Cannabinoids and cell fate

Cannabinoids and cell fate

Manuel Guzman*, Cristina Sanchez, Ismael Galve-Roperh

Department of Biochemistry and Molecular Biology I, School of Biology, Complutense University, 28040 Madrid, Spain

Abstract

Cannabinoids recently have been shown to control the cell survival/death decision. Thus, cannabinoids induce growth arrest or apoptosis

in a number of transformed neural and non-neural cells in culture. In addition, cannabinoid administration induces regression of malignant

gliomas in rodents by a mechanism that may involve sustained ceramide generation and extracellular signal-regulated kinase activation. In

contrast, most of the experimental evidence indicates that cannabinoids may protect normal neurons from toxic insults, such as glutamatergic

overstimulation, ischaemia, and oxidative damage. Regarding immune cells, low doses of cannabinoids may enhance proliferation, whereas

high doses of cannabinoids usually induce growth arrest or apoptosis. The potential therapeutic applications of these findings are discussed.

D 2002 Elsevier Science Inc. All rights reserved.

Keywords: Cannabinoids; Cell proliferation; Apoptosis; Neuroprotection; Cancer

Abbreviations: AC, adenylyl cyclase; 2-AG, 2-arachidonoylglycerol; AEA, anandamide, arachidonoylethanolamide; cAMP, cyclic AMP; CBD, cannabidiol;

ERK, extracellular signal-regulated kinase; NMDA, N-methyl-D-aspartate; PKA, protein kinase A; PKB, protein kinase B; SM, sphingomyelin; THC,

tetrahydrocannabinol; VSCC, voltage-sensitive Ca2+ channel.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 175

2. Cannabinoid signalling pathways and cell fate . . . . . . . . . . . . . . . . . . . . . . . . . . 176

3. Transformed neural cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 177

3.1. Mechanism of cannabinoid action . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 178

4. Non-transformed neural cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 178


5. Immune cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 180


6. Other peripheral cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 180


7. Potential therapeutic applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 181

Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 182

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 182

1. Introduction

Cannabinoids, the active components of Cannabis sativa

L. (marijuana), exert a wide array of effects on the CNS, as

well as on peripheral sites, such as the immune, cardiovas-

cular, respiratory, digestive, reproductive, and ocular sys-

tems (Pertwee, 2000; Porter & Felder, 2001). Nowadays, it

is widely accepted that most of these effects of marijuana

are mediated by the binding of its cannabinoid constituents

to specific receptors that are normally bound by a family of

endogenous ligands—the endocannabinoids (Di Marzo et

al., 1998; Piomelli et al., 2000). During the last few years,

cannabinoids have been shown to control the cell survival/

death decision by inhibiting or stimulating cell growth.

0163-7258/02/$ – see front matter D 2002 Elsevier Science Inc. All rights reserved.

PII: S0163 -7258 (02 )00256 -5

* Corresponding author. Tel.: +34-913944668; fax: +34-913944672.

E-mail address: [email protected] (M. Guzman).

Pharmacology & Therapeutics 95 (2002) 175–184

Most of these studies have focused on neural and immune

cells, although some reports also exist on other cell types.

This review summarises the current status of this intriguing

topic, including the putative molecular mechanisms impli-

cated, and its potential clinical implications.

2. Cannabinoid signalling pathways and cell fate

Before their specific receptors were described, it was

already known that cannabinoids inhibit adenylyl cyclase

(AC), with the consequent decrease in intracellular cyclic

AMP (cAMP) levels (Howlett, 1984). Since then, further

extensive investigations have shown different signal trans-

duction pathways involved in the action of these com-

pounds. Most of the effects of cannabinoids are mediated

by their specific receptors CB1 and CB2, which are coupled

to AC through heterotrimeric Gi/o-proteins (Matsuda et al.,

1990; Munro et al., 1993). The CB1 receptor also exerts

modulation of ion channels, inducing inhibition of N- and P/

Q-type voltage-sensitive Ca2 + channels (VSCCs) and

activation of rectifying K + channels (Fig. 1). These two

effects could be responsible for the inhibition of the release

of glutamate and other neurotransmitters by blunting mem-

brane depolarisation and exocytosis (Pertwee, 2000; Porter

& Felder, 2001).

