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Istituto per la Chimica di Molecole di Interesse Biologico (D.M., T.B., V.D.M.), Istituto di Cibernetica (L.D.P.), and Istituto di Biochimica delle Proteine ed Enzimologia (P.O.), Consiglio Nazionale delle Ricerche, 80072 Arco Felice (NA); and Centro di Endocrinologia e Oncologia Sperimentale, Consiglio Nazionale delle Ricerche, and Dipartimento di Biologia e Patologia Cellulare e Molecolare, Università di Napoli Federico II (C.L., M.B.), 80131 Naples, Italy
Address all correspondence and requests for reprints to: Dr. Vincenzo Di Marzo, Istituto per la Chimica di Molecole di Interesse Biologico, Consiglio Nazionale delle Ricerche, 80072 Arco Felice (NA), Italy. E-mail: vdm{at}trinc.icmib.na.cnr.it
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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9-tetrahydrocannabinol, one, named CB1,
most abundant in the brain and some peripheral tissues, and the other,
named CB2, almost exclusively expressed in immune cells, have been
characterized to date (see Ref. 1 for review). These findings together
with the discovery of two endogenous ligands for these receptors,
N-arachidonoyl-ethanolamine (anandamide) (2) and
2-arachidonoylglycerol (2-AG) (3, 4), named endocannabinoids (ECs), led
to the suggestion of the existence in mammals of an endogenous
cannabinoid system. The metabolism and pharmacological properties of
the ECs have been thoroughly investigated (see Refs. 5, 6, 7, 8, 9, 10 for recent
reviews), and yet the physiological role of these metabolites is still
a matter for speculation. Anandamide and/or 2-AG were suggested to
participate in several physiopathological situations, ranging from the
modulation of neuronal (6) and immune cell (7) function to
neuroprotection (8), control of cardiovascular and endocrine functions
(9, 10), and antinociception (11, 12). We recently reported that
anandamide arrests selectively the proliferation of human breast cancer
cells (HBCCs) by inhibiting DNA synthesis and the
G1/S transition of the cell cycle (13). This
effect is exerted through the activation of as yet unidentified
cannabinoid-binding sites and via the down-modulation of the expression
of the long form of the receptor (PRLr) of PRL, one of the endogenous
hormones necessary to these cells to proliferate and differentiate
(14). We also reported that anandamide together with its putative
phospholipid biosynthetic precursor (5, 6, 15) and the amidohydrolase
responsible for anandamide degradation (for a review, see Ref. 16) are
present in HBCCs. Cis-9-octadecenoamide, a bioactive
compound that inhibits anandamide hydrolysis (5), also exerts a weak
antimitogenic action, probably by raising the levels of endogenous
anandamide in HBCCs (17). These data suggest that anandamide may
function as a local down-modulator of HBCC proliferation. The present
study was aimed at providing answers to questions raised by our
previous investigations. Are ECs also capable of inhibiting the
mitogenic action induced by other growth factors? Is the proliferation
of other PRL-responsive cancer cells also inhibited by these endogenous
mediators? Is the antiproliferative effect of ECs mediated by the CB1
or the CB2 subtype of cannabinoid receptors? We have investigated
whether and through what mechanism anandamide and 2-AG also inhibit the
nerve growth factor (NGF)-induced proliferation of HBCCs and the
PRL-induced proliferation of human prostate cancer cells. |
Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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RT-PCR amplification of CB1/CB2 messenger RNA (mRNA)
Total RNA was prepared from MCF-7, T-47D, and DU-145 cells by
the acid guanidium thiocyanate-phenol-chloroform method. Complementary
