Endocannabinoids In Endocrine And Related Tumours

[Jacob Bell]

Maurizio Bifulco, Anna Maria Malfitano, Simona Pisanti and Chiara Laezza1
Dipartimento di Scienze Farmaceutiche, Universita` di Salerno, 84084 Fisciano (Salerno), Italy
1IEOS, CNR Napoli, 80131 Napoli, Italy
(Correspondence should be addressed to M Bifulco; Email: maubiful@unisa.it)

Abstract
The 'endocannabinoid system', comprising the cannabinoid CB1 and CB2 receptors, their
endogenous ligands, endocannabinoids and the enzymes that regulate their biosynthesis and
degradation, has drawn a great deal of scientist attention during the last two decades. The
endocannabinoid system is involved in a broad range of functions and in a growing number of
physiopathological conditions. Indeed, recent evidence indicates that endocannabinoids influence
the intracellular events controlling the proliferation of numerous types of endocrine and related
cancer cells, thereby leading to both in vitro and in vivo antitumour effects. In particular, they are
able to inhibit cell growth, invasion and metastasis of thyroid, breast and prostate tumours. The
chief events of endocannabinoids in cancer cell proliferation are reported highlighting the
correspondent signalling involved in tumour processes: regulation of adenylyl cyclase, cyclic
AMP-protein kinase-A pathway and MEK-extracellular signal-regulated kinase signalling cascade.
Endocrine-Related Cancer (2008) 15 391—408

Introduction
Up to date since the isolation and characterisation of the
psychoactive component of Cannabis sativa, D9-tetrahydrocannabinol
(D9-THC), about 60 different plant
terpeno-phenols more or less related to THC have been
isolated and defined cannabinoids. They include cannabidiol
(CBD), cannabinol, cannabigerol and cannabichromene.
The discovery of these principles stimulated
the generation of a whole range of synthetic analogues
that included not only compounds structurally similar to
phytocannabinoids, but also analogues with different
chemical structures, including classic and non-classic
cannabinoids and aminoalkylindoles (Howlett et al.
2002) aswell as the subsequently discovered endogenous
arachidonic acid derivatives or endocannabinoids. The
discovery of this family of endogenous cannabinoids
(Devane et al. 1992, Mechoulam et al. 1995, Sugiura
et al. 1995) has focused much attention on cannabinoids
and their pharmacological properties during the last few
years (Di Marzo et al. 2004). The best-known
endogenous cannabimimetics are N-arachidonoyl-ethanolamine
(AEA also called anandamide), another
arachidonate derivate, 2-arachidonoyl-glycerol (2-AG)
and an ether-type endocannabinoid, 2-arachidonoylglyceryl
ether (Noladin ether) (Devane et al. 1992,
Mechoulam et al.1995, Sugiura et al. 1995, 2002, Hanus
et al. 2001). Moreover, compounds called 'endocannabinoid-
like' are present in human, rat and mouse brain
where they might inhibit the degradation of AEA or 2-
AG and, consequently, increase their activity
(Mechoulam et al. 2002). So far, N-palmitoylethanolamine
(PEA), N-oleoylethanolamine and N-stearoylethanolamine
exhibit this endocannabinoid-like activity
(Di Marzo 1998, Maccarrone & Finazzi-Agro` 2002).
Two different cannabinoid receptors (CBs) have
been identified so far and cloned from mammalian
tissues: CB1, or central receptor (Matsuda et al. 1990)
and CB2, or peripheral receptor (Munro et al. 1993).
Whereas the CB1 is preferentially expressed in the
central nervous system (Matsuda et al. 1990), the
CB2 has been described as the predominant form
expressed by peripheral immune cells (Munro et al.
1993, Galiegue et al. 1995). An ever increasing number
of reports and a lot of pharmacological evidence
suggest that endocannabinoids might exert their
biological effects also through non-CB1/CB2 receptors
which, however, have not yet been cloned except for
transient receptor potential vanilloid type 1 (TRPV1),
the TRPV1 ion channel, which is activated by various
lipids including anandamide (Begg et al. 2005).
Endocannabinoids show variable selectivity for the
two receptors (McAllister & Glass 2002, Mechoulam
et al. 2002). Both the CB1 and CB2 genes encode
a seven-transmembrane domain protein belonging to
the Gi/o-protein-coupled receptor family (Munro et al.
1993). CB1 receptors were found to efficiently couple
and activate both Gi and Go, whereas CB2 only Go, also
showing an agonist-selective G-protein signalling
(Glass & Northup 1999).
