Modulation of P-glycoprotein-mediated Drug Transport by Alterations ...

THEJOURNAL OF BIOLOGICAL CHEMISTRY 0 1992 by The American Society forBiochemistry and Molecular Biology, Inc.

Vol. 267. No. 35, Issue of December 15, pp. 24995-25002.1992 Printed in U.S.A.

Modulation of P-glycoprotein-mediated Drug Transport by Alterations in Lipid Fluidity of Rat LiverCanalicular MembraneVesicles* (Received for publication, April 6, 1992)

Frank A. Sinicrope, PradeepK. DudejaS, Bruce M. Bissonnette, AhmadR. Safa, and Thomas A. Brasitus4 From the Department of Medicine, The University of Chicago, Chicago, Illinois 60637

P-glycoprotein (P-gp) is believed to function as an Multidrug resistance (MDR)’ is characterized by the develATP-dependent efflux pump for natural product anti- opment of tumor cell resistance to multiple natural product cancer drugs in multidrug-resistant (MDR) tumor cells anticancer drugsincluding Vinca alkaloids and anthracyclines and in certain normal tissues. P-gp has been localized (1,2). The mechanism of MDR in mammalian cells involves to theapical plasma membrane of the bile canaliculus the overexpression of a 150-170-kDa plasma membrane inwhere it has been shown to transport [’H]daunomycin. tegral protein, known as P-glycoprotein (P-gp) (2-4). FullI n this study, we investigated whether alterations in length cDNAs encoding P-gp have been cloned and characmembrane lipid fluidityof canalicular membraneves- terized andreveal that this protein has two nucleotide-binding icles (CMV) could modulate the P-gp-mediated accusites which are homologous to ATP-binding sitesof bacterial mulation of [‘H]daunomycin and [’Hlvinblastine. Accumulation of both cytotoxic agents was stimulated bytransport proteins (5-7). P-gp is believed to reduce intracelATP, exhibited temperature dependence and osmotic lular drug accumulationin resistant cells as compared to their drug-sensitive parental cell lines by functioning as an ATPsensitivity, and followed Michaelis-Menten kinetics. Alterations in CMV lipid fluidity wereinduced by the dependent, unidirectional drug efflux pump with broad subknown fluidizers, 2-(2-methoxyethoxy)ethyl8-(cis-2- strate specificity (4,8). Genetic analysissuggests that P-gpis n-octylcyclopropy1)octanoate(A&) and benzylalcohol, a member of a family of membrane-associated transport proteins which are involved inmultidrugresistance, malarial and were assessed by fluorescence polarization techniquesusingthefluorescentprobe, 1,6-diphenyl- chloroquine resistance by Plasmodium fakiparum, cystic fibrosis, and bacterial transportprocesses (9). ) 1,3,5-hexatriene (DPH). Both A& (2.5-5.0 p ~ and Interestingly, P-gp has not only been found in multidrugbenzyl alcohol (10-20 mM) produced a dose-dependent increase inCMV lipid fluidity. Moreover, both fluidiz- resistant tumor cells, but also in a number of normal human ers, at the above doses, significantly inhibited ( p c and other mammalian tissues(10-12). In polarized epithelial 0.05) the ATP-dependent accumulationof [’Hldaunocells, P-gp has beenlocalized to theapical plasma membrane mycin. [‘HIVinblastine accumulation was also inhibof the bile canaliculus, small intestine and colon, and proximal ited by A& ( p c 0.05). Lower doses of A& (0.6 p ~ )renal tubules. P-gp is also found in the adrenal cortex and and benzyl alcohol (1 mM) failed to influence either (11).To capillary endothelial cells of the brain and testes lipid fluidityor P-gp-mediated drug accumulation. Ki- date, no naturally occurring substrates have been identified ) netic analysis revealed thatA& (5.0 p ~ noncompetifor P-gp in these normal tissues. Recently, however, Kamitively inhibited [‘Hldaunomycin accumulation and un- mot0 et al. (13) have shown that P-gp localized to the bile competitively inhibited [‘Hlvinblastine accumulation canaliculus of normal rat liver appears to be responsible for 2 respecwith apparent Ki values of -1.5 and ~ 1 . pM, the ATP-dependent transportof the cytotoxic agent, daunotively.VerapamilcompetitivelyinhibitedP-gp-memycin. Furthermore, their study represents the first characdiated accumulation of [‘Hldaunomycin but failed to terization of the functionof P-gp ina normal tissue. alter thefluidity of CMV. Taken together, the present results demonstrate that while increases in membrane Drugs involved in the MDR phenotype are predominantly fluidity of CMV are not necessarily required to inhibithydrophobic, positively charged molecules (at physiological pH) which contain two planar aromatic rings (14) and bind P-gp-mediated drug accumulation,theycaninhibit specifically to P-gp (15-18). Several reports have shown that these processes, at least in CMV. Alterations in the physical state of CMV, therefore, appear to be at least a number of lipophilic compounds are able to reverse MDR in vitro (4, 5) and that these agents may modulate MDR by one important modulatorof P-gpfunction. interacting with anticancer drug-binding site(s) of P-gp (19* This work was supported by the Samuel Freedman Laboratories 24). Specifically, Tamai and Safa (23) and others (24) have for Cancer Research at the University of Chicago and United States shown that Vinca alkaloids, cyclosporin A, and verapamil Public Health Service Grants CA-36745 (to T. A. B.), CA-47652 and competitively interact with a common drug-binding site on CA-56078 (to A. R. S.), and DK42086 (from the Digestive Diseases P-gp and thereby inhibit drug transport. Research Center). The casts of publication of this article were dePrior studies from our laboratory (25-28) and others (29frayed in part by the payment ofpage charges. This article must physical state of plasma therefore be hereby marked “aduertisement” in accordance with 18 36) have shownthat alterations in the membrane lipids can influence a number of important carrierU.S.C. Section 1734 solely to indicate this fact, $ Current address: Dept. of Medicine, University of Illinois, Chimediated transport processes, including transport of various cago, IL 60612. J Recipient of a Merit Award from the National Cancer Institute, National Institutes of Health. To whom correspondence and reprint requests should be addressed University of Chicago Hospitals and Clinics, Box 400,5841 South Maryland Ave., Chicago, IL 60637. Tel.: 312-702-9898.Fax: 312-702-2182.

