Lecithins, mainly composed of the phospholipids phosphatidylcholines (PC), have many different uses in the pharmaceutical and clinical field. PC are involved in structural and biological functions as membrane trafficking processes and cellular signaling. Considering the increasing applications of lecithin-based nanosystems for the delivery of therapeutic agents, the aim of the present work was to determine the effects of phosphatidylcholine nanoparticles over breast cancer cellular proliferation and signaling. PC dispersions at 0.01 and 0.1% (w/v) prepared in buffer pH 7.0 and 5.0 were studied in the MCF-7 breast cancer cell line. Neutral 0.1% PC-derived nanoparticles induced the activation of the MEK-ERK1/2 pathway, increased cell viability and induced a 1.2 fold raise in proliferation. These biological effects correlated with the increase of epidermal growth factor receptor (EGFR) content and its altered cellular localization. Results suggest that nanoparticles derived from PC dispersion prepared in buffer pH 7.0 may induce physicochemical changes in the plasma membrane of cancer cells which may affect EGFR cellular localization and/or activity, increasing activation of the MEK-ERK1/2 pathway and inducing proliferation. Results from the present study suggest that possible biological effects of delivery systems based on lecithin nanoparticles should be taken into account in pharmaceutical formulation design.
Lecithins are a mixture of phospholipids where phosphatidylcholines are the main components (up to 98% w/w). Egg or soy lecithin as well as purified phospholipids is used for pharmaceutical purposes as dispersing, emulsifying, and stabilizing agents included in intramuscular and intravenous injectables or parenteral nutrition [
Phosphatidylcholines, the main components of lecithins, are glycerophospholipids that incorporate choline as the head group. The fatty acids bound to the glycerophosphatidic acid can vary but generally one of them is unsaturated and the other one is saturated. Phosphatidylcholine (PC) is a major constituent of the cell membranes which is more commonly found in the exoplasmic or outer leaflet of the plasma membrane. PC also plays a role in membrane-mediated cell signaling. The phospholipase D-mediated catabolism of PC yields phosphatidic acid (PA) and choline, which are important lipid second messengers involved in several signaling pathways [
Phosphatidylcholine is also a substrate of the phosphatidylcholine-specific phospholipase C (PC-PLC). This enzyme has been implicated in proliferation, differentiation, and apoptosis of mammalian cells. PC-PLC-mediated hydrolysis of PC yields PC-derived diacylglycerol (DAG) and phosphocholine (P-chol) [
The lipid second messengers PA and DAG that are generated as a result of PLD and PC-PLC activity, respectively, can also affect membrane trafficking, directly by altering membrane curvature or indirectly by recruiting and/or activating signaling mediators [
Membrane phospholipids as well as their fatty acid profile are altered in tumor cells. The choline metabolite profile of cancer cells is characterized by an elevation of phosphocholine and total choline-containing compounds. Indeed, total cellular phosphatidylcholine (PC) can be used as a marker for membrane proliferation in neoplastic mammary gland tissues [
Phosphatidylcholines are therefore not inert vehicles but biological active compounds; phospholipids and their derived second messengers are involved in cell proliferation and trafficking, and the increase of phosphocholine and choline-containing compounds has been described in tumor cells. It has been recently highlighted that certain excipients have a role as active pharmaceutical components of formulations because they can modify the pharmacological activity of an active drug or produce biological effects [
Purified phosphatidylcholine from soybean lecithin (Phospholipon 90G, CAS-number 97281-47-5) was purchased from Lipoid (Ludwigshafen, Germany). Trizma base, HEPES, Tween 20, Triton X-100, sodium dodecyl sulfate (SDS), glycine, ammonium persulfate, aprotinin, phenylmethylsulfonyl fluoride (PMSF), sodium orthovanadate, 2-mercaptoethanol, Hoechst 33258, and BSA-fraction V were obtained from Sigma Chemical Co. (St. Louis, MO, USA). PVDF membranes, high performance chemiluminescence film, and enhanced chemiluminescence- (ECL-) Plus are from Amersham Biosciences (GE Healthcare, Piscataway, NY, USA). Mini-Protean apparatus for SDS-polyacrylamide electrophoresis, miniature transfer apparatus, acrylamide, bis-acrylamide, and TEMED were obtained from Bio-Rad Laboratories (Hercules, CA, USA). Anti-EGFR (1005) antibody and secondary antibodies conjugated with HRP were purchased from Santa Cruz Biotechnology Laboratories (Santa Cruz, CA, USA). Antibodies anti-phospho-mTOR Ser2448, anti-mTOR, anti-p44/42 MAP kinase (ERK 1/2), and anti-phospho-p44/42 MAP kinase Thr202/Tyr204 were from Cell Signaling Technology Inc. (Beverly, MA, USA). Cy3-conjugated secondary antibody against rabbit polyclonal immunoglobulins was from Jackson ImmunoResearch Laboratories, Inc. Bicinchoninic acid (BCA) protein assay kit was obtained from Thermo Scientific, Pierce Protein Research Products (Rockford, IL, USA).
