BJP British Journal of DOI:10.1111/j.1476-5381.2012.02103.xwww.brjpharmacol.orgPharmacology
RESEARCH PAPER Correspondencebph_2103 1431..1447
Dr Chiara Riganti, Department of
Oncology, Via Santena 5/bis,
The association of statins 10126 Turin, Italy. E-mail:chiara.riganti@unito.it
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plus LDL receptor-targeted Keywordsblood–brain barrier; statins;
ATP-binding cassette transporters;
liposome-encapsulated doxorubicin; nitric oxide;low-density lipoproteins receptor;
liposomes; central nervous
doxorubicin increases in system tumours----------------------------------------------------------------
Received
vitro drug delivery across 8 January 2012Revised
blood–brain barrier cells 20 May 2012Accepted
22 June 2012
ML Pinzón-Daza1,2, R Garzón2, PO Couraud3, IA Romero4, B Weksler5,
D Ghigo1,6, A Bosia1,6 and C Riganti1,6
1Department of Oncology, Faculty of Medicine, University of Turin, Turin, Italy, 2Unidad de
Bioquímica, Facultad de Ciencias Naturales y Matemáticas, Universidad del Rosario, Bogotá,
Colombia, 3Institut Cochin, Centre National de la Recherche Scientifique UMR 8104, Institut
National de la Santé et de la Recherche Médicale (INSERM) U567, Université René Descartes,
Paris, France, 4Department of Biological Sciences, The Open University, Milton Keynes, UK,
5Department of Medicine, Weill Medical College, New York, USA, and 6Center for Experimental
Research and Medical Studies, University of Turin, Turin, Italy
BACKGROUND AND PURPOSE
The passage of drugs across the blood–brain barrier (BBB) limits the efficacy of chemotherapy in brain tumours. For instance,
the anticancer drug doxorubicin, which is effective against glioblastoma in vitro, has poor efficacy in vivo, because it is
extruded by P-glycoprotein (Pgp/ABCB1), multidrug resistance-related proteins and breast cancer resistance protein
(BCRP/ABCG2) in BBB cells. The aim of this study was to convert poorly permeant drugs like doxorubicin into drugs able to
cross the BBB.
EXPERIMENTAL APPROACH
Experiments were performed on primary human cerebral microvascular endothelial hCMEC/D3 cells, alone and co-cultured
with human brain and epithelial tumour cells.
KEY RESULTS
Statins reduced the efflux activity of Pgp/ABCB1 and BCRP/ABCG2 in hCMEC/D3 cells by increasing the synthesis of NO,
which elicits the nitration of critical tyrosine residues on these transporters. Statins also increased the number of low-density
lipoprotein (LDL) receptors exposed on the surface of BBB cells, as well as on tumour cells like human glioblastoma. We
showed that the association of statins plus drug-loaded nanoparticles engineered as LDLs was effective as a vehicle for
non-permeant drugs like doxorubicin to cross the BBB, allowing its delivery into primary and metastatic brain tumour cells
and to achieve significant anti-tumour cytotoxicity.
CONCLUSIONS AND IMPLICATIONS
We suggest that our ‘Trojan horse’ approach, based on the administration of statins plus a LDL receptor-targeted liposomal
drug, might have potential applications in the pharmacological therapy of different brain diseases for which the BBB
represents an obstacle.
© 2012 The Authors British Journal of Pharmacology (2012) 167 1431–1447 1431
British Journal of Pharmacology © 2012 The British Pharmacological Society
BJP ML Pinzón-Daza et al.
Abbreviations
ABC, ATP-binding cassette; AMC, amino-4-methylcumarine; BBB, blood–brain barrier; BCRP/ABCG2, breast cancer
resistance protein; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; GGPP, geranylgeranyl pyrophosphate; LDL,
low-density lipoprotein; MRPs/ABCCs, multidrug resistance-related proteins; PEG, polyethylene glycol; Pgp/ABCB1,
P-glycoprotein; PTIO, phenyl-4,4,5,5,tetramethylimidazoline-1-oxyl 3-oxide; TBS, Tris-buffered saline
Introduction with LDL receptor-targeted liposome-encapsulated doxoru-
bicin simultaneously increases the drug permeability across
Primary CNS tumours, like glioblastoma, and CNS metastasis the BBB and the cytotoxicity against CNS tumour cells, which
of solid tumours are poorly responsive to chemotherapy, as a would meet the two requisites of an optimal chemotherapy
consequence of the intrinsic tumour resistance and of the low for brain tumours.
penetration of many anticancer drugs across the blood–brain
barrier (BBB). Both tumour cells and BBB endothelial cells
are rich in ATP-binding cassette (ABC) transporters, like Methods
P-glycoprotein (Pgp/ABCB1), multidrug resistance-related
proteins (MRPs/ABCCs) and breast cancer resistance protein Chemicals
(BCRP/ABCG2) that extrude drugs from the luminal side of Plasticware for cell cultures was from Falcon (Becton Dickin-
endothelial cells back into the blood and out of the tumour son, Franklin Lakes, NJ, USA). Mevastatin, simvastatin, rhod-
cells (Declèves et al., 2006; Mercer et al., 2009; Robey et al., amine 123 and Hoechst 33342 were purchased from
2010). Doxorubicin, for instance, is a very effective anticancer Calbiochem (San Diego, CA, USA). Electrophoresis reagents
drug against glioblastoma cells in vitro, but since it is a sub- were obtained from Bio-Rad Laboratories (Hercules, CA, USA);
strate of Pgp/ABCB1, MRP1/ABCC1 and BCRP/ABCG2, its the protein content of cell monolayers and lysates was
delivery across the BBB is hampered and it is ineffective assessed with the BCA kit from Sigma Chemical Co (St. Louis,
clinically at treating this tumour. MO, USA). When not otherwise specified, all the other rea-
The effects of a pegylated liposomal doxorubicin (Doxil or gents were purchased from Sigma Chemical Co.
Caelyx) have recently been investigated in glioblastoma, but
the results obtained are unconvincing (Glas et al., 2007; Beier
et al., 2009; Ananda et al., 2011). As alternative strategy, Cell lines
liposome-encapsulated drugs that have shown selective intra- The hCMEC/D3 cells, a human brain microvascular endothe-
tumour drug delivery, lower side effects and particular effi- lial cell line, were cultured as previously described (Weksler
cacy against aggressive and chemoresistant tumours (Jabr- et al., 2005). Human glioblastoma U87-MG cells were cultured
Milane et al., 2008; Riganti et al., 2011), have been proposed in DMEM medium, human neuroblastoma SJKNP cells and
as vehicles able to cross the BBB (through a diffusion process human breast cancer MDA-MB-231 cells were grown in RPMI-
or a receptor-mediated endocytosis) and deliver their cargo 1640 medium, human lung cancer A549 cells were grown in
into the CNS (Deelen and Loscher, 2007). Certain types of Ham’s F12 medium. These cell lines (purchased from Ameri-
-1
drug-containing nanoparticles adsorb apoB-100 and apo-E can Type Cells Collection) were maintained with 1% v v
proteins from human low-density lipoprotein (LDLs; Kim penicillin-streptomycin and 10% v v
-1 FBS, at 37°C and 5% CO2
et al., 2007), are recognized by the LDL receptor and achieve and were preliminarily characterized for the amount of Pgp,
an efficient drug delivery into brain parenchyma (Michaelis MRP1, BCRP and LDL receptor and for drug-resistance param-
et al., 2006; Nikanjam et al., 2007) because both neurons and eters (intracellular doxorubicin accumulation, extracellular
glial cells express LDL receptors (Ambruosi et al., 2006). release of lactate dehydrogenase, LDH) as reported below.
In addition to increasing the delivery across the BBB, an
‘ideal’ chemotherapy for primary and metastatic tumours of Cholesterol and geranylgeranyl pyrophosphate
CNS must simultaneously overcome the ABC transporters- de novo synthesis
dependent drug resistance of tumour cells. To meet these two Cells grown to confluence in 35-mm diameter Petri dishes,
requisites, in the present study we have investigated a incubated as reported in the Results section, were labelled for
nanoparticle-based approach – that is, the association of 24 h with 1 mCi·mL-1 [3H]-acetate (3600 mCi·mmol-1; Amer-
cholesterol-lowering drugs like statin plus a LDL receptor- sham Bioscience, Piscataway, NJ, USA), then washed and sub-
targeting liposomal doxorubicin (termed ‘apo-Lipodox’; jected to lipid extraction with the methanol/hexane method
Kopecka et al., 2011). (Kopecka et al., 2011). Samples were resuspended in 30 mL
Previous works from our group has shown that statins chloroform and separated by TLC, using 1:1 v v-1 ether/
have chemosensitizing properties in epithelial tumours hexane as the mobile phase. Solutions of 10 mg·mL-1 choles-
(Riganti et al., 2005; Riganti et al., 2006; Kopecka et al., 2011). terol and 10 mg·mL-1 geranylgeranyl pyrophosphate (GGPP)
Moreover statins are reported to exert cytotoxic effects were used as standards. After exposure for 1 h to an iodine-
against CNS tumour cells (Bababeygy et al., 2009; Yanae et al., saturated atmosphere, the migrated spots were cut out and
2011). To our knowledge, no data exist on the effects of their radioactivity was measured by liquid scintillation, using
statins on the metabolism and permeability of BBB cells. In a Tri-Carb Liquid Scintillation Analyzer (PerkinElmer,
this study, we investigated whether the association of statin Waltham, MA, USA). Cholesterol and GGPP synthesis were
1432 British Journal of Pharmacology (2012) 167 1431–1447
New strategy for drug delivery into brain tumours BJP
expressed as pmol [3H]-cholesterol per 106 cells or [3H]-GGPP Results are expressed as nmol nitrite·min-1·mg-1 cell proteins.
per 106 cells, according to the corresponding calibration Nitrite production was measured by adding 0.15 mL of cell
curve. culture medium to 0.15 mL of Griess reagent in a 96-well
plate. After a 10 min incubation at 37°C in the dark, the
Spectrophotometric measurement of absorbance was detected at 540 nm with a Packard EL340
membrane cholesterol microplate reader. A blank was prepared for each experiment
Cells were rinsed with 0.5 mL PBS, sonicated on ice with two in the absence of cells, and its absorbance was subtracted
bursts of 10 s and centrifuged at 100 000¥ g for 1 h at 4°C. from that obtained in the presence of cells. Nitrite concen-
-1
The pellets (cell membrane fractions) were resuspended in tration was expressed as nmol nitrite·mg cell protein.