In addition to these well-defined cannabinoid receptor-

coupled signalling pathways, more recent data provide

evidence that other transducing systems may be involved

in cannabinoid effects (Fig. 1). Thus, cannabinoid recep-

tors are coupled to activation of extracellular signal-regu-

lated kinase (ERK) (Wartmann et al., 1995; Bouaboula et

al., 1995a, 1995b, 1996) and of c-Jun N-terminal kinase and

p38 mitogen-activated protein kinase (Liu et al., 2000;

Rueda et al., 2000). The CB1 receptor is also coupled to

the activation of protein kinase B (PKB) (Gomez del Pulgar

et al., 2000). Cannabinoids modulate as well sphingolipid-

metabolising pathways by inducing sphingomyelin (SM)

breakdown and by acutely increasing the levels of the

second messenger ceramide (Sanchez et al., 1998b; Galve-

Roperh et al., 2000). Recent data indicate that this effect is

independent of G-proteins and is mediated by the adaptor

protein designated as factor associated with neutral SMase

activation (Sanchez et al., 2001b). Cannabinoid receptor

activation can also generate a sustained peak of ceramide

accumulation via enhanced synthesis de novo that plays an

important role in the induction of apoptosis (Galve-Roperh

et al., 2000; Guzman et al., 2001a). Because the different

Fig. 1. CB1 receptor-coupled signalling pathways potentially involved in the control of cell fate. Cannabinoids produce their effects by binding to specific Gi/o-

protein-coupled plasma membrane receptors. In particular, the CB1 receptor signals inhibition of AC and of N- and P/Q-type VSCC, release of Ca2 + from

intracellular stores, as well as activation of mitogen- and stress-activated protein kinase cascades (ERK, JNK, p38), and of PKB. Acute ceramide generation

may be a G-protein-independent process involving the adaptor protein FAN. The link between CB1 receptor activation and sustained ceramide generation via

enhanced synthesis de novo is unknown as yet. The cross-talk among the different pathways has been omitted for simplification. FAN, factor associated with

neutral SMase activation; JNK, c-Jun N-terminal kinase.

M. Guzman et al. / Pharmacology & Therapeutics 95 (2002) 175–184176

mitogen- and stress-activated protein kinase cascades ,PKB,

and ceramide are widely accepted to be involved in the

control of the fate of different cells, cannabinoids, as

discussed in this review, may play a general role in the

commitment of the cell survival/death decision.

3. Transformed neural cells

As shown in Table 1, various cannabinoids have been

shown to induce the death of glioma (Sanchez et al.,

1998a; Jacobsson et al., 2000), astrocytoma (Sanchez et

al., 1998a), neuroblastoma (Sanchez et al., 1998a; Mac-

carrone et al., 2000) and pheochromocytoma cells (Sarker

et al., 2000) in culture, and, most interestingly, the

regression of malignant gliomas in vivo (Galve-Roperh

et al., 2000; Sanchez et al., 2001a). Thus, rats bearing

malignant gliomas, when treated intratumourally with D9-

tetrahydrocannabinol (THC) or WIN-55,212-2 for 1 week,

survived significantly longer than untreated animals.

Moreover, a complete eradication of the tumours was

evidenced in 20–35% of the treated animals. Similar

experiments carried out on immune-deficient mice also

showed that tumour growth was significantly lower in

cannabinoid-treated animals than in control animals

(Galve-Roperh et al., 2000; Sanchez et al., 2001a).

