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Vol. 299, Issue 3, 951-959, December 2001
Departments of Pharmacology and Clinical Neuroscience (S.O.P.J., T.W., C.J.F.) and Odontology (S.O.P.J.), Umeå University, Umeå, Sweden
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Abstract |
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 Top
 Abstract
 Introduction
Experimental Procedures
Results
Discussion
References
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The effects of the endocannabinoids anandamide (AEA) and
2-arachidonoylglycerol (2-AG) upon rat C6 glioma cell proliferation were examined and compared with a series of synthetic cannabinoids and
related compounds. Cells were treated with the compounds each day and
cell proliferation was monitored for up to 5 days of exposure. AEA
time- and concentration-dependently inhibited C6 cell proliferation. After 4 days of treatment, AEA and 2-AG inhibited C6 cell proliferation with similar potencies (IC50 values of 1.6 and 1.8 µM,
respectively), whereas palmitoylethanolamide showed no significant
antiproliferative effects at concentrations up to 10 µM. The
antiproliferative effects of both AEA and 2-AG were blocked completely
by a combination of antagonists at cannabinoid receptors (SR141716A and
SR144528 or AM251 and AM630) and vanilloid receptors (capsazepine) as
well as by 
-tocopherol (0.1 and 10 µM), and reduced by calpeptin
(10 µM) and fumonisin B1 (10 µM), but not by
L-cycloserine (1 and 100 µM). CP 55,940, JW015, olvanil,
and arachidonoyl-serotonin were all found to affect C6 glioma cell
proliferation (IC50 values of 5.6, 3.2, 5.5, and 1.6 µM,
respectively), but the inhibition could not be blocked by cannabinoid + vanilloid receptor antagonists. It is concluded that the
antiproliferative effects of the endocannabinoids upon C6 cells are
brought about by a mechanism involving combined activation of both
vanilloid receptors and to a lesser extent cannabinoid receptors, and
leading to oxidative stress and calpain activation. However, there is
at present no obvious universal mechanism whereby plant-derived,
synthetic, and endogenous cannabinoids affect cell viability and proliferation.
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Introduction |
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Top
Abstract
Introduction
Experimental Procedures
Results
Discussion
References
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The
antimitotic effects of the cannabinoid
9-tetrahydrocannabinol
(9-THC), the principal psychoactive component
of hashish and marijuana, have been known since the 1970s (Munson et
al., 1975
). More recent studies have shown that
9-THC and/or the synthetic cannabinoid (CB)
receptor agonist WIN 55,212-2 induces apoptosis in various mouse, rat,
and human cells (Sánchez et al., 1998; Zhu et al., 1998; Ruiz et
al., 1999; Jacobsson et al., 2000; Sarker et al., 2000) and have been
shown to induce regression of malignant gliomas in Wistar rats and mice
(Galve-Roperh et al., 2000), a result also seen with the
CB2 receptor-selective agonist JWH-133
(Sánchez et al., 2001). In rat C6 glioma cells, the effect of
9-THC upon cell viability, which takes ~5
days to become apparent (Sánchez et al., 1998), is brought about
by a pathway involving CB receptors followed by sustained ceramide
accumulation (Galve-Roperh et al., 2000). Blockade of both
CB1 and CB2 receptors by
the selective antagonists SR141716A and SR144528 is required to
protect the cells against the deleterious effects of
9-THC upon cell viability (Galve-Roperh et
al., 2000). In contrast, in human PC-3 prostate cells, the apoptotic
effects of 9-THC are not mimicked by WIN
55,212-2 and are not prevented by pertussis toxin treatment of the
cells, suggestive of a CB receptor-independent mechanism (Ruiz et al.,
1999).
The endogenous cannabinoid ("endocannabinoid")
arachidonoylethanolamide (anandamide, AEA) is a partial agonist with
similar affinity at CB1 and
CB2 receptors but lower efficacy at the
CB2 receptor. In addition, AEA can activate
vanilloid receptors (Zygmunt et al., 1999; Smart et al., 2000). AEA has
been shown to induce apoptosis in several cell types, but the molecular
mechanism behind this action appears to be rather different from that
of 9-THC. Thus, the proapoptotic effects of
AEA on human CHP-100 neuroblastoma and U-937 lymphoma cells (Maccarrone
et al., 2000) were suggested to be mediated via vanilloid receptors.
