Chemico-Biological Interactions 151 (2005) 83–94

Impact of exogenous lysolipids on sensitive and multidrugresistant K562 cells:1H NMR studies

Mounir Traıkia, Carole Marbeuf-Gueye, Edith Hantz, Laurence Le Moyec∗

LPBC-CSSB, UMR CNRS 7033, UFR SMBH, 74 rue Marcel Cachin, 93017 Bobigny, France

Received 17 December 2004; accepted 20 December 2004

Abstract

The ability of lysolipids to enter into a membrane bi-layer and disturb the membrane structure was used to study the behaviorof K562 erythroleukemic cells, K562 wild type (K562wt) as well as the multidrug resistant cells K562adr. Both types of cells,when analyzed by proton NMR spectroscopy exhibit the high resolution signals assigned to so-called “mobile lipid” signals,which, in most cases, are located outside the lipid bi-layer as lipid droplets. In order to perform these studies, the K562wt andK562adr cells were treated for 48 h with lysophosphatidylcholine oleoyl (LPC18), lysophosphatidylcholine palmitoyl (LPC16)andl-�-lysophosphatidyslerine (LPS).

After evaluating toxicity of lysolipids, proton NMR of whole treated cells was used to analyze the mobile lipid content.Nile red staining and fluorescence microscopy were used to detect the presence of intracellular lipid droplets. Membrane lipida scopy thef ysolipids.

a mobilel ntensee ning oft©

K

1

c

f

s orveleser-

elllism

0

symmetry perturbation was estimated by annexin V staining with use of flow cytometry. Using fluorescence spectrounctioning of P-glycoprotein (P-gp) responsible for multidrug resistance was also evaluated after the treatment with l

Lysolipids were found to be more toxic for K562wt than for K562adr cells. LPS and LPC16 produced an increased ofipid NMR signal and amount of lipid droplets in K562wt cells only. LPC18, with the lowest toxicity, has shown more iffects on NMR spectra with a large increase of lipid NMR signal without changes in lipid droplet staining. The functio

he P-gp pump and membrane asymmetry were not modified by any of the lysolipids used.2004 Elsevier Ireland Ltd. All rights reserved.

eywords:Cell membrane; Lipid droplets; Lysolipids; Nuclear magnetic resonance; Multidrug resistance

. Introduction

Most anticancer drugs are directed towards intra-ellular targets like DNA or the cytoskeleton. There

∗ Corresponding author. Tel.: +33 1 48 38 7323;ax: +33 1 48 38 7777.

E-mail address:lemoyec@smbh.univ-paris13.fr (L.L. Moyec).

are, however, chemotherapeutics like ether–lipidalkyl-lysolipids acting at the cell membrane le[1,2]. Lysolipids are known for their lytic properti[3] and inhibition of phospholipid synthesis, pturbing the membrane lipids’ turnover[4]. Smallamounts of lysolipids are usually found in the cmembranes where they are produced by metabo

009-2797/$ – see front matter © 2004 Elsevier Ireland Ltd. All rights reserved.doi:10.1016/j.cbi.2004.12.005

84 M. Traıkia et al. / Chemico-Biological Interactions 151 (2005) 83–94

of diacylphospholipids[5]. Lysolipids are able toinduce a positive curvature in the membrane bi-layerand, as a consequence, introduce a packing stress.These molecules affecting the cell membranes areable to produce apoptosis for some types of cells[6].Apoptosis is indicated early by the externalizationof phosphatidylserine (PdtSer). PdtSer is normallylocated within the inner part of the plasma membraneand the detection of this lipid by annexin V outsidethe membrane of the intact cells is a good indicationof the early stage of apoptosis.

P-glycoprotein (P-gp) is responsible for multidrugresistance (MDR) phenotype, which is the main causeof the intracellular chemotherapeutics failure. Expres-sion of P-gp modifies the membrane lipid organization[7]. P-gp, called also ABCB4, is able to transport a largevariety of exogenous molecules including fluorescentlipids having one short chain and one long chain[8].P-gp plays also a role in the distribution of several phys-iological molecules such as phosphatidylserine[9] andcholesterol[10].