The CB1 receptor is known to be coupled with the
inhibition of adenylyl cyclase, inhibition of voltagedependent
CaCC channels and activation of G-protein
regulated inwardly rectifying KC currents (Howlett
1995, Porter & Felder 2001). Furthermore, the CB1
receptor has been shown to regulate different members
of mitogen-activated protein kinase (MAPK), such as
extracellular signal-regulated kinase (ERK; Bouaboula
et al. 1995, Pertwee et al. 1997), c-Jun N-terminal
kinase (Liu et al. 2000, Rueda et al. 2000), p-38
(Galve-Roperh et al. 2000, Rueda et al. 2000) and
p42/44 (Bouaboula et al. 1995). It is also reported that
the CB1 receptor activates phosphatidylinositol-3
kinase (PI3K), which in turn mediates tyrosine
phosphorylation, activation of Raf and may also signal
via phosphokinase B (PKB) in an SR141716-sensitive
manner (Gomez del Pulgar et al. 2002a,b). It was
shown that anandamide, via the CB1 receptor,
increases the tyrosine protein phosphorylation of
several proteins including focal adhesion kinase
(FAK) in normal neurons of the rat hippocampus, by
inhibiting adenylyl cyclase and phosphokinase A
(PKA; Derkinderen et al. 1996). In addition, the CB1
receptor regulates the sphingolipid metabolism, leading
to enhanced ceramide levels by either activating
sphingomyelin hydrolysis (Sanchez et al. 1998, 2001)
or increasing ceramide synthesis de novo (Gomez del
Pulgar et al. 2002a,b).
CB2 receptors, similar to CB1, through their ability
to couple to Gi/o, can inhibit adenylyl cyclase and
activate MAP kinase and Krox-24 pathways through a
phosphokinase C (PKC)-dependent activation of
MAPK (Bouaboula et al. 1996). However, in contrast
to CB1, CB2 receptors do not seem to modulate ion
channels directly (Felder et al. 1995). Evidence
suggests the involvement of the CB2 receptor in the
activation of the PI3K/PKB pathway, which in turn
induces the translocation of Raf-1 to the membrane and
phosphorylation of p42/p44 MAP kinase (Sanchez
et al. 2003a,b). It is also suggested that a cannabinoidmediated
reduction of MAP kinase may inhibit
interleukin-2 (IL-2) production in mouse splenocytes
and contribute a mechanism for immunosuppression
by cannabinoids (Kaplan et al. 2003). Although it was
not determined that the CB subtype was involved in
mediating this response, it is likely to be CB2-mediated
as this is the most abundantly expressed cannabinoid
receptor subtype in the immune system (Parolaro et al.
2002). Collected evidence suggests that different
structural classes of CB agonists have the unique
ability to activate different signalling cascades which,
in turn, influences agonist efficacy.
In the central nervous system, endocannabinoids
act as modulator compounds as well as neurotransmitters
(MacDonald & Vaughan 2001, Wilson & Nicoll
2002); in the peripheral and neural tissues, they have
been shown to modulate as paracrine or autocrine
mediators, protein and nuclear factors involved in cell
proliferation, differentiation and apoptosis. These data
suggest that the endocannabinoid signalling system
could be involved, among other effects, in the control
of cell survival, death and neoplastic transformation
(Guzman et al. 2001, Bifulco & Di Marzo 2002,
Bifulco et al. 2006).
The fundamental aspects of tumorigenesis widely
accepted are deregulation of cell survival pathways and
resistance to apoptosis. The aberrant growth and
survival of tumour cells is dependent upon a small
number of highly activated signalling pathways, the
inhibition of which elicits potent growth inhibitory or
apoptotic responses in tumour cells. Accordingly, there
is a considerable interest in therapeutics that can
modulate survival signalling pathways and target
cancer cells for death.
Accumulated evidence indicates that CBs could be
an important target for the treatment of cancer due to
their ability to regulate signalling pathways critical for
cell growth and survival. Several studies have
produced exciting new leads in the search for anticancer
treatments with cannabinoid-related drugs.
Natural, THC, synthetic, HU210, WIN-55,212-2 and
endogenous, 2-AG, AEA cannabinoids are nowadays
known to control various cancer types by modulating
tumour growth, apoptosis, migration and blood supply
to tumours (Bifulco & Di Marzo 2002, Guzman et al.
2002). In this review, we have tried to summarise the
importance of CB expression and modulation to induce
antitumour effects.
Cannabinoids and breast cancer
Breast cancer is the number one cause of cancer in
women (Glass et al. 2007). For the past few years, there
has been an increasing interest in the development of
agents targeted against molecular pathways considered
to be involved in the process of malignant transformation
or tumour progression. However, it is well
known that many of the signalling molecules required
for normal mammary gland development and lactation
are also involved in breast carcinogenesis, including
those activated downstream of the oestrogen receptor
(ER) and the human epidermal growth factor receptor
family (EGFR and erbB; Visvader & Lindeman 2003).
