Cholesterol Interaction with the Daunorubicin Binding Site of P-Glycoprotein

Biochemical and Biophysical Research Communications 276, 909 –916 (2000) doi:10.1006/bbrc.2000.3554, available online at http://www.idealibrary.com on

Cholesterol Interaction with the Daunorubicin Binding Site of P-Glycoprotein Er-jia Wang, Christopher N. Casciano, Robert P. Clement, and William W. Johnson Drug Metabolism and Pharmacokinetics, Schering-Plough Research Institute, Lafayette, New Jersey 07848

Received August 23, 2000

The inherent complexities of cholesterol disposition and metabolism preclude a single transmembrane active transport avenue for this steroid-precursor, cellmembrane 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 ␮M 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 ␮M. 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. © 2000 Academic Press Key Words: cholesterol; P-glycoprotein; MDR; transport.

Biliary secretion is controlled by the active canalicular secretion of bile salts by transmembrane enzymes that couple ATP hydrolysis to the transport of a wide variety of substrates (1). These transporters are members of the superfamily of ABC (ATP-binding cassette) membrane transport proteins, which currently includes P-gp (P-glycoprotein) or MDR1 (multidrug resistance protein), MDR2, SPGP (sister P-glycoprotein), Abbreviations used: P-gp, P-glycoprotein; ABC, ATP-binding cassette; MDR, multidrug resistance; HMG-CoA, 3-hydroxy-3-methylglutaryl-CoA; DNR, daunorubicin; Rho, rhodamine 123.

and MRP1–MRP5 (multidrug resistance-associated proteins). Because P-gp exhibits a remarkably broad substrate recognition including many lipophilic compounds ranging in size from 200 –900 MW this protein causes vectorial efflux of many xenobiotics and drugs from normal as well as transformed cells (2–5). And since it is expressed on the apical surface of intestinal, hepatic, kidney, and brain epithelial cells, it has been postulated that P-gp functions to excrete xenobiotics into the lumen and reduce accumulation of toxic compounds in these tissues (2). The xenobiotic defense role is even more complex than this paradigm proposes. Some physiologic substrates for P-gp have been identified, such as phosphatidylethanolamine and phosphatidylcholine (6, 7) and sphingomyelin (8), also induced cholestasis (9) as well as HMG-CoA reductase inhibitor (statin) administration (10) will elevate production of the MDR1 gene product. Additionally, other members of the MDR subfamily (i.e., MDR2) translocate phosphatidylcholine out of hepatocytes and into the bile, a process necessary for normal bile formation yet this protein is not expressed in the intestine (11). The heterozygous knockout mdr2⫺/⫺ mice have not only a defect in biliary phospholipid secretion but also an accompanying defect in intestinal triglyceride and cholesterol transport. Indeed, all classes of lipoproteins were reduced in mdr2⫺/⫺ mice, in particular highdensity lipoprotein (HDL) levels (12). Another ABC transporter, “sister P-glycoprotein” (SPGP), is an effective bile acid transporter and is evidently the bile salt export pump (BSEP) (13). The MRP subfamily also contains several members that transport bile salts and bile conjugates and are regulated in response to cholestasis (14). The recently revealed link of Tangier disease (TD, familial hypoalphalipoproteinemia) with a defective mutant of ABC-A1 transporter suggested a direct role in cholesterol disposition of an ABC transporter (15– 17). Abc1 knockout (⫺/⫺) mice had about a 70% reduction in plasma cholesterol, significantly reduced plasma phospholipids, and low density lipoprotein (LDL) cholesterol (LDL-C), and an almost complete

