Modulation of P-glycoprotein-mediated multidrug resistance by acceleration of passive drug permeation across the plasma membrane Ronit Regev*, Hagar Katzir*, Daniella Yeheskely-Hayon and Gera D Eytan Department of Biology, the Technion ) Israel Institute of Technology, Haifa, Israel Keywords anesthetics; modulators; multidrug resistance; P-glycoprotein; propofol Correspondence G D Eytan, Department of Biology, The Technion ) Israel Institute of Technology, Haifa, Israel Fax: +972 8225153 Tel: +972 8293406 E-mail: eytan@tx.technion.ac.il Website: http://biology.technion.ac.il *These authors contributed equally to this work (Received 22 July 2007, revised September 2007, accepted 11 October 2007) doi:10.1111/j.1742-4658.2007.06140.x The drug concentration inside multidrug-resistant cells is the outcome of competition between the active export of drugs by drug efflux pumps, such as P-glycoprotein (Pgp), and the passive permeation of drugs across the plasma membrane Thus, reversal of multidrug resistance (MDR) can occur either by inhibition of the efflux pumps or by acceleration of the drug permeation Among the hundreds of established modulators of Pgp-mediated MDR, there are numerous surface-active agents potentially capable of accelerating drug transbilayer movement The aim of the present study was to determine whether these agents modulate MDR by interfering with the active efflux of drugs or by allowing for accelerated passive permeation across the plasma membrane Whereas Pluronic P85, Tween-20, Triton X100 and Cremophor EL modulated MDR by inhibition of Pgp-mediated efflux, with no appreciable effect on transbilayer movement of drugs, the anesthetics chloroform, benzyl alcohol, diethyl ether and propofol modulated MDR by accelerating transbilayer movement of drugs, with no concomitant inhibition of Pgp-mediated efflux At higher concentrations than those required for modulation, the anesthetics accelerated the passive permeation to such an extent that it was not possible to estimate Pgp activity The capacity of the surface-active agents to accelerate passive drug transbilayer movement was not correlated with their fluidizing characteristics, measured as fluorescence anisotropy of 1-(4-trimethylammonium)-6-phenyl1,3,5-hexatriene This compound is located among the headgroups of the phospholipids and does not reflect the fluidity in the lipid core of the membranes where the limiting step of drug permeation, namely drug flip-flop, occurs Classic multidrug resistance (MDR) is attributed to the elevated expression of the ATP-dependent drug efflux pumps ABCB1 [also known as P-glycoprotein (Pgp)], ABCC1 (also known as multidrug resistanceassociated protein) and ABCG2 (also known as breast cancer resistance protein and mitoxantrone resistance protein), all of which belong to the superfamily of ATP-binding cassette (ABC) transporters [1] Efflux mediated by ABC drug transporters leads to decreased cellular accumulation of anticancer drugs, which is a main cause of the limited success of the currently applied chemotherapy regimens Pgp, a product of the ABCB1 (previously known as MDR1) gene, is the most extensively studied ABC drug transporter Pgp transports chemically dissimilar drugs that act on diverse targets [2] The intracellular drug concentration in drug-resistant cells is the outcome of competition between the active export of drugs by the efflux pumps Abbreviations ABC, ATP-binding cassette; CCCP, carbonyl cyanide m-chlorophenylhydrazone; MDR, multidrug resistance; Pgp, P-glycoprotein; TMA-DPH, 1-(4-trimethylammonium)-6-phenyl-1,3,5-hexatriene; TMRM, tetramethylrhodamine methyl ester 6204 FEBS Journal 274 (2007) 6204–6214 ª 2007 The Authors Journal compilation ª 2007 FEBS R Regev et al and the passive entry of drugs by permeation across the plasma membrane [3] One of the characteristics of the MDR phenotype initially described by Skovsgaard [4] and later by Tsuruo et al [5] is that MDR can be reversed by cotreatment of resistant cells with nontoxic concentrations of hydrophobic compounds known as chemosensitizers, modulators, or resistance modifiers These include calcium channel blockers (such as verapamil), calmodulin antagonists and antiarrhythmia agents, antihistamines, lysosomotropic amines, immunosuppressants, steroid hormones and modified steroids, nonionic detergents, anesthetics, and various amphipathic drugs MDR can be reversed by inhibition of the active export of drugs either by binding of inhibitors