Liposomal Sphingomyelin Influences the Cellular Lipid Profile of

Article pubs.acs.org/molecularpharmaceutics

Liposomal Sphingomyelin Influences the Cellular Lipid Profile of Human Lymphoblastic Leukemia Cells without Effect on P‑Glycoprotein Activity Nadine C. L. Zembruski,† Chi D. L. Nguyen,† Dirk Theile,† Ramadan M. M. Ali,† Melanie Herzog,† Götz Hofhaus,‡ Udo Heintz,† Jürgen Burhenne,† Walter E. Haefeli,† and Johanna Weiss*,† †

Department of Clinical Pharmacology and Pharmacoepidemiology, University of Heidelberg, Im Neuenheimer Feld 410, 69120 Heidelberg, Germany ‡ CryoEM, CellNetWorks, University of Heidelberg, Im Neuenheimer Feld 267, 69120 Heidelberg, Germany

ABSTRACT: Sphingomyelin (SM)/cholesterol liposomes are currently investigated as drug carriers in cancer therapy. However, no data is available on the influence of SM itself on P-glycoprotein (P-gp) mediated multidrug resistance. P-gp is at least partly located in sphingolipid-enriched lipid raft domains of the plasma membrane, and its activity depends on the lipid profile of the membrane, which could be altered by therapeutical SM liposomes. Therefore, the aim of this study was to analyze the effect of liposomal SM on P-gp activity, P-gp distribution in microdomains, SM content of the membrane domains, and sensitivity of human lymphoblastic CEM cells toward cytotoxic drugs in vitro. Assays were conducted in CEM and multidrug resistant CEM/ADR5000 cells. SM-only liposomes were prepared by a newly developed ethanol injection protocol and thoroughly characterized. Inclusion of SM into the membrane was analyzed by fluorescence microscopy and flow cytometry. Influence of SM liposomes on P-gp activity was assessed by rhodamine efflux and calcein assay, and sensitivity toward cytotoxic drugs was analyzed by flow cytometric 7-AAD staining. Influence on P-gp distribution was analyzed by Western blot after density gradient centrifugation. SM 16:0, 18:0, and 24:1 were quantified by liquid chromatography coupled to tandem mass spectrometry. P-gp was mainly located in nonraft fractions, which did not change upon liposome treatment. Liposomes increased SM 16:0 and SM 24:1 content in nonraft domains, but not in raft domains of multidrug resistant cells. SM-only liposomes did not influence P-gp activity and chemosensitivity. In conclusion, SM-only liposomes in therapeutic amounts did not influence Pgp mediated multidrug resistance in CEM cells. KEYWORDS: P-glycoprotein, multidrug resistance, SM, liposomes, lipid rafts

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INTRODUCTION Sphingomyelin (SM) is an important sphingolipid of the cellular membrane.1 Within the plasma membrane SM is mainly located in lipid rafts, which are ordered microdomains of the cellular membrane.2 Lipid rafts mainly contain lipids with saturated longer fatty acid chains and are enriched with SM and cholesterol, resulting in tight packing3 and reduced fluidity of the membrane. Detergent-resistant membranes (DRMs) are methodological correlates of lipid rafts, and enrichment of proteins in DRMs is suggested to indicate affinity to lipid rafts.4 The physiological function of ABC transporters is the transport of endogenous or exogenous compounds across membranes. Transport may participate in all pharmacokinetic steps and thus modify the distribution of substrates within the © 2013 American Chemical Society

body and facilitate or limit access to organs and thereby protect against xenobiotics.5−8 Overexpression of ABC transporters in cancer cells can lead to increased efflux of cytotoxic drugs and subsequent resistance of cancer cells toward a variety of structurally and functionally diverse cytotoxic drugs (multidrug resistance, MDR).6 Multiple studies have demonstrated partial, main, or no localization of P-gp in DRMs depending on the cell line and extraction protocol.9 In CEM cells two populations of P-gp Received: Revised: Accepted: Published: 1020

August 31, 2012 January 18, 2013 February 4, 2013 February 4, 2013 dx.doi.org/10.1021/mp300485j | Mol. Pharmaceutics 2013, 10, 1020−1034

Molecular Pharmaceutics

Article

trile and methanol were obtained from Carl Roth, (Karlsruhe, Germany). Anti-P-gp antibody (Clone C219), saponine, DLserine, TBME, and vincristine sulfate were obtained from Merck (Darmstadt, Germany). Pefabloc was obtained from Serva (Heidelberg, Germany), and leupeptin, pepstatin, and bromphenol blue were obtained from Biomol (Hamburg, Germany). Nitrocellulose membranes (Optitran BA-S 85) were obtained from Schleicher & Schuell BioScience (Dassel, Germany). Fetal calf serum (FCS) and HEPES were purchased from PAA Laboratories (Pasching, Austria) and TRIS and NaCl from AppliChem (Darmstadt, Germany). Pheophorbide A (PhA) was purchased from Frontier Scientific Europe (Carnforth, U.K.) and calcein acetoxymethyl ester (calcein AM) from Invitrogen (Karlsruhe, Germany). Egg SM, SM 16:0, SM 18:0, SM 24:0, and N-[6-[(7-nitro-2-1,3-benzoxadiazol-4yl)amino]chexanoyl]sphingosine-1-phosphocholine (C6-NBD SM) were obtained from Avanti Polar Lipids (Alabaster, AL, USA). Rhodamine 123 (rhodamine) was from Calbiochem (San Diego, CA, USA) and LY335979 (zosuquidar) from Lilly (Bad Homburg, Germany). Camptothecin and doxorubicin were purchased from Molekula (Taufkirchen, Germany), and Slim Fast was purchased from Slim Fast (Messel, Germany). RNeasy Mini-Kit was obtained from Qiagen (Hilden, Germany) and the RevertAid H Minus First Strand cDNA Synthesis Kit from Fermentas (St. Leon-Rot, Germany). Materials for real-time PCR (LightCycler capillaries and LightCycler FastStart DNA Master SYBR Green I kit), the Cytotoxicity Detection KitPlus LDH, and Triton X-100 were from Roche Applied Science (Mannheim, Germany). The Absolute QPCR SYBR Green Mix was obtained from Abgene (Hamburg, Germany). Primers were synthesized by Eurofins MWG Operon (Ebersberg, Germany). Doxorubicin and daunorubicin were supplied by Toronto Research Chemicals (Toronto, Canada). CCRF-CEM and CEM/ADR5000 Cells. The human T lymphoblast cell line CCRF-CEM (CEM) (ATCC, Manassas, VA, USA) and the multidrug resistant subline CEM/ADR5000 (kindly provided by Professor Dr. Thomas Efferth, Mainz, Germany) overexpressing human multidrug resistance gene 1 (MDR1/ABCB1) encoding for P-gp27 were cultivated under standard cell culture conditions. RPMI medium supplemented with 10% FCS, 100 units/mL penicillin, and 100 μg/mL streptomycin was used as culture medium. To maintain P-gp overexpression the culture medium for CEM/ADR5000 cells was supplemented with 0.43 μM doxorubicin. Cells were counted with an electronic cell counter (CASY, Schärfe System, Reutlingen, Germany). Quantification of mRNA Expression by Real-Time RTPCR. RNA isolation, cDNA synthesis, and real-time RT-PCR were conducted as published previously.28 Primer sequences were also published previously.28−31 The most suitable housekeeping gene for normalization was identified using geNorm (Center for Medical Genetics, Ghent, Belgium), which determines the most stable reference gene from a set of tested genes in a given cDNA sample panel.32,33 Hypoxanthine-guanine phosphoribosyltransferase (hPRT) proved to be the most stable housekeeping gene in CEM cells among the tested housekeeping genes (ß2-microglobulin, glucose-6-phosphate dehydrogenase, glucuronidase beta, HPRT, human acidic ribosomal protein, ribosomal protein L13) under the experimental conditions. Data was evaluated by calibrator-normalized relative quantification with efficiency correction using the RelQuant software version 1.01 or the LightCycler 480 software version 1.5 (Roche