DNA (cDNA) synthesis was performed in a 20-µl reaction mixture
containing 75 mM KCl, 3 mM
MgCl2, 10 mM dithiothreitol, 1
mM deoxy-NTPs, 50 mM Tris-HCl (pH 8.3), 5 µg
total RNA, 20 U ribonuclease inhibitor (Roche Molecular Biochemicals, Indianapolis, IN), 0.125
A260 units hexanucleotide mixture (Roche Molecular Biochemicals) for random priming, and 200 U Moloney
murine leukemia virus reverse transcriptase (Superscript, Life Technologies, Inc., Gaithersburg, MD). The cDNA reaction mixture
was incubated at room temperature for 10 min and at 37 C for 90 min,
and the reaction stopped by heating at 98 C for 5 min followed by
lowering the temperature to 4 C. RT-PCR amplification was performed
using 0.5–3 µl cDNA reaction mixture and 2 U Thermus
icelandicus DNA polymerase (Red-Hot, Advanced Biotechnologies,
Columbia, MD) in a 25-µl PCR reaction mixture containing 25
mM Tris-[hydroxymethyl]-methyl-propane-sulfonic
acid, sodium salt buffer (pH 9.3), 50 mM KCl, 1.5
mM MgCl2, 1
mM ß-mercaptoethanol, 12% dimethylsulfoxide,
250 µM of each deoxy-NTP, and 0.5
µM each of 5'- and 3'-primers. Reactions were
performed in a PE Gene Amp PCR System 9600 thermocycler
(Perkin-Elmer Corp., Norwalk, CT). The amplification
protocol consisted of an initial denaturation of 5 min at 95 C,
followed by 25–40 cycles of 1 min at 95 C, 1 min at 55 C, and 2 min at
75 C. A final cycle of 10 min was carried out at 75 C. The primers used
were: CB1 sense primer, 5'-CGC AAA GAT AGC CGC AAC GTG T-3'; CB1
antisense primer, 5'-CAG ATT GCA GTT TCT CGC AGT T-3'; CB2 sense
primer, 5'-TTT CCC ACT GAT CCC CAA TG-3'; CB2 antisense primer, 5'-AGT
TGA TGA GGC ACA GCA TG-3'; ß2-microglobulin
sense primer, 5'-CCA GCA GAG AAT GGA AAG TC-3'; and
ß2-microglobulin antisense primer, 5'-GAT
GCT GCT TAC ATG TCT CG-3'. The expected sizes of the amplicons were 244
bp for CB1, 337 bp for CB2, and 268 bp for
ß2-microglobulin. The
ß2-microglobulin housekeeping gene expression
was used to evaluate any variation in the RNA content and cDNA
synthesis in the different preparations. Furthermore, the PCR primers
for ß2-microglobulin were selected on the basis
of the sequence of the ß2-microglobulin gene
(NCBI accession no. M17987) by including the intron 402-1017. These
primers, in the presence of contaminant DNA, would have generated an
amplicon of the expected size of 886 bp. Ten microliters of the PCR
products were analyzed by electrophoresis on 2% agarose gels (MS
agarose, Roche Molecular Biochemicals) in 1 x
Tris-acetate-EDTA buffer at 4 V/cm for 4 h. Ethidium
bromide (0.1 µg/ml) was included in both the gel and the
electrophoresis buffer, and the PCR products were detected by exposure
under UV light. No PCR product was detected in the absence of cDNA,
primers, or Red-Hot DNA polymerase.
Binding assays
Binding assays in MCF-7, T-47D, and DU-145 cells were carried
out using the filtration procedure reported previously (13), and
membranes were prepared as therein described, except for the absence of
phenylmethylsulfonylfluoride from the binding buffer. The binding of
increasing concentrations (100–10000 pM) of
[3H]SR141716A to aliquots (0.4 mg total
proteins) of these membranes and the displacement of a fixed
concentration (300 pM) of
[3H]SR141716A by increasing concentrations
(0.025, 0.1, 0.5, 1.0, and 5.0 µM) of anandamide
or arvanil were measured in equilibrium assays. SR141716A (10
µM) was used to determine nonspecific binding. Receptor
binding results were analyzed with GraphPad software (GraphPad Software, Inc., San Diego, CA). Scatchard curves for the binding
of [3H]SR141716A were used to calculate the
binding capacity (Bmax) and
Kd for this ligand using nonlinear regression,
and one- and two-site analyses were compared to determine better
fit values (r2 = 0.88 for one-site binding).