on sites located on the efflux pumps or by indirect inhibition of Pgp either through modulation of its lipid environment or as a result of depletion of cellular ATP Alternatively, MDR can be reversed by acceleration of the passive permeation of the drugs through modulation of the lipid environment in the plasma membrane Among the hundreds of established modulators of Pgp-mediated MDR, there are numerous surface-active agents capable of affecting the lipid environment of membranes They include nonionic detergents, such as Tween-20 and Triton X-100 [6,7], excipients serving as nontoxic diluents of drugs, such as Cremophor EL, poly(ethyleneglycol) 300 and Pluronic block copolymers [6,8,9], and anesthetics, such as benzyl alcohol, chloroform and diethyl ether [10,11] Sinicrope et al have shown that chloroform and benzyl alcohol inhibit uptake of drugs into Pgp-containing vesicles [11] Pluronic P85 [12], chloroform, benzyl alcohol, Tween-20, Nonidet P-40 and Triton X-100 [10] inhibited the ATPase activity of Pgp located in either microsomes or reconstituted vesicles On the other hand, surfaceactive modulators, such as Pluronic block copolymers [13,14], benzyl alcohol, chloroform and various nonionic detergents [10,15] accelerate passive movement of doxorubicin across artificial membranes As the inhibition of Pgp ATPase activity exerted by the various agents was more pronounced than their effect on the flip-flop of drugs, it has been assumed that anesthetics and nonionic detergents modulate Pgp-mediated MDR by inhibition of Pgp [10] The aim of the present study was to elucidate the mechanism by which the various surface-active modulators reverse MDR in Pgp-overexpressing cells: whether they inhibit the Pgp-mediated active export, or allow for accelerated passive uptake of drugs The effect of these agents on Pgp-mediated activity was estimated as the active efflux of daunorubicin upon MDR modulation by acceleration of drug permeation replenishment of cellular ATP in drug-resistant cells Under these conditions, the initial efflux is due to active transport of the anthracycline without the contribution of passive transbilayer drug movement Whereas the nonionic detergents, excipients and Pluronic block copolymers inhibited Pgp-mediated drug export, the anesthetics modulated MDR by acceleration of passive drug uptake, with no concomitant inhibition of Pgp-mediated drug efflux Results Effect of modulators on cellular uptake and efflux of daunorubicin Certain modulators of Pgp-mediated MDR, such as detergents, excipients, and anesthetics, can affect membrane characteristics such as fluidity In order to determine whether these modulators enhance drug uptake into drug-resistant cells by inhibition of Pgp or by allowing for accelerated passive movement of the drugs across the plasma membrane, the active efflux of daunorubicin upon replenishment of cellular ATP to drug-resistant cells was measured using spectrofluorometry Measurement of the intracellular presence of daunorubicin inside the cells was based on the observation that its fluorescence is quenched upon intercalation between the DNA base pairs in the nucleus The affinity of binding of daunorubicin to DNA is high, and thus cellular uptake of daunorubicin can be measured as the quenching of its fluorescence by the nuclear DNA [16] Daunorubicin enters rapidly into drug-sensitive cells, presumably by passive transport across the plasma membrane (Fig 1, trace a) As expected, depletion of cellular ATP did not affect daunorubicin uptake into these cells (Fig 1, trace b) On the other hand, daunorubicin uptake into drug-resistant cells was very limited Apparently, as the intracellular concentration of daunorubicin rises, Pgp prevents further net drug uptake Thus, after an initial period of rapid daunorubicin uptake, further uptake was largely prevented (Fig 1, trace c) Depletion of cellular ATP by glucose deprivation combined with poisoning of mitochondrial respiration by sodium azide resulted in an intracellular ATP concentration equal to 0.2 mm Under these conditions, cellular Pgp with a Km of 0.8 mm [17] is expected to operate at 20% of its capacity This allows for increased uptake of daunorubicin, but not to the levels observed in drug-sensitive cells Upon subsequent addition of glucose to these cells, cellular ATP levels were replenished and Pgp actively removed the excess daunorubicin from the cells (Fig 1, trace d) FEBS Journal 274 (2007) 6204–6214 ª 2007 The