Identification and targeting of selective vulnerability rendered by tamoxifen resistance.

BACKGROUND: The estrogen receptor (ER)-positive breast cancer represents over 80% of all breast cancer cases. Even though adjuvant hormone therapy with tamoxifen (TMX) is saving lives of patients with ER-positive breast cancer, the acquired resistance to TMX anti-estrogen therapy is the main hurdle for successful TMX therapy. Here we address the mechanism for TMX resistance and explore the ways to eradicate TMX-resistant breast cancer in both in vitro and ex vivo experiments. EXPERIMENTAL DESIGN: To identify compounds able to overcome TMX resistance, we used short-term and long-term viability assays in cancer cells in vitro and in patient samples in 3D ex vivo, analysis of gene expression profiles and cell line pharmacology database, shRNA screen, CRISPR-Cas9 genome editing, real-time PCR, immunofluorescent analysis, western blot, measurement of oxidative stress using flow cytometry, and thioredoxin reductase 1 enzymatic activity. RESULTS: Here, for the first time, we provide an ample evidence that a high level of the detoxifying enzyme SULT1A1 confers resistance to TMX therapy in both in vitro and ex vivo models and correlates with TMX resistance in metastatic samples in relapsed patients. Based on the data from different approaches, we identified three anticancer compounds, RITA (Reactivation of p53 and Induction of Tumor cell Apoptosis), aminoflavone (AF), and oncrasin-1 (ONC-1), whose tumor cell inhibition activity is dependent on SULT1A1. We discovered thioredoxin reductase 1 (TrxR1, encoded by TXNRD1) as a target of bio-activated RITA, AF, and ONC-1. SULT1A1 depletion prevented the inhibition of TrxR1, induction of oxidative stress, DNA damage signaling, and apoptosis triggered by the compounds. Notably, RITA efficiently suppressed TMX-unresponsive patient-derived breast cancer cells ex vivo. CONCLUSION: We have identified a mechanism of resistance to TMX via hyperactivated SULT1A1, which renders selective vulnerability to anticancer compounds RITA, AF, and ONC-1, and provide a rationale for a new combination therapy to overcome TMX resistance in breast cancer patients. Our novel findings may provide a strategy to circumvent TMX resistance and suggest that this approach could be developed further for the benefit of relapsed breast cancer patients.

New Treatments in Renal Cancer: The AhR Ligands.

Kidney cancer rapidly acquires resistance to antiangiogenic agents, such as sunitinib, developing an aggressive migratory phenotype (facilitated by c-Metsignal transduction). The Aryl hydrocarbon receptor (AhR) has recently been postulated as a molecular target for cancer treatment. Currently, there are two antitumor agent AhR ligands, with activity against renal cancer, that have been tested clinically: aminoflavone (AFP 464, NSC710464) and the benzothiazole (5F 203) prodrug Phortress. Our studies investigated the action of AFP 464, the aminoflavone pro-drug currently used in clinical trials, and 5F 203 on renal cancer cells, specifically examining their effects on cell cycle progression, apoptosis and cell migration. Both compounds caused cell cycle arrest and apoptosis but only 5F 203 potently inhibited the migration of TK-10, Caki-1 and SN12C cells as well as the migration signal transduction cascade, involving c-Met signaling, in TK-10 cells. Current investigations are focused on the development of nano-delivery vehicles, apoferritin-encapsulated benzothiazoles 5F 203 and GW610, for the treatment of renal cancer. These compounds have shown improved antitumor effects against TK-10 cells in vitro at lower concentrations compared with a naked agent.

Differential effects of MEK inhibitors on rat neural stem cell differentiation: Repressive roles of MEK2 in neurogenesis and induction of astrocytogenesis by PD98059.