Displacement curves (calculated by means of Pharm/PCS software
[Microcomputer Specialists (MCS), Philadelphia, PA)] were used to
calculate the Ki values for anandamide by
inserting the corresponding IC50 values from the
best-fitting curves into the Cheng-Prusoff equation.
Cell proliferation assays
Cell proliferation assays were carried out according to the
method previously described (13) in 6-well dishes containing
subconfluent cells (at a density of about 50,000 cells/well). With
MCF-7 and T-47D cells test substances were introduced 3 h after
cell seeding and then daily at each change of medium. Cells were
treated with trypsin and counted by a hemocytometer 4 days after the
addition of test substances. No significant decrease in cell viability
(as assessed by trypan blue) was observed with up to 100
µM anandamide. With DU-145 cells, we used a previously
described procedure (18). Substances were added 3 h after cell
seeding (50,000 cells/well). PRL (1 mIU/ml) or vehicle was then added
after 24 h with the change of medium, in the presence of the test
substances or vehicle. After 72 h, cells were treated with trypsin
and counted by a hemocytometer. To study the effect of NGF on MCF-7
cell proliferation, we used the previously described procedure (19).
Twenty-four hours after cell seeding (50,000 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 ß-NGF (100
ng/ml) plus test substances or vehicle and treated with trypsin, then
counted after 48 h. Means were compared by means of unpaired
Student’s t test, using P < 0.05 as the
threshold for statistical significance.
Western immunoblotting
Cells in 100-mm petri dishes were treated with test substances
under the same conditions those described above for cell proliferation
assays. Cells were then washed twice with 137 mM NaCl, 3
mM KCl, 12 mM
Na2HPO4, and 2
mM KH2PO4 (pH
7.4) and lysed with a lysis buffer consisting of 50 mM
Tris-HCl (pH 7.4), 1 mM EDTA, 150 mM NaCl, 1
mM Na3VO4, 1
mM NaF, 1% Nonidet P-40, 0.25% sodium deoxycholate, 1
mM phenylmethylsulfonylfluoride, 1% Triton X-100 and 1
µg/ml each of aprotinin, leupeptin, and pepstatin A. Lysates were
loaded onto gels containing, respectively, 10% and 7.5%
polyacrylamide for blotting of PRLr and CB1 or Trk, respectively.
Proteins were transferred to nitrocellulose membranes, which were then
incubated first for 1 h at room temperature with the first
antibody, i.e. either antihuman PRL receptor monoclonal
antibody (U5, purchased from Affinity BioReagents, Inc.,
Golden, CO; 1:1000), antimouse Trk monoclonal antibody (B-3,
Santa Cruz Biotechnology, Inc., Santa Cruz, CA; 1:500), or
CB1 polyclonal antibody (Cayman, Ann Arbor, MI; 1:800) and then with
the appropriate horseradish peroxidase-labeled second antibody
conjugates (1:5000; Bio-Rad Laboratories, Inc., Hercules,
CA). Bands were visualized by the enhanced chemiluminescence technique
(Bio-Rad Laboratories, Inc.). The anti-Trk and CB1
antibodies cross-react with human Trk and CB1.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The effects of anandamide, the synthetic cannabinoid HU-210, the
metabolically stable anandamide analog (R)-methanandamide,
2-AG, and the anandamide congener, palmitoylethanolamide, on human
EFM-19 breast cancer cells and of anandamide on MCF-7 and T-47D cells
have been described previously (13). As shown in Table 2, the IC50 values
for the antiproliferative effect of anandamide on MCF-7 and T-47D
cells, after only a few subculturing passages, were 1.4 and 1.9
µM, respectively. IC50
values as high as 7 µM were instead found for
anandamide when using MCF-7 cells that had undergone between 10–15
subculturing passages. 2-AG was less active than anandamide in T-47D,
but not in MCF-7 (Table 2) or EFM-19 (13), cells. As shown in Fig. 1A, the selective CB2 agonist BML-190 (20)
did not exert any antiproliferative action on HBCCs up to a
10-µM concentration, whereas arvanil, a CB1
agonist (see below), was a potent inhibitor of HBCC proliferation. The
selective CB2 antagonist SR144528 (21) did not reverse the
antiproliferative action of anandamide and arvanil, whereas the CB1
antagonist SR141716A did, in agreement with our previous report (13).