These authors proposed that AEA induces a rise in intracellular
calcium, mitochondrial uncoupling, and cytochrome c release,
and activation of the caspase cascade. Furthermore, Sarker et al.
(2000) found that AEA induces apoptosis in rat PC-12 pheochromocytoma
cells by increasing the superoxide anion formation and caspase-3
activation. AEA has also been shown to inhibit the proliferation of
human breast and prostate cancer cell lines in vitro (De Petrocellis et
al., 1998) secondary to a CB1 receptor-mediated
inhibition of prolactin action, at the level of the prolactin receptor.
The different mechanisms proposed for the effects of, on the one hand plant-derived and synthetic cannabinoids, and on the other hand AEA, upon cell survival present a somewhat confusing picture. In the present study, we have investigated, in the same cells and under the same conditions, the receptors involved in the antiproliferative effects of synthetic and endogenous cannabinoids, including compounds that are selective for the CB1 or CB2 receptors. In addition, we have investigated whether the antiproliferative effects of AEA are mimicked by the endocannabinoid 2-arachidonoylglycerol (2-AG) and the metabolically stable AEA analog R-(+)-methanandamide.
Rat glioma C6 cells are ideal for this study because they express both
functional cannabinoid and vanilloid receptors (Sánchez et al.,
1997; Bíró et al., 1998) and respond to AEA (Maccarrone et al., 2000). These cells, however, do not in our hands show a
mitogenic response to prolactin (T. Wallin and S.O.P Jacobsson, unpublished data), thereby precluding investigation into effects of AEA
upon proliferation stimulated by this hormone. We reported previously
that single administrations of AEA (
10 µM) failed to affect C6
glioma cell viability (Jacobsson et al., 2000), although this result
presumably reflected the rapid removal of AEA from the culture medium.
To reduce the cellular metabolism of AEA, we have used a sensitive
assay to minimize the number of cells required per well, and in
addition treated the cells daily with AEA (or the other test
compounds). Because the effects of 9-THC upon
cell viability are more apparent at low culturing serum contents
(Jacobsson et al., 2000), we have elected to investigate the effects of
the compounds upon cell proliferation at 1% fetal bovine serum.
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Experimental Procedures |
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Top
Abstract
Introduction
Experimental Procedures
Results
Discussion
References
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Materials.
AEA, quinacrine (mepacrine),
-tocopherol, fumonisin B1, and
L-cycloserine were all purchased from Sigma Chemical Co.
(St. Louis, MO). Olvanil, palmitoyletanolamide (PEA),
arachidonoylserotonin (AA-5-HT), and 2-AG were obtained from the Cayman
Chemical Co. (Ann Arbor, MI). Capsaicin, capsazepine, CP 55,940 [(
)-cis-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-trans-4-(3-hydroxypropyl) cyclohexanol], JWH 015 [(2-methyl-1-propyl-1H-indol-3-yl)-1-naphthalenylmethanone], R-(+)-methanandamide
[R-(all-Z)]-N-(2-hydroxy-1-methylethyl)-5,8,11,14-eicosatetraenamide; MeAEA], WIN 55,212-2 [(R)-(+)-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl) pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl]-1 naphthalenylmethanone mesylate]; AM 630 [6-lodo-2-methyl-1-[2-(4-morpholinyl)ethyl]-1H-indol-3-yl](4-methoxyphenyl) methanone]; AM 251 [N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide], and ACEA [arachidonyl-2'-chloroethylamide/(all
Z)-N-(2-cycloethyl)-5,8,11,14-eicosatetraenamide] were
obtained from Tocris Cookson (Bristol, UK). CyQUANT cell proliferation assay kits and calcein-AM were bought from Molecular Probes (Eugene, OR). SR141716A
(N-piperidino-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-3-pyrazole-carboxamide) and SR144528
(N-[(1S)-endo-1,3,3-trimethyl bicyclo
[2.2.1]
heptan-2-yl]-5-(4-chloro-3-methylphenyl)-1-(4-methylbenzyl)-pyrazole-3-carboxamide) were kind gifts from Sanofi Recherche (Montpellier, France).