There are two main pathways involved in the tumour
phenotype: the Ras/Raf—MAPK ERK1/2 pathway, and
the PI3K/AKT pathway. Together, these pathways
regulate cell survival, proliferation, growth and
motility. Ras signalling is often enhanced in breast
cancers, due to the increased expression of erbB
receptors, signalling intermediates and/or Ras proteins
themselves (Malaney & Daly 2001). An important Ras
effector pathway resulting in mitogenic signalling is
the Raf/MEK/Erk cascade, which influences multiple
end points including increased transcription of cyclin
D1 (Coleman et al. 2004). Similarly, oestrogens and
progestins also activate cytoplasmic signalling
pathways including Src/Ras/Erk signalling (Edwards
2005). There is also some evidence of cross-talk
between the ErbB family of receptors and ER
signalling in breast cancer. The overexpression of
EGFR and heregulin receptor 2 (HER2) with the
subsequent downstream activation of MAPKs is
implicated in the mechanisms responsible for resistance
to hormonal treatment during prolonged endocrine
therapy or by long-term oestrogen deprivation
(Nicholson et al. 2004). Targeted therapies against all
the above-mentioned pathways have recently become
one of the most active and promising areas of
development in oncology. Therefore, new drugs
affecting multiple points along these pathways are
increasingly needed.
For instance, cannabinoidsmodulateMAPK/ERKand
PI3K/AKT survival pathways, which have a prominent
role in the control of cell fate (Guzman 2003). In 1998,
De Petrocellis et al. demonstrated for the first time the
antimitogenic action of CB1 receptor stimulation in
human breast cancer cell lines, EFM-19 and MCF-7,
known to express oestrogen and prolactin (PRL)
receptors and proliferate in response to the treatment
with steroid or lactogenic hormones (Clevenger et al.
1995). Anandamide inhibited the expression of PRL
receptor, induced downregulation of the breast cancer
associated antigen (brca1) gene product and the highaffinity
neurotrophin receptors trk (Melck et al. 1999a,b;
Fig. 1). The dose-dependent antiproliferative effect was
proportional to the degree of hormone dependency of
breast cancer cell lines. Themechanism involved in such
an effect has been ascribed to the inhibition of adenylyl
cyclase, cyclic AMP (cAMP) protein kinase-A (PKA)
pathway and, consequently, to the activation of MAPK
(Fig. 1). Cannabinoids prevent the inhibition of RAF1
(caused by PKA-induced Raf-phosphorylation) and
induce a prolonged activation of the RAF1-MEK-ERK
cascade (Melck et al. 1999a,b), leading to the downregulation
of the PRL receptor and trk levels. Moreover,
compounds like palmitoylethanolamide (PEA)
might act as 'entourage' substance for AEA, enhancing
cannabinoid biological actions. Di Marzo et al. (2001)
reported that chronic treatment with PEA enhances the
AEA-induced inhibition of cell proliferation through
decreased expression of fatty acid amide hydrolase
(FAAH), the enzyme chiefly responsible for AEA
degradation. Similar results, obtained with arvanil,
a more stable AEA analogue (Melck et al. 1999a,b),
and HU210, which cannot be hydrolysed by FAAH,
suggested that PEA could also enhance the vanilloid
receptor (VR1)-mediated effects of AEA on calcium
influx into cells (De Petrocellis et al. 2000, 2002).
The cell cycle machinery was deregulated at
multiple levels in breast cancer (Caldon et al. 2006).
Cyclins, Cdk and Cdk inhibitors, have been extensively
studied as cell cycle regulators in breast cancer cells, as
putative mammary oncogenes or tumour suppressor
genes and as potential markers of therapeutic response
or outcome. We reported that anandamide arrests the
proliferation of human breast cancer cells MDA-MB-
231 in the S phase of the cell cycle as a consequence of
the specific loss in Cdk2 activity, upregulation of
p21waf and a reduced formation of the active complex
cyclin E/Cdk2 kinase (Laezza et al. 2006). Recently, it
has been demonstrated that the checkpoint kinase Chk1
mediates both intra-S and G2 phase checkpoints by
targeting the Cdc25A phosphatase to proteolysis
following DNA damage, being also periodically
activated in every S phase of the unperturbed cell
cycle (Sorensen et al. 2003, Uto et al. 2004). It has
been observed that anandamide activates a cell cycle
checkpoint, through Chk1 activation and Cdc25A
proteolysis, thereby preventing Cdk2 activation by
dephosphorylation on critical inhibitory residues
(Thr14/Tyr15), which arrests cells in S phase. This
entails that the endocannabinoid system could be
involved in the regulation of cell cycle, the main
process controlling cell fate. Moreover, this could be of
great medical interest, since it has been proposed that
DNA damage checkpoints might become activated
during the early stages of tumorigenesis leading to cell
cycle blockade or apoptosis and could act as a barrier
against genomic instability and tumour progression
(Bartkova et al. 2005).
D9-THC was reported to reduce human breast cancer
cell proliferation by blocking the progression of cell
cycle in G2/M phase via the downregulation of Cdc2
and by inducing apoptosis. In this case, the effects were
mediated by CB2 receptors (Fig. 1). However, CB2-
selective antagonists significantly but not totally
prevented such effects, pointing to the existence of
CB receptor-independent mechanism (Caffarel et al.