The vanilloid receptor antagonist, capsazepine (not shown), also
counteracted the effect of arvanil, in agreement with the capability of
this compound to activate both CB1 and vanilloid receptors, whose
presence in HBCCs was recently suggested (17A ).
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). The
antiproliferative effects of anandamide and arvanil were attenuated by
SR141716A, but not by SR144528 (Fig. 1C). Capsazepine also counteracted
the effect of arvanil (data not shown).
Endogenous and synthetic cannabimimetics suppress Trk levels in
MCF-7 cells
MCF-7 cells express both low (p75) and high (Trk) affinity NGF
receptors (19). We detected the latter receptor as a single band of
approximately 140 kDa using the Western immunoblotting technique
carried out by means of a monoclonal anti-Trk antibody. As shown in
Fig. 2, anandamide,
(R)-methanandamide, HU-210, and arvanil, but not
palmitoylethanolamide and BML-190, suppress the expression of Trk in
starved, ß-NGF-treated MCF-7 cells. The effect of
anandamide was dose related (IC50 = 0.6
µM) and, like that of HU-210, was
reversed by SR141716A. The effect of arvanil was partly counteracted by
either SR141716A or capsazepine.
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, the two HBCC
lines studied express the CB1 mRNA as detected from the finding of a
RT-PCR-amplified cDNA fragment of the expected size obtained by using
oligonucleotide primers with the same sequence as human CB1 cDNA (see
Ref. 22 for a review). HBCCs also express CB2 mRNA as detected by using
the same technique (23). When using the same amount of total RNA (from
the same preparation), and the same nonsaturating number of
amplification cycles, the CB2 signal was reproducibly much less intense
than the CB1 signal, and, in T-47D cells, hardly visible. Using the
same procedure, probes and amplification conditions, CB2 and CB1
transcripts of the same size were found in human lymphocytes, the
former always in higher amounts than the latter (data not shown). The
presence of CB1-like receptors in MCF-7 cells was confirmed by Western
immunoblotting, which showed the presence of a single band with an
apparent molecular mass of approximately 57 kDa (Fig. 3B). This value
is compatible with the human CB1 receptor molecular mass if
glycosylation of this protein (as described in Ref. 22) is taken into
account. This value is very similar to the molecular mass (
58 kDa)
of CB1-immunoreactive proteins detected in various rat and human cells
that express CB1 receptors (24). We found that the abundance of the CB1
transcript, compared with that of the
ß2-microglobulin transcript, as well as the
intensity of the CB1-immunoreactive band decreased when MCF-7 cells
underwent 10–15 subculturing passages (Fig. 3b and data not
shown).
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shows the Bmax and
Kd values for
[3H]SR141716A binding sites in each HBCC line,
calculated from the corresponding Scatchard plots.
[3H]SR141716A specific binding in these two
cell lines, determined in the presence of 10 µM
SR141716A, depended on the number of subculturing passages and varied
between 10–40% for MCF-7 cells and 15–30% for T-47D cells.
Anandamide Ki values for the displacement of
[3H]SR141716A specific binding were 1.50
± 0.31 and 0.85 ± 0.25 µM (n = 3) for T-47D
and MCF-7 cells, respectively. The Ki
value for arvanil in MCF-7 cells was 0.25 ± 0.10 µM
(n = 3).
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).
(R)-Methanandamide and arvanil were not less potent than
anandamide. The effect of anandamide was reversed by SR141716A, but not
by SR144528, and was not mimicked by BML-190 and palmitoylethanolamide
(Fig. 4, A and B). Both SR141716A and capsazepine, but not SR144528,
counteracted the effect of arvanil (Fig. 4B and data not shown).
