Jacob Bell
New Member
Palmitoylethanolamide inhibits the expression of fatty acid amide hydrolase and enhances the anti-proliferative effect of anandamide in human breast cancer cells
Vincenzo DI MARZO*1, Dominique MELCK*, Pierangelo ORLANDO, Tiziana BISOGNO*, Orna ZAGOORY, Maurizio BIFULCO, Zvi VOGEL and Luciano DE PETROCELLISs
*Istituto per la Chimica di Molecole di Interesse Biologico, Via Toiano 6, 80072, Arco Felice, Napoli, Italy, Istituto di Biochimica delle Proteine ed Enzimologia, Via Toiano 6, 80072, Arco Felice, Napoli, Italy, Department of Neurobiology, Weizmann Institute of Science, Rehovot 76100, Israel, Centro di Endocrinologia e Oncologia Sperimentale, Consiglio Nazionale delle Ricerche, and Dipartimento di Biologia e Patologia Cellulare e Molecolare, Universitadi Napoli Federico II, 80131, Napoli, Italy, and sIstituto di Cibernetica, Consiglio Nazionale delle Ricerche, Via Toiano 6, 80072, Arco Felice, Napoli, Italy
Palmitoylethanolamide (PEA) has been shown to act in synergy with anandamide (arachidonoylethanolamide; AEA), an en¬dogenous agonist of cannabinoid receptor type 1 (CB"). This synergistic effect was reduced by the CB# cannabinoid receptor antagonist SR144528, although PEA does not activate either CB" or CB# receptors. Here we show that PEA potently enhances the anti-proliferative effects of AEA on human breast cancer cells (HBCCs), in part by inhibiting the expression of fatty acid amide hydrolase (FAAH), the major enzyme catalysing AEA degradation. PEA (110 M) enhanced in a dose-related manner the inhibitory effect of AEA on both basal and nerve growth factor (NGF)-induced HBCC proliferation, without inducing any cytostatic effect by itself. PEA (5 M) decreased the IC&! values for AEA inhibitory effects by 36-fold. This effect was not blocked by the CB# receptor antagonist SR144528, and was not mimicked by a selective agonist of CB# receptors. PEA enhanced AEA-evoked inhibition of the expression of NGF Trk receptors, which underlies the anti-proliferative effect of the endocannabinoid on NGF-stimulated MCF-7 cells. The effect of PEA was due in part to inhibition of AEA degradation, since treatment of MCF-7 cells with 5 M PEA caused a C 3040% down-regulation of FAAH expression and activity. However, PEA also enhanced the cytostatic effect of the cannabinoid receptor agonist HU-210, although less potently than with AEA. PEA did not modify the affinity of ligands for CB" or CB# receptors, and neither did it alter the CB"}CB#-mediated inhi¬bitory effect of AEA on adenylate cyclase type V, nor the expression of CB" and CB# receptors in MCF-7 cells. We suggest that long-term PEA treatment of cells may positively affect the pharmacological activity of AEA, in part by inhibiting FAAH expression.
Key words: 2-arachidonoylglycerol, arvanil, cell proliferation, endocannabinoids, receptors.
INTRODUCTION
Anandamide (arachidonoylethanolamide; AEA) was the rst endogenous substance to be proposed as an agonist for the cannabinoid receptor type 1 (CB" receptor) [1,2]. AEA belongs to a family of lipids, the N-acylethanolamines (NAEs) (see [3] for a review), whose most well known component, palmitoylethanol¬amide (PEA), was described as early as 1957 as a potent anti¬inammatory compound in egg yolk [4]. More recently, the anti-inammatory properties of PEA and other saturated NAEs have been revisited [5], and it was proposed [6] that this lipid could act as an agonist for the CB# receptor [7]. Since these ndings, PEA has been seen as a rather enigmatic molecule (see [8] for a review), in as much as it is capable of inducing numerous cannabinoid-like pharmacological actions both in itro [6,9] and in io [1012], in some cases in a manner that is sensitive to a CB# receptor antagonist, SR144528 [11]. Yet PEA exhibits very little, if any, affinity for the cloned CB" and CB# receptors from rat, mouse or human [1315]. While some authors have proposed the existence of CB#-like cannabinoid pound [11], this hypothesis has not found any molecular support to date. Other authors have suggested that PEA may act as an entouragecompound, i.e. inhibiting the inactivation of en dogenous cannabinoids such as AEA, thereby increasing their levels [8]. Indeed, PEA can be hydrolysed by the enzyme that is mostly responsible for AEA degradation [16,17], fatty acid amide hydrolase (FAAH) (see [18], and [19] for a recent review). However, PEA is not a very efficacious inhibitor of AEA hydrolysis [20,21], possibly because FAAH appears to prefer as substrates unsaturated rather than saturated NAEs. The recent discovery of another enzyme catalysing the hydrolysis of AEA and PEA at similar rates [22] re-opens the possibility that the latter compound acts as an enhancerof the activity of th former, as shown for another endocannabinoid, 2-arachidonoyl-glycerol (2-AG) and some of its congeners [23]. PEA is co-synthesized with AEA in most of the cells analysed so far, and in amounts 510-fold higher than those of the endocannabinoid [8,16,24], and could thus also play a role as an entourage substance for AEA when the two substances are produced endogenously.
We have reported previously that 24 days of treatment with AEA and 2-AG, but not PEA, can potently inhibit the pro¬liferation of human breast cancer cells (HBCCs) via CB"-like receptors [25]. This effect was mediated by inhibition of cAMP production and protein kinase A, and by dis-inhibition of mitogen-activated protein kinase [26], and was due to suppression of the expression of the receptor for prolactin, a hormone used by HBCCs as an autacoid mitogen [25]. In fact, AEA and 2-AG, but not PEA, also inhibit the proliferation of human prostate cancer cells induced by exogenous prolactin [27]. We also found that AEA and 2-AG, but not PEA, inhibit the nerve growth factor (NGF)-induced proliferation of MCF-7 cells, a HBCC line, by blocking, through the same intracellular signalling pathways, expression of the high-affinity NGF receptors Trk [27]. In the present study we have investigated whether PEA enhances the anti-proliferative effects of AEA in MCF-7 cells, and, if so, through which mechanism. We report that chronic treatment with PEA, at concentrations 510-fold higher than those of AEA (which reect the PEA}AEA ratio of concen¬trations in tissues), signicantly enhances the inhibitory effect of the endocannabinoid on the basal and NGF-induced prolifer-ation of MCF-7 cells, as well as on Trk expression. We demonstrate that this entourage effect of PEA is not due to direct interaction with CB", CB# or FAAH, or to interference with cannabinoid-receptor-coupled signalling, but can be explained in part by inhibition of the expression of FAAH.
