Fluorescent substrates for flow cytometric evaluation of efflux inhibition in ABCB1, ABCC1, and ABCG2 transporters.

ATP binding cassette (ABC) transmembrane efflux pumps such as P-glycoprotein (ABCB1), multidrug resistance protein 1 (ABCC1), and breast cancer resistance protein (ABCG2) play an important role in anticancer drug resistance. A large number of structurally and functionally diverse compounds act as substrates or modulators of these pumps. In vitro assessment of the affinity of drug candidates for multidrug resistance proteins is central to predict in vivo pharmacokinetics and drug-drug interactions. The objective of this study was to identify and characterize new substrates for these transporters. As part of a collaborative project with Life Technologies, 102 fluorescent probes were investigated in a flow cytometric screen of ABC transporters. The primary screen compared substrate efflux activity in parental cell lines with their corresponding highly expressing resistant counterparts. The fluorescent compound library included a range of excitation/emission profiles and required dual laser excitation as well as multiple fluorescence detection channels. A total of 31 substrates with active efflux in one or more pumps and practical fluorescence response ranges were identified and tested for interaction with eight known inhibitors. This screening approach provides an efficient tool for identification and characterization of new fluorescent substrates for ABCB1, ABCC1, and ABCG2.

The monoamine oxidase A inhibitor clorgyline is a broad-spectrum inhibitor of fungal ABC and MFS transporter efflux pump activities which reverses the azole resistance of Candida albicans and Candida glabrata clinical isolates.

Resistance to the commonly used azole antifungal fluconazole (FLC) can develop due to overexpression of ATP-binding cassette (ABC) and major facilitator superfamily (MFS) plasma membrane transporters. An approach to overcoming this resistance is to identify inhibitors of these efflux pumps. We have developed a pump assay suitable for high-throughput screening (HTS) that uses recombinant Saccharomyces cerevisiae strains hyperexpressing individual transporters from the opportunistic fungal pathogen Candida albicans. The recombinant strains possess greater resistance to azoles and other pump substrates than the parental host strain. A flow cytometry-based HTS, which measured increased intracellular retention of the fluorescent pump substrate rhodamine 6G (R6G) within yeast cells, was used to screen the Prestwick Chemical Library (PCL) of 1,200 marketed drugs. Nine compounds were identified as hits, and the monoamine oxidase A inhibitor (MAOI) clorgyline was identified as an inhibitor of two C. albicans ABC efflux pumps, CaCdr1p and CaCdr2p. Secondary in vitro assays confirmed inhibition of pump-mediated efflux by clorgyline. Clorgyline also reversed the FLC resistance of S. cerevisiae strains expressing other individual fungal ABC transporters (Candida glabrata Cdr1p or Candida krusei Abc1p) or the C. albicans MFS transporter Mdr1p. Recombinant strains were also chemosensitized by clorgyline to other azoles (itraconazole and miconazole). Importantly, clorgyline showed synergy with FLC against FLC-resistant C. albicans clinical isolates and a C. glabrata strain and inhibited R6G efflux from a FLC-resistant C. albicans clinical isolate. Clorgyline is a novel broad-spectrum inhibitor of two classes of fungal efflux pumps that acts synergistically with azoles against azole-resistant C. albicans and C. glabrata strains.

Identification of Nile red as a fluorescent substrate of the Candida albicans ATP-binding cassette transporters Cdr1p and Cdr2p and the major facilitator superfamily transporter Mdr1p.

Clinically relevant azole resistance in the fungal pathogen Candida albicans is most often associated with the increased expression of plasma membrane efflux pumps, specifically the ATP-binding cassette (ABC) transporters CaCdr1p and CaCdr2p and the major facilitator superfamily (MFS) transporter CaMdr1p. Development of potent pump inhibitors that chemosensitize cells to azoles is a promising approach to overcome antifungal resistance. Here we identify Nile red as a new fluorescent substrate for CaCdr1p, CaCdr2p, and CaMdr1p. Nile red was effluxed efficiently from Saccharomyces cerevisiae cells heterologously expressing these transporters. Enniatin selectively inhibited the efflux of Nile red from S. cerevisiae cells expressing CaCdr1p or CaMdr1p but not from cells expressing CaCdr2p. This indicates that Nile red can be used for the identification of inhibitors specific for particular transporters mediating antifungal resistance in pathogenic yeast.

High-throughput flow cytometry to detect selective inhibitors of ABCB1, ABCC1, and ABCG2 transporters.