When compared to the wild type cells, resistant cellmembranes contain a different proportion of phospho-lipids. Some phospholipids are needed for the func-tioning of P-gp, in particular, when P-gp is isolatedin vesicles[11,12]. It should be mentioned here, how-ever, that the consequences of a P-gp expression onthe membrane fluidity are still not well understood, assome authors have reported an increase in fluidity whilethe others concluded a fluidity decrease[13–15]. It alsos ym-m em-b ranep

idsc m-b ignal[ olif-e ions attya ids( acidci ig-n umorce sisp s

generally attributed as fatty acid chains included in dis-organized regions containing lipids[17,25,26]. Amongsub-cellular fractions evoked for these lipids are lipiddroplets, membrane microdomains and/or apoptoticbodies. It has been reported that when lipid dropletscan be detected in tumors, for example, then ML sig-nals are present in NMR spectra[26,27], but the con-verse is not always verified[26]. The apoptotic processmay also be responsible for generation of ML[21,24].It has also been suggested that ML may be related tolipid traffic within cells and at the membrane levels[7]. In resistant and sensitive cells, proton NMR spec-troscopy showed that lipids were not inserted in simi-larly structured domains[18] as ML signals appearedwith different intensity.

The main aim of this work was to investigate theconsequence of lysolipid treatment on cellular lipidicorganization. The leukaemia cell line K562 was used ascellular model and was compared to the MDR K562adrcell line, which expresses the P-gp protein. The cellularlipids were studied by proton NMR spectroscopy, Nilered staining and fluorescence microscopy. Membraneasymmetry perturbation was followed by annexin Vand flow cytometry. P-gp functioning was also followedby fluorescence spectroscopy.

This study compares the consequences resultingfrom the addition of lysolipids containing different po-lar head groups, serine or choline with 16 or 18 carbonchain lengths on the erythroleukemia cell lines, K562and K562adr, during 48 h.

larl , al-t ro-l ing.

2

2

9 yl( reds ce).l ums ids( ndf

eems to be clear that the lipid organization and asetry within the membrane and between the two mrane leaflets are under regulation of several membroteins including ABC proteins[16].

When liquid NMR spectroscopy is used, lipontained within the crystal–liquid ordered merane should not produce any observable proton s

17,18]. Nevertheless, there are cells like cancer prrating cells, which produce relatively high resolutignals, corresponding to methylene protons of fcids (at 1.2 ppm) and methyl functions of fatty acat 0.8 ppm). These signals derived from the fattyhains called mobile lipids (ML)[19]. Cellular local-zation of ML is not yet clearly established. ML sals have been described in several types of cells: tells in a first place[20], activated lymphocytes[21,22],mbryonic cells[23] and cells undergoing apoptohenomena[21,24]. The origin of the ML signal i

Our results show that lysolipids modify the celluipid metabolism as seen by NMR spectroscopyhough this lipid modification does not affect cell piferation, membrane asymmetry and P-gp function

. Material and methods

.1. Drugs and chemicals

l-�-Lysophosphatidylcholine oleoyl (C18:1, [cis]-) (LPC18), l-�-lysophosphatidylcholine palmitoC16:0) (LPC16). Nile red and phosphate buffealine (PBS) were purchased from Sigma (Fran-�-Lysophosphatidyserine palmitoyl (Brain-sodialt) (LPS) was obtained from Avanti Polar-LipUSA). RPMI 1640 Medium with Glutamax-1 aoetal bovine serum were obtained from GibcoTM

M. Traıkia et al. / Chemico-Biological Interactions 151 (2005) 83–94 85

(Paisley, Scotland, UK). Adriamycin (adriblastine®)and Daunorubicin were from Carlo Erba (Italy). An-nexin V fluorescein conjugate (FITC annexin V) andpropidium iodide were from Molecular Probes (Ore-gon, USA). Deuterium oxide (D2O) was from Euriso-Top (Saclay, France).

2.2. Cell culture

The K562 wild type (K562wt) cell line is a hu-man erythroleukaemia cell line and K562adr is itsadriamycin-resistant derived cell line, both were a giftfrom Prof. F. Calvo (Universite Paris 7). K562 cellswere cultured in RPMI 1640 with Glutamax, supple-mented with 10% foetal bovine serum at 37◦C in a hu-midified atmosphere of 95% air and 5% CO2. The resis-tant K562adr cells were cultured in the same mediumcontaining 0.5�g/mL adriamycin. Frozen stock cellswere thawed every 30–35 passages. The proton NMRspectra aspect of whole cells were regularly controlled.

