Adenovirus type 7 induces interleukin-8 in a lung slice model and requires activation of Erk

Adenovirus (Ad), particularly Ad type 7 (Ad7), causes severe lung infection and pneumonia. Initially, Ad causes neutrophilic inflammation of the distal airways and alveoli. Interleukin-8 (IL-8) is the major lung neutrophil chemotaxin, and we have shown that Ad7 induces IL-8 release from the A549 alveolar epithelial cell line. We sought to determine whether ex vivo human and bovine lung tissue containing primary pneumocytes could be used as a more accurate and relevant model to study Ad acute inflammation. We found that cultured lung tissue preserved normal lung architecture for more than 10 days. IL-8 was generated upon exposure of the lung organ culture to Ad7. IL-8 production required activation of the Ras/Erk pathway, since a pharmacological inhibitor blocked the appearance of IL-8 in the medium. Both human and bovine lung explants supported replication of Ad7, and immunohistochemistry experiments demonstrated the presence of the Ad hexon antigen within alveolar epithelial cells. These findings show that our novel human lung organ culture accurately reproduces the in vivo infectious disease process. Thus, this organ culture model represents a valuable tool for studying the acute innate immune response to respiratory infections.     

A novel flavonoid from Fissistigma cupreonitens, 5‑hydroxy‑7,8‑dimethoxyflavanone,competitively inhibited the efflux function of human P-glycoprotein and reversed cancer multi-drug resistance

BackgroundP-glycoprotein (P-gp) over-expression plays a vital role in not only systemic drug bioavailability but also cancer multi-drug resistance (MDR). Develop functional inhibitors of P-gp can conquer both problems.Purpose and study designThe aim of the present study was to research the P-gp modulating effects and MDR reversing ability of a novel flavonoid from Fissistigma cupreonitens, the underlying inhibitory mechanisms were further elucidated as well.MethodsCalcein-AM, rhodamine 123, and doxorubicin were fluorescent substrates for the evaluation of P-gp inhibitory function and detailed drug binding modes. Docking simulation was performed to reveal the in silico molecular bonding. ATPase assay and MDR1 shift assay were adopted to reveal the ATP consumption and conformational change of P-gp. The MDR reversing effects were demonstrated through cytotoxicity, cell cycle, and apoptosis analyses.Results5‑hydroxy‑7,8‑dimethoxyflavanone inhibited the efflux of rhodamine 123 and doxorubicin in a competitive manner, and increased the intracellular fluorescence of calcein at a concentration as low as 2.5 μg/ml. 5‑hydroxy‑7,8‑dimethoxyflavanone slightly changed P-gp's conformation and only stimulated ATPase at very high concentration (100 μg/ml). The docking results showed that 5‑hydroxy‑7,8‑dimethoxyflavanone and verapamil exhibited similar binding affinity to P-gp. The MDR reversing effects were prominent in the vincristine group, the reversal folds were 23.01 and 13.03 when combined with 10 μg/ml 5‑hydroxy‑7,8‑dimethoxyflavanone in the P-gp over-expressing cell line (ABCB1/Flp-In™-293) and MDR cancer cell line (KB/VIN), respectively.ConclusionThe present study demonstrated that 5‑hydroxy‑7,8‑dimethoxyflavanone was a novel effective flavonoid in the P-gp efflux inhibition and in vitro cancer MDR reversion.     

Inhibition of multidrug resistance by adamantylgb3, a globotriaosylceramide analog