Arachidonyl-ethanolamide-[1-3H]
([3H]AEA) (specific activity 60 Ci
mmol
1) was purchased from American Radiolabeled
Chemicals (St. Louis, MO). Tissue culture media and all supplements
were obtained from Invitrogen (Sweden). Calpeptin was obtained
from Calbiochem (San Diego, CA).
Cell Cultures.
Rat C6 glioma cells were obtained from
American Type Culture Collection (Manassas, VA) and used over a passage
range of 41 to 54. Cells were cultured in 75-cm2
culturing flasks in Ham's F10 medium supplemented with 25 mM HEPES
buffer, L-glutamine, 10% fetal bovine serum (FBS), and 100 units ml1 penicillin + 100 µg
ml1 streptomycin (PEST). The cells were
maintained at 37°C in a humidified atmosphere with 5%
CO2 in air and the medium was changed every 3 to
4 days.
C6 Cell Proliferation Assay.
C6 glioma cells were seeded in
96-well flat bottom microplates at a density of 2500 cells
well1 in Ham's F10 medium, supplemented with
penicillin-streptomycin. In all experiments (except in the initial AEA
experiments, where serum-free conditions were also tested) the culture
medium was also supplemented with 1% FBS. Substances to be tested were
introduced 6 h after seeding. The total assay volume was 200 µl.
Test substances were serial diluted in appropriate solvents. AEA,
olvanil, AA-5-HT, R-(+)-methanandamide, PEA, ACEA, JWH015,
arachidonic acid, capsaicin, and capsazepine were dissolved in ethanol.
2-AG was dissolved in acetonitrile, whereas SR141716A, SR144528, AM251,
AM630, CP 55,940, and WIN 55,212-2 were dissolved in dimethyl
sulfoxide. The total solvent concentration was kept constant at
0.5% in all assays. Prolactin was dissolved in phosphate-buffered
saline (pH 7.2). Test substances were administered daily, by replacing
100 µl of medium with fresh medium containing test substances at the indicated concentration. After incubation for the desired time, the
cell density was determined, using the CyQUANT cell proliferation assay
kit, which quantifies the total amount of nucleic acid in the sample.
The medium was removed by gently inverting the microplate, and blotting
it onto a paper towel. The plate was then frozen (80°C). At the day
of analysis, plates were thawed over a period of 30 min at room
temperature. Fluorescence reagents were then added. After 5-min
incubation at room temperature, the sample fluorescence was measured
(excitation/emission: 495/520 nm) in a FLUOstar Galaxy microplate
reader (BMG Labtechnologies GmbH, Germany). Sample fluorescence
values were converted into cell numbers from a C6 cell reference
standard curve.
Statistical Analyses. The concentration-dependent effects of the test compounds upon C6 cell proliferation were analyzed for statistical significant differences using one-way analysis of variance (ANOVA) with post hoc Bonferroni's, or where appropriate Dunnett's, multiple comparisons test between each concentration and the corresponding control data. The time- and concentration-dependent effects of AEA upon C6 cell proliferation were analyzed using two-way ANOVA. Apparent IC50 values were determined by fitting the data to a sigmoidal dose-response equation by using nonlinear regression. All statistical analyses were undertaken using GraphPad Prism 3.00 (GraphPad Software, San Diego, CA). All data are presented as the means ± S.E.M. of at least three separate experiments.
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Results |
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Top
Abstract
Introduction
Experimental Procedures
Results
Discussion
References
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Inhibition of C6 Cell Proliferation by Synthetic and Endogenous
Cannabinoids.