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lack of high density lipoproteins (HDL) (18). Notably, these Abc1 knockout (⫺/⫺) mice exhibited increased absorption of cholesterol and accumulation of lipidladen macrophages and pneumocytes in the lungs—an indication of the transport of dietary cholesterol by the ABC-A1 transporter. Therefore, cholesterol disposition appears to be inextricably linked with several members of the ABC transporter superfamily. Several recent studies suggest a role for P-gp in the trafficking of sterols within cells. Compounds known to inhibit P-gp nonspecifically also inhibited esterification of plasma membrane cholesterol in rat hepatoma cells (19), CaCo-2 cells (20), and a human hepatoma cell line (21). Since these compounds do not significantly inhibit acyl-CoA:cholesterol acyltransferase (ACAT), these nonspecific inhibitors of P-gp are suggested to affect cholesterol movement from the plasma membrane to the endoplasmic reticulum (ER) (22). Recent experiments demonstrated that greater expression of P-gp was correlated with increased esterification of plasma membrane cholesterol, a result that indicates a direct link between P-gp and cholesterol trafficking within cells (23). That the level of MDR1 expression in intestinal cells influences the amount of cholesterol taken up by those cells provides additional direct evidence for a P-gp-mediated cholesterol disposition (24) (though this reaction could be explained by transport of a regulatory molecule). Moreover, a correlation among cholesterol ester levels, acyl-CoA:cholesterol acyltransferase, MDR1 (Pg-p), and advanced atherosclerotic lesions was demonstrated, while the dramatic decrease in mRNA levels of HMG CoA-reductase and lowdensity lipoprotein receptor (LDL-R) suggested homeostatic regulation of cholesterol (25). Our objective in this study was to investigate the suggested direct interaction of cholesterol with P-gp. Herein we show that increasing cholesterol concentrations dramatically inhibits active transport of the P-gp marker substrates daunorubicin (DNR) yet does not effect rhodamine 123 (Rho) transport. Moreover, we show a cholesterol concentration-dependent hydrolysis of ATP, the required energy source for P-gp function. MATERIALS AND METHODS Chemicals. Daunorubicin (DNR), rhodamine 123 (Rho), cholesterol, verapamil, colchicine, cyclosporin A, mannitol, dithiothreitol, ATP disodium, ammonium molybdate, ascorbic acid, sodium metaarsenite, aprotinin, leupeptin, EGTA, EDTA, HEPES, ouabain, phenylmethylsulfonyl fluoride, and TRIZMA base were purchased from Sigma Chemical Co. (St. Louis, MO). Hanks’ balanced salt solution, Alpha Minimum Essential Medium, DMEM, penicillin/streptomycin, fetal bovine serum (FBS), and trypsin-EDTA were obtained from Life Technologies, Inc. (Rockville, MD). Microplates (Costar 96-well), plastic tubes, and cell culture flasks (75 cm 2) were purchased from Corning Inc. (Corning, NY). All other reagents were of the highest grade commercially available. Cell lines. CR1R12 cell line, provided by Dr. Alan Senior (Univ. of Rochester), was maintained in complete ␣-minimum essential me-

dium (␣-MEM) supplemented with 10% FBS, penicillin/streptomycin (50 units/50 ␮g/ml) in a 5% CO 2–95% air atmosphere at 37°C. Colchicine (0.5 ␮g/ml) was added to the culture medium. Cells were grown to 80 –90% confluency and treated with trypsin–EDTA before subculturing. The 3T3 G185 cell line presenting the gene product of human MDR1 was licensed from NIH and maintained in DMEM. FACS flow cytometry. Fluorescence measurements of individual cells were performed using a Becton-Dickinson FACScalibur fluorescence-activated cell sorter (San Jose, CA), equipped with an ultraviolet argon laser (excitation at 488 nm, emission at 530/30 and 570/30 nm band-pass filters). Analysis was gated to include single cells on the basis of forward and side light-scatter and was based on acquisition of data from 10,000 cells. Log fluorescence was collected and displayed as single-parameter histograms. A direct functional assay for the P-gp efflux pump in CR1R12 or NIH-3T3-G185 cells was performed with the flow cytometer (26). Cell viability test. Cell viability was assessed using propidium iodide staining. Dead cells in which propidium iodide was bound to double strands of DNA or RNA were detected in certain regions of the cytometry dot plots and not included in the final data calculations. Calculation of relative fluorescence. The DNR or Rho fluorescence intensity of individual cells was recorded as histograms. The mean fluorescence intensity of 10,000 cells was used for comparison among different conditions. Verapamil or CSA were selected as positive controls to normalize the measurements because they are known P-gp efflux pump inhibitors. Relative fluorescence was used for quantitation and comparison among different compounds. The relative fluorescence (% inactivation) represents a ratio obtained through the following formula: the geometric mean fluorescence of a discrete sample divided by the geometric mean fluorescence in the presence 100 ␮M verapamil or 50 ␮M CSA, times 100, or expressed as