2006). In contrast, a previous paper (McKallip et al.
2005) demonstrated that human breast cancer cell
lines, MCF-7 and MDA-MB-231, and the carcinoma
induced in mice by mouse mammary 4T1 cells
injection, are resistant to the D9-THC-induced cytotoxicity.
Furthermore, mice exposure to D9-THC led to
significantly elevated 4T1 tumour growth and metastasis,
probably due to inhibition of the specific
antitumour immune response. Indeed, the well-known
immunosuppressive properties of cannabinoids,
through CB2 receptors, have to be taken in regard,
since they may compromise antitumour immune
responses (Klein 2005). In contrast, McKallip reported
that the breast cancer cell lines express low levels of
CB1/CB2 receptors, hypothesising that the degree of
sensitivity of a tumour to D9-THC may be related to the
level of CB1/CB2 expression and that D9-THC
exposure may lead to increased growth and metastasis
of tumours with low or no expression of CBs
(McKallip et al. 2005). It is an unsurprising data that
different clones of the same cell lines showed very
variable levels of receptors as well as different
responsivity to hormones and growth factors; moreover,
the CB receptor expression could be modulated,
at least in part, by culture conditions and the number of
subculturing passages, even in the absence of specific
ligands (Melck et al. 2000). In addition, 4T1 cells
express high levels of VR1, and this could be a very
interesting data because these breast cancer cells may
be more sensitive to AEA, a potent agonist for the VR1
rather than D9-THC (Melck et al. 1999a,b, Zygmunt
et al. 2000). Caffarel et al. (2006) reported a correlation
between CB2 expression and the histologic grade of
human breast tumours. The overexpression of the
growth factor receptors and the ER negative receptor
status has been linked to a poor prognosis and a more
aggressive breast tumour phenotype. CB2 expression
was reported to be higher in those tumours with poor
prognosis and predicted low response to conventional
therapies, for instance estrogen receptor-negative
(ER—) and progesterone receptor-negative (PR—)
tumours, which are weakly responsive to the adjuvant
tamoxifen (Glass et al. 2007). Noteworthy, the main
limitation of the possible future use of D9-THC in
cancer therapy might be represented by its psychotropic
properties. An alternative could be represented
by the non-psychotropic CBD, which has recently
become a highly attractive therapeutic entity for a
plethora of pharmacological positive effects not
limited to cancer. It was reported to inhibit breast
cancer growth both in vitro and in vivo in xenograft
tumours, inducing apoptosis via direct or indirect
activation of CB2 and/or VR1 and increasing intracellular
calcium and reactive oxygen species (Ligresti
et al. 2006). The antitumour mechanism of action is
somewhat puzzling, since the modulation of a distinct
signalling pathway has not been identified. CBD has a
very low affinity for both CB1 and CB2 receptors, in
some models being antagonist at CB1 receptors
(Mechoulam et al. 2007). A very recent paper reported
that CBD was able to inhibit the invasiveness of highly
malignant breast cancer cells through the inhibition, at
the promoter level, of Id-1 an inhibitor of basic helixloop-
helix transcription factors strongly involved in
tumour progression (McAllister et al. 2007).
The CB1 receptor signalling has been reported to be
involved in metastatic processes. Indeed, anandamide
inhibited breast cancer cell migration, downregulating
FAK and Src phosphorylation/activation (Grimaldi
et al. 2006; Fig. 1). All these effects correlated with an
inhibitory effect on breast cancer metastasis in vivo,
since anandamide reduced the formation of lung
metastatic nodules in mice, and were all attenuated
by the CB1 receptor antagonist SR141716. CB1
receptors might be a target for therapeutic strategies
not only to slow down the growth of breast carcinoma
but also to inhibit its metastatic diffusion in vivo.
Considering the antitumour properties of the CB
agonists, it could be expected that CB antagonists/
inverse agonists like SR141716 (rimonabant,
Acomplia, Sanofi-Aventis) introduced in the clinic as
anti-obesity drug, if used alone, could instead enhance
proliferation of normal and malignant cells leading to
cancer. Collected data excluded this possibility,
reporting rather that not only agonists to CBs but
also antagonists, when used alone, are able to inhibit
tumour growth (Bifulco et al. 2004, 2007a,b, Pisanti
et al. 2006) or induce apoptosis in cancer cells (Derocq
et al. 1998, Powles et al. 2005). Indeed, we recently
provided evidence of antiproliferative effect exerted by
the CB1 cannabinoid antagonist SR141716 in breast
cancer cells (Sarnataro et al. 2006). We reported that
rimonabant exerts antitumour effects on breast cancer
in vitro, through G1/S phase arrest and in vivo in
xenograft tumours, providing a new mechanism of
action for this drug. Rimonabant, at nanomolar
concentrations, inhibits human breast cancer cell
proliferation, being more effective in highly invasive
metastatic cells, depending on both the presence and
the different expression levels of the CB1 receptor and
the ER status. The molecular mechanism at the basis of
rimonabant function implicates an inhibition of downstream
ERK1/2 signalling inside lipid rafts/caveolae.
