FASEB J. Avanti Polar Lipids
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Published as doi: 10.1096/fj.07-102855.
 (The FASEB Journal. 2008;22:3358-3369.)
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Human tumor nanoparticles induce apoptosis of pancreatic cancer cells

Elodie Ristorcelli, Evelyne Beraud, Patrick Verrando, Claude Villard, Daniel Lafitte, Véronique Sbarra, Dominique Lombardo1 and Alain Verine

INSERM UMR 911; Aix-Marseille Université, Marseille, France

1 Corresponding author: Faculté de Médecine, 27 Bld Jean Moulin, 13385 Marseille Cedex 05, France. E-mail: dominique.lombardo{at}medecine.univ-mrs.fr


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Exosomes are vesicles secreted by most hematopoietic cells on fusion of multivesicular endosomes with the plasma membrane. Many studies have reported that exosomes may also be released by tumor cells. Exosomes are believed to play an antitumor role through immune cells. We asked whether tumor exosomes have biological activities on tumor cells. We report that human pancreatic tumor nanoparticles, exosome-like as characterized by proteomic analyses and rich in lipid rafts, decreased tumor cell proliferation. Nanoparticles increased Bax and decreased Bcl-2 expressions. Caspase-3 and -9 but not caspase-8 inhibitors impaired apoptosis, which implicates the mitochondria apoptotic pathway. The ceramide-sphingomyelin apoptotic pathway was inoperative. Moreover, nanoparticles induced phosphatase and tensin homolog (PTEN) and glycogen synthase kinase (GSK) -3β activation and decreased pyruvate dehydrogenase activity. In nanoparticle-treated cells, PTEN formed complexes with actin, β-catenin, and GSK-3β. Thus, β-catenin may no longer be available to activate the survival pathway. Nanoparticles triggered the down-regulation of cyclin D1 and poly(ADP-ribose) polymerase. Hence, nanoparticles counteracted the constitutively activated phosphatidylinositol 3-kinase/Akt survival pathway to drive tumor cells toward apoptosis. Our study provides the first evidence of an apoptotic function of tumor-derived nanoparticles on tumor cells. We propose a new role for nanoparticles, i.e., as signal carriers for interaction between cells, which may have implications in physiopathological situations.—Ristorcelli, E., Beraud, E., Verrando, P., Villard, C., Lafitte, D., Sbarra, V., Lombardo, D., Verine, A. Human tumor nanoparticles induce apoptosis of pancreatic cancer cells.


Key Words: exosomes • cell death • proliferation


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
NANOPARTICLES DIFFER FROM MICROPARTICLES in size and protein and lipid composition (1) . Nanoparticles arise from the inward budding of endosomal membranes, which generates multivesicular bodies. These multivesicular bodies fuse with the plasma membrane (2) , leading to the secretion of internal vesicles. Exosomes are released as nanoparticles into the extracellular environment by various cells, including reticulocytes (3) , platelets (4) , B and T lymphocytes (5) , and dendritic cells (6) . In addition to differences in their protein contents, exosomes also differ in lipid composition according to their parent cells (7) . Many studies have provided evidence that human tumor cells and tumor cell lines secrete membrane vesicles morphologically analogous to the exosomes released by hematopoietic cells (8 9 10) . Nanoparticles were described as being the product of reticulocyte maturation, providing the means to sort obsolete proteins (10) . Antigen-presenting cells exploit exosomes to elicit antitumor T-cell responses (9) . Exosomes expressed by dendritic cells pulsed with tumor peptides elicit T cell-dependent rejection of established tumors (11) . Furthermore, exosomes generated by pulsed dendritic cells may sensitize adjacent dendritic cells, thereby amplifying the immune response. Conversely, exosomes help tumor cells escape from destruction by cytotoxic T-lymphocytes (12) . Lastly, nanoparticles of tumor cell origin are thought to play an antitumor role as a source of tumor antigens (8 , 9) . Exosomes are thus not simple garbage bags; they provide mechanisms to the organism by mediating intercellular communications and shipping messages through the transfer of a panel of proteins from one cell to another (13) .

We therefore asked whether tumor-derived nanoparticles may exert biological effects on tumor cells themselves. For that, we characterized exosome-like nanoparticles rich in lipids forming rafts secreted by human pancreatic tumor cells. Our study provides the first evidence that nanoparticles from tumor cells lead to apoptosis of these cells.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Materials
Peroxidase-labeled antibodies to rabbit and mouse immunoglobulins and to goat immunoglobulins and antibodies to cyclin D1 were from Roche Diagnostics (Basel, Switzerland) or from Calbiochem (San Diego, CA, USA). Antibodies to Bcl-2 and to actin were from DakoCytomation A/S (Glostrup, Denmark) and Sigma-Aldrich Corp. (St. Louis, MO, USA), respectively. A cocktail of phosphatase inhibitors, wortmannin and C48/80, endoglycoceramidase, filipin, fumonisin B2, nocodazole, chloroquine, bafilomycine, sphingosine-1-phosphate, imipramine, desipramine, LiCl, and goat immunoglobulin G (IgG) fluorescein isothiocyanate (FITC) -labeled antibodies to biotin were from Sigma-Aldrich Corp. Agarose-immobilized phosphatase and tensin homolog (PTEN) antibodies and antibodies to phospho-Akt (Thr-308) and to bax were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Other antibodies to cleaved caspase-3 (Asp-175), caspase-3, cleaved caspase-9 (Asp-330), caspase-9, cleaved poly(ADP-ribose) polymerase (PARP) (Asp-214), PARP, phospho-Akt (Ser-473), phospho-glycogen synthase kinase (GSK) -3β (Ser-9), phospho-PTEN (Ser-380), phospho-PDK1 (Ser-241), PTEN, phospho β-catenin (Ser-33/37, Thr41), and Akt were from Cell Signaling (Beverly, MA, USA), and antibodies to β-catenin were provided by Abcam (Cambridge, UK). RPMI 1640 and Dulbecco’s modified Eagle’s medium cell culture media, penicillin, streptomycin, and trypsin-EDTA were from Cambrex Corp. (Walkersville, MD, USA). Streptavidin-colloidal gold was from British Biocell International (Cardiff, UK). Polyclonal antibodies (pAbL64), directed against human bile salt-dependent lipase (BSDL), were homemade. Sequencing grade porcine trypsin was from Promega Corp. (Madison, WI, USA). Caspase inhibitors and potassium bisperoxo(1,10-phenanthroline)oxovanadate [bpV(phen)] were from Alexis (San Diego, CA, USA).

