Calcein AM Assay Research Articles

Development of Decision Tree Models for Substrates, Inhibitors, and Inducers of P-Glycoprotein

In silico classification of new compounds for certain properties is a useful tool to guide further experiments or compound selection. Interaction of new compounds with the efflux pump P-glycoprotein (P-gp) is an important drug property determining tissue distribution and the potential for drug-drug interactions. We present three datasets on substrate, inhibitor, and inducer activities for P-gp (n = 471) obtained from a literature search which we compared to an existing evaluation of the Prestwick Chemical Library with the calcein-AM assay (retrieved from PubMed). Additionally, we present decision tree models of these activities with predictive accuracies of 77.7 % (substrates), 86.9 % (inhibitors), and 90.3 % (inducers) using three algorithms (CHAID, CART, and C4.5). We also present decision tree models of the calcein-AM assay (79.9 %). Apart from a comprehensive dataset of P-gp interacting compounds, our study provides evidence of the efficacy of logD descriptors and of two algorithms not commonly used in pharmacological QSAR studies (CART and CHAID).

Synthesis and biological evaluation of a small molecule library of 3rd generation multidrug resistance modulators

The development of new modulators possessing high efficacy, low toxicity and high selectivity is a pivotal approach to overcoming P-glycoprotein (P-gp) mediated multidrug resistance (MDR) in tumour cells. In this study 39 compounds are presented which have been synthesized and pharmacologically investigated in our laboratory. Similarly to the potent 3rd generation MDR modulator tariquidar (XR9576) the compounds contain a tetrahydroisoquinoline–ethyl-phenylamine substructure that, in contrast to XR9576, is connected to a smaller hydrophobic part, thus leading to molecules of lower molecular weight. The connection between the tetrahydroisoquinoline–ethyl-phenylamine substructure and the hydrophobic part was achieved through four different types of linkers: amide, urea, amide-ether and amide-styryl. A number of structural modifications in the hydrophobic part were created. The calcein AM assay served as test system to determine the P-gp transport inhibitory potencies of the compounds. For the amide linker derivatives a structure–activity relationship analysis was performed outlining which structural modifications contributed to the inhibitory potency. The compounds containing a bicyclic hydrophobic part with a particular substituent in a specific orientation were identified as the most potent amide derivatives. Among the urea derivatives the compounds with highest inhibitory potency possessed an ortho-nitro substituent. The conformational analysis revealed that this position enables the formation of a hydrogen bond to the urea linker thus stabilizing the conformation. Regarding the amide-styryl derivatives the elongation of the amide linker seemed to be most decisive for the observed increase in activity. The most promising candidate in the whole library possess an amide-ether linker and an ortho-nitro substituent in the hydrophobic part. This compound inhibites P-gp slightly less than tariquidar and can serve as a lead structure for new potent P-gp modulators.

Limitations and relative utility of screening assays to assess engineered nanoparticle toxicity in a human cell line

Single-walled carbon nanotubes (SWCNT), fullerenes (C 60), carbon black (CB), nC 60, and quantum dots (QD) have been studied in vitro to determine their toxicity in a number of cell types. Here, we report that classical dye-based assays such as MTT and neutral red (NR) that determine cell viability produce invalid results with some NM (nanomaterials) due to NM/dye interactions and/or NM adsorption of the dye/dye products. In this study, human epidermal keratinocytes (HEK) were exposed in vitro to CB, SWCNT, C 60, nC 60, and QD to assess viability with calcein AM (CAM), Live/Dead (LD), NR, MTT, Celltiter 96 ® AQueous One (96 AQ), alamar Blue (aB), Celltiter-Blue ® (CTB), CytoTox One™ (CTO), and flow cytometry. In addition, trypan blue (TB) was quantitated by light microscopy. Assay linearity (R 2 value) was determined with HEK plated at concentrations from 0 to 25,000 cells per well in 96-well plates. HEK were treated with serial dilutions of each NM for 24 h and assessed with each of the viability assays. TB, CAM and LD assays, which depend on direct staining of living and/or dead cells, were difficult to interpret due to physical interference of the NM with cells. Results of the dye-based assays varied a great deal, depending on the interactions of the dye/dye product with the carbon nanomaterials (CNM). Results show the optimal high throughput assay for use with carbon and noncarbon NM was 96 AQ. This study shows that, unlike small molecules, CNM interact with assay markers to cause variable results with classical toxicology assays and may not be suitable for assessing nanoparticle cytotoxicity. Therefore, more than one assay may be required when determining nanoparticle toxicity for risk assessment.