Up-regulation of pump (transporter) expression and selection of resistant cancer cells result in cancer multidrug resistance to diverse substrates of these transporters. While more than 48 members of the ATP binding cassette (ABC) transporter superfamily have been identified, up to now only three human ABC transporters-ABCB1, ABCC1, and ABCG2-have unambiguously been shown to contribute to cancer multidrug resistance. The use of low-toxicity and high-specificity agents as a targeted transporter inhibition strategy is necessary to effectively overcome multiple drug resistance. An objective of the present studies was to develop and validate HyperCyt (IntelliCyt, Albuquerque, NM) flow cytometry high-throughput screeening assays to assess the specificity of test compounds that inhibited transporters as an integral part of the screen. Two separate duplex assays were constructed: one in which ABCB1 and ABCG2 transporters were evaluated in parallel using fluorescent J-aggregate-forming lipophilic cation 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolcarbocyanine iodide as substrate, and the other in which ABCB1 and ABCC1 transporters were evaluated in parallel using fluorescent calcein acetoxymethyl ester as substrate. ABCB1-expressing cells were color-coded to allow their distinction from cells expressing the alternate transporter. The assays were validated in a screen of the Prestwick Chemical Library (Illkirch, France). Three novel selective inhibitors of the ABCC1 transporter were identified in the screen, and the activity of each was confirmed in follow-up chemosensitivity shift and reversal studies. This high-throughput screening assay provides an efficient approach for identifying selective inhibitors of individual ABC transporters, promising as probes of transporter function and therapeutic tools for treating chemotherapy-resistant cancers.

Targeting efflux pumps to overcome antifungal drug resistance.

Resistance to antifungal drugs is an increasingly significant clinical problem. The most common antifungal resistance encountered is efflux pump-mediated resistance of Candida species to azole drugs. One approach to overcome this resistance is to inhibit the pumps and chemosensitize resistant strains to azole drugs. Drug discovery targeting fungal efflux pumps could thus result in the development of azole-enhancing combination therapy. Heterologous expression of fungal efflux pumps in Saccharomyces cerevisiae provides a versatile system for screening for pump inhibitors. Fungal efflux pumps transport a range of xenobiotics including fluorescent compounds. This enables the use of fluorescence-based detection, as well as growth inhibition assays, in screens to discover compounds targeting efflux-mediated antifungal drug resistance. A variety of medium- and high-throughput screens have been used to identify a number of chemical entities that inhibit fungal efflux pumps.

Inhibition of neovascularization by environmental agents.

The formation of new blood vessels, neovascularization, occurs by two unique processes: vasculogenesis, the de novo assembly of blood vessels from angioblast precursors, and angiogenesis, the formation of new capillary sprouts from preexisting vessels. There are many potential targets by which environmental pollutants may inhibit neovascularization and thus there are many possible phenotypic outcomes. Two examples of environmental pollutants that have been demonstrated to inhibit neovascularization include 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a prototypical halogenated aromatic hydrocarbon, and constituents found in environmental tobacco smoke. Studies have shown that TCDD disrupts neoangiogenesis by inhibiting the expression of angiogenic stimuli as well as by reducing the responsiveness of endothelial cells to those stimuli. Additionally, studies have shown that constituents of environmental tobacco smoke, including pyradine and pyrazine derivatives, can potently inhibit the angiogenic process of branching as well as the vasculogenic process involved in capillary plexus formation. Further, the inhibition of neovascularization by either TCDD or environmental tobacco smoke constituents is associated with reduced endothelial cell proliferation and altered expression of extracellular matrix proteins. Future research that identifies the specific angiogenic signaling pathways that are disrupted by these pollutants will improve our ability to assess their risk to human health. Finally, it is likely that many other environmental pollutants impact neovascularization; however, very few have been studied in sufficient detail. Thus, additional research also is needed to identify those environmental agents that mediate their toxicity by disrupting neovascularization.

A selective ATP-binding cassette subfamily G member 2 efflux inhibitor revealed via high-throughput flow cytometry.