0.25 mL PBS and the cholesterol concentration was measured
with an enzymatic colorimetric assay kit (OSR6516, Olympus Western blot analysis
System Reagent, Olympus Europe Holding GmbH, Hamburg, Cells were rinsed with lysis buffer (50 mmol·L-1 Tris,
Germany), following the manufacturer’s instructions. The 10 mmol·L-1 EDTA, 1% v v-1 Triton-X100), supplemented
absorbance was measured at 540/600 nm by an Olympus with the protease inhibitor cocktail set III (80 mmol·L-1
Analyzers spectrophotometer (Olympus Europe Holding aprotinin, 5 mmol·L-1 bestatin, 1.5 mmol·L-1 leupeptin,
GmbH). b-Methyl-cyclodextrin (10 mmol·L-1 for 3 h) was 1 mmol·L-1 pepstatin; Calbiochem), 2 mmol·L-1 phenylmeth-
used as a cholesterol chelator (Kopecka et al., 2011). A 50 mL ylsulfonyl fluoride (PMSF) and 1 mmol·L-1 sodium
aliquot was used to determine the protein content with the orthovanadate, then sonicated and centrifuged at 13 000¥ g
BCA kit. The results are expressed in mg cholesterol·mg-1 for 10 min at 4°C. Extracts of protein, 20 mg, were subjected
membrane proteins, according to a previously prepared titra- to SDS-PAGE and probed with the following antibodies: anti-
tion curve. phospho-Ser(176/180)-IKKa/b (Cell Signaling Technology
Inc, Danvers, MA, USA), anti-IKKa/b (Santa Cruz Biotechnol-
RhoA and RhoA kinase activity ogy Inc., Santa Cruz, CA, USA), anti-IkB-a (Santa Cruz
To evaluate RhoA activity, the GTP-bound fraction, taken as Biotechnology Inc.), anti-neuronal NOS (nNOS/NOS I, Trans-
an index of monomeric G-proteins activation (Laufs and duction Laboratories, Lexington, KY, USA), anti-inducible
Liao, 2000), was measured using the G-LISA™ RhoA Activa- NOS (iNOS/NOS II, Transduction Laboratories), anti-
tion Assay Biochem Kit (Cytoskeleton Inc, Denver, CO, USA), endothelial NOS (eNOS/NOS III, Transduction Laboratories),
according to the manufacturer’s instructions. Absorbance was anti-phospho-(Ser 1177) eNOS (Cell Signaling Technology
read at 450 nm, using a Packard EL340 microplate reader Inc), anti-Pgp/ABCB1 (Santa Cruz Biotechnology Inc.), anti-
(Bio-Tek Instruments, Winooski, VT, USA). For each set of MRP1/ABCC1 (Abcam, Cambridge, MA, USA), anti-BCRP/
experiments, a titration curve was prepared, using serial dilu- ABCG2 (Santa Cruz Biotechnology Inc.), anti-glyceraldehyde
tion of the Rho-GTP positive control of the kit. Data are 3-phosphate dehydrogenase (GAPDH, Santa Cruz Biotechnol-
expressed as absorbance units·mg-1 cell proteins. RhoA kinase ogy Inc.) This procedure was followed by exposure to a
activity was measured using the CycLex Rho Kinase Assay Kit peroxidase-conjugated secondary antibody (Bio-Rad). The
(CycLex Co, Nagano, Japan), following the manufacturer’s membranes were washed with Tris-buffered saline (TBS)-
instructions. The titration curve was prepared with serial dilu- Tween 0.1% v v-1, and proteins were detected by enhanced
tions of recombinant RhoA kinase (Rock2, MBL Inc, Woburn, chemiluminescence (PerkinElmer).
MA, USA). Data are expressed as absorbance units·mg-1 cell To assess the presence of nitrated proteins, the whole cell
proteins. extract was immunoprecipitated with a rabbit polyclonal
anti-nitrotyrosine antibody (Millipore, Billerica, MA, USA),
NF-kB activity using the PureProteome Protein A and Protein G Magnetic
Nuclear proteins were extracted using the Nuclear Extract Kit Beads (Millipore). Immunoprecipitated proteins were sepa-
(Active Motif, Rixensart, Belgium) and quantified. The activ- rated by SDS-PAGE and probed with anti-Pgp/ABCB1, anti-
ity of NF-kB was assessed by the TransAM™ Flexi NFkB Family MRP1/ABCC1 or anti-BCRP/ABCG2 antibody. Whole cells
kit (Active Motif), by adding 1 pmol of the biotinylated probe lysates, 30 mg, were probed with the same antibodies before
containing the NF-kB consensus site 5′-GGGACTTTCC-3′ to the immunoprecipitation step to check the total amount of
10 mg of nuclear extract proteins. The absorbance at 450 nm Pgp, MRP1 and BCRP.
was measured with a Packard EL340 microplate reader (Bio-
Tek Instruments). For each set of experiments, a blank was ABC transporters activity
prepared with bi-distilled water, and its absorbance was sub- To measure the ATPase activity of Pgp/ABCB1, MRP1/ABCC1
tracted from that obtained in the presence of nuclear extracts. and BCRP/ABCG2, cells were lysed in buffer A (50 mmol·L-1
Data are expressed as absorbance units·mg-1 cell proteins. HEPES, 750 mmol·L-1 KCl, 200 mmol·L-1 sucrose,
10 mmol·L-1 NaHCO3; pH 7.4), supplemented with protease
NOS activity and nitrite measurement inhibitor cocktail set III, centrifuged at 13 000¥ g for 5 min,
Cells were detached by trypsin/EDTA, resuspended in 0.3 mL then at 100 000¥ g for 1 h at 4°C. The pellet was resuspended
of assay buffer (20 mmol·L-1 HEPES, 0.5 mmol·L-1 EDTA, and in 1 mL buffer B (20 mmol·L-1 HEPES, 160 mmol·L-1 KCl,
1 mmol·L-1 dithiothreitol DTT; pH 7.2) and sonicated. NOS 1 mmol·L-1 MgCl , 1 mmol·L-12 CaCl2, 0.5% v v-1 Triton X-100;
(EC 1.14.13.49) activity was measured in 100 mg of cell lysates pH 7.4). To obtain membrane fractions enriched in Pgp/
with the Ultrasensitive Colorimetric Assay for Nitric Oxide ABCB1, MRP1/ABCC1 and BCRP/ABCG2, 100 mg of mem-
Synthase kit (Oxford Biomedical Research, Oxford, MI, USA). brane proteins were immunoprecipitated overnight with the
British Journal of Pharmacology (2012) 167 1431–1447 1433
BJP ML Pinzón-Daza et al.
specific primary antibodies, then washed twice with 1 mL phosphocoline, 1,2 dipalmitoyl-sn-glycero-3-phosphogly-
buffer B, supplemented with 2 mmol·L-1 DTT; 50 mg of each cerol, at a molar ratio 4.2:11.4:15.2:11.4. For each prepara-
sample were mixed with 2 mmol·L-1 ATP, 2.5 mmol·L-1 phos- tion, 30 mg desiccated liposomes were incubated with
phoenolpyruvate, 7.5 U pyruvate kinase and 8.0 U LDH to 1.5 mmol·L-1 doxorubicin in sterile aqueous solution, accord-
check ATPase activity, as previously described (Doublier et al., ing to the manufacturer’s instructions. The residual non-
2008). The reaction was started by adding 0.25 mmol·L-1 encapsulated drug was removed by gel filtration in a
NADH and was followed for 10 min, measuring the absorb- Sephadex G-50 (Amersham Bioscience) column. The amount
ance at 340 nm with a Packard EL340 microplate reader. The of encapsulated doxorubicin was quantified by diluting 50 mL
reaction kinetics was linear throughout the time of measure- of liposomal suspension in 0.5 mL of 1:1 v v-1 ethanol/HCl
ment. The NADH oxidation rate (expressed as mmol NADH 0.3 N, sonicating the liposomes and measuring the fluores-
oxidized·min-1·mg-1 cell proteins) of each sample was sub- cence emitted by the drug with a LS-5 spectrofluorimeter
tracted from the oxidation obtained in the absence of (PerkinElmer). Excitation and emission wavelengths were
immunoprecipitated proteins. The ATP hydrolysis rate was 475 nm and 553 nm respectively. The encapsulation effi-
calculated stoichiometrically and ATPase activity was ciency was calculated as described (Wong et al., 2004). The
expressed as mmol ATP hydrolysed·min-1·mg-1 cell proteins. liposomes with an encapsulation efficiency higher than 85%
The efflux of rhodamine 123, taken as an index of Pgp/ (termed ‘Lipodox’) were collected, stored at a doxorubicin
ABCB1 and MRP1/ABCC1 activity, and the intracellular accu- concentration of 0.5 mmol·L-1 and used to produce apoB100-
mulation of Hoechst 33342, taken as an index of BCRP/ conjugated doxorubicin-loaded liposomes (termed ‘apo-
ABCG2 activity, were measured as reported previously Lipodox’). With this aim, the following recombinant peptide
(Riganti et al., 2011). from human apoB100 was used: DWLKAFYDKVAEKLKEA-
FRLTRKRGLKLA (LDL receptor-binding site is underlined;
LDL receptor expression GenScript, Piscataway, NJ, USA). An aqueous solution of the-1
Total RNA was extracted and reverse transcribed using peptide, 2.5 mmol·L , was added to liposomal doxorubicin,
the QuantiTect Reverse Transcription Kit (Qiagen, Hilden, vortexed and incubated for 30 min at room temperature,
Germany). RT-PCR was carried out using IQ™ SYBR Green with mild agitation. The unbound peptide was removed by
Supermix (Bio-Rad), according to the manufacturer’s instruc- dialysis as described previously (Nikanjam et al., 2007). The
tions. The same cDNA preparation was used for the quanti- amount of peptide attached to the liposomes was detected by
fication of LDL receptor and GAPDH, used as a housekeeping the QuantiPro BCA Assay kit (Sigma).