MATERIALS AND METHODS Materials
MCF-7, T-47D and DU-145 cells were purchased from A.T.C.C. (Manassas, VA, U.S.A), and EFM-19 cells were from DSM; cells were cultured as advised by the manufacturers. COS-7 cells were cultured in Dulbeccos modied Eagles medium supple mented with 5% (v}v) fetal calf serum, 100 units}ml penicillin and 100 g}ml streptomycin in a humidied atmosphere con¬sisting of 5 % CO# and 95% air, at 37 °C. ["%C]AEA (5 mCi}mmol), AEA and PEA were synthesized as described in [1,16]. Arvanil [N-(3-methoxy-4-hydroxybenzyl)arachidonoyl¬amide] was synthesized as described in [28]. The synthetic cannabinoid HU-210 and linoleoylethanolamide were kindly donated by Professor R. Mechoulam (Hebrew University of Jerusalem, Israel). [2-$H]Adenine (18.0 Ci}mmol) was purchased from American Radiolabeled Chemicals (St. Louis, MO, U.S.A.). The phosphodiesterase inhibitors 3-isobutyl-1 -methylxanthine and RO-20-1724 were from Calbiochem (La Jolla, CA, U.S.A.). Forskolin and cAMP were from Sigma (St. Louis, MO, U.S.A.). SR144528 was a gift from SanoRecherche (Montpellier, France). BML-190 was purchased from Biomol (Plymouth Meeting, PA, U.S.A.), and human prolactin and recombinant -NGF were from Sigma. [$H]SR141716A (55 Ci}mmol) was purchased from Amersham. For cell trans¬fection, the plasmid consisting of rabbit adenylate cyclase type V (AC-V) cDNA in the pXMD1 vector [29] was as described previously [30]. Human CB# cDNA was kindly provided by Dr S. Munro (University of Cambridge, U.K.), and human CB" cDNA was kindly provided by Dr M. Parmentier (University of Bruxelles, Belgium).
Cell proliferation assays
Cell proliferation assays were carried out according to the method described previously [2527] in six-well dishes containing subcon-uent cells (at a density of approx. 50000 cells}well). With MCF-7, EFM-19 and T-47D cells, test substances were introduced 3 h
after cell seeding and then daily at each change of medium. Cells were trypsinized and counted in a haemocytometer 4 days after the addition of test substances. No signicant decrease in cell viability (as assessed by Trypan Blue exclusion) was observed with up to 100 M AEA. With DU-145 cells the effect was studied after stimulation with prolactin, as AEA does not inhibit the basal proliferation of these cells. Prolactin (1 m-unit}ml) or vehicle was added 24 h after seeding with the change of medium, in the presence of the test substances or vehicle. After 72 h, cells were trypsinized and counted in a haemocytometer. In order to study the effect of NGF on MCF-7 cell proliferation, we used a different procedure [27]. At 24 h after cell seeding (50000 cells}well) the medium was changed to serum-free medium and cells were starved for 24 h. Cells were then treated with serum-free medium containing -NGF (100 ng}ml) plus test substances or vehicle, and trypsinized and counted after 48 h. Means were compared using the unpaired Studentst test, with P ! 0.05 as the threshold for statistical signicance.
Western immunoblotting
After treatment with test substances, which was carried out under the same conditions as described above for cell pro¬liferation assays, but in 100 mm Petri dishes, cells were washed twice with buffer containing 137 mM NaCl, 3 mM KCl, 12 mM Na#HPO% and 2 mM KH#PO% (pH 7.4), and then lysed with a lysis buffer consisting of 50 mM Tris}HCl, pH 7.4, 1 mM EDTA, 150 mM NaCl, 1 mM Na$VO%, 1 mM NaF, 1% Nonidet P40, 0.25% sodium deoxycholate, 1 mM PMSF, 1% Triton X-100 and 1g}ml each of aprotinin, leupeptin and pepstatin A. Lysates were loaded on to gels containing 7.5 % (w}v) poly¬acrylamide for blotting of Trk. Proteins were transferred to nitrocellulose membranes, which were then incubated rst for 1 h at room temperature with the rst antibody, i.e. anti-(mouse Trk) monoclonal antibody (B-3; Santa Cruz Biotechnologies, Inc., Santa Cruz, CA, U.S.A.; diluted 1:500), and then with the appropriate horseradish peroxidase-labelled second antibody conjugates (1:5000; Bio-Rad, Hercules, CA, U.S.A.). Bands were visualized by the enhanced chemiluminescence technique (Bio-Rad). The anti-Trk antibody cross-reacts with human Trk.
Binding assays
Binding assays were carried out as described in [25,27] on membranes prepared from either MCF-7 cells or male CD rat brains, as described therein. The binding of increasing concen¬trations (0.110 nM) of [$H]SR141716A to aliquots (0.4mg of total protein) of these membranes, and the displacement of a xed concentration (300 pM) of [$H]SR141716A by increasing concentrations (0.025, 0.1, 0.5, 1.0, 5.0) of AEA in the presence or absence of PEA, were measured in equilibrium assays. SR141716A (10 M) was used to determine non-specic binding. Receptor binding results were analysed with GraphPad software (San Diego, CA, U.S.A.). Scatchard curves for the binding of [$H]SR141716A were used to calculate Bmax and Kd values for this ligand by using non-linear regression, and one- and two-site analyses were compared to determine better-t values (r# ¯ 0.88 for one-site binding). Displacement curves (calculated by means of Pharm}PCS software) were used to calculate the Ki values for AEA by inserting the corresponding IC&! values from the best¬tting curves into the ChengPrusoff equation.
COS cell transfection with AC-V and cAMP assay
At 24 h before transfection, a 10 cm plate of conuent COS-7 cells was trypsinized and cells were divided into ve 10 cm plates.
The cells were transfected, using the DEAE-dextran chloroquine method [30], with 2 g}plate human CB" or CB# cDNA and 2 g}plate AC-V cDNA. The cells were trypsinized after 24 h and re-cultured in 24-well plates, and after an additional 24 h the cells were assayed for AC activity.
The cAMP assay was performed as described previously [30]. In brief, cells cultured in 24-well plates were incubated for 2 h with 0.25 ml}well fresh growth medium containing 5 Ci}ml [2-$H]adenine. This medium was replaced with Dulbeccos modi¬ed Eagles medium containing 20 mM Hepes (pH 7.4) and the phosphodiesterase inhibitors RO-20-1724 (0.5 mM) and 3-iso¬butyl-1-methylxanthine (0.5 mM). Substances diluted in 20 mg} ml fatty-acid-free BSA were then added. AC activity was stimulated in the presence or absence of test compounds by the addition of forskolin. After 10 min at 37 °C the medium was removed, and the reaction was terminated by adding to the cell layer 1 ml of 2.5% perchloric acid containing 0.1 mM unlabelled cAMP. Aliquots of 0.9 ml of the acidic extract were neutralized with 100 l of 3.8 M KOH}0.16 M K#CO$ and applied to a two-step column separation procedure, following which the [$H]-cAMP was eluted into scintillation vials and radioactivity was counted. Unless otherwise described, background levels (cAMP accumulation in the absence of stimulant) were subtracted from all values. AEA or PEA, alone or in combination, were added together with the forskolin for the 10-min assay period (acute treatment) or incubated with the cells for 18 h (or for the times indicated) prior to the 10-min assay (started by the addition of forskolin) (chronic treatment). All experiments were performed in triplicate. In MCF-7 cells, the cAMP assay was per-formed using a kit (Amersham), as described previously [26].