Cell growth
Cell lines originated from human pancreatic cancer (SOJ-6, BxPC-3, MiaPaCa-2, and Panc-1), from prostatic cancer (PC3), and from umbilical vein endothelial cells (HUVECs). Cells (8000 cells/well) grown in required medium with 10% fetal calf serum (FCS) unless otherwise stated were seeded in a 96-well culture plates and treated with increasing amounts of nanoparticles. Cell growth was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) assay. All determinations were compared with those of cell controls without added nanoparticles.

Nanoparticle purification
SOJ-6 cells were grown up to 80% confluence in standard medium. The medium was removed. Cells were rinsed 3 times in PBS and maintained for 24–48 h in a serum-free medium (13 ml for a 20-cm-diameter dish). During this last step, the cell viability remained higher than 90%, as estimated by a trypan blue test. The supernatant was collected and centrifuged (1000 g at 4°C for 20 min) before percolation through 0.22-µm filters (14) . Finally, the medium was submitted to ultracentrifugation (200,000 g at 4°C for 16 h) by using an SW41 rotor (Beckman Instruments, Fullerton, CA, USA). The pellet, containing nanoparticles, was resuspended in 0.5 ml of buffer (PBS or HEPES, depending on analyses).

Sucrose density gradient
Nanoparticles were resuspended in 0.5 ml of HEPES buffer (20 mM, pH 7.4) supplemented with sucrose to reach a density of 2.5 g/ml (15) . The solution was placed at the bottom of a centrifuge tube (13 ml). Twelve milliliters of a linear gradient of sucrose (0.25–1 M) density was layered on the nanoparticle suspension and ultracentrifuged as described above. Fractions (1 ml) were collected and their content was analyzed by Western blotting. The density of each fraction was determined by refractometry.

Treatment of nanoparticles with trypsin or endoglycoceramidase
Nanoparticles were incubated at 37°C for 15 min with trypsin 0.05% in PBS or for 7 h with endoglycoceramidase (20 mU) in 20 mM sodium acetate buffer (pH 5) containing 0.2% BSA and 0.1% Lubrol PX. The reaction mixture (500 µl) was diluted to 11 ml in cold PBS, and the excess of enzyme was removed by ultracentrifugation as described above. The effects of trypsin-, endoglycoceramidase-, or mock-treated nanoparticles were estimated by cell proliferation assessment.

Pyruvate dehydrogenase (PDH) activity
PDH activity was determined using the MSP30 Dipstick Assay Kit (Mitosciences, Eugene, OR, USA).

Biotinylation of nanoparticles
Nanoparticles in PBS were incubated (1 h at room temperature) with 20 mg/ml biotinamidocaproate N-hydroxysuccinimide (Molecular Probes, Eugene, OR, USA) in dimethylformamide. At the end of the incubation, the excess reagent was removed by dialyzing the sample against PBS (4 L for 4 h).

SDS-PAGE and Western blotting
SOJ-6 cells were grown in 6-well culture plates. At subconfluence, the medium was removed and replaced for 24 h by fresh medium without FCS. SOJ-6 cells were then incubated with nanoparticles for various times. At the end of incubation, cells were washed 3 times with ice-cold PBS (without Ca2+ and Mg2+). Cell pellets were lysed at 4°C in 0.5 ml of lysis buffer [10 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% Triton X-100, 1 mM benzamidine, and phosphatase inhibitor cocktail]. Homogenates were then cleared by centrifugation (10,000 g at 4°C for 10 min). An aliquot was saved for protein determination (MicroBCA kit; Pierce Chemical, Rockford, IL, USA). Proteins in reducing SDS buffer were separated onto 7.5, 10, or 12% polyacrylamide with 0.1% SDS. After migration, proteins were either silver stained or transferred onto nitrocellulose membranes (Mini Transblot; Bio-Rad, Hercules, CA, USA). Transferred proteins were immunodetected by using appropriate primary and secondary antibodies. After washes, membranes were developed with a chemoluminescent substrate (Roche Diagnostics). In each experiment, a control was included by omitting the primary antibodies.

Immunoprecipitation
Cells were either treated or not with nanoparticles and lysed. Lysates were cleared and incubated overnight at 4°C with 25 µg of an agarose-immobilized PTEN antibody suspension. The antibody-antigen complex was recovered by centrifugation (10,000 g for 20 min). Pellets were washed according to the manufacturer’s protocol. The final supernatant was analyzed by SDS-PAGE. Proteins were then electrotransferred onto nitrocellulose membranes and immunodetected with adequate antibodies.

Mass spectrometry
Large (20x20 cm) SDS-PAGE was performed to optimize the protein separation. The migration was run overnight (60 mA at 4°C) on an SDS-PAGE device (Fisher, Illkirch, France). After migration, gels were silver stained, and the different protein bands were excised, destained, and in-gel digested (16) with trypsin (20 µg/ml in 10 mM Tris/HCl, pH 9, buffer). Peptides were extracted, dried in a vacuum centrifuge, and redissolved in 20 µl of 5% formic acid before mass analyses (17) . The digest was analyzed on an EttanPro spectrometer (GE Healthcare, Little Chalfont, Buckinghamshire, UK). Proteins were identified by Profound (ProteoMetrics, New York, NY, USA) and Mascot software programs (MatrixScience, London, UK) that correlate mass spectrometry data with comprehensive sequence databases.