have been described of which one with high ATPase activity was located in raft fractions whereas the other population was less active and located in nonraft fractions.10 P-gp activity depends on the specific lipid profile of surrounding membrane regions.11−14 Moreover, P-gp reconstitution in liposomes with tightly packed SM and cholesterol or phosphatidylcholine (PC) 16:0/16:0 and cholesterol increased P-gp activity, suggesting that lipid raft domains could enhance P-gp activity.15 Additionally, the amount of esterified cholesterol has been described to correlate with the degree of MDR in CEM cells. Taken together, cholesterol and other raft-associated lipids such as SM with its high affinity to cholesterol appear to influence MDR.16 Liposomes are small vesicles that are used as drug carriers in cancer treatment to increase the concentration of the encapsulated drug at the tumor site and hence increase effectiveness and tolerability. The majority of liposomes contain PC and cholesterol as main components.17 In addition, formulations containing SM have been developed. SM-based liposomes are supposed to have the advantage of high drug retention and increased circulation time as shown for vincristine.18 Several SM-based liposomal formulations containing, e.g., vinorelbin, topotecan, or vincristine are currently investigated in preclinical or clinical trials in the treatment of several cancer diseases,19−24 and it has been suggested that SMbased vincristine liposomes might be a salvage option in relapsed or refractory ALL.22 Liposomal formulations used in drug therapy usually contain further excipients such as cholesterol in addition to the major lipid component. Cholesterol, however, has been demonstrated previously to influence P-gp activity25,14,26 and was therefore excluded from the liposomal preparation in this study. For the evaluation of the effect of liposomal SM on P-gp mediated MDR a model for SM-based liposomes without further excipients is required. We therefore developed a simple preparation method for SM-only liposomes combining ethanol injection with sonication and optimized process parameters to control liposome characteristics and stability. This method is simple and robust and can easily be applied in pharmacological or toxicological laboratories without pharmaceutical apparatus. Although SM liposomes are tested in clinical trials in the treatment of leukemia and although the lipid profile of the plasma membrane has been described to influence P-gp activity, no data is available on the influence of liposomal SM on P-gp activity in lymphoblast cells. We therefore treated human acute lymphoblastic leukemia (CEM) cells and their multidrug resistant subline with SM-only liposomes and assessed their influence on P-gp activity, P-gp distribution in raft and nonraft membrane domains, SM concentration in membrane domains, and sensitivity of parental and multidrug resistant CEM cells toward the cytotoxic drugs camptothecin, vincristine, and doxorubicin.

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MATERIALS AND METHODS Materials. RPMI medium, Hanks balanced salt solution (HBSS), phosphate buffered saline (PBS), dimethyl sulfoxide (DMSO), anti-β-actin antibody (Clone AC-74), penicillin− streptomycin (10 000 units of penicillin and 10 mg of streptomycin per mL), bovine serum albumin (BSA), ethanol, Tween 20, ammonium acetate, and 7-aminoactinomycin D (7AAD) were purchased from Sigma-Aldrich (Taufkirchen, Germany). Anticaveolin 1 antibody (Clone 2297) was obtained from Becton Dickinson (Heidelberg, Germany), and acetoni1021

dx.doi.org/10.1021/mp300485j | Mol. Pharmaceutics 2013, 10, 1020−1034


Article

Cytotoxicity was expressed as a percentage of the effect obtained with the positive control (Triton X-100 1%; >30% = cytotoxic). Furthermore, cytotoxicity of doxorubicin (1−50 μM) in CEM and CEM/ADR5000 cells was determined prior to quantification of intracellular doxorubicin concentrations to ensure that intracellular concentrations are not modified by acute cytotoxic effects. The assay was conducted as described by the manufacturer’s instructions. Treatment of Cells with Liposomes. Cells were counted and adjusted to the desired cell concentration. Concentration was set to 2 × 105 to 2 × 106 cells/mL (concentration range for cultivation), if not stated otherwise. The cell suspension was centrifuged at 400 g and room temperature for 5 min, and the supernatant was discarded. The cell pellet was resuspended in the liposome suspension with the desired concentration and incubated at 37 °C on a shaker for 30 min if not stated otherwise. After incubation, cells were washed and further analyzed. Treatment with Cytotoxic Drugs. CEM and CEM/ ADR5000 cells were treated with camptothecin, vincristine, or doxorubicin subsequent to liposome treatment to determine the impact of liposomes on the sensitivity of CEM and CEM/ ADR5000 cells toward cytotoxic drugs. In brief, CEM and CEM/ADR5000 cells (adjusted to a concentration of 2 × 105 to 2 × 106 cells/mL, pretreated with liposomes, and washed) were resuspended in cell culture medium containing 100−400 μM vincristine, 0.01−50 μM doxorubicin, or 0.01−50 μM camptothecin (different for parental and overexpressing cells). Camptothecin was incubated for 16 h, doxorubicin and vincristine for 24 h under standard cell culture conditions. After incubation, cell suspensions were washed with RPMI with 2% FCS and stained with rhodamine and 7-AAD. Isolation of DRMs. DRMs of 4 × 107 CEM/ADR5000 cells per sample were isolated by density gradient centrifugation to analyze nonraft and raft fractions. Isolation was conducted as described previously.37 Samples were centrifuged in a SW-40 rotor (Beckman Coulter, Krefeld, Germany) at 39,000 rpm and 4 °C for 18 h with low acceleration and without break. Fractions were either analyzed directly (protein content, cholesterol content, SM content, and Western blot analysis) or stored at −20 °C until analysis. Protein Quantification. Protein quantification was conducted in cell lysates to enable loading of equal protein amounts on the SDS−PAGE and in fractions of DRM isolation for characterization of the protein distribution in the fractions. Quantification was conducted using the BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions. Western Blot Analysis. Western blot analysis was conducted to characterize protein expression in CEM and CEM/ADR5000 cells and to characterize protein content of raft and nonraft fractions after density gradient centrifugation as described previously with minor modifications.30 Unspecific binding sites were blocked by incubation for 1 h with 5% Slim Fast (w/v) in phosphate buffered saline containing 0.1% Tween 20. Immunoblot analysis was carried out with monoclonal antibodies raised against Cav-1 (1:250; overnight at 4 °C), βactin (1:40 000; 30 min at room temperature), and P-gp (1:100, overnight at 4 °C). Cholesterol Quantification. Cholesterol was quantified with the Amplex Red Cholesterol Assay Kit according to the manufacturer’s instructions.