Authors Journal compilation ª 2007 FEBS 6205 MDR modulation by acceleration of drug permeation R Regev et al Inhibitors of Pgp known to interact directly with the transporter, such as verapamil and cyclosporin A, interfered with the Pgp-mediated efflux of daunorubicin In the presence of these modulators, the Pgp-mediated efflux observed upon replenishment of glucose was largely eliminated, and daunorubicin uptake into these cells was similar to that observed in drug-sensitive cells (Fig 1, trace e, and Fig 2) There was a clear correlation between the degree of inhibition of Pgpmediated efflux and the increase in daunorubicin uptake into the drug-resistant cells (Fig 3) As expected, these inhibitors had no effect on the uptake of daunorubicin into drug-sensitive cells Using a similar methodology; we studied MDR modulation by eight membrane-active agents The detergents Tween80 and Triton X-100, the excipient Cremophor EL and the block copolymer Pluronic P85 modulated Pgpmediated resistance by a mechanism similar to that observed with the Pgp inhibitors verapamil and cyclosporin A They had no effect on the passive uptake of daunorubicin into drug-sensitive cells They enhanced daunorubicin uptake into drug-resistant cells as a result, and in correlation with inhibition of the active export of daunorubicin by Pgp (Fig 4) On the other hand, the general anesthetics chloroform, propofol, and diethyl ether, and the local anesthetic benzyl alcohol, appeared to modulate Pgpmediated MDR by a different mechanism They accelerated the passive uptake of the drug, rather than interfering with the Pgp-mediated activity These drugs enhanced daunorubicin uptake into drug-sensitive cells as well as into drug-resistant cells The enhanced Daunorubicin Glucose Daunorubicin Fluorescence a Cells b c d e f g 30 minutes Fig Daunorubicin uptake and efflux into and out of drug-sensitive and drug-resistant K562 cells Drug-sensitive cells (traces a and b) or drug-resistant cells (traces c–g) were incubated for 30 either in a medium containing mM glucose (traces a and c) or in ATP depletion medium containing 10 mM sodium azide and no glucose (traces b and d–g) Subsequently, lM daunorubicin (thin arrows) and lM cyclosporin A (trace e), 0.2 mM propofol (trace f) or propofol and cyclosporin A (trace g) were added At the time points marked by the thick arrows, mM glucose were added Daunorubicin fluorescence was monitored continuously Resistant Cells Sensitive Cells Daunorubicin Daunorubicin Fluorescence Daunorubicin Glucose 0 5 Cells Cells 10 10 20 20 30 60 Time [minutes] 6206 30 60 Fig Verapamil effect on uptake and influx of daunorubicin into and out of drug-resistant and drug-sensitive cells Drug-resistant and drug-sensitive K562 cells were incubated for 30 under ATP-depleting conditions in the presence of 10 mM sodium azide and the absence of glucose Subsequently, various concentrations of verapamil (marked next to the traces in terms of micromolar concentrations) were added together with lM daunorubicin Daunorubicin was taken up by the cells, and subsequently, at the time points marked by the empty arrows, mM glucose was added, allowing Pgp to expel the drug from the drug-resistant cells Daunorubicin fluorescence was monitored continuously FEBS Journal 274 (2007) 6204–6214 ª 2007 The Authors Journal compilation ª 2007 FEBS R Regev et al MDR modulation by acceleration of drug permeation 0.2 0.0 1.0 10 20 [nmoles] 0.1 Daunorubicin Uptake Daunorubcin Efflux [nmoles min–1] 2.0 0.0 Verapamil [µM] Fig Quantitative effect of verapamil on uptake and efflux of daunorubicin into and out of drug-resistant and drug-sensitive cells The effects of verapamil on daunorubicin transport in drug-resistant and drug-sensitive cells was measured as described in Fig As no curve could be fitted to the kinetics of daunorubicin uptake into the resistant cells, the uptake rate was measured as the amount of drug taken up within 15 after addition of daunorubicin either to drug-resistant cells (squares) or to drug-sensitive cells (triangles) The amount of daunorubicin pumped out of the resistant cells by Pgp (circles) was assessed by a linear regression to the efflux kinetics exhibited during the period subsequent to the first minute after the addition of glucose to the cells uptake of daunorubicin in their presence was not correlated with inhibition of daunorubicin export by Pgp Moreover, at low concentrations, these agents even accelerated the Pgp-mediated export of