Relative fluorescence ⫽

Fluorescence of sample geometric mean ⫻ 100. Fluorescence of reference std. geometric mean

Membrane microsome preparations. CR1R12 cell membranes enriched with the MDR1 gene product transport enzyme were used for preparation of membrane microsomes. Cells were washed with complete Hanks’ buffer before being resuspended in 10 ml lysis buffer (Tris–HCl, 50 mM; mannitol, 50 mM; EGTA, 2 mM; and dithiothreitol, 2 mM; pH 7.0 at 25°C) containing protease inhibitors (phenylmethylsulfonyl fluoride, 1 mM; aprotinin, 10 ␮g/ml; leupeptin, 10 ␮g/ml). All subsequent steps were performed at 4°C. The cells were lysed by nitrogen cavitation (Parr Instrument Co., Moline, IL) at 500 psi for 15 min twice. Nuclei and mitochondria were sedimented by centrifugation at 4000g for 10 min. The microsomal membrane fraction was then sedimented by centrifugation at 100,000g for 60 min. The pellet was resuspended in 0.25 M sucrose buffer (10 mM Tris– HCl, 1 mM EDTA, pH 7.5) and homogenized using a Potter-Elvehjem homogenizer. Aliquots of membrane microsomes were rapidly frozen and stored at ⫺80°C until analysis. ATP hydrolysis and phosphate release. The consumption of ATP was determined by the liberated inorganic orthophosphate, which forms a color complex with molybdate (27). We have developed an ATP hydrolysis assay based on phosphate-release determination using membrane microsome preparations (28 –30). The method was modified to be carried out in a 96-well microplate. The microsomes were thawed on ice prior to diluting to 3.5 ␮g protein per well in ice-cold ATPase buffer (sodium ATP, 3 mM; KCl, 50 mM; MgSO4, 10 mM; dithiothreitol, 3 mM; Tris–HCl, 50 mM; pH 7.0) containing 0.5 mM EGTA (to inhibit Ca-ATPase), 0.5 mM ouabain (to inhibit the Na/K-ATPase), and 3 mM sodium azide (to inhibit the mitochondrial ATPase). The total incubation volume including the various inhibi-

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tors was 100 ␮l. The incubation reaction was initiated by transferring the plate from ice to 37°C and incubating for 30 min; the reaction was terminated by the addition of 50 ␮l 12% SDS solution at room temperature, followed by the addition of 50 ␮l of a mixture solution of (equal volumes) 18% fresh ascorbic acid in 1 N HCl and 3% ammonium molybdate in 1 N HCl. After 4 min, 100 ␮l of a solution of 2% sodium citrate and 2% sodium meta-arsenite in 2% acetic acid was added to fix the color formation. After 30 min incubation at room temperature, the fixed released phosphate was quantitated colorimetrically in a microplate reader (Bio-Tek FL600, VT) at 750 nm. The respective values for background with ATPase assay buffer alone were obtained in parallel and subtracted from the values for experimental samples. By comparison to a standard curve, the amount of phosphate released—and hence ATP consumed—was quantified. Water-insoluble drugs were dissolved in methanol; the maximum methanol concentration (2% v/v) was shown not to affect the ATPase activity.