The abbreviations used are: MDR, multidrug resistance; P-gp, Pglycoprotein; CMV, canalicular membrane vesicles; A&, 2-(2-methDPH, 1,6-di0xyethoxy)ethyl 8-(cis-2-n-octylcyclopropyl)octanoate; phenyl-1,3,5-hexatriene; AMP-PNP, adenyl-5”yl imidodiphosphate; ATPyS, adenosine 5’-0-(3-thiotriphosphate).

24995

24996

Fluidity AltersP-glycoprotein Function

nutrients and electrolytes, as well as membrane-bound enzy- ing sodium dodecyl sulfate-polyacrylamide (7.5%) gel electrophoresis matic activities. In rat hepatocyte plasma membranes, the (38), the proteins were electrophoretically transferred to an ImobilonP sheet (0.45-pm pore size). The blot was then blocked for 2 h in local anestheticand lipid fluidizing agent, benzyl alcohol, Tris-buffered saline containing 5% (v/v) powdered milk. Incubation increased the activity of glucagon and fluoride-stimulated of the blot was subsequently performed with an anti-P-gp monoclonal adenylylcyclase and 5"nucleotidase (30). In addition, in antibody (C219, 1 pg/ml) (3) for 2 h at 25 "C followed by incubation CMV, the carrier-mediated transport of the bile acid tauro- with goat anti-mouse antibody conjugated to horseradish peroxidase cholate has been shown to be modulated by alterations in (0.3 pl/ml) for 1 h at 25 "C. Antigen was detected by xerography lipid fluidity (36).' To date, however, studies examining the following exposure of the blot to Kodak XAR-5 film. Vesicle Drug Accumulation Studies-The accumulation potential effect of alterations in membrane lipid fluidity on of Membrane [3H]daunomycin and [3H]vinblastine byCMV was measured at P-gp-mediated transport have not been performed. Therefore, 30 "C in the presence and absence of the known fluidizers, AzC (0.6the present experiments were conducted to determine whether 5.0 p ~final , concentration) or benzyl alcohol (1,10, and 20 mM, final alterations in membrane lipid fluidity could modulate the P- concentration), using a rapid Millipore filtration technique as degp-mediated accumulation of [3H]daunomycin and [3H]vin- scribed previously (43,44). Thereaction medium contained either 12 blastine in normal rat liver CMV and, if so, to characterize p~ [3H]daunomycin or 12 p~ [3H]vinblastine, 1.2 mM ATP or 1.2 mM AMP, and an ATP regenerating system (3 mM creatine phosthe modeof interaction of fluidizing agents with the accu- phate, 3.6 pg of creatine phosphokinase) in buffer B (10 mM Hepesmulation of these drugs. Tris buffer, pH 7.4, containing 0.25 M sucrose, 0.2 mM CaCl,, and 10 The results of these experiments demonstrate that P-gp is mM MgClz).CMV were added to test tubes in aliquots of 20 pl (25responsible for the ATP-dependent accumulation of [3H] 50 pg of protein) and preincubated for 5 min at 30 "C. The reaction daunomycin and [3H]vinblastine in rat liver CMV. Moreover, medium was also preincubated for 5 min at 30 "C, and then a 0.1-ml these studies demonstrate for the first time that increases in aliquot was added to theCMV to initiate transport. The reaction was at theappropriate time interval(s) by adding 5 ml of icethe fluidity of CMV induced by the known fluidizing agents, terminated cold buffer A. The diluted sample was immediately filtered through a A2C (2-(2-methoxyethoxy)ethyl 8-(cis-2-n-octylpropyl)oc- glass microfiber filter (Whatman, 0.65 pM pore size) using a Millipore tanoate) and benzyl alcohol, inhibit P-gp-mediated accumu- manifold filtration assembly. The filters were further washed twice lation of both Vinca alkaloids and anthracyclines. These with 5 ml of ice-cold buffer A and placed in liquid scintillation fluid, findings, as well as adiscussion of their potential significance and the radioactivity was counted in a liquid scintillation counter in themultidrug resistance phenomenon, are thebasis for the (Packard Tri-Carb1500). All values were corrected for radioactivity present in 0-minvesicle blanks. present report. [3H]Daunomycin and [3H]vinblastine accumulation by CMV was measured in the presence of ATP, AMP, and in separate experiments with the nonhydrolyzable ATP analogs ATPyS and AMP-PNP. ATP-dependent accumulation of these radiolabeled drugs was deterMaterials-[[3H]Daun~mycin (11.7 Ci/mmol) and[T-~'P]ATP mined by subtracting values obtained in the presence of AMP. All (6000 Ci/mmol) were obtained from Du Pont-New England Nuclear. experiments represent three or more determinations performed in [3H]Vinblastine (16 Ci/mmol) was purchased from Amersham. Dau- triplicate. nomycin hydrochloride, vinblastine sulfate, verapamil hydrochloride, Additional experiments were performed to assess the accumulation ATP, AMP-PNP, ATPyS, AMP, creatine phosphate, creatinephos- of the