Lipid analysis
Lipids were extracted in chloroform-methanol-water (1:2:0.9, v/v/v) in a Dounce homogenizer in the presence of standards and then analyzed by gas liquid chromatography for phospholipids, cholesterol, sphingomyelin, and ceramide mass content (18) .

Effects of drugs on cell growth
SOJ-6 cells were preincubated with drugs (60 min in medium supplemented with 0.5% FCS). Drug concentrations used were those commonly described to affect specific cell functions with minimal toxicity. Then drugs (at the same concentration as that of the preincubation) and nanoparticles were incubated for another 24 h. The cell growth was estimated by using an MTT assay. All determinations were compared with those of cells incubated with drug only (control cells) or without drug in the presence or absence of nanoparticles.

Apoptosis determination
SOJ-6 cells grown in 8-well plates (BD Falcon, Bedford, MA, USA) were treated with nanoparticles for 24 h before the addition of a CaspACE FITC-VAD-fmk in situ marker (Promega Corp.) at a final concentration of 10 µM in the culture medium according to the manufacturer’s instructions. Then cells were washed in PBS, fixed (15 min) in 2% paraformaldehyde, and washed once again. The number of apoptotic fluorescent cells was determined in triplicate on collections of fields randomly examined under a fluorescence microscope (Carl Zeiss, New York, NY, USA).

Electron microscopy
After biotin labeling, nanoparticles were incubated 30 min with streptavidin-colloidal gold complexes (5 nm). SOJ-6 cells were washed twice with fresh culture medium and incubated (24 h) with biotin-labeled nanoparticles. The last washing was performed with cold 0.1 M cacodylate (pH 7.4) buffer. The cells were fixed with 1% (v/v) glutaraldehyde in sodium cacodylate buffer (1 h, 4°C). Cells were then postfixed for 1 h in 1% osmium tetroxide at 4°C. Subsequently cells were dehydrated by graded ethanol solutions and embedded in Epon. Thin sections were mounted on Parlodion and carbon-treated nickel grids. The cell-material grids were thoroughly washed with PBS and with distilled water, stained with uranyl acetate and lead acetate, and examined with an electron microscope (JEOL Inc., Peabody, MA, USA). Control experiments were performed by omitting biotin prelabeling of nanoparticle-streptavidin-gold complexes.

Nanoparticles were also examined by electron microscopy. Approximately 20 µg of freshly prepared nanoparticles was disposed on top of Formvar-coated 300-mesh gold grids. They were then fixed (15 min at 20°C) in PBS containing paraformaldehyde and glutaraldehyde (0.2% each). After washings with PBS and water (twice each), a negative staining was performed with 1% phosphotungstic acid in water (1 min at 20°C). Nanoparticles on grids were examined, and pictures were treated with Image J software [National Institutes of Health (NIH); http://rsb.info.nih.gov/nih-image/] to measure nanoparticle diameters.

Immunofluorescence microscopy
SOJ-6 cells grown in 8-chamber tissue culture-treated glass slides (BD Falcon) were incubated with biotinylated nanoparticles for 24 h. Cells were washed in PBS, fixed (15 min) in 2% paraformaldehyde, and washed once again. Cells were then incubated with 1% BSA (15 min) and further with antibiotin FITC-labeled antibodies diluted at 1:100 in PBS and 1% BSA (30 min). Finally, cells on slides were washed with PBS and mounted on glass cover slides in 10% Dabco and 50% glycerol in PBS. Cells were observed and photographed using a confocal microscope (Zeiss; LSM510).

Statistical analysis
Results are expressed as means ± SD. Differences between experimental groups were analyzed with the Mann-Whitney test. A value of P < 0.05 was considered significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Human pancreatic tumor SOJ-6 cells secrete nanoparticles
Six 20-cm-diameter dishes of confluent SOJ-6 cells (150–200x106 cells) were necessary to obtain 2.0 ± 0.18 mg (proteins) of nanoparticles. Electron microscopy shows that SOJ-6 cell nanoparticles are spherical vesicles with a mean diameter of 39 ± 7 nm. The box plot representation indicates that 50% of the particles had a diameter between 34 and 45 nm (Fig. 1 A). The size of these nanovesicles correlated with that of exosomes expressed by dendritic cells (15) .


Figure 1
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  		Figure 1. Characterization of nanoparticles produced by human pancreatic tumor SOJ-6 cells. A) After isolation, nanoparticles from SOJ-6 cells were examined by electron microscopy. Several fields (inset) were photographed and were used to determine the diameter of nanoparticles. The range of observed diameters is represented statistically by the box plot. The line inside the box represents the mean diameter of nanoparticles (n=100). The box represents the interquartile range "50% of values." Tails extend to values within 1.5x the interquartile range. B) The characterization of SOJ-6 cell-derived nanoparticle density was done by centrifugation on a sucrose gradient density (from 1.05 to 1.26 g/ml). Fractions (1 ml) were collected from the top to the bottom of centrifuge tube. An aliquot of each fraction, diluted with an equal volume of 20 mM HEPES buffer, was subjected to SDS-PAGE. After electrotransfer onto nitrocellulose membrane, proteins were probed with antibodies to BSDL (top). After scanning, the black intensity of each lane was quantitated with NIH image software (middle). The slope (bottom) allowed the determination of density (g/ml) at which nanoparticles were collected. C) After separation and migration of SOJ-6 nanoparticle proteins on SDS-PAGE and silver staining, the most-colored bands, S1 to S35, were excised and analyzed by MALDI-TOF mass spectrometry.