Applied Science, Mannheim, Germany) as described previously.34 All samples were amplified at least in duplicate. Preparation of SM-Only Liposomes. Liposomes were prepared by ethanol injection as described for phosphatidylcholines with some modifications.35,36 In brief, preheated ethanolic egg SM was rapidly injected from a Hamilton syringe into RPMI medium (45 °C), which was vigorously stirred on a magnetic stirrer, followed by sonication for 20 s. 25 mM and 50 mM stock solutions were investigated at injection ratios of 1:20 (one volume of stock solution into 19 volumes of RPMI medium) and 1:50 (n = 3). Z-average and polydispersity index (PDI) were assessed by photon correlation spectroscopy (measurement angle 173° backscatter) on the day of preparation, and after 1, 2, 3, and 4 weeks of storage at 4 °C (n = 3). Reproducibility was tested by the analysis of mean and standard deviation (SD) of 20 independent preparations performed by five blinded laboratory members for the 50 mM/1:20 preparation protocol. Stability upon dilution from a 2.5 mM liposome to a 20 μM liposome suspension with cell culture medium was evaluated as alteration in z-average and PDI. Stability under cell culture conditions (37 °C, 5% CO2) was tested at the concentration of 20 μM for four days. Preparation of Fluorescent C6-NBD-SM Liposomes. Fluorescent SM-only liposomes for analysis of SM distribution in CEM cells upon exposure were prepared identically to regular SM-only liposomes using egg SM (50 mM) containing 1% (mol/mol) fluorescent C6-NBD-SM. Cryoelectron Microscopy of Liposomes. To assess the structure of SM-only liposomes and fluorescence labeled SM liposomes cryoelectron microscopy was conducted. Liposomes (2.5 mM) were applied to a glow discharged specimen support grid and blotted from one side in a humidified atmosphere for 2 s. The support grid was plunged into liquid ethane and mounted under liquid nitrogen on a Gatan 350 cold stage (Gatan, Munich, Germany). The stage was transferred on a Zeiss 923 electron microscope (Carl Zeiss, Gö ttingen, Germany). The microscope was equipped with a field emission gun operated at 200 kV and an Omega filter (corrected in column) with a slit width of 50 eV. Zero loss images were recorded with a 4 k Tietz camera at about 10 m under focus at magnifications of 50 kx. Cytotoxicity Testing. The cytotoxic effect of SM-only liposomes (2 μM, 20 μM, 200 μM, and 2.5 mM) in CEM and CEM/ADR5000 cells was tested with the Cytotoxicity Detection Kit (Roche Applied Science). The assay was conducted according to the manufacturer’s instructions with the modification that RPMI medium (solvent of liposomes) was used as background control. CEM/ADR5000 cells were cultivated in culture medium without doxorubicin for at least one day prior to the assay. In brief, 6.5 × 104 cells in culture medium were pipetted into the wells of a 96-well plate. Cells were washed with Hanks balanced salt solution, and the supernatant was aspirated. Cells were resuspended in liposomal preparations (2 μM, 20 μM, 200 μM, and 2.5 mM in RPMI1640 medium, each in quadruplicate) and incubated at 37 °C under shaking for 2 h. RPMI-1640 medium served as solvent for the background and low control. After incubation, 100 μL of freshly prepared reaction mixture was added to the wells and the mixture incubated at room temperature for 30 min in darkness. Absorption was measured at 492 nm by a Multiskan RC microplate reader. Cytotoxicity was expressed as percentage of the effect obtained with the positive control (Triton X-100 1%). 1022



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SM-only liposomes (20 μM) or cell culture medium (negative control) in quadruplicate under standard cell culture conditions for four days. On day four, cells were harvested for quantification of mRNA. Inclusion of Liposomal SM into CEM Cells. CEM cells (n = 3) were incubated with 20 μM SM-only or 20 μM C6NBD-SM liposomes for 30 min at 37 °C. SM-only liposomes served as a negative control. After incubation, cells were washed twice with PBS (4 °C) and incubated with 0.5 μM pheophorbide A on ice for 15 min for cytosol staining. Afterward cells were washed twice with PBS (4 °C) and analyzed immediately by flow cytometry and fluorescence microscopy. For fluorescence microscopy the cell suspension (250 μL) was pipetted on a coated cover glass and incubated for 15 min. After incubation the cover glass was carefully rinsed with PBS (4 °C) and mounted on a microscope slide. Microscope slides were analyzed by fluorescence microscopy. Flow cytometric analysis was conducted on a LSR II flow cytometer (BD, Heidelberg, Germany). NBD fluorescence of 30 000 events was measured at 530/30 nm. CEM cells were gated in the forward scatter versus side scatter plot. Inclusion of liposomal SM was evaluated as percentage of NBD-positive cells. Quantification of Apoptosis by Flow Cytometric 7AAD Staining. Quantification of apoptosis by flow cytometric 7-AAD staining was conducted as described previously.40 In brief, CEM and CEM/ADR5000 cells (105−106 cells per sample) were washed with RPMI with 2% FCS and centrifuged at 400g and room temperature for 5 min. Each sample was resuspended in 250−500 μL of RPMI with 2% FCS and 5 μg/ mL 7-AAD. Samples were incubated for 20 min on ice in darkness. After incubation, samples were centrifuged (400g, 5 min, 4 °C), washed once with 1 mL of PBS with 2% FCS (4 °C), centrifuged again (400g, 5 min, 4 °C), and resuspended in 200−500 μL of PBS with 2% FCS (4 °C). Samples were stored on ice and analyzed by flow cytometry within one hour. Viable, apoptotic, and late apoptotic/dead cells were analyzed in a forward scatter versus 7-AAD dot plot. Two control samples were used for standardized gating of cell populations. Untreated and unstained cells served as a control for viable cells. To accurately and reproducibly set the gates of apoptotic and late apoptotic/dead cells we measured a second control sample that was stained with 5 μg/mL 7-AAD and saponine. Saponines as surface-active glycosides permeabilize cellular membranes41 thus imitating leakiness of late apoptotic/dead cells. The gate defined by saponine treated cells represented late apoptotic/ dead cells. Fluorescence intensity values between late apoptotic/dead and viable cells were considered to represent apoptotic cells. Calcein Assay. The calcein assay was used to assess P-gp activity in CEM and CEM/ADR5000 cells treated with liposomes to confirm the results obtained by rhodamine efflux analysis, which also measures P-gp activity. The assay was conducted as described previously with minor modifications.27,42,43 CEM/ADR5000 cells were cultivated in doxorubicin-free culture medium for at least one day prior to the assay. Cells were adjusted to a concentration of 3 × 106 cells/ mL, and 100 μL of the cell suspension was aliquoted. The suspension was centrifuged at 400g and room temperature for 5 min, and the supernatant was discarded. The pellets were resuspended in 50 μL of the liposome suspensions at concentrations of 0.02−200 μM and incubated at 37 °C for