Table 1

Studies on the effect of cannabinoids on neural cell fate

Cell System Cannabinoid Effect on cell fate CB receptor

implicated

Reference

Neurons In vivo WIN-55,212-2 Protection from

ischaemia

CB1 Nagayama et al., 1999

THC Protection from

excitotoxicity

CB1 Van der Stelt et al., 2001

THC (chronic) Neurotoxicity N.D. Scallet et al., 1987

THC (chronic) Absence of

neurotoxicity

– Chan et al., 1996

Culture PEA, D8-THC,

11-hydroxy-THC,

WIN-55,212-2,

nabilone

Protection from

excitotoxicity

CB2-like Skaper et al., 1996

WIN-55,212-2,

CP-55,940

Protection from

excitotoxicity

CB1 Shen & Thayer, 1998

THC Protection from

excitotoxicity

CB1 Abood et al., 2001

CP-55,940 Protection from

excitotoxicity

CB1 Hampson & Grimaldi, 2001

WIN-55,212-2,

AEA

Protection from

hypoxia and

hypoglycemia

None Nagayama et al., 1999

AEA, 2-AG,

MeAEA

Protection from

ischaemia

None Sinor et al., 2000

THC, CBD Protection from

oxidative damage

None Hampson et al., 1998b

THC, D8-THC,

CBN, CBD

Protection from

oxidative damage

None Chen & Buck, 2000

THC Apoptosis CB1 Chan et al., 1998

THC No effect on viability – Sanchez et al., 1998a

Astrocytes Culture THC No effect on viability – Sanchez et al., 1998a

Human grade IV

astrocytoma

In vivo JWH-133 Decreased tumour size CB2 Sanchez et al., 2001a

Glioma C6 In vivo THC,

WIN-55,212-2,

JWH-133

Decreased tumour size CB1, CB2 Galve-Roperh et al., 2000;

Sanchez et al., 2001a

Culture THC,

WIN-55,212-2,

HU-210, CP-55,940,

JWH-133

Decreased viability,

apoptosis

CB1, CB2 Sanchez et al., 1998a, 2001a;

Galve-Roperh et al., 2000

THC, CBD Decreased viability N.D. Jacobsson et al., 2000

Astrocytoma U373 MG Culture THC Decreased viability N.D. Sanchez et al., 1998a

Neuroblastoma N18 TG2 Culture THC Decreased viability N.D. Sanchez et al., 1998a

Neuroblastoma CHP100 Culture AEA Apoptosis None (VR1) Maccarrone et al., 2000

Pheochromocytoma PC12 Culture AEA,

WIN-55,212-2

Apoptosis CB1 Sarker et al., 2000

CBN, cannabinol; MeAEA, methanandamide; N.D., not determined; PEA, palmitoylethanolamide.

M. Guzman et al. / Pharmacology & Therapeutics 95 (2002) 175–184 177

Using different biochemical and pharmacological

approaches, the implication of the CB1 receptor was shown

in C6 glioma and PC-12 pheochromocytoma cells. More-

over, by the use of selective CB1 and CB2 antagonists in

vitro (Galve-Roperh et al., 2000) and in vivo (Sanchez et al.,

2001a), the involvement of the CB2 receptor in cannabi-

noid-induced apoptosis has also been observed in C6 glioma

cells, which could provide the basis for the management of

gliomas without psychotropic side-effects (see Section 7).

Of interest, a role for the VR1 vanilloid receptor in ananda-

mide (AEA)-induced apoptosis of CHP100 neuroblastoma

cells has been put forward recently (Maccarrone et al.,

2000).

3.1. Mechanism of cannabinoid action

Several signalling pathways have been implicated in

cannabinoid-induced apoptosis of transformed neural cells.

The apoptotic death of C6 glioma cells seems to depend on

the sustained generation of ceramide. This lipid second

messenger is believed to be important in the regulation of

cell function in the CNS (Goswami & Dawson, 2000). The

increased ceramide levels observed in glioma cells upon

cannabinoid challenge would lead to the activation of the

ERK cascade mediated by Raf-1 (Galve-Roperh et al.,

2000). It is generally accepted that the activation of the

ERK cascade leads to cell proliferation. However, recent

investigations have begun to define situations in which ERK

mediates growth arrest, as well as apoptotic and non-

apoptotic death, in many cells, including neural cells

(Grewal et al., 1999). The relationship between activation

of the ERK cascade and cell fate depends on the duration of

the stimulus. Following cannabinoid receptor activation,

two peaks of ceramide generation are observed in C6 glioma

cells that have different kinetics (minute versus day range),

magnitude (2- vs. 4-fold), mechanistic origin (SM hydro-

lysis versus ceramide synthesis de novo), and function

(metabolic regulation versus induction of apoptosis) (Guz-

man et al., 2001a). The data obtained by Galve-Roperh et al.

(2000) show that the apoptotic action of THC relies on the

long-term peak of ceramide generation and ERK activation.