two radiolabeled compounds by these CMV preparations in the phokinase, and AzC were obtained from Sigma. Benzyl alcohol was presence and absence of a known inhibitor of P-gp function, verapapurchased from Fisher Scientific Co. 1,6-Diphenyl-1,3-5-hexatriene mil (50 p ~ final , concentration), as described above. Experiments (DPH) was obtained from Molecular Probes Inc. Anti-P-gp mono- were also performed using [y-3'P]ATP to exclude the possibility that clonal antibody C219 was purchased from Centocor (Malvern, PA). the membrane fluidizers were increasing vesicle permeability to ATP ECL Western Blotting Detection System was obtained from Amer- (see below). Uptake of [T-~'P]ATPwas measured in thepresence and sham and Imobilon-P was purchased from Millipore Corp. The MDR absence ofAzC (5.0 p ~ and ) benzyl alcohol (20 mM) as described cell line, KB-GSV2,wasgrown and maintained as previously de- above. scribed (37). A detailed kinetic analysis was performed in CMV using varying Membrane Vesicle Preparations-Canalicular membrane vesicles concentrations of [3H]daunomycin(0-100 p ~ or) [3H]vinblastine(0were isolated from liver homogenates of male Sprague-Dawley rats 60 p ~ in) the presence and absence of AzC (5.0 pM, final concentraweighing 200-300 g, using .nitrogen cavitation and calcium precipi- tion). Kinetic parameters were then determined from Eadie-Hofstee tation techniques, as described previously by Inoue et al. (39). The plots of initial rate uersus drug concentrationsas described (45). The purity of these preparations was determined by assaying appropriate inhibitory constant (K,)of A2C was estimated by using the following marker enzymes a t 37 "C(39,40). The specific activity ratios (purified equation: l/Vmaxi= l/Vmax(1 + [i]/K,) where Vmex,and V,, are canalicular membrane/crude homogenate) for CMV marker enzymes maximal velocity values obtained in the presence ( VmsrJand absence demonstrated an -60-fold enrichment in y-glutamyltransferase and ( Vmm)of the inhibitor AzC and [i] isthe concentration ofAZC in the a -40-fold enrichment in alkaline phosphatase (p-nitrophosphatase) reaction medium. activities. The enrichment ratios between homogenate and purified Similar kinetic parameter analyses were also performed using vesicles for succinic dehydrogenase, NADPH cytochrome c reductase varying concentrations of [3H]daunomycin(0-100 p M ) in the presence and sodium/potassium-dependent adenosine triphosphatase, and and absence of verapamil(25 pM). marker enzymes for mitochondrial, microsomal, and sinusoidal memSteady State Fluorescence Polarization Studies-All fluorescence branes, respectively, ranged from 0.30 to 1.50 in each preparation. measurements were performed in the presence and absence of AzC CMV were suspended in buffer A (10 mM Hepes-Tris buffer, pH 7.4, (0.6-5.0 p M ) or benzyl alcohol (1-30 mM) or verapamil (25-50 p M ) at containing 0.25 M sucrose and 0.2 mM CaCl2) and stored at -70 "c 25 "C. Steady state fluorescence polarization studies were routinely prior to use. performed with an SLM-4800C spectrofluorometer in the T-format In certain experiments (see below), KB-GSV2 cell surface mem- using the lipid-soluble fluorophore DPH. The techniques used to load brane vesicleswere prepared by nitrogen cavitation and sucrose the preparations with this probe and thequantitation of fluorescence differential centrifugation procedures as previously described (19,41). have previously been described in detail by our laboratory and others Membrane protein concentration was measured as described by (27, 46, 47). Lowry et al. (42), using bovine serum albumin as standard. Corrections for light scattering and for fluorescence in the ambient Identification of P-gp in CMV-Identification of P-gp in CMV by mediumwere routinely performed as described (48). Final molar immunoblotting was performed using an enhanced chemilumines- ratios of the probe/lipid ranged from 0.001 to 0.002, and the anisocence technique (ECL Western Blotting Detection System). Follow- tropy ( r ) differences noted in theseexperiments could not be ascribed to differences in the probe concentrations in these preparations(48). 'The term lipid fluidity as applied to anisotropic bilayer lipids is The lifetimes of the excited-state of DPH in each preparation were used to denote the relative motional freedom of lipid molecules or obtained at 30 MHz and analyzed using an IBM PC/XT interfaced substituents thereof. A more detailed description of the term is given with the spectrofluorometer and software provided by SLM (SLM Aminco, Urbana, IL) as previously described (49). Differences in the in Ref. 29. EXPERIMENTALPROCEDURES