Pathogenesis of osteoarthritis: Chondrocyte replicative senescence or apoptosis?

The aim of this study was to investigate by flow cytometry cellular viability and apoptosis of human chondrocytes isolated from osteoarthritic cartilage and to correlate replicative senescence with apoptosis of these cells. To understand the mechanisms underlying the process of cell death in cartilage destruction, we investigated by flow cytometry cellular viability (Cell viability calcein-AM assay) and apoptosis (Light scattering properties of chondrocytes, study of chondrocyte death using Annexin-V-FITC and propidium iodide double-labeling, caspase-3 activity determination) of human chondrocytes isolated from osteoarthritic and nonosteoarthritic cartilage. Senescent cells were characterized using the senescence-associated-beta-galactosidase marker (SA-beta-Gal marker) by staining with chromogenic substrate (X-Gal) to produce blue coloration of SA-beta-Gal-positive cells and microscopy analysis. The results we obtained show that between 25 and 40% of chondrocytes were in apoptosis and all of them were SA-beta-Gal-positive. These results demonstrate that the death of osteoarthritic chondrocytes is an apoptotic phenomenon which is preceded by an accelerated mechanism of replicative senescence.

Functional assay and structure–activity relationships of new third-generation P-glycoprotein inhibitors

Twenty-eight compounds, including 24 structurally related derivatives of tariquidar synthesized in our laboratory, and four XR compounds, reported by Xenova group Ltd, were investigated by the Hoechst 33342 and Calcein AM functional assays for estimation of their inhibitory effects on the transport activity of P-glycoprotein (P-gp). A high correlation between the effects obtained in both assays was observed at the substrate concentrations used. The analyses of kinetics data from experiments at different substrate concentrations revealed non-competitive inhibition in the Calcein AM assay and competitive inhibition in the Hoechst 33342 assay. The 3D structures of the compounds were further aligned on Hoechst 33342 using flexible and pharmacophore alignments. The results suggested that inhibitors could interact with the H-binding site of P-gp and this could potentially be achieved by different ways of binding. The best 3D-QSAR models, generated by CoMFA and CoMSIA, yielded an internal predictive squared correlation coefficient higher than 0.8 and included electrostatic, steric, hydrogen bond acceptor, and hydrophobic fields. Validation of the models on an external test set of 30 XR compounds gave predictive squared correlation coefficients of up to 0.66. An excellent correspondence between the experimental and modeled activities of the test compounds was observed. The models can be used for prediction and rational design of new P-gp inhibitors.

Resveratrol Exerts Antiproliferative Effect and Induces Apoptosis in Waldenstrom's Macroglobulinemia.