Dispersions of Phospholipon 90G 0.01 and 0.1% (w/v) in two different diluents (66 mM isotonic phosphate buffer pH 7.0 and 50 mM isotonic acetate buffer pH 5.0) were prepared. Buffers were isotonized by adding sodium chloride when necessary according to Sörensen and White-Vincent methods. Phosphatidylcholine was first dispersed in the appropriate diluent with means of extensive mixing at 60°C by use of a thermostated magnetic stirrer in order to obtain good hydration. Next, the dispersion was stirred for 2 minutes at the same temperature with a high-shear mixer (Ultra-Turrax T18 basic, IKA Werke, Staufen, Germany) and sonicated at 20 kHz for 10 minutes. It was then sterilized by autoclaving (121°C, 15 minutes). The sizes of the resulting particles in the dispersions were determined by photon correlation spectroscopy (PCS) using a Zetasizer (Malvern Nano ZS, Malvern Instruments Ltd., UK). The zeta potential of the samples was measured by the same instrument and the zeta potential values were calculated according to Smoluchowski equation (Table
Effect of pH on the particle size and zeta potential of the phosphatidylcholine nanoparticles.
Formulation | Particle size ( |
PdI | Z-Pot (mV) ± SD |
---|---|---|---|
pH 5.0 |
|
0.494 |
|
pH 7.0 |
|
0.544 |
|
Phosphatidylcholine (PC) nanoparticles were prepared in pH 5.0 and pH 7.0 buffers and analyzed by Dynamic Light Scattering (DLS). The size and zeta potential of the particles were measured and reported as mean ± S.E.M. (
MCF-7 human breast cancer cell line was obtained from the American Type Culture Collection (ATCC) (Rockville, MD, USA). Cells were maintained in Dulbecco’s minimum essential medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 50
To perform immunoblotting assays, cells were seeded in clear 6-well plates (Corning Costar, Fisher Scientific, USA) at a density of 300,000 cells/well, while for immunofluorescence assays cells were seeded at a density of 20,000 cells/well in covers placed in 24-well plates. Phosphatidylcholine nanoparticles at 0.1 and 0.01% were added in the presence or absence of serum. Cells were further incubated at 37°C for 24 hours in a 5% CO2 atmosphere. After incubation, cells were washed with phosphate saline buffer and dishes were kept at −80°C until cell solubilization to prepare cells extracts, while covers were immediately processed for specific immunofluorescence labeling.
Cells were homogenized in buffer composed of 1% v/v Triton, 0.1 M Hepes, 0.1 M sodium pyrophosphate, 0.1 M sodium fluoride, 0.01 M EDTA, 0.01 M sodium vanadate, 0.002 M PMSF, and 0.035 trypsin inhibitory units/mL aprotinin (pH 7.0) at 4°C. Cell homogenates were centrifuged at 15,000 ×g for 40 minutes at 4°C to remove insoluble material. Protein concentration of supernatants was determined by the BCA protein assay kit. Equal protein aliquots of solubilized cells were diluted in Laemmli buffer, boiled for 5 minutes, and stored at −20°C until electrophoresis.