For NMR experiments cells were mass cultured un-der the same conditions after seeding 105 cells/mL in75 cm2 flasks at day 0 and were used after 2 days forexperiments, which represents two doubling times forthese cell lines. The cells were used in the exponentialphase of the growth curve, far before confluence. Theproton NMR spectra aspect of whole cells were reg-ularly controlled and showed no significant differencein their ML signal level at this stage (D2) of the growthc

2

di-l estv 0%o terc -i ialc -c ithi inedb ul-t per-f reee celll

2.4. NMR analysis

Cells (1–2× 107) were collected by a 10 min cen-trifugation at 1000 rpm. The pellet was washed withPBS/H2O and three times with 2 mL PBS/D2O. Thefinal pellet was suspended in 350�L of PBS/D2O andplaced in a 5 mm Shigemi NMR tube (Shigemi Co., Al-lison Park, PA, USA). In each experiment, the percent-age of viable cells was checked prior to NMR analysisand was higher than 95%. This percentage was alsochecked after the 3 h and 30 min NMR analysis andwas higher than 60% giving rise to an identical one-dimensional (1D) proton spectrum.

The spectra of whole cells were obtained at 25◦Con a Unity Inova (Varian, France) spectrometer work-ing at 500 MHz for proton. One-dimensional exper-iments were performed by accumulating 128 tran-sients of 90◦ pulse and 2 s relaxation delay. The sig-nal from the residual water was suppressed by the pre-saturation technique with an irradiation of 0.03 mWduring 2 s. Acquisition time was 1.34 s on 16 K datapoints corresponding to a spectral width of 6 kHz. TheFourier transform was applied without zero-filling withan exponential window function multiplication corre-sponding to 1 Hz line broadening. Two-dimensional(2D) COSY experiments were performed with 2 K datapoints in the F2 direction and 256 data points in the F1direction. The two-dimensional Fourier transformationwas applied after zero filling to 512 data points in the F1direction with a sine-bell function in both directions.E (1D)a

xter-n entsw

d bym re.T ine,a t of� tan-d re-p thec ue( ea-s tion( alled“ at-m l res-

urve.

.3. Inhibition of cell proliferation

The stock solution of lysolipids, in water, wasuted into the cell culture medium so that the higholume of lysolipid water solution was less than 1f total volume. Cells were plated in 96-well microtiulture plates for 48 h at 37◦C in 5% CO2 with increasng concentration of lysolipids. In each well, the initell concentration was 105 cells/mL and the quantifiation of surviving cells after 2 days incubation wncreasing concentrations of lysolipids was determy counting cells with a cell Coulter (Beckman Co

er Counter Z1 Inc., USA). The experiments wereormed in triplicate and the points obtained for thxperiments with the same lysolipid and the same

ine were pooled to calculate the IC50.

ach experiment consisted of one-dimensionalcquisition and two-dimensional COSY spectrum.

The chemical shifts scale was referenced to eal TMS (trimethylsilane) at 0 ppm. Peak assignmere made based on data from literature.In 1D spectra, peak surfaces were measure

anual integration in the Bruker WINNMR softwahe region from 2.85 to 3.10 ppm including creats the major peak, creatinine and the multipleCH2 protons of lysine was used as an internal sard called “Ct”. The other peak surfaces wereorted to the creatine peak surface. This requiredalculation of the ratio of the “Ct” integration valfrom normalized spectra) to the number of cells mured for each experiment. This previous calculadata not shown) demonstrated that the peak cCt” was not affected by any of the lysolipid treents. The values obtained for the other spectra

86 M. Traıkia et al. / Chemico-Biological Interactions 151 (2005) 83–94

onances with different treatments of the two cell lineswere compared by an ANOVA analysis followed by aStudent–Neumann–Keuls test for group-to-group com-parison.P< 0.05 was considered as a significant value.