Background: Resveratrol is a polyphenolic natural product, synthesized by a wide variety of plant species including grapes. It has gained considerable attention because of its anti-cancer properties, as demonstrated in solid and haematological malignancies. We therefore examined Resveratrol for its anti-tumor activity in Waldenstrom's Macroglobulinemia (WM).Methods: We examined the effect of increasing concentrations of resveratrol (5–80 μM) on WM cell lines (BCWM.1), IgM secreting low-grade lymphoma cell lines (WM-WSU, MEC-1, RL), peripheral blood mononuclear cells (PBMCs) isolated from healthy donors, primary CD19+ WM cells and bone marrow stromal cells (BMSCs) isolated from bone marrow of patients with WM. [3H]-thymidine uptake and calcein-AM assay were used to evaluate the effect of resveratrol on proliferation and cytotoxicity respectively. Apoptosis and cell cycle analysis were investigated at 24h by flow cytometry using Annexin V-propidium iodide (PI) staining and PI-staining respectively. Apoptotic and cell signaling pathways targeted by resveratrol were investigated by Western Blot at 24 h and 6 h respectively. Since BMCSc confer growth and resistance to conventional treatments, we also tested the effect of resveratrol on WM cells co-cultured with BMSCs. Gene expression analysis has been performed on BCWM.1 cultured in presence or absence of resveratrol.Results: Resveratrol induced significant cytotoxicity and inhibition of DNA synthesis at 24 and 48 h on BCWM.1 with an IC50 of 10–20μM. Similar data was obtained with primary CD19+ WM cells. In contrast, resveratrol did not trigger significant reduction of proliferation of PBMCs. Resveratrol induced apoptosis in BCWM.1, as demonstrated by flow cytometry. Dose-dependent apoptosis at 24h with induction of JNK followed by caspases 3, 8, 9 and PARP cleavage was also observed. Resveratrol induced reduction of Mcl-1 and increase of p53, p63 and p73, as shown by gene expression analysis and western blot, providing an alternative mechanism of cell growth arrest in absence or mutation of p53. In parallel, resveratrol induced down-regulation of cyclin-D1, -D2, -E1, cdk-2, -4, -6 and up-regulation of p21Cip1 and p27Kip1, demonstrated in terms of transcript by gene expression analysis and protein levels by western blotting. We next observed that resveratrol inhibited ERK and Akt phosphorylation in BCWM.1 in a dose-dependent manner, as well as Akt activity, as shown by the in vitro Akt kinase assay. Phosphorylation of GSK3α/β and ribosomal protein-S6, downstream target proteins of Akt, were also markedly inhibited. Resveratrol also down-regulated Wnt signaling pathway with a reduction of nuclear β-catenin levels and a decrease of myc and survivin, both downstream target proteins of β-catenin. Lastly, adherence to BMSCs did not confer protection to WM cells against resveratrol-induced cytotoxicity Furthermore, resveratrol demonstrated synergistic cytotoxicity when combined with dexamethasone, fludarabine and bortezomib.Conclusion: These in vitro data demonstrated that resveratrol has significant antitumor activity in WM, providing the framework for clinical trials in WM patients.

New functional assay of P-glycoprotein activity using Hoechst 33342

In this study we describe a simplified, HTS-capable functional assay for the multidrug resistance (MDR) transporter P-glycoprotein (P-gp) based on its substrate Hoechst 33342. The physicochemical properties of Hoechst 33342 and the enormous milieu dependency of its fluorescence intensity allowed performing the assay in a homogeneous manner. This new assay served as an effective tool to estimate the potency of 10 well recognized P-gp substrates and modulators. Further, the potency of these compounds was also estimated in the calcein AM assay. The Hoechst 33342 and calcein AM assays yielded significantly comparable results for all compounds tested. Principal component analysis (PCA) applied to literature data on inhibition of P-gp activity and our results obtained in the Hoechst 33342 and calcein AM assay indicated similarity of compared functional transport assays. However, no correlation could be detected between these functional assays and the ATPase activity assay.