Because exosomes are rich in raft-forming lipids (13) , we used the raft-associated pancreatic BSDL (19) as a nanoparticle marker. The different fractions obtained on a continuous sucrose gradient were submitted to SDS-PAGE followed by Western blotting (Fig. 1B , top) using pAbL64 antibodies to BSDL. The amount of nanoparticles in each gradient fraction was estimated by determining the intensity of bands corresponding to BSDL (Fig. 1B , middle). The amount of nanoparticles was maximum in fractions 4 to 6, corresponding to a peak density between 1.06 and 1.15 g/ml (Fig. 1B , bottom), as reported previously (8) . Their low density agrees with their high content in cholesterol and sphingomyelin (see below).

Characterization of proteins carried by pancreatic nanoparticles derived from SOJ-6 cells
Molecule separation on SDS-PAGE and silver staining of bands showed that nanoparticles contained many proteins (Fig. 1C ). Well-resolved bands were excised and analyzed by matrix-assisted laser desorption ionization/time of flight (MALDI-TOF). For protein band identification, several criteria were taken into consideration, such as the sequence coverage, the expectation values, and the number of sequenced peptides (Table 1 ). Among proteins thus identified, several had already been described in nanoparticles prepared from other cells (20) , which shows that isolated vesicles were not microparticles or apoptotic corpses (21) .


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  		Table 1. Proteins analyzed by silver staining, SDS-PAGE visualization (Fig. 1) , and mass spectroscopy

In SOJ-6 nanoparticles, the proteins detected included cytosolic proteins, such as actin (band S23) (Fig. 1C ) and annexin A2 (band S12), and cytoplasmic metabolic enzymes, such as phosphoglycerate mutase (band S19), glucose-6-phosphate isomerase (band S20), lactate dehydrogenase (band S17), and enolase-1 (band S21). Also present were histones (band S8); chaperones, such as Hsp90 (band S29); peptidylprolyl-isomerase A (band S5); galectin-3-binding protein (band S31); and lipocalin (band S22), an extracellular protein implicated in cell proliferation and differentiation. SOJ-6 nanoparticles also contained Cbr1 (band S17), a member of the family of short-chain dehydrogenases/reductases, and calsyntenin (band S30), a neuronal membrane protein of the cadherin superfamily involved in calcium signaling. The serine protease urokinase plasminogen activator (band S18) promotes tumor cell adhesion, migration, and proliferation, as well as extracellular matrix degradation, which facilitates tumor cell invasion and metastasis. BSDL, a pancreatic proangiogenic factor, was also detected by MALDI-TOF and Western blot. Endosomal protein (Rab4), chaperone (Hsc70), and GPI-anchored protein (CD14) were also detected by Western blot (not shown).

Lipid composition of pancreatic nanoparticles derived from SOJ-6 cells
Compared with the whole-cell phospholipid content, the SOJ-6 cell nanoparticle content was depleted in phospholipids such as phosphatidylserine (PS) (Table 2 ), which is highly represented in microparticles. Lysophospholipids and triglycerides were also present. Note, however, that these vesicles are enriched in sphingomyelin, ceramides, and cholesterol, lipids that are characteristic of membrane lipid rafts (22) .


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  		Table 2. Lipid composition of nanoparticles and of parent SOJ-6 cells

Nanoparticles induce apoptosis of SOJ-6 cells
SOJ-6 cell treatment with nanoparticles isolated from pancreatic SOJ-6 cells resulted in a dose-dependent inhibition of cell proliferation 24 h after treatment (Fig. 2 A). The nanoparticle-mediated decrease in cell proliferation correlated with the induction of apoptosis (Fig. 2A ). Nanoparticles derived from SOJ-6 cells also reduced the proliferation of other pancreatic tumor cells, but their effects differed from line to line (Fig. 2B ). However, they did not affect HUVECs taken as "normal" control cells.


Figure 2
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  		Figure 2. Proliferation of human cell lines in the presence of SOJ-6 cell-derived nanoparticles. A) Comparative representation of cell proliferation and apoptosis. SOJ-6 cells were incubated with increasing concentrations of SOJ-6 cell-derived nanoparticles (up to 20 µg/well, or 200 µg/ml in terms of protein). After 24 h of incubation, proliferation was assessed by MTT assay. Results are expressed as a percentage relative to controls (no added nanoparticles). Data are means ± SD of 3 independent experiments. Apoptosis was determined with the CaspACE FITC-VAD-fmk kit ({blacksquare}, percentage of proliferation; {square}, apoptotic cells/cm2). B) Cells were incubated with increasing concentrations (up to 20 µg/well) of nanoparticles: SOJ-6 (•), BxPC-3 ({diamondsuit}), MiaPaCa-2 ({blacksquare}), Panc-1({blacktriangleup}), HUVEC ({triangleup}), and PC3 ({square}).

Ceramide apoptotic pathway is not implicated in SOJ-6 cell apoptosis
Nanoparticles are lipid-rich particles, including sphingomyelin, cholesterol, and ceramides (Table 2) that can be internalized by the cell to reach the endolysosomal compartment, where they can activate the ceramide-dependent apoptotic pathway via sphingomyelinase activation (23) . It can be ruled out that sphingomyelinase produces ceramides, because imipramine and desipramine, two potent inhibitors of acid sphingomyelinase, had no reverse effect on the inhibition of the proliferation induced by nanoparticles (Fig. 3 A). Furthermore, sphingosine-1-phosphate, which inhibits ceramide-induced apoptosis (24) did not affect the decrease in SOJ-6 proliferation in the presence of nanoparticles. Last, nanoparticles by themselves did not activate the de novo synthesis of ceramides, as shown by the ineffectiveness of fumonisin B2 in reversing the decrease in proliferation promoted by nanoparticles. We then examined the effects of nanoparticles on SOJ-6 cell proliferation in the presence of drugs affecting the endolysosomal route. Nocodazole, which promotes tubulin depolymerization and disrupts microtubules, also decreased SOJ-6 cell proliferation (Fig. 3A ). Despite the presence of the drug, nanoparticles still significantly decreased cell proliferation. Drugs increasing the lysosomal pH (bafilomycine A and chloroquine) did not affect the nanoparticle antiproliferative effects. These data rule out the implication of the apoptotic pathway depending on the sphingomyelin/ceramide metabolism in the death of SOJ-6 cells promoted by nanoparticles.