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, DU-145 cells express CB1 and, to a lesser
extent, CB2 mRNA, as determined from the finding of RT-PCR-amplified
cDNA fragments. DU-145 cells also contain specific binding sites for
[3H]SR141716A, whose Bmax
and Kd values are shown in Table 3, and an approximately 57-kDa protein detected by
Western immunoblotting (Fig. 3B). Anandamide displaced
[3H]SR141716A from DU-145 cell membranes with a
Ki of 160 ± 19 nM (n =
3).
Anandamide inhibits PRLr expression in DU-145 cells
Using the Western immunoblotting technique carried out with a
specific monoclonal antibody, DU-145 cells were shown to contain
measurable levels of the high molecular mass (100 kDa) form of the
PRLr. Anandamide inhibited the expression of this receptor in a fashion
sensitive to the presence of SR141716A (Fig. 4D).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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100 kDa) form of the PRLr (13). With the
present investigation we wanted to determine whether the antimitogenic
effect of ECs is also observed when cell proliferation is induced by
hormones or growth factors other than PRL. This issue is important
because cancer cell growth in vivo is subject to the control
of several of these mediators. An earlier report had shown that
synthetic cannabinoids do not interfere with estrogen-induced MCF-7
cell proliferation (25). However, recent studies clearly pointed to NGF
as a possible mitogenic agent for HBCCs and prostate cancer cells, both
in vitro and in vivo (19, 26, 27). Therefore, we
decided to study the effects of anandamide and 2-AG on NGF-induced HBCC
proliferation. We used a well established protocol with MCF-7 cells
(19) and observed selectively the effects of drugs on the mitogenic
action induced by NGF. We found that submicromolar concentrations of
anandamide and 2-AG inhibit NGF-induced MCF-7 cell proliferation in a
fashion that was 1) mimicked by metabolically stable cannabimimetic
compounds or CB1, but not CB2, receptor-selective agonists; and 2)
reversed by CB1, but not CB2, receptor antagonists. Of the two types of
NGF-binding sites described to date, the low affinity p75 receptor and
the high affinity tyrosine kinase Trk receptors (see Ref. 28 for a
recent review), only the latter have been implicated in the tumorigenic
activity of NGF. Conversely, coexpression of the p75 receptor with Trk
was recently shown to counteract the Trk-mediated proliferative effect
of NGF (29). We found here that the inhibition by endogenous and
synthetic cannabinoids of NGF-induced MCF-7 cell proliferation was due
to down-regulation of Trk receptors. In fact, using the same assay
conditions and the same drug concentrations as those necessary to
observe the antiproliferative effect, a strong suppression of the
levels of the approximately 140-kDa Trk proteins was found with
anandamide and other cannabimimetic agents. This effect was reversed by
coincubation with a CB1 antagonist. We observed a full correlation
between the effects of drugs (or of their combinations) on NGF-induced
proliferation and their modulation of Trk levels in MCF-7 cells. This
represents the first report of the cannabinoid-induced inhibition of
Trk receptors, a family of proteins that is the target of several