Multidrug resistance (MDR) via the ABC drug transporter (ABCB1), P-glycoprotein (P-gp/MDR1) overexpression, is a major obstacle in cancer chemotherapy. Many inhibitors reverse MDR but, like cyclosporin A (CsA), have significant toxicities. MDR1 is also a translocase that flips glucosylceramide inside the Golgi to enhance neutral glycosphingolipid (GSL) synthesis. We observed partial MDR1/globotriaosylceramide (Gb3) cell surface co-localization, and GSL removal depleted cell surface MDR1. MDR1 may therefore interact with GSLs. AdamantylGb3, a water-soluble Gb3 mimic, but not other GSL analogs, reversed MDR1-MDCK cell drug resistance. Cell surface MDR1 was up-regulated 1 h after treatment with CsA or adaGb3, but at 72 h, cell surface expression was lost. Intracellular MDR1 accumulated throughout, suggesting long term defects in plasma membrane MDR1 trafficking. AdaGb3 or CsA rapidly reduced rhodamine 123 cellular efflux. MDR1 also mediates gastrointestinal epithelial drug efflux, restricting oral bioavailability. Vinblastine apical-to-basal transport in polarized human intestinal C2BBe1 cells was significantly increased when adaGb3 was added to both sides, or to the apical side only, comparable with verapamil, a standard MDR1 inhibitor. Disulfide cross-linking of mutant MDR1s showed no binding of adaGb3 to the MDR1 verapamil/cyclosporin-binding site between surface proximal helices of transmembrane segments (TM) 6 and TM7, but rather to an adjacent site nearer the center of TM6 and the TM7 extracellular face, i.e. close to the bilayer leaflet interface. Verotoxin-mediated Gb3 endocytosis also up-regulated total MDR1 and inhibited drug efflux. Thus, a functional interplay between membrane Gb3 and MDR1 provides a more physiologically based approach to MDR1 regulation to increase the bioavailability of chemotherapeutic drugs.     

Suppression of multidrug resistance by treatment with TRAIL in human ovarian and breast cancer cells with high level of c-Myc

In this study, we investigated the role of c-Myc in overcoming multidrug resistance (MDR) in human ovarian and breast cancer cells by TRAIL. We showed that P-gp expressing MDR variants (Hey A8-MDR and MCF7-MDR cells) with high level of c-Myc were highly susceptible to TRAIL treatment when compared to their drug-sensitive parental human ovarian cancer Hey A8 and breast MCF-7 cells, respectively. Up-regulation of DR5 TRAIL receptor and down-regulation of c-FLIP and the promotion of caspase-dependent cell death, which contribute to TRAIL sensitization of MDR cells, were regulated by the over-expressed c-Myc in the MDR cells. After targeted inhibition of c-Myc with specific siRNA, these responses to TRAIL disappeared and TRAIL-induced apoptosis was also suppressed in MCF7-MDR cells. Treatment with TRAIL significantly reduced P-glycoprotein (P-gp)-mediated efflux of rhodamine123 in both Hey A8-MDR and MCF7-MDR cells. Furthermore, TRAIL significantly potentiated the cytotoxicity of vinblastine, vincristine, doxorubicin and VP-16 that are P-gp substrate anticancer drugs in both MDR cells, which resulted in the reversal effect of TRAIL on the MDR phenotype. The present study shows for the first time that elevated c-Myc expression in the MDR cells plays a critical role in overcoming MDR by TRAIL that can act as a specific sensitizer for P-gp substrate anticancer drug.     

Evaluation of the Role of P-Glycoprotein in Ivermectin Uptake by Primary Cultures of Bovine Brain Microvessel Endothelial Cells

The P-glycoprotein efflux system located on the apical membrane of brain capillary endothelial cells functions as part of the blood-brain barrier. In this study, primary cultures of bovine brain microvessel endothelial cells (BMECs) were investigated for the presence of a P-glycoprotein system and its contribution in regulating ivermectin distribution across the blood-brain barrier. Results of rhodamine 123 uptake studies with cyclosporin A and verapamil as substrates indicated that a functional efflux system was present on BMECs. Immunoblot analysis with the C219 monoclonal antibody to the product of the multidrug resistant member 1(MDR1) gene also confirmed the expression of MDR1 in the BMECs. Unbound ivermectin was shown to significantly increase the uptake of rhodamine 123 in BMECs, however, the drug only modestly enhanced the transcellular passage of rhodamine. The results of these studies affirmed that unbound ivermectin is an inhibitor of the MDR1 efflux system in BMECs.     

Assessment of P-glycoprotein-dependent drug transport in isolated rat hepatocytes using rhodamine 123

To test the activity of P-170 glycoprotein in isolated hepatocytes, a method has been developed employing the fluorescent dye rhodamine 123 (R-123). The uptake of R-123 by both freshly isolated and 4-hr-plated hepatocytes depends on dye concentration, time of incubation, and cell number. The efflux of R-123 from cells is inhibited by sodium azide and by verapamil. In standard conditions the efficiency of efflux of R-123 from cells correlates with the relative amount of immunoblottable glycoprotein. The method has been applied to detection of P-170 activity in hepatocytes from animals of different ages as well as from carcinogen-treated animals. The proposed assay appears a simple and adequate tool for the functional assessment of multidrug transporter in liver.Abbreviations AAF acetaminofluorene - MDR multidrug resistance - Pgp P-glycoprotein - R-123 rhodamine 123     