Quantification of Intracellular Doxorubicin in CEM and CEM/ADR5000 Cells after SM-Only Liposome Treatment. Intracellular doxorubicin concentrations were measured to analyze the effect of SM-only liposomes on doxorubicin uptake. CEM and CEM/ADR5000 cells were adjusted to 1.5 × 106 cells/mL and treated with SM-only liposomes or RPMI medium (negative control) for 30 min (conducted in quadruplicate). Cells were washed with RPMI medium, centrifuged (800g, 5 °C, 5 min), and incubated in 0.1−5 μM (CEM) or 1−50 μM (CEM/ADR5000) doxorubicin for 2 h under standard cell culture conditions. Cytotoxicity testing was done in advance to ensure that doxorubicin did not have cytotoxic effects under these conditions. After incubation, cell suspensions were centrifuged (800g, 4 °C, 5 min) and washed twice with PBS (4 °C) to ensure removal of extracellular doxorubicin. Cell pellets were resuspended in 115 μL of PBS (4 °C), and 100 μL was transferred to plastic tubes for doxorubicin extraction. 10 μL was used for determination of the cell concentration in each sample. The cell suspension was stored at −20 °C until analysis. Detection and quantification of intracellular doxorubicin concentrations was performed using protein precipitation and high performance liquid chromatography with fluorescence detection on a Thermo Electron HPLC system (P4000 pump, AS3000 sampler, and FL3000 detector; Thermo Electron Scientific, Waltham, MA). The method was established as described previously with further optimization of sample extraction and chromatography.38 In brief, calibration, quality control (QC), and unknown samples were spiked with internal standard (daunorubicin 500 ng/mL), borate buffer (pH 10.0, 100 μL) was added, and the cell suspension was homogenized by sonication. For extraction 2-propanol (2 mL) and tert-butyl methyl ether (TBME, 3 mL) were added and the samples were shaken overhead (10 min). The samples were subsequently centrifuged at 3000g for 10 min, and the organic layer (4 mL) was transferred to a separate tube. The organic phase was evaporated in a water bath at 40 °C under a stream of nitrogen, and the dried residue was reconstituted in 200 μL of the HPLC eluent. The injection volume was 20 μL. Chromatographic separation was done on a Fusion-RP column (150 mm × 2.1 mm, 80 Å pore size, 4 μm particle size (Phenomenex, Aschaffenburg, Germany) using isocratic conditions of 80% ammonium acetate buffer (5 mM adjusted to pH 3.5 with acetic acid), 10% methanol, and 10% acetonitrile from min 0−5. Thereafter, the organic content was increased to 50% methanol and 50% acetonitrile (min 5−7) and run isocratically for a further 9 min (min 7−16). The flow rate was 0.35 mL/min, and the column was heated to 40 °C. The analytes were detected at an excitation wavelength of 460 nm and emission wavelength of 550 nm. Calibration for doxorubicin in cells was done in the range of 0.09 ng/100 000 cells to 183.37 ng/100 000 cells. The lower limit of quantification was 0.09 ng/100 000 cells. The assay was validated using QC samples in the upper, middle, and lower calibration range according to the procedures recommended by the U.S. Food and Drug Administration.39 The overall accuracy varied from 1.4 to 8.2%, and the overall precision ranged from 4.2 to 7.3% (% CV batch-to-batch). Induction Assay in CEM Cells. The induction assay was used to test whether SM-only liposomes induce the mRNA expression of MDR1/ABCB1 or cav-1 because both proteins are relevant for MDR. CEM cells (1 × 106 cells) were treated with 1023



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a temperature of 50 °C. HPLC was established as described previously47 with further optimizations. HPLC was run at a flow rate of 0.4 mL/min with eluent B (methanol/acetonitrile 6:4 with 5% chloroform, 2.5 mM ammonium acetate, and 10 μM serine) and eluent A (H2O with 2.5 mM ammonium acetate and 10 μM serine) at the following gradient modus: 0− 1 min 35% A, 1−10 min 35−2% A, 10−30 min 2% A. SMs were detected by tandem mass spectrometry on a TSQ 7000 mass spectrometer (Thermo Fisher, Dreiech, Germany). ESI interface parameters were as follows: middle position, spray voltage 4.5 kV, sheath gas (N2) 90 psi, auxiliary gas (N2), and capillary heater temperature 350 °C. Collision voltage was set at −36 V and the multiplier voltage at 1.6 kV. The LC/MS/MS system was tuned using Xcalibur system software version 1.2 to SM 16:0, SM 18:0, and SM 24:1 using stock solutions and direct infusion via a syringe pump in the selected reaction monitoring mode (SRM). The following MS/MS transitions [mass/charge] were monitored: SM 16:0 (703.57−184.1), SM 18:0 (731.59−184.1), SM 24:1 (813.68−184.1). Statistical Analysis. All statistical analyses were performed with GraphPad InStat Version 3.10 or GraphPad Prism Version 5.0 (GraphPad, La Jolla, CA, USA). Statistical significance of differences in MDR1/ABCB1 mRNA expression and of P-gp activity in CEM and CEM/ADR5000 cells was evaluated by two-tailed t test. Differences in protein and cholesterol distribution in density gradient centrifugation fractions and influence of liposome treatment on sensitivity toward cytotoxic drugs were evaluated by two-way ANOVA with Bonferroni post hoc test. The rhodamine ratio in liposomes and drug treated cells was evaluated with repeated measures ANOVA with post hoc test for linear trend between column means and column number.

30 min under shaking. The suspension was centrifuged and the supernatant discarded, and the cell pellets were washed with PBS. The cell pellets were resuspended in 50 μL of calcein AM (2 μM) and incubated at 37 °C for 30 min under shaking. Afterward, the uptake was stopped on ice and cells were washed twice with precooled HEPES buffered HBSS. Cells were lysed with 1% Triton X-100 for 15 min, and calcein fluorescence was measured with 485 nm excitation and 535 nm emission filters on a Fluoroskan Ascent Fluorometer (Labsystems, Helsinki, Finland). Each concentration was tested in quadruplicate in 5− 6 experiments on two different days. Rhodamine Efflux. The rhodamine efflux analysis was conducted as described previously.44 P-gp activity was determined as the ratio of median fluorescence of cells incubated with the specific P-gp inhibitor LY335979 (inhibited) and cells incubated with medium (not inhibited).45 Quantification of P-gp Activity in 7-AAD Stained Cells. To assess P-gp activity in viable, apoptotic, and late apoptotic/dead cell populations, CEM and CEM/ADR5000 cells were stained with rhodamine and 7-AAD. Samples were first stained with rhodamine and LY335979 (inhibited) or medium alone (not inhibited). Subsequent to incubation with LY335979, samples were washed with RPMI with 2% FCS and resuspended in RPMI with 2% FCS containing 5 μg/mL 7AAD (4 °C) and further processed as described above. Quantification of SM. CEM cells were centrifuged at 800− 1000 g and 5 °C for 5 min. Cell pellets were washed twice with PBS (4 °C), and the supernatant was removed completely. Each sample (5 × 105 cells, if not stated otherwise) was resuspended in 200 μL of water for injection and transferred to glass centrifugation tubes. Calibration samples were prepared by spiking blank matrix with the calibration solutions yielding calibration samples with 0, 5, 12.38, 27.5, 88.75, 162.5, and 250 nM of SM 16:0, SM 18:0, and SM 24:1. QC samples were prepared by spiking blank matrix with 25 μL of the internal standard and 25 μL of QCA, QCB, or QCC solutions to yield QC samples with 8.12 nM, 81.24 nM, and 187.5 nM. Matrix consisted of BSA solution for quantification in CEM cells (16.5 μg/200 μL, protein concentration of 1 × 106 CEM cells). For the analysis of raft and nonraft fractions 15% sucrose in TNE buffer (TRIS, NaCl, EDTA) and 40% sucrose in TNE buffer with 2 μL/mL Triton-X 100 were used as matrix, respectively. The liquid−liquid extraction was conducted as described previously with modifications.46 In brief, samples were homogenized by sonication and subsequent vortexing. Methanol (1.5 mL) was added, and the mixture was vigorously vortexed. 5 mL of TBME was added, and the glass tubes were rotated for one hour in an overhead shaker. Afterward 1.25 mL of water for mass spectrometry was added and the tubes were rotated for a further 10 min in an overhead shaker. For phase separation the tubes were centrifuged at 1000g and room temperature for 10 min. 4 mL of the upper organic phase was transferred to a fresh glass tube and evaporated in a water bath at 40 °C under a nitrogen stream. The dried residue was dissolved in 1.0 mL of methanol and stored at −20 °C until analysis. Lipids were separated by high performance liquid chromatography (HPLC). The HPLC system consisted of a HTC PAL autosampler (CTC Analytics, Zwingen, Switzerland) and a SpectraSystem P4000 HPLC System with ERC 310SP degasser (Thermo Fisher, Dreieich, Germany). Chromatographic separation was done on a Synergi Max-RP column (C12, 150 mm × 2.0 mm, 80 Å pore size, 4 μm particle size) (Phenomenex, Aschaffenburg, Germany) with guard column at