neurotropins (28). In a previous study (13) we suggested that anandamide inhibition of basal HBCC proliferation was due to interaction with selective cannabinoid-binding sites and not to the formation of arachidonic acid, a metabolite known to modulate cancer cell proliferation and differentiation (30). The presence of cannabinoid receptors was suggested by binding assays carried out using a radioligand, [3H]CP55,940, that does not discriminate between CB1 and CB2 receptors (1). Therefore, we provided no molecular information as to which cannabinoid receptor subtype was involved in the EC antiproliferative action. In the present study, binding sites for the selective CB1 antagonist [3H]SR141716A were identified, and anandamide shown to bind to these proteins with an affinity comparable to that reported for other CB1-containing tissues and cells (1). Evidence for the presence of CB1 and, to a lesser extent, CB2 mRNA was provided by the RT-PCR technique. Western immunoblotting confirmed the presence of CB1-like receptors in MCF-7 cells. These data taken together with the lack of agonistic or antagonistic effects described above for selective CB2 agonists and antagonists point to the involvement of CB1-like receptors in EC action on both basal [i.e. induced by endogenous PRL (13)] and NGF-induced HBCC proliferation. In further support of this conclusion we observed that when MCF-7 cells undergo several subculturing passages they express increasingly lower levels of CB1 mRNA, CB1-immunoreactive protein, and [3H]SR141716A specific binding, and become correspondingly less responsive to anandamide. Furthermore, the Ki values for anandamide displacement of [3H]SR141716A from HBCC membranes correlate well with the IC50 values for its inhibition of cell proliferation. Although more potent in EFM-19 cells (13), 2-AG was equipotent to or less potent than anandamide in MCF-7 and T-47D cells, respectively. This fact may be due to different contents of anandamide or 2-AG hydrolases in HBCC lines. Indeed, we showed that 1) such enzymes are present in HBCCs and limit the action of endocannabinoids (13, 17); and 2) ECs are equipotent to or, more frequently, less potent than their metabolically stable or nonhydrolyzable analogs in all HBCC lines studied (this study and data not shown).
A corollary to our previous finding of an involvement of PRLr in anandamide antimitogenic actions on HBCCs is that ECs may inhibit the proliferation of other PRL-responsive cells that express functional cannabinoid receptors. In keeping with this hypothesis, here we have shown that when the proliferation of the prostate DU-145 cell line is induced by exogenous PRL, anandamide and 2-AG are again capable of exerting a potent antiproliferative action, and that anandamide inhibits the expression of PRLr in these cells. Little or no effect was observed on basal DU-145 cell proliferation, which probably depends on other factors present in the cell culture medium. As in the case of HBCCs, this antimitogenic effect was not due to arachidonic acid formed from anandamide or 2-AG hydrolysis, as metabolically stable anandamide analogs were equipotent or more potent than the two ECs. In fact, the effects of anandamide and 2-AG on DU-145 cell proliferation were due to the interaction with CB1 receptors, as we found in these cells specific [3H]SR141716A binding displaceable by anandamide with a Ki consistent with the IC50 for its antiproliferative effect. A CB1 mRNA transcript and an abundant CB1-immunoreactive protein were also identified. Moreover, as shown above for HBCCs, selective CB1 and CB2 agonists and antagonists were either very effective or ineffective by either mimicking or counteracting, respectively, the anandamide antiproliferative effect on DU-145 cells. Given the slight (20–30%) stimulatory effect of exogenous PRL on DU-145 cell proliferation in vitro observed by us and others (18), further studies are necessary before suggesting that the inhibitory effects of ECs on these cells may have a biological relevance in vivo. However, our data with DU-145 cells confirm that both endogenous and synthetic cannabinoids are potent negative effectors of PRL receptor levels in tumor cells that coexpress this protein and CB1 cannabinoid receptors.