In the case of PC-12 pheochromocytoma cells, AEA has

been shown to induce superoxide generation, which could

trigger downstream uncharacterised signals that culminate

in caspase-3 activation, thereby resulting in apoptosis

(Sarker et al., 2000). The involvement of superoxide in

AEA-induced apoptosis was supported by the preventive

action of the antioxidant N-acetylcysteine (Sarker et al.,

2000). In the case of CHP100 neuroblastoma cells, VR1

receptor activation by AEA triggers apoptosis through a

cascade of events that seems to involve a rise in cytosolic-

free Ca2 + concentration, cyclo-oxygenase, and lipoxyge-

nase activation, and a drop in mitochondrial membrane

potential, cytochrome c release, and caspase activation

(Maccarrone et al., 2000). The possibility that AEA binds

to VR1 capsaicin receptors (Zygmunt et al., 1999), however,

is conflictive and subject to ample debate (Szolcsanyi, 2000;

Piomelli, 2001). Moreover, capsaicin has been shown to

induce apoptosis in A172 glioma cells by a VR1-independ-

ent process (Lee et al., 2000).

4. Non-transformed neural cells

One of the most exciting aspects of current cannabinoid

research is the possibility that cannabinoids play a role as

neuroprotective agents, both pharmacologically and physio-

logically via the endocannabinoid system (Di Marzo et al.,

1998; Piomelli et al., 2000). Thus, most of the experimental

evidence indicates that cannabinoids may protect neurons

from insults, such as glutamatergic excitotoxicity, isch-

aemia, and oxidative damage (Table 1). The neuroprotective

action of cannabinoids has been shown in vivo. Thus,

neurotoxicity induced by global cerebral ischaemia in vivo

following cerebral artery occlusion was reduced in the CA1

region of the hippocampus by previous intraperitoneal

injection of WIN-55,212-2 via a CB1-dependent mechanism

(Nagayama et al., 1999). Cannabinoid treatment also

reduced the infarct volume caused by focal cerebral isch-

aemia, leading to enhanced neuronal survival of penumbral

cortical tissue (Nagayama et al., 1999). Likewise, in an in

vivo model of ouabain-induced excitotoxicity, THC exerted

a CB1-mediated reduction of the infarct volume, as deter-

mined by magnetic resonance imaging (Van der Stelt et al.,

2001).

Experiments conducted on cultured neurons have also

evidenced a cytoprotective action of cannabinoids, although

variable results have been obtained on the possible implica-

tion of cannabinoid receptors. A protective action of can-

nabinoids in a model of delayed postglutamate excitotoxic

death in cerebellar granule neurons was first reported by

Skaper et al. (1996). This study showed that palmitoyletha-

nolamide and some cannabinoid ligands (D8-THC, 11-

hydroxy-THC, WIN-55,212-2, nabilone), but not others

[AEA, cannabidiol (CBD)], protected these cells independ-

ently of N-methyl-D-aspartate (NMDA) and kainate receptor

antagonism, and this was attributed to a CB2-like-mediated

process. Although cerebellar granule neurons were shown to

express both CB1 and CB2 receptors, no direct proof linking

cannabinoid receptor activation with the neuroprotective

action was reported. WIN-55,212-2 and CP-55,940 were

later shown to protect hippocampal neurons from presynap-

tically evoked glutamate excitotoxicity by a CB1-mediated

process (Shen & Thayer, 1998). Again, the cannabinoid

neuroprotective action was not a consequence of direct

NMDA receptor antagonism. Likewise, two recent reports

have shown that CP-55,940 (Hampson & Grimaldi, 2001)

and THC (Abood et al., 2001) protect cultured neurons

against glutamatergic excitotoxicity via CB1 receptor activa-

tion. In contrast, WIN-55,212-2 and AEA protective action

in cortical neuron cultures subjected to hypoxia and glucose

deprivation has been shown to be independent of CB1 and


CB2 receptor activation (Nagayama et al., 1999). Similarly,

the protective action of nanomolar concentrations of AEA,

methanandamide, and 2-arachidonoylglycerol (2-AG) on

cortical neuron cultures is independent of CB1 and CB2

receptor activation (Sinor et al., 2000).

In addition, THC and CBD have been shown to be

protective in an ischaemic model of glutamate-mediated

toxicity in primary cortical neurons (Hampson et al.,

1998b). These two cannabinoids prevented cell death inde-

pendently of CB1 receptor activation, with an EC50 in the

micromolar range. This effect was attributed to their anti-

oxidant properties, cyclic voltametry showing that their

oxidation potentials are even higher than those of other

well-known antioxidants, such as ascorbate and tocopherol.

Although others have been unable to reproduce such an

effect (Nagayama et al., 1999), submicromolar concentra-

tions of plant-derived cannabinoids as well are protective

against oxidative cell death via a cannabinoid receptor-

independent mechanism (Chen & Buck, 2000).