2.5. Nile red staining

The glycerol–dye solution was prepared as de-scribed previously[28]. A stock solution of Nile redwas prepared in acetone (500�g/mL). The dye solu-tion was made of 10 mL glycerol 75% in a phosphatebuffer solution added to 50�L of the Nile red stock so-lution. A small amount of washed cell pellet was placedon a glass slide and left to dry at room temperature. Thecells were then fixed for 15 min in 4% formaldehyde,stained with a drop of the glycerol–Nile red solutionand covered by a glass coverslip. The slides were ex-amined by fluorescence light microscopy with a poly-var Reichert-Jung® microscope. The Nile red fluores-cence can be examined at two different wavelengths.With the fluorescein filter set, a yellow-gold fluores-cence corresponds to neutral lipid structures. With therhodamine filter set, a red fluorescence corresponds tophospholipids, other amphiphatic lipids and hydropho-bic proteins, when negative for the yellow-gold flu-orescence. Several fields from each preparation werephotographed and digitized.

2.6. Flow-cytometry

-t os-p tedt ly-s reem -l 0o MNt d1 ellsw with5 anfl w,C sis.

ncea -r

used as a control of apoptosis as it was reported to becytotoxic on K562 cells with a IC50 of 2�M with-out producing high level of apoptosis in this cell line[29].

2.7. P-gp efflux of daunorubicin

The rationale and validation of the experimentalset-up for measuring the kinetics of active transportof anthracyclins from tumor cells has been exten-sively described and discussed in the literature (see[30] and references herein). The method uses con-tinuous spectrofluorometric monitoring (Perkin-ElmerLS50B spectrofluorometer) of the anthracycline fluo-rescence signal decrease at 590 nm (here the daunoru-bicin, λex = 480 nm) after incubation with cells. Thedrug fluorescence decreases during cellular anthracy-clin uptake and this quenching is reduced when the drugis transported by P-gp in extracellular medium. Thus,cells expressing a functional P-gp present a modifiedfluorescent kinetic.

For each kinetic, 2× 106 cells were suspended in2 mL Hepes with 5 mM glucose. Daunorubicin (1�Mfinal concentration) was added. The decrease of fluo-rescence intensity was followed over at least 1 h 30 min.

3. Results

3.1. Inhibition of cell proliferation

S),l car-b 2wta sedcd crit-i -t del(

ofl r.K 16t df 18.T in-d redt

The TACSTM

Annexin V-FITC Apoptosis Detecion kit was used for detection of cell surface phhtidylserine. The kit includes annexin V conjuga

o FITC and propidium iodide. For cytometric anais, K562adr cells were grown in a doxorubicin-fedium. For each sample, 105 or 106 cells were col

ected, washed in PBS and gently suspended in 10�Lf binding buffer (10 mM HEPES pH 7.4, 150 maCl, 5 mM KCl, 1 mM MgCl2, 1.8 mM CaCl2) con-

aining 0.25�g annexin V-FTIC. Incubation laste5 min in the dark at room temperature. Finally, cere washed and suspended in the binding buffer�g propidium iodide and analyzed with a FACScow cytometer (Becton Dickinson, Mountain VieA). At least 10,000 cells were counted per analyDot plots represent annexin V-FITC fluoresce

long theX-axis (FL1) and propidium iodide fluoescence alongY-axis (FL2). Arsenate (As2O3) was

Cytotoxic effects of lysophosphatidylserine (LPysophosphatidylcholine with a 16 (LPC16) and 18ons chain (LPC18), were measured for both K56nd K562adr cells by culturing cells with an increaoncentration of lysolipids ranging from 25 to 300�Muring 48 h. The concentrations used are above the

cal micellar concentration (CMC). The IC50 were deermined by fitting the curves to a Boltzman moTable 1).

These results showed clearly that toxicityysolipids may differ distinctly from each othe562wt cells were more sensitive to LPS and LPC

han the K562adr cells. Similar IC50 values were founor both K562wt and K562adr cells in case of LPChe lower IC50 values obtained for LPS and LPC16icated higher toxicity of these lipids when compa

o LPC18.

M. Traıkia et al. / Chemico-Biological Interactions 151 (2005) 83–94 87

Table 1Concentration inhibiting 50% (IC50) of the cell proliferation of K562wt and K562adr cell lines

Cell line IC50 of the lysolipids (mean± S.D.)�M Proliferation rate (% of the control) ofcells for a concentration of 75�M

LPS LPC16 LPC18 LPS LPC16 LPC18

K562wt 85 ± 2 73± 2 203± 5 64 50 100K562adr 116 ± 4 90± 2 187± 13 93 78 100

Means were obtained from at least four experiment data sets used to fit the sigmoid model. From the calculated curves, proliferation rates werededuced for each lysolipids at 75�M as used in further experiments.