Reverse transcriptasePCR (RT-PCR) amplication of CB1/CB2 mRNA
Total RNA was prepared from MCF-7 cells by the Trizol2 method (Life Technologies) according to the manufacturers instructions. To remove contaminating DNA, 12 g RNA samples were digested with DNase according to the DNA-free (Ambion) protocol. Retro-transcription of mRNA into cDNA was performed in a 20 l reaction mixture containing 75 mM KCl, 3 mM MgCl#, 10 mM dithiothreitol, 1 mM dNTPs, 50 mM Tris}HCl, pH 8.3, 5 g of total RNA, 20 units of RNase inhibitor (BoehringerRoche), 0.125 A#'! unit of hexanucleotide mixture (BoehringerRoche) for random priming and 200 units of Moloney murine leukaemia virus reverse transcriptase (Superscript; GIBCO). The reaction mixture was incubated at room temperature for 10 min and then at 37 °C for 90 min, and was stopped by heating at 98 °C for 5 min, cooled in ice, and stored at ®20 °C. Control samples were prepared by omitting reverse transcriptase from the retro-transcription mixture.
DNA amplication was performed in a 50 l PCR reaction mixture containing 0.52 l of the retro-transcription mixture, 1¬PCR buffer (supplied as a component of the DNA polymerase kit), 2 mM MgCl#, 250 M dNTPs, 0.5 M each of 5« and 3« primers and 2.5 units of Platinum !$!& Taq DNA polymerase (Life Technologies). The mixtures were amplied in a PE Gene Amp PCR System 2400 thermocycler (Perkin Elmer). The primers used were: CB" sense primer, 5«-CGCAAAGATAGCCGCAA¬CGTGT-3«; CB" antisense primer, 5«-CAGATTGCAGTT¬TCTCGCAGTT-3«; CB# sense primer 5«-TTTCCCACTGA¬TCCCCAATG-3«; CB# antisense primer, 5«-AGTTGATGAG¬GCACAGCATG-3«; FAAH sense primer, 5«-GCCTGGGAA-GTGAACAAAGGGACC-3«; FAAH antisense primer, 5«-CCACTACGCTGTCGCACTCCGCCG-3«; #-microglobulin sense primer, 5«-CCAGCAGAGAATGGAAAGTC-3«; # microglobulin antisense primer, 5«-GATGCTGCTTACATGT¬CTCG-3«. The amplication prole consisted of an initial de¬naturation of 2 min at 95 °C, and then 2035 cycles of 30 s at 95 °C, annealing for 30 s at 55 °C (CB" and #-microglobulin) or at 60 °C (CB2 and FAAH) and elongation for 1 min at 72 °C. A nal extension of 10 min was carried out at 72 °C. The expected sizes of the amplicons were 244 bp for CB", 337 bp for CB#, 202 bp for FAAH and 268 bp for #-microglobulin. #-Micro-globulin was used as a housekeeping gene in order to evaluate variations in mRNA quality and content and to monitor cDNA synthesis in the different preparations. Furthermore, the PCR primers for FAAH and #-microglobulin were selected by including an intron sequence; subsequently, in the presence of contaminating genomic DNA, the expected sizes of the amplicon would be 425 bp and 886 bp for FAAH and #-microglobulin respectively. Quantication of expression levels was performed in the exponential phase of amplication determined, for a xed quantity of retro-transcription mixture, by analysing the amount of amplicon synthesized at different numbers of amplication cycles. Aliquots of 1020 l of PCR products were electro¬phoresed on a 2 % (w}v) agarose gel (MS agarose; Boehringer Roche) in 1¬TAE buffer at 4 V}cm for 4 h. Ethidium bromide (0.1 g}ml) was included in both the gel and electrophoresis buffer, and PCR products were detected by UV visualisation.
AEA hydrolysis in intact cells
After 4 days of treatment with either vehicle or 5 M PEA, MCF-7 cells in 10 cm Petri dishes were washed with serum-free medium and incubated with ["%C]AEA (10000 c.p.m.}ml) in 10 ml of serum-free medium for up to 1 h. After various intervals of time, aliquots were taken and extracted with chloroform}water (2:1, v}v), and the radioactivity of the aqueous phase, due uniquely to ["%C]ethanolamine produced from ["%C]AEA hy¬drolysis, was measured by scintillation spectrometry.
RESULTS AND DISCUSSION
We have shown previously that PEA, up to a concentration of 10 M, is not capable of mimicking the anti-proliferative effect of AEA on HBCCs and human prostate cells [25,27]. This early nding was not surprising, since this anti-proliferative effect was shown to be due to interaction with CB" receptors, for which PEA has very little, if any, affinity [1315]. Based on the proposed role of PEA as an entourage substance for AEA [23], on the previously synergistic action of this compound on the anti¬hyperaglesic effects of AEA [11], and on the observation that MCF-7 cells do synthesize PEA [31], we wanted to investigate whether this compound could enhance the AEA-induced in¬hibition of HBCC proliferation. As PEA is usually 510 times more abundant than AEA in HBCCs [8], and since the IC&! values for the anti-proliferative effects of AEA are in the 0.2 2 M concentration range [2527], we tested the effects of 110 M PEA. We found that co-incubation of cells with PEA in this concentration range enhanced the AEA-evoked inhibition of EFM- 19 and MCF-7 cell basal proliferation with an EC&! of C 2 M (Figure 1A). When 5 M PEA was tested with increasing concentrations of AEA, the IC&! for the inhibition of cell proliferation was lowered from 1.1 ³0.2 to 0.3 ³0.1 M in MCF-7 cells, and from 2.3 ³0.3 to 0.3 ³0.1 M in T-47D cells (Figure 1B). These differences were signicant (P ! 0.01 by ANOVA). When MCF-7 cell proliferation was stimulated by NGF, we found that PEA (5 M) again enhanced the anti-proliferative effect of AEA, the IC&! of which was reduced from 0.55 ³0.11 to 0.15 ³0.05 M (Figure 1C; P ! 0.01). We have shown previously that the inhibitory effect of AEA on the NGF-induced pro¬liferation of MCF-7 cells is due to suppression of the high-affinity Trk receptors for NGF [27]. Here we found that PEA (5 M) also signicantly enhanced the down-regulatory action of AEA on Trk expression, as assessed by Western immunoblotting of MCF-7 cell total proteins carried out with a monoclonal antibody against Trk (Figure 1D). Finally, we ran an experiment in DU-145 human prostate cancer cells, in which AEA blocks prolactin-induced proliferation [27]. Again, PEA (2.5 M) signicantly enhanced the inhibitory action of AEA (0.1 M), from 42.6% to 100% inhibition (Figure 1C, inset). These effects of PEA were not due to prevention of AEA adhesion to the plasticware used for the assays. In fact, when the plates were incubated with ["%C]AEA in the absence or presence of PEA (5 M), no difference was found in the recovery of radioactivity in the incubation medium after up to 1 h of incubation (results not shown). PEA was suggested previously to act as an agonist at CB#-like receptors [6,11], although this hypothesis is not supported by more recent data [1315]. A CB# receptor RNA transcript was indeed identied in HBCCs by RT-PCR [27]. However, here we found that the effect of PEA on the inhibitory actions of AEA in basal or NGF-induced HBCC proliferation was not mimicked by BML-190, a selective agonist of CB# receptors (Table 1). Furthermore, this effect of PEA was not inuenced by SR144528, a selective antagonist of CB# receptors (Table 2). Finally, the stimulatory effect of PEA on the AEA-induced down-regulation of Trk receptors was not mimicked by use of BML-190 (Figure 1D). These ndings indicate that CB# receptors are not involved in the effects of PEA on HBCC proliferation described here.