In initial experiments, AEA was administered daily
and the cell growth was followed during 5 days in either serum-free
(Fig. 1A) or 1% serum (Fig. 1B)
conditions. When cultured in the presence of 1% FBS, 10 µM AEA
decreased the cell density from 34 ± 1.3 × 103 cells/well in the control cultures to
9.4 ± 1.0 × 103 cells/well
(p < 0.001; n = 4). Even in serum-free
conditions, AEA showed antiproliferative effects (Fig. 1A), but the low
growth rate and the general status of the C6 cells when deprived of
serum during 5 days ruled out further experiments in this extreme
condition. In consequence, the remaining experiments were undertaken
using 1% FBS in the culture medium.
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-tocopherol (0.1 and 10 µM), and greatly reduced with the
cell-permeable calpain inhibitor calpeptin (10 µM) (Table
1). In contrast, the phospholipase
A2 inhibitor quinacrine (1 µM) did not affect
the antiproliferative response to AEA (Table 1). The effects of two
inhibitors of cellular ceramide biosynthesis,
L-cycloserine and fumonisin
B1, on the antiproliferative effect of the
endocannabinoids are shown in Fig. 3.
L-Cycloserine had no effect on the cellular
response to endocannabinoids, although a concentration of 100 µM
L-cycloserine caused a significant decrease in
cell number per se (Fig. 3). Fumonisin B1
significantly inhibited the endocannabinoid effect upon C6 cells at a
concentration of 10 µM, whereas 0.1 µM was without effect (Fig. 3).
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), showed no significant inhibitory effect upon C6 cell
proliferation at concentrations up to 10 µM (Fig. 2A). Further
experiments have, however, found a 40% reduction in cell viability at
a PEA concentration of 30 µM (K.-O. Jonsson, S.O.P. Jacobsson, and
C. J. Fowler, unpublished data). The metabolically stable analog
of AEA, meAEA, showed no effect on cell proliferation at concentrations
up to 3 µM, but a small decrease was obtained at 10 µM (Fig. 2B;
n = 4). In a subsequent experiment (see below), 1 µM
meAEA was without effect, whereas 10 µM showed a greater degree of
inhibition (56%).
Four synthetic CB receptor agonists were investigated. The nonselective
agonists CP 55,940 and WIN 55,212-2 both inhibited cell proliferation,
although the latter appeared to show a "threshold"-like profile
with little or no inhibition 3 µM and maximal inhibition at 10 µM
(Fig. 2C). CP 55,940 and the CB2
receptor-selective agonist JWH015 (Showalter et al., 1996) were roughly
equipotent inhibitors of cell proliferation (IC50
values of 5.6 and 3.2 µM, respectively; Fig. 2, B and C;
n = 4). The CB1
receptor-selective agonist ACEA (Hillard et al., 1999) was also tested,
but inconsistent results were found in different experimental series,
precluding conclusions to be made as to its antiproliferative effects
(data not shown).
Two vanilloid receptor agonists were investigated. Olvanil produced an
inhibition of C6 cell proliferation, with an IC50
value of 5.5 µM (n = 4), whereas capsaicin had modest
effects (Fig. 2D). AA-5-HT, a synthetic inhibitor of fatty acid amide
hydrolase reported as inactive at CB1 receptors
in vivo (Bisogno et al., 1998), inhibited the cell proliferation with
an IC50 value of 1.6 µM (Fig. 2B;
n = 7), a potency also seen with arachidonic acid (Fig.
2A). Representative photomicrographs of the effects of 3 µM AEA and 3 µM olvanil upon C6 cell morphology after 4 days of incubation are
presented in Fig. 4, A-C.
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Effects of Cannabinoid and Vanilloid Receptor Antagonists upon
Antiproliferative Effects of Synthetic and Endogenous
Cannabinoids.
SR141716A (1 µM), a CB receptor antagonist with
marked selectivity for CB1 receptors
(Rinaldi-Carmona et al., 1994), significantly attenuated the
antiproliferative effect of 3 µM 2-AG (Fig.