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RESULTS Characterization of CEM and CEM/ADR5000 Cells. Western blot analysis of CEM (3 technical replicates) and CEM/ADR5000 (4 technical replicates) cells demonstrated that P-gp expression was profoundly higher in CEM/ADR5000 than in parental cells. Cav-1 protein expression was low in both cell lines (Figure 1). mRNA expression of the ABC-transporters that are relevant for the pharmacokinetics of camptothecin, vincristine, and doxorubicin (ABCB1, ABCC1, ABCC2, ABCC3, ABCC4,

Figure 1. Western blot analysis of P-gp and cav-1 in CEM (3 technical replicates) and CEM/ADR5000 cells (4 technical replicates). 1024



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ABCC6, ABCC10, and ABCG2) was quantified in CEM and CEM/ADR5000 cells. ABCC2 could not be detected in either cell line, and ABCC3 and ABCC6 were detected in all samples but expression was too low to be quantified (expression at the lower limit of detection). ABCB1 mRNA was increased approximately 650-fold in the MDR cell line compared to the parental cell line (p < 0.0001), confirming results of Western blot analysis. CEM and CEM/ADR5000 did not differ in the expression of ABCC1, ABCC4, ABCC10, or ABCG2. Rhodamine ratio was more than three times higher in CEM/ ADR5000 (4.3 ± 0.9) than in parental CEM cells (1.3 ± 0.3) (p < 0.0001, n = 18 experiments), confirming the functional relevance of the PCR and Western blot results. Development and Characterization of SM-Only Liposomes Prepared by Ethanol Injection. The liposomes obtained by the described preparation protocols had a size determined as z-average of 71 ± 3 nm (25 mM ethanolic egg SM injected at a ratio of 1:20 (volume/volume) into medium), 85 ± 5 nm (50 mM/1:20), 76 ± 8 nm (25 mM/1:50), and 77 ± 5 nm (50 mM/1:50). Statistical analysis of the differences revealed that the concentration of the stock solution and the injection ratio significantly influenced the size. The liposomes prepared by the 50 mM/1:20 protocol differed significantly from all other tested protocols (p < 0.001, 25 mM/1:20 liposomes; p < 0.05, 25 mM/1:50 liposomes; p < 0.01, 50 mM/ 1:50 liposomes). The size distribution assessed as PDI was 0.348 ± 0.050 (25 mM/1:20), 0.215 ± 0.052 (50 mM/1:20), 0.505 ± 0.174 (25 mM/1:50), and 0.243 ± 0.036 (50 mM/ 1:50). The size distribution also differed significantly and was best for the protocol injecting 50 mM SM at a ratio of 1:20. Liposomes prepared by injecting 25 mM at a ratio of 1:50 were not stable at 4 °C and formed larger aggregates in contrast to the other liposomes that were stable for at least 28 days (Figure 2).

0.05 after dilution. Stability under cell culture conditions was likewise good: z-average and PDI were 58 ± 1 nm and 0.168 ± 0.047 before incubation, 57 ± 2 nm and 0.113 ± 0.015 after 24 h, and 56 ± 2 nm and 0.123 ± 0.028 after 96 h (p > 0.05). Z-average and PDI of NBD-labeled SM liposomes were 87 ± 11 nm and 0.32 ± 0.13 (n = 3). Cryoelectron Microscopy. Cryoelectron microscopy documented that SM-only liposomes and NBD-labeled SM liposomes were unilamellar vesicles and comparable in structure (Figure 3).

Figure 3. Cryoelectron microscopy of 2.5 mM SM-only (A) and NBD-labeled SM liposomes (B).

Cytotoxicity of SM-Only Liposomes. SM-only liposomes were not cytotoxic in CEM/ADR5000 cells at all tested concentrations. In the parental CEM cell line liposomes up to the concentration of 200 μM were not cytotoxic, whereas the 2.5 mM liposome suspension was cytotoxic (38%), i.e., at a liposome concentration not applied in subsequent experiments. Inclusion of Liposomal SM into CEM Cells. CEM cells were treated with SM-only liposomes containing 1% (mol/ mol) fluorescent C6-NBD-SM. Samples for fluorescence microscopy were further stained with 0.5 μM pheophorbide A for cytosol staining. Figure 4 shows a representative fluorescence microscopy image of CEM cells (n = 3) and illustrates that fluorescent SM predominantly accumulated in the plasma membrane of CEM cells.

Figure 2. Z-average of SM-only liposomes prepared by different ethanol injection protocols during storage at 4 °C for 28 days. Results are displayed as mean ± SD of 3 preparations on different days.

The ethanol injection protocol injecting 50 mM SM at a ratio of 1:20 yielded stable liposomes with the narrowest size distribution and was therefore further characterized with regard to suitability for cell culture experiments. The preparation method showed a low interday and interpersonal variability as shown by the z-average and PDI of 20 independent preparations (79 ± 10 nm and 0.24 ± 0.06). To test the usability for cell culture experiments it was demonstrated that further dilution of the 2.5 mM suspension (25 mM ethanolic egg SM injected at a ratio of 1:20) to a 20 μM suspension did not change z-average and PDI considerably. Z-average and PDI were 60 nm and 0.186 before and 58 ± 1.4 nm and 0.168 ±

Figure 4. Fluorescence microscopy of CEM cells stained with C6NBD-SM liposomes (green) and pheophorbide A (red) for cytosol staining.

Analysis by flow cytometry revealed that 98.1 ± 0.4% of CEM cells treated with C6-NBD-SM liposomes showed high NBD-fluorescence (detected at 530/30 nm) and thus incorporated C6-NBD-SM. Influence of SM-Only Liposomes on SM Content of CEM and CEM/ADR5000 Cells. CEM and CEM/ADR5000 cells were treated with SM-only liposomes, and SM content was 1025



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Figure 5. SM content in CEM (A) and CEM/ADR5000 (B) cells as quantified by LC/MS/MS. Results are expressed as mean ± SD (n = 6−8). ** p < 0.01.