Samples were subjected to electrophoresis in SDS-polyacrylamide gels. Electrotransference of proteins from gel to PVDF membranes and incubation with antibodies were performed as already described [
To reprobe with other antibodies, the membranes were washed with acetonitrile for 10 minutes and then incubated in stripping buffer (2% w/v SDS, 0.100 M 2-mercaptoethanol, 0.0625 M Tris/HCl, pH 6.7) for 40 minutes at 50°C while shaking, washed with deionized water, and blocked with BSA.
Cells were seeded in clear 96-well plates (Corning Costar, Fisher Scientific, USA) at a density of 10,000 cells/well. Phosphatidylcholine at 0.1 and 0.01% was added in 100
DNA synthesis in proliferating cells was determined by measuring BrdU incorporation with a BrdU ELISA assay [
Cells were washed twice in PBS, pH 7.0, fixed in 2% formaldehyde in PBS for 10 minutes at room temperature. After three washes with PBS (5 minutes each), fixed cells were permeabilized with 0.5% Triton X-100 in PBS for 15 minutes and incubated in blocking solution (10% FBS in PBS) for 30 minutes to decrease nonspecific binding of the antibodies. Cells were then incubated for 1 hour at 37°C with anti-EGFR, then washed, and incubated with Cy3-conjugated secondary antibody against rabbit polyclonal immunoglobulins. After a final washing step (3 washes 5 minutes each in PBS), cells were incubated with Hoechst 33258 (2
Experiments were performed analyzing the phosphatidylcholine dispersions and vehicle (control) in parallel,
Previous results from our research group have demonstrated that nanoparticles prepared from phosphatidylcholine dispersed at 0.01 and 0.1% (w/v) in buffer pH 5.0 and buffer pH 7.0 are able to bind oligonucleotides and deliver them to breast cancer cells [
Akt is activated by many types of cellular stimuli and regulates fundamental cellular functions such as transcription, translation, proliferation, growth, and survival. Its dysregulation has been associated with the development of diseases such as cancer [
Akt and mTOR phosphorylation and protein content. MCF-7 breast cancer cells were incubated for 24 hs with PC nanoparticles dispersed at 0.1 and 0.01% (w/v) in buffer pH 5.0 and buffer pH 7.0 or vehicle (Ct) in the absence ((a) and (c)) or presence ((b) and (d)) of serum. Representative results of immunoblots with anti-Akt and anti-phospho-Akt S473 ((a) and (b)) and anti-mTOR and anti-phospho-mTOR S2448 ((c) and (d)) are shown. Reprobing with anti-actin antibody demonstrated uniformity of protein loading in all lanes. Quantification of phosphorylated proteins was performed by scanning densitometry and expressed as percent of values measured for control, nonstimulated breast cancer cells (Ct). Data are expressed as the mean ± S.E.M. of the indicated number (
Mammalian target of rapamycin complex is a Ser/Thr kinase of the phosphatidylinositol 3-kinase-related kinase protein family. Akt phosphorylates and activates mTOR, thus inducing protein synthesis and cell growth [
The Ras/Raf/MEK/ERK cascade couples signals from cell surface receptors to transcription factors, which can regulate cell cycle progression, apoptosis, or differentiation [
MAP kinase kinase (MEK) is a dual-specificity kinase that phosphorylates tyrosine and threonine residues on extracellular-signal-regulated kinases 1 and 2 (ERK 1/2) [
Phosphorylation of MEK1/2 and ERK1/2 was studied in the MCF-7 cells incubated with PC nanoparticles. Results showed that MEK1/2 and ERK1/2 phosphorylation was significantly increased when cells were treated with phosphatidylcholine dispersed in pH 7.0 solution at high concentration independently of the absence (Figures
ERK1/2 and MEK1/2 phosphorylation and protein content. MCF-7 breast cancer cells were incubated for 24 hs with PC nanoparticles dispersed at 0.01 and 0.01% (w/v) in buffer pH 5.0 and buffer pH 7.0 or vehicle (Ct) in absence ((a) and (c)) or presence ((b) and (d)) of serum. Representative results of immunoblots with anti-p44/42 MAP kinase (ERK1/2) and anti-phospho-p44/42 MAP kinase Thr202/Tyr204 ((a) and (b)) and anti-MEK1/2 and antiphospho MEK ((c) and (d)) are shown. Reprobing with anti-actin antibody demonstrated uniformity of protein loading in all lanes. Quantification of phosphorylated proteins was performed by scanning densitometry and expressed as percent of values measured for control, nonstimulated breast cancer cells (Ct). Data are expressed as the mean ± S.E.M. of the indicated number (
As it was previously mentioned, MEK1/2-ERK 1/2 signaling pathway is involved in cell growth and proliferation promotion, so the effects of phosphatidylcholine nanoparticles over breast cancer cell viability were studied. For this purpose, MCF-7 cells were seeded in 96-well plates and incubated during 24 hours (Figures