24997

Fluidity Alters P-glycoprotein Function anisotropy values noted in these preparations after treatment with A& or benzyl alcoholcould not be ascribed to differences in the lifetimes of the excited-stateof DPH in these preparations(49). Statistical Methods-All results areexpressed as the meanvalue f S.E.Paired or unpaired t tests were used for all statistical analysis. A value of p < 0.05 was consideredsignificant.

i

RESULTS

Identification of P-gp in Rat Liver CMV-As assessed by protein immunoblots using the anti-P-gp monoclonal anti0.04 . . , . , . I /body C219, a proteinband waslabeledinCMVwith an 00 IO 20 3 0 4 0 60 apparent molecular weight of -150,000 (Fig. 1, lane I ) , conTime(min) a protein sistent with the known M , of P-gp (4). Furthermore, FIG.2. Time course of ATP-dependent [3H]daunomycinacof similar M, was also identified with this antibody in KB- cumulation by CMV. ATP-dependent daunomycin accumulation GSV2 cell homogenates (Fig. 1, lane 2), an MDR cell line by CMV wasmeasured as a function of time. Reaction medium known to contain functional P-gp (50). A doublet was noted contained 12 p~ ['HHJdaunomycin, 1.2 mM ATP, and an ATP regenin the KB-GSV2 immunoblot which may represent glycosy- erating system (3 mM creatine, 3.6 pg of creatinine phosphokinase) in Buffer B (see text). Addition of reaction medium (100 pl) to 20 PI latedand nonglycosylated forms of P-gp. A parallelblot of CMV (25-50 pg of protein) initiated thereaction, and daunomycin incubated with preimmune sera showed no reactivity (data accumulation was measured asdescribed in the text. ATP-dependent not shown). These data, therefore, are in agreement with prior accumulation was determined by subtracting values obtained in the studies which have localized P-gp to the canalicular domain presence of AMP. Data points represent the mean f S.E. of three experiments performed in triplicate. of normal rat liver (13). Drug Accumulation Studies-P-gp has been shown to be an 1 ATP-dependent drug efflux pump in MDR tumorcells (4,8) as well as in membranevesicles prepared from such cells (45). Recently, the P-gp-mediated transport of ['H]daunomycin wasdemonstratedinnormalrat liverCMV (13). I t was, therefore, of interest to examine and compare the characteristics of ['Hldaunomycin and ['HHIvinblastine accumulation by CMV in the presence (1.2 mM ATP) and absence(1.2 mM AMP) of ATP. As shown in Fig.2, a t eachtimepoint examined up to 3 min, ATP stimulated the accumulation of ['H]daunomycin by the vesicles (approximately 1.5-fold a t 3 min). At equilibrium (60 min), however, accumulation was comparable in these preparations. This latter finding, there+ I 60 fore, aspreviously described (13,45), indicates that the averageintravesicular volumes of ATP-andAMP-containing Time (min) vesicles were comparable, indicating that the differences in FIG.3. Time course of ATP-dependent [3H]vinblastine acthe ratesof accumulation of ['H]daunomycin in the presence cumulation by CMV. ['H]vinblastine (12 /IM) accumulation by of ATP were not due to alterationsvesicular in size. Moreover, CMV was measured as a function of time. ATP-dependent accumusince similar aliquots of membrane proteinwere used in these lation was determined by subtracting values obtained in thepresence experiments, changes invesicular number are notresponsible of AMP. The procedures were as described in Fig. 2 and in the text. f S.E. of five experiments performed Data points represent the mean for thedifferences seen in the present studies. in triplicate. ATP also stimulated an approximately1.5-fold increase in t h e accumulation of ['Hlvinblastine by rat liver CMV (Fig. 3). However, the effect of ATP on ['Hlvinblastine accumula- P-gp for vinblastine (45, 51) and is supported by our kinetic tion was more rapid (maximalat 15-30 s) than that notedfor data (see below). Separate experimentswere conducted to assess the effect(s) [:'H]daunomycin (maximal a t 3 min) and was dissipated after of the nonhydrolyzable ATP analogs ATPrS and AMP-PNP 2 min. This finding may reflect the known higher affinity of on P-gp-mediated accumulation of both cytotoxic agents. As with AMP, both nonhydrolyzable ATP analogs failed to stimk Da ulate drug accumulation (data not shown) (13,45). 205 To further characterize theseprocesses in normal rat liver CMV, experiments were conducted to determine theconcen) ['HI tration dependence of ['H]daunomycin (0-100 p ~ and 116 p ~ accumulation. ) Kinetic analysis revealed vinblastine (0-60 no thattheaccumulation of both ['H]daunomycin (data not shown) and ['HJvinblastine (Fig. 4) were saturable and con49 formed to Michaelis-Mentenkinetics. When analyzed by Eadie-Hofstee plots (Fig. 5 and Table I), the apparent K , for , its ['H]daunomycin was estimated tobe 44.20 f 4.70 p ~ and apparent maximum velocity (V,,,) was 1.60 f 0.16 nmol/mg of protein/min (Fig. 5A). For ['HHIvinblastine, the apparent K , was 25.70 f 5.14 p ~ and , its V,,, 1.47 f 0.21 nmol/mg of FIG.1. Identificationof P-gp in CMV. Immunoblotting of CMV (100 pg of protein, lane I ) and KB-GSV2 cell homogenates protein/min (Fig. 5B). In order to ensure that the aforementioned transport stud(100 pg of protein, lane 2 ) with anti-P-gp monoclonal antibody, C219 (see "Experimental Procedures"). ies truly involved the accumulation of these radiolabeled I