We next investigated whether the stimulatory effect of PEA on the cytostatic actions of AEA could indeed be due to an entourage effect, i.e. to inhibition of intracellular AEA degradation by cells.
In fact, HBCCs express FAAH [31], the enzyme mostly re¬sponsible for AEA degradation in cells, which is also capable of recognizing PEA as a substrate. However, we have shown previously that PEA does not effectively inhibit the hydrolysis of ["%C]AEA by intact EFM-19 cells during short (030 min) incubations of cells with both compounds [31], in agreement with previous observations that this lipid is a worse FAAH substrate than AEA and is not capable of inhibiting AEA uptake by cells [16,20,21,31]. Therefore we investigated here whether a 4-day treatment of MCF-7 cells with PEA (i.e. the same conditions necessary to observe potentiation of AEA cytostatic effects) would lead to modulation of FAAH expression, as assessed by quantitative RT-PCR of a FAAH RNA transcript. We found that PEA (5 M) signicantly decreased (by approx. 3040%) the expression of FAAH at the transcriptional level (Figures 2A and 2B) (the values from densitometric scans of the bands decreased from 1590 ³51 to 1093 ³59 arbitrary units; means ³ S.E.M.; n ¯ 8; P ! 0.01 by ANOVA). This effect was accom¬panied by a corresponding decrease in FAAH activity, as assessed by the decreased ability of intact MCF-7 cells to hydrolyse exogenous ["%C]AEA (from 4099 ³331 to 3006 ³250 c.p.m.}h per 10' cells; mean³S.E.M.; n ¯ 8; P ! 0.05 by ANOVA). These data indicate that the stimulatory action of PEA on the cytostatic effects of AEA is due, at least in part, to inhibition of AEA degradation by FAAH, potentially resulting in higher levels of AEA during the proliferation experiments and, sub¬sequently, in a potentiation of AEA activity. We then challenged this hypothesis by examining the effects of PEA in the presence of: (1) an AEA analogue, arvanil [28], which is more stable than AEA to enzymic hydrolysis, and (2) HU-210, a synthetic CB" receptor agonist and cytostatic agent for HBCCs, which cannot be hydrolysed by FAAH. We found that PEA (5 M) also potentiated the anti-proliferative effects of both arvanil and HU-210 in all cell lines under study, and under all conditions used (Figure 3 and results not shown), although less potently than with AEA. In MCF-7 cells, for example, the IC&! values were decreased only 2-fold (from 0.6 ³0.1 to 0.3 ³0.1 M with arvanil and from 3.6 ³0.3 to 1.8 ³0.2 M with HU-210; n ¯ 3; P ! 0.05 by ANOVA), compared with 36-fold with AEA (see above). These data suggest that PEA is also capable of enhancing the cytostatic action of AEA through mechanisms other than in¬hibition of its enzymic hydrolysis.
Since the anti-proliferative effects of AEA are mediated by CB" receptors and through the inhibition of AC [2527], we studied the possibility that PEA enhances AEA action by exerting either short- or long-term stimulatory actions on the expression, affinity for ligands or coupling to AC of CB" receptors. We started by examining the possibility of an allosteric effect of PEA on [$H]SR141716A binding to rat brain cell membranes, or on displacement of [$H]SR141716A binding by AEA. We found no stimulatory action on [$H]SR14171 6A binding (Bmax 1805³121 and 2082³150 fmol}mg of protein; Kd 1.45 ³018 and 1.69³ 0.20 nM; without and with 5 M PEA respectively; means³ S.E.M.; n ¯ 3). We found a non-statistically signicant effect on the Ki values for displacement of [$H]SR141716A by AEA (1.0³0.2 and 0.6³0.2M without and with 5M PEA re¬spectively; means ³S.E.M.; n ¯ 3). Likewise, no signicant effect of PEA was found when using membrane preparations from MCF-7 cells (results not shown), which contain much less specic [$H]SR141716A binding sites than rat brain [27]. The effect on cannabinoid receptor expression of long-term treatment of cells with PEA was studied by means of RT-PCR, as described above for FAAH. We found that the same 4-day treatment of MCF-7 cells with PEA (5 M) that led to inhibition of FAAH expression did not signicantly affect the expression of RNA transcripts for either CB" or CB# receptors (results not shown). Finally, we examined whether either short- or long-term treatment of cells with PEA could inuence CB receptor coupling to AC. We found no effect of PEA (10 M) on AEA-induced inhibition of forskolin-stimulated cAMP production in MCF-7 cells (results not shown). As we reasoned that the level of expression of either CB" receptors or AC in these cells might depend on several factors, such as the number of subculturing passages [27], we repeated these experiments under more controlled and repro¬ducible conditions, i.e. in COS cells co-transfected with CB" receptors and the AC-V isoform. We found no effect of either acute (Figure 4) or chronic (results not shown) treatment with PEA on the AEA-induced inhibition of cAMP formation in COS cells transfected with AC-V and either CB" or CB# cDNAs.
In summary, we have found that PEA is capable of inhibiting the expression of FAAH in HBCCs, and that this regulatory effect is responsible, at least in part, for the PEA-induced enhancement of the anti-proliferative effects of AEA. Although there has been previous evidence for effects of fatty acid amides on FAAH activity [32,3 3], this is, to the best of our knowledge, the rst time that a regulatory action on FAAH expression (and at the transcriptional level) has been reported for a member of this class of bioactive lipids. Our ndings have several potentially important implications. Firstly, our data strengthen the previous hypothesis [31] that NAEs might play a role as tumour growth suppressors in HBCCs. While members of this family of lipids that activate CB" receptors, such as AEA, might inhibit pro¬liferation directly, the saturated and monounsaturated NAEs, such as PEA, which are usually more abundant than AEA, might enhance this effect. Secondly, our present ndings might be exploited pharmaceutically by leading to the development of tumour-suppressing cocktailswhose potency might be greater than that of simple CB" receptor agonists. In fact, there is increasing evidence that substances that activate cannabinoid receptors can also be used as anti-neoplastic drugs in io([34,35]; M. Bifulco and V. Di Marzo, unpublished work). However, this therapeutic application might be limited by the undesired psychotropic side effects expected from these drugs. This limitation might be overcome by the administration of endocannabinoids, which seem to have much lower potential for dependence [36], in combination with a non-psychotropic sub-stance, such as PEA, which would lower the concentrations necessary to observe the tumour-suppressing effect. Finally, the present study improves our knowledge of the molecular mechan¬sims through which PEA acts synergistically with AEA [8]. It is possible that these synergistic effects, at least when they are observed after chronic PEA treatment, may be due in part to modulation of the expression of FAAH and a subsequent increase in the amount of AEA available for cannabinoid receptor activation. However, since PEA is also capable of increasing the anti-proliferative effects ofHU-210, additional molecular mecha-nisms are likely to underlie the synergistic effects of this NAE on cannabinoid-receptor-mediated biological actions. Indeed, we have preliminary data showing that PEA, independently of FAAH, can signicantly enhance acute effects of AEA that are mediated by vanilloid VR" receptors [37] (L. De Petrocellis and V. Di Marzo, unpublished work). Here we have presented data that argue against possible regulatory effects of PEA on CB"}CB# receptor expression, ligand affinity and functional activity. Future studies will aim at nding other molecular targets involved in the biochemical and pharmacological actions of PEA.