gene. The sequences of LDL receptor primers were 5′-TG To assess the role of PEG in the peptide attachment,
AACTGGTGAGAGGACCAC-3′, 5′-TGTTCTTAAGCCGCCAG anionic non-pegylated liposomes (COATSOME EL01A,
TTGTT-3′; the sequences of the GAPDH primers were NOF Corporation), with the following composition: 1,2
5′-TGGTCACCAGGGCTGCTT-3′, 5′-AGCTTCCCGTTCTCAG dipalmitoyl-sn-glycero-3-phosphocoline, cholesterol, 1,2
CCTT-3′. The relative quantification of each sample was dipalmitoyl-sn-glycero-3-phosphoglycerol (molar ratio of
obtained by comparing the LDL receptor PCR product with 3:4:3), were used in control experiments. To investigate the
the GAPDH PCR product, with the Bio-Rad Software Gene effects of liposomal shell alone, empty anionic pegylated
Expression Quantitation (Bio-Rad). liposomes (COATSOME EL-01-PA series), conjugated or not
For flow cytometry detection of surface LDL receptors, with the LDL receptor-targeted peptide, were used.
cells were washed with PBS, detached with Cell Dissociation The size of the liposomes was evaluated by dynamic light
Solution (Sigma) and resuspended at 5 ¥ 105 cells·mL-1 in scattering: 10 mL of liposome suspension were diluted in 1 mL-1
1 mL RPMI medium containing 5% v v-1 FBS. Samples were of 120 mmol·L NaCl solution and analysed with an ALV-
washed with 0.25% w v-1 BSA-PBS, incubated with the NIBS dynamic light scattering instrument (Langen, Germany)
primary antibody for LDL receptors (Abcam) for 45 min at provided with a Ne–He laser and an ALV-5000 multiple tau
4°C, then washed twice and incubated with secondary FITC- digital correlator. The scattered light intensity was recorded
conjugated antibody for 30 min at 4°C. After washing and for 30 s on suspensions at 37°C. The hydrodynamic radius of
fixation with paraformaldehyde 2% w v-1, the number of LDL liposomes was evaluated by using both the cumulant method
receptors expressed on the surface was detected on 100 000 and the CONTIN algorithm (Provencher, 1982). The mean
cells by a FACSCalibur system (Becton Dickinson), using the radius of each liposomal preparation is presented in Table 1.
Cell Quest software (Becton Dickinson). Control experiments The morphological analysis of apo-Lipodox was per-
included incubation of cells with non-immune isotypic formed on diluted samples (5 mg·mL
-1 liposomes in
-1
antibody, followed by the appropriate labelled secondary 120 mmol·L NaCl solution), using a Philips CM10 transmis-
antibody. sion electron microscope (TEM; Philips Amsterdam, The
Netherlands), at an acceleration voltage of 80 kV. For each
sample, a minimum of three microscopic fields was
Synthesis of LDL receptor-targeted examined.
liposome-encapsulated doxorubicin
LDL receptor-targeted liposome-encapsulated doxorubicin Permeability coefficient across the BBB cells
was prepared using anionic pegylated liposomes The permeability to inulin, taken as a parameter of the integ-
(COATSOME EL-01-PA, NOF Corporation, Tokyo, Japan) of rity of the tight junctions (Monnaert et al., 2004), was meas-
the following composition: 1,2 distearoyl-sn-3-glycero-3- ured on hCMEC/D3 cells seeded at 50 000·cm-2 and grown
phosphoethanolamine conjugated with polyethylene for 7 days up to confluence in 6-multiwell Transwell devices
glycol (PEG), cholesterol, 1,2 dipalmitoyl-sn-glycero-3- (0.4 mm diameter pores-size, Corning Life Sciences, Chorges,
1434 British Journal of Pharmacology (2012) 167 1431–1447
New strategy for drug delivery into brain tumours BJP
Table 1
Dynamic light scatter analysis of liposomes
Liposomesa Acronymb Doxorubicin PEG Peptide Radius (mean  SD) P.I.c
COATSOME EL01A – Yes No Yes 70.05 nm  22.45 nm 0.187
COATSOME EL-01-PA Lipodox Yes Yes No 70.63 nm  28.55 nm 0.183
COATSOME EL-01-PA apo-Lipodox Yes Yes Yes 72.05 nm  23.14 nm 0.180
COATSOME EL-01-PA EL No Yes No 69.57 nm  27.35 nm 0.171
COATSOME EL-01-PA apoEL No Yes Yes 71.77 nm  18.71 nm 0.177
aCommercial name (NOF Corporation).
bAcronyms used in text and figures.
cPolydispersity Index.
France). Cells were incubated with or without mevastatin or (20 mmol·L-1 HEPES/KOH, 10 mmol·L-1 KCl, 1.5 mmol·L-1
simvastatin (0.1 mmol·L-1 for 24 h) or b-methyl-ciclodextrin MgCl2, 1 mmol·L-1 EGTA, 1 mmol·L-1 EDTA, 1 mmol·L-1 DTT,
(10 mmol·L-1 for 3 h), then the culture medium was replaced 1 mmol·L-1 PMSF, 10 mg·mL-1 leupeptin; pH 7.5). Then 20 mg
in the upper and lower chambers and 2 mCi·mL-1 [14C]-inulin cell lysates were incubated for 1 h at 37°C with 20 mmol·L-1
(10 mCi·mmol-1; PerkinElmer) was added to the upper of the fluorogenic substrate of caspase 3, DEVD-7-amino-
chamber of Transwell. After 3 h, the medium in the lower 4-methylcumarine (DEVD-AMC), in 0.25 mL caspase
chamber was collected and the amount of [14C]-inulin was assay buffer (25 mmol·L-1 HEPES, 0.1% w v-1 3-[(3-
measured using a Tri-Carb Liquid Scintillation Analyzer (Perk- cholamidopropyl ) dimethylammonio ] - 1 - propanesulfonate
inElmer). Radioactivity was converted to nmol inulin·cm-2, CHAPS, 10% w v-1 sucrose, 10 mmol·L-1 DTT, 0.01% w v-1 egg
using a calibration curve prepared previously. albumin; pH 7.5). The reaction was stopped by adding
For doxorubicin permeability, hCMEC/D3 cells seeded as 0.75 mL ice-cold 0.1% w v-1 trichloroacetic acid and the fluo-
reported above, were incubated with or without mevastatin rescence of the AMC fragment released by active caspase-3
or simvastatin (0.1 mmol·L-1 for 24 o 48 h). After this period, was read using a LS-5 spectrofluorimeter (PerkinElmer). Exci-
the culture medium was replaced in the upper and lower tation and emission wavelengths were 380 nm and 460 nm
chambers and 5 mmol·L-1 doxorubicin or apo-Lipodox were respectively. Fluorescence was converted in pmol·mg-1 cell
added in the upper chamber of Transwell for 3 h, then the protein using a calibration curve prepared previously with
medium in lower chamber was collected and the amount of standard solutions of AMC.
doxorubicin was measured fluorimetrically, using a LS-5 spec- For fluorescence microscope analysis, tumour cells in the
trofluorimeter (PerkinElmer). Excitation and emission wave- lower chamber were seeded on sterile glass coverslips and
lengths were 475 nm and 553 nm, respectively. Fluorescence treated as reported above. At the end of the incubation time,
was converted in nmol doxorubicin·cm-2, using a calibration samples were rinsed with PBS, fixed with 4% w v-1 parafor-
curve previously set. maldehyde for 15 min, washed three times with PBS and
The permeability coefficients were calculated as described incubated with 4′,6-diamidino-2-phenylindole dihydrochlo-
previously (Siflinger-Birnboim et al., 1987). ride (DAPI) for 3 min at room temperature in the dark. Cells
were washed three times with PBS and once with water, then
Intratumour drug accumulation and toxicity the slides were mounted with 4 mL of Gel Mount Aqueous
in co-culture models Mounting and examined with a Leica DC100 fluorescence
The hCMEC/D3 cells (50 000·cm-2) were grown for 7 days up microscope (Leica Microsystems GmbH, Wetzlar, Germany).
to confluence in 6-multiwell Transwell devices (0.4 mm diam- For each experimental point, a minimum of five microscopic
eter pores-size), whereas in the lower chamber, 500 000 cells fields were examined.