Figure 6. Western blot analysis of density gradient centrifugation fractions of CEM/ADR5000 cells after medium (A) or SM-only liposome (B) treatment. One exemplary plot of three experiments is displayed.

subsequently quantified by LC/MS/MS to assess whether liposome treatment increased cellular SM content. SM-only liposomes were prepared with egg SM that contains 86% SM 16:0, 6% SM 18:0, 3% SM 24:1, 3% SM 22:0, and 2% unknown lipids. Therefore SM 16:0, SM 18:0, and SM 24:1 were quantified. SM 22:0 was not available as standard for LC/MS/ MS calibration and could therefore not be quantified. The results of SM quantification are displayed in Figure 5. SM-only liposomes did not influence the absolute amount of SM 16:0 and SM 24:1 in CEM cells (A). SM 18:0 was detected, but concentrations were below the limit of quantification (2.67 ng/ 500 000 cells). SM-only liposomes significantly increased the concentration of SM 18:0 and SM 24:1 in CEM/ADR5000

cells (p < 0.01) (B) to a small extent. SM 16:0 content was not influenced by liposomes (B). Induction of MDR1/ABCB1 and Cav-1 mRNA Expression by SM-Only Liposomes. CEM cells were treated with SM-only liposomes for four days to assess whether liposomes influence MDR via induction of MDR1/ABCB1 or cav-1. mRNA expression of MDR1/ABCB1 (normalized ratio to medium control) was 1.2 ± 0.8 in SM-only liposome treated CEM cells. Cav-1 mRNA expression was 0.9 ± 0.5 in SM-only liposome treated CEM cells. Statistical analysis revealed no significant induction of MDR1/ABCB1 or cav-1 mRNA expression by SM-only liposomes (p > 0.05, n = 4). Protein and Cholesterol Distribution in Density Gradient Centrifugation Fractions. Protein content was 1026



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Figure 7. Absolute amount of SM 16:0, SM 18:0, and SM 24:1 in raft fractions (fraction 10 + 11) (A) and nonraft fractions (fraction 3 + 4) (B) of CEM/ADR5000 cells as quantified by LC/MS/MS. Results are expressed as mean ± SD (n = 4). * p < 0.05, ** p < 0.01.

Figure 8. Percentage of viable (A), apoptotic (B), and late apoptotic/dead (C) CEM and CEM/ADR5000 (D) cells that have been treated with SMonly liposomes and subsequently with camptothecin. Results are expressed as mean ± SD for four experiments on two different days. Viable, apoptotic, and late apoptotic CEM cells are displayed in three separate plots, because illustration in one image is unclear due to crossing curves of the populations.

Western Blot Analysis of Density Gradient Centrifugation Fractions. P-gp and cav-1 protein distribution in density gradient fractions (n = 3) were analyzed by Western blot analysis. A representative Western blot analysis is shown in Figure 6 (n = 3). The raft marker cav-1 is only weakly expressed in CEM/ADR5000 cells (see also Figure 1) and could only be clearly detected in fractions 1−4 with the highest total protein amount when images were overexposed (not shown). P-gp was primarily found in fractions 1−5, whereas fractions 9−11 only

highest in nonraft fractions (fractions 1−4, bottom of ultracentrifuge tube) and lowest in raft fractions (fractions 9− 11). The distribution was similar as published previously.37 SMonly liposomes did not influence the protein distribution (p > 0.05, n = 3). Cholesterol was predominantly found in nonraft fractions and to a small extent in raft fractions and followed the typical bimodal distribution.37 SM-only liposomes did not influence cholesterol distribution (p > 0.05, n = 3). 1027



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Figure 9. P-gp activity determined as rhodamine ratio in viable CEM (A) and CEM/ADR5000 (B) cells that have been treated with SM-only liposomes or medium and subsequently with camptothecin. Results are expressed as mean ± SD for four experiments on two different days.

Figure 10. Percentage of viable (A), apoptotic (B), and late apoptotic/dead (C) CEM and CEM/ADR5000 (D) cells that have been treated with SM-only liposomes and subsequently with vincristine. Results are expressed as mean ± SD for three experiments on different days. Viable, apoptotic, and late apoptotic CEM cells are displayed in three separate plots, because illustration in one image is unclear due to crossing curves of the populations.

contained low amounts of P-gp compared to nonraft fractions. SM-only liposomes did not provoke a significant displacement of P-gp (Figure 6). SM Concentration in Density Gradient Centrifugation Fractions. The absolute content of SM in raft and nonraft fractions of CEM/ADR5000 cells was determined by LC/MS/ MS. For the analysis of raft fractions 100 μL of fraction 3 and 100 μL of fraction 4 were combined and SM content was quantified. For the analysis of nonraft fractions 100 μL of fraction 10 and 100 μL of fraction 11 were combined. Results are expressed as total amount of SM (ng) in fractions 3 + 4 and

10 + 11 (n = 4, 2 density gradient centrifugations quantified in duplicate) and are shown in Figure 7. SM-only liposomes significantly increased the content of SM 16:0 and 24:1 in nonraft fractions, but not in raft fractions. P-gp Activity after Liposome Treatment. To assess P-gp activity after liposome treatment two functional assays, the calcein assay and rhodamine efflux, were conducted. In the calcein assay, fluorescence intensity was not changed in CEM/ ADR5000 cells, indicating that SM-only liposomes did not influence P-gp activity. Rhodamine ratio in CEM/ADR5000 cells was 4.3 ± 0.9 in untreated and 4.2 ± 1.2 in liposome 1028



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Figure 11. P-gp activity determined as rhodamine ratio in viable CEM (A) and CEM/ADR5000 (B) cells that have been treated with SM-only liposomes and vincristine. Results are expressed as mean ± SD for three experiments on different days.

Figure 12. Percentage of viable (A), apoptotic (B), and late apoptotic/dead (C) CEM and CEM/ADR5000 (D) cells that have been treated with SM-only liposomes and subsequently with doxorubicin. Results are expressed as mean ± SD for three experiments on different days in CEM cells. The experiment was conducted only once in CEM/ADR5000 cells, because CEM/ADR5000 were not sensitive toward doxorubicin (culture medium also contains doxorubicin). Viable, apoptotic, and late apoptotic CEM cells are displayed in three separate plots, because illustration in one image is unclear due to crossing curves of the populations.

differences of fluorescence values between liposome treated and untreated cells in CEM/ADR5000 cells and inhibited samples of parental CEM cells (p > 0.05). The difference between liposome-treated and untreated not inhibited parental CEM cells was significant (two-tailed t test, p < 0.05). The difference, however, is small and only significant due to the large number of samples (n = 18). The difference is not relevant and is not confirmed by the comparison of the values either in inhibited parental CEM cells or in inhibited or not inhibited CEM/ ADR5000 cells. Sensitivity toward Cytotoxic Drugs and P-gp Activity after Liposome Treatment in CEM and CEM/ADR5000

treated cells, and 1.3 ± 0.3 and 1.3 ± 0.2 in untreated and liposome treated parental CEM cells (n = 18), again suggesting that liposomes did not change P-gp activity. The absolute fluorescence values detected in parental CEM cells were 1965 ± 276 in liposome-untreated not inhibited cells, 1756 ± 219 in liposome-treated not inhibited cells, 2637 ± 868 in liposomeuntreated inhibited cells, and 2220 ± 502 in liposome-treated inhibited cells. Absolute fluorescence values in CEM/ADR5000 cells were 197 ± 47 in liposome-untreated not inhibited cells, 193 ± 43 in liposome-treated not inhibited cells, 834 ± 254 in liposome-untreated inhibited cells, and 830 ± 353 in liposometreated inhibited cells. Statistical analysis revealed no significant 1029



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Figure 13. Intracellular doxorubicin concentration in liposome treated CEM (A) and CEM/ADR5000 (B) cells and relative uptake of doxorubicin into CEM (C) and CEM/ADR5000 (D) cells after treatment with 0.1−5 μM (CEM cells) or 1−50 μM doxorubicin (CEM/ADR5000 cells). Results are expressed as intracellular doxorubicin concentration (ng/100 000 cells) (A and B) or intracellular doxorubicin as percentage of exposed doxorubicin normalized to cell count (C and D) (mean ± SD; n = 4).