Figure 3
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  		Figure 3. Interaction of nanoparticles with pancreatic tumor cells. A) SOJ-6 cells were incubated with nanoparticles (10 µg/well in terms of protein) for 24 h in the presence or absence (control) of drugs at indicated concentration. At the end of incubation, proliferation was measured by MTT assay and expressed as a percentage of control. B) Biotin-labeled nanoparticles were first incubated 30 min with streptavidin colloidal-gold complexes (5 nm) and with SOJ-6 cells. After 24 h of incubation, cells were treated for electron microscopy. Negative controls were performed with nanoparticles incubated with streptavidin colloidal-gold complexes without prelabeling with biotin. C) SOJ-6 cells were incubated for 24 h with trypsin-treated or mock-treated nanoparticles or with supernatants collected after centrifugation of trypsin or mock-treated nanoparticles. D) SOJ-6 cells were incubated for 24-h with heat-denatured (60°C for 20 min) or mock-treated nanoparticles. At the end of incubation, proliferation in C and D was measured by MTT assay and expressed as a percentage of control. E) SOJ-6 cells were incubated with biotin-labeled nanoparticles for 24 h. Cells were washed, fixed with paraformaldehyde, and saturated with 1% BSA in PBS. Cells were further incubated with FITC-labeled antibodies to biotin and observed with a confocal microscope. All data are means ± SD of 3 independent determinations; ***P < 0.001.

Interaction of nanoparticles with SOJ-6 cells
Nanoparticles can interact with a target cell by endocytic uptake, binding to a cell surface receptor, or fusion with plasma membrane. Uptake via macropinocytose and phagocytosis can be ruled out because amiloride, cytochalasin, and latrunculin B did not affect the inhibition of cell proliferation due to nanoparticle treatment (data not shown). Electron microscopy data on SOJ-6 cells challenged with colloidal gold-labeled nanoparticles suggest that these vesicles may be endocytosed by clathrin-dependent (Fig. 3B , arrow) or -independent (arrowhead) mechanisms. However, sucrose, which inhibits clathrin-dependent endocytosis, had no effect, suggesting that endocytosis may not be the major pathway involved in the interaction of nanoparticles with cells. To determine whether proteins on the nanoparticle surface may be involved in a receptor-dependent interaction, potential ligand surface proteins of vesicles were stripped by trypsin. Trypsin- and mock-treated nanoparticles were still able to promote the decrease in SOJ-6 cell proliferation, whereas supernatants showed residual activity (Fig. 3C ). Nanoparticles were also treated with endoglycoceramidase or warmed. These treatments did not affect the decrease in proliferation induced by nanoparticles (Fig. 3D ). These results suggest that neither ligand (glyco) proteins nor glycolipids are involved in the interaction of nanoparticles with pancreatic cells.

In line with the hypothesis that nanoparticles fuse with the plasma membrane, the interaction was localized at particular membrane sites. Nanoparticles contained characteristic lipids (Table 2) , and they were rich in lipids associated with membrane raft microdomains (22) . Therefore, nanoparticles may interact with these domains, which Février and Raposo (13) suggested are cell signaling platforms promoting apoptosis. Filipin, a sterol-binding drug, disrupts the structure of lipid rafts. At 0.5 µM, filipin decreased the proliferation of SOJ-6 cells (Fig. 3A ), corroborating the sensitivity of pancreatic tumor cells to raft disorganization (25) . However, this decrease was potentiated by nanoparticle addition.

After a 24-h incubation with biotin-labeled nanoparticles and with FITC-labeled antibodies to biotin, fluorescent spots were visualized at the surface of cells. Such spots were undetectable when cells were incubated with unlabeled nanoparticles as controls (Fig. 3E ). Images indicated that nanoparticles were not extensively internalized and were most likely to be associated with the SOJ-6 cell membrane to promote inhibition of cell proliferation. However, the dotted figure indicated that the nanoparticle materials were localized at discrete sites or at specific areas of plasma membrane. Taken as a whole, these data support the hypothesis that nanoparticles may interact with raft microdomains to perturb cell proliferation.

Nanoparticles affect the PI3K/Akt/GSK-3β survival pathway
We investigated whether nanoparticles affected the survival of SOJ-6 cells via the PI3K/Akt signaling pathway, which is constitutively active in pancreatic tumors (26) . Wortmannin decreased SOJ-6 cell growth in a dose-dependent manner (Fig. 4 A). However, cotreatment of cells with nanoparticles still led to a significant decrease in cell proliferation compared with that obtained with wortmannin only. In agreement with studies monitoring C48/80 effects, which show that C48/80 at high concentration decreases Akt activity (27) , this compound (used here at 10 µg/ml) had inhibitory input on cell proliferation. Conversely, we activated PI3K with the C48/80 effector used at low concentration (2 µg/ml). At this concentration, C48/80 did not rescue proliferation when nanoparticles were added to cell cultures.


Figure 4
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  		Figure 4. PI3K/Akt pathway involvement in SOJ-6 cells treated with nanoparticles. A) SOJ-6 cells were cultured in 96-well plates and incubated with nanoparticles (10 µg/well in terms of protein) for 24 h in the presence or absence (control) of drugs at indicated concentrations. At the end of incubation, proliferation was measured by MTT assay and expressed as a percentage of control. B) SOJ-6 cells were treated for 24 or 48 h with nanoparticles (10 µg/well) and lysed. Cell lysate proteins were separated on SDS-PAGE (40 µg of protein/lane) and electrotransferred onto nitrocellulose membrane. The level of kinases and phosphorylated kinases was appreciated by probing membrane with specific antibodies and compared with controls (without nanoparticles). C) SOJ-6 cells were treated with or without nanoparticles and lysed. Lysates were immunoprecipitated with agarose-immobilized antibodies to PTEN. The antibody-antigen complex recovered by centrifugation was washed in SDS buffer, and the final supernatant was analyzed on SDS-PAGE. Proteins were then electrotransferred on nitrocellulose membrane and probed with antibodies as mentioned. Western blots are representative of 3 independent experiments. D) SOJ-6 cells were cultured in 96-well plates and incubated for 1 h in the presence of drugs at indicated concentrations without (control) or with nanoparticles (10 µg/well) for 24 h. At the end of incubation, proliferation was measured by MTT assay and expressed as a percentage of control. Data are means ± SD of 3 independent determinations; ***P < 0.001.