Multidrug-resistance phenotype of a subpopulation of T-lymphocytes without drug selection

Multidrug-resistant (MDR) cells demonstrate the increased activity of the membrane transport system performing efflux of diverse lipophylic drugs and fluorescent dyes from the cells. In order to detect MDR cells we have developed a simple test consisting of three steps: staining of the cells with fluorescent dye rhodamine 123, incubation in the dye-free medium and, finally, detection by fluorescence microscopy of the cells that have lost accumulated dye. The experiments with B-lymphoma cell lines with different degrees of MDR have shown that the cell fluorescence after the poststaining incubation is indeed inversely proportional to the degree of resistance. Application of this testing procedure to normal human or mouse leukocytes revealed the presence of the cells rapidly losing the dye in these populations. Cell fractionation experiments have shown that there are T-lymphocytes (most T-killers/suppressors and a part of T-helpers) that demonstrate rapid efflux of rhodamine 123. This characteristic was detected also in T-killer clones and cell line and in some T-lymphomas. The inhibitors of the MDR transport system, reserpine and verapamil, blocked the efflux of the dye from these cells. Rhodamine-losing T-lymphoma contained large amounts of the mRNA coding P-glycoprotein, the MDR efflux pump, and demonstrated increased resistance to rhodamine 123, gramicidin D, colchicine, and vincristine, the drugs belonging to the cross-resistance group for the MDR cells. The role of the increased activity of the MDR membrane transport system in T-lymphocytes is discussed.     

Type I cell-like morphology in tight alveolar epithelial monolayers

The pulmonary alveolar epithelium separates air spaces from a fluid-filled interstitium and might be expected to exhibit high resistance to fluid and solute movement. Previous studies of alveolar epithelial barrier properties have been limited due to the complex anatomy of adult mammalian lung. In this study, we characterized a model of isolated alveolar epithelium with respect to barrier transport properties and cell morphology. Alveolar epithelial cells were isolated from rat lungs and grown as monolayers on tissue culture-treated Nuclepore filters. On Days 2-6 in primary culture, monolayers were analyzed for transepithelial resistance (Rt) and processed for electron microscopy. Mean cell surface area and arithmetic mean thickness (AMT) were determined using morphometric techniques. By Day 5, alveolar epithelial cells in vitro exhibited morphologic characteristics of type I alveolar pneumocytes, with thin cytoplasmic extensions and protruding nuclei. Morphometric data demonstrated that alveolar pneumocytes in vitro develop increased surface area and decreased cytoplasmic AMT similar to young type I cells in vivo. Concurrent with the appearance of type I cell-like morphology, monolayers exhibited high Rt (greater than 1000 omega.cm2), consistent with the development of tight barrier properties. These monolayers of isolated alveolar epithelial cells may reflect the physiological and morphological properties of the alveolar epithelium in vivo.     

Repopulation of a human alveolar matrix by adult rat type II pneumocytes in vitro : A novel system for type II pneumocyte culture

This paper describes the preparation of lung acellular alveolar matrix fragments and culture of rat type II pneumocytes directly on the alveolar epithelial basement membrane, thereby permitting study of the effect of lung basement membrane on the morphology and function of type II cells. Collagen types I, III, IV and V, laminin and fibronectin were located by immunofluorescence in the lung matrix with the same patterns as those described for the normal human lung. Transmission electron microscopy (TEM) of the fragments revealed intact epithelial and endothelial basement membranes. The matrix maintained the normal three-dimensional alveolar architecture. Glycosaminoglycans were still present by Alcian Blue staining. Isolated adult rat type II pneumocytes cultured on 150 micron thick fragments of acellular human alveolar extracellular matrix undergo gradual cytoplasmic flattening, with loss of lamellar bodies, mitochondria, and surface microvilli. These changes are similar to the in vivo differentiation of type II pneumocytes into type I pneumocytes. The type II pneumocyte behaviour on the lung epithelial basement membrane contrasted sharply with that of the same cell type cultured on a human amnionic basement membrane. On the latter surface the cells retained their cuboidal shape, lamellar bodies and surface microvilli for up to 8 days. These observations suggest that the basement membranes from different organ systems exert differing influences on the morphology and function of type II pneumocytes and that the alveolar and amnionic basement membranes may have differing three-dimensional organizations. The technique of direct culture of type II cells on the lung basement membrane provides a useful tool for studying the modulating effect of the basement membrane on alveolar epithelial cells.     