daunorubicin (Fig 1, trace f, and Fig 5) As expected, the active export from the drug-resistant cells was largely eliminated by the Pgp inhibitor cyclosporin A (Fig 1, traces e and g) When applied at higher concentrations, the anesthetics further enhanced the rate of passive drug permeation and interfered with the active export of the drug by Pgp Effect of modulators on the efflux of the rhodamine analog tetramethylrhodamine methyl ester (TMRM) Daunorubicin uptake, measured as described above, reflects cellular uptake of the drug, its transfer into the nucleus, and binding to the DNA To assess the effect of modulators specifically on drug transport across the cell plasma membrane, we characterized the effect of the modulators on the cellular pharmacokinetics of the rhodamine analog TMRM TMRM taken up by cells accumulates in the mitochondria in response to the mitochondrial electrochemical potential [18] This Fig Quantitative effects of Pluronic P85, Tween-20, Chremophor EL and Triton X-100 on uptake and efflux of daunorubicin into and out of drug-resistant and drug-sensitive cells The effects of the above-mentioned agents on daunorubicin uptake and efflux into and out of drug-resistant and drug-sensitive cells was quantitated as described in Fig FEBS Journal 274 (2007) 6204–6214 ª 2007 The Authors Journal compilation ª 2007 FEBS 6207 MDR modulation by acceleration of drug permeation R Regev et al Fig Quantitative effects of propofol, chloroform, diethyl ether and benzyl alcohol on uptake and efflux of daunorubicin into and out of drug-resistant and drugsensitive cells The effect of the abovementioned agents on daunorubicin uptake and efflux into and out of drug-resistant and drug-sensitive cells was quantitated as described in Fig accumulation can be eliminated by dissipation of this potential with the uncoupler carbonyl cyanide m-chlorophenylhydrazone (CCCP), thus allowing for the measurement of transport into and out of the cell cytoplasm without involvement of the mitochondrial sink Under these conditions, the transport rate of the dye is limited by the plasma membrane The observed effects of the various modulators on TMRM efflux were in accord with the results obtained with daunorubicin The Pgp inhibitors cyclosporin A and verapamil, as well as Tween-80, Triton X-100, Cremophor EL, and Pluronic P85, inhibited efflux of TMRM preloaded into drug-resistant cells and had little effect on efflux of TMRM preloaded into drug-sensitive cells (Fig and data not shown) In contrast, the anesthetics (chloroform, benzyl alcohol, diethyl ether, and propofol) accelerated the efflux of TMRM preloaded into drug-resistant and drug-sensitive cells (Fig and data not shown) The effect was much more pronounced in drug-sensitive cells, but was also significant in drug-resistant cells In the absence of the uncoupler, the dye TMRM accumulated in the mitochondria of K562 cells The amount of TMRM taken up by the cells was high, and the time course of the efflux was longer Thus, it was possible to measure uptake and efflux by rapid separa6208 tion of the cells from the incubation medium All the Pgp modulators mentioned above enhanced TMRM uptake into resistant cells The Pgp inhibitors cyclosporin A and verapamil, and Cremophor EL, Tween20, and Pluronic 85, inhibited Pgp-mediated efflux of TMRM (Fig 7) On the other hand, the anesthetics benzyl alcohol, chloroform and propofol accelerated the efflux of TMRM (Fig 7) Lack of correlation between acceleration of drug permeation and membrane fluidity as measured with 1-(4-trimethylammonium)-6-phenyl-1,3,5hexatriene (TMA-DPH) It has been suggested that the characteristic of certain anesthetics and surface agents relevant to the modulation of Pgp-mediated MDR is their capacity to alter the fluidity of the cell plasma membranes [6,10,11,19] Therefore, the effect of the above-mentioned Pgp modulators on membrane fluidity was studied by measuring the steady-state anisotropy fluorescence of TMA-DPH TMA-DPH is a short-chain lipid analog with a hydrophilic head consisting of a constitutively positively charged quaternary amine and a hydrophobic tail This dye is localized among the headgroups of the phospholipids located in the cell plasma membrane FEBS Journal 274 (2007) 6204–6214 ª 2007 The Authors Journal compilation ª 2007 FEBS R Regev et al Fig Effects of propofol and Pluronic P85 on TMRM efflux from cells Drug-resistant (A, C) and drug-sensitive (B, D) K562 cells were loaded with TMRM in the