Cells. CEM and CEM/ADR5000 cells were treated with liposomes and subsequently incubated with camptothecin (16 h), vincristine (24 h), or doxorubicin (24 h). After incubation, cells were stained with rhodamine to assess P-gp activity and subsequently with 7-AAD to assess viability. Camptothecin. Figure 8 shows the percentage of viable (A), apoptotic (B), and late apoptotic/dead (C) CEM and CEM/ ADR5000 (D) cells at several camptothecin concentrations in SM-only liposome treated or untreated control cells. SM-only liposomes did not influence the sensitivity of CEM cells toward camptothecin (p > 0.05). SM-only liposomes did not influence P-gp activity of viable CEM (A) and CEM/ADR5000 (B) cells under camptothecin treatment (Figure 9). The ratio of rhodamine fluorescence did not differ significantly in liposome treated or untreated cells (p > 0.05). The linear increasing trend of the rhodamine ratio in liposome-untreated CEM cells (0.01−5 μM camptothecin) and liposome-untreated CEM/ADR5000 cells (0.1−25 μM camptothecin), however, was significant (p < 0.05). Rhodamine ratios of apoptotic and late apoptotic/dead cells are not displayed, because increased permeability of the plasma membrane impeded calculation of rhodamine ratios. Vincristine. Figure 10 shows the percentage of viable (A), apoptotic (B), and late apoptotic/dead (C) CEM cells at several vincristine concentrations in SM-only liposome treated CEM cells or CEM/ADR5000 cells (D). SM-only liposomes did not influence the sensitivity of CEM or CEM/ADR5000 cells toward vincristine (p > 0.05). SM-only liposomes also did not influence P-gp activity in viable CEM and CEM/ADR5000 cells under vincristine treatment (Figure 11). The ratio of rhodamine fluorescence did not differ significantly in liposome treated or untreated cells (p > 0.05). The linear increasing trend of rhodamine ratio in

liposome treated (but not in medium treated) CEM/ADR5000 cells (0−400 μM vincristine), however, was significant (p < 0.05). Doxorubicin. Figure 12 shows the percentage of viable (A), apoptotic (B), and late apoptotic/dead (C) CEM and CEM/ ADR5000 (D) cells at several doxorubicin concentrations in SM-only liposome treated or untreated control cells. SM-only liposomes did not influence the sensitivity of CEM cells (p > 0.05). SM-only liposomes significantly influenced the rhodamine ratio in doxorubicin treated CEM cells (data not shown) as determined by two-way repeated measure ANOVA (p < 0.05). The effect, however, was small and rather scattering around the ratio of 1 and therefore not relevant. Intracellular Doxorubicin Concentration in CEM and CEM/ADR5000 Cells. CEM and CEM/ADR5000 cells were treated with the same doxorubicin concentrations used for assessing drug sensitivity toward doxorubicin upon SM liposome treatment. Figure 13 displays the intracellular doxorubicin concentration normalized to cell count (A and B) and the percentage uptake of doxorubicin (C and D) with increasing exposure. Relative uptake was substantially lower in multidrug resistant CEM/ADR5000 cells compared to sensitive CEM cells and was not influenced by SM-only liposomes.

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DISCUSSION Evidence has accumulated that P-gp activity is affected by the specific lipid microenvironment of the plasma membrane.11−14 Biochemical reconstitution of P-gp into SM/cholesterol liposomes, for example, has been demonstrated to enhance transport activity,15 suggesting that therapeutic SM liposomes could influence MDR of tumor cells by alteration of P-gp activity. Considering that several SM-based liposomal for1030



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analysis. In contrast, expression of other ABC transporters did not differ significantly, making CEM/ADR5000 cells a suitable resistant leukemic cell line for the investigation of the influence of SM liposomes on P-gp. To confirm the hypothesis that liposomal SM could influence MDR in leukemic cells it was first investigated by fluorescence microscopy whether liposomal SM is included into CEM cells. The analysis revealed that liposomal SM preferentially accumulated in the plasma membrane of nearly all CEM cells: the site of P-gp activity alteration. We therefore quantified relevant SM species that were contained in SM liposomes (SM 16:0 (86%), SM 18:0 (6%), SM 24:1 (3%)) in liposome treated and untreated cells and found that SM content normalized to cell count was roughly three times higher in multidrug resistant CEM/ADR5000 cells than in sensitive cells without liposome treatment. CEM/ADR5000 were only slightly larger (roughly 15−20%) than CEM cells as assessed by cell analysis (electronic cell counter, data not shown), indicating that the absolute content of SM was increased in CEM/ADR5000 cells. These results are consistent with previous studies demonstrating that SM concentration was increased in multidrug resistant murine leukemia P388/dx cells and that the plasma membrane showed a higher degree of structural order compared to sensitive P388 cells.54,55 In combination with previous studies, demonstrating that reconstitution of P-gp into SM/cholesterol liposomes increased P-gp activity,15 these data suggest that SM is involved in P-gp mediated MDR. The content of SM 18:0 and SM 24:1 was furthermore significantly increased by liposome treatment in CEM/ADR5000 cells, but not in sensitive CEM cells. The reason for this remarkable difference is unclear. Exposure of CEM and CEM/ADR5000 cells with SM-only liposomes did not increase the concentration of SM 16:0, even though this species accounts for 86% of the lipid content of the liposomal preparation. In the cellular sphingolipid cycle SMs are hydrolyzed by SMases to ceramides that are highly bioactive molecules involved in cellular processes (e.g., apoptosis, cell proliferation, differentiation, and angiogenesis),56,57 and in particular ceramide 16:0 has been proposed to be a strong inducing agent in TNFα induced apoptosis in rat and mouse hepatocytes.56,58 A manipulation of the SM cycle could influence the whole network of bioactive sphingolipids,58 and one might therefore speculate that regulation of the precursor molecule SM 16:0 is more rigorous compared to other SM species, resulting in the persistence of SM 16:0 levels in the total cell membrane and the raft region that is involved in signal transduction. Regulation of sphingolipid pathways and lipid homeostasis, however, is highly complex and unresolved57 and warrants further investigation. To examine the effect of liposome exposure in more detail SM content was furthermore quantified in raft and nonraft fractions of CEM/ADR5000 cells. The content of SM 16:0 and SM 24:1 was significantly increased in nonraft fractions, but not in raft fractions. SM 18:0 was not increased in either fraction. It is open, why the total content of SM 18:0 was increased in CEM/ ADR5000 cells, but not in raft and nonraft fractions of CEM/ ADR5000 cells. In addition, it is unclear why SM-only liposomes influenced SM content of nonraft fractions, but did not have any effect on raft fractions. One might speculate that SM levels were unchanged in raft fractions due to rigorous regulation of lipid raft homeostasis. Recently, evidence has accumulated that lipid rafts are involved in signal transduction (e.g., immunoglobulin E signaling and T-cell antigen receptor signaling) and protein and lipid transport59,60 coupled with a