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Source: Palmitoylethanolamide inhibits the expression of fatty acid amide hydrolase and enhances the anti-proliferative effect of anandamide in human breast cancer cells
Vincenzo DI MARZO*1, Dominique MELCK*, Pierangelo ORLANDO, Tiziana BISOGNO*, Orna ZAGOORY, Maurizio BIFULCO, Zvi VOGEL and Luciano DE PETROCELLISs
*Istituto per la Chimica di Molecole di Interesse Biologico, Via Toiano 6, 80072, Arco Felice, Napoli, Italy, Istituto di Biochimica delle Proteine ed Enzimologia, Via Toiano 6, 80072, Arco Felice, Napoli, Italy, Department of Neurobiology, Weizmann Institute of Science, Rehovot 76100, Israel, Centro di Endocrinologia e Oncologia Sperimentale, Consiglio Nazionale delle Ricerche, and Dipartimento di Biologia e Patologia Cellulare e Molecolare, Universitadi Napoli Federico II, 80131, Napoli, Italy, and sIstituto di Cibernetica, Consiglio Nazionale delle Ricerche, Via Toiano 6, 80072, Arco Felice, Napoli, Italy
Palmitoylethanolamide (PEA) has been shown to act in synergy with anandamide (arachidonoylethanolamide; AEA), an en¬dogenous agonist of cannabinoid receptor type 1 (CB"). This synergistic effect was reduced by the CB# cannabinoid receptor antagonist SR144528, although PEA does not activate either CB" or CB# receptors. Here we show that PEA potently enhances the anti-proliferative effects of AEA on human breast cancer cells (HBCCs), in part by inhibiting the expression of fatty acid amide hydrolase (FAAH), the major enzyme catalysing AEA degradation. PEA (110 M) enhanced in a dose-related manner the inhibitory effect of AEA on both basal and nerve growth factor (NGF)-induced HBCC proliferation, without inducing any cytostatic effect by itself. PEA (5 M) decreased the IC&! values for AEA inhibitory effects by 36-fold. This effect was not blocked by the CB# receptor antagonist SR144528, and was not mimicked by a selective agonist of CB# receptors. PEA enhanced AEA-evoked inhibition of the expression of NGF Trk receptors, which underlies the anti-proliferative effect of the endocannabinoid on NGF-stimulated MCF-7 cells. The effect of PEA was due in part to inhibition of AEA degradation, since treatment of MCF-7 cells with 5 M PEA caused a C 3040% down-regulation of FAAH expression and activity. However, PEA also enhanced the cytostatic effect of the cannabinoid receptor agonist HU-210, although less potently than with AEA. PEA did not modify the affinity of ligands for CB" or CB# receptors, and neither did it alter the CB"}CB#-mediated inhi¬bitory effect of AEA on adenylate cyclase type V, nor the expression of CB" and CB# receptors in MCF-7 cells. We suggest that long-term PEA treatment of cells may positively affect the pharmacological activity of AEA, in part by inhibiting FAAH expression.
Key words: 2-arachidonoylglycerol, arvanil, cell proliferation, endocannabinoids, receptors.
INTRODUCTION
Anandamide (arachidonoylethanolamide; AEA) was the rst endogenous substance to be proposed as an agonist for the cannabinoid receptor type 1 (CB" receptor) [1,2]. AEA belongs to a family of lipids, the N-acylethanolamines (NAEs) (see [3] for a review), whose most well known component, palmitoylethanol¬amide (PEA), was described as early as 1957 as a potent anti¬inammatory compound in egg yolk [4]. More recently, the anti-inammatory properties of PEA and other saturated NAEs have been revisited [5], and it was proposed [6] that this lipid could act as an agonist for the CB# receptor [7]. Since these ndings, PEA has been seen as a rather enigmatic molecule (see [8] for a review), in as much as it is capable of inducing numerous cannabinoid-like pharmacological actions both in itro [6,9] and in io [1012], in some cases in a manner that is sensitive to a CB# receptor antagonist, SR144528 [11]. Yet PEA exhibits very little, if any, affinity for the cloned CB" and CB# receptors from rat, mouse or human [1315]. While some authors have proposed the existence of CB#-like cannabinoid pound [11], this hypothesis has not found any molecular support to date. Other authors have suggested that PEA may act as an entouragecompound, i.e. inhibiting the inactivation of en dogenous cannabinoids such as AEA, thereby increasing their levels [8]. Indeed, PEA can be hydrolysed by the enzyme that is mostly responsible for AEA degradation [16,17], fatty acid amide hydrolase (FAAH) (see [18], and [19] for a recent review). However, PEA is not a very efficacious inhibitor of AEA hydrolysis [20,21], possibly because FAAH appears to prefer as substrates unsaturated rather than saturated NAEs. The recent discovery of another enzyme catalysing the hydrolysis of AEA and PEA at similar rates [22] re-opens the possibility that the latter compound acts as an enhancerof the activity of th former, as shown for another endocannabinoid, 2-arachidonoyl-glycerol (2-AG) and some of its congeners [23]. PEA is co-synthesized with AEA in most of the cells analysed so far, and in amounts 510-fold higher than those of the endocannabinoid [8,16,24], and could thus also play a role as an entourage substance for AEA when the two substances are produced endogenously.
We have reported previously that 24 days of treatment with AEA and 2-AG, but not PEA, can potently inhibit the pro¬liferation of human breast cancer cells (HBCCs) via CB"-like receptors [25]. This effect was mediated by inhibition of cAMP production and protein kinase A, and by dis-inhibition of mitogen-activated protein kinase [26], and was due to suppression of the expression of the receptor for prolactin, a hormone used by HBCCs as an autacoid mitogen [25]. In fact, AEA and 2-AG, but not PEA, also inhibit the proliferation of human prostate cancer cells induced by exogenous prolactin [27]. We also found that AEA and 2-AG, but not PEA, inhibit the nerve growth factor (NGF)-induced proliferation of MCF-7 cells, a HBCC line, by blocking, through the same intracellular signalling pathways, expression of the high-affinity NGF receptors Trk [27]. In the present study we have investigated whether PEA enhances the anti-proliferative effects of AEA in MCF-7 cells, and, if so, through which mechanism. We report that chronic treatment with PEA, at concentrations 510-fold higher than those of AEA (which reect the PEA}AEA ratio of concen¬trations in tissues), signicantly enhances the inhibitory effect of the endocannabinoid on the basal and NGF-induced prolifer-ation of MCF-7 cells, as well as on Trk expression. We demonstrate that this entourage effect of PEA is not due to direct interaction with CB", CB# or FAAH, or to interference with cannabinoid-receptor-coupled signalling, but can be explained in part by inhibition of the expression of FAAH.