Efficacy of Laser-Activated Gold Nanoshells in Ablating Prostate Cancer Cells in Vitro

Nanoshells (NS) are nanoparticles consisting of a dielectric silica core covered by a thin gold shell. Nanoshells can be designed to absorb near-infrared (NIR) light strongly to generate heat and provide optically guided hyperthermic ablation. Laser-activated gold nanoshells (LAGN) may offer a minimally invasive targeted ablative treatment for prostate cancer. We studied the in-vitro effectiveness of LAGN ablation on human prostate cancer cells. Two human prostate cancer (PCa) cell lines, PC-3 and C4-2, were grown to 80% confluency in T medium with 5% fetal bovine serum. In order to determine a threshold concentration of gold nanoshells (GNS) needed to achieve full cellular ablation, dose titration was performed. In subsequent experiments, GNS were added to PCa cells in phosphate-buffered saline at concentrations above the predetermined threshold. The cells were then exposed to NIR light (810 nm, 88 W/cm2) for 5 minutes and stained immediately for viability using the Calcein AM assay. For determining long-term cell survival, the crystal violet assay was employed. The GNS could be evenly distributed across the culture plates. A ratio of 5000 GNS per PCa cell was critical for achieving cell kill. Cells treated with GNS + NIR demonstrated a laser-specific zone of cell death. The crystal violet viability assay confirmed consistent cell death rather than induction of cell dormancy. Cells treated with GNS alone or with NIR light alone demonstrated no toxicity. Laser-activated gold nanoshells can ablate human PCa cells in vitro. This nanoparticle technology is an attractive therapeutic agent for selective tumor ablation.

Rapid assessment of p-glycoprotein–drug interactions at the blood–brain barrier

We report on a cost- and time-effective fluorescent dye (calcein–acetoxymethylester; calcein–AM)-based assay to screen compounds for p-glycoprotein (p-gp) interactions at the blood–brain barrier. This assay enables the use of freshly isolated or freshly defrosted endothelial cells without prior cell culture. Isolated porcine brain capillary endothelial cells (PBCECs) in suspension were exposed to calcein–AM in absence and presence of substrates and inhibitors of p-gp. Accumulation of intracellular calcein fluorescence was measured using a 96-well plate reader or flow cytometry. P-gp substrates and inhibitors significantly increased intracellular calcein fluorescence in freshly isolated suspended PBCEC and in suspended PBCECs after storage in liquid nitrogen. In both models, intracellular calcein accumulation was similar. Relative concentration-dependent fluorescence profiles of cell suspensions were comparable to those of confluent cell monolayers. Western blot analysis showed highest p-gp expression in freshly isolated cells and cells cultured for 7 d. Importantly, using freshly isolated PBCECs in suspension in combination with fluorescence-activated cell sorting analysis reduced experimental time to 4 hrs vs 7 d with cultured PBCECs monolayers, while retaining and even improving feasibility, reliability, specificity, and sensitivity of the assay. In summary, an improved calcein–AM assay using suspended PBCECs for screening of drug–p-gp interactions is presented . This assay is more cost and time effective and more sensitive than other calcein–AM-based methods and can be performed under nonaseptic working conditions. Therefore, this assay is a useful tool in preclinical high-throughput screening.

IL-17A promotes the growth of airway epithelial cells through ERK-dependent signaling pathway

The effects of IL-17A on mucin production and growth of airway epithelial cells were examined. Histological and immunohistochemical analyses revealed that IL-17A increased the mucin production and number of tracheal epithelial cells in air–liquid interface cultures. The biological property of IL-17A to stimulate the mucin production by tracheal epithelial cells was determined using an ELISA. The mitogenic effect of IL-17A on tracheal epithelial cells was confirmed with Calcein-AM assay. The growth-stimulatory effect of IL-17A was dose-dependent and mediated via the ERK MAP kinase pathway. Inhibitors of MEK abrogated the mitogenic effect of IL-17A, whereas an inhibitor of p38 or JNK displayed no significant inhibitory effect. Moreover, relatively lower doses of IL-13 also significantly increased the growth of tracheal epithelial cells through a distinct signaling pathway from that of IL-17A. These findings provide the first evidence that IL-17A stimulates the growth of airway epithelial cells through the ERK MAP kinase pathway.