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P-glycoprotein Function Alters Fluidity

24998 1.2

r

A.

i .5

[ ' H I Vinblastine IpMl

FIG. 4. Concentration dependence of ['Hlvinblastine accumulation by CMV and inhibition by the membrane fluidizer, A&. ATP-dependent drug accumulationwas measured in a medium containing 12 p M [3H]vinblastine and various concentrationsof vinblastine (0-60 FM). After incubation of CMV with the reaction medium (see text), vinblastine accumulation was measuredin the presence (closed symbols)and absence(open symbols)of A2C (5.0 p ~ ) after 20 s. ATP-dependent accumulationwas calculated by subtracting values obtained in the presence of AMP. Each point represents the mean f S.E. of four experiments performedin triplicate. agents into the intravesicularspace rather than just binding t o these preparations, experiments were performed to examine the effects of increasing sucrose concentrations (0.25-1.0 M) on drug accumulation. As seen in Fig. 6, the ATP-dependent accumulation of [3H]daunomycin (Fig. 6A) and [3H]vinblastine (Fig. 6B) by CMV decreased withincreased medium osmolarity. These data indicated that vesicles the were intact, were responsive to changes in media osmolarity, and, most importantly, that these compoundswere largely accumulated in the intravesicular space, albeit withsome binding. In this regard, extrapolation of the lines to infinite osmolarity revealeda binding component of-31% for[3H]daunomycin and -14% for [3H]vinblastine. In order to furtherconfirm that the accumulationof these drugs by CMV was, indeed, due to a P-gp-mediated process, CMV (20 pg) were preincubated with either the anti-P-gpmonoclonal antibody C219 (25 pg) or with rabbit anti-mouse IgG (25 pg) for 10 min at 30 "C. ATP-dependent [3H]daunomycin accumulation by CMV was then determined after90 s. I n two separate experiments, incubation of CMV with C219 antibodies reduced ATP-dependent[3H]daunomycin accumulation by -80% (control, 0.59 nmol/mg/min; C219-treated, 0.11 nmol/mg/min). This finding strongly supports our contention that accumulationof these drugs by CMV was due t o a P-gp-mediated process. Fluidity Studies-As previously stated, prior studies have shown that alterations in rat hepatocyte plasma membrane fluidity caninfluence a number of important transmembrane transport processes (29-36). In the present experiments, it was of interest to determine whether the known membrane fluidizers, A2C and benzyl alcohol, influenced the ATP-dependent accumulation of [3H]daunomycin and [3H]vinblastine by rat liver CMV. As assessed by steady-state fluorescence polarization techniques, using the lipid-soluble fluorophore DPH, bothA2C (1.25 to 5 p ~final , concentration) and benzyl alcohol (10 to 30 mM, final concentration)significantly reduced the anisotropy ( r ) values of this probe, e.g. increased the fluidity of CMV (Fig. 7). At lower concentrations (1p~ A&; 1 mM benzyl alcohol),however, these agents had no effect on membrane CMV (data not shown). Moreover, in agreement with our prior observations (52), methyl alcohol (50 mM, final concentration), whichserved as an osmotic

nmol/mg/min

FIG. 5. Kinetic analysis of ['H]daunomycin and ['Hlvinblastine accumulation by CMV and its inhibition by A&. ATPdependent daunomycin (A) and vinblastine ( B ) accumulation were measured as described in the legend to Fig. 4 and in the text. EadieHofstee plots of uptake in the presence (closed symbols) or absence (open symbols)of A& (5.0 p M ) indicate that A2C is a noncompetitive and an uncompetitive inhibitor of daunomycin (A) and vinblastine ( B )accumulation, respectively.The units of the abscissa and ordinate are nmol/mg of protein/min and nmol/mg of protein/pM, respectively. Each point represents the mean f S.E. of four experiments performed in triplicate. TABLEI Kinetic parameters for drug accumulation by CMV in presence and absence of A2C Kinetic analysisof the effect of the fluidizer AzC on ATP-dependent daunomycin and vinblastine accumulation in CMV. A2C produced a reduction in V,,, for daunomycin without altering the K , value, consistent with noncompetitive inhibition. For vinblastine, boththe VmaXand K,,, values were reduced by A2C indicating that the mechanism of inhibition was uncompetitive. Eachvalue represents the mean f S.E. of 4 experiments performed in triplicate. Kinetic parameters were estimated from Eadie-Hofstee plots.