Other studies, however, have put forward a neurotoxic

effect of THC. Thus, morphological changes in the hip-

pocampus; decreases in the mean volume, synaptic density,

and dendritic length of CA3 pyramidal neurons; and

reduced neuronal density in rat hippocampus associated

with chronic THC oral administration (60 mg/kg/day, 90

days) have been described (Scallet et al., 1987; Scallet,

1991). In contrast, Chan et al. (1996) treated rats and mice

orally with THC 5 days a week for 2 years, and did not find

any significant histopathological alterations in the brain,

even at doses of 50 mg/kg for rats and 250 mg/kg for mice.

Likewise, direct intracranial administration of THC (1.0 mg/

kg/day) or WIN-55,212-2 (0.1 mg/kg/day) to rats for 7 days

did not induce neural cell apoptosis, as assessed by TUNEL

staining (Galve-Roperh et al., 2000).

An in vitro study has also shown a decrease in the

viability of hippocampal neurons of 50% after 5 days

following pretreatment with 1-mM THC (Chan et al.,

1998). This effect results from CB1-mediated apoptosis,

and might involve a phospholipase A2-induced release of

arachidonic acid, which promotes the activation of cyclo-

oxygenases and lipoxygenases. Both pathways generate free

radicals that can lead to lipid peroxidation and cell death. In

contrast, in another report, the viability of cortical neurons

and astrocytes in culture did not decrease after exposure to

1-mM THC for 15 days (Sanchez et al., 1998a).


In ischaemia, when oxygen and nutrient deprivation

occurs, there is a large synaptic accumulation of glutamate,

which may have excitotoxic consequences, at least in part,

by Ca2 + overload through NMDA receptors (Dirnagl et

al., 1999). Regulation of glutamatergic synaptic transmis-

sion and Ca2 + homeostasis is believed to constitute the

main explanation for the neuroprotective action of canna-

binoids (Piomelli et al., 2000; Guzman et al., 2001b).

Glutamatergic transmission is regulated by cannabinoids

basically at the presynaptic level through inhibition of

glutamate release (Shen et al., 1996; Shen & Thayer,

1998; Misner & Sullivan, 1999). Thus, while cannabinoids

did not inhibit excitatory postsynaptic currents elicited by

exogenous application of NMDA or kainate, they pre-

vented currents evoked by stimulation of presynaptic

neurons (Shen et al., 1996). Cannabinoid-induced inhibi-

tion of presynaptic glutamate release is a consequence of

their inhibitory effect on Ca2 + influx through different

types of voltage-sensitive channels, including N- (Caulfield

& Brown, 1992; Mackie & Hille, 1992) and P/Q-type

channels (Twitchell et al., 1997; Hampson et al., 1998a).

Modulation of presynaptic K + channels may also contrib-

ute to this cannabinoid action (Robbe et al., 2001). Other

mechanisms potentially involved in cannabinoid-induced

neuroprotection include abrogation of transglial gap junc-

tion-dependent Ca2 + signalling (Venance et al., 1995),

inhibition of brain nitric oxide (Hillard et al., 1999) and

pro-inflammatory cytokine production (Lyman et al.,

1989), activation of the cytoprotective phosphoinositide

30-kinase/PKB pathway (Gomez del Pulgar et al., 2000),

and an enhanced supply of energy substrates from astro-

cytes to neurons (Guzman & Blazquez, 2001).

The observation that AEA (Di Marzo et al., 1994) and

2-AG (Stella et al., 1997) synthesis is activated by Ca2 +

may constitute a mechanism of feedback regulation, with

the generated cannabinoids preventing excessive Ca2 +

influx by their inhibitory action on VSCC (Piomelli et

al., 2000). For example, the levels of AEA and its

precursor N-arachidonoylphosphatidylethanolamine are

increased upon toxic insults such as glutamatergic over-

stimulation, hydrogen peroxide exposure, and blockade of

mitochondrial respiration with azide (Hansen, H. S. et al.,

1998; Hansen, H. H. et al., 2000). Because elevation of

intracellular Ca2 + concentration in neurons is associated

with ischaemic conditions and endocannabinoid synthesis

is increased upon Ca2 + overload, it is tempting to specu-

late that endocannabinoids act as neuromodulators to

induce brain protection. However, regulation of endocan-

nabinoid levels in vivo appears to be a complex issue, with

differences within specific neuronal populations and

among different endocannabinoids (Giuffrida et al., 1999;