MATERIALS AND METHODS Materials
MCF-7, T-47D and DU-145 cells were purchased from A.T.C.C. (Manassas, VA, U.S.A), and EFM-19 cells were from DSM; cells were cultured as advised by the manufacturers. COS-7 cells were cultured in Dulbeccos modied Eagles medium supple mented with 5% (v}v) fetal calf serum, 100 units}ml penicillin and 100 g}ml streptomycin in a humidied atmosphere con¬sisting of 5 % CO# and 95% air, at 37 °C. ["%C]AEA (5 mCi}mmol), AEA and PEA were synthesized as described in [1,16]. Arvanil [N-(3-methoxy-4-hydroxybenzyl)arachidonoyl¬amide] was synthesized as described in [28]. The synthetic cannabinoid HU-210 and linoleoylethanolamide were kindly donated by Professor R. Mechoulam (Hebrew University of Jerusalem, Israel). [2-$H]Adenine (18.0 Ci}mmol) was purchased from American Radiolabeled Chemicals (St. Louis, MO, U.S.A.). The phosphodiesterase inhibitors 3-isobutyl-1 -methylxanthine and RO-20-1724 were from Calbiochem (La Jolla, CA, U.S.A.). Forskolin and cAMP were from Sigma (St. Louis, MO, U.S.A.). SR144528 was a gift from SanoRecherche (Montpellier, France). BML-190 was purchased from Biomol (Plymouth Meeting, PA, U.S.A.), and human prolactin and recombinant -NGF were from Sigma. [$H]SR141716A (55 Ci}mmol) was purchased from Amersham. For cell trans¬fection, the plasmid consisting of rabbit adenylate cyclase type V (AC-V) cDNA in the pXMD1 vector [29] was as described previously [30]. Human CB# cDNA was kindly provided by Dr S. Munro (University of Cambridge, U.K.), and human CB" cDNA was kindly provided by Dr M. Parmentier (University of Bruxelles, Belgium).
Cell proliferation assays
Cell proliferation assays were carried out according to the method described previously [2527] in six-well dishes containing subcon-uent cells (at a density of approx. 50000 cells}well). With MCF-7, EFM-19 and T-47D cells, test substances were introduced 3 h
after cell seeding and then daily at each change of medium. Cells were trypsinized and counted in a haemocytometer 4 days after the addition of test substances. No signicant decrease in cell viability (as assessed by Trypan Blue exclusion) was observed with up to 100 M AEA. With DU-145 cells the effect was studied after stimulation with prolactin, as AEA does not inhibit the basal proliferation of these cells. Prolactin (1 m-unit}ml) or vehicle was added 24 h after seeding with the change of medium, in the presence of the test substances or vehicle. After 72 h, cells were trypsinized and counted in a haemocytometer. In order to study the effect of NGF on MCF-7 cell proliferation, we used a different procedure [27]. At 24 h after cell seeding (50000 cells}well) the medium was changed to serum-free medium and cells were starved for 24 h. Cells were then treated with serum-free medium containing -NGF (100 ng}ml) plus test substances or vehicle, and trypsinized and counted after 48 h. Means were compared using the unpaired Studentst test, with P ! 0.05 as the threshold for statistical signicance.
Western immunoblotting
After treatment with test substances, which was carried out under the same conditions as described above for cell pro¬liferation assays, but in 100 mm Petri dishes, cells were washed twice with buffer containing 137 mM NaCl, 3 mM KCl, 12 mM Na#HPO% and 2 mM KH#PO% (pH 7.4), and then lysed with a lysis buffer consisting of 50 mM Tris}HCl, pH 7.4, 1 mM EDTA, 150 mM NaCl, 1 mM Na$VO%, 1 mM NaF, 1% Nonidet P40, 0.25% sodium deoxycholate, 1 mM PMSF, 1% Triton X-100 and 1g}ml each of aprotinin, leupeptin and pepstatin A. Lysates were loaded on to gels containing 7.5 % (w}v) poly¬acrylamide for blotting of Trk. Proteins were transferred to nitrocellulose membranes, which were then incubated rst for 1 h at room temperature with the rst antibody, i.e. anti-(mouse Trk) monoclonal antibody (B-3; Santa Cruz Biotechnologies, Inc., Santa Cruz, CA, U.S.A.; diluted 1:500), and then with the appropriate horseradish peroxidase-labelled second antibody conjugates (1:5000; Bio-Rad, Hercules, CA, U.S.A.). Bands were visualized by the enhanced chemiluminescence technique (Bio-Rad). The anti-Trk antibody cross-reacts with human Trk.
Binding assays
Binding assays were carried out as described in [25,27] on membranes prepared from either MCF-7 cells or male CD rat brains, as described therein. The binding of increasing concen¬trations (0.110 nM) of [$H]SR141716A to aliquots (0.4mg of total protein) of these membranes, and the displacement of a xed concentration (300 pM) of [$H]SR141716A by increasing concentrations (0.025, 0.1, 0.5, 1.0, 5.0) of AEA in the presence or absence of PEA, were measured in equilibrium assays. SR141716A (10 M) was used to determine non-specic binding. Receptor binding results were analysed with GraphPad software (San Diego, CA, U.S.A.). Scatchard curves for the binding of [$H]SR141716A were used to calculate Bmax and Kd values for this ligand by using non-linear regression, and one- and two-site analyses were compared to determine better-t values (r# ¯ 0.88 for one-site binding). Displacement curves (calculated by means of Pharm}PCS software) were used to calculate the Ki values for AEA by inserting the corresponding IC&! values from the best¬tting curves into the ChengPrusoff equation.
COS cell transfection with AC-V and cAMP assay
At 24 h before transfection, a 10 cm plate of conuent COS-7 cells was trypsinized and cells were divided into ve 10 cm plates.
The cells were transfected, using the DEAE-dextran chloroquine method [30], with 2 g}plate human CB" or CB# cDNA and 2 g}plate AC-V cDNA. The cells were trypsinized after 24 h and re-cultured in 24-well plates, and after an additional 24 h the cells were assayed for AC activity.
The cAMP assay was performed as described previously [30]. In brief, cells cultured in 24-well plates were incubated for 2 h with 0.25 ml}well fresh growth medium containing 5 Ci}ml [2-$H]adenine. This medium was replaced with Dulbeccos modi¬ed Eagles medium containing 20 mM Hepes (pH 7.4) and the phosphodiesterase inhibitors RO-20-1724 (0.5 mM) and 3-iso¬butyl-1-methylxanthine (0.5 mM). Substances diluted in 20 mg} ml fatty-acid-free BSA were then added. AC activity was stimulated in the presence or absence of test compounds by the addition of forskolin. After 10 min at 37 °C the medium was removed, and the reaction was terminated by adding to the cell layer 1 ml of 2.5% perchloric acid containing 0.1 mM unlabelled cAMP. Aliquots of 0.9 ml of the acidic extract were neutralized with 100 l of 3.8 M KOH}0.16 M K#CO$ and applied to a two-step column separation procedure, following which the [$H]-cAMP was eluted into scintillation vials and radioactivity was counted. Unless otherwise described, background levels (cAMP accumulation in the absence of stimulant) were subtracted from all values. AEA or PEA, alone or in combination, were added together with the forskolin for the 10-min assay period (acute treatment) or incubated with the cells for 18 h (or for the times indicated) prior to the 10-min assay (started by the addition of forskolin) (chronic treatment). All experiments were performed in triplicate. In MCF-7 cells, the cAMP assay was per-formed using a kit (Amersham), as described previously [26].