(U87-MG, SJKNP, A549 or MDA-MB-231 cells) were seeded at
day 4. At day 7, 0.1 mmol·L-1 simvastatin was added to the Statistical analysis
upper chamber medium for 48 h, when indicated. During the All data in the text and figures are presented as means  SD.
final 24 h, 5 mmol·L-1 doxorubicin or apo-Lipodox were added The results were analysed by a one-way ANOVA. A P < 0.05
into the upper chamber of Transwell, then the inserts were was considered significant.
removed and the extracellular medium of cells in the lower
chamber was checked for the LDH activity, taken as an index
of cytotoxicity and necrosis (Kopecka et al., 2011). Cells in Results
the lower chamber were detached, and divided into two aliq-
uots of 250 000 cells each: the first aliquot was lysed in Statins induce the synthesis of NO in human
0.5 mL ethanol/HCl 0.3 N (1:1 v v-1) and analysed for the primary BBB cells by a RhoA/RhoA
intracellular content of doxorubicin, as reported above. kinase/NF-kB-dependent mechanism
Results are expressed as nmol doxorubicin·mg-1 cell proteins. As inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A
The second aliquot was lysed in 0.5 mL of caspase lysis buffer reductase enzyme, statins decrease the synthesis of cholesterol
British Journal of Pharmacology (2012) 167 1431–1447 1435
BJP ML Pinzón-Daza et al.
and isoprenoids like farnesyl pyrophosphate and GGPP in ABCG2 (Figure 2A). The immunoprecipitation of membrane
mammalian cells (Liao and Laufs, 2005). We observed in solid extracts with an anti-nitrotyrosine antibody, followed by the
tumours that a decrease in GGPP reduces the activity of small Western blot detection with the specific primary antibodies,
GTPase of the RhoA family, activates the NF-kB transcription revealed that mevastatin and simvastatin promoted a nitra-
factor and up-regulates the inducible NO synthase (iNOS/NOS tion on tyrosine of Pgp/ABCB1 and BCRP/ABCG2, similar to
II) gene (Riganti et al., 2006; Riganti et al., 2008). The human the one produced by the NO donor sodium nitroprusside
brain microvascular endothelial hCMEC/D3 cells were sensi- (Figure 2A). Both these transporters also showed a low level of
tive to both mevastatin and simvastatin, two lipophilic statins nitration in untreated cells, whereas no nitration was detect-
that reduced the endogenous synthesis of cholesterol and able on MRP1/ABCC1 in each experimental condition. In
GGPP in a dose- and time-dependent manner (Figure 1A). The parallel the ATPase activity of Pgp/ABCB1 and BCRP/ABCG2,
lowest concentration that significantly decreased cholesterol but not that MRP1/ABCC1, was decreased in the presence of
and GGPP level after 24 h was 0.1 mmol·L-1 and this was the statins (Figure 2B), suggesting that the nitration probably
chosen for all the subsequent experiments. The cholesterol impairs the catalytic cycle of the pumps. Indeed, the intrac-
chelator b-methyl-cyclodextrin significantly depleted the ellular retention of rhodamine 123 (Figure 2C) that is
cell membrane of cholesterol (Supporting information inversely related to the activity of Pgp/ABCB1, and the intra-
Figure S1A) and in parallel disrupted the integrity of the tight cellular retention of Hoechst 33342 (Figure 2D) that is
junctions in hCMEC/D3 cells, as demonstrated by the inversely related to the activity of BCRP/ABCG2, were both
increased permeability coefficient of inulin (Supporting infor- increased by the statins. Although the tyrosine nitration and
mation Figure S1B). In the experimental conditions used in the decrease in ATPase activity in Pgp/ABCB1 and BCRP/
this study, mevastatin and simvastatin reduced the amount of ABCG2 were already detectable after 24 h of incubation with
cholesterol in the cell membrane by a lesser extent than mevastatin or simvastatin (Figure 2A and B) and were not lost
b-methyl-cyclodextrin (Supporting information Figure S1A) after 48 h (data not shown), the effects of these changes on
and did not affect the permeability to inulin (Supporting the activity of Pgp/ABCB1 and BCRP/ABCG2 were stronger
information Figure S1B). after 48 h (Figure 2C and D).
In keeping with the decrease in GGPP, the activity of the As doxorubicin is transported by Pgp/ABCB1, MRP1/
geranylgeranylated protein RhoA, measured as the amount of ABCC1 and BCRP/ABCG2, its ability to cross the BBB cells is
active GTP-bound protein, was reduced by both statins, poor in vivo; also in hCMEC/D3 cells, the permeability coef-
resulting in a lower activation of the downstream effector ficient was low (Figure 2E). However, the treatment with
RhoA kinase (Figure 1B). In parallel, the amount of IKKa/b statins for 48 h significantly increased the permeability of
complex phosphorylated on serine 176/180, which was the drug. The presence of the NO scavenger 2-phenyl-
nearly undetectable in untreated hCMEC/D3 cells, was 4,4,5,5,tetramethylimidazoline-1-oxyl 3-oxide (PTIO) pre-
increased by mevastatin and simvastatin, without significant vented the increase in permeability to doxorubicin
changes in the expression of IKKa/b protein (Figure 1C). Such (Figure 2E), suggesting that NO levels play a crucial role in
an increase was accompanied by a decrease in the inhibitory regulating the activity of Pgp/ABCB1 and BCRP/ABCG2 in
protein Ik-Ba (Figure 1C) and by the activation of NF-kB, hCMEC/D3 cells.
measured as the ability of the transcription factor translo-
cated into the nucleus to bind its specific target sequence
(Figure 1D). The association of statins plus a LDL
Differently from the constitutive NOS isoforms (neuronal receptor-targeted liposome-encapsulated
NOS/NOS I and endothelial NOS/NOS III), the inducible doxorubicin further increases the drug
iNOS/NOS II is usually absent in non-stimulated cells and is transport across BBB cells
up-regulated by different stimuli, like cytokines and bacterial By lowering the de novo synthesis of cholesterol, statins force
lipopolysaccharide that induce the activation of NF-kB (Pautz mammalian cells to expose the LDL receptor on their surface
et al., 2010). Statins increased the expression of NOS II; this (Liao and Laufs, 2005). In hCMEC/D3 cells treated with mev-
was undetectable in untreated hCMEC/D3 cells (Figure 1E). astatin and simvastatin, the mRNA level for LDL receptors
The statins did not induce NOS I and did not change the was significantly increased after 24 h (Figure 3A), a time point
expression of NOS III, but a slight increase in the amount of at which the synthesis of cholesterol was decreased
the active phospho-(Ser1177)-NOS III was detected following (Figure 1A). The amount of LDL receptor protein on the cell
statins treatment (Figure 1E). As a consequence of these surface was further increased by statins after 48 h (Figure 3B).
changes, the global enzymatic activity of NOS in cell lysates We have previously shown that anionic pegylated lipo-
and the synthesis of NO, measured as the amount of the somes loaded with doxorubicin and conjugated with a syn-
stable NO-derivative nitrite in culture supernatant, were sig- thetic peptide containing the LDL receptor-binding site from
nificantly increased in hCMEC/D3 cells treated with mevas- human apo-B100 (the so-called ‘apo-Lipodox’) was internal-
tatin or simvastatin (Figure 1F). ized efficiently via a LDL receptor-mediated endocytosis and
was less extruded by Pgp/ABCB1 in solid tumour cells
Statins reduce the activity of Pgp and BCRP (Kopecka et al., 2011). We thus applied apo-Lipodox that
and increase the permeability of doxorubicin appeared as round-shaped particles with electron-dense areas
across the BBB cells by a NO-dependent due to the doxorubicin packed inside the liposomal shell
mechanism (Figure 3C), to hCMEC/D3 cells untreated or treated with
hCMEC/D3 cells constitutively express several ABC transport- mevastatin and simvastatin for 48 h. In control cells, the
ers (Carl et al., 2010), like Pgp/ABCB1, MRP1/ABCC1, BCRP/ permeability coefficient of apo-Lipodox was higher than that
1436 British Journal of Pharmacology (2012) 167 1431–1447
New strategy for drug delivery into brain tumours BJP
Figure 1
Effects of statins on cholesterol and GGPP synthesis, RhoA/RhoA kinase activity, NF-kB pathway and NO synthesis in BBB cells. (A) Cholesterol and
GGPP synthesis. hCMEC/D3 cells were incubated in the absence (CTRL) or presence of different concentrations of 0.1, 1, 10 mmol·L-1 mevastatin
(MVS) and simvastatin (SIM) for 24 h (left panel), or in the presence of 0.1 mmol·L-1statin for 12, 24 or 48 h (right panel). In the subsequent 24 h,
the cells were labelled with [3H]-acetate, then cholesterol (open columns) and GGPP (hatched columns) synthesis was measured as reported under
Methods. Data are presented as means  SD (n = 3). Versus CTRL: *P < 0.05. For the subsequent experiments, 0.1 mmol·L-1MVS or SIM for 24 h
was used. (B) RhoA/RhoA kinase activity. Samples were subjected to ELISA assays to measure the amount of RhoA-GTP (open bars) and the activity
of RhoA kinase (hatched bars). The experiments were performed in duplicate, as described in the Methods section. Data are presented as means
 SD (n = 3). Versus CTRL: *P < 0.05. (C) Western blot detection of phospho-Ser(176/180)-IKK a/b, IKK a/b and IkB-a protein in extracts from
hCMEC/D3 cells. The expression of GAPDH was used to check the equal protein loading. The figure is representative of three experiments with
superimposable results. (D) NF-kB activity. The activity of NF-kB was detected in the nuclear extracts measuring the DNA-binding capacity of NF-kB
on its target sequence (see Methods). Measurements were performed in duplicate and data are presented as means  SD (n = 3). Versus CTRL:
*P < 0.05. (E) Western blot detection of NOS isoforms (nNOS/NOS I; iNOS/NOS II; eNOS/NOS III) and of phospho-(Ser 1177)eNOS/NOS III
protein in extracts from hCMEC/D3 cells. The expression of GAPDH was used to check the equal protein loading. The figure is representative of
three experiments with superimposable results. (F) NO synthesis. NOS activity in cell lysates and nitrite accumulation in the extracellular medium
were measured by use of spectrophotometric assays, as reported in Methods. Data are presented as means  SD (n = 3). Versus CTRL: *P < 0.05.