Di Marzo et al., 2000). In this context, the levels of

oleoylethanolamide, a compound structurally related to

AEA, but which does not bind to cannabinoid receptors,

are also expected to increase during neurodegenerative

insults, since it is synthesised by the same biosynthetic

pathway as AEA (Hansen, H. S. et al., 1998). Although it

has been shown that oleoylethanolamide inhibits acid

ceramidase in vitro, which, in turn, might reinforce ceram-

ide-induced effects on neural cells (Wiesner & Dawson,

1996; Sastry & Rao, 2000), the latter oleoylethanolamide

effect occurs only in the high micromolar range and may

involve as well SM hydrolysis (I. Galve-Roperh, M. Guz-

man, and T. Levade, unpublished observations).


5. Immune cells

Many in vitro and in vivo studies have shown that

cannabinoids are immunosuppressive agents (Cabral &

Dove Pettit, 1998; Klein et al., 1998). However, a careful

examination of the literature pertaining to this field

indicates that cannabinoids may either stimulate or inhibit

the function of a variety of immune cells. This variation

in drug effects depends on experimental factors, such as

drug concentration, timing of drug delivery, and type of

cell function examined. In addition, some of these can-

nabinoid effects are also evident upon treatment with

non-psychoactive cannabinoid analogues, and their pre-

vention by selective CB2 antagonists either was not tested

(SR144528 is available only since 1998) or did not

occur.

Regarding the control of immune-cell fate, THC at 10–

30 mM has been shown to induce apoptosis of human

peripheral blood mononuclear cells (Schwarz et al., 1994),

as well as of mouse macrophages and lymphocytes (Zhu et

al., 1998). In contrast, Derocq et al. (1995) demonstrated

for the first time that human B-cell proliferation is stimu-

lated synergistically by antibodies directed to surface

immunoglobulins or CD40 plus plant-derived or synthetic

cannabinoids at nanomolar concentrations. A similar effect

of cannabinoids was observed when the metabolic status of

mouse splenocytes was examined (Sanchez et al., 1997).

Likewise, Valk et al. (1997b) showed that a number of

murine hematopoietic growth factor-dependent cell lines

require the presence of AEA for optimal growth in serum-

free medium. AEA also enhanced the number and size of

interleukin 3-induced myeloid colonies from mouse bone

marrow. Moreover, following retroviral insertional muta-

genesis, these authors have identified a virus integration

site, denominated Evi11, within the gene encoding CB2,

suggesting that CB2 represents a proto-oncogene involved

in leukomogenesis (Valk et al., 1997a). This is also

supported by the observation that in 12% of Cas-Br-M

murine leukaemia virus-induced tumours, retroviral inte-

grations occur either at 50 or 30 regions of the CB2 gene

(Valk et al., 1999). Although the growth-inducing effect of

AEA may partially rely, at least in vitro, on a cannabinoid

receptor-independent mechanism (Derocq et al., 1998), all

these findings indicate that cannabinoids should be con-

sidered ‘‘immunomodulators’’ rather than just ‘‘immuno-

suppressors,’’ because of their ability to either suppress or

enhance immune-cell growth.

The possibility that the CB2 receptor plays a role in the

control of immune-cell fate under the physiological setting

is supported by recent studies on the gene expression

profile of the human promyelocytic cell line HL60 acti-

vated with CP-55,940 using nucleic acid microarrays

spotted with different cDNA probes. CB2 activation

induced the up-regulation of 9 genes involved in cytokine

synthesis, transcriptional regulation, and cell differenti-

ation. The reported gene-induction pattern and the pheno-

typic properties of cannabinoid-exposed HL-60 cells

seemed related to a cell differentiation program (Jbilo et

al., 1999; Derocq et al., 2000). Most of these induced

genes are under the control of natural factor-kB, a ubi-

quitous transcription factor that modulates immune-cell

fate. These data, together with the changes in CB2 expres-

sion during the differentiation and CD40-induced activa-

tion of human B cells (Carayon et al., 1998), point to an

involvement of the CB2 receptor in the onset of immune-

cell maturation.


The transduction systems responsible for CB2 receptor

signalling are as yet unclear. It has been traditionally

assumed that inhibition of the cAMP/protein kinase A

(PKA) pathway may be responsible for the immunosup-

pressive action of cannabinoids by decreasing the expres-

sion of cAMP-responsive genes (Kaminski, 1998).