Reverse transcriptasePCR (RT-PCR) amplication of CB1/CB2 mRNA
Total RNA was prepared from MCF-7 cells by the Trizol2 method (Life Technologies) according to the manufacturers instructions. To remove contaminating DNA, 12 g RNA samples were digested with DNase according to the DNA-free (Ambion) protocol. Retro-transcription of mRNA into cDNA was performed in a 20 l reaction mixture containing 75 mM KCl, 3 mM MgCl#, 10 mM dithiothreitol, 1 mM dNTPs, 50 mM Tris}HCl, pH 8.3, 5 g of total RNA, 20 units of RNase inhibitor (BoehringerRoche), 0.125 A#'! unit of hexanucleotide mixture (BoehringerRoche) for random priming and 200 units of Moloney murine leukaemia virus reverse transcriptase (Superscript; GIBCO). The reaction mixture was incubated at room temperature for 10 min and then at 37 °C for 90 min, and was stopped by heating at 98 °C for 5 min, cooled in ice, and stored at ®20 °C. Control samples were prepared by omitting reverse transcriptase from the retro-transcription mixture.
DNA amplication was performed in a 50 l PCR reaction mixture containing 0.52 l of the retro-transcription mixture, 1¬PCR buffer (supplied as a component of the DNA polymerase kit), 2 mM MgCl#, 250 M dNTPs, 0.5 M each of 5« and 3« primers and 2.5 units of Platinum !$!& Taq DNA polymerase (Life Technologies). The mixtures were amplied in a PE Gene Amp PCR System 2400 thermocycler (Perkin Elmer). The primers used were: CB" sense primer, 5«-CGCAAAGATAGCCGCAA¬CGTGT-3«; CB" antisense primer, 5«-CAGATTGCAGTT¬TCTCGCAGTT-3«; CB# sense primer 5«-TTTCCCACTGA¬TCCCCAATG-3«; CB# antisense primer, 5«-AGTTGATGAG¬GCACAGCATG-3«; FAAH sense primer, 5«-GCCTGGGAA-GTGAACAAAGGGACC-3«; FAAH antisense primer, 5«-CCACTACGCTGTCGCACTCCGCCG-3«; #-microglobulin sense primer, 5«-CCAGCAGAGAATGGAAAGTC-3«; # microglobulin antisense primer, 5«-GATGCTGCTTACATGT¬CTCG-3«. The amplication prole consisted of an initial de¬naturation of 2 min at 95 °C, and then 2035 cycles of 30 s at 95 °C, annealing for 30 s at 55 °C (CB" and #-microglobulin) or at 60 °C (CB2 and FAAH) and elongation for 1 min at 72 °C. A nal extension of 10 min was carried out at 72 °C. The expected sizes of the amplicons were 244 bp for CB", 337 bp for CB#, 202 bp for FAAH and 268 bp for #-microglobulin. #-Micro-globulin was used as a housekeeping gene in order to evaluate variations in mRNA quality and content and to monitor cDNA synthesis in the different preparations. Furthermore, the PCR primers for FAAH and #-microglobulin were selected by including an intron sequence; subsequently, in the presence of contaminating genomic DNA, the expected sizes of the amplicon would be 425 bp and 886 bp for FAAH and #-microglobulin respectively. Quantication of expression levels was performed in the exponential phase of amplication determined, for a xed quantity of retro-transcription mixture, by analysing the amount of amplicon synthesized at different numbers of amplication cycles. Aliquots of 1020 l of PCR products were electro¬phoresed on a 2 % (w}v) agarose gel (MS agarose; Boehringer Roche) in 1¬TAE buffer at 4 V}cm for 4 h. Ethidium bromide (0.1 g}ml) was included in both the gel and electrophoresis buffer, and PCR products were detected by UV visualisation.
AEA hydrolysis in intact cells
After 4 days of treatment with either vehicle or 5 M PEA, MCF-7 cells in 10 cm Petri dishes were washed with serum-free medium and incubated with ["%C]AEA (10000 c.p.m.}ml) in 10 ml of serum-free medium for up to 1 h. After various intervals of time, aliquots were taken and extracted with chloroform}water (2:1, v}v), and the radioactivity of the aqueous phase, due uniquely to ["%C]ethanolamine produced from ["%C]AEA hy¬drolysis, was measured by scintillation spectrometry.
RESULTS AND DISCUSSION
We have shown previously that PEA, up to a concentration of 10 M, is not capable of mimicking the anti-proliferative effect of AEA on HBCCs and human prostate cells [25,27]. This early nding was not surprising, since this anti-proliferative effect was shown to be due to interaction with CB" receptors, for which PEA has very little, if any, affinity [1315]. Based on the proposed role of PEA as an entourage substance for AEA [23], on the previously synergistic action of this compound on the anti¬hyperaglesic effects of AEA [11], and on the observation that MCF-7 cells do synthesize PEA [31], we wanted to investigate whether this compound could enhance the AEA-induced in¬hibition of HBCC proliferation. As PEA is usually 510 times more abundant than AEA in HBCCs [8], and since the IC&! values for the anti-proliferative effects of AEA are in the 0.2 2 M concentration range [2527], we tested the effects of 110 M PEA. We found that co-incubation of cells with PEA in this concentration range enhanced the AEA-evoked inhibition of EFM- 19 and MCF-7 cell basal proliferation with an EC&! of C 2 M (Figure 1A). When 5 M PEA was tested with increasing concentrations of AEA, the IC&! for the inhibition of cell proliferation was lowered from 1.1 ³0.2 to 0.3 ³0.1 M in MCF-7 cells, and from 2.3 ³0.3 to 0.3 ³0.1 M in T-47D cells (Figure 1B). These differences were signicant (P ! 0.01 by ANOVA). When MCF-7 cell proliferation was stimulated by NGF, we found that PEA (5 M) again enhanced the anti-proliferative effect of AEA, the IC&! of which was reduced from 0.55 ³0.11 to 0.15 ³0.05 M (Figure 1C; P ! 0.01). We have shown previously that the inhibitory effect of AEA on the NGF-induced pro¬liferation of MCF-7 cells is due to suppression of the high-affinity Trk receptors for NGF [27]. Here we found that PEA (5 M) also signicantly enhanced the down-regulatory action of AEA on Trk expression, as assessed by Western immunoblotting of MCF-7 cell total proteins carried out with a monoclonal antibody against Trk (Figure 1D). Finally, we ran an experiment in DU-145 human prostate cancer cells, in which AEA blocks prolactin-induced proliferation [27]. Again, PEA (2.5 M) signicantly enhanced the inhibitory action of AEA (0.1 M), from 42.6% to 100% inhibition (Figure 1C, inset). These effects of PEA were not due to prevention of AEA adhesion to the plasticware used for the assays. In fact, when the plates were incubated with ["%C]AEA in the absence or presence of PEA (5 M), no difference was found in the recovery of radioactivity in the incubation medium after up to 1 h of incubation (results not shown). PEA was suggested previously to act as an agonist at CB#-like receptors [6,11], although this hypothesis is not supported by more recent data [1315]. A CB# receptor RNA transcript was indeed identied in HBCCs by RT-PCR [27]. However, here we found that the effect of PEA on the inhibitory actions of AEA in basal or NGF-induced HBCC proliferation was not mimicked by BML-190, a selective agonist of CB# receptors (Table 1). Furthermore, this effect of PEA was not inuenced by SR144528, a selective antagonist of CB# receptors (Table 2). Finally, the stimulatory effect of PEA on the AEA-induced down-regulation of Trk receptors was not mimicked by use of BML-190 (Figure 1D). These ndings indicate that CB# receptors are not involved in the effects of PEA on HBCC proliferation described here.
We next investigated whether the stimulatory effect of PEA on the cytostatic actions of AEA could indeed be due to an entourage effect, i.e. to inhibition of intracellular AEA degradation by cells.