The antiproliferative effect requires lipid rafts integrity
and the presence of CB1 receptor in lipid rafts,
previously reported to be highly localised in this
compartment and regulated in its trafficking by agonist
binding (Sarnataro et al. 2005). Interestingly, lipid rafts
and caveolin 1, a protein enriched in rafts, play a
critical role in breast tumour growth and metastasis
(Sloan et al. 2004, Williams et al. 2004). Perturbation
of lipid rafts/caveolae may represent a useful antitumoural
tool to control CB1 signalling in breast
cancer (Sarnataro et al. 2006).
Cannabinoids and prostatic cancer
Prostate cancer is the most commonly diagnosed
malignancy in men and the second leading cause of
cancer death in males (Society American Cancer
2005). Most early tumours are androgen-dependent,
thus depriving the tumour of androgens via surgical or
medical castration (Gnanapragasam et al. 2003) has
proven to have significant effects at the initial stages of
prostate cancer. Despite the early efficacy of androgen
ablation, advanced prostate cancer is resilient to such
treatments and eventually relapses into a hormone
refractory (androgen-independent) disease, with devastating
results on morbidity and mortality rates (Isaacs
1994, Lara et al. 2004). In spite of being insensitive to
hormone-withdrawal therapy, a majority of these
tumours continue to express the androgen receptor
(AR) and androgen-regulated genes like prostatespecific
antigen (PSA), indicating that the AR pathway
is active (Denmeade et al. 2003).
The AR activity seems to be tightly regulated by the
activation of distinct growth factor cascades that can
induce the AR modifications, including phosphorylation
and acetylation or changes in interactions of AR
with cofactors (Culig et al. 2004, Taplin & Balk 2004)
such as EGFR, insulin-like growth factor-I (IGF-I),
keratinocyte growth factor, IL-6 and oncostatin M.
IGF-I, which is produced by prostatic stromal cells in
response to androgen stimulation, works in a paracrine
manner by stimulating the surrounding prostatic
epithelial cells, resulting in an increased proliferation
(Moschos & Mantzoros 2002, Garrison & Kyprianou
2004). The proliferation of prostate cancer cells is
stimulated by an activated IGF-I signalling pathway
(Stattin et al. 2004). The primary cell survival pathway
activated by IGF-I is the PI3/Akt signalling
pathway. The binding of the IGF-I ligand to the IGF-I
receptor (IGF-IR) results in the activation of phosphoinositol-
3 kinase (PI3) that further activates the Akt
pathway, resulting in the phosphorylation (deactivation)
of the proapoptotic Bad protein and effectively
blocking apoptosis (Moschos & Mantzoros 2002).
IGF-I also induces the activation of the MAPK
pathway via the Ras protein, deactivating the downstream
target Bad protein and, leading to cell survival
and proliferation (Moschos & Mantzoros 2002).
Fibroblast growth factors (FGFs) play a significant
role in the development of prostate cancer; FGF-2 acts
as a mitogen for prostatic stromal cells, exerts its effect
mainly in an autocrine manner (Ropiquet et al. 1999,
Garrison & Kyprianou 2004) and also contributes to
angiogenesis (Mydlo et al. 1988). In contrast, FGF-7
acts in a paracrine manner as a mitogen for prostatic
epithelial cells (Ittman & Mansukhani 1997). FGF-8 is
thought to play a role in carcinogenesis due to its
overexpression in prostate cancer cells. Once activated,
the FGFRs target the downstream MAPK pathway,
resulting in cell survival, proliferation and angiogenesis
(Tsang & Dawid 2004, Yamada et al. 2004).
Transforming growth factor-b (TGF-b) is released
from prostatic stromal cells and works in a paracrine
manner, inhibiting prostatic epithelial cell growth and
inducing apoptosis (Wu et al. 2001, Bhowmick et al.
2004). SMAD proteins, primary intracellular effectors
of TGF-b signalling, trigger the activation of a series of
transcription factors that dictate the proliferative and/
or apoptotic outcomes of the cells (Bello-DeOcampo &
Tindall 2003). The SMAD-activated transcription
factors downregulate the transcription of the cell
survival factor Bcl-2 (Guo & Kyprianou 1999).
Further, the cell cycle is effectively halted by the
increased expression of the cyclin-dependent kinase
inhibitor p27Kip1 (Guo & Kyprianou 1999). Transcription
activated by the TGF-b/SMAD signalling
pathway leads to an increased expression of IGF
binding protein-3 (IGFBP-3), the primary binding
protein involved in sequestering IGF-I (Nickerson
et al. 1997, Motyl & Gajewska 2004). Finally, the
activated SMAD also has an effect on cytosol,
activating the apoptosis initiation factor caspase-1
(Guo & Kyprianou 1999).