Accordingly to the data obtained above, the prolif-eration rate of cells incubated with a 75�M lysolipidconcentration were calculated (Table 1). At this dose,LPC18 has not modified the proliferation rate ofK562wt and K562adr cells, while LPC16 has reducedmore effectively the cellular proliferation rate whencompared to LPS. The sensitive K562wt cells haveshown lower proliferation rates than K562adr withthe same lysolipid concentration. Thus, the selectedconcentration of 75�M during 48 h has produced ef-fects ranging from 50% of proliferation rate (withLPC16 in K562wt) to no effect (LPC18 in K562wt andK562adr).

3.2. 1H NMR spectra

1H NMR spectra obtained for the K562wt andK562adr intact cells are shown inFig. 1. Accordingto the earlier experiments[18] and 2D COSY spec-tra, several resonances could be assigned as indicatedin Fig. 1. The signal surfaces measured were: methyl(CH3), between 0.7 and 1 ppm, from aminoacids andpeptide chain terminaisons; methylene (CH2), between1.1 and 1.5 ppm, arising from ML fatty acids; the largeregion including glutamine, glutamate resonances andallylic protons (CH2 CH ) from fatty chains (Glu),between 1.8 and 2.4 ppm; CH3 of creatine and�CH3of lysine (Ct) between 3 and 3.1 ppm;N-trimethyl fromcholine (CHO) between 3.1 and 3.3 ppm; peaks frominositol (Ino) between 3.5 and 3.7 ppm and ML fattya d5

(Ct)tba thel

The data obtained for K562wt and K562adr cellswere compared by an ANOVA analysis. The analysishas shown that, at day 2 (D2) after seeding, the sensi-tive and the resistant cells did not show significant MLsignal levels contrary to what is found at D4 after seed-ing [28]. However, the Ino/Ct ratio was significantlydifferent between K562wt and K562adr.

In the case of K562wt cells, the effects inducedby LPS, LPC16 and LPC18 on the CH3/Ct, CH2/Ct,Glu/Ct andRratios were significantly higher than thosein the non-treated cells. The changes in the signal in-tensity concern mainly the fatty acids. The increaseof the signal intensity produced by LPC18 was higherthan that induced by LPS and LPC16 (Fig. 1). LPC18treatment increases significantly (Table 1andFig. 1)the double bond to Ct ratio when compared to controlK562wt cells. The two choline-containing lysolipids,LPC16 and LPC18, significantly increased the Cho/Ctand Ino/Ct ratios when compared to control and LPStreated K562wt cells. In the case of K562adr cells,LPS treatment has not induced any distinct modifi-cation of the spectra. LPC16 produced an increase ofIno/Ct and Chol/Ct ratios when compared to the con-trol cells, but the resonances from fatty acids were notmodified. LPC18 treatment results in the increase ofall ratios when compared to control, LPS and LPC16treated cells.

3.3. Nile red staining

enceo w-iK uldn cedt eat-m adr

cid double bonds signals (CHCH) between 5.2 an.4 ppm.

These surfaces were divided by that of creatineo form the ratios listed inTable 2. Finally, theR ratio,etween CH2 of ML fatty acid chains and CH3 of ML,nd amino acids, which is often used to describe

evel of ML signals, was calculated.

The Nile red staining was able to detect the presf lipid droplets with the yellow fluorescence sho

ng the neutral lipid content of droplets (Fig. 2). In the562wt and K562adr control cells, lipid droplets coot be detected. In K562wt cells, the lysolipids indu

he appearance of the lipid droplets with LPC18 trent being the most effective. In the case of K562

88 M. Traıkia et al. / Chemico-Biological Interactions 151 (2005) 83–94

Fig. 1. 1H NMR spectra obtained on K562cells, region between 0.5 and 6 ppm. Peak assignment: (1) CH3 from fatty acids and amino acids, (2)CH2 from fatty acids, (3) alanine, (4) Glutamine, glutamate and CH2 from fatty acids, (5) creatine and lysine, (6)N-trimethyl from choline, (7)inositol, (8) CH CH from fatty acids, (9) lactate. The K562wt cell spectra are presented on the left side and the K562 adr cell spectra on theright side of the figure. The upper spectra are control cell spectra, the middle spectra are from LPS treated cells and lower spectra from LPC18treated cells.

cells, none of the lysolipids, including LPC18, couldinduce lipid droplets to be detectable by a Nile redstaining and optic microscopy.Fig. 2summarizes theseresults by showing the images of control K562wt cellswithout lipid droplets (upper image) and effects ofLPC18 on K562wt showing the appearance of droplets(middle) and on K562adr cells demonstrating the ab-sence of droplets (bottom). Images of droplets detectedon lysolipid-treated K562wt cells are not shown butwere comparable to the image obtained with LPC18.