Effects of plant sterols on human multidrug transporters ABCB1 and ABCC1

The effects of dietary plant sterols on human drug efflux transporters P-glycoprotein (P-gp, ABCB1) and multidrug resistance protein 1 (MRP1, ABCC1) were investigated using P-gp-overexpressing human carcinoma KB-C2 cells and human MRP1 gene-transfected KB/MRP cells. The effects of natural phytosterols found in foods, herbs, and dietary supplements such as β-sitosterol, campesterol, stigmasterol, fucosterol, and z-guggulsterone were investigated. The accumulation of daunorubicin or rhodamine 123, fluorescent substrates of P-gp, increased in the presence of guggulsterone in KB-C2 cells. The efflux of rhodamine 123 from KB-C2 cells was inhibited by guggulsterone. Guggulsterone also increased the accumulation of calcein, a fluorescent substrate of MRP1, in KB/MRP cells. The ATPase activities of P-gp and MRP1 were stimulated by guggulsterone. These results suggest that guggulsterone, a natural dietary hypolipidemic agent have dual inhibitory effects on P-gp and MRP1 and the potencies to cause food-drug interactions.     

Stereoselective regulation of MDR1 expression in Caco-2 cells by cetirizine enantiomers

MDR1-encoded P-glycoprotein (P-gp) is a drug efflux transporter mainly expressed in liver, kidney, intestine, brain (at the level of the blood-brain barrier), and placenta. It thus plays important roles in drug absorption, distribution, and excretion. Cetirizine is a second-generation nonsedating antihistamine used to treat allergic disease of respiratory system, skin and eyes. To evaluate P-gp expression and function in Caco-2 cells pretreated with cetirizine enantiomers, we assessed the sensitivity of Caco-2 cells to paclitaxel using the MTT assay and the polarized transport of rhodamine-123 and doxorubicin across Caco-2 monolayers. RT-PCR and flow cytometry were used to assay MDR1 mRNA and P-gp protein respectively. The sensitivity of Caco-2 cells to paclitaxel decreased significantly after cells were pretreated with 100 microM R-cetirizine but increased upon treatment with S-cetirizine. The efflux of rhodamine-123 and doxorubicin was enhanced significantly after Caco-2 monolayers were pretreated with 100 microM R-cetirizine but was reduced by S-cetirizine. The MDR1 mRNA and P-gp levels in Caco-2 cells were increased by 100 microM R-cetirizine and decreased by 100 microM S-cetirizine. These results suggest that R-cetirizine up-regulates MDR1 expression while S-cetirizine down-regulates MDR1 expression.     

Influence of combinations of digitonin with selected phenolics,terpenoids, and alkaloids on the expression and activity of P-glycoprotein in leukaemia and colon cancer cells

P-glycoprotein (P-gp or MDR1) is an ATP-binding cassette (ABC) transporter. It is involved in the efflux of several anticancer drugs, which leads to chemotherapy failure and multidrug resistance (MDR) in cancer cells. Representative secondary metabolites (SM) including phenolics (EGCG and thymol), terpenoids (menthol, aromadendrene, β-sitosterol-O-glucoside, and β-carotene), and alkaloids (glaucine, harmine, and sanguinarine) were evaluated as potential P-gp inhibitors (transporter activity and expression level) in P-gp expressing Caco-2 and CEM/ADR5000 cancer cell lines. Selected SM increased the accumulation of the rhodamine 123 (Rho123) and calcein-AM (CAM) in a dose dependent manner in Caco-2 cells, indicating that they act as competitive inhibitors of P-gp. Non-toxic concentrations of β-carotene (40 μM) and sanguinarine (1 μM) significantly inhibited Rho123 and CAM efflux in CEM/ADR5000 cells by 222.42% and 259.25% and by 244.02% and 290.16%, respectively relative to verapamil (100%). Combination of the saponin digitonin (5 μM), which also inhibits P-gp, with SM significantly enhanced the inhibition of P-gp activity. The results were correlated with the data obtained from a quantitative analysis of MDR1 expression. Both compounds significantly decreased mRNA levels of the MDR1 gene to 48% (p < 0.01) and 46% (p < 0.01) in Caco-2, and to 61% (p < 0.05) and 1% (p < 0.001) in CEM/ADR5000 cells, respectively as compared to the untreated control (100%). Combinations of digitonin with SM resulted in a significant down-regulation of MDR1. Our findings provide evidence that the selected SM interfere directly and/or indirectly with P-gp function. Combinations of different P-gp substrates, such as digitonin alone and together with the set of SM, can mediate MDR reversal in cancer cells.     