The progression of prostate cancer is dependent on
angiogenesis, mediated primarily via the increased
expression of vascular endothelial growth factor
(VEGF). Once VEGF is released, it binds to VEGF
receptors on adjacent endothelial cells and induces a
series of cell survival and mitogenic pathways,
primarily through the PI3/Akt pathway and the Rasmediated
MAP kinase pathway. VEGF may also exert
its action by positively feeding back on the Src protein
in the cytosol, maintaining the VEGF promoting
stimulus. Thus, Src, hypoxia-inducible factor 1a, and
signal transducer/activator of transcription-3 act to
regulate cell survival (Semenza 2003).
Several prostatic intraepithelial neoplasia and invasive
prostatic cancer show an increased expression of
EGFR tyrosine kinase, EGF and TGF-a (Kim et al.
1999). Moreover, androgen-independent human prostate
cancer cell lines, PC3 and DU145, overexpress
EGFR, which, through selective interaction with
autocrine- and paracrine-secreted EGF and TGF-a,
promotes cell proliferation. In these models, androgen
and EGF downregulate p27kip, inhibitor of the cyclindependent
protein kinase (Ye et al. 1999). Activated
EGFR may induce the stimulation of distinct mitotic
cascades, including Shc, MAPK, PI3K/Akt, nuclear
factor-kappa B (NF-kB) and phospholipase Cg (PCg)
signalling pathways, which participate in the stimulation
of proliferation, survival, motility and invasion
of PC cells (Mimeault et al. 2003a,b, Torring et al.
2003, Bonaccorsi et al. 2004, Mimeault et al. 2006).
Recent investigations also revealed that the EGF—
EGFR signalling elements could play a pivotal role
during different stages of PC progression by modulating
several other signalling pathways including AR,
hedgehog and Wnt/b-catenin cascades (Mimeault et al.
2003a,b, 2006, Torring et al. 2003). EGF may induce
the activation of AR synergistically in the presence of
low androgen levels or in the absence of androgens in
a cell type-dependent manner (Culig et al. 1994, Orio
et al. 2002, Gregory et al. 2004, Festuccia et al. 2005).
It has also been reported that EGF may induce the AR
nuclear translocation and enhance the growth of the
CWR22R 2152 cell subline (Festuccia et al. 2005).
In prostate tumour cells, an upregulated expression
of hedgehog signalling components also appears to
occur. In particular, the enhanced expression level of
sonic hedgehog ligand, SHH, in PC cells may lead to
the activation of the GLI-1 transcription factor. This
results in the expression of numerous tumorigenic
genes, including cyclin D1 and c-Myc, which
participate in the sustained growth of PC cells (Fan
et al. 2004, Karhadkar et al. 2004, Olsen et al. 2004,
Sanchez et al. 2004).
Several Wnt ligands are expressed at significant
levels in prostatic stromal cells, androgen-dependent
and -independent PC cell lines and tumoural tissues
(Chen et al. 2004, Zhu et al. 2004). Wnt1 and b-catenin
are also highly expressed in the metastatic LNCaP,
DU145 and PC3 cells (Chen et al. 2004). It has been
reported that Wnt3a induces AR transcriptional
activity in the absence or in the presence of low
concentrations of androgens, at least in part, through an
increase in the cytosolic and nuclear b-catenin levels in
AR-positive CWR22Rv1 and LNCaP cells. This effect
was accompanied by an enhanced rate of cell growth
(Verras et al. 2004).
It has been reported that the PC cell lines, including
LNCaP, CWR22Rv1, DU145 and PC3 cells, express
the receptor IL-6R, showing a high affinity for IL-6
(Okamoto et al. 1997, Culig et al. 2005). Moreover,
IL-6 is also secreted by highly metastatic CWR22Rv1,
DU145 and PC3 cells, while LNCaP cells did not
produce a significant IL-6 level. The treatment with the
exogenous IL-6 of diverse PC cells has revealed that
this cytokine may modulate AR activity. It has been
reported that IL-6 may enhance AR activity in
AR-transfected DU145 and PC3 cells as well as
AR-mutant LNCaP cells synergistically in the presence
of low levels of androgen and/or in a ligandindependent
manner (Yang et al. 2003, Culig et al.
2005). Similarly, it has also been reported that the
IL-6-type cytokine, oncostatin M, may induce in a
paracrine fashion, the activation of AR and growth
stimulation in DU145-AR and CWR22Rv1 cells
(Godoy-Tundidor et al. 2005).
A molecular dissection of the deregulation of growth
factor signalling pathways in prostate tumorigenesismay
provide promising new therapeutic targets for prostate
cancer. We report here the emerging findings providing
evidence that cannabinoids should be considered
effective agents for the treatment of prostate cancer.