3.4. Flow-cytometry

The dot–plot analysis of the experimental data ob-tained for the control and LPS treated K562wt are

shown onFig. 3. Unlabelled, viable cells that are neg-ative for both annexin V and propidium iodide testsappear in the dot–plot within the lower left quadrant.Early apoptotic cells, externalizing PdtSer and bind-ing annexin V, appear in the lower right quadrant; lateapoptotic or necrotic cells, binding both annexin V andpropidium iodide are located in the upper right quad-rant; and a population of the necrotic cells, which arenegative to annexin V and positive to propidium iodidetests appear in the upper left quadrant. The data col-lected inTable 3indicate the percentage of cells in anearly apoptotic stage.

In the case of K562wt control cells the annexin Vbinding occurs in less than 1% of cells. When K562wtcells were treated with lysolipids the proportions were

M. Traıkia et al. / Chemico-Biological Interactions 151 (2005) 83–94 89

Table 2NMR signals integration reported to creatin-containing peak integration

n CH CH/Ct Ino/Ct Cho/Ct Glu/Ct CH2/Ct CH3/Ct R

K562wtControl

Mean 9 0.083 0.926 3.020 3.075 2.617 1.647 1.6S.D. 0.107 0.235 0.511 0.320 0.814 0.292 0.3

LPSMean 7 0.300 0.967 3.074 4.273c 7.248c 2.363c 2.9c

S.D. 0.395 0.286 0.587 0.791 3.654 0.541 0.7

LPC16Mean 4 0.356 1.454b 4.736b 5.030c 8.572c 2.763c 3.1c

S.D. 0.087 0.200 0.550 0.066 1.475 0.283 0.2

LPC18Mean 4 6.201a 1.810b 5.021b 19.247a 52.006a 9.466a 5.5a

S.D. 2.019 0.599 1.762 5.235 16.300 2.793 0.1

K562adrControl

Mean 8 0.050 1.352 3.616 2.253 1.950 1.397 1.4S.D. 0.052 0.133 0.342 0.593 0.504 0.164 0.2

LPSMean 6 0.070 1.262 3.599 2.600 2.691 1.622 1.6S.D. 0.059 0.111 0.302 0.491 0.731 0.186 0.3

LPC16Mean 4 0.026 1.575b 4.837b 2.859 2.165 1.603 1.4S.D. 0.005 0.110 0.273 0.068 0.116 0.113 0.1

LPC18Mean 4 1.038a 1.819b 4.868b 5.900a 10.475a 3.094a 3.3a

S.D. 0.288 0.184 0.419 0.769 2.236 0.422 0.3

R ratio is CH2 upon CH3 ratio. Presented are mean values and their standard deviations (S.D.) for each cell line and each lysolipid after 48 htreatment. The effects of treatments on K562wt and K562adr cells were compared to control untreated cells by ANOVA.P< 0.05 was consideredas significantly different.

a Significantly different from control, LPS and LPC16.b Significantly different from control and LPS.c Significantly different from control.

Table 3Percentage of cells in lower right dot plot quadrant, obtained by flowcytometry after staining with annexin V and propidium iodide

Controln= 4

Asn= 4

LPSn= 4

LPC16n= 3

LPC18n= 2

K562wtMean 0.38 2.50 2.25 1.83 2.50S.D. 0.22 1.66 1.68 1.55

K562adrMean 3.63 6.25 2.00 4.17 2.75S.D. 4.29 3.61 1.00 2.72

This is the percentage of cells externalizing PdtSer in their outer lipidmembrane leaflet.

not significantly modified. In K562adr cells, despitea higher average of cells in the LR quadrant of con-trol cells when compared to K562wt cells, the differ-ence was not significant because of a large S.D. value(Table 3). Treated K562adr cells were not significantlydifferent from the control value also because of a largevariation of the values calculated.