Tetrandrine and fangchinoline,bisbenzylisoquinoline alkaloids from Stephania tetrandra can reverse multidrug resistance by inhibiting P-glycoprotein activity in multidrug resistant human cancer cells

The overexpression of ABC transporters is a common reason for multidrug resistance (MDR) in cancer cells. In this study, we found that the isoquinoline alkaloids tetrandrine and fangchinoline from Stephania tetrandra showed a significant synergistic cytotoxic effect in MDR Caco-2 and CEM/ADR5000 cancer cells in combination with doxorubicin, a common cancer chemotherapeutic agent. Furthermore, tetrandrine and fangchinoline increased the intracellular accumulation of the fluorescent P-glycoprotein (P-gp) substrate rhodamine 123 (Rho123) and inhibited its efflux in Caco-2 and CEM/ADR5000 cells. In addition, tetrandrine and fangchinoline significantly reduced P-gp expression in a concentration-dependent manner. These results suggest that tetrandrine and fangchinoline can reverse MDR by increasing the intracellular concentration of anticancer drugs, and thus they could serve as a lead for developing new drugs to overcome P-gp mediated drug resistance in clinic cancer therapy.     

Bivalent probes of the human multidrug transporter P-glycoprotein

A small library of bivalent agents was designed to probe the substrate binding sites of the human multidrug transporter P-glycoprotein (P-gp). The bivalent agents were composed of two copies of the P-gp substrate emetine, linked by tethers of varied composition. An optimum distance between the emetine molecules of approximately 10 A was found to be necessary for blocking transport of the known fluorescent substrate rhodamine 123. Additionally, it was determined that hydrophobic tethers were optimal for bridging the bivalent compounds; hydrophilic or cationic moieties within the tether had a detrimental effect on inhibition of transport. In addition to acting as probes of P-gp's drug binding sites, these agents were also potent inhibitors of P-gp. One agent, EmeC5, had IC50 values of 2.9 microM for inhibiting transport of rhodamine 123 and approximately 5 nM for inhibiting the binding of a known P-gp substrate, [125I]iodoarylazidoprazosin. Although EmeC5 is an inhibitor of P-gp and was shown to interact directly with P-gp in one or more of the substrate binding sites, our data suggest that it is either not a P-gp transport substrate itself or a poor one. Most significantly, EmeC5 was shown to reverse the MDR phenotype of MCF-7/DX1 cells when co-administered with a cytotoxic agent, such as doxorubicin.     

Identification of a multidrug resistance-like system in Tetrahymena pyriformis: evidence for a new detoxication mechanism in freshwater ciliates.

The freshwater ciliate Tetrahymena pyriformis is an ubiquitous organism that is present in all aquatic ecosystems. This protozoan showed a clear resistance against some polycyclic aromatic hydrocarbons which can be attributed to an efflux pump probably of the multidrug resistance (MDR) type. Immunocytochemical detection showed a positive stain of ciliate cells using the monoclonal antibodies 4E3, raised against P-glycoprotein (P-gp). The kinetics of P-gp expression were studied for control cultures and cultures treated with 15 microM benzo(a)pyrene. Western blot analysis using the Ab1, anti-P-gp polyclonal antibodies indicates the presence of two bands of 66 and 96 kDa of which the intensity increased with time in benzo(a)pyrene-treated ciliates. Uptake experiments with target compounds for the MDR pump, namely adriamycin, rhodamine 123 and two polycyclic aromatic hydrocarbons, benzo(a)pyrene and 7,12-dimethylbenzanthracene, were carried out by flow cytometry, in the presence or absence of cyclosporin (an inhibitor of the multidrug resistant pump). The data indicate that the accumulation of these compounds by ciliate cells is significantly enhanced in the presence of cyclosporin. This suggests that Tetrahymena is provided with a P-gp-like system that is functionally active in a way similar to that of the mammalian P-gp.     