Neural stem cells (NSCs) proliferate and differentiate into neurons and glia depending on the culture environment. However, the underlying mechanisms determining the fate of NSCs are not fully understood. Growth factors facilitate NSC proliferation through mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) kinase (MEK) and MAPK activation, and NSCs differentiate into neurons, astrocytes, or oligodendrocytes when mitogens are withdrawn from the culture media. Here, we aimed to identify the effects and roles of MEK signaling on the determination of NSC fate. MEK inhibitors, U0126, SL327, and PD98059, had differential effects on NSC differentiation. U0126 and SL327, which are known to inhibit MEK1 and MEK2, induced neuronal differentiation, whereas PD98059, which is reported to preferentially inhibit MEK1 at higher concentrations, increased astrocytogenesis. Knockdown of MEK2 using small interfering RNA increased neurogenesis and over-expression of wild type (WT) MEK2 inhibited neurogenesis, suggesting a repressive role of MEK2 in neuronal differentiation. The chemical structure of PD98059 appears to be important for induction of astrocytogenesis because not only PD98059 (2'-amino-3'-methoxyflavone) but also its chemical structural mimetic, 3'-methoxyflavone, enhanced astrocytogenesis. Therefore, in our study, we suggest that MEK inhibitors have distinct functions in determining NSC fate. Inhibition of MEK2 is important for induction of neurogenesis in NSCs. U0126 and SL327 increase neurogenesis through MEK2 inhibition, whereas PD98059 induced astrocytogenesis in NSCs, which is mediated by the chemical structure, particularly the 3'-methoxy group rather than its renowned MEK1 inhibition.

Fluorimetric Properties of 3-Aminoflavone Biomolecule (3-AF). X-ray Crystal Structure of New Polymorph of 3-AF.

The crystal structure of the new polymorphic form of 3-aminoflavone (3-AF) has been determined by single crystal X-ray diffraction. This report presents results of fluorimetric studies on 3-AF in methanol and aquatic solvents. Based on 3D fluorescence emission spectra, optimal values for excitation (λex) and emission/analytical (λem) wavelength, the analytical concentration range as well as the range of concentration quenching for the studied compound were established. Moreover, the limit of detection (LOD) and the limit of quantification (LOQ) were determined. The results were compared with those obtained using the standard UV-Vis absorption spectrophotometric method. The effect of acidity (pH) and the concentration of halide anions (chlorides, bromides, iodides and fluorides) on fluorescence quenching were analysed.

Biotransformations of Flavones and an Isoflavone (Daidzein) in Cultures of Entomopathogenic Filamentous Fungi.

Entomopathogenic filamentous fungi of the genus Isaria are effective biocatalysts in the biotransformation of flavonoids as well as steroids. In the present study, the species Isariafumosorosea and Isariafarinosa isolated from the environment were used. Their catalytic capacity to carry out biotransformations of flavones-unsubstituted, with hydroxy- and amino-substituents as well as a hydroxylated isoflavone-was investigated. Biotransformations of flavone, 5-hydroxyflavone, 6-hydroxyflavone, 7-hydroxyflavone, and daidzein resulted in the formation of O-methylglucosides, in the case of flavone and 5-hydroxyflavone with additional hydroxylations. 7-Aminoflavone was transformed into two acetamido derivatives. The following products were obtained: From flavone⁻flavone 2'-O-ß-d-(4''-O-methyl)-glucopyranoside, flavone 4'-O-ß-d-(4''-O-methyl)-glucopyranoside and 3'-hydroxyflavone 4'-O-ß-d-(4''-O-methyl)-glucopyranoside; from 5-hydroxyflavone⁻5-hydroxyflavone 4'-O-ß-d-(4''-O-methyl)-glucopyranoside; from 6-hydroxyflavone⁻flavone 6-O-ß-d-(4''-O-methyl)-glucopyranoside; from 7-hydroxyflavone⁻flavone 7-O-ß-d-(4''-O-methyl)-glucopyranoside; from daidzein⁻daidzein 7-O-ß-d-(4''-O-methyl)-glucopyranoside; and from 7-aminoflavone⁻7-acetamidoflavone and 7-acetamido-4'-hydroxyflavone. Seven of the products obtained by us have not been previously reported in the literature.

Ex Vivo and In Silico Approaches of Tracheal Relaxation through Calcium Channel Blockade of 6-Aminoflavone and Its Toxicological Studies in Murine Models.