mulations containing, for example, vinorelbin, topotecan, or vincristine are currently investigated in preclinical or clinical trials19 and that SM liposomes get into direct contact with tumor cells, it is crucial to know potential cellular effects of liposomal SM themselves. However, the potential influence of liposomal SM on P-gp activity in ALL cells is unknown. ALL was chosen as a prototype disease for leadoff investigations because SM-based liposomes loaded with vincristine are currently being studied in clinical trials in ALL.20 Furthermore cellular effects of SM liposomes can be expected to be particularly relevant in hematological malignancies, because intravenously applied liposomes get into direct contact with circulating cancer cells, such as lymphoblast cells in ALL. The aim of this study was to develop and characterize SM-only liposomes prepared by ethanol injection and to comprehensively investigate the effects of SM-only liposomes on P-gp activity, on P-gp distribution within the membrane microdomains, on the lipid profile in sensitive and multidrug resistant lymphoblastic CEM cells, and on sensitivity toward cytotoxic drugs. SM-Only Liposomes Prepared by Ethanol Injection. Liposomes prepared by all injection protocols are categorized as small vesicles (∼0.02 μm to ∼0.2 μm),48 and size of the liposomes prepared by the 50 mM/1:20 protocol is comparable to SM/cholesterol liposomes described previously (100 ± 30 nm;49 106 ± 36 nm50). Injection of 50 mM egg SM at a ratio of 1:20 yielded liposomes with the narrowest size distribution, an important quality criterion for liposomes. These results are in contrast to a previous study that investigated the influence of the concentration of the initial stock solution for the phospholipid 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC).35 Narrow size distribution with small liposomes (40− 70 nm) were only obtained when injecting POPC at low concentrations of ≤25 mM.35 The concentration of 50 mM that proved to be best for SM was unsuitable for POPC liposomes.35 Considering that the optimal concentration tremendously varies for different lipids, it is evident that process parameters should be optimized for each individual liposome model. Stability of SM-only liposomes varied between injection protocols. The preparation protocol injecting 25 mM SM at a ratio of 1:50 yielded unstable liposomes. One possible reason could be that this protocol adds up to the lowest final lipid concentration of 0.5 mM that could favor aggregation. The interday and interpersonal variability was very low. Thus, the interlaboratory transferability of the protocol is expected to be good. The cryoelectron microscopy image could only be taken with few liposomes, because the majority of liposomes accumulated on the grid material, which impedes the capture of a good quality image due to high background. Liposomes on the grid also were unilamellar vesicles similar to the illustrated vesicles. SM-only liposomes were not cytotoxic to CEM cells and hence are a suitable liposome model for the evaluation of the influence of liposomal SM on P-glycoprotein mediated multidrug resistance. Influence of Liposomal SM on P-Glycoprotein Mediated Multidrug Resistance. CEM cells are a widely accepted and intensively described cell model for ALL51−53 and therefore were used for the evaluation of cellular effects of SMonly liposomes in this study. In addition a P-gp overexpressing cell line (CEM/ADR5000) was used to analyze the effects on P-gp activity. CEM/ADR5000 cells differed by a much higher expression and activity of P-gp as confirmed by Western blot analysis, quantification of mRNA expression, and functional 1031



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distinct lipid environment.59 Regulation mechanisms of lipid raft composition, however, are until now poorly understood.61,62 Further investigations on regulatory mechanisms are required to explain the phenomena observed in this study with certainty. SM liposomes did not change cholesterol or protein distribution in density gradient centrifugation fractions. In addition, P-gp Western blot suggests that SM liposomes did not substantially change the distribution of P-gp within the membrane, which is mainly located in nonraft fractions. These results are in line with data published previously describing that P-gp was located in raft and nonraft fractions in resistant CEM cells.10 Although liposome treatment significantly influenced the SM content, it did not have any effect on P-gp activity. Consecutive treatment of CEM and CEM/ ADR5000 cells with SM-only liposomes and camptothecin, vincristine, or doxorubicin did not show any effect of liposomal SM on drug sensitivity of sensitive or resistant cells. Investigation of P-gp activity in viable CEM/ADR5000 cells treated with camptothecin and vincristine demonstrated that Pgp activity increased parallel to drug concentration. The assumed reason is that cells naturally expressing higher amounts of P-gp were selected. Transcriptional induction of P-gp can be ruled out, because the effect was only observed in MDR, but not in parental cells. Intracellular doxorubicin concentration was measured in parental and resistant cells and compared to the sensitivity of cells. As expected, intracellular uptake of the P-gp substrate doxorubicin8 was much lower in MDR cells. Absolute intracellular drug concentrations, however, were comparable in CEM and CEM/ADR5000 cells, because CEM/ADR5000 were exposed to ten times higher drug concentrations for comparability with drug sensitivity testing. SM liposomes did not affect intracellular doxorubicin concentrations. Interestingly, intracellular concentrations of approximately >3 ng/100 000 cells did induce apoptosis in CEM cells but not in its resistant counterpart (CEM/ADR5000) as shown by the 7AAD sensitivity assay. These results illustrate that MDR is a complex mechanism that also involves cellular repair mechanisms beyond overexpression of efflux transporters.6 Doxorubicin was used over a wide concentration range in this in vitro study. According to the summary of product characteristics of a liposomal doxorubicin preparation, the maximum plasma concentration of 8.34 ± 0.49 μg/mL was observed in 23 patients treated for Kaposi’s sarcoma who received single doses of 20 mg/m2 administered by a 30 min infusion.62 The range of doxorubicin that was applied in this study (0.1−50 μM) covers the observed plasma concentrations of 8.34 μg/mL (approximately 14 μM) in patients. Maximum vincristine concentrations were 1132 ± 308 ng/mL in 12 metastatic melanoma patients after injection of vincristine sulfate liposomes (2.0 mg/m2) at the first cycle of therapy.24 The concentration range of vincristine that was tested in this study was higher than the observed maximum plasma concentration of 1132 ng/mL (approximately 1.2 μM). Vincristine concentrations of 100−400 μM were required to exert cytotoxic effects on CEM and CEM/ADR5000 cells under the experimental conditions of this study. It is likely that cells of different origin demonstrate varying and specific sensitivity of cytotoxic drugs. Thus, the results of this study should not be readily extrapolated to other cell lines or clinical samples.

The plasma concentration of liposomal SM in treated patients has been estimated in advance to ensure application of a reasonable concentration reflecting actual practice. The plasma concentration was calculated using pharmacokinetic data of vincristine loaded SM/cholesterol liposomes in nonHodgkin lymphoma patients63 (for a vincristine dosage of 2 mg/m2 and a body surface area of 1.7 m2). The volume of distribution of SM/cholesterol liposomes (58/42, mol/mol) has been described to be 2.7 l.24 On the basis of these data the plasma concentration was calculated to be approximately 25 μM. Therefore, all effects that were observed in this study (applying 20 μM for only 30 min) could have clinical relevance in patients. It is important to keep in mind that sphingolipids, in general, not only are structural components of cellular membranes but also act as bioactive molecules and have been associated with various diseases.61 Cellular effects of liposomes containing the sphingolipid SM on relevant cells and tissues should therefore be carefully evaluated in further studies. In conclusion, we found that SM-only liposomes prepared with egg SM significantly altered the cell membrane’s lipid profile confirming integration into the membrane. This did, however, not modify P-gp activity and drug sensitivity toward camptothecin, vincristine, or doxorubicin in sensitive or multidrug resistant human lymphoblastic CEM cells.

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AUTHOR INFORMATION

Corresponding Author

*Department of Clinical Pharmacology and Pharmacoepidemiology, Molecular Biology/Biochemistry Laboratory. Phone: +49-6221-5639402. Fax: +49-6221-564642. E-mail: johanna. [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors thank Corina Mueller for excellent technical assistance, Thomas Efferth for kindly providing CEM/ ADR5000 cells, Eli Lilly for kindly providing LY335979, Johannes Poeschl for the access to the flow cytometer, and Gert Fricker for access to photon correlation spectroscopy.

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