[3H]Daunomycin Control AzC'

[3H]Vinblastine

Control

A&

K,

Vmax

clM

nrnollrnglmin 1.60 f 0.16 0.76 f 0.21 1.47 f 0.21 0.66 f 0.07

44.20 f 4.70 40.51 f 6.15 25.70 f 5.14 16.40 f 2.58

"A& (5.0 pM).

control for benzyl alcohol, also had no effect on the fluidity of CMV (Fig. 7). As shown in Table 11, both benzyl alcohol and A2C, in a concentration-dependent manner, also increased the fluidity of cell surface membranes prepared from KB-GSV2 cells. It was also of interest to determine whether verapamil, a

Fluidity Alters P-glycoprotein Function A.

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FIG.6. Osmotic sensitivity of ATP-dependent [3H]daunomycin and [3H]vinblastine accumulation by CMV. CMV were incubated for 60 min at 25 "C in buffer containing 0.25-1.0M sucrose. Accumulation studies were performed in reaction medium (see text) which was adjusted to the osmolarity of the vesicle buffer. ATPdependent daunomycin ( A ) and vinblastine ( B ) accumulation were measured following a 1-min incubationof CMV and reaction medium and were calculated by subtracting values obtained in the presence of AMP. Data points represent the mean value of three experiments performed in triplicate.

0.23 control

10.0

20.0

30.0

Benzyl alcohol h M ) FIG.7. Increase in membranelipid fluidity of CMV induced by A2C and benzyl alcohol. Anisotropy ( r ) values were measured by steady state fluorescence polarization at 25 "C using the fluores(DPH). Both A2C ( A ) and cent probe 1,6-diphenyl-1,3,5-hexatriene benzyl alcohol ( B )produced a dose-dependent increase in membrane lipidfluidity compared to control values (50 mM methyl alcohol). f S.E. of three separate preparations. Data points represent the mean

calcium channel blocker and a known inhibitor of P-gpmediated transport processes (45), could influence CMV fluTABLEI1 idity. In contrast to our findings with A2C and benzyl alcohol, The effects of benzylalcohol and A2Con the fluidity ofcell surface verapamil (25 and 50 p ~ final , concentrations)failed to alter membranes prepared fromKB-GSV2 Cells the fluidity of CMV, as assessed by r values of DPH (data not Both benzyl alcohol and A& decreased the r values, e.g. increased shown). the fluidity, of KB-GSV2 membranes in a concentration-dependent The Effects of A2C, Benzyl Alcohol, and Verapamil on Drug manner. Each value represents the mean k S.E. of four experiments Accumulation by CMV-As shown in Fig. 8, both A2C and Derformed in triulicate. benzyl alcohol, a t concentrations which fluidized the memAgent Concentration r values of DPH at 25 "C branes, significantly inhibited the ATP-dependent accumuNone 0.204 f 0.001 lation of [3H]daunomycin by rat liver CMV. Lower concenBenzyl alcohol 10 mM 0.197f 0.001" trations ofA2C and benzylalcohol,whichfailed toalter Benzyl alcohol 20 mM 0.183 -t 0.002" membrane fluidity of CMV, also failed to influence the accuBenzyl alcohol 30 mM 0.173 f 0.001" 2.5 p M 0.184 f 0.002" AzC mulation of these radiolabeled drugs.A2C (Fig. 8C) and benzyl 0.171 f 0.002" 5.0p M A2C alcohol (data not shown)were also found to inhibit the ATP" p < 0.05or less compared to no additionvalue. dependent accumulation of [3H]vinblastine in these preparations. Furthermore, methylalcohol (50 mM) was not found to affect the ATP-dependent accumulation of either of these CMV, kinetic analyses were performed to determine whether radiolabeled drugs (labeled as benzyl alcohol control). This A& influenced the K,,, and/or Vmaxvalues of these processes. latter finding notonly lends further support to the contention As shown in Fig. 5 and Table I, this agent reduced the V,,, that fluidity changes may inhibit these processes, but also of ATP-dependent[3H]daunomycinaccumulationwithout indicates that changes in the osmolarity of the buffer, second- significantly altering theK, value of this process as assessed ary to benzyl alcohol, were not responsible for its inhibitory by an Eadie-Hofstee plot. In contrast, A2C (5.0 /IM) reduced effects on drug accumulation. It should be noted, however, both the V,,, and K,,, values for [3H] vinblastine accumulathat,ascan be seen inthis figure, while benzylalcohol tion. The same kinetic parameters were obtained when the inhibited drug accumulation by CMV in a dose-dependent data were examined by Lineweaver-Burk analysis. These findmanner, A2C did not. The reasons for this latter result are ings indicated thatA2C noncompetitively inhibited the ATPunclear at this time andwill require further studies toclarify dependent accumulation of [3H]daunomycin and uncompetithis issue. tively inhibited [3H]vinblastine accumulation by P-gp in rat To further elucidate the mechanism of interaction between liver CMV. Furthermore, the apparent inhibitory constants these fluidizing agents andradiolabeled drug accumulationby ( K J for A2C on both [3H]daunomycin and [3H]vinblastine