In conclusion, our data suggest that ECs may exert a general inhibition of the mitogenic action of PRL and NGF by interfering with the expression of their respective receptors. PRLr and Trk have been shown to be involved in the onset and establishment of breast and prostate carcinomas that seem to be facilitated by endogenous PRL and NGF (18, 19, 26, 27, 31, 32). However, our findings may have important implications not only for the possible development of new, therapeutically useful, antitumor drugs, but also for understanding of the general physiological role played by ECs in relation to PRL and NGF biological responses. Although PRL is an important mediator in the regulation of reproduction and lactogenesis as well as the immune response (reviewed in Refs. 33, 34), NGF has been implicated not only in neuronal differentiation (28), but also in neurogenic inflammation, chronic pain, and the neuroendocrine/immune axis (reviewed in Ref. 35). It is possible that down-regulation of PRLr and Trk expression by ECs is also found in other tissues and cells that respond to either of the two growth factors (e.g. pituitary lactotroph cells, epithelial mammary duct cells, reproductive organs, lymphocytes, mastocytes, and undifferentiated nerve cells). This would support the hypothesis of a modulatory role of these lipids in situations as diverse as reproduction and lactogenesis, neuronal development, inflammation, antinociception, and the immune response (5, 6, 7, 8, 9, 10, 11, 12), which is suggested, inter alia, by the occurrence of ECs in the pituitary (36), immune cells (37, 38), and milk (39). It is also possible that the regulatory mechanisms described here are restricted to cancer cells. Therefore, further studies are required to assess in what other cell types and under what conditions the inhibitory effect of ECs on PRLr and Trk levels can be observed. Also, an investigation of the possible mechanism through which activation of CB1 receptors leads to down-regulation of PRLr and Trk should be carried out. It is possible that a typical intracellular event triggered by CB1 receptor activation, adenylyl cyclase inhibition (1), underlies the suppression of Trk, as trk gene expression was recently shown to be up-regulated by cAMP in human monocytes, rat brain, and pancreatic ß-cells (40, 41, 42).
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Acknowledgments |
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Footnotes |
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Received July 21, 1999.
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H. Li, T. J. Ahonen, K. Alanen, J. Xie, M. J. LeBaron, T. G. Pretlow, E. L. Ealley, Y. Zhang, M. Nurmi, B. Singh, et al. Activation of Signal Transducer and Activator of Transcription 5 in Human Prostate Cancer Is Associated with High Histological Grade Cancer Res., July 15, 2004; 64(14): 4774 - 4782. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Maccarrone, M. DeFelici, F.G. Klinger, N. Battista, F. Fezza, E. Dainese, G. Siracusa, and A. Finazzi-Agro Mouse blastocysts release a lipid which activates anandamide hydrolase in intact uterus Mol. Hum. Reprod., April 1, 2004; 10(4): 215 - 221. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. L. Lannon, M. J. Martin, C. E. Tognon, W. Jin, S.-J. Kim, and P. H. B. Sorensen A Highly Conserved NTRK3 C-terminal Sequence in the ETV6-NTRK3 Oncoprotein Binds the Phosphotyrosine Binding Domain of Insulin Receptor Substrate-1: AN ESSENTIAL INTERACTION FOR TRANSFORMATION J. Biol. Chem., February 20, 2004; 279(8): 6225 - 6234. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. W. Klein, C. Newton, K. Larsen, L. Lu, I. Perkins, L. Nong, and H. Friedman The cannabinoid system and immune modulation J. Leukoc. Biol., October 1, 2003; 74(4): 486 - 496. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Maccarrone, S. Cecconi, G. Rossi, N. Battista, R. Pauselli, and A. Finazzi-Agro Anandamide Activity and Degradation Are Regulated by Early Postnatal Aging and Follicle-Stimulating Hormone in Mouse Sertoli Cells Endocrinology, January 1, 2003; 144(1): 20 - 28. [Abstract] [Full Text] [PDF]
|
|
|
|
|
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T. J. Ahonen, P. L. Harkonen, H. Rui, and M. T. Nevalainen PRL Signal Transduction in the Epithelial Compartment of Rat Prostate Maintained as Long-Term Organ Cultures in Vitro Endocrinology, January 1, 2002; 143(1): 228 - 238. [Abstract] [Full Text] [PDF]
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10%) inhibition of basal DU-145 cell
proliferation only at high (
5 µM) doses. The effects of
only 1 µM BML-190 and palmitoylethanolamide are shown,
but these compounds were also tested at a higher (10 µM)
concentration, and no significant effect was found. SD bars
(never higher than 10–15% of the mean) are not shown for the sake of
clarity. B, Effects of CB1 and CB2 selective antagonists on anandamide
and arvanil inhibition of PRL-induced DU-145 cell proliferation. C,
Identification of CB1 and CB2 transcripts in DU-145 cell RNA by RT-PCR.
For details, see Fig. 3A
,
P < 0.05;