These data did not allow us to conclude whether nanoparticles act independently of PI3K. We therefore examined the downstream partners of PI3K along the PI3K/Akt pathway. The phosphorylation of pyruvate dehydrogenase kinase 1 (PDK1) at Ser-241 increased on cell challenging with nanoparticles compared with that of controls without nanoparticles (Fig. 4B ). Total Akt was not affected by nanoparticle cell treatment; however, Akt Ser-473 phosphorylation rose in the presence of nanoparticles. These data also show that PDK1 and Akt are both phosphorylated in controls, ascertaining that this pathway is constitutively operative in pancreatic cancer cells. Among survival mechanisms, activation of the Akt pathway leads to phosphorylation and inactivation of GSK-3β (28) . However, Ser-9 phosphorylation of phospho-GSK-3β decreased after treatment of cells with nanoparticles (Fig. 4B ). LiCl, a GSK-3β inhibitor, decreases the inhibitory effect on proliferation induced by nanoparticles (Fig. 4D ). These data suggest that nanoparticles induced GSK-3β dephosphorylation and activation. This activation of GSK-3β, in turn, could stop the activation of the survival pathway involving PDK1 and Akt phosphorylation resulting from cell challenging with nanoparticles.

Nanoparticles promote PTEN dephosphorylation
GSK-3β has been implicated in the feedback regulation of the PI3K/Akt pathway via the phosphorylation and inactivation of the tumor suppressor PTEN deleted on chromosome 10 (29) . PTEN was phosphorylated (i.e., inactive) in controls without nanoparticles. This phosphorylation decreased with time in cells treated with nanoparticles (Fig. 4B ). Therefore, PTEN as well as GSK-3β was activated in cells incubated with nanoparticles. This dephosphorylation (or lack of phosphorylation) did not result from mutation in proteins, as determined by reverse transcriptase-polymerase chain reaction experiments and sequencing. On note, PC3 cells deficient in PTEN (30) are insensitive to nanoparticles (Fig. 2B ). Further, the PTEN inhibitor bpV(phen) decreased the effects of nanoparticles on SOJ-6 cell proliferation (Fig. 4D ). These results suggested that PTEN and GSK-3β may be functionally linked. To answer this point, PTEN was immunoprecipitated from lysates obtained after nanoparticle treatment of SOJ-6 cells. GSK-3β coprecipitated with PTEN and was mainly phosphorylated (Fig. 4C ). β-Catenin and actin were also immunoprecipitated with PTEN. In the complex, β-catenin appeared unphosphorylated. Therefore, it seems that treatment of SOJ-6 cells induced the capture of inactive GSK-3β and of active β-catenin to form complexes with PTEN and actin. Such complexes, except that of actin-PTEN, did not take place in nanoparticle mock-treated cells. Moreover positive modulation of GSK-3β and of PTEN results in cyclin D1 down-regulation and cell arrest in the G1/S phase (31 , 32) . Cyclin D1 expression decreased by 50% in nanoparticle-treated cells (Fig. 4B ).

Nanoparticles affect Bax and Bcl-2 expression
GSK-3β and PTEN, through the down-regulation of antiapoptotic Bcl-2 or through the up-regulation of proapoptotic Bax, activate the death machinery involving caspases and mitochondria (33 , 34) . We recorded the expression of Bax and of Bcl-2 on nanoparticle treatment of cells. Bax expression increased with time, whereas Bcl-2 expression decreased after 48 h of treatment (Fig. 5 A).


Figure 5
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  		Figure 5. SOJ-6 cell derived nanoparticles induce tumor cell apoptosis. A) SOJ-6 cells were treated for 24 or 48 h with nanoparticles and lysed. Cell lysate proteins were separated on SDS-PAGE (40 µg protein/lane) and electrotransferred onto nitrocellulose membrane. The levels of Bax, Bcl-2, (cleaved) caspase-9, (cleaved) PARP, and actin were appreciated by probing membranes with specific antibodies as indicated. Western blots are representative of 3 independent experiments. B) SOJ-6 cells were treated for 24 or 48 h with nanoparticles and lysed. PDH activity of lysate was determined. Data are means ± SD of 3 independent determinations; **P < 0.01. C) Activities of different caspases were determined with the CaspACE kit. SOJ-6 cells were incubated for 4 h with caspase inhibitors. After addition of 10 µg/well fresh nanoparticles, incubation was carried on for another 24 h. After washing and fixation, cells were observed and counted under fluorescent microscopy. Results, expressed as a percentage compared with controls (assays conducted without inhibitor) are mean cell counts of 10 fields per assay; **P < 0.01. N.S., not significant.

Nanoparticles decrease PDH activity, activate caspases, and induce PARP cleavage
Nanoparticles induced the capture of inactive GSK-3β as a complex with PTEN and actin; however, active GSK-3β, which is generated on cell treatment with nanoparticles (Fig. 4B ), may be available for further signaling. GSK-3β is known to inactivate PDH, an enzyme involved in the tricarboxylic acid cycle into which the major organic fuel molecules of the cell converge during catabolism. Effectively, PDH activity was reduced on nanoparticle cell treatment (Fig. 5B ).