British Journal of Pharmacology (2012) 167 1431–1447 1437
BJP ML Pinzón-Daza et al.
Figure 2
Effects of statins on ABC transporters activity and doxorubicin permeability across BBB cells. The hCMEC/D3 cells were incubated in the absence
(CTRL) or presence of 0.1 mmol·L-1mevastatin (MVS) or simvastatin (SIM) for 24 h or 48 h, then subjected to the following investigations. (A)
Nitration of ABC transporters. After a 24 h incubation, cells were lysed and the whole cell extracts were immunoprecipitated with an anti-
nitrotyrosine polyclonal antibody. The immunoprecipitated proteins were subjected to Western blotting, using an anti-Pgp, an anti-MRP1 or an
anti-BCRP antibody (see Methods). The NO donor sodium nitroprusside (100 mmol·L-1 for 24 h, SNP) was used as a positive control of nitration.
The figure is representative of three experiments with similar results. (B) ATPase activity was measured spectrophotometrically after immunopre-
cipitation of Pgp, MRP1, BCRP from membrane fractions, as described in the Methods. Measurements were performed in duplicate and data are
presented as means  SD (n = 3). Versus CTRL: *P < 0.05. (C) Rhodamine assay. Cells were incubated for 20 min at 37°C with the fluorescent Pgp
substrate rhodamine 123. The intracellular fluorescence was assessed by flow cytometry analysis in untreated cells (grey peak) and in cells treated
with statins for 24 h (dotted line) or 48 h (continuous line). The figures shown here are representative of three similar experiments performed in
duplicate. (D) Hoechst 33342 assay. Cells were incubated for 15 min at 37°C with Hoechst 33342, lysed and analysed fluorimetrically for the
intracellular content of the dye. Measurements were performed in duplicate and data are presented as mean  SD (n = 3). Versus CTRL: *P < 0.05.
(E) Transport of doxorubicin across BBB monolayer. hCMEC/D3 cells were grown up to the confluence in Transwell insert, in fresh medium or in
the presence of statins for 48 h, alone or co-incubated with the NO scavenger PTIO (100 mmol·L-1). 5 mmol·L-1doxorubicin was then added in the
upper chamber. After 3 h, the medium was recovered by the lower chamber and the amount of doxorubicin was measured fluorimetrically.
Measurements were performed in duplicate and data are presented as means  SD (n = 4). Versus CTRL: *P < 0.05; versus condition without PTIO:
°P < 0.001.
1438 British Journal of Pharmacology (2012) 167 1431–1447
New strategy for drug delivery into brain tumours BJP
Figure 3
Effects of statins on the expression of LDL receptors and on the permeability of LDL receptor-targeted liposomal doxorubicin across the BBB. The
hCMEC/D3 cells were incubated in the absence (CTRL) or presence of 0.1 mmol·L-1mevastatin (MVS) or simvastatin (SIM) for 24 h (A, B), 48 h (B,
D and E), then subjected to the following investigations. (A) RT-PCR of LDL receptors. Total RNA was extracted, reverse transcribed and amplified
by RT-PCR, as indicated in Methods. Measurements were performed in triplicate and data are presented as means  SD (n = 3). Versus CTRL:
*P < 0.005. (B) Flow cytometry analysis of surface LDL receptors in untreated cells (grey peak) or treated with MVS (continuous line) or SIM (dotted
line) for 48 h. The figures shown here are representative of 3 similar experiments, performed in triplicate. (C) Apo-Lipodox imaging by TEM. The
micrographs (28 500¥ magnification) are representative of three similar experiments. (D) Transport of apo-Lipodox across the BBB monolayer. The
hCMEC/D3 cells were grown to the confluence in Transwell insert, in fresh medium or in the presence of statins or of the NO scavenger PTIO
(100 mmol·L-1 for 48 h), then 5 mmol·L-1 apo-Lipodox was added in the upper chamber. After 3 h, the amount of the drug recovered by the lower
chamber medium was measured fluorimetrically. Measurements were performed in duplicate and data are presented as means  SD (n = 4).
Versus CTRL: *P < 0.005; versus condition without PTIO: °P < 0.001. (E) Transport of Lipodox and apo-Lipodox across the BBB monolayer. The
hCMEC/D3 cells were grown up to the confluence in Transwell insert, in fresh medium or in the presence of SIM, then 5 mmol·L-1 Lipodox or
apo-Lipodox was added in the upper chamber. When indicated, the free LDL receptor-targeted apoB100 peptide (the same that was conjugated
with apo-Lipodox), was co-incubated with the liposomes (100 mmol·L-1; B100). After 3 h, the amount of doxorubicin recovered by the lower
chamber medium was measured fluorimetrically. Measurements were performed in duplicate and data are presented as means  SD (n = 3).
Versus CTRL: *P < 0.005; versus SIM: °P < 0.05.
British Journal of Pharmacology (2012) 167 1431–1447 1439
BJP ML Pinzón-Daza et al.
of free doxorubicin (see Figures 2E and 3D; comparing these information Figure S3D). We thus excluded a permeabilizing
data the significance of apo-Lipodox vs. doxorubicin was P < effect exerted by the liposomal shell itself.
0.005). The transport of apo-Lipodox across the BBB monol-
ayer was even higher than that of free doxorubicin in cells
exposed to statins (Figures 2E and 3D; significance of apo- The association of statins plus LDL
Lipodox vs. doxorubicin in statin-treated cells: P < 0.001). receptor-targeted liposome-encapsulated
Again, the addition of PTIO dramatically decreased the per- doxorubicin is effective at delivering
meability of apo-Lipodox, suggesting that the NO levels – and doxorubicin into tumour cells co-cultured
the consequent status of nitration on ABC transporters – are with BBB cells
also critical for the transport of apo-Lipodox in hCMEC/D3 The abundance of ABC transporters on primary CNS
cells. tumours, on cerebral metastasis of epithelial solid tumours
In addition to the increased intracellular retention due to and on BBB cells determines a poor response to chemo-
the inhibition of ABC transporters, we also investigated therapy. To clarify if the association of statins and apo-
whether an increased uptake by LDL receptor-mediated endo- Lipodox can overcome such resistance, we produced
cytosis was critical in determining the greater permeability of co-culture models by growing hCMEC/D3 cells on Transwell
apo-Lipodox. Apo-Lipodox exhibited a mean concentration insert and tumour cells – that is, human glioblastoma multi-
of LDL receptor-targeted peptide of 20  1.6 mg·mL-1, corre- forme U87-MG cells, human neuroblastoma SJKNP cells,
sponding to about 3 ¥ 1012 peptides·mL-1 (n = 3). human invasive breast cancer MD-MBA-231 cells and human
The permeabilities of Lipodox (liposomal doxorubicin non-small cells lung cancer A549 cells – in the lower chamber.
without the LDL receptor-targeted peptide) and apo-Lipodox A preliminary characterization of these cell lines showed
were similar in untreated hCMEC/D3 cells and were unaf- that they all expressed at various levels Pgp/ABCB1, MRP1/
fected by an excess of free LDL receptor-targeted peptide ABCC1 and BCRP/ABCG2 (Figure 4A), and that simvastatin
under basal conditions (Figure 3E). However, apo-Lipodox did not modify the amount of these transporters (Figure 4A)
crossed the BBB monolayer at a significantly higher rate when but increased the surface LDL receptors (Figure 4B). Whereas
cells where pre-incubated with simvastatin, which increased the intracellular content of free doxorubicin was low in all
the amount of LDL receptors (Figure 3A and B); this transport the cell lines (giving a fluorescence similar to the autofluo-
was dramatically decreased by the competing free LDL rescence of untreated cells, Figure 4C) and not sufficient to
receptor-targeted peptide (Figure 3E). In contrast, Lipodox elicit cytotoxicity (Figure 4C), apo-Lipodox was accumulated
permeability was the same in the presence of simvastatin or to higher extent, exerting significant cell damage, as revealed
of the competing peptide (Figure 3E), suggesting that the by the release of LDH in the culture supernatant. The addi-
uptake of non-targeted liposomes was not dependent on the tion of simvastatin to free doxorubicin enhanced the drug
LDL receptor-mediated endocytosis. retention and the LDH release compared to doxorubicin
To assess whether the presence of PEG influences the alone. For all the cell lines, the maximal increase in these
attachment of the LDL receptor-targeted peptide to the lipo- parameters was achieved by the combination of simvastatin
somal shell and/or the binding to the LDL receptor, parallel plus apo-Lipodox (Figure 4C).
experiments were performed with anionic non-pegylated When added to the upper chamber of the Transwell con-
liposomes. These particles did not significantly differ in their taining confluent hCMEC/D3 cells on the insert and U87-MG
radius (Table 1) or in the amount of peptide attached (22  in the lower chamber, doxorubicin poorly entered glioblas-
1.9 mg·mL-1; n = 3). The permeability of pegylated and non- toma cells, as shown by the low red fluorescence in U87-MG
pegylated apo-Lipodox was superimposable in both untreated cells recovered by the lower chamber (Figure 5A); the drug
and simvastatin-treated hCMEC/D3 cells (Supporting infor- delivery into tumour cells was improved by pretreating BBB
mation Figure S2), suggesting that the presence of PEG did cells with simvastatin for 48 h followed by doxorubicin or
not interfere with the attachment of LDL receptor-targeted apo-Lipodox in the last 24 h. The strongest fluorescent signal
peptide or with the liposome uptake, either by simple endo- was achieved by the association of simvastatin (for 48 h) plus
cytosis or LDL receptor-mediated endocytosis. apo-Lipodox (in the last 24 h; Figure 5A).
