Carboxy-2',7'-dichlorofluorescein Research Articles

Functional Alterations of Multidrug Resistance-Associated Proteins 2 and 5, and Breast Cancer Resistance Protein upon Snail-Induced Epithelial-Mesenchymal Transition in HCC827 Cells.

Our previous report indicated that Snail-induced epithelial-mesenchymal transition (EMT) enhanced P-glycoprotein (P-gp) function and drug resistance to P-gp substrate anticancer drug in a human non-small cell lung cancer (NSCLC) cell line, HCC827. Our objective is to evaluate the changes in the mRNA and protein expression levels and the functions of multidrug resistance-associated protein (MRP) 2, MRP5 and breast cancer resistance protein (BCRP). Snail-expressing HCC827 cells showed increased mRNA levels of Snail and a mesenchymal marker vimentin, and decreased mRNA levels of an epithelial marker E-cadherin after transduction, indicating that Snail had induced EMT consistent with our previous reports. The mRNA level of MRP2 was significantly decreased, while that of MRP5 remained unchanged, in Snail-expressing cells. The expression levels of MRP2 and MRP5 proteins in whole-cell homogenate were unchanged in Snail-expressing cells, but MRP5 protein showed significantly increased membrane localization. Snail-transduction increased the efflux transport of 5-(and-6)-carboxy-2',7'-dichlorofluorescein (CDCF), a substrate of MRP2, 3 and 5. This increase was blocked by MK571, which inhibits MRP1, 2, and 5. Toxicity of cisplatin, a substrate of MRP2 and 5, was significantly decreased in Snail-expressing cells. BCRP mRNA and protein levels were both decreased in Snail-expressing cells, which showed an increase in the intracellular accumulation of 7-ethyl-10-hydroxycamptothecin (SN-38), a BCRP substrate, resulting in reduced viability. These results suggested that MRP5 function appears to be increased via an increase in membrane localization, whereas the BCRP function is decreased via a decrease in the expression level in HCC827 cells with Snail-induced EMT.

Endogenous, cholesterol-activated ATP-dependent transport in membrane vesicles from Spodoptera frugiperda cells

Transport proteins of the ATP-binding cassette (ABC) family are found in all kingdoms of life. In humans, several ABC efflux transporters play a role in drug disposition and excretion. Therefore, in vitro methods have been developed to characterize the substrate and inhibitor properties of drugs with respect to these transporters. In the vesicular transport assay, transport is studied using inverted membrane vesicles produced from transporter overexpressing cell lines of both mammalian and insect origin. Insect cell expression systems benefit from a higher expression compared to background, but are not as well characterized as their mammalian counterparts regarding endogenous transport. Therefore, the contribution of this transport in the assay might be underappreciated. In this study, endogenous transport in membrane vesicles from Spodoptera frugiperda -derived Sf9 cells was characterized using four typical substrates of human ABC transporters: 5(6)-carboxy-2,′7′-dichlorofluorescein (CDCF), estradiol-17β-glucuronide, estrone sulfate and N-methyl-quinidine. Significant ATP-dependent transport was observed for three of the substrates with cholesterol-loading of the vesicles, which is sometimes used to improve the activity of human transporters expressed in Sf9 cells. The highest effect of cholesterol was on CDCF transport, and this transport in the cholesterol-loaded Sf9 vesicles was time and concentration dependent with a Km of 8.06 ± 1.11 μM. The observed CDCF transport was inhibited by known inhibitors of human ABCC transporters, but not by ABCB1 and ABCG2 inhibitors verapamil and Ko143, respectively. Two candidate genes for ABCC-type transporters in the S. frugiperda genome (SfABCC2 and SfABCC3) were identified based on sequence analysis as a hypothesis to explain the observed endogenous ABCC-type transport in Sf9 vesicles. Although further studies are needed to verify the role of SfABCC2 and SfABCC3 in Sf9 vesicles, the findings of this study highlight the need to carefully characterize background transport in Sf9 derived membrane vesicles to avoid false positive substrate findings for human ABC transporters studied with this overexpression system.

The Effect of Albumin on MRP2 and BCRP in the Vesicular Transport Assay.