To determine which caspases are activated on cell treatment with nanoparticles, a complementary study was performed using caspase inhibitors. SOJ-6 cells were preincubated for 4 h with Z-DEVD-fmk (a caspase-3 inhibitor), Z-IETD-fmk (a caspase-8 inhibitor), Z-LEHD-fmk (a caspase-9 inhibitor), or Z-VAD-fmk (a pancaspase inhibitor), and then nanoparticles were added and the cells were incubated for another 24 h. The results demonstrated that the inhibitors of caspases-3 and -9 and of pancaspase significantly decreased the amount of apoptotic cells (Fig. 5C ). In contrast, the inhibitor of caspase-8 did not significantly affect the amount of apoptotic cells. Furthermore, caspase-9 was cleaved into its 37-kDa active form, which increased with time (Fig. 5A ). An ultimate effector of caspase-3 is PARP, which plays an essential role in DNA damage repair and cell death. During apoptosis, PARP, a 116-kDa protein, was cleaved into 85- and 24-kDa fragments with reduced activity (35) . On SOJ-6 cell challenging with nanoparticles, PARP was cleaved to yield the 85-kDa inactive fragment (Fig. 5A ).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Many studies have been done on nanoparticles derived from dendritic cells (36) and cancer cells such as breast, liver, ovarian, prostatic, glioma, melanoma, and others (refs. 8 , 14 , 37 , and 38 and references therein), but few have dealt with the role of these particles on tumor cells. Our study shows for the first time that tumor-derived nanoparticles induce apoptosis in tumor cells. These vesicles rich in cholesterol and sphingomyelin trigger cell apoptosis by inducing the activation of the mitochondrial apoptotic pathway, which results in caspase-3 and -9 activation. Our study also documents the protein and lipid composition of SOJ-6 pancreatic cell-derived nanoparticles. Electron microscopy showed homogeneous vesicles ~40 nm in diameter. The density of these nanoparticles, close to 1.15 g/ml, is in the lower range of that described (36) and correlates with their lipid composition, in which cholesterol and sphingomyelin predominate.

Nanoparticles expressed by human pancreatic tumor SOJ-6 cells contain many proteins, which we characterized by mass spectrometry. Among them are annexins, chaperones (notably Hsp90), and cytoplasmic and cytoskeleton proteins. Histones were also found as reported by others (21) . Most of these proteins were already described in the proteome of exosomes (36) . The membrane orientation of nanoparticles is identical to that of cells, and during their formation cytoplasm is included in the nanoparticle. This cytoplasm inclusion is reflected by the presence of metabolic enzymes, such as enolase-1 in SOJ-6 cell-derived nanoparticles. Such parameters are characteristic of nanoparticles referred to as exosomes (39) and differ from those in microparticles or apoptotic corpses (21) . The term "exosomes" testifies to their origin in multivesicular bodies and the presence of marker proteins such as Hsc70 (40) . Although the chaperone and endosomal Rab4 were actually detected, the term "nanoparticles" rather than "exosomes" was preferred here, as the aim of this study was to define the role and not to provide a full characterization of particles isolated from human pancreatic tumor cells.

Nanoparticles expressed by SOJ-6 are partly depleted in phospholipids containing 2- to 20-fold less phosphatidylcholine and phosphatidylethanolamine than the parent cells. Contrarily to microparticles, PS was poorly represented, whereas phosphatidylinositol was not detected. However, sphingomyelin, ceramides, and free cholesterol contents were ~4x higher in nanoparticles than in whole-cell lipids. This lipid composition suggests that nanoparticles produced by pancreatic cancer cells exhibit high lipid raft microdomains, a common feature of exosomes (41) . Since the recent work of Laulagnier et al. (42) , it is clear that the lipid composition of nanoparticles is heterogeneous and varies according to cell origin. Therefore, it is possible that we have isolated sphingomyelin-cholesterol-rich nanoparticle subfractions. To our knowledge, this is the first analysis of the lipid composition of nanoparticles derived from tumor cells.

Our results demonstrate that nanoparticles expressed by tumor cells induce a decrease in cancer cell proliferation but not in that of "normal" HUVECs, which do not harbor constitutively activated PI3K/Akt pathway (43) . This decrease results from the activation of the apoptotic pathway. As shown here, the expression of proapoptotic Bax increases, whereas that of antiapoptotic Bcl-2 is repressed. When Bax predominates, programmed cell death is accelerated, and the death-repressor activity of Bcl-2 is counteracted (44) . On overexpression consecutive to an apoptotic signal, Bax is believed to translocate to the mitochondrial membrane to initiate cytochrome c release and caspase activation (45) . The apoptosis of SOJ-6 cells promoted by nanoparticles is inhibited by caspase inhibitors, indicating that the cell death effectively involves caspase activities, more specifically those of caspase-9 and -3. Caspase-8 inhibition did not affect the apoptosis induced by nanoparticles. This scheme of apoptosis with Bax overexpression and caspases-9 and -3 activation correlates with the activation of the mitochondrial apoptotic pathway.

The question now is how these nanoparticles interacted with cells. Although nanoparticles are endocytosed by dendritic (46) and pancreatic cells (see Fig. 3B ), endocytosis is not the major way for these latter cells, because inhibitors of the endolysosomal pathway do not affect the decrease in proliferation observed on nanoparticle treatment of SOJ-6 cells. Our results also show that the metabolism of sphingomyelin and ceramide consecutive to endocytosis is not responsible for nanoparticle-induced apoptosis. Furthermore, stripping of surface glycoproteins and glycolipids and heat denaturation does not affect nanoparticle effects on SOJ-6 cell proliferation, which consequently rules out any role in cell death mediated by a receptor ligand (such as Fas/Fas ligand). Once cells are incubated with labeled nanoparticles, the labeling evokes dots at the cell surface, meaning that vesicles interact at discrete and/or specific sites on the cell membrane. Taking into account the lipid composition of such vesicles and the additive effects of drugs affecting raft organization in presence of nanoparticles, one may hypothesize that nanoparticles fuse or at least interact with the cell membrane at the level of raft lipid microdomains (13) . Because lipid rafts are signaling platforms (47) , this interaction in turn perturbs cell signaling and drives cells toward apoptosis. This point needs further studies to be confirmed. Interestingly PTEN and β-catenin, whose phosphorylation is modified on cell challenging with nanoparticles, are linked to rafts.