AltersFluidity

25000 A.

control

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Benzyl alcohol (mM)

R .L

P-glycoprotein Function [3H]daunomycin (control, 45.1 f 7.2 pbi; verapamil-treated, 78.7 f 12.4 p ~ n ,= 4, p < 0.05), while V,,, values remained unchanged (control, 1.59 f 0.27; verapamil-treated, 1.65 f 0.18 nmol/mg of protein/min, n = 3). Verapamil, therefore, inhibits P-gp-mediated drug accumulation by these vesicles in a competitive manner. Moreover, as shown in Table 111, verapamil(50 p ~ significantly ) inhibited the ATP-dependent accumulation of both [3H]daunomycin (-67%) and [3H]vinblastine (-82%) by CMV. Prior studies by Kamimoto et al. (13) have established that only although -80% of CMV areorientedright-sideout, inside-out CMV demonstrate ATP-dependent drug transport by P-gp. It was, therefore, theoreticallypossible that A& and/ or benzyl alcohol increased the permeability of these preparations to ATP, thus permitting ATP to enter the right-sideout populationof CMV. This, in turn, could leadto anincrease in efflux of the radiolabeled drugs by P-gp, and potentially account for the present findings. To address this possibility, the accumulation of [Y-~*P]ATP was assessed in CMV in the ) benzyl alcohol (20 presence and absenceof A& (5.0 p ~ and mM). No differences were found in the accumulation of [y32P] under these conditions (data not shown), indicating that this possibility was not responsible for the inhibitory effects of these fluidizers on [3H]daunomycin and [3H]vinblastine accumulation by CMV. DISCUSSION

C.

Our results demonstrate an ATP-dependent accumulation process for both [3H]daunomycin and [3H]vinblastineby rat liver CMV which is saturable,osmotically sensitive, and temperature-dependent. ATP hydrolysis appears to be required for drug accumulationas shown by the fact that nonhydrolyzable ATP analogs, like AMP, failed to stimulate drug accumulation (13, 45). Accumulation studieswithbothdrugs revealed an overshoot phenomenon, and thedecline in ATPdependent drug accumulation to controlvalues at later time points suggestsdepletion of ATPand/oritsregenerating mnlrd 2.5 5.0 system (13) as well as possible diffusion of drugs out of the intravesicular space (45). AzC (kW These aforementioned characteristics are consistent with FIG. 8. Inhibition of ATP-dependent ['Hldaunomycin ac- P-gp mediationof these accumulationprocesses by CMV (13). cumulation by A& and benzyl alcohol and ['Hlvinblastine Moreover, severaladditional linesof evidence support therole accumulation by A,C in CMV. Bars refer to percent accumulation of P-gp in theseprocesses. These include: 1)identification of as comparedwith control which was arbitrarily defined as 100%. P-gp in rat liver CMV by immunoblotting techniques; 2) the Various concentrations of membranefluidizers were added to the reaction medium, and uptake was measured as described in the text. inhibition of drug accumulationby the anti-P-gpmonoclonal Inhibition of daunomycin accumulation by the membrane fluidizer antibody C219; and 3) the competitive inhibition of these A& ( A )and benzyl alcohol( B )occurred at all but the lowest dosages. accumulation processes by verapamil. Taken together, these C demonstrates the inhibition of vinblastine accumulation by A&. findings indicate that the drug accumulation processes studied ATP-dependent accumulation was calculated by subtracting values in the present experimentswere, indeed, mediated by P-gp. obtained in the presence of AMP. Each point representsthe mean f It is known that multiple structurally unrelated and highly S.E. of four to eight experiments performedin triplicate. TABLEI11 accumulation were ~ 1 . and 5 ~ 1 . pM, 2 respectively. Inhibition of ATP-dependent fH]daunomycin and PHIvinblastine Prior studies haveshown that verapamil competes with accumulation inCMV by verapamil vinblastine for a common drug-binding site(s) on P-gp (24), The effect of the MDR reversing agent verapamil on daunomycin and, therefore, is a highly specific and potent competitive and vinblastine accumulation by CMV. Verapamil (50 p M ) signifiinhibitor of ATP-dependent, P-gp-mediated transport. Fur- cantly inhibited the ATP-dependent uptake of both drugs consistent drug transport mediated by P-gp. Values represent the mean f thermore, Horio et al. (45) have shown that verapamil com- with S.E. of 3 experiments performed in triplicate at 3 min for daunomycin petitively inhibited the accumulation of vinblastine by plasma and at 20 sec for Vinblastine. membrane vesicles of KB-V1 cells. Based on these studies, it [3H]Daunomycin Inhibition [3HiVinbiastine Inhibition was of interest to determine whether verapamil inhibited drug accumulation accumulation accumulation by CMV and, if so, by what mechanism(s). To nmollmglmin % nmol/mg/min % address this issue, kinetic studies were performed on [3H] Control 1.50 f 0.39 0 1.47 f 0.11 0 daunomycin accumulation in the presence and absenceof 25 Verapamil 0.50 & 0.27 0.17" 67 f 0.04" 82 (50 uM) p~ verapamil. Analysis of the Eadie-Hofstee plot revealed " p < 0.05 compared to control values. t h a t verapamil significantly increased the apparent K,,, for