Empty liposomes may affect different intracellular path- Quantitative fluorimetric analysis of intratumour doxoru-
ways modulating the activity of Pgp/ABCB1 in epithelial cells bicin confirmed that the drug content was significantly
(Riganti et al., 2011). To clarify whether empty liposomes higher when simvastatin was combined with apo-Lipodox
affected the activation of NF-kB, the synthesis of nitrite, the rather than with doxorubicin in all the cell lines co-cultured
activity of NOS enzyme, the induction of necrosis or apop- with hCMEC/D3 cells (Figure 5B). In keeping with these
tosis, we measured these parameters (Supporting information results, doxorubicin alone did not exert any relevant cyto-
Figure S3A–C) in hCMEC/D3 cells, exposed to the same toxic effect in tumour cells, in terms of LDH release
empty anionic pegylated liposomes used for apo-Lipodox, (Figure 5B) and caspase-3 activation (Figure 5C). The associa-
conjugated or not with the LDL receptor-targeted peptide. tion of free doxorubicin plus statin or the use of apo-Lipodox,
None of the liposomal formulations modified the above two experimental conditions that increased the intracellular
parameters compared to the untreated cells. Furthermore, amount of doxorubicin in the cell lines without a BBB mon-
when we co-incubated free doxorubicin with empty lipo- olayer (Figure 4C), were significantly less effective in the pres-
somes, with or without the LDL receptor-targeted peptide, in ence of BBB monolayer (Figure 5B), although apo-Lipodox
hCMEC/D3 cells pretreated or not with simvastatin increased the release of LDH in all the cell lines and induced
(0.1 mmol·L-1 for 48 h), we did not detect a different perme- the activation of caspase-3 in A549 and SJKNP cells
ability compared with free doxorubicin alone (Supporting (Figure 5C). Also in BBB-tumour cells co-cultures, the
1440 British Journal of Pharmacology (2012) 167 1431–1447
New strategy for drug delivery into brain tumours BJP
Figure 4
Characterization of different CNS- and non-CNS-derived tumour cells for ABC transporters expression, surface LDL receptors and resistance to
doxorubicin. Human glioblastoma U87-MG cells, human neuroblastoma SJKNP cells, human breast cancer MDA-MB-231, human lung cancer
A549 cells were grown in the absence (CTRL) or presence of simvastatin (0.1 mmol·L-1for 48 h; SIM). When indicated, 5 mmol·L-1doxorubicin
(DOXO) or apo-Lipodox (APOLIPO) were added in the last 24 h. (A) Western blot analysis of ABC transporters. The expression of Pgp, MRP1 and
BCRP on whole cells extracts was detected by Western blotting. The expression of GAPDH was used to check the equal protein loading. The figure
is representative of three experiments with superimposable results. (B) Flow cytometry analysis of surface LDL receptors in untreated cells (dotted
line) or SIM-treated cells (continuous line). Negative controls, with non-immune isotypic antibodies, are represented by the grey peak. The figures
shown here are representative of two similar experiments, each performed in duplicate. (C) Intracellular doxorubicin accumulation and toxicity.
Culture supernatant was checked for the extracellular activity of LDH; cells were detached and lysed to quantify the intracellular amount of
doxorubicin, as described in Methods. The hatched columns in the conditions ‘CTRL’ and ‘SIM’ refer to the cell autofluorescence, measured in
the absence of doxorubicin or apo-Lipodox administration. Measurements were performed in duplicate and data are presented as means  SD
(n = 3). Versus CTRL: *P < 0.05.
British Journal of Pharmacology (2012) 167 1431–1447 1441
BJP ML Pinzón-Daza et al.
Figure 5
Drug delivery and antitumour efficacy of statins plus LDL receptor-targeted liposomal doxorubicin in co-culture models. The hCMEC/D3 cells were
grown for 7 days up to confluence in Transwell inserts, whereas U87-MG, SJKNP, A549 or MDA-MB-231 cells were seeded at day 4 in the lower
chamber. At day 0, supernatant in the upper chamber was replaced with fresh medium without (CTRL) or with simvastatin (0.1 mmol·L-1for 48 h;
SIM). 5 mmol·L-1doxorubicin (DOXO) or apo-Lipodox (APOLIPO) were added in the upper chamber of Transwell in the last 24 h, then the following
investigations were performed. (A) Microscope analysis of doxorubicin accumulation. U87-MG cells were seeded on sterile glass coverslips, treated
as reported above, then analysed by fluorescence microscopy to detect the intracellular accumulation of doxorubicin. The cells were also
counterstained with the nuclear fluorescent probe DAPI. The micrographs are representative of three experiments with similar results. (B) Culture
supernatant of tumour cells was checked for the extracellular activity of LDH, cells were detached and lysed to quantify the intracellular amount of
doxorubicin, as described in Methods. The hatched columns of the conditions ‘CTRL’ and ‘SIM’ refer to the cell autofluorescence, measured in the
absence of doxorubicin or apo-Lipodox administration. Measurements were performed in duplicate and data are presented as means  SD (n = 4).
Versus CTRL: *P < 0.05. (C) Apoptosis induction. The activation of caspase-3 in tumour cells lysates was measured fluorimetrically as described in the
Methods section. Measurements were performed in duplicate and data are presented as means  SD (n = 3). Versus CTRL: *P < 0.05.
1442 British Journal of Pharmacology (2012) 167 1431–1447
New strategy for drug delivery into brain tumours BJP
association of simvastatin plus apo-Lipodox was the most integrity of tight junctions and to the increased paracellular
effective at achieving a significant delivery of doxorubicin leakage of drugs across the BBB monolayer (Monnaert et al.,
(Figure 5B) and inducing cell death by both necrosis 2004). In our work, we observed that a strong decrease in
(Figure 5B) and apoptosis (Figure 5C). Free doxorubicin membrane cholesterol, caused by b-methyl-cyclodextrin,
co-incubated with empty liposomes, with or without the LDL actually impaired the integrity of tight junctions in
receptor-targeted peptide, did not accumulate within hCMEC/D3 cells, but the smaller variations of membrane
U87-MG cells more than doxorubicin alone, either in the cholesterol produced by statins did not. According to these
absence or in the presence of simvastatin (Supporting infor- data, it seems unlikely that statins – at least at the concen-
mation Figure S4A). Similarly, the death of glioblastoma cells, trations used in this study – increase the paracellular trans-
measured as release of LDH and activation of caspase-3, was port across BBB. At low (micromolar) concentrations, that is,
not increased by the addition of free doxorubicin to empty in keeping with the Ki of statins for 3-hydroxy-3-
liposomes (Supporting information Figure S4B). methylglutaryl coenzyme A reductase (Liao and Laufs, 2005),
mevastatin and simvastatin lowered the synthesis of isopre-
noids in hCMEC/D3 cells and decreased the activity of
Discussion and conclusions RhoGTPase. In parallel, they increased the synthesis of NO,
by up-regulating the IKK/NF-kB-mediated expression of
The therapeutic management of CNS tumours, currently iNOS/NOS II and by enhancing the phosphorylation on
based on surgery, radiotherapy and chemotherapy, is not serine 1177 of eNOS/NOS III. The effects exerted by RhoA
completely safe and compatible with an acceptable quality of on NF-kB and NO synthesis are highly variable and depend
life. Chemotherapy is the first choice in disseminated on cell type (Nakata et al., 2007; Ahn et al., 2008; 32: Ye et al.,
tumours, such as invasive glioblastoma, high-risk medullob- 2008). Lovastatin has been reported to inhibit RhoA, stimu-
lastoma or multiple metastasis, but the percentage of success late the activity of IKK-a and NF-kB and enhance the expres-
remains low. New targeted therapies, anti-angiogenic thera- sion of iNOS in rat glioma cells (33: Rattan et al., 2003); we
pies or gene therapies show a real benefit only in limited hypothesize that such a mechanism occurs also in our model.
groups of patients with known specific molecular defects It has been also shown that RhoA kinase reduces the stability
(Sathornsumetee et al., 2007; Rossi et al., 2008). Thereby, the of eNOS mRNA and prevents the Akt-dependent phosphor-
development of new pharmacological therapies for CNS ylation of eNOS (Rikitake and Liao, 2005). In accord with
tumours is still needed. these observations, we detected an increased phosphoryla-
The low drug delivery across BBB and the low drug accu- tion of eNOS in BBB cells treated with statins that inhibited
mulation within the tumour are among the main factors that RhoA kinase. This event, together with the up-regulation of
decrease the efficacy of chemotherapy in primary and meta- iNOS isoform, led to a significant increase in NO levels and
static brain tumours. In addition, having no fenestrations this suggests that this is the mechanism by which statins
and many tight junctions, the brain microvascular endothe- reduce the vascular tone and increase cerebral flow, as inhibi-
lium has high levels of drug efflux pumps of the ABC trans- tors of Rho kinase produce similar effects (Rikitake et al.,
porter family (Declèves et al., 2006; Mercer et al., 2009; Robey 2005).
et al., 2010). The latter have also been identified in CNS A high blood flow usually ensures better delivery of anti-
tumours (Nakagawa et al., 2009), where they contribute to cancer drugs within tumour areas and better tumour oxy-
the frequently observed chemoresistance. Therefore, an effec- genation enhances the cytotoxicity of many anticancer drugs
tive pharmacological therapy for CNS malignancies should including doxorubicin. In addition to these theoretical ben-
overcome the extrusion of the drugs from both endothelial efits, the increased production of NO elicited by the statins
and tumour cells. In this perspective, we suggest a new com- reduced the activity of at least two proteins – Pgp/ABCB1 and
bination approach, based on the association of statins plus BCRP/ABCG2 – that are involved in the apical extrusion of
LDL receptor-targeting liposomal drug, starting from two drugs in hCMEC/D3 cells (Tai et al., 2009) and in vivo
considerations: (i) both statins and liposomal drugs have (Declèves et al., 2006; Mercer et al., 2009; Robey et al., 2010).