Computational Models for Identifying Potential P-Glycoprotein Substrates and Inhibitors

Multidrug resistance mediated by ATP binding cassette (ABC) transporters such as P-glycoprotein (P-gp) represents a serious problem for the development of effective anticancer drugs. In addition, P-gp has been shown to reduce oral absorption, modulate hepatic, renal, or intestinal elimination, and restrict blood-brain barrier penetration of several drugs. Consequently, there is a great interest in anticipating whether drug candidates are P-gp substrates or inhibitors. In this respect, two different computational models have been developed. A method for discriminating P-gp substrates and nonsubstrates has been set up based on calculated molecular descriptors and multivariate analysis using a training set of 53 diverse drugs. These compounds were previously classified as P-gp substrates or nonsubstrates on the basis of the efflux ratio from Caco-2 permeability measurements. The program Volsurf was used to compute the compounds' molecular descriptors. The descriptors were correlated to the experimental classes using partial least squares discriminant analysis (PLSD). The model was able to predict correctly the behavior of 72% of an external set of 272 proprietary compounds. Thirty of the 53 previously mentioned drugs were also evaluated for P-gp inhibition using a calcein-AM (CAM) assay. On the basis of these additional P-gp functional data, a PLSD analysis using GRIND-pharmacophore-based descriptors was performed to model P-gp substrates having poor or no inhibitory activity versus inhibitors having no evidence of significant transport. The model was able to discriminate between 69 substrates and 56 inhibitors taken from the literature with an average accuracy of 82%. The model allowed also the identification of some key molecular features that differentiate a substrate from an inhibitor, which should be taken into consideration in the design of new candidate drugs. These two models can be implemented in a virtual screening funnel.

Synthesis, characterization and in vitro cytotoxicity of Co(II) complexes with N6-substituted adenine derivatives: X-ray structures of 6-(4-chlorobenzylamino)purin-di-ium diperchlorate dihydrate and [Co 6(μ-L 6) 4Cl 8(DMSO) 10] · 4DMSO

Cobalt(II) complexes of the composition [Co(L 1)Cl(H 2O) 2] · H 2O ( 1), [Co(L 2)Cl(H 2O) 2] · 2H 2O ( 2), [Co(L 3)Cl(H 2O) 2] · H 2O ( 3), [Co(L 4)Cl(H 2O) 2] · H 2O ( 4), [Co(L 5)Cl(H 2O) 2] · H 2O ( 5) and [Co(L 6)Cl(H 2O) 2] · H 2O ( 6), where HL 1 = 6-(3-chlorobenzylamino)purine, HL 2 = 6-(4-chlorobenzylamino)purine, HL 3 = 6-(2,3-dimethoxybenzylamino)purine, HL 4 = 6-(3,4-dimethoxybenzylamino)purine, HL 5 = 6-(3-fluorobenzylamino)purine and HL 6 = 6-(4-fluorobenzylamino)purine, have been prepared. The compounds have been characterized by elemental analyses, ES+ mass, UV–Vis, IR and NMR spectroscopies, magnetic measurements, molar conductances and thermogravimetric analysis as mononuclear tetrahedral high-spin cobalt(II) complexes. In vitro cytotoxicities of the complexes were tested by a Calcein AM assay against the following human tumour cell lines: malignant melanoma ( G361), chronic myelogenous erythroleukemia ( K562), osteogenic sarcoma ( HOS) and breast adenocarcinoma ( MCF7). The molecular structures of 6-(4-chlorobenzylamino)purin-di-ium diperchlorate dihydrate, [H 2+L 2](ClO 4) 2 · 2H 2O, and a hexanuclear Co(II) complex, [Co 6(μ-L 6) 4Cl 8(DMSO) 10] · 4DMSO ( 7), have been determined by a single crystal X-ray analysis. To elucidate the manner of the organic ligand coordination to cobalt, the geometry of the complex 2 was optimized using DFT calculations at the B3LYP/6-311+G* level of the theory.