The ABC transporters multidrug resistance associated protein 2 (MRP2) and breast cancer resistance protein (BCRP) are of interest in drug development, since they affect the pharmacokinetics of several drugs. Membrane vesicle transport assays are widely used to study interactions with these proteins. Since albumin has been found to affect the kinetics of metabolic enzymes in similar membrane preparations, we investigated whether albumin affects the kinetic parameters of efflux transport. We found that albumin increased the Vmax of 5(6)-carboxy-2’,7’-dichlorofluorescein (CDCF) and estradiol-17-β-D-glucuronide uptake into MRP2 vesicles in the presence of 0.1% bovine serum albumin (BSA) by 2 and 1.5-fold, respectively, while BSA increased Lucifer yellow uptake by 30% in BCRP vesicles. Km values increased slightly, but the change was not statistically significant. The effect of BSA on substrate uptake was dependent on the vesicle amount, while increasing BSA concentration did not significantly improve substrate uptake. These results indicate a minor effect of albumin on MRP2 and BCRP, but it should be considered if albumin is added to transporter assays for example as a solubilizer, since the effect may be substrate or transporter specific.

Drug Distribution to Retinal Pigment Epithelium: Studies on Melanin Binding, Cellular Kinetics, and Single Photon Emission Computed Tomography/Computed Tomography Imaging

Melanin binding is known to affect the distribution and elimination of ocular drugs. The purpose of this study was to evaluate if the extent of drug uptake to primary retinal pigment epithelial (RPE) cells could be estimated based on in vitro binding studies with isolated melanin and evaluate the suitability of single photon emission computed tomography/computed tomography (SPECT/CT) in studying pigment binding in vivo with pigmented and albino rats. Binding of five compounds, basic molecules timolol, chloroquine, and nadolol and acidic molecules methotrexate and 5(6)-carboxy-2',7'-dichlorofluorescein (CDCF), was studied using isolated melanin from porcine choroid-RPE at pH 5.0 and 7.4. The uptake to primary porcine RPE cells was studied with timolol, chloroquine, methotrexate, and CDCF. The cell study setting was modeled using parameters from the in vitro binding study. In vivo kinetics of 3-[I-123]-iodochloroquine was studied by the SPECT/CT method in albino and pigmented rats. All basic compounds bound to melanin at both pH values, whereas the acidic compounds bound more at pH 5.0 than at pH 7.4. The basic compounds (chloroquine, timolol) showed significant cellular uptake, unlike the acidic compounds (methotrexate, CDCF). On the basis of the modeling, melanin binding was a major factor governing the overall drug distribution to the RPE cells. Likewise, melanin binding explained distribution of 3-[I-123]-iodochloroquine in the pigmented RPE, whereas drug accumulation was not seen in the albino rat. This study demonstrates the suitability of noninvasive SPECT/CT imaging in monitoring ocular melanin binding in vivo. These studies are a useful step toward understanding the pharmacokinetic impact of melanin binding.

A membrane vesicle-based assay to enable prediction of human biliary excretion

1. Prediction of biliary excretion is a challenge due to the lack of in vitro assays. Our laboratory previously demonstrated a highly significant correlation between in vitro IC50 values against mrp2 using rat canalicular liver plasma membrane vesicles and in vivo biliary excretion (Colombo et al., 2012). This study explores the possibility of predicting in vivo biliary excretion in human using membrane vesicles prepared from MDCKII cells transfected with human ABCC2.2. In vitro MRP2 activity was determined by measuring the ATP-dependent uptake of 5(6)-carboxy-2′,7′-dichlorofluorescein (CDCF) in inside-out membrane vesicles isolated from MDCK-ABCC2 cells. CDCF uptake was time- and concentration-dependent (Km of 4.0 ± 1.2 µM and a Vmax of 7.8 ± 0.9 pmol/mg/min) and inhibited by benzbromarone and MK-571 with IC50 values of 1.2 and 7.6 µM, respectively.3. A significant linear correlation (r2 = 0.790) between the in vitro IC50 values from the described MRP2 assay and in vivo biliary excretion in humans was observed using 11 well-documented drugs covering low to high biliary excretions.4. This study demonstrates, for the first time, that inhibition of CDCF uptake in MDCKII-ABCC2 vesicles not only provides a screening assay to assess MRP2 drug–drug interaction potential, but is also predictive of human MRP2-mediated biliary excretion.