RESULTS As fluorescent substrates transported by P-gp, DNR and rhodamine 123 are markers for active transport function by quantifying cell fluorescence (26). Herein we show that cholesterol can effectively inhibit the P-gp-mediated transport of DNR but not Rho. The IC 50 (concentration at half maximum inhibition) can be determined from a simple function, as shown in Fig. 1, where the retained fluorescence is measured for samples of viable cells at varying concentrations of cholesterol. The concentration dependency of inhibition displayed a sigmoidal response curve (Fig. 1a), a consequence of cooperativity, with the Hill equation for allosteric interaction enzymes therefore being the appropriate function for fitting to the data: v ⫽ V maxS n/(K⬘ ⫹ S n). The IC 50 of DNR transport in the NIH-3T3-G185 cell line (expressing the gene product of human MDR1) is ⬇8 ␮M, and cholesterol can achieve about 95% of verapamil-mediated transport inhibition. As shown in Fig. 1b, cholesterol has no effect on Rho transport out of G185, in dramatic contrast to the results with the other marker substrate, DNR (and most other known P-gp substrates). Because these cell lines overexpress the respective transporter enzymes, the IC 50 would be expected to be higher than under in vivo conditions, where far fewer copies of the enzyme would be contained per cell. Importantly, a control experiment performed with the parent (nontransfected) cell line, NIH3T3, showed that supplemental cholesterol had no significant effect on cellular retention of DNR over the concentration range used above (Fig. 1c). In similar experiments with CR1R12 cells from hamster ovary, the presence of added cholesterol had no impact on the transport of DNR or Rho (data not shown). Hence, the effects from supplemented cholesterol were not indirect results of altered protein/ membrane interface or changes in passive permeation but rather direct consequences of cholesterol/P-gp interaction at the DNR binding site. ATP hydrolysis. As ATP is consumed at a purported rate of about 1 per transport event, the hydro-

FIG. 1. (a– c) Intracellular retention of daunorubicin (a) or rhodamine 123 (b) in G185 cells or nontransfected parent cell line NIH-3T3 (c) versus competing cholesterol concentration. DNR fluorescence intensity is expressed as relative fluorescence. The efflux phase or incubation was 30 min in all cases. The average number of cells per assay was 10,000. The function for the line through the data is the Hill equation: v ⫽ V maxS n/(K⬘ ⫹ S n). The parameters IC 50 and the Hill coefficient along with the standard deviation are shown on the respective graph.

lysis of ATP represents transport function turnover rate or activity assay (31–35). The presence of cholesterol causes a concentration-dependent increase in the rate of ATP hydrolysis relative to baseline rate, which is consistent with it being a comparatively rapid substrate for P-gp (Fig. 2). The K m is ⬃58 ␮M, and the V max is ⬃1.6-fold above baseline, as indicated in Fig. 2. The ATP hydrolysis activity assay results are known to be in a contrived environment in that the enzyme is associated with microsomes and no longer in a viable cell membrane. However, of the typical ways to perform the ATP hydrolysis assay the microsomal preparation

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FIG. 2. P-gp-mediated ATP hydrolysis rates in the presence of cholesterol. The data is fit to a hyperbola and the V max ⫽ 35 ⫾ 1.4 nmol/min/mg membrane protein with a K m ⫽ 58 ⫾ 22 ␮M.