Novel fluorescence assay using calcein‐AM for the determination of human erythrocyte viability and aging

A highly sensitive, fast, and simple flow cytometric assay to assess human red blood cell (RBCs) viability and aging is reported. The assay described in this report is based on the use of acetoxymethyl ester of calcein (calcein-AM), a fluorescein derivative and nonfluorescent vital dye that passively crosses the cell membrane of viable cells and is converted by cytosolic esterases into green fluorescent calcein, which is retained by cells with intact membranes and inactive multidrug resistance protein. The loss of calcein can be easily determined by flow cytometry, and the cytosolic localization of esterases was demonstrated by spectrofluorometric analyses. We found that RBCs incubated with Ca(2+), which induces a rapid and modulated self-death that shares several features with apoptosis (Bratosin et al., Cell Death Differ 2001;8:1143-1156), externalized phosphatidylserine and lost calcein staining and cytosolic adenosine triphosphate content. Double labeling using phycoerythrin-labeled annexin-V and calcein-AM showed that the decrease of esterase activity is an early event that precedes the externalization of phosphatidylserine residues. In addition, this assay allowed us to distinguish young and aged RBCs isolated by ultracentrifugation in a self-forming Percoll gradient and can be considered as a reliable marker of RBC aging. Calcein-AM assay may represent a wide application for assessing RBC viability, particularly in blood banks.

A Pharmacophore Hypothesis for P-Glycoprotein Substrate Recognition Using GRIND-Based 3D-QSAR

Trying to understand the complex interactions that substrates and inhibitors have with the efflux transporter P-glycoprotein has been the subject of various publications. In this work, we have confined our study to substrates by picking a diverse set of 129 compounds based on the efflux ratios from Caco-2 permeability measurements. These compounds were then evaluated for P-glycoprotein inhibition using a calcein-AM assay. The subsequent data was used in a 3D-QSAR analysis using GRIND pharmacophore-based and physicochemical descriptors. Pharmacophore-based descriptors produced a much more robust model than the one obtained from physicochemical-based descriptors. This supports the process proposed by Seelig and co-workers previously published whereby the substrate enters the membrane as the first step and is then recognized by P-glycoprotein in a second step. The strong correlation, highlighted by PLS statistical analysis, between pharmacophoric descriptors and inhibition values suggests that substrate interaction, with perhaps the mouth of the protein or another binding site, plays a key role in the efflux process, yielding a model in which diffusion across the membrane is less important than substrate-protein interaction. One pharmacophore emerged from the analysis of the model. We pose that the recognition elements, at least determined by the molecules used in this study, are two hydrophobic groups 16.5 A apart and two hydrogen-bond-acceptor groups 11.5 A apart and that the dimensions of the molecule also plays a role in its recognition as a substrate.

Increased functional expression of P-glycoprotein in Caco-2 TC7 cells exposed long-term to cadmium

The objective of this study was to investigate whether P-glycoprotein (P-gp) functional expression in intestinal cells is modified after long-term exposure to the food contaminant cadmium (Cd). The Caco-2 cell line, clone TC7, was first validated as a cellular model for long-term exposure to cadmium. Cytotoxicity tests after acute exposure of 24 h showed a significant concentration-dependent decrease in cellular viability at cadmium levels higher than 10 μM and led us to select the cadmium ranges for long-term exposure: 1, 5, and 10 μM. Intestinal cells were exposed to these cadmium concentrations for four consecutive weeks without inducing DNA condensation or fragmentation. In the second part of this work, we studied the functional expression of the drug efflux pump multidrug resistance P-glycoprotein after long-term exposure to cadmium by immunoblotting with the monoclonal antibody F4 and measurement of calcein-AM ± the P-gp inhibitor verapamil. Western blot analysis with the F4 antibody detected a single band of 170 to 180 kDa which is the size previously reported for P-gp. Calcein-AM assay showed that four weeks exposure of intestinal cells to 1, 5, and 10 μM Cd increased P-gp functional expression in proportion to the Cd concentration.