chemosensitizer properties in solid tumours; (ii) liposomal Activators of endogenous NO synthesis, such as lipopoly-
drugs are delivered across the BBB more easily than free drugs. saccharide, TNF-a and endothelin-1, as well as the NO donor
We have previously observed that in cancer cells, statins SNP, are known to affect Pgp/ABCB1 in rat brain capillaries,
increase the synthesis of NO, which inhibits ABC transporters producing a decrease in the transporter expression and activ-
efflux (Riganti et al., 2005; Riganti et al., 2006; Riganti et al., ity after a 3 h incubation (Bauer et al., 2007; Hartz et al., 2007)
2008), and decrease the amount of membrane cholesterol, followed by an increase at 6 h (Bauer et al., 2007). Since the
which also lowers the activity of Pgp/ABCB1 (Kopecka et al., expression of Pgp/ABCB1 is subjected to rapid changes in BBB
2011). cells (Bauer et al., 2007; Hawkins et al., 2010), we cannot
In the past few years, statins have been proposed as drugs exclude the possibility that the NO synthesis induced by
with potential benefits in cerebrovascular and neurodegen- statins produces a decrease in Pgp/ABCB1 expression at
erative diseases, but to our knowledge, whether they have earlier time points (e.g. less than 6 h), followed by a return to
effects on the transport functions of brain microvascular a ‘steady state’ at 24 h. Furthermore, the effects of NO are
endothelium has not been investigated. We found here that highly dependent on the species studied and on the amount
in human BBB cells, the lipophilic statins mevastatin and and rate of NO released (Huerta et al., 2008). This variability
simvastatin decreased the synthesis of cholesterol and isopre- may explain some apparently contrasting findings, that is, a
noids and reduced the amount of cholesterol in plasma mem- high concentration of exogenous NO for 6 h increased the
brane. This event has been previously linked to the loss of expression of Pgp/ABCB1 in rat endothelial cells (Bauer et al.,
British Journal of Pharmacology (2012) 167 1431–1447 1443
BJP ML Pinzón-Daza et al.
2007), whereas weak inducers of NOS II like statins did not efflux of doxorubicin, increasing the trans-cellular transport
produce any change of Pgp/ABCB1 expression in human of the drug. Empty liposomal shells, which inhibited the
hCMEC/D3 cells after 24 h (Figure 2A). Such low levels of activity of Pgp/ABCB1 in chemoresistant tumours (Riganti
endogenous NO, however, were sufficient to elicit a detect- et al., 2011), did not exert any permeabilizing activity on the
able nitration of the transporter, which decreased its activity. delivery of doxorubicin across the BBB, suggesting that the
The reduction was even more pronounced after 48 h, indi- effects of the lipidic envelope alone may be tissue-dependent.
cating that statins induce a persistent inhibition of Pgp/ We show here that simvastatin enhanced the number of
ABCB1 activity after a longer time. As we considered the LDL receptors exposed and induced chemosensitization in
prolonged half-life of Pgp/ABCB1 in low-serum mediums different human tumour (glioblastoma, neuroblastoma, non-
(Zhang and Ling, 2000), the standard culture condition for small cell lung cancer, breast cancer) cells with constitutive
hCMEC/D3 cells, we expected the reduction in activity of the expression of Pgp/ABCB1, MRP1/ABCC1 and BCRP/ABCG2
ABC pumps to be higher at 48 h than at 24 h, although a and different degrees of resistance to doxorubicin. Pretreat-
clear nitration of the pump was already detectable after 24 h. ment of the tumour cells with simvastatin followed by the
The reversal of statins’ effects by the NO scavenger PTIO apo-Lipodox showed the greatest cytotoxic efficacy not only
suggests that the persistent inhibition of Pgp/ABCB1 and in primary CNS tumours like glioblastoma, but also in epi-
BCRP/ABCG2 activity was due to a continuous synthesis of thelial tumours, such as lung and breast cancers cells, whose
NO by hCMEC/D3 cells. metastasis within the CNS are often unresponsive to therapy.
Since in other models of BBB, the rate of drug efflux was In co-culture models, the application of free doxorubicin on
NO independent (Salkeni et al., 2009), we cannot exclude a the luminal side of hCMEC/D3 monolayer did not yield any
priori that statins also reduce the activity of ABC transporters strong drug delivery into the tumour cells growing under the
by other mechanisms, for example, by decreasing the Pgp/ BBB. Apo-Lipodox alone or the combination of simvastatin
ABCB1 expression and glycosylation (Sieczkowski et al., plus free doxorubicin increased drug delivery and toxicity.
2010) or by reducing the amount of cholesterol in the plasma However, the extent of this increase was variable, depending
membrane, an event that impairs the activity of Pgp/ABCB1 on the different levels and activity of ABC transporters and
(Troost et al., 2004; Kopecka et al., 2011). Whatever the pleio- LDL receptors, and on the existence of other mechanisms of
tropic effects of statins on ABC transporters are, the final resistance characterizing each cell line. The presence of spe-
result of treating BBB cells with statins was a dramatic cific tumour cells on the basal side of hCMEC/D3 cells may
increase in the permeability of doxorubicin, a substrate of also affect the permeability of the BBB differently, determin-
both Pgp/ABCB1 and BCRP/ABCG2. ing different rate and kinetics of drug delivery. Only the
In all mammalian cells, statins also up-regulate the tran- pretreatment of the co-cultures with simvastatin followed by
scription and surface exposure of LDL receptors, offering a apo-Lipodox elicited a clear increase in doxorubicin delivery
flexible method of designing specific, nanoparticle-based tar- and toxicity, consisting of both necrotic and apoptotic death,
geted therapies. We have previously validated the efficacy of in all the models investigated, including the highly chemore-
an engineered LDL receptor-targeted liposome-encapsulated sistant glioblastoma.
doxorubicin (‘apo-Lipodox’) in solid tumours with high A direct anti-tumour activity of statins against glioblas-
expression of LDL receptors (Kopecka et al., 2011). Besides toma cells has been reported previously (Bababeygy et al.,
being taken up more by LDL receptor-driven endocytosis 2009; Yanae et al., 2011): for instance, in rat C6 glioma cells,
(Kopecka et al., 2011), liposomal particles alter the lipid com- 5 mmol·L-1 mevastatin and simvastatin induced apoptosis by
position of the plasma membrane microdomains (i.e. lipid decreasing the intracellular availability of GGPP and the acti-
rafts) where Pgp/ABCB1 is active, interfere with ATP hydroly- vation of ERK1/2 and Akt (Yanae et al., 2011), suggesting that
sis and substrate binding, and elicit a lower extrusion of the effect could be mediated by a decrease in the activity of a
doxorubicin in Pgp-overexpressing cells (Riganti et al., 2011). geranylgeranylated protein. We did not detect direct cyto-
In untreated hCMEC/D3 cells, the permeability for apo- toxic effects of statins in co-cultured glioblastoma cells,
Lipodox was significantly greater than that of free doxoru- perhaps because we used simvastatin at lower dose. It is
bicin, probably as a consequence of the decreased efflux via noteworthy, however, that statins, at concentrations that are
Pgp/ABCB1 and of the increased uptake. Since both the non- found in the blood of patients receiving anti-cholesterolemic
LDL receptor-targeted Lipodox and the LDL receptor-targeted therapy, enhanced the efficacy of a chemotherapeutic drug
apo-Lipodox had a higher uptake than free doxorubicin in like doxorubicin against glioblastoma cells.
the absence of statins, it is likely that liposomes have a facili- Phase I/II trials with pegylated liposomal doxorubicin
tated entry within BBB cells by simple endocytosis. The have shown that the drug has very few side effects and is well
uptake of apo-Lipodox was unaffected by an excess of the free tolerated, but does not offer a significant advantage in terms
LDL receptor-binding apoB100 peptide under basal condi- of overall survival or progression-free survival in comparison
tions, suggesting that the LDL receptors present on the to the standard protocols based on radio-chemotherapy (Glas
surface of untreated hCMEC/D3 cells were too few to play a et al., 2007; Beier et al., 2009; Ananda et al., 2011). For this
critical role in the liposome uptake. In contrast, the LDL reason, chemical modifications and improvements of drug-
receptor became a helpful tool to increase the endocytosis of loaded nanoparticles are under intensive investigations
targeted liposomes when its levels increased on the cells’ (Michaelis et al., 2006; Nikanjam et al., 2007; Guo et al., 2011;
surface, that is, after exposure to statins. Simvastatin, which Wohlfart et al., 2011). The approach proposed in our work
forced BBB cells to expose more LDL receptors and simulta- differs from the other studies on CNS-penetrating nanoparti-
neously decreased the activity of Pgp/ABCB1, indeed cles because it is more ‘physiological’: it takes advantage of
enhanced the apo-Lipodox uptake and reduced the apical the usual metabolic effects of therapeutic doses of statins –
1444 British Journal of Pharmacology (2012) 167 1431–1447
New strategy for drug delivery into brain tumours BJP
the increase in LDL receptors exposed and the synthesis of Ananda S, Nowak AK, Cher L, Dowling A, Brown C, Simes J et al.