presence of CCCP (1 lM) and in the presence or absence of verapamil (10 lM), respectively The apparent intracellular TMRM concentrations in drug-sensitive and drug-resistant cells were about 30 and 12 times the concentration in the medium, respectively TMRM was removed by pelleting the cells and suspending them in CCCP-containing fresh medium in the absence (empty circles) or presence of the anesthetic propofol at 0.1 mM (empty squares), 0.2 mM (full circles), or 0.5 mM (full squares), or the surface-active agent Pluronic P85 at 0.0001% (empty squares), 0.001% (full circles), 0.01% (triangles), or 0.1% (full squares) The amount of TMRM associated with the cells was determined by flow cytometry as described in Experimental procedures MDR modulation by acceleration of drug permeation A B C D [20–22] The modulators had a variable effect on the fluorescence anisotropy of TMA-DPH, and no clear correlation could be observed between this effect and the efficacy of the agents in the modulation of Pgpmediated TMRM efflux (Fig 8) Thus, whereas the anesthetics chloroform, benzyl alcohol and diethyl ether decreased the fluorescence anisotropy of TMADPH, propofol had little effect on the fluorescence anisotropy Yet, all these anesthetics reversed Pgpmediated MDR by acceleration of the passive transmembrane movement The effect of the other surface-active agents on membrane fluidity, measured as TMA-DPH fluorescence anisotropy, varied between no effect and marked effect (Fig 8) However, none of the other agents accelerated the transbilayer permeation of the drugs and dyes Discussion Analysis of the reversal of Pgp-mediated MDR by various membrane-active agents revealed two apparently conflicting mechanisms of MDR modulation The detergents, excipients and Pluronic block copolymers reversed the resistance by inhibition of Pgp-mediated active transport On the other hand, low concentrations of the anesthetics benzyl alcohol, propofol, chloroform and diethyl ether reversed the resistance by acceleration of the passive transport of the drug or dye across the plasma membrane At these concentrations, the anesthetics did not inhibit the Pgp-mediated efflux, but even accelerated it The acceleration of drug permeation across the plasma membrane was not due to permeabilization of the cells, as the cells were not stained with membrane-impermeable stains, such as propidium iodide More significantly, the cells retained the capacity of Pgp to pump drugs out of the cells Interestingly, the results obtained with living cells differ from the data obtained when the effects of the various agents were analyzed using liposomes and plasma membrane preparations [6,10,11] In cell-free systems, the anesthetics had a much more pronounced effect on Pgp ATPase activity in comparison to their effect on the drug flip-flop across the membrane In contrast, in K562 cells, low concentrations of anesthetics affected exclusively the permeation rate of the drugs On the other hand, the nonionic detergents accelerated flipflop in liposomes, but had little effect on permeation in the cells Only the simultaneous analysis of Pgpmediated efflux and permeation in the cells led to the conclusion that the anesthetics modulate MDR by acceleration of the permeation, whereas the other agents modulate MDR by inhibition of the active efflux Net activity of Pgp without interference of the passive permeation of the drugs was observed as daunorubicin efflux from drug-resistant cells In these experiments, Pgp-mediated net transport was observed after replenishment of cellular ATP levels by glucose FEBS Journal 274 (2007) 6204–6214 ª 2007 The Authors Journal compilation ª 2007 FEBS 6209 MDR modulation by acceleration of drug permeation R Regev et al A B C D E F supplement Before the addition of glucose, the cellular content of daunorubicin was in quasi-equilibrium resulting from equal rates of the passive inward transport of the drug and the residual active drug efflux catalyzed by Pgp Upon replenishment of ATP, the initial outward drug transport, away from this equilibrium, is due only to the activity of Pgp Thus, the acceleration of the efflux observed in the presence of the anesthetics is mediated by Pgp activity and is not due to faster drug permeation A plausible cause for this acceleration is faster passive movement of the drugs to the active site of the Pgp It has been suggested that Pgpmediated dye or drug export involves incorporation of the drugs into the inner monolayer