Viability of MCF-7 breast cancer cells incubated with phosphatidylcholine (PC) nanoparticles. Breast cancer cells were incubated for 24 hours ((a) and (b)) and 48 hours ((c) and (d)) with PC nanoparticles dispersed at 0.01 and 0.1% (w/v) or vehicle (Ct) in buffer pH 5.0 and buffer pH 7.0 in the absence ((a) and (c)) or the presence ((b) and (d)) of serum. After incubation, cell viability was evaluated using the CellTiter 96 aqueous nonradioactive cell proliferation assay (Promega). Triplicates were run for each treatment. Values were expressed in terms of percent of untreated control cells set as 100%. Data are expressed as the mean ± S.E.M. of the indicated number (
To ascertain if the increased cell viability induced by phosphatidylcholine nanoparticles 0.1% at pH 7.0 was a consequence of cell proliferation induction, BrdU incorporation assay was performed. Considering that the differences between viability of basal cells and PC-treated cells were better evidenced when cells were treated in the medium without serum, BrdU incorporation was assessed after 48 hours of treatment with phosphatidylcholine nanoparticles in absence of serum. Results demonstrated that increased cell viability correlated with increased incorporation of BrdU (Figure
Proliferation of MCF-7 breast cancer cells incubated with phosphatidylcholine (PC) nanoparticles. Breast cancer cells were incubated for 48 hours with PC nanoparticle prepared in buffer pH 7.0 at 0.01 and 0.1% (w/v) or vehicle (Ct) in the absence of serum (a). Proliferation was determined by measuring BrdU incorporation with a BrdU ELISA. Triplicates were run for each treatment. Values were expressed in terms of percent of untreated control cells set as 100% (a). Data are expressed as the mean ± S.E.M. of the indicated number (
The effects of phosphatidylcholine 0.1% dispersed in buffer pH 7.0 over MCF-7 cell proliferation correlated with the increased phosphorylation levels observed for MEK 1/2 and ERK1/2. Results suggest that high concentration of phosphatidylcholine nanoparticles at pH 7.0 induces activation of the MEK1/2-ERK1/2 pathway and cell proliferation of the breast cancer cells.
Molecular aspects of cell signaling are controlled by receptor/ligand localization and trafficking [
As previously mentioned, PC and second messengers derived from these phospholipids are fundamental components of the cell membrane and affect its dynamics and protein trafficking. Particularly, previous studies have demonstrated that phospholipid membrane composition affects EGF receptor endocytosis and signaling [
Endosomal trafficking of EGFR is crucial for determining the amplitude and duration of EGFR signaling. Actually, endocytosis of the EGFR is required for EGF-induced MAP kinase activation. This was evidenced in experiments in which EGF induction of MAPKs was reduced in dynamin mutant cells which showed defects in clathrin-dependent receptor-mediated endocytosis [
Results showed that EGFR levels increased when cells were treated with PC nanoparticles dispersed in buffer pH 7.0 at high concentration both in absence or presence of serum (Figures
EGFR protein content and immunocytochemistry. MCF-7 breast cancer cells were incubated for 24 hours with PC nanoparticles dispersed at 0.1 and 0.01% (w/v) in buffer pH 5.0 and buffer pH 7.0 or vehicle (Ct) in absence (a) or presence (b) of serum for immunoblot studies, but only with PC dispersions at 0.1%, pH 7.0, or vehicle (control) in absence of serum for immunocytochemistry (c). Western blotting was performed as described in M & M. Membranes were reprobed to asses actin content and demonstrate equal protein loading in all lanes. Representative immunoblots are shown ((a) and (b)). EGFR quantification was performed by scanning densitometry and expressed as percent of values measured for control, nonstimulated breast cancer cells. Data are expressed as the mean ± S.E.M. of the indicated number (
EGFR immunocytochemistry. MCF-7 breast cancer cells were incubated for 24 hours with PC nanoparticles dispersed at 0.1 in buffer pH 7.0 or vehicle (Ct) in absence of serum. For EGFR immunocytochemistry, cells were washed, fixed, permeabilized, blocked, and incubated with the anti-EGFR antibody, the Cy3-conjugated secondary antibody, and Hoechst. Finally covers were mounted and examined by confocal microscopy. Images were obtained using sequential scanning. Representative images of EGFR, nuclear staining, merge, and bright field are shown.