from MDR1 overexpressing cell lines is the closest to native environment. A few known substrates, such as H33342, cause ATP hydrolysis rates to be lower than baseline ATP consumption. This effect is ostensibly due to competition with an endogenous substrate with a rate of transport approximately that of the baseline ATP hydrolysis rate. If cholesterol is a substrate for P-gp, then it will compete with other substrates (i.e., H33342) for the active transport site and act as an inhibitor. To test this hypothesis, an experiment was performed at various fixed concentrations of cholesterol and H33342, and a K i determined for the interaction with H33342 (36). As shown by the Cornish-Bowden plot analysis of the data (Fig. 3), a K⬘i (or ␣K i, noncompetitive or mixedtype inhibition) of 90 ␮M was determined, which indicated that H33342 inhibited cholesterol transport activity. The Lineweaver-Burk plots in Fig. 4 also show a pattern of mixed-type inhibition (or nearly noncompetitive). A replot of slope versus [H33343] (Fig. 4, inset) indicates an estimated K i of about 38 ␮M. The intercept replot (Fig. 4b) indicates an ␣K i of about 90 ␮M, where ␣ is a factor by which the binding of cholesterol affects the K s of H33342 binding, and vice versa. The findings of these two graphical analyses described above are in reasonable agreement. The K i estimated here is for the interaction of H33342 with cholesterol transport activity and not the reverse. The most reasonable approximation of the concentration with which cholesterol interacts with the transport enzyme under these conditions is the K s for the cholesterol-induced ATP hydrolysis activity of about 5 ␮M. DISCUSSION The physiologic disposition of cholesterol is complex and probably involves various active transporters as

well as transport facilitated by lipoproteins and cellular constituent microdomains. Recent evidence from several fronts has indicated important cholesterol disposition roles for some ABC transporters, including the MDR1 gene product P-glycoprotein (see Introduction). Particularly relevant is the role of P-gp for esterification of cholesterol in the ER lumen (23), and more recently, evidence suggests that P-gp affects the cellular uptake of cholesterol (24). The active transport of cytosolic cholesterol to the ER lumen could result in a net cellular uptake of cholesterol. In order to evaluate the potential direct motivation of cholesterol by P-gp, it is important to evaluate the effect of cholesterol on P-gp function. This was done through both monitoring active transport of a P-gp substrate and assessing transporter activity by the consumption of the energy source, ATP. Herein we show that cholesterol has a dramatic quantifiable effect on the P-gp-mediated transport of the substrate DNR at comparatively low concentrations. Cholesterol inhibits P-gp transport of DNR in a saturable manner with an IC 50 of 8 ␮M in the G185 cell line over-expressing the human MDR1 transporter enzyme. There is ultimately a 6-fold increase in retention of the substrate DNR, essentially equal to the extent of DNR transport inhibition caused by verapamil. The absence of an impact on Rho transport caused by the presence of added cholesterol, however, indicates that the cholesterol supplementation in these experiments is not significantly affecting the permeability or membrane/protein interface. In addition, this result indicates that cholesterol binding is selective for a nonrhodamine site that does recognize DNR (37). Notably, the control experiment with the parent cell line of the G185 cells showed that supplemental cholesterol had no significant effect on DNR retention and further sup-

FIG. 3. Cornish-Bowden plot at various fixed concentrations of cholesterol. The intersection below the [H33342] axis is characteristic of mixed-type (or noncompetitive) inhibition. The corresponding [H33342] at the line intercept represents ⫺K⬘i, as shown on the graph, and is about 90 ␮M.

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FIG. 4. (a) The reciprocal 1/v versus 1/[cholesterol] plot in the presence of different fixed concentrations of H33342. The characteristics of the intersection are of a mixed-type (or noncompetitive) inhibitor. The K s can be estimated at 5 ␮M and the V max ⬇ 50. Inset: Slope replot of reciprocal plot data revealing a K i of about 38 ␮M. A K s was estimated from the double reciprocal plot at about 6 ␮M. (b) Intercept of 1/v axis replot of the reciprocal plot data. Characteristic of mixed-type or noncompetitive inhibition, this analysis indicates an ␣ ⬇ 2.4. The V max is estimated at about 42 (nmol/min/mg) by the intersection of the line with the vertical axis and ␣K i is estimated by the intersection with the [H33342] axis to be about 90 ␮M.