Evaluation and comparison of MRP1 activity with three fluorescent dyes and three modulators in leukemic cell lines

MRP1 activity was evaluated and compared in 11 cell lines with different levels of MRP1 expression using functional assays of calcein acetoxymethyl ester (calcein-AM), carboxyfluorescein diacetate (CFDA) and Rhodamine 123 (Rh123) in combination with the modulators cyclosporin A (CsA), probenecid and MK571. A good correlation was found between MRP1 expression and the modulatory effect of MK571 on calcein-AM uptake ( P=0.01) and probenecid effect on CFDA uptake ( P=0.02). Additionally, the combined modulatory effect of MK571 and probenecid on CFDA uptake ( P<0.0001) and on calcein-AM uptake ( P=0.0001) were highly significant. No correlation was found between MRP1 expression and the effects of three modulators on Rh123 uptake or efflux. In conclusion, calcein-AM and CFDA uptake assays are the best choices to probe MRP1 activity and combination of two modulators may improve the efficiency of these assays.

Valproic acid, a mood stabilizer and anticonvulsant, protects rat cerebral cortical neurons from spontaneous cell death: a role of histone deacetylase inhibition

We studied the neuroprotective effects of valproic acid (VPA), a primary mood stabilizer and anticonvulsant, in cultured rat cerebral cortical neurons (CCNs). CCNs underwent spontaneous cell death when their age increased in culture. As shown by mitochondrial activity and calcein-AM assays, treatment of CCNs with VPA starting from day 9 in vitro markedly increased viability and prolonged the life span of the cultures. The neuroprotective action of VPA was time-dependent and occurred at therapeutic levels with a maximal effect at about 0.5 mM. LiCl (1 mM) also protected CCNs from aging-induced, spontaneous cell death but less effectively. VPA-induced neuroprotection in aging CCN cultures was associated with a robust increase in histone H3 acetylation levels and the protective effect was mimicked by treatment with a histone deacetylase inhibitor, trichostatin A, but not by VPA analogs which are inactive in blocking histone deacetylase. Our results suggest a role of histone deacetylase inhibition in mediating the neuroprotective action of VPA.

Interaction of Cytochrome P450 3A Inhibitors with P-Glycoprotein

Many clinically important drug interactions occur due to inhibition of human liver cytochrome P450 3A (CYP3A) metabolism. The drug efflux pump P-glycoprotein (Pgp) can be an additional locus contributing to these drug interactions because there is overlap in drugs that are substrates for both proteins. We screened a number of CYP3A inhibitors (macrolide antibiotics, azole antifungals, and ergotpeptides) for their ability to interact with Pgp, compared with prototypical Pgp inhibitors. We used cell lines expressing human, mouse, and rat mdr1 genes. Pgp antagonism was defined by interactions of the drugs with four cell lines (LLC-PK1, L-MDR1, L-mdr1a, and L-mdr1b) using a microfluorometric calcein-AM assay and characterized for their inhibitor constant (K(i)) toward calcein-AM. The compounds were further defined for their ability to inhibit MDR1 by their effect on vinblastine accumulation into L-MDR1 cells. Representative compounds from each class of drugs were further tested as Pgp substrates, defined by the ability of human Pgp or mouse mdr1a/Pgp to transport them across a polarized kidney epithelial cell in vitro. These same compounds were administered radiolabeled in vivo to mdr1a (+/+) and (-/-) mice and the distribution of radioactivity compared. The results are summarized as follows: 1) Some drug interactions with Pgp were substrate- and/or assay-dependent. 2) Ergot alkaloids were identified as a class of MDR1/Pgp chemosensitizers. 3) The Ergot alkaloids revealed species differences in the structure-activity relationships for inhibition of Pgp. Simultaneous inhibition of Pgp by many CYP3A inhibitors contributes to human variation in the extent of drug-drug interactions.