However, extremely high doses of plant-derived cannabi-

noids and AEA are necessary to lower the basal and/or

forskolin-stimulated intracellular cAMP concentration in

CB2-transfected cells (Bayewitch et al., 1995; Slipetz et

al., 1995). Moreover, THC may behave as an antagonist in

the CB2-mediated AC inhibition (Bayewitch et al., 1996).

In contrast, THC and AEA do inhibit AC through CB1

(Pertwee, 2000). Other differences between CB1- and CB2-

coupled signalling include the ability of the former to

modulate Ca2 + and K + channels (Pertwee, 2000), ERK

independently of protein kinase C (Bouaboula et al.,

1996), and PKB (Gomez del Pulgar et al., 2000). These

findings indicate that pathways distinct from the cAMP/

PKA route may be responsible for the control of cell fate

through the CB2 receptor. It is possible that the CB2

receptor might control immune-cell proliferation by coup-

ling to ERK activation, a process that is dependent on Gi/o-

proteins, but that is independent of cAMP (Bouaboula et

al., 1996).

6. Other peripheral cells

The effect of cannabinoids on human breast cancer cell

growth has been studied. In particular, AEA exerts a

remarkable antiproliferative action on MCF-7, EFM-19,

and T-47D cells through a CB1-mediated mechanism (De

Petrocellis et al., 1998; Melck et al., 2000). Unlike

cannabinoid-induced apoptosis of C6 glioma cells (San-

chez et al., 1998a), cannabinoid-induced inhibition of

breast cancer cell growth does not involve apoptosis, but

cell cycle arrest, at the G1/S transition (De Petrocellis et

al., 1998). Cannabinoids also inhibit human prostate cancer

cell growth. A CB1-dependent process has been shown for

cannabinoid antiproliferative action on DU-145 cells

(Melck et al., 2000), whereas CB1-independent apoptosis

is triggered by THC on PC-3 cells (Ruiz et al., 1999).



CB1 receptor activation in MCF-7 cells has been shown

to inhibit AC and to activate the Raf-1/ERK cascade (Melck

et al., 1999). These two signalling events seem to mediate—

at least in part—the antiproliferative action of AEA by

lowering the expression of the high-molecular mass (100

kDa) form of the prolactin receptor and of the high-affinity

trk neurotrophin receptor (Melck et al., 1999, 2000). Modu-

lation of the synthesis of neurotrophins (Velasco et al.,

2001) and their receptors (Melck et al., 1999) via the

ERK cascade may be an additional factor involved in the

control of cell fate by cannabinoids.

It is worth noting that human breast cancer cells produce

significant amounts of acylethanolamides, as well as of

oleamide, a primary fatty acid amide that does not bind to

cannabinoid receptors, but that potentiates the antiprolifer-

ative action of AEA. This process has been suggested to rely

on the oleamide-mediated competitive inhibition of fatty

acid amide hydrolase, which blunts AEA degradation and

thereby enhances AEA biological activity (Bisogno et al.,

1998). However, the hypothesis that AEA and oleamide are

autacoid suppressors of human breast cancer cell prolifera-

tion awaits in vivo confirmation.

7. Potential therapeutic applications

As discussed in this review, recent studies have dealt

with the antiproliferative effect of cannabinoids on different

transformed cells. However, this property of marijuana

compounds was first reported 25 years ago by Munson et

al. (1975), who showed that cannabinoids may inhibit the

growth of Lewis lung adenocarcinoma, B-tropic Friend

leukaemia virus-induced splenomegaly, and L1210 leuk-

aemia cells in vivo. Although these observations were

actually promising, further investigations were not per-

formed in this area until a few years ago. The case of

gliomas is of particular interest because they are one of the

most malignant forms of cancer, resulting in the death of

affected patients within months after diagnosis. Conven-

tional therapies, including surgery, radiotherapy, chemother-

apy, and immunotherapy, are usually ineffective or just

palliative (Avgeropoulos & Batchelor, 1999; Holland,

2000). The most recent strategies for glioma treatment are

focused on gene therapy, but no trial performed so far has

been significantly successful. Therefore, it is essential to

develop new therapeutic strategies for the management of

gliomas, and most likely to combine some of them to obtain

significant clinical results (Avgeropoulos & Batchelor,

1999; Holland, 2000). One of these alternative therapeutic

approaches might be based on the use of cannabinoid

agonists, since these compounds induce apoptosis in vitro

and inhibition of tumour growth without significant collat-

eral effects in vivo (Galve-Roperh et al., 2000; Sanchez et

al., 2001a). Of interest, cannabinoid apoptotic action relies

on the generation of ceramide, a lipid second messenger that

may have antitumoural properties, either alone (Schmelz et

al., 1999) or in combined therapies (Radin, 2001).