of the plasma membrane, its lateral movement toward the active site of Pgp, and active extrusion by the latter directly from the lipid phase of the inner leaflet of the plasma membrane [23] This model is supported by experimental 6210 Fig Effects of various agents on TMRM efflux from K562 cells Drug-resistant cells (A, C, E) and drug-sensitive K562 cells (B, D, F) were loaded with TMRM in the presence or absence of verapamil (10 lM), respectively The apparent intracellular TMRM concentrations in drug-sensitive and drug-resistant cells were about 200 and 40 times, respectively, the concentration in the medium The cells were separated from the TMRM-containing medium by centrifugation and suspension in fresh medium in the absence (empty circles) or presence of the following additions: (A, B) the Pgp inhibitors verapamil (30 lM, squares) or cyclosporine A (10 lM, full circles); (C, D) the surfaceactive agents Cremophor EL (0.05%, empty squares), Tween-20 (0.01%, full circles), or Pluronic P85 (0.01%, full squares); (E, F) the anesthetics chloroform (10 mM, empty squares), benzyl alcohol (10 mM, full circles), or propofol (0.2 mM, full squares) Cell samples were obtained after various further incubation periods, and the amount of TMRM associated with them was determined after centrifugation through oil cushions as described in Experimental procedures data, including observation of suitable side entrances from the lipid matrix into the protein that may permit access of substrates to the core of the protein [24–28] The anesthetics could facilitate the incorporation of drugs into the inner leaflet of the plasma membrane and ⁄ or accelerate its lateral transport to the Pgp Tran et al [29] have analyzed the kinetic parameters of Pgpmediated transport across a confluent monolayer of canine cells, and concluded that the association of the drugs with Pgp was rate-limited by their lateral diffusion in the plasma membrane In contrast to the situation when daunorubicin efflux was measured, the anesthetic-accelerated efflux of preloaded TMRM was due mainly to acceleration of passive transport of the dye In this case, the efflux is due to Pgp activity and passive transport of the dye However, the acceleration of the passive transport by itself could account for the observed enhanced efflux rate FEBS Journal 274 (2007) 6204–6214 ª 2007 The Authors Journal compilation ª 2007 FEBS R Regev et al MDR modulation by acceleration of drug permeation Fig Effect of the various anesthetics on membrane lipid fluidity measured with TMA-DPH Anisotropy (r) values were measured by steady-state fluorescence polarization at 25 °C using the fluorescent probe TMA-DPH Passive transbilayer movement of MDR-type drugs and dyes involves the incorporation of the agent into the proximal membrane leaflet, transbilayer flip-flop, and release from the opposing leaflet The incorporation and release of these agents is very fast, and they are practically in equilibrium between the liquid phase and the membranes [29,30] Thus, the anesthetic-mediated acceleration of transbilayer drug movement is due either to enhanced rate of drug flip-flop across the lipid core of the membranes or to greater affinity of the drugs for the plasma membrane Anesthetics and nonionic detergents have been shown to accelerate flip-flop of doxorubicin and mitoxantrone across liposome membranes [10,15] As Breuzrd et al have observed that modulation of mitoxantrone resistance in Pgp-overexpressing cells reduces the amount of drug incorporated in the plasma membranes [31], it seems that the anesthetics accelerate the permeation by enhancing the drug flip-flop rate across the lipid core of the membrane Benzyl alcohol, diethyl ether and other fluidizing agents inhibit the activity of Pgp in cell-free systems [6,10,11] The inhibition of Pgp activity could be due either to a direct effect on the enzyme or to an indirect effect mediated by alterations in membrane structure As the inhibitory concentrations of fluidizers are similar to those required for membrane fluidization, it has been suggested that Pgp-mediated modulation of MDR is due to increased membrane fluidity, evident as reduced fluorescence anisotropy of probes, such as DPH [6,11] However, Dudeja et al have observed that polyoxyethylene surfactants reverse MDR in KB8-5-11 drug-resistant cells by decreasing membrane fluidity measured as fluorescence anisotropy of a variety of membrane probes [32] Similarly, Woodcock et al [6] have ascribed modulation of MDR by Chremophor EL to a reduction in membrane fluidity Hugger et al evaluated the activity of Pgp in cell monolayers and found no correlation between the inhibition of Pgp activity and fluidity of the membranes measured as fluorescence anisotropy of TMA-DPH [33] In the present study, there was no correlation between the capacity of the various membrane-active agents to reverse MDR and their effect on membrane fluidity as measured with TMA-DPH TMA-DPH is localized among the headgroups of the phospholipids at the outer surface of the plasma membrane [20–22] The membrane characteristics relevant to the effect of the anesthetics on MDR are probably located at the hydrophobic core of the membrane, where drug flipflop across the membrane occurs and the side entrances of Pgp are located [28] The apparent contradiction between the anestheticmediated inhibition of Pgp ATPase activity in cell-free systems and their mode of modulation in K562 cells could be due to the combined effect of the anesthetics, inhibition of Pgp activity and acceleration of passive drug permeation In living cells, the anesthetics accelerate drug permeation at concentrations lower than FEBS Journal 274 (2007) 6204–6214 ª 2007 The Authors Journal compilation ª 2007 FEBS 6211 MDR modulation by acceleration of drug permeation R Regev et al those required to inhibit Pgp ATPase activity Likewise, the agents tested here, other than the anesthetics, inhibited Pgp-mediated efflux and had no effect on the rate of passive transbilayer movement Yet, it has been shown that diblock polymers accelerate movement of drugs, including doxorubicin, across artificial membranes [13] At higher concentrations and in other cells, these agents accelerate the transbilayer movement of drugs Thus, diblock polymers, such as Pluronic P85, inhibited Pgp-mediated transport across cell monolayers, but had no effect on the passive transbilayer movement of drugs Higher concentrations of the copolymers accelerated the passive transport across the cell monolayers [34,35] A similar conclusion was reached by Bogman et al., who measured rhodamine 123 efflux from leukemia cells [36] On the other hand, poly(ethylene glycol) modulated resistance in Caco-2 cell monolayers by concomitant inhibition of Pgp-mediated active transport and acceleration of passive movement of drugs [33] The capacity of anesthetics to accelerate passive drug permeation without membrane permeabilization could play a role in various functions besides modulation of MDR, such as modulation of pharmacokinetics in anesthetized patients, especially enhancement of passive transport of drugs across the blood–brain barrier The present study demonstrates that anesthetics modulate MDR by accelerating passive drug permeation This mechanism of MDR modulation is not necessarily limited to anesthetics, and could be operative in MDR modulation by other modulators and under other circumstances Experimental procedures Daunorubicin, TMRM, TMA-DPH, 12-AS and mineral oil were purchased from Sigma (Rehovot, Israel) K562, a human leukemia cell line established from a patient with a chronic myelogeneous leukemia in blast transformation [37], was purchased from ATCC (Rockville, MD) and maintained in Iscove’s medium supplemented with 100 lgỈmL)1 penicillin G, 100 lgỈmL)1 streptomycin, mm glutamine, and 10% fetal bovine serum (Biological Industries, Beit-haemmek, Israel) The K562 Pgp-overexpressing subline was obtained by sequential exposure of cells to increasing concentrations of doxorubicin The resistant subline was maintained in the presence of 0.5 lm doxorubicin Continuous monitoring of daunorubicin cellular transport Uptake and efflux of the anthracycline daunorubicin was assayed by monitoring the quenching of the drug’s fluores- 6212 cence upon intercalation between base pairs of the nuclear DNA, essentially as previously described [38] The drug fluorescence was monitored continuously in a Cary eclipse fluorescence spectrophotometer (Varian Inc., Palo Alto, CA), with the temperature kept at 37 °C In a typical experiment, 106 cells were partially depleted of ATP by incubation with stirring for 30 in mL of medium composed of NaCl (132 mm), KCl (3.5 mm), CaCl2 (1 mm), MgCl2 (0.5 mm), sodium azide (1 mm), and Hepes ⁄ Tris buffer (20 mm, pH 7.4) Subsequently, the agent whose effect was being tested was added, and after a further of incubation, daunorubicin (5 lm) was added Finally, cellular ATP was replenished