Catalase immunocytochemistry allows automatic detection of lung type II alveolar cells

In mammalian lung, type II pneumocytes are especially critical in normal alveolar functioning, as they are the major source of surfactant and the progenitors of type I alveolar cells. Moreover, they undergo proliferation and transformation into type I cells in most types of cellular injury, where flattened type I pneumocytes are selectively destroyed. Hyperplasia of alveolar type II cells has also been described in some human chronic lung diseases. In lung, type II pneumocytes and non-ciliated bronchiolar cells are the unique cell types that contain a considerable amount of peroxisomes. Due to the presence of dihydroxyacetone phosphate acyltransferase and non-specific lipid-transfer protein, these organelles have been suggested to be involved in the synthesis and/or transport of the lipid moiety of surfactant. In the present research, the peroxisomal marker enzyme catalase was immunolocalised at the light microscopic level, utilising the avidin-biotin complex method, in lung specimens excised from newborn, adult and aged rats. In all the examined stages the immunoreactivity was so selective for type II pneumocytes it allowed quantitation of these cells by an automated detection system. This was accomplished on specimens from newborn rat lung, in which labelled alveolar cells were counted by a grey level-based procedure and their main morphometric parameters were determined.     

Upregulation of P-glycoprotein by probiotics in intestinal epithelial cells and in the dextran sulfate sodium model of colitis in mice

P-glycoprotein (P-gp) mediates efflux of xenobiotics and bacterial toxins from the intestinal mucosa into the lumen. Dysregulation of P-gp has been implicated in inflammatory bowel disease. Certain probiotics have been shown to be effective in treating inflammatory bowel disease. However, direct effects of probiotics on P-gp are not known. Current studies examined the effects of Lactobacilli on P-gp function and expression in intestinal epithelial cells. Caco-2 monolayers and a mouse model of dextran sulfate sodium-induced colitis were utilized. P-gp activity was measured as verapamil-sensitive [(3)H]digoxin transepithelial flux. Multidrug resistant 1 (MDR1)/P-gp expression was measured by real-time quantitative PCR and immunoblotting. Culture supernatant (CS; 1:10 or 1:50, 24 h) of Lactobacillus acidophilus or Lactobacillus rhamnosus treatment of differentiated Caco-2 monolayers (21 days postplating) increased (～3-fold) MDR1/P-gp mRNA and protein levels. L. acidophilus or L. rhamnosus CS stimulated P-gp activity (～2-fold, P < 0.05) via phosphoinositide 3-kinase and ERK1/2 MAPK pathways. In mice, L. acidophilus or L. rhamnosus treatment (3 × 10(9) colony-forming units) increased mdr1a/P-gp mRNA and protein expression in the ileum and colon (2- to 3-fold). In the dextran sulfate sodium (DSS)-induced colitis model (3% DSS in drinking water for 7 days), the degree of colitis as judged by histological damage and myeloperoxidase activity was reduced by L. acidophilus. L. acidophilus treatment to DSS-treated mice blocked the reduced expression of mdr1a/P-gp mRNA and protein in the distal colon. These findings suggest that Lactobacilli or their soluble factors stimulate P-gp expression and function under normal and inflammatory conditions. These data provide insights into a novel mechanism involving P-gp upregulation in beneficial effects of probiotics in intestinal inflammatory disorders.     

Cholesterol interaction with the daunorubicin binding site of P-glycoprotein

The inherent complexities of cholesterol disposition and metabolism preclude a single transmembrane active transport avenue for this steroid-precursor, cell-membrane constituent. Yet the ABC (ATP binding cassette) transporters are inextricably linked to elements of cholesterol disposition. Recent observations have suggested that, under certain settings, the ABC transporter P-glycoprotein (P-gp) performs a direct role in cholesterol disposition. The gene product of MDR1 (multidrug resistance transporter), P-glycoprotein also confers protection against xenobiotics. Using a whole cell assay in which the retention of a marker substrate is evaluated and quantified, we studied the ability of cholesterol to inhibit directly the function of this transporter. In a NIH-G185 cell line presenting an overexpressed amount of the human transporter P-gp, cholesterol caused dramatic inhibition of daunorubicin transport with an IC(50) of about 8 microM yet had no effect on the parent cell line nor rhodamine 123 transport. Additionally, using the ATP-hydrolysis assay, we showed that cholesterol increases P-gp-mediated ATP hydrolysis by approximately 1.6-fold with a K(s) of 5 microM. Suggesting that cholesterol directly interacts with the substrate binding site of P-gp, these results are consistent with cholesterol being transported by MDR1 P-gp.