5), but was less effective in blocking
the 3 µM AEA-induced decrease in cell number. Essentially the same
results were obtained when SR144528 (1 µM), a CB receptor antagonist
with a 700-fold higher affinity for the CB2
receptor than the CB1 receptor (Rinaldi-Carmona et al., 1998), was used (Fig. 5). However, both the 2-AG- and AEA-mediated effects were significantly reduced by incubating the
cultures with a combination of 1 µM SR141716A and 1 µM SR144528 (Fig. 5). In contrast, the cannabinoid receptor antagonists had no
effect upon the decrease in cell number produced by 3 µM olvanil (Fig. 5). AA-5-HT (3 µM) produced a significant (p < 0.001) reduction in cell number per se, but this reduction was not
affected by the cannabinoid antagonists. Thus, mean (±S.E.M.,
n = 3) densities (× 103
well1) for cells treated with 3 µM AA-5-HT in
the absence and presence of SR141716A, SR144528, and SR141716A + SR144528 were 20.1 ± 0.40, 20.6 ± 0.38, 21.3 ± 0.61, and 20.6 ± 0.67, respectively.
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; Lan et al., 1999; Ross et al.,
1999). The antiproliferative effect of 3 µM AEA was not affected by
AM630, but was completely blocked by AM251 (and by capsazepine) (Fig.
6). In contrast, the antiproliferative effects of 3 µM CP 55,940, JWH015, and AA-5-HT were not inhibited by
AM251, AM630, and capsazepine either alone or in combination; indeed,
the combination appeared to increase the antiproliferative effects of
these compounds (Fig. 6).
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Combination of Effects of meAEA and Capsaicin upon C6 Glioma Cell
Proliferation.
The effects of the combination of meAEA (1 and 10 µM) and capsaicin (1 and 10 µM) upon the proliferation of C6 cells
is shown in Table 3. Essentially additive
effects that could not be blocked by the combination of AM251, AM630,
and capsazepine were found for the two compounds.
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Discussion |
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Top
Abstract
Introduction
Experimental Procedures
Results
Discussion
References
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In the present study, the effects of endogenous and synthetic
cannibinoids upon the proliferation of C6 glioma cells were investigated to resolve some of the conflicting data in the literature concerning the receptor systems involved in the effects of these compounds. The choice of a glioma cell line was prompted by the finding
that cannabinoids reduce their cell proliferation in vivo (Galve-Roperh
et al., 2000). Indeed, the recent finding by this group of a high
frequency of CB2 receptor expression in human astrocytomas, and that the expression correlated with tumor malignancy (Sánchez et al., 2001), raises the possibility that modulation of
cannabinoid receptors in vivo may provide a possible avenue for
treatment of patients with glioma and motivate per se further experimentation in glioma cells. We have previously shown that the
effects of 9-THC upon C6 glioma cell viability
are highly dependent upon the assay serum concentration, with no
deleterious effects being seen after a 6-day treatment with 1 µM
9-THC at a FBS concentration of 10%. In
contrast, in serum-free medium, sensitivity to
9-THC and cannabidiol was seen (Jacobsson et
al., 2000). C6 glioma cells, however, proliferate poorly in serum-free
medium, and the FBS concentration in the present study was chosen as a
balance between sensitivity to cannabinoids and rate of cell proliferation.
Robust effects of AEA and 2-AG upon cell proliferation were seen after
their repeated administration over 4 days. The protective effects of
-tocopherol and calpeptin found in the present study implicate
oxidative stress and excessive intracellular calcium to be involved in
the antiproliferative effects of AEA and 2-AG toward C6 cells. These
results are perhaps not surprising given that 1) vanilloid receptors
are directly coupled to cation channels, with a high permeability to
calcium (Szallasi and Blumberg, 1999); and 2) calpain overexpression
and apoptosis is a commonly used pathway for toxic stimuli in C6 glioma
cells (Ray et al., 1999). Cannabinoid receptor-mediated activation of
the ceramide pathway is well characterized (for review, see
Guzmán et al., 2001), and the finding that fumonisin
B1, an inhibitor of ceramide synthase (Wang et
al., 1991), reduces the antiproliferative effects of AEA and 2-AG is
consistent with the hypothesis described by others (Galve-Roperh et
al., 2000) that the proapoptotic actions of ceramide are involved in
cannabinoid actions in C6 glioma cells. Although the lack of effect in
the present study of the serine palmitoyltransferase inhibitor
L-cycloserine is a surprising result, it is not
unreasonable to conclude that the antiproliferative effects of AEA and
2-AG in our C6 glioma cells require a combination of cannabinoid- and vanilloid-receptor-mediated mechanisms, including influx of calcium and
activation of the ceramide pathway. However, this conclusion must be
validated in further experiments.