In fact, HBCCs express FAAH [31], the enzyme mostly re¬sponsible for AEA degradation in cells, which is also capable of recognizing PEA as a substrate. However, we have shown previously that PEA does not effectively inhibit the hydrolysis of ["%C]AEA by intact EFM-19 cells during short (030 min) incubations of cells with both compounds [31], in agreement with previous observations that this lipid is a worse FAAH substrate than AEA and is not capable of inhibiting AEA uptake by cells [16,20,21,31]. Therefore we investigated here whether a 4-day treatment of MCF-7 cells with PEA (i.e. the same conditions necessary to observe potentiation of AEA cytostatic effects) would lead to modulation of FAAH expression, as assessed by quantitative RT-PCR of a FAAH RNA transcript. We found that PEA (5 M) signicantly decreased (by approx. 3040%) the expression of FAAH at the transcriptional level (Figures 2A and 2B) (the values from densitometric scans of the bands decreased from 1590 ³51 to 1093 ³59 arbitrary units; means ³ S.E.M.; n ¯ 8; P ! 0.01 by ANOVA). This effect was accom¬panied by a corresponding decrease in FAAH activity, as assessed by the decreased ability of intact MCF-7 cells to hydrolyse exogenous ["%C]AEA (from 4099 ³331 to 3006 ³250 c.p.m.}h per 10' cells; mean³S.E.M.; n ¯ 8; P ! 0.05 by ANOVA). These data indicate that the stimulatory action of PEA on the cytostatic effects of AEA is due, at least in part, to inhibition of AEA degradation by FAAH, potentially resulting in higher levels of AEA during the proliferation experiments and, sub¬sequently, in a potentiation of AEA activity. We then challenged this hypothesis by examining the effects of PEA in the presence of: (1) an AEA analogue, arvanil [28], which is more stable than AEA to enzymic hydrolysis, and (2) HU-210, a synthetic CB" receptor agonist and cytostatic agent for HBCCs, which cannot be hydrolysed by FAAH. We found that PEA (5 M) also potentiated the anti-proliferative effects of both arvanil and HU-210 in all cell lines under study, and under all conditions used (Figure 3 and results not shown), although less potently than with AEA. In MCF-7 cells, for example, the IC&! values were decreased only 2-fold (from 0.6 ³0.1 to 0.3 ³0.1 M with arvanil and from 3.6 ³0.3 to 1.8 ³0.2 M with HU-210; n ¯ 3; P ! 0.05 by ANOVA), compared with 36-fold with AEA (see above). These data suggest that PEA is also capable of enhancing the cytostatic action of AEA through mechanisms other than in¬hibition of its enzymic hydrolysis.
Since the anti-proliferative effects of AEA are mediated by CB" receptors and through the inhibition of AC [2527], we studied the possibility that PEA enhances AEA action by exerting either short- or long-term stimulatory actions on the expression, affinity for ligands or coupling to AC of CB" receptors. We started by examining the possibility of an allosteric effect of PEA on [$H]SR141716A binding to rat brain cell membranes, or on displacement of [$H]SR141716A binding by AEA. We found no stimulatory action on [$H]SR14171 6A binding (Bmax 1805³121 and 2082³150 fmol}mg of protein; Kd 1.45 ³018 and 1.69³ 0.20 nM; without and with 5 M PEA respectively; means³ S.E.M.; n ¯ 3). We found a non-statistically signicant effect on the Ki values for displacement of [$H]SR141716A by AEA (1.0³0.2 and 0.6³0.2M without and with 5M PEA re¬spectively; means ³S.E.M.; n ¯ 3). Likewise, no signicant effect of PEA was found when using membrane preparations from MCF-7 cells (results not shown), which contain much less specic [$H]SR141716A binding sites than rat brain [27]. The effect on cannabinoid receptor expression of long-term treatment of cells with PEA was studied by means of RT-PCR, as described above for FAAH. We found that the same 4-day treatment of MCF-7 cells with PEA (5 M) that led to inhibition of FAAH expression did not signicantly affect the expression of RNA transcripts for either CB" or CB# receptors (results not shown). Finally, we examined whether either short- or long-term treatment of cells with PEA could inuence CB receptor coupling to AC. We found no effect of PEA (10 M) on AEA-induced inhibition of forskolin-stimulated cAMP production in MCF-7 cells (results not shown). As we reasoned that the level of expression of either CB" receptors or AC in these cells might depend on several factors, such as the number of subculturing passages [27], we repeated these experiments under more controlled and repro¬ducible conditions, i.e. in COS cells co-transfected with CB" receptors and the AC-V isoform. We found no effect of either acute (Figure 4) or chronic (results not shown) treatment with PEA on the AEA-induced inhibition of cAMP formation in COS cells transfected with AC-V and either CB" or CB# cDNAs.
In summary, we have found that PEA is capable of inhibiting the expression of FAAH in HBCCs, and that this regulatory effect is responsible, at least in part, for the PEA-induced enhancement of the anti-proliferative effects of AEA. Although there has been previous evidence for effects of fatty acid amides on FAAH activity [32,3 3], this is, to the best of our knowledge, the rst time that a regulatory action on FAAH expression (and at the transcriptional level) has been reported for a member of this class of bioactive lipids. Our ndings have several potentially important implications. Firstly, our data strengthen the previous hypothesis [31] that NAEs might play a role as tumour growth suppressors in HBCCs. While members of this family of lipids that activate CB" receptors, such as AEA, might inhibit pro¬liferation directly, the saturated and monounsaturated NAEs, such as PEA, which are usually more abundant than AEA, might enhance this effect. Secondly, our present ndings might be exploited pharmaceutically by leading to the development of tumour-suppressing cocktailswhose potency might be greater than that of simple CB" receptor agonists. In fact, there is increasing evidence that substances that activate cannabinoid receptors can also be used as anti-neoplastic drugs in io([34,35]; M. Bifulco and V. Di Marzo, unpublished work). However, this therapeutic application might be limited by the undesired psychotropic side effects expected from these drugs. This limitation might be overcome by the administration of endocannabinoids, which seem to have much lower potential for dependence [36], in combination with a non-psychotropic sub-stance, such as PEA, which would lower the concentrations necessary to observe the tumour-suppressing effect. Finally, the present study improves our knowledge of the molecular mechan¬sims through which PEA acts synergistically with AEA [8]. It is possible that these synergistic effects, at least when they are observed after chronic PEA treatment, may be due in part to modulation of the expression of FAAH and a subsequent increase in the amount of AEA available for cannabinoid receptor activation. However, since PEA is also capable of increasing the anti-proliferative effects ofHU-210, additional molecular mecha-nisms are likely to underlie the synergistic effects of this NAE on cannabinoid-receptor-mediated biological actions. Indeed, we have preliminary data showing that PEA, independently of FAAH, can signicantly enhance acute effects of AEA that are mediated by vanilloid VR" receptors [37] (L. De Petrocellis and V. Di Marzo, unpublished work). Here we have presented data that argue against possible regulatory effects of PEA on CB"}CB# receptor expression, ligand affinity and functional activity. Future studies will aim at nding other molecular targets involved in the biochemical and pharmacological actions of PEA.
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Source: Palmitoylethanolamide inhibits the expression of fatty acid amide hydrolase and enhances the anti-proliferative effect of anandamide in human breast cancer cells