Concomitant to the induction of apoptosis by nanoparticles, we observed Akt phosphorylation. However, the survival pathway was overbalanced by apoptotic processes. Interestingly, the Ser-9-phosphorylated inactive form of GSK-3β coprecipitates with PTEN. Active β-catenin, known to be involved in a functional complex with GSK-3β, also coprecipitates along with actin in PTEN immunoprecipitation. On one hand, this result suggests that inactive GSK-3β and PTEN form complexes with the actin cytoskeleton where active β-catenin is sequestered. Another possibility is that the formation of this complex may hinder the β-catenin activity. In these complexes, β-catenin cannot be phosphorylated and inactivated by GSK-3β (itself inactive). Consequently, complexed β-catenin may not translocate to the nucleus to activate target proliferation genes such as that of cyclin D1, which may explain the decrease in cyclin D1 expression. This complex may not form in PTEN-deficient PC3 cells, resulting in insensitivity to nanoparticles. Activated PTEN and GSK-3β can also participate in cyclin D1 down-regulation (31 , 32) . On the other hand, nanoparticles induce GSK-3β Ser-9 dephosphorylation, resulting in active kinase that does not complex to PTEN. Therefore, the active GSK-3β is available for inactivating the PDH by phosphorylation (48) . This PDH inhibition probably evokes an increase in the pyruvate concentration in cells and the damaging of mitochondria, thereby driving cells toward apoptosis (49) .

This preponderance of apoptosis over survival induced by nanoparticles results in particular from the activation of GSK-3β and PTEN by their dephosphorylation at Ser-9 and Ser-380, respectively. Inhibiting these kinases by specific drugs results in partial reversion of cell proliferation inhibition, thus confirming the fact that their activation is essential for the inhibition of the proliferation evoked by nanoparticles. Although we do not know whether other phosphorylation sites, notably on PTEN (50) , are affected by nanoparticles, many questions arise. Among them, do nanoparticles activate Ser-protein phosphatases that may dephosphorylate PTEN and GSK-3β, thus overriding the survival PI3K/Akt pathway? Another possibility lies in the inhibition by nanoparticles of Ser-protein kinases. Casein kinase (CK) 2 is described as being responsible for PTEN phosphorylation at many sites, except on Ser-380 (50) . Its phosphorylation depends on a not yet identified protein kinase, which could be CK1 but not CK2 (51) or GSK-3β (31) . Our results are in line with this possibility, as the kinase phosphorylating PTEN seems to be inhibited by nanoparticles. Further investigations are needed to identify protein phosphatases or kinases whose activities are dysregulated by nanoparticles.

Thus, nanoparticles produced by tumor cells exert pleiotropic effects on cells and may have autocrine control of tumor growth, as on the one hand, they may activate the PI3K/Akt survival pathway, and on the other hand, they overbalance the survival by restoring PTEN and GSK-3β activities, driving cells toward apoptosis. A putative scheme giving a comprehensive overview of this mechanism is shown in Fig. 6 .


Figure 6
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  		Figure 6. Putative mechanism of action of nanoparticle on pancreatic tumor cells. Nanoparticles expressed by tumor cells interact with membrane lipid raft domains of tumor cells to activate both PTEN and GSK-3β. PTEN and inactive pSer9-GSK-3β form complexes linked to the actin cytoskeleton that sequester active β-catenin. β-Catenin may no longer be able to translocate to the nucleus to activate target genes such as cyclin D1 to promote survival. Challenging tumor cells with nanoparticles also triggers successively 1) activation of GSK-3β, 2) PDH inhibition and probable damaging of mitochondria, 3) increase in proapoptotic Bax vs. antiapoptotic Bcl-2 proteins, 4) activation of caspase-9 and -3, leading to PARP cleavage, and 5) down-regulation of cyclin D1. Such GSK-3β activation counteracts the constitutively activated PI3K/Akt survival pathway to drive tumor cells toward apoptosis.

During development, cycling between proliferation and apoptosis is a common process that helps organogenesis (52) . In this sense, tumors may control their own growth to favor the recruitment of endothelial cells and the establishment of the neovasculature. Going further, nanoparticles expressed by pancreatic tumor cells may also participate in tumor angiogenesis, as they carry BSDL, a proangiogenic factor (53) . Assuming that the main features relative to nanoparticle effects on tumor cell signaling, evidenced in vitro, mimic those occurring in vivo, one may ask whether the apoptotic activity of nanoparticles is addressed to tumor cells themselves to ultimately regulate tumor growth rate and/or to other cells, notably immune cells, to escape the immune system.


   ACKNOWLEDGMENTS
 
The authors are grateful to J. Courageot (electron microscopy core facility, School of Medicine, Marseille, France) and C. Franceschi [INSERM Unité Mixte de Recherche (UMR) 911] for microscopy experiments. J. Bertrand-Michel (INSERM UMR563, Toulouse, France) is gratefully acknowledged for lipid analyses. This work was supported by institutional funding from INSERM (Paris, France) and from Université de la Méditerranée (Marseille, France).

Received for publication December 11, 2007. Accepted for publication May 2, 2008.


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B. M. Stenson, M. Ryden, K. R. Steffensen, K. Wahlen, A. T. Pettersson, J. W. Jocken, P. Arner, and J. Laurencikiene
Activation of Liver X Receptor Regulates Substrate Oxidation in White Adipocytes
Endocrinology, September 1, 2009; 150(9): 4104 - 4113.
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