Substrate translocation and stimulated ATP hydrolysis of human ABC transporter MRP3 show positive cooperativity and are half-coupled

ABC transporters are involved in countless processes from lipid excretion over cellular detoxification to multidrug resistance of cancer cells. The latter is especially conferred by the ABCC subfamily also called multidrug resistance-associated proteins (MRPs) that excrete a variety of amphipathics including anticancer drugs by ATP-dependent transport. As the mechanisms of substrate translocation and ATP hydrolysis are still unclear for MRPs, we investigated the kinetics of both processes with focus on cooperativity and coupling between ATPase activity and substrate transport using purified MRP3 in proteoliposomes. Although the ATP-dependent uptake of amphipathics and the hydrophilic 5(6)-carboxy-2′-7′-dichlorofluorescein (CDCF) into the lumen of proteoliposomes showed affinity constants similar to those reported for cell-based assays, the maximal uptake rates were up to 250 times higher. Moreover, all substrates showed cooperative interactions of two subunits. Upon stimulation with amphipathics, ATPase activity of MRP3 increased from 80nmol/(mgmin) to 180nmol/(mgmin) showing positive cooperativity with a Hill coefficient of 2. While Hill coefficient and maximal ATPase activity were found to be substrate independent, the affinity constants are characteristic for a given substrate and correspond to the value for transport. Therefore, cooperative interactions of the two nucleotide binding domains (NBDs) in MRP3 are mediated by substrate binding to the transmembrane domains (TMDs). In contrast to amphipathic substrates, CDCF did not stimulate ATPase activity despite being transported in an ATP-dependent manner. This indicates that ATP hydrolysis and substrate translocation are half-coupled in MRP3 as CDCF shuttles on a basal TMD activity resulting from the basal ATPase activity.

A high throughput in vitro mrp2 assay to predict in vivo biliary excretion

Prediction of biliary excretion is a challenge for drug discovery scientists due to the lack of in vitro assays. This study explores the possibility of establishing a simple assay to predict in vivo biliary excretion via the mrp2 transport system.In vitro mrp2 activity was determined by measuring the ATP-dependent uptake of 5(6)-carboxy-2′,7′-dichlorofluorescein (CDCF) in canalicular plasma membrane vesicles (cLPM) from rat livers. The CDCF uptake was time- and concentration-dependent (Km of 2.2 ± 0.3 µM and Vmax of 115 ± 26 pmol/mg/min) and strongly inhibited by the mrp2 inhibitors, benzbromarone, MK-571, and cyclosporine A, with IC50 values ≤ 1.1 µM.Low inhibition of CDCF uptake by taurocholate (BSEP inhibitor; 57 µM) and digoxin (P-gp inhibitor; 101 µM) demonstrated assay specificity towards mrp2.A highly significant correlation (r2 = 0.959) between the in vitro IC50 values from the described mrp2 assay and in vivo biliary excretion in rats was observed using 10 literature compounds.This study demonstrated, for the first time, that a high throughput assay could be established with the capability of predicting biliary excretion in the rat using CDCF as a substrate.

Characterization of rhodamine-123, calcein and 5(6)-carboxy-2′,7′-dichlorofluorescein (CDCF) export via MRP2 (ABCC2) in MES-SA and A549 cells

Based on our initial results on the effects of several ATP-binding cassette (ABC) transporter inhibitors on rhodamine-123 efflux from A549, a human lung carcinoma, and MES-SA, a human uterine sarcoma cell line, the aim of this study was to identify the transporter responsible for this export. Export of two fluorescent dyes, rhodamine-123 and calcein, was investigated in both cell lines by testing five commonly used inhibitors of ABC transporters: verapamil, cyclosporin A, MK571, GF129018 and fumitremorgin C. A very high degree of correlation ( R 2 = 0.91–0.99) between results obtained in the two cell lines suggested that the same transporter was involved in the export of tested fluorescent substrates in both cell lines. Expression analysis and gene silencing techniques, as well as transport of additional substrate 5(6)-carboxy-2′,7′-dichlorofluorescein (CDCF) on membrane vesicles revealed that the transporter was multidrug resistance related protein 2 (MRP2, ABCC2). Furthermore, it was found that the tested modulators showed very diverse effects on the export of three fluorescent substrates via MRP2, with some modulators being inhibitory in one, while having no effect or even stimulating the transport in the other fluorescent dye assay. Verapamil inhibited rhodamine-123, but stimulated CDCF transport and did not affect calcein export. GF129018 did not affect calcein and CDCF transport, but it inhibited rhodamine-123 transport. These results demonstrate the importance of studying various combinations of potential substrates and modulators of MRP2 in order to estimate possible drug–drug interactions in living organisms. In addition, A549 and MES-SA cells were shown to be good cell models for studying interactions of compounds with human MRP2.