Exposure of PC3 cells, both to THC and to R-(C)-
methanandamide (MET) stimulated the PI3K/PKB
pathways, via CB1/CB2 activation, which increased
phosphorylation of PKB, induced translocation of
Raf1 to the membrane and phosphorylation of p44/42
Erk-kinase (Sanchez et al. 2003a,b). The treatment with
AEA at micromolar concentration for 48 h (Mimeault
et al. 2003a,b) results in the inhibition of EGF-induced
proliferation of DU145 and PC3 cells as well as
androgen-stimulated LNCaP, via G1 arrest, and downregulated
EGFR levels (Fig. 2). Both phenomena were
CB1-mediated. A similar growth arrest and receptor
modulation was also reported for PRL and nerve growth
factor-stimulated DU145 (De Petrocellis et al. 1998,
Melck et al. 2000), via the same AEA-modulated signal
transduction pathways described in breast cancer cells
(Melck et al. 2000). Importantly, a longer incubation
time (5—6 days) with AEA was able to induce massive
apoptotic effects, via cellular ceramide accumulation,
CB1/CB2-mediated, in DU145 and PC3, whereas in
LNcaP cells AEA did not exert similar effects (Fig. 2).
Intriguingly, 4 days of treatment with MET or
exogenous cannabinoids, at submicromolar concentrations,
increased the proliferation rate of LNCap
cells and the expression of AR; long-lasting incubation
periods led to differentiation (Sanchez et al. 2003a,b).
Met-induced mitogenic effect seems PKC, rather
than cAMP-pathway dependent; furthermore, in
this cellular model the androgen receptor expression
is CB1- and, partially, CB2-mediated (Sanchez et al.
2003a,b, Sarfaraz et al. 2005, 2006). In a recent study,
WIN-55,212-2 treatment significantly decreased
LNCaP cell viability and AR expression in a dose-
(micromolar) and time-dependent manner, with
maximal effect at 72h (Sarfaraz et al. 2005);
concomitantly, the authors showed a decrease in the
intracellular as well as the secreted levels of the PSA, a
glycoprotein androgen-receptor regulated (Henttu et al.
1990, Montgomery et al. 1992, Lee et al. 1995) and,
presently the most accepted marker of assessment of
prostate cancer progression (Stamey et al. 1987; Fig. 2).
Their results showed that treatment of LNCaP with
WIN-55,212-2 also inhibits VEGF protein expression, a
ubiquitous cytokine that plays a key role in angiogenesis
(Blazquez et al. 2003). Finally, 2AG inhibits the
invasion of androgen-independent prostate cancer cells
PC3 and DU145 through CB1-dependent inhibition of
adenylyl cyclase and decreased activity of PKA
(Nithipatikom et al. 2004; Fig. 2). Recently, Sarfaraz
et al. (2006) showed that treatment of human prostate
cancer LNCaP cells with CB agonist WIN-55,212-2
caused an arrest of the cells in the G0/G1 phase of the
cell cycle, sustained by the activation of ERK1/2,
induction of p27/KIP1 and inhibition of cyclin D1
(Fig. 2). G0/G1 arrest upregulated the Bax/BCl-2 ratio
and activated caspases resulting in an induction of
apoptosis. The blocking of both cannabinoid receptors
CB1 and CB2 by their specific antagonist resulted in the
inhibition of ERK1/2 activation. The inhibition of
ERK1/2 signalling by the ERK1/2 inhibitor PD98059
reversed the distribution of cells in the G1 phase of the
cell cycle and also decreased the percentage of apoptotic
cells when compared with WIN-55,212-2 treatment
alone. The ERK1/2 inhibitor also reversed the effects of
WIN-55,212-2 on p27/KIP1 and cyclin D1 proteins
operative in the G1 phase of the cell cycle and Bcl-2, an
important pro-apoptotic protein. Similar results were
observed when ERK1/2 was silenced using siRNA.
Moreover,WIN-55,212-2 treatment of the cells resulted
in a dose-dependent decrease in protein expression of
cyclin D1, cyclin D2 and cyclin E, as well as cdk2, cdk4
and cdk6. Downregulation of cdk4/6 has been shown to
be associated with a decrease in the expression of
retinoblastoma (pRb) tumour suppressor protein, a key
regulator of the G1/S phase transition in the cell
cycle (24, 25). The authors have observed that the
treatment with this agonist resulted in a decrease in the
protein expression of pRb and its molecular partner, the
transcriptional factor E2F. Because the activity of E2F is
known to be dependent on its heterodimeric association
with members of the DP family of proteins, they also
evaluated the effect of WIN-55,212-2 treatment on
both the members of DP family viz. DP-1 and DP-2.