Stimulation of CB1 receptors results in
arachidonic acid production secondary to phospholipase
A2 activation (see Shivachar et al., 1996, for
data with AEA), and this pathway is responsible for the neurotoxic
effects of 9-THC in cultured hippocampal
neurons (Chan et al., 1998). Although the lack of effect of SR141716A
per se (see below) would argue against such a mechanism being important
for the antiproliferative effects of AEA in C6 glioma cells, we
investigated the phospholipase A2 inhibitor
quinacrine upon the antiproliferation produced by endocannabinoids. No
antagonism of the effects of either AEA or 2-AG was found with either
0.01 or 1 µM quinacrine. Although higher concentrations of quinacrine
are often used in short-term experiments, a concentration of 1 µM is
sufficient to produce a large functional inhibition of phospholipase
A2 in fibroblast cells (Kim et al., 1998). In any
case, higher concentrations of quinacrine were found to produce toxic
effects per se on C6 cell proliferation under the long incubation times
used (data not shown).
It is well established that AEA (and 2-AG) activates vanilloid
receptors (VR, Zygmunt et al., 1999; De Petrocellis et al., 2000; Smart
et al., 2000). We found that the VR antagonist capsazepine significantly blocked the effects of AEA and 2-AG. This result is
consistent with the effects of this compound upon the proapoptotic actions of AEA on suspensions of several cell lines, including C6
glioma cells (Maccarrone et al., 2000) and suggests involvement of
vanilloid receptors. Maccarrone et al. (2000) found that 10 µM
capsazepine reduced the number of apoptotic bodies produced by AEA
treatment of C6 cell suspensions by 57%. In our hands, this
concentration of capsazepine was toxic per se, but intriguingly the
toxicity of the combination of 10 µM capsazepine and AEA (and 2-AG)
was lower than found for either compound per se.
AEA is removed from the medium by cellular uptake followed by fatty
acid amidohydrolase (FAAH)-catalyzed metabolism, raising the
possibility that compounds that block FAAH may increase endogenous AEA
to a level sufficient to cause effects upon cell proliferation. Indeed,
in a recent study, it was demonstrated that the antiproliferative effect of AEA upon breast cancer cells was enhanced by PEA, which not
only competes for FAAH but also produced a down-regulation of this
enzyme (Di Marzo et al., 2001). The FAAH inhibitor AA-5-HT did produce
an antiproliferative effect in the present study, but this effect is
most probably due to a nonspecific cell toxicity rather than an
increased cellular level of AEA because it was not prevented by CB + VR antagonists.
The experiments with CB receptor antagonists were more complex. In the
first experimental series, significant attenuation of the
antiproliferative effect was seen with the combination of the
CB1 receptor antagonist SR141716A and the
CB2 receptor antagonist SR144528, whereas modest
(in the case of 2-AG) or no (in the case of AEA) attenuation was seen
when the compounds were given separately. In the second series of
experiments, the CB2 receptor antagonist AM630
was without effect upon the antiproliferative effects of AEA, whereas
the CB1 receptor antagonist AM251 completely blocked the effect. In both experiments, however, a combination of CB
receptor and VR antagonists completely blocked the antiproliferative effects of AEA. In a recent study, it was reported that although 1 µM
SR141716A did not affect the vanilloid receptor-mediated calcium
mobilization response to AEA and capsaicin in human embryonic kidney
cells transfected with the human VR1 receptor,
concentrations of 2.5 and 5 µM SR141716A antagonized the response (De
Petrocellis et al., 2001). Given that AM251 is structurally related to
SR141716A (Lan et al., 1999), it is possible that the compound affects
the function of vanilloid receptors at the concentration used (0.3 µM), thereby contributing to its antagonism of the antiproliferative effect of AEA. An alternate explanation for the different results with
the CB1 receptor antagonists in the two series of
experiments, which were undertaken 4 months apart, is that they reflect
the difficulties associated with the use of a heterogeneous population of cells where the cannabinoid receptor component of the
antiproliferative effect may vary (Galve-Roperh et al., 2000). However,
were this the case, antagonism of the effects of CP 55,940 by the CB
antagonists would have been expected.