Divergent Effects of Nitric Oxide Donors on the Biliary Efflux Transporters in Isolated Perfused Rat Livers: Nitric Oxide-Independent Inhibition of ABC Transporters by Sodium Nitroprusside

Rhodamine 123 (RH-123) and its glucuronidated metabolite (RH-Glu) are excreted into the bile via the ABC efflux transporters P-glycoprotein (P-gp) and multidrug resistance-related protein type 2 (Mrp2), respectively. In this study, we investigated the short-term (2 h) effects of a low or high concentration of nitric oxide (NO) donors sodium nitroprusside (SNP) and isosorbide dinitrate (ISDN) on the hepatobiliary disposition of RH-123 and its metabolite in an isolated perfused rat liver model. Additionally, the effects of ISDN on the hepatobiliary disposition of 5 (and 6)-carboxy-2', 7'- dichlorofluorescein (CDF), a specific marker of Mrp2, were investigated in the same model. Whereas SNP caused a substantial (85-90%) reduction in the P-gp- and Mrp2-mediated transport of RH-123 and RH-Glu, respectively, ISDN did not affect either of these transporters. However, ISDN reduced the biliary recovery of RH-Glu, most likely because of inhibition of the formation of the metabolite. Further studies showed that the effects of SNP on these transporters are due to a substantial (88%) depletion of hepatic ATP levels by this NO donor. Additionally, studies using CDF revealed an almost identical hepatobiliary disposition of this Mrp2 marker in the presence or absence of ISDN. It is concluded that short-term exposure of rat livers to NO does not affect the functions of the efflux transporters P-gp and Mrp2. The observed inhibitory effects of SNP on the functions of both P-gp and Mrp2 are via an NO-independent mechanism.

Characterization of 5(6)-Carboxy-2,′7′-Dichlorofluorescein Transport by MRP2 and Utilization of this Substrate as a Fluorescent Surrogate for LTC4

MRP2 (ABCC2) is an efflux transporter expressed on the apical membrane of polarized cells. This protein has a major role in the biliary elimination of toxic compounds from the liver. As MRP2 transports many endogenous compounds, including LTC4 as well as xenobiotics and toxic phase II metabolites, blockade of this transporter may cause the accumulation of these compounds in the hepatocyte, resulting in hepatotoxicity. The vesicular transport assay is a great tool to study drug-drug and drug-endogenous compound interactions of ABC transporters. In this assay, inside-out membrane vesicles are used, so the test compound can readily access the transporter. As MRP2 transports many ionic compounds that are difficult to investigate in a whole-cell system because of permeability reasons, the vesicular transport assay is a good choice for screening MRP2-mediated interactions. LTC4 is not an optimal substrate for high-throughput screening for MRP2 interactors, even though it is an important MRP2 substrate. Therefore, the transport of a drug surrogate, 5(6)-carboxy-2,'7'-dichlorofluorescein (CDCF), by MRP2 was characterized using the vesicular transport assay. The data indicate that CDCF proves to be an ideal substrate for MRP2 vesicular transport assay with its optimal detection and transport properties.

MODULATION OF HEPATIC CANALICULAR OR BASOLATERAL TRANSPORT PROTEINS ALTERS HEPATOBILIARY DISPOSITION OF A MODEL ORGANIC ANION IN THE ISOLATED PERFUSED RAT LIVER

This study examined the impact of hepatic transport protein modulation on the hepatobiliary disposition of a nonmetabolized probe substrate, 5- (and 6)-carboxy-2',7'dichlorofluorescein (CDF) in rat isolated perfused livers (IPLs). In vivo treatment with modulators (100 and 200 mg/kg/day clofibric acid, 80 mg/kg/day phenobarbital, and 25 mg/kg/day dexamethasone) was used to alter the expression of hepatic transport proteins [organic anion transporting polypeptide 1a1, multidrug resistance-associated protein (Mrp) 3, and Mrp2] governing the disposition of CDF. The basolateral and biliary excretion of CDF was measured in single-pass IPLs from control and treated rats. Modulators increased the percentage of CDF eliminated into perfusate of IPLs from treated rats ( approximately 20-35%) compared with controls ( approximately 10%); CDF biliary excretion was decreased in the treated groups. These observations are consistent with modulator-associated increases in the first-order rate constant governing CDF excretion from the hepatocytes into perfusate (k(perfusate)) or decreases in the first-order rate constant governing CDF excretion into bile (k(bile)). Pharmacokinetic modeling of the data and subsequent simulations revealed that the routes of CDF excretion were most sensitive to changes in k(perfusate). In contrast, hepatic accumulation of CDF was most sensitive to k(bile). The differential sensitivity of CDF excretory routes and hepatic accumulation to these rate constants is a function of intrahepatic distribution kinetics, which must be taken into consideration in assessing the potential impact of altered hepatobiliary transport processes.