by addition of glucose (10 mm) In experiments not involving ATP depletion, the medium contained glucose (10 mm) instead of sodium azide Daunorubicin fluorescence was monitored using an excitation wavelength of 490 nm and an emission wavelength of 595 nm The extent of fluorescence quenching upon binding of daunorubicin to DNA was determined by exposure of daunorubicin to saturating amounts of DNA, which resulted in quenching of 94% of the fluorescence The quenching of daunorubicin bound to cellular DNA was converted to amount of daunorubicin by using a factor equal to the fluorescence of an equivalent daunorubicin solution in the absence of cells divided by the amount of daunorubicin multiplied by 0.94 Measurement of TMRM efflux from cells TMRM efflux was monitored in cells whose mitochondrial membrane potential was dissipated by CCCP Under these conditions, the dye efflux was rapid and was monitored by flow cytometry K562 cells were loaded with TMRM as follows: the cells were washed once with a medium composed of Dulbecco’s NaCl ⁄ Pi supplemented with CCCP (1 lm), MgCl2 (1 mm), CaCl2 (1 mm), and glucose (10 mm), suspended to a density of · 105 cellsỈmL)1 in the same medium, also containing TMRM (10 lm), and incubated for 30 at 37 °C Cell aliquots were pelleted and kept on ice until the assay was begun by their suspension in dye-free medium pre-equilibrated at 15 °C Samples of 2000 cells each were analyzed by flow cytometry measurements in a Becton Dickinson (Franklin Lakes, NJ) FACScan flow cytometer equipped with an argon ion laser and a thermostated jacket of the assay tube The fluorescence signal was detected through the standard FL2 channel, and was gated using the forward and side scatterings to exclude dead cells and debris from the analysis TMRM efflux from cells not exposed to the uncoupler CCCP was measured by a quantitative assay based on the rapid separation of the cells from the external medium Apart from the absence of CCCP, the experimental setup was similar to that described above To measure the TMRM amount associated with the cells, 0.4 mL samples were withdrawn and placed in an Eppendorf-style microfuge FEBS Journal 274 (2007) 6204–6214 ª 2007 The Authors Journal compilation ª 2007 FEBS R Regev et al above a 0.2 mL cushion composed of 95 parts of silicone oil AR 200 (d20 ¼ 1.049) and five parts of mineral oil (d20 ¼ 0.89) After centrifugation for at 16 060 g at room temperature, the oil cushion was washed three times with water by suction, and all the upper phase, including part of the oil cushion but leaving a fraction of the oil above the cell pellets, was removed The cell pellets were dissolved by addition of 0.05 mL of guanidine hydrochloride (5 m) buffered with Hepes ⁄ Tris (50 mm, pH 7.4), centrifugation for min, and incubation for at least h at room temperature The dissolved samples were mixed thoroughly with 0.5 mL of water and centrifuged for Samples (0.4 mL) were withdrawn from the pellets dissolved in the aqueous phase The TMRM fluorescence was determined using an excitation wavelength of 552 nm and an emission wavelength of 580 nm To ensure fidelity of the assay, dye-free cell samples were mixed with known amounts of TMRM and processed as above The TMRM yield thus obtained matched the amount expected It was determined that the time required to separate cells from the external medium was 1.5 All curves were adjusted accordingly Membrane fluidity measurements K562 cells were labeled with TMA-DPH (1 lm) by incubation for at 24 °C Steady-state fluorescence anisotropy (r) was determined using excitation and emission wavelengths of 358 nm and 428 nm, respectively Fluorescence anisotropy was calculated as described by Shinitzky & Barenholz [39] According to these authors, the fluorescence anisotropy values are inversely proportional to cell membrane fluidity A high degree of fluorescence anisotropy represents a high structural order or low cell membrane fluidity Studies on membrane fluidity performed with the fluorescent probe TMA-DPH yielded information about the rigidity of the cell membrane near the lipid polar heads [40] All the fluorescence measurements were repeated six times and corrected for the contribution of light scattering by performing control experiments on cells without fluorescent probes References Gottesman MM, Fojo T & Bates SE (2002) Multidrug resistance in cancer: role of ATP-dependent transporters Nat Rev 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