Asthma is a condition in which a person's airways become inflamed, narrowed, and produce greater amounts of mucus than normal. It can cause shortness of breath, chest pain, coughing, or wheezing. In some cases, symptoms may be exacerbated. Thus, the current study was designed to determine the mechanism of action of 6-aminoflavone (6-NH2F) in ex vivo experiments, as well as to determine its toxicity in acute and sub-chronic murine models. Tissues were pre-incubated with 6-NH2F, and concentration-response curves to carbachol-induced contraction were constructed. Therefore, tracheal rings pre-treated with glibenclamide, 2-aminopyridine, or isoproterenol were contracted with carbachol (1 µM), then 6-NH2F relaxation curves were obtained. In other sets of experiments, to explore the calcium channel role in the 6-NH2F relaxant action, tissues were contracted with KCl (80 mM), and 6-NH2F was cumulatively added to induce relaxation. On the other hand, tissues were pre-incubated with the test sample, and after that, CaCl2 concentration-response curves were developed. In this context, 6-NH2F induced significant relaxation in ex vivo assays, and the effect showed a non-competitive antagonism pattern. In addition, 6-NH2F significantly relaxed the contraction induced by KCl and CaCl2, suggesting a potential calcium channel blockade, which was corroborated by in silico molecular docking that was used to approximate the mode of interaction with the L-type Ca2+ channel, where 6-NH2F showed lower affinity energy when compared with nifedipine. Finally, toxicological studies revealed that 6-NH2F possesses pharmacological safety, since it did not produce any toxic effect in both acute and sub-acute murine models. In conclusion, 6-aminoflavone exerted significant relaxation through calcium channel blockade, and the compound seems to be safe.

Aminoflavone upregulates putative tumor suppressor miR-125b-2-3p to inhibit luminal A breast cancer stem cell-like properties.

Metastatic breast cancer is incurable and often due to breast cancer stem cell (CSC)-mediated self-renewal. We previously determined that the aryl hydrocarbon receptor (AhR) agonist aminoflavone (AF) inhibits the expression of the CSC biomarker α6-integrin (ITGA6) to disrupt the formation of luminal (hormone receptor-positive) mammospheres (3D breast cancer spheroids). In this study, we performed miRNA-sequencing analysis of luminal A MCF-7 mammospheres treated with AF to gain further insight into the mechanism of AF-mediated anti-cancer and anti-breast CSC activity. AF significantly induced the expression of >70 microRNAs (miRNAs) including miR125b-2-3p, a predicted stemness gene regulator. AF-mediated miR125b-2-3p induction was validated in MCF-7 mammospheres and cells. miR125b-2-3p levels were low in breast cancer tissues irrespective of subtype compared to normal breast tissues. While miR125b-2-3p levels were low in MCF-7 cells, they were much lower in AHR100 cells (MCF-7 cells made unresponsive to AhR agonists). The miR125b-2-3p mimic decreased, while the antagomiR125b-2-3p increased the expression of stemness genes ITGA6 and SOX2 in MCF-7 cells. In MCF-7 mammospheres, the miR125b-2-3p mimic decreased only ITGA6 expression although the antagomiR125b-2-3p increased ITGA6, SOX2 and MYC expression. AntagomiR125b-2-3p reversed AF-mediated suppression of ITGA6. The miR125b-2-3p mimic decreased proliferation, migration, and mammosphere formation while the antagomiR125b-2-3p increased proliferation and mammosphere formation in MCF-7 cells. The miR125b-2-3p mimic also inhibited proliferation, mammosphere formation, and migration in AHR100 cells. AF induced AhR- and miR125b2-3p-dependent anti-proliferation, anti-migration, and mammosphere disruption in MCF-7 cells. Our findings suggest that miR125b-2-3p is a tumor suppressor and AF upregulates miR125b-2-3p to disrupt mammospheres via mechanisms that rely at least partially on AhR in luminal A breast cancer cells.

Influence of amide versus ester linkages on the anticancer properties of the new flavone-biotin conjugates.