NO – and from the properties of liposomes, that induce (2011). Phase 2 trial of temozolomide and pegylated liposomal
permeability across the BBB and efficacy against drug- doxorubicin in the treatment of patients with glioblastoma
resistant tumours. We are currently testing this association in multiforme following concurrent radiotherapy and chemotherapy.
animal models bearing primary and metastatic CNS tumours, J Clin Neurosci 18: 1444–1448.
in order to determine the pharmacokinetic profile, the Bababeygy SR, Polevaya NV, Youssef S, Sun A, Xiong A,
optimal administration regimen, the immunogenicity of Prugpichailers T et al. (2009). HMG-CoA reductase inhibition causes
apoLipodox, the antitumour effect and the presence of side increased necrosis and apoptosis in an in vivo mouse glioblastoma
effects. Testing our strategy in animals will add further impor- multiforme model. Anticancer Res 29: 4901–4908.
tant details about the molecular mechanisms of apo-Lipodox, Bauer B, Hartz AMS, Miller DS (2007). Tumor necrosis factor a and
inferred by these in vitro experiments, in order to validate the endothelin-1 increase P-glycoprotein expression and transport
feasibility and efficacy of the association proposed. activity at the blood-brain barrier. Mol Pharmacol 71: 667–675.
The problem of poor permeability across the BBB is not
Beier CP, Schmid C, Gorlia T, Kleinletzenberger C, Beier D,
limited to anticancer drugs, but also affects the delivery of Grauer O et al. (2009). RNOP-09: pegylated liposomal doxorubicine
agents used in epilepsy and neurodegenerative diseases. In a and prolonged temozolomide in addition to radiotherapy in newly
general perspective, our ‘Trojan horse’ approach, based on diagnosed glioblastoma-a phase II study. BMC Cancer 9: 308–318.
the administration of statins followed by a LDL receptor-
targeting liposomal drug, might have potential applications Carl SM, Lindley DJ, Couraud PO, Weksler BB, Romero I,
in the pharmacological therapy of different CNS diseases. Mowery SA et al. (2010). ABC and SLC transporter expression and
pot substrate characterization across the human CMEC/D3
blood-brain barrier cell line. Mol Pharm 7: 1057–1068.
Declèves X, Amiel A, Delattre JY, Scherrmann JM (2006). Role of
Acknowledgements ABC transporters in the chemoresistance of human gliomas. Curr
Cancer Drug Targets 6: 433–445.
We thank Mr Costanzo Costamagna, Department of Genet- Deelen JF, Loscher W (2007). The blood-brain barrier and cancer:
ics, Biology and Biochemistry, University of Turin, for the transporters, treatment, and Trojan horses. Clin Cancer Res 13:
technical assistance and Mr Silvano Panero, Department of 1663–1674.
Vegetal Biology, University of Turin, for the help with the
Doublier S, Riganti C, Voena C, Costamagna C, Aldieri E,
TEM analysis. We are grateful to Dr Marco Minella and Prof
Pescarmona G et al. (2008). RhoA silencing reverts the resistance to
Claudio Minero, Department of Analytical Chemistry, Uni- doxorubicin in human colon cancer cells. Mol Cancer Res 6:
versity of Turin, for having provided the dynamic light scat- 1607–1620.
tering facility and for the valuable advices in the
interpretation of the data. We are indebted with Prof Davide Glas M, Koch H, Hirschmann B, Jauch T, Steinbrecher A,
Schiffer, Neuro-Bio-Oncology Center, Vercelli, Italy, for the Herrlinger U et al. (2007). Pegylated liposomal doxorubicin in
recurrent malignant glioma: analysis of a case series. Oncology 72:
helpful discussion and the critical revision of the manuscript.
302–307.
This work has been supported by grants from Compagnia
di San Paolo, Italy (Neuroscience Program; grant 2008.1136) Guo L, Fan L, Pang Z, Ren J, Ren Y, Li J et al. (2011). TRAIL and
and Italian Association for Cancer Research (AIRC; MFAG doxorubicin combination enhances anti-glioblastoma effect based
11475) to CR. M L P-D is a recipient of an ERACOL Erasmus on passive tumor targeting of liposomes. J Control Release 154:
Mundus fellowship. 93–102.
Hartz AMS, Bauer B, Fricker G, Miller DS (2007). Rapid modulation
of P-glycoprotein-mediated transport at the blood-brain barrier by
tumor necrosis factor-a and lipopolysaccharide. Mol Pharmacol 69:
Conflict of in interest 462–470.
Hawkins BT, Sykes DB, Miller DS (2010). Rapid, reversible
None. modulation of blood-brain barrier P-glycoprotein transport activity
by vascular endothelial growth factor. J Neurosci 30: 1417–1425.
Huerta S, Chilka S, Bonavida B (2008). Nitric oxide donors: novel
cancer therapeutics. Int J Oncol 33: 909–927.
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online version of this article:
Rikitake Y, Kim HH, Huang Z, Seto M, Yano K, Asano T et al.
(2005). Inhibition of Rho kinase (ROCK) leads to increased cerebral Figure S1 Effects of statins on membrane cholesterol and
blood flow and stroke protection. Stroke 36: 2251–2257. tight junctions integrity in hCMEC/D3 cells. (A) Cells were
1446 British Journal of Pharmacology (2012) 167 1431–1447
New strategy for drug delivery into brain tumours BJP
incubated in the absence (CTRL) or presence of 0.1 mmol·L-1 Methods section. Data are presented as means  SD (n = 3). (C)
mevastatin (MVS) or simvastatin (SIM) for 24 h or Cytotoxicity. Culture supernatant of cells was checked for the
10 mmol·L-1 b-methyl cyclodextrin (MCD) for 3 h, then lysed extracellular activity of LDH (open bars), cells were detached
and subjected to cell membranes isolation. The cholesterol and lysed to measure the activity of caspase-3 (hatched bars),
content in membrane extracts was measured spectrophoto- as described in the Methods section. Measurements were per-
metrically as described in the Methods section. Measure- formed in duplicate and data are presented as means  SD
ments were performed in duplicate and data are presented as (n = 3). (D) Transport of doxorubicin across BBB monolayer in
means  SD (n = 3). Vs CTRL: *P < 0.01. (B) Cells were seeded the presence of empty liposomes. The hCMEC/D3 cells were
on Transwell insert, grown up to confluence for 7 days and grown up to the confluence for 7 days in Transwell insert, in
incubated as reported in A; 2 mCi·mL-1 [14C]-inulin was then fresh medium or in the presence of simvastatin (5 mmol·L-1 for
added in the upper chamber. After 3 h the medium was recov- 48 h; SIM); then 5 mmol·L-1 doxorubicin (DOXO), alone or
ered by the lower chamber and the amount of [14C]-inulin co-incubated with 5 mmol·L-1 empty anionic pegylated lipo-
was measured by liquid scintillation. Measurements were per- somes (EL) or empty anionic pegylated LDL receptor-targeted
formed in duplicate and data are presented as means  SD conjugated-liposomes (apoEL), were added in the upper
(n = 4). Vs CTRL: *P < 0.05. chamber. After 3 h the amount of the drug recovered by the
Figure S2 Effects of pegylation on the permeability of LDL lower chamber medium was measured fluorimetrically. Meas-
receptor-targeted liposomal doxorubicin across the BBB. Cells urements were performed in duplicate and data are presented
were grown up to the confluence for 7 days in Transwell as means  SD (n = 3). Vs the corresponding condition
insert, then incubated in the absence (CTRL) or presence of without SIM: * P < 0.005.
0.1 mmol·L-1 simvastatin (SIM) for 48 h. 5 mmol·L-1 apo- Figure S4 Effects of empty liposomes on the delivery and
Lipodox obtained from anionic non-pegylated liposomes cytotoxicity of free doxorubicin in co-culture models. The
(COATSOME EL01A series; - PEG) or from anionic pegylated hCMEC/D3 cells were grown for 7 days up to confluence in
liposomes (COATSOME EL-01-PA series; + PEG) was added in Transwell inserts, whereas U87-MG cells were seeded at day 4
the upper chamber and after 3 h the amount of the drug in the lower chamber. At day 0, supernatant in the upper
recovered by the lower chamber medium was measured fluori- chamber was replaced with fresh medium without
metrically. Measurements were performed in duplicate and (CTRL) or with simvastatin (0.1 mmol·L-1for 48 h; SIM).
data are presented as means  SD (n = 3). Vs CTRL: * P < 0.005. 5 mmol·L-1doxorubicin (DOXO), alone or co-incubated
Figure S3 Effects of empty liposomes on NF-kB activity, NO with 5 mmol·L-1 empty anionic pegylated liposomes (EL) or
synthesis, cytotoxicity and permeability in hCMEC/D3 cells. empty anionic pegylated LDL receptor-targeted conjugated-
The hCMEC/D3 cells were incubated in the absence (CTRL) or liposomes (apoEL), were added in the upper chamber of Tran-
presence of 5 mmol·L-1 empty anionic pegylated liposomes swell in the last 24 h, then the following investigations were
(EL) or empty anionic pegylated LDL receptor-targeted performed. (A) U87-MG cells were lysed in ethanol/HCl and
conjugated-liposomes (apoEL) for 24 h, then subjected to the the intracellular amount of doxorubicin was measured fluori-
following investigations. (A) NF-kB activity. The activity of metrically (see Methods section). Measurements were per-
NF-kB was detected in the nuclear extracts measuring the formed in duplicate and data are presented as means  SD
DNA-binding capacity of NF-kB on its target sequence (see (n = 3). (B) The culture supernatant of tumor cells was
Methods). Measurements were performed in duplicate and checked for the extracellular activity of LDH (open bars), cells
data are presented as means  SD (n = 3). (B) NO synthesis. were detached and lysed to measure the activity of caspase-3
NOS activity in cell lysates (open bars) and nitrite accumula- (hatched bars), as described in the Methods section. Measure-
tion in the extracellular medium (hatched bars) were meas- ments were performed in duplicate and data are presented as
ured with spectrophotometric assays, as reported in the means  SD (n = 3).
British Journal of Pharmacology (2012) 167 1431–1447 1447