WIN-55,212-2 caused a dose-dependent decrease in the
protein expression of DP-1 and DP-2. Finally, the
authors suggested that the CB agonist should be
considered an effective agent for the treatment of
prostate cancer but this hypothesis must be supported by
in vivo experiments.
Cannabinoids and thyroid cancers
Thyroid cancer is the most common endocrine malignancy.
It is characterised by genetic alterations resulting
in a dysregulation of cell growth and death.Alterations in
key signalling effectors seem to be the hallmark of
distinct forms of thyroid neoplasia. The overexpression
and/or uncontrolled activation of receptor tyrosine
kinases, downstream signalling molecules and the
inhibition of programmed cell death (apoptosis) have
all been demonstrated to occur in thyroid cancer. Several
compounds presently being tested in preclinical and
clinical studies target intracellular molecules involved in
these processes. These agents are not tumour-specific, as
these pathways are active in normal and malignant cells,
but are thought to be tumour-selective because the
cancers demonstrate higher levels of pathway activation,
making more sensitive than normal cells at lower
concentrations (Braga-Basaria & Ringel 2003, Kondo
et al. 2006). Ras activation is central to the pathogenesis
of some thyroid cancers, and it can occur through
mutations in the genes encoding Ras or through
activation of upstream regulators. In thyroid carcinoma,
activating mutations of ras genes (N-,K- orH-ras) can be
found in asmany as 30%of cases (Mizukami et al. 1988).
Ras is a small GTP-binding protein (G-protein) regularly
expressed in normal thyroid cells, and its protein product
is involved in several important functions, including
proliferation, differentiation and cell survival. In thyroid
cancer, the overactivation of Ras may occur through
activating mutations in the ras gene or by the overactivation
of receptor tyrosine kinase receptors.
Mutations in the gene encoding Ras can result in the
expression of Ras proteins that are constitutively bound
toGTP, i.e. once they are activated they are not able to be
turned off (Lemoine et al. 1989, Namba et al. 1990,
Karga et al. 1991). In addition to activating mutations,
Ras overactivation can occur secondary to receptor
overactivation. The enhanced signalling of receptor
tyrosine kinases is a common event in thyroid cancer,
particularly papillary thyroid cancer. For these reasons,
Ras is a reasonable molecular target to consider for novel
forms of thyroid cancer therapy. Moreover, overexpression
of receptor tyrosine kinases, including FGF, EGF,
hepatocyte growth factor (c-Met), VEGF, insulin and
IGF-I receptors are commonly identified in thyroid
cancers. Several of these receptors are common to many
cancers and they are expressed at very low levels in nonneoplastic
tissues and may be associated with angiogenesis
or progression, making them excellent therapeutic
targets. It is known that the formation of new blood
vessels is a crucial step in determining tumour expansion
and is greatly dependent on proangiogenic factors that are
produced in a paracrine fashion by tumour cells
undergoing hypoxia ormechanical compression. Several
growth factors are involved in the process of angiogenesis
in malignant tumours; among them, VEGF appears
to be the most prominent, being the functional of
stimulating vascular proliferation and permeability, and
inducing metastasis. Significantly increased levels of
VEGF have recently been demonstrated in the serum of
patients with well-differentiated metastatic thyroid
tumours when compared with lower levels found in
patients considered to be in a complete remission (Tuttle
et al. 2002). In patients with other malignancies, a worse
prognosis was observed in those who expressed higher
levels of VEGF in their tumours, probably due to an
increased vessel formation and development of metastasis
(Huang et al. 2001, Klein et al. 2001, Lennard et al.
2001). Themolecular alterations identified in this disease
represent targets for early clinical trials that are aimed at
tailoring multimodal approaches to the treatment of
thyroid cancers. Recently, it has been described that
endo/cannabinoids are able to affect the activity or
expression of these molecules. In a recent paper, Bifulco
et al. (2001) showed the effect of 2-methyl-arachidonyl-
2-fluoro-ethylamide (Met-F-AEA), a stable analogue of
the endocannabinoid anandamide, on a rat thyroid
epithelial cell line (FRTL-5) transformed by the K-ras
oncogene (KiMol), and on epithelial tumours derived
from these cells. Met-F-AEA induced a dose-dependent
(IC50Z5 mM) arrest of the cell cycle of these cells at the
G0/G1 phase, associated with a significant reduction of
cells in the S andG2/Mphase.The antiproliferative effect
was accompanied by a striking reduction in the p21ras
activity (Fig. 3). All these effects were attenuated
significantly by SR141716. The Met-F-AEA cytostatic
action was significantly smaller in non-transformed
FRTL-5 cells than in KiMol cells. The treatment with
Met-F-AEAexerted opposite effects on the expression of
CB1 receptors in KiMol and FRTL-5 cells, with a strong
upregulation in the former case and a suppression in nontransformed
cells.The authors evaluated theMet-F-AEA
effect in a nude mouse xenograft model, where K-rastransformed
(KiMol) cