Interfering; Transcription, Genetic; Xenograft Model Antitumor Assays

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), Gamma-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, alpha-Naphthoflavone (alpha-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.

Topics: Acetylcysteine; Antineoplastic Agents; Apoptosis; Blotting, Western; Breast Neoplasms; Caspases; Cytochrome P-450 CYP1A1; Cytochrome P-450 CYP1A2; DNA Damage; DNA, Neoplasm; Enzyme Activation; Female; Flavonoids; Free Radical Scavengers; Humans; Reactive Oxygen Species; Receptors, Estrogen; S Phase

Aminoflavone (5-amino-2,3-fluorophenyl)-6,8-difluoro-7-methyl-4H-1-benzopyran-4-one; AF; NSC 686288) is a novel anticancer agent with a unique pattern of growth inhibitory activity in the National Cancer Institute (NCI) 60 tumor cell line screen. Phase I clinical trials with AF will begin soon. We previously demonstrated extensive metabolism of AF by cytochrome P450 (CYP) 1A1 and CYP1A2, metabolic activation, formation of irreversible protein and DNA adducts and p53 stabilization in sensitive, but not resistant, human tumor cell lines treated with AF [9]. The present studies focus on the effects of AF on cellular DNA and cellular responses to DNA damage.. Phosphorylation of H2AX in MCF7 cells treated with AF was determined with immunofluorescence. MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4- sulfophenyl)-2H-tetrazolium) assays were used to determine the effect of cotreatment with caffeine or wortmannin, inhibitors of ataxia-telangiectasia-mutated protein (ATM) and ATR (ATM- and rad3-related protein), on the AF IC(50) values for MCF7 cells. DNA damage in MCF7 cells treated with AF was determined by alkaline elution. DNA-topoisomerase complex stabilization was ascertained by the ICE (in vitro complex of enzyme) assay.. Treatment of sensitive cells with AF resulted in phosphorylation of H2AX, a histone 2A variant that is phosphorylated in response to DNA damage. AF IC(50) values for MCF7 cells were lowered by cotreatment with caffeine or wortmannin, further implicating DNA damage in AF cytotoxicity. There was no evidence of DNA-DNA cross-linking in sensitive cells, but protein-associated single-strand breaks were observed after AF treatment. Although this pattern of DNA damage is commonly associated with topoisomerase poisons, there was no evidence for AF-induced stabilization of either topoisomerase I- or II-DNA complexes.. These studies further implicate DNA damage in the cytotoxicity of AF and identify biochemical features of that damage including formation of protein-associated single-strand breaks not involving topoisomerase I or II.

Topics: Androstadienes; Ataxia Telangiectasia Mutated Proteins; Breast Neoplasms; Caffeine; Cell Cycle Proteins; Cell Line, Tumor; Cytochrome P-450 CYP1A1; DNA Damage; DNA Topoisomerases, Type I; DNA Topoisomerases, Type II; DNA-Binding Proteins; DNA, Single-Stranded; Female; Flavonoids; Fluorescent Antibody Technique; Histones; Humans; Phosphodiesterase Inhibitors; Phosphoinositide-3 Kinase Inhibitors; Phosphorylation; Protein Serine-Threonine Kinases; Tumor Suppressor Proteins; Wortmannin

Aminoflavone (AF) is entering clinical trials. We recently reported that AF induces DNA-protein cross-links (DPC) and gamma-H2AX in MCF-7 human breast cancer cells. To elucidate the mechanism of action of AF and provide biomarkers indicative of AF activity, we correlated AF activity profile (GI(50)) with gene expression patterns in the NCI-60 cell lines. Sulfotransferases (SULT) showed the highest positive correlation coefficients among approximately 14,000 probe sets analyzed (r = 0.537, P < 0.001). Stable transfection of SULT1A1 into AF-resistant MDA-MB-231 cells sensitized these cells to AF. AF produced DPCs, gamma-H2AX foci, and S-phase arrest in the SULT1A1-transfected but not in the parent MDA-MB-231 cells. Conversely, cells in which SULT1A1 was knocked down by small interfering RNA failed to induce gamma-H2AX. Inhibition of SULTs and cytochrome P450 (CYP) enzymes by natural flavonoids blocked the antiproliferative activity of AF and the formation of AF-DNA adducts. AF also induces SULT1A1 and CYP expression in MCF-7 cells, suggesting the existence of an aryl hydrocarbon receptor-mediated positive feedback for AF activation by CYP and SULT1A1. Metabolism studies showed that AF can be oxidized by CYP at two amino groups to form N-hydroxyl metabolites that are substrates for bioactivation by SULTs. We propose that both N-sulfoxy-groups can be further converted to nitrenium ions that form adducts with DNA and proteins. The results reported here show the importance of SULT1A1 and CYP for AF activation and anticancer activity. They also suggest using SULT1A1 and gamma-H2AX as biomarkers for prediction of AF activity during patient selection and monitoring of clinical trials.