Chemotherapeutics tumor resistance is a principal reason for treatment failure, and clinical and experimental data indicate that multidrug transporters such as ATP-binding cassette (ABC) B1 and ABCG2 play a leading role by preventing cytotoxic intracellular drug concentrations. Functional efflux inhibition of existing chemotherapeutics by these pumps continues to present a promising approach for treatment. A contributing factor to the failure of existing inhibitors in clinical applications is limited understanding of specific substrate/inhibitor/pump interactions. We have identified selective efflux inhibitors by profiling multiple ABC transporters against a library of small molecules to find molecular probes to further explore such interactions. In our primary screening protocol using JC-1 as a dual-pump fluorescent reporter substrate, we identified a piperazine-substituted pyrazolo[1,5-a]pyrimidine substructure with promise for selective efflux inhibition. As a result of a focused structure-activity relationship (SAR)-driven chemistry effort, we describe compound 1 (CID44640177), an efflux inhibitor with selectivity toward ABCG2 over ABCB1. Compound 1 is also shown to potentiate the activity of mitoxantrone in vitro as well as preliminarily in vivo in an ABCG2-overexpressing tumor model. At least two analogues significantly reduce tumor size in combination with the chemotherapeutic topotecan. To our knowledge, low nanomolar chemoreversal activity coupled with direct evidence of efflux inhibition for ABCG2 is unprecedented.

High-throughput cytotoxicity screening by propidium iodide staining.

This unit describes a system for the automated high-throughput analysis of cell cytotoxicity in 96-well and 384-well microplates. Discrete cell cultures are analyzed at rates of 40/min (approximately 2.5 min/96 wells, approximately 10 min/384 wells) and cytotoxicity is quantified on the basis of a combination of propidium iodide (PI) fluorescence analysis and cell counting performed by the flow cytometer. Only 2 microl is aspirated from a culture for analysis so that assays can be performed in small volumes to minimize reagent cost and usage.

PYK2 mediates anti-apoptotic AKT signaling in response to benzo[a]pyrene diol epoxide in mammary epithelial cells.

Polycyclic aromatic hydrocarbons, such as benzo[a]pyrene (BaP), are known mammary carcinogens in rodents and may be involved in human breast cancer. The carcinogenicity of BaP has been partially attributed to the formation of the BaP diol epoxide (BPDE), which has been shown to stably bind DNA and act as an initiator. BaP is a complete carcinogen, but the mechanisms for tumor promotion are less well characterized. Previous studies have demonstrated that BPDE enhanced anti-apoptotic signaling through Akt; however, mechanisms for Akt activation by BPDE are not well defined. In the current studies, we found that BPDE increased intracellular Ca2+ concentration in the human mammary epithelial cell line MCF-10A. A peak in Ca2+ concentration at 20 min was followed by increased phosphorylation of Pyk2 at Tyr881 and increased total tyrosine phosphorylation of the epidermal growth factor receptor (EGFR). Consistent with activation of the EGFR, Akt and ERK1/2 phosphorylation was detected in MCF-10A cells treated with BPDE. Pharmacological methods to prevent Ca2+ elevation and EGFR activity, and small-interfering RNA against Pyk2, prevented Akt phosphorylation by BPDE, which suggested that Ca2+, Pyk2 and EGFR activation lay upstream of Akt. In addition, we found that BPDE increased p53 activity and apoptosis in MCF-10A; however, transient transfection of constitutively active Akt attenuated both BPDE-dependent apoptosis and p53 activity. In contrast, apoptosis was enhanced by inhibitors of phosphatidyl inositol 3-kinase (PI3-K). This work demonstrates a novel mechanism for Akt activation by BPDE that occurs through increased Ca2+ concentration, and implicates Ca2+, Pyk2, EGFR and Akt as a potential pathway by which BPDE can inhibit apoptosis and act as a promoter of carcinogenesis.

2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) inhibition of coronary vasculogenesis is mediated, in part, by reduced responsiveness to endogenous angiogenic stimuli, including vascular endothelial growth factor A (VEGF-A).

BACKGROUND: 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) exposure prior to chick embryo incubation (GD 0) induces dilated cardiomyopathy, and reduces myocardial hypoxia, vascular endothelial growth factor A (VEGF-A) expression, and coronary vascularization. We investigated whether reduced coronary vascularization 1) occurs in the absence of changes in cardiac morphology and 2) is associated with altered secretion of VEGF-A and/or an antivasculogenic factor. METHODS: Chicken eggs were treated with control (corn oil) or TCDD (0.075-0.3 pmol of TCDD/gm) on GD 5. In vivo cardiac morphology and artery number were determined on GD 10, while in vitro vascular outgrowth and VEGF-A secretion were determined from cardiac explants on GD 6. Effects of recombinant VEGF-A (rcVEGF-A), soluble flt-1 (sFlt-1) receptor plus rcVEGF-A, and control conditioned media were assessed in TCDD explants, while effects of TCDD-conditioned media was assessed in control explants. RESULTS: TCDD reduced coronary artery number in vivo by 53 +/- 8% and induced a dose-related reduction in tube outgrowth in vitro, but had no effect on cardiac morphology. All TCDD doses reduced explant VEGF-A secretion equally (43 +/- 3%), compared to control. sFlt-1 blocked outgrowth in control cultures and blocked rcVEGF-A-mediated rescue of outgrowth in TCDD explants. Control conditioned media partially rescued outgrowth from TCDD explants, while conditioned media from TCDD explants had no effect on controls. CONCLUSIONS: TCDD inhibition of coronary vascularization can occur in the absence of changes in cardiac morphology and is associated with reduced VEGF-A secretion but not an antivasculogenic factor. Since control media only partly rescues TCDD's inhibitory effect, we suggest that TCDD-exposed endothelial cells are less responsive to vasculogenic stimuli.