Taken together, these data would suggest that the antiproliferative
effects of AEA and 2-AG toward our C6 cells under the experimental
conditions used here are a consequence of a combination of effects on
both cannabinoid and vanilloid receptors, and that the effect upon
vanilloid receptors predominates. If this result can be extended to the
synthetic cannabinoids, it can be predicted that CP 55,940 and WIN
55,212-2 should not affect cell proliferation to any large extent,
given that these compounds do not activate vanilloid receptors (Zygmunt
et al., 1999; Smart et al., 2000). Indeed, given the potencies of these
compounds functionally to activate CB receptors in intact cells
(Hillard et al., 1999; Ross et al., 1999), antiproliferative effects of
CP 55,940 and WIN 55,212-2 in the submicromolar range would have been
predicted whether effects upon CB receptors alone were sufficient to
produce antiproliferative effects. Similarly, the VR receptor activator capsaicin would not be expected to produce as good antiproliferative effects as found with AEA, given its lack of effects at CB receptors (Di Marzo et al., 1998). At these submicromolar concentrations, no
effects were seen in our hands, and the antiproliferative effects seen
at the higher concentrations of CP 55,940 were presumably nonspecific
effects of this compound, because they could not be antagonized by the
CB (or VR) antagonists. Such nonspecific effects may also account for
the antiproliferative actions of WIN 55,212-2 and JWH015. In this
respect it should be noted that the induction of apoptosis produced by
high concentrations (200-250 µM) of capsaicin in human glioblastoma
cells is not related to actions upon vanilloid receptors (Lee et al.,
2000).
Olvanil and meAEA are agonists at both vanilloid and cannabinoid
receptors over the concentration ranges used (Di Marzo et al., 1998)
and would thus have been expected to produce effects similar to those
seen with AEA. In the case of olvanil, antiproliferative effects were
observed but could not be blocked by CB receptor and VR antagonists
either alone or in combination, suggestive of a nonspecific mode of
action at the relatively high concentrations used here. No
antiproliferative effects at low concentrations of olvanil (i.e., those
giving a selective activation of VR) were seen, consistent with the
data with capsaicin. meAEA produced a weaker inhibition of
proliferation than AEA, again despite the fact that meAEA interacts
with both receptor systems (Ralevic et al., 2000). Although we have no
firm explanation for these anomalous results, one possibility is that a
certain balance of agonist effects is required on the two receptor
systems for antiproliferative effects to be observed. In the case of
AEA, concentrations required for activation of VR receptors are
approximately 10-fold higher than required for activation of CB
receptors (Zygmunt et al., 1999; Smart et al., 2000), whereas olvanil
is highly VR-selective (Di Marzo et al., 1998; De Petrocellis et al.,
2000). meAEA is approximately 10 times less potent than AEA at
inhibiting the binding of [3H]resinoferatoxin
to Chinese hamster ovary cells transfected with rVR1 (Ross et al.,
2001). In this assay, capsaicin and AEA are equipotent (Ross et al.,
2001), raising the possibility that the combination of meAEA and
capsaicin may mimic AEA with respect to antiproliferative effects in C6
cells. Although the antiproliferative effects of meAEA + capsaicin were
greater than for meAEA alone, the combination of the two compounds
produced additive effects and could not be antagonized by CR receptor + VR receptor antagonists.
In conclusion, the present study suggests that AEA and 2-AG, but not
meAEA or olvanil, produce antiproliferative effects upon C6 glioma
cells by a mechanism that involves both cannabinoid and vanilloid
receptors, followed by oxidative stress and calpain activation. In our
hands, compounds activating either CB receptors or VR alone do not
produce antiproliferative effects at relevant concentrations. The
protective effects of CB receptor antagonists (either SR14116A + SR144528 or AM251) are in contrast to the report by Maccarrone et al.