ports a lack of indirect effects on cell membrane passive permeability or DNR sequestration. ATP hydrolysis kinetics using microsomes show that cholesterol “transport” by P-gp has a K m of ⬃58 ␮M and a K s of ⬃5 ␮M. Cholesterol “transport” is also inhibited by H33342, a known substrate for P-gp. It may not be surprising that the P-gp active site recognizes cholesterol, for this promiscuous trans-

porter nonspecifically moves diverse lipophilic structures of ⬃200 –900 MW. Indeed, the hydroxylated heptahydronaphthalene moiety typifies the “type II unit” of 2 electron donor groups with a spatial separation of 4.6 ⫾ 0.6Å suggested by Seelig (38) as one of two general patterns for substrate recognition by P-gp (constructed from a structure activity relationship study of known substrates of P-gp). Moreover, the inclusion of

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steroids among P-gp substrates and inhibitors (39), and the expression of P-gp in the adrenal gland (40) and choroid plexus (41) indicates that the function is beyond xenobiotic defense. Although lipids and cholesterol have often been characterized for their various nonspecific or indirect effects on P-gp, the observations described here are not consistent with the known indirect effects on MDR. Lipid-layer physical properties modified by indirect lipid interactions of P-gp modulators affect the membrane binding of rhodamine 6G and liposome permeability (42), and cholesterol can affect drug binding to the membrane bilayer (43). For example, binding of daunomycin to PC liposomes was inversely correlated with their cholesterol content (44). The effect, however, was not as dramatic as the observations described herein since high membrane concentrations (9 –33%) only achieved a maximum 50% decrease in binding. Similarly, cholesterol derivatives (“rigidifiers”) caused a ⬃1.6-fold maximum increase in rhodamine 123 retention (45). In contrast, vincristine retention (or “influx”) was inversely correlated with cholesterol concentration in the membrane (46), and known fluidizers inhibited DNR transport in canalicular membrane vesicles (47). Membrane environment alterations caused by cholesterol are known to affect P-gp interaction (48) with substrates such as azidopine, whose binding increases by ultimately 3-fold at 20% cholesterol in liposomes before decreasing at higher concentrations (49). The lipid environment also influences characteristics of ATP hydrolysis activity (50). Since the membrane lipid environment is well characterized for modulating drug (substrate) interactions with P-gp (48), and the host lipid bilayer where the substrate-binding site(s) apparently accesses substrates is critical to binding (51), it has been argued that P-gp may perform differently or exhibit unique selectivities in different tissue cells (52). For example, the P-gp inhibitor PSC388 did not alter cholesterol esterification in MDR1-transfected 3T3 fibroblasts, but could inhibit cholesterol esterification in Dox 6 cells, a drug-resistant myeloma cell line that overexpresses MDR1 (23). This result is similar to the observations described here in that cholesterol has no effect on DNR transport in the MDR CR1R12 cell line from hamster ovary. Even within a cell, P-gp is localized to apical membranes, where sphingolipids and cholesterol are enriched compared to basolateral surfaces (53). Indeed, P-gp preferentially localizes to low-density cholesterolenriched membrane domains, but acute depletion of cholesterol impacts P-gp-mediated drug transport in a substrate- and cell-type-specific manner (54, 55). Notably, the hepatic canalicular membrane is comparatively rich in free cholesterol, phosphatidylserine and sphingomyelin, hence more viscous (56 –58). In summary, P-gp could exhibit differing profiles of substrate selectivity in various membrane lipid environments,

and cholesterol disposition is very complicated and probably has many mediators and perhaps redundancy in transport mechanisms. P-gp may perform a selective role in cholesterol disposition in certain tissue cell types, such as the small intestine or liver. Cells in the small intestine play a uniquely major role in cholesterol metabolism since this organ regulates absorption of dietary and biliary cholesterol (59). Approximately 50% of the cholesterol in blood is believed to be derived from the absorption of dietary and biliary cholesterol in the gut. In conclusion, cholesterol appears to have a direct effect on the substrate binding site of P-gp—a result consistent with cholesterol being transported by MDR1 P-gp. P-gp may not perform this function equally in all cell types and would likely be one of many mediators of cholesterol disposition and metabolism. ACKNOWLEDGMENTS The authors are grateful to Professor Adriane L. Stewart for editorial assistance and Eleanor Johnson for her comments.

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