2,3,7,8-tetrachlorodibenzo-p-dioxin reduces myocardial hypoxia and vascular endothelial growth factor expression during chick embryo development.

BACKGROUND: Previous research has demonstrated that 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) induces cardiomyocyte growth arrest, thinner ventricle walls, and reduced number and size of coronary arteries during chick embryogenesis. Coronary vascular development is believed to be mediated, in part, by myocardial oxygen gradients and a subsequent increase in hypoxia-inducible factor 1alpha (HIF-1alpha) and vascular endothelial growth factor-A (VEGF-A) expression. We investigated whether TCDD inhibition of coronary development was associated with altered myocardial oxygen status and reduced cardiac HIF-1alpha and VEGF-A. METHODS: Chick embryos were exposed to 15% or 20% O2 for 24 hr from incubation days 9-10 or were injected with control (corn oil) or 0.24 pmol TCDD/gm egg on day 0. On day 9, embryos were injected with control (0.9% NaCl) or EF5, a tissue hypoxia marker, and cardiac binding of EF5 was determined by immunohistochemistry on day 10. In addition, embryo hearts were analyzed for VEGF-A mRNA by in situ hybridization and quantitative RT-PCR, and for HIF-1alpha mRNA by quantitative RT-PCR. RESULTS: Cardiac binding of EF5 was significantly increased in embryos exposed to 15% O2, compared to embryos exposed to 20% O2. In contrast, TCDD-exposed embryos exhibited significantly reduced binding of EF5 in the heart, compared to controls. Similarly, cardiac expression of HIF-1alpha and VEGF-A were increased following hypoxia and tended to be decreased following TCDD exposure. CONCLUSIONS: These results suggest that the myocardium may be a target of TCDD toxicity, resulting in reduced myocardial hypoxia, and HIF-1alpha and VEGF-A expression believed necessary for normal coronary development.

Vascular endothelial growth factor rescues 2,3,7,8-tetrachlorodibenzo-p-dioxin inhibition of coronary vasculogenesis.

BACKGROUND: We previously demonstrated that the environmental pollutant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) reduces coronary vascular development in chick embryos in vivo. In the current study, we assessed whether TCDD inhibits early events in coronary endothelial tube formation and outgrowth, and whether this inhibition occurs through a vascular endothelial growth factor (VEGF)-dependent mechanism. METHODS: Fertile chicken eggs were treated with control (corn oil) or TCDD (0.3 pmol TCDD/g) on incubation day 0. On embryonic day 6, cardiac ventricle explants were cultured on a three-dimensional collagen gel, when coronary angioblasts are present, but prior to their assembly into endothelial tubes. Endothelial cells migrating out from explants were identified by immunohistochemistry, and endothelial tube number and length were quantitated. In addition, on incubation days 6 and 8, cardiac VEGF mRNA and protein were measured by reverse transcriptase-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA), respectively. RESULTS: Endothelial tube length and number were significantly reduced (40% +/- 1.7% and 36% +/- 3%, respectively) in TCDD explants, compared to controls. Recombinant exogenous VEGF, as well as hypoxic stimulation with CoCl2 or 10% O2, significantly increased the length and number of outgrowing tubes in TCDD cultures, and this stimulation was prevented by a VEGF neutralizing antibody. In contrast, VEGF neutralizing antibody reduced the length and number of tubes only in control cultures, and had no inhibitory effect on tube outgrowth from TCDD explants. Finally, hearts from TCDD-treated embryos exhibited a significant reduction in both VEGF mRNA and protein, compared to controls. CONCLUSIONS: These data suggest that TCDD inhibits early coronary vascular outgrowth via a VEGF-dependent mechanism.