Different neurological disorders, such as ischaemia, Par-

kinson’s and Huntington’s diseases, and multiple sclerosis,

are accompanied by excitotoxicity, oxidative stress, Ca2 +

imbalance, and/or inflammatory responses that lead to

progressive neuronal death (Dirnagl et al., 1999; Mehler

& Gokhan, 2000). As discussed in Section 4, cannabinoids

may interfere with these processes. In addition, cannabi-

noids modulate the release and/or action of neurotransmit-

ters, such as glutamate, dopamine, acetylcholine, and g-

aminobutyric acid, and they are usually considered as

neuromodulators (Di Marzo et al., 1998; Piomelli et al.,

2000; Wilson & Nicoll, 2001)—although, because they are

not stored in vesicles, perhaps they should be considered as

‘‘atypical’’ messengers (Baranano et al., 2001). These

effects could provide the basis for potential therapeutic

applications of cannabinoids in the treatment of diverse

neurodegenerative disorders, such as Huntington’s disease

(Glass et al., 2000) and multiple sclerosis (Baker et al.,

2000). In addition, the existence of an inhibitory loop by

which endocannabinoids blunt dopaminergic control of

voluntary movement (Giuffrida et al., 1999) point to the

use of CB1 antagonists as potential candidates for the

management of Parkinson’s disease (Piomelli et al., 2000).

Furthermore, clinical trials are being conducted with the

non-psychoactive cannabinoid HU-211 (dexanabinol), a

noncompetitive NMDA receptor antagonist (Feigenbaum

et al., 1989). This compound reduces the inflammatory

response after closed-head injury or lipopolysaccharide-

induced septic shock, both in vivo and in vitro, and prevents

typical NMDA-induced responses, such as tremor, seizures,

and lethality, in rodents (Gallily et al., 1997; Shohami et al.,

1997). Clinical studies have been carried out up to Phase II

in patients with severe head trauma, with encouraging

results showing a better neurological outcome compared

with the placebo group (Pop, 2000).

Recent cannabinoid investigations focus on therapeutic

strategies aimed at avoiding non-desired CB1-mediated

psychotropic effects. Thus, the possible implication of

CB2 in the protection of neurons from excitotoxicity

(Skaper et al., 1996), the growth-inhibiting action on

gliomas (Galve-Roperh et al., 2000; Sanchez et al.,

2001a), and the control of spasticity in multiple sclerosis

(Baker et al., 2000) opens an attractive clinical challenge.

Of interest, highly selective CB2 agonists, e.g., JWH-133

(Huffman et al., 1999) and HU-308 (Hanus et al., 1999),

have been synthesised recently. Alternatively, an approach

based on the achievement of high endocannabinoid levels

by selective blockade of the AEA transporter (Beltramo et

al., 1997, 2000) or the AEA-degrading enzyme fatty acid

amide hydrolase (Boger et al., 2000) can be envisaged. Of

importance, these types of approaches would interfere with

endocannabinoid levels mildly and in a neuronal activity-

dependent mechanism. Furthermore, non-psychotropic can-


nabinoids, such as HU-211 (Pop, 2000) and CBD (Malfait

et al., 2000), which bind to non-cannabinoid receptors, may

also be potential therapeutic agents. New results are

expected to arise from more extensive studies regarding

the array of cannabinoid actions reviewed herein.

Acknowledgements

We are indebted to Cristina Blazquez, Marıa L. de

Ceballos, Marisa Cortes, Andres Daza, Teresa Gomez del

Pulgar, Marta Izquierdo, Daniel Rueda, and Guillermo

Velasco for the support that made possible all our

investigations included herein. Work in the authors’

laboratory is supported by grants from Comision Intermi-

nisterial de Ciencia y Tecnologıa (PM 98/0079), Comunidad

Autonoma de Madrid (CAM 08.1/0079/2000), and Funda-

cion Ramon Areces.

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Cannabinoids and cell fate

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