3.5. Daunorubicin efflux by P-gp

Treatment of K562wt cells with LPS, LPC16 orLPC18 has not modified the kinetics of drug trans-port. In the case of K562adr cells, treatment during

90 M. Traıkia et al. / Chemico-Biological Interactions 151 (2005) 83–94

Fig. 2. Nile red staining of lipid droplets as detected by yellow flu-orescence. (Upper figure): Control K562wt cells, (middle figure):LPC18:1 treated K562 cells and (bottom figure): LPC 18:1 treatedK562adr cells. The 10�m bar in the bottom figure can be used in thefigure pictures.

2 days with LPC18 did not modify the transport ki-netics (Fig. 3b). This result demonstrates that the P-gp function was not affected by the treatments withLPS, LPC16 and LPC18 lysolipids. Furthermore, thelysolipids added to the fluorescence cuvette to mea-sure the immediate effect on cells did not modify theanthracycline fluorescence steady state kinetics.

4. Discussion

Lipid droplets were described in eukaryotic cellsas neutral lipid storage in cytoplasm. They are par-

ticularly developed in adipocytes[31]. It seems thatlipid droplets bud from the endoplasmic reticulum (ER)[32] where they are embedded in one or two phos-pholipid bilayers. Some proteins seem to be includedin these structures, such as adipocyte differentiation-related protein (ADRP), which protects lipid dropletsin adipocytes from hydrolysis by lipase. Caveolin isalso transported by lipid droplets to the plasma mem-brane[33]. It is hypothesized that lipid droplets play arole in cholesterol homeostasis[34].

Outside adipocytes, lipid droplets can be found inother cells and were described in fibroblast[31] in can-cer cells[35] and were also described in cells whereML signals were increased such as C6 cells cultured instressing conditions[36] or cells undergoing apotosis[37]. Usually lipid droplets are stained with the fluores-cent Nile red dye or bodipiy probe and detected withfluorescent microscopy with confocal laser scanning[35] or not [28] or with electronic microscopy (EM)[37].

While the presence of lipid droplet is reasonable inadipocytes, their role in other cell types is still unclear.Lipid droplets certainly interfere with lipid metabolismand in particular in the transport of lipids and phos-pholipids from ER to the plasma membrane throughcytoplasma[32].

The aim of this work was to further investigate therelationship between intracellular lipid metabolism andthe lipid membrane bi-layer. We have tried to provokea perturbation of the lipid metabolism in order to inves-t for-m veu ownt ighM don im,l ls byd

byW c-t ormm fa-v thep hel tiono tiono ane tty

igate the possible consequences of lipid dropletsation and of NMR ML signals. Therefore, we hased the K562 cell line, as we have previously sh

hat K562wt cells easily produce lipid droplets and hL signals on the contrary to K562adr cells whichot easily induce high ML signals. To achieve this a

ysolipids were used, as they are able to enter celirect interference with the plasma membrane.

The properties of lysolipids were discussedeltzien earlier[3]. Lysophospholipids have a lytic a

ion on the cells. In an aqueous medium lysolipids ficelles and their interactions with cell membranes

or their incorporation within the external leaflet oflasmic membrane leading to their lytic activity. T

ysis occurs above the critical micellar concentraf the lysolipids. The length and the level of saturaf the lysolipid fatty acyl chain were shown to bessential parameter for inducing cellular lysis. A fa

M. Traıkia et al. / Chemico-Biological Interactions 151 (2005) 83–94 91

Fig. 3. Cytofluorescence dot plots of K562wt cells (left side) and LPS treated K562wt cells (right side) with FITC-annexin V fluorescence alongtheX-axis and propidium iodide flourescence along theY-axis.

acyl chain length of 16 carbons is more efficient thanthat of the 18 carbons and unsaturation diminishes theefficiency of the lytic action.

The IC50 found in the experiments performed in thiswork were 50–100-fold higher than the CMC obtainedin pure water with LPS, LPC16 and LPC18[3]. Thisis probably an effect of the interactions between thelysolipids and large molecules present in a cell culturemedium such as proteins from fetal serum. The IC50values found in this study were similar to those ob-tained for LPC16 with vascular smooth muscle cells[6]. The experiments presented here used lysolipid atdoses lower than their IC50, excluding the lytic effectscaused by these molecules.