Despite the multiple and different uses of lecithin with pharmaceutical and therapeutic purposes, the possible biological consequences of phosphatidylcholine administration should be considered. They are important phospholipids involved not only in structural functions in the cell but also in membrane trafficking processes and signaling. Moreover, increased levels of phosphocholine and choline-containing compounds have been associated with progression and bad prognosis of tumors. Considering the increasing use of lecithin-based formulations for the delivery of antineoplastic agents, the biological effects of nanoparticles derived from aqueous phosphatidylcholine dispersions over breast cancer cells proliferation and signaling were studied. Previously characterized phosphatidylcholine nanoparticles proposed as oligonucleotide delivery systems were used for that purpose [
Incubation with the phosphatidylcholine nanoparticles prepared in neutral buffer was associated with increased EGFR content in the cancer cells and with its altered cellular localization. High phosphatidylcholine concentrations might induce physicochemical changes in the plasma membrane that affect receptor trafficking and turnover. Moreover, a process has been recently described, dependent on sustained stimulation of cPCK and PLD activities, that leads to EGFR sequestration near the perinuclear region, in the pericentrion [
Increased concentration of phosphatidylcholine nanoparticles dispersed in buffer pH 7.0 had significant effects over cell proliferation, EGFR levels, and activation of the MEK1/2-ERK1/2 pathways; however, such effects were not observed for PC nanoparticles dispersed in pH 5.0 buffer. The main differences between both PC preparations is the charge associated with the particles (zeta potential) (Table
In spite of the studies accomplished using lipid-based nanocarriers for drug and gene delivery, the relationship between their physicochemical characteristics and activation of membrane receptors remains as an area of knowledge with incipient development. In this regard, cationic liposomal lipids have been described to modify cellular pathways and stimulate immune or anti-inflammatory responses [
Results from the present study suggest that high phosphatidylcholine concentrations, assembled in negatively charged nanoparticles, may induce physicochemical changes in the plasma membrane that affect EGFR cellular localization and/or its activity, therefore facilitating accumulation of the receptor in the cytoplasm, which would be associated with increased activation of the MEK-ERK1/2 pathway and induction of cell cycle progression. It is interesting that the described effects were specifically observed for the phosphatidylcholine nanoparticles prepared in pH 7 buffer but not at pH 5; so we propose that this might be related to the different net charge and morphology associated with the particles, as no significant differences in size between nanoparticles obtained from dispersion at pH 7.0 and 5.0 were observed. Considering that the PC nanoparticles preformed in a pH 5.0 buffer showed no significant biological effects over the breast cancer cells, these would be safer than those prepared in a pH 7.0 buffer to deliver antimitotic agents.
The interpretation of the interaction between nanocarriers with membrane receptors is a matter that must be elucidated for a more appropriate understanding of the biological effects that are promoted. The present study highlights the importance of the research on the effects of vehicles broadly used in the pharmaceutical area and demonstrates that possible biological effects of formulations based on phosphatidylcholine nanoparticles should be considered. Moreover, studies about the possible biological action of PC nanoparticles on normal cells would be useful to expand our knowledge about their potential pharmaceutical uses. Excipient effects over normal physiology and cell biology represent important factors to be concerned about in rational formulation design.
The authors declare that there is no conflict of interests regarding the publication of this paper.
Support for these studies was provided by the National Agency of Scientific and Technological Promotion (ANPCyT, PICT 595) and the University of Buenos Aires (UBACYT 20020090200186).