Novel biotinylated C-6 substituted flavones were synthesised by a one-step method that connects biotin to 6-hydroxyflavone and 6-aminoflavone by esterification and amidation of hydroxyl and amino groups, respectively. The obtained compounds, 6-O-biotinylflavone and 6-biotinylamidoflavone, are the bifunctional molecules composed of a flavone moiety as a fluorescent reporter and biotin as a cancer-targeting unit. Antiproliferative activity was evaluated using SRB assays in MCF-7, MCF-10A, HepG2, MDA-MB-231, 4T1, and Balb/3T3 cell lines. In vitro evaluation revealed that compounds with biotin moiety displayed better cell selectivity between the cancer and normal cells than the parental substrates. These results indicate that anticancer effect is not related to the position of biotin moiety, but it is related to the presence of ester or amide bond. 6-O-Biotinylflavone was more active than 6-hydroxyflavone against human breast (MDA-MB-231) and liver (HepG2) cancer cells with IC50 (concentration of tested agent that inhibits proliferation of the cell population by 50%) values equal to 78.5 ± 18.8 µM and 133.2 ± 14.2 µM, respectively. Non biotinylated 6-aminoflavone was more active than 6-biotinylamidoflavone against all tested cell lines, with IC50 values between 34.3 ± 9.1 µM (4T1) and 173.86 ± 24.3 µM (MCF-7).

AhR ligand Aminoflavone inhibits α6-integrin expression and breast cancer sphere-initiating capacity.

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.

6-Acetamidoflavone obtained by microbiological and chemical methods and its antioxidant activity.

The aim of this research was to select microbial strains capable of transforming flavonoid compounds containing an amino group. 6-Aminoflavone (1) was used as a model compound. Biotransformation of 1 by the cultures of Rhodococcus sp. DSM 364 and Gordonia sp. DSM 44456 bacteria led to 6-acetamidoflavone (2) with the isolated yields in 86% and 93%, respectively. The structure of the product was determined on the basis of spectroscopic data, including NMR spectra (1H, 13C, 1H-1H, 1H-13C, and DEPT 135°), a UV and IR spectrum and HR ESI-MS analysis. The developed method of microbial replacement of the amino group in 6-aminoflavone (1) by a acetamido one may find application in chemical industry, being an alternative to the reactions involving acetic anhydride. The antioxidant activity of flavone, and its C-6 substituted derivatives: 6-aminoflavone (1), 6-hydroxyflavone and isolated metabolite 6-acetamidoflavone (2) has been examined using the ABTS and DPPH methods.

Aminoflavone-loaded EGFR-targeted unimolecular micelle nanoparticles exhibit anti-cancer effects in triple negative breast cancer.

Triple negative breast cancer (TNBC) is an aggressive subtype of breast cancer for which there is no available targeted therapy. TNBC cases contribute disproportionately to breast cancer-related mortality, thus the need for novel and effective therapeutic methods is urgent. We have previously shown that a National Cancer Institute (NCI) investigational drug aminoflavone (AF) exhibits strong growth inhibitory effects in TNBC cells. However, in vivo pulmonary toxicity resulted in withdrawal or termination of several human clinical trials for AF. Herein we report the in vivo efficacy of a nanoformulation of AF that enhances the therapeutic index of AF in TNBC. We engineered a unique unimolecular micelle nanoparticle (NP) loaded with AF and conjugated with GE11, a 12 amino acid peptide targeting epidermal growth factor receptor (EGFR), since EGFR amplification is frequently observed in TNBC tumors. These unimolecular micelles possessed excellent stability and preferentially released drug payload at endosomal pH levels rather than blood pH levels. Use of the GE11 targeting peptide resulted in enhanced cellular uptake and strong growth inhibitory effects in TNBC cells. Further, AF-loaded, GE11-conjugated (targeted) unimolecular micelle NPs significantly inhibit orthotopic TNBC tumor growth in a xenograft model, compared to treatment with AF-loaded, GE11-lacking (non-targeted) unimolecular micelle NPs or free AF. Interestingly, the animals treated with AF-loaded, targeted NPs had the highest plasma and tumor level of AF among different treatment groups yet exhibited no increase in plasma aspartate aminotransferase (AST) activity level or observable tissue damage at the time of sacrifice. Together, these results highlight AF-loaded, EGFR-targeted unimolecular micelle NPs as an effective therapeutic option for EGFR-overexpressing TNBC.