After an insertion of lysolipids into the outerleaflet, they are redistributed actively into the in-ner leaflet by aminophospholipids translocase[38].Lysolipid translocation is slower than the lipid translo-cation and it depends on the nature of the head-group and the fatty acid chain length and unsatura-tion. Consequently, some differences in the effects ofthe three lysolipids used in the present study wereexpected.

The cytotoxicity of the three lysolipids used in thisstudy depended on:- cell lines with the wild type cells being more sen-

sitive; thus, as lysolipid targets are the cell mem-branes, this indicates the membrane differences be-tween K562wt and K562adr cells;

- polarity of a head group, LPS being more toxic than

- han

Besides differences in cytotoxicity, lysolipid treat-ments have also produced different NMR spectra. Themajor changes were observed for the mobile lipid sig-nal intensities. ML signals of K562wt cells were moreaffected than those of K562adr cells. The nature ofthe polar head group did not influence the mobile lipidsignal intensity, but it changed the amount of cytoso-lic metabolite proportion seen with the increase of theCho/Ct ratio. This effect was found for both K562wtand K562adr cells.

Despite its lowest toxicity, LPC18 caused the high-est mobile lipid signal increase. On the contrary, LPC16with the highest toxicity, affected the mobile lipid sig-nals only for K562wt cells and the observed signalmodifications were weaker than those observed forLPC18.

The experiment with Nile red staining has shownthat only the K562wt cells could produce the lipiddroplets. For K562adr cells the lipid droplets werenot detected even after treatment with LPC18 induc-ing the highest ML signal increase in spectra. In otherworks, lipid droplets have been detected by electronicmicroscopy[17,21]or with confocal microscopy[39].The size of lipid bodies detected or even estimated is notin contradiction with a possible detection with simpleoptic microscopy[31]. However, it cannot be excludedthat the size of the lipid droplets formed in K562adrcells would be smaller than in K562wt cells and othertypes of cells. This could be produced by a partial ex-trusion of lysolipids by P-gp resulting in the formationo glys ith-o

the LPC;length of the acyl chain, LPC18 being less toxic tLPC16.

f fewer and smaller lipid droplets. This result stronuggests that mobile lipid signals may be detected wut possible detection of lipid droplets.

92 M. Traıkia et al. / Chemico-Biological Interactions 151 (2005) 83–94

The annexin V and PI staining were used to verifywhether the membrane asymmetry was perturbed bythe lysolipid insertion into cell membranes. AnnexinV binding is used to detect apoptosis and should beinterpreted in relation to the PI staining showing theintracellular passage of the fluorescent probe observedin case of necrotic cell death. Our results showed thatthe distribution of phosphatidylserine in the membranewas not affected by lysolipid treatments even when thecellular lipids were distinctly modified.

In control K562wt and K562adr cells, phos-phatidylserine distribution was also identical, contraryto what was shown in another cell line[9]. This differ-ence was used to establish the role of P-gp in membraneasymmetry maintenance. As already reported by oth-ers, K562 cells are known to be resistant to apoptosis asthey lack the BCL-2 factor[40]. To induce apoptosis inthis cell line, some authors have used a tyrosine kinaseinhibitor to modulate BCL-2 and BCL-abl apoptosispathway[24]. The resistance to apoptosis is confirmedhere by arsenate, which was reported to be able to pro-voke apoptosis on HL60 cells, but not in K562 cells[29].

It is generally accepted that P-gp function, i.e., itsability to extrude drugs outside the cell, depends onthe nature and the organization of this membrane pro-tein lipid surrounding. This hypothesis is supported bythe fact that isolated P-gp needs particular types oflipids to maintain its transport activity in vitro[12,41].Moreover, cholesterol and sphingolipids, lipids iso-l ain-t dt ithm tiv-i thec tionp linet

y in-d ab-s alsw lipidd roupa ef-f lipidd 18.T im-p ted

against the cell plasma membrane. Even in the absenceof cytotoxic effects some molecules, such as lysolipids,produce modification of lipid distribution within cells.Such an effect on lipid content could be taken into ac-count when screening drugs aimed against the cellularmembrane such as ether–lipids in order to avoid exclu-sion of these type of drugs from further investigationswith cytotoxicity as the only criterion.

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