(2000) that the number of apoptotic bodies produced 48 h after
incubation of C6 glioma cell suspensions with 10 µM AEA was increased
by 77% in the presence of SR141716A and by 11% in the presence of
SR144528 (both 1 µM). Interestingly, these authors demonstrated that
the addition of AM404 further increased the number of apoptotic bodies
produced by the combination of AEA and SR141716A (and of AEA alone), a
result that is presumably a combination of the ability of this compound
to prevent AEA metabolism (Beltramo et al., 1997) and to activate
vanilloid receptors (De Petrocellis et al., 2000, 2001; Zygmunt et al.,
2000). Such differences between the data of Maccarrone et al. (2000)
and the present study may reflect differences in the properties of the
C6 cells used in the two laboratories, given that these cells are
rather heterogeneous in nature. In this respect, Galve-Roperh et al.
(2000) demonstrated that two subclones of C6 cells showed a dramatic
difference in the sensitivity to the effects of
9-THC upon cell viability. Clearly, there is
at present no obvious universal mechanism whereby plant-derived,
synthetic, and endogenous cannabinoids affect cell viability and proliferation.
| |
Acknowledgments |
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We thank Ingrid Persson for excellent technical assistance.
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Footnotes |
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Accepted for publication August 28, 2001.
Received for publication May 30, 2001.
This work was supported by MFR Grant 12158 from the Swedish Research Council and research funds of the Medical Odontological Faculty, Umeå University, Umeå, Sweden.
Address correspondence to: Stig Jacobsson, Ph.D., Department of Pharmacology and Clinical Neuroscience, Umeå University, SE-901 87 Umeå, Sweden. E-mail: stig.jacobsson{at}pharm.umu.se
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Abbreviations |
|---|
9-THC, 9-tetrahydrocannabinol;
CB, cannabinoid;
AEA, N-arachidonoylethanolamide (anandamide);
2-AG, 2-arachidonoylglycerol;
PEA, palmitoylethanolamide;
AA-5-HT, arachidonoyl-serotonin;
ACEA, arachidonyl-2'-chloroethylamide/(all
Z)-N-(2-cycloethyl)-5,8,11,14-eicosatetraenamide;
ANOVA, analysis of variance;
meAEA, R-(+)-methanandamide;
VR, vanilloid receptor;
FAAH, fatty acid amidohydrolase.
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References |
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Top
Abstract
Introduction
Experimental Procedures
Results
Discussion
References
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9-tetrahydrocannabinol.
J Neurosci
18:
5322-53329-Tetrahydrocannabinol induces apoptosis in human prostate PC-3 cells via a receptor-independent mechanism.
FEBS Lett
458:
400-404[Medline].9-Tetrahydrocannabinol induces apoptosis in C6 glioma cells.
FEBS Lett
436:
6-10[Medline].9-Tetrahydrocannabinol stimulates glucose utilization in C6 glioma cells.
FEBS Lett
436:
6-10.9-tetrahydrocannabinol-evoked arachidonic acid mobilization and blockade by SR141716A [N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(24-dichlorophenyl)-4-methyl-1Hpyrazole-3-carboximide hydrochloride].
Biochem Pharmacol
51:
669-676[Medline].9-Tetrahydrocannabinol induces apoptosis in macrophages and lymphocytes: involvement of bcl-2 and caspase-1.
J Pharmacol Exp Ther
286:
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, 1 µM; 
, 100 µM) and fumonisin B1 (
, 0.1 µM; 
,
10 µM) upon the sensitivity to the antiproliferative effects of AEA
and 2-AG. C6 glioma cells were treated daily for 4 days with the
concentrations of compounds shown, as described under
Experimental Procedures. 
, no additions ("none").
, SR141716A (1 µM); 
, SR144528 (1 µM); 
, capsazepine (1 µM);
, no additions
("none").