Light-induced pH changes in leaves of C4 plants

Light-induced changes in the fluorescence of the pH-indicating dyes pyranine or 5-(and 6-)carboxy-2′, 7′-dichlorofluorescein (CDCF) which had been fed to leaves were examined to monitor cellular pH changes. After short-term feeding of pyranine (pK 7.3) to leaves of Amaranthus caudatus L., a NAD-malic-enzyme-type C4 plant, vascular bundles and surrounding cells became fluorescent. Fluorescence emission from mesophyll cells required longer feeding times. In CO2-free air, pyranine fluorescence increased much more on illumination after mesophyll cells had become fluorescent than when only the vascular bundles and the bundle sheath of Amaranthus leaves had been stained. After short feeding times and in the absence of actinic illumination, CO2 decreased pyranine fluorescence very slowly in Amaranthus and rapidly in C3 leaves. After prolonged feeding times, the extent of the light-dependent increase in pyranine fluorescence was several times greater in different C4 plants than in C3 species. The kinetics of the fluorescence changes were also remarkably different in C3 and C4 plants. Carbon dioxide (500 μl · l−1) suppressed the light-induced increase in pyranine fluorescence more in C4 than in C3 leaves. Light-dependent changes in light scattering, which are indicative of chloroplast energization, and in 410-nm transmission, which indicate chloroplast movement, differed kinetically from those of the changes in pyranine fluorescence. Available evidence indicated that light-dependent changes in pyranine fluorescence did not originate from the apoplast of leaf cells. Microscopic observation led to the conclusion that, after prolonged feeding times or prolonged incubation, changes in pyranine fluorescence emitted from C4 leaves reflect pH changes mainly in the cytosol of mesophyll cells. A transient acidification reaction indicated by quenching of pyranine fluorescence in the dark-light transient and not observed in C3 species is attributed to the carboxylation of phosphoenolpyruvate. After short feeding times and in the absence of actinic illumination, CO2 (250 μl μ l−1) decreased pyranine fluorescence very slowly in Amaranthus and more rapidly in C3 leaves. After prolonged feeding times, both the rate and the extent of CO2-dependent quenching of pyranine fluorescence increased, but the increase was insufficient to indicate the presence of highly active carbonic anhydrase in the compartment from which pyranine fluorescence was emitted. In contrast to pyranine, CDCF (pK 4.8) did not increase but rather decreased its fluorescence on illumination of an Amaranthus leaf, indicating acidification of an acidic compartment, most probably the vacuole of green leaf cells. The pattern of the acidification reaction was similar in C4 and C3 leaves. The remarkably large extent of the light-dependent increase in pyranine fluorescence from leaves of C4 species and its slow kinetics are proposed to be caused by an alkalization of the cytosol which in the absence of CO2 is larger in the mesophyll than in the bundle sheath. It gives rise to deprotonation of dye originally located in the mesophyll and, in addition, of dye which diffuses from the bundle sheath into the mesophyll following a pH gradient. Implications of slow diffusional transport of pyranine and CO2 between mesophyll and bundle-sheath cells and the fast metabolite transport required in C4 photosynthesis are discussed.

Dicarboxy-dichlorofluorescein: A new fluorescent probe for measuring acidic intracellular pH

Derivatives of fluorescein sensitive to pH are extensively utilized for the determination of intracellular pH (pH i). Available dyes have p K a values of approximately 7.0, and are not well suited for measuring acidic pH i. We examined the fluorescein derivative, 5 (and 6)-carboxy-2′,7′-dichlorofluorescein (CDCF) for its potential in the microspectrofluorometric measurement of pH i during acidic conditions. CDCF showed intense fluorescence and pH sensitivity near its “effective” p K a value of 4.2, using a 495 440 nm dual excitation wavelength ratio method. Protein interactions caused fluorescence ratio deviations which were most pronounced at the extremes of pH, whereas calcium and magnesium concentrations had little effect on the fluorescent ratio intensity. Intracellular calibration performed using nigericin in the presence of high potassium eliminated the need to correct for protein interactions, and the ratio method minimized any variations due to dye concentration differences or instrument fluctuation. Intracellular retention of the dye was high, and 95% of the initial signal remained after 1 h. Fluorescence bleaching was 14.5% after 1 h of continuous excitation and cell survival was not affected by dye loading. We conclude that CDCF is an excellent intracellular pH indicator in the pH range of 4–5.