Mitochondrial dysfunction is an early indicator of doxorubicin-induced apoptosis

Generation of reactive oxygen species and mitochondrial dysfunction has been implicated in doxorubicin-induced cardiotoxicity. This study examined pro-apoptotic mitochondrial cell death signals in an H9C2 myocyte rat cell line and in isolated rat heart mitochondria exposed to doxorubicin. Mitochondrial and cellular viability were assessed using an MTT viability assay (formazan product formed by functional mitochondrial dehydrogenases) and calcein AM dye (fluoresces upon cleavage by cytosolic esterases). Mitochondrial dysfunction followed by cell death was observed using nM concentrations of doxorubicin. Significant doxorubicin-induced cell death was not apparent until after 6 h following doxorubicin exposure using the calcein AM assay. The involvement of apoptosis is evidenced by an increase in TUNEL (terminal (TdT)-mediated dUTP-biotin nick end labeling)-positive nuclei following doxorubicin treatment. Furthermore, doxorubicin administered to isolated mitochondria induced a rapid increase in superoxide production, which persisted for at least 1 h and was followed by increased cytochrome c efflux. In addition, caspase-3 activity was increased with doxorubicin administration in the H9C2 myocyte cell line. An oxidant-mediated threshold of mitochondrial death may be required for doxorubicin-induced apoptosis.

Antidisialoganglioside/granulocyte macrophage–colony-stimulating factor fusion protein facilitates neutrophil antibody-dependent cellular cytotoxicity and depends on FcγRII (CD32) and Mac-1 (CD11b/CD18) for enhanced effector cell adhesion and azurophil granule exocytosis

Polymorphonuclear leukocytes (PMNs) mediate antibody-dependent cellular cytotoxicity (ADCC), which is increased by the addition of granulocyte-macrophage colony-stimulating factor (GM-CSF). We sought to determine whether PMN ADCC also would be increased by the addition of an antibody/GM-CSF fusion protein and whether this would be associated with the up-regulation and activation of Mac-1 (CD11b/CD18) and with azurophil granule exocytosis. ADCC against LA-N-1 human neuroblastoma cells was evaluated with 4-hour calcein acetoxymethyl ester (calcein-AM) microcytotoxicity assay, electron microscopy, and multi-parameter flow cytometry. With the calcein-AM assay, LA-N-1 cell survival was 10%, 55%, and 75% when PMN ADCC was mediated by the antidisialoganglioside (anti-GD2) immunocytokine hu14.18/GM-CSF, by monoclonal antibody (mAb) hu14.18 mixed with GM-CSF, and by hu14.18 alone. Function-blocking mAbs demonstrated that FcgammaRII and FcgammaRIII were required for ADCC with hu14.18 alone or mixed with GM-CSF, but that only FcgammaRII was required for ADCC with hu14.18/GM-CSF. ADCC mediated by hu14.18 and hu14.18/GM-CSF was Mac-1 dependent. Electron microscopy demonstrated the greatest PMN adhesion, spreading, and lysis of targets with hu14.18/GM-CSF. Monoclonal antibodies blocking Mac-1 function allowed the tethering of PMN to targets with hu14.18/GM-CSF but prevented adhesion, spreading, and cytolysis. Flow cytometry showed that hu14.18 with or without GM-CSF and hu14.18/GM-CSF all mediated Mac-1-dependent PMN-target cell conjugate formation but that GM-CSF was required for the highest expression and activation of Mac-1, as evidenced by the mAb24-defined beta(2)-integrin activation epitope. Hu14.18/GM-CSF induced the highest sustained azurophil granule exocytosis, almost exclusively in PMNs with activated Mac-1. Thus, hu14.18/GM-CSF facilitates PMN ADCC against neuroblastoma cells associated with FcgammaRII and Mac-1-dependent enhanced adhesion and degranulation.