Topics: Adenocarcinoma; Aryl Hydrocarbon Hydroxylases; Arylsulfotransferase; Biotransformation; Breast Neoplasms; Cell Line, Tumor; Cross-Linking Reagents; Cytochrome P-450 CYP1A1; Cytochrome P-450 CYP1B1; Cytochrome P-450 Enzyme System; DNA Adducts; DNA, Neoplasm; Drug Resistance, Neoplasm; Feedback, Physiological; Female; Flavonoids; Gene Expression Regulation, Neoplastic; Histones; Humans; Microsomes, Liver; Neoplasm Proteins; Neoplasms; Prodrugs; Receptors, Aryl Hydrocarbon; Recombinant Fusion Proteins; RNA, Small Interfering

Aminoflavone (5-amino-2,3-fluorophenyl)-6,8-difluoro-7-methyl-4H-1-benzopyran-4-one) (NSC 686288) is a candidate for possible advancement to phase I clinical trial. Aminoflavone has a unique activity profile in the NCI 60 cell lines (COMPARE analysis; http://www.dtp.nci.nih.gov/docs/dtp_search.html), and exhibits potent cellular and animal antitumor activity. To elucidate the mechanism of action of aminoflavone, we studied DNA damage in MCF-7 cells. Aminoflavone induced DNA-protein cross-links (DPC) and DNA single-strand breaks (SSB). Aminoflavone induced high levels of DPC and much lower level of SSB than camptothecin, which induces equal levels of DPC and SSB due to the trapping topoisomerase I-DNA complexes. Accordingly, neither topoisomerase I nor topoisomerase II were detectable in the aminoflavone-induced DPC. Aminoflavone also induced dose- and time-dependent histone H2AX phosphorylation (gamma-H2AX). Gamma-H2AX foci occurred with DPC formation, and like DPC, persisted after aminoflavone removal. Aphidicolin prevented gamma-H2AX formation, suggesting that gamma-H2AX foci correspond to replication-associated DNA double-strand breaks. Accordingly, no gamma-H2AX foci were found in proliferating cell nuclear antigen-negative or in mitotic cells. Bromodeoxyuridine incorporation and fluorescence-activated cell sorting analyses showed DNA synthesis inhibition uniformly throughout the S phase after exposure to aminoflavone. Aminoflavone also induced RPA2 and p53 phosphorylation, and induced p21(Waf1/Cip1) and MDM2, demonstrating S-phase checkpoint activation. These studies suggest that aminoflavone produces replication-dependent DNA lesions and S-phase checkpoint activation following DPC formation. Gamma-H2AX may be a useful clinical marker for monitoring the efficacy of aminoflavone in tumor therapies.

Topics: Breast Neoplasms; Cell Cycle Proteins; Cell Line, Tumor; Cross-Linking Reagents; Cyclin-Dependent Kinase Inhibitor p21; DNA Damage; DNA Replication; DNA-Binding Proteins; DNA, Neoplasm; DNA, Single-Stranded; Flavonoids; Histones; Humans; Nuclear Proteins; Phosphorylation; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-mdm2; Replication Protein A; S Phase; Tumor Suppressor Protein p53

Aminoflavone (4H-1-benzopyran-4-one, 5-amino-2-(4-amino-3-fluorophenyl)-6,8-difluoro-7-methyl; NSC 686288) demonstrates differential antiproliferative activity in the National Cancer Institute's anticancer drug screen. We demonstrate here that MCF-7 human breast cancer cells are sensitive to aminoflavone both in vitro and when grown in vivo as xenografts in athymic mice. As previous work has indicated that aminoflavone requires metabolic activation by cytochrome P450 1A1 (CYP1A1), we investigated the effect of aminoflavone on CYP1A1 expression and on the aryl hydrocarbon receptor (AhR), a transcriptional regulator of CYP1A1. In aminoflavone-sensitive but not aminoflavone-resistant cells, the drug caused a 100-fold induction of CYP1A1 mRNA and a corresponding increase in ethoxyresorufin-O-deethylase activity. An AhR-deficient variant of the MCF-7 breast carcinoma, AH(R100), with diminished CYP1A1 inducibility, exhibits cellular resistance to aminoflavone and is refractory to CYP1A1 mRNA induction by the drug. The increase in CYP1A1 mRNA in the aminoflavone-sensitive MCF-7 breast tumor cell results from transcriptional activation of xenobiotic-responsive element (XRE)-controlled transcription. Aminoflavone treatment causes a translocation of the AhR from the cytoplasm to the nucleus with subsequent formation of AhR-XRE protein DNA complexes. In contrast to the aminoflavone-sensitive MCF-7 cells, the resistant cell lines (MDA-MB-435, PC-3, and AH(R100)) demonstrated constitutive nuclear localization of AhR. Additionally, aminoflavone failed to induce ethoxyresorufin-O-deethylase activity, CYP1A1 transcription, AhR-XRE complex formation, and apoptosis in aminoflavone-resistant cells. These results suggest that the cytotoxicity of aminoflavone in a sensitive breast tumor cell line is the result of the engagement of AhR-mediated signal transduction.

Topics: Animals; Antineoplastic Agents; Apoptosis; Aryl Hydrocarbon Hydroxylases; Breast Neoplasms; Cell Line, Tumor; Cell Nucleus; Cell Proliferation; Cytochrome P-450 CYP1A1; Cytochrome P-450 CYP1B1; Enzyme Induction; Flavonoids; Humans; Mice; Mice, Nude; Promoter Regions, Genetic; Protein Transport; Receptors, Aryl Hydrocarbon; Response Elements; Transcription, Genetic; Transcriptional Activation; Xenograft Model Antitumor Assays