AltersFluidity

P-glycoprotein Function

25001

lipophilic cytotoxic drugs are substrates for P-gp (4, 17, 45). these studies, particularly those by Guffy et al. (56), suggest I n addition, a variety of lipophilic agents have beenshown to that alterations influidity may modulate P-gp-mediated drug reverse MDR, presumably by interacting with drug binding accumulation notonly in normal ratliver plasma membranes, sites on P-gp (19-24). Given that P-gp resides in the plasma but also in MDR tumorcells. It should, however, be noted that other investigators have membrane and has six transmembrane hydrophobic domains, we sought to alter membrane lipid fluidity in an attempt to failed to showa direct relationship between alterations in modulate P-gp function. Our results demonstrate for the firstfluidity and the MDR phenomenon (58, 59). For example, time that increases in the fluidity of rat liver CMV induced Montaudon et al. (58), based on fluorescence polarization studiesusing doxorubicin-sensitive and-resistantrat CG by two different fluidizing agents, A2C and benzyl alcohol, mouse can inhibit the P-gp-mediated accumulation of radiolabeled glioblastomacells and simian virus 40-transformed liver cells, concluded that alterations in membrane fluidity daunomycin and vinblastine. The kineticsof the inhibitoryeffect of A2Con daunomycin were neither necessary nor sufficient for the expression of accumulation showed a noncompetitive interaction between resistance in these cells. Kessel (59) also showed that verapthese two compounds, indicating that the rate of daunomycin ami1 could partially reverse anthracycline resistance inP388/ accumulation was reduced without affecting its binding affin- ADR cells, but that this was not accompanied by alterations ity toP-gp. This suggests that A2C may directly bind to P-gp in membrane fluidity. In the present studies, in agreement with the findings of at a site which is distinct from that where Vinca alkaloids and anthracyclines interact. Similarly, A2C reduced the rate Kessel(59), verapamilwas alsofound tocompetitively inhibit fluidity ( VmaX) ofvinblastine accumulation in CMV, but, in contrast drug accumulationby CMV and yetfailed to alter the t o its effect on daunomycin accumulation, it also altered the of these preparations. Based on these results, it is clear that alterations in fluidity are not necessary to inhibit P-gp-me(K,) to P-gp consistent with uncomapparent binding affinity petitive inhibition. These datasuggest that alterations in the diateddrugaccumulation by CMV. Based on the present experiments with A2C and benzyl alcohol, however, alteralipid microenvironment surrounding P-gp may change the protein's conformation, therebydecreasing its ability to func- tions in fluidity do appear to be sufficient to inhibit P-gption as a drug transporter. The finding that low doses of both mediated drug accumulationby CMV. In summary, our data demonstrate for the first time that fluidizers failed t o influence either membrane fluidity or drug accumulation suggest that increases in lipid fluidityper se are increases in membranefluidity induced by the known fluidizresponsible for inhibition of drug accumulation by P-gp. To ers, A& and benzyl alcohol, can inhibit P-gp-mediated accudate, the only other known noncompetitive interaction be- mulation of both Vinca alkaloids and anthracyclines in nortweenanMDR reversing agentand a cytotoxic drug for mal ratliver canalicular membranes. Kinetic analysisrevealed binding to P-gp was recently described by Tamai and Safa that the mode of interaction between A& and both radiola(22). Furthermore, our results represent the first description beled daunomycin and vinblastine were noncompetitive and of an uncompetitive interactionbetween a n inhibitor of P-gp uncompetitive, respectively,suggesting that A2C may alter the conformationof P-gp and thereby preventVinca alkaloid and a cytotoxic drug. Attempts at the reversal of MDR in uiuo, i.e. in clinical and anthracycline accumulation. While our results were obtrials, have been fraught by the dose-limiting toxicity of the tained in normal ratliver canalicular membranes which conMDR reversing agent, verapamil (53). Therefore, elucidation tain P-gp, further studies are required to determine whether cell plasma of the kinetic mechanism of inhibition by MDR reversing P-gp-mediated drug accumulation in other normal membranes as well as in MDR tumor cell plasma membranes agents affords the opportunity to use both competitive and noncompetitive or uncompetitive inhibitors of P-gp in com- can be modulated by alterations in lipid fluidity. bination with cytotoxic drug(s). This approachmay allow the Acknowledgment-We thank Lynn Nelson for excellent secretarial use of lower concentrations of P-gp inhibitors with a syner- support. gistic effect and, thereby, avoid the toxicity of a single MDR reversing agent. REFERENCES The present findings are also of interest in view of prior 1. Biedler, J. L., and Riehm, H. (1970) Cancer Res. 30,1174-1184 2. Juliano, R. L., and Ling, V. (1976) Biochim. Biophys. Acta 4 5 5 , 152-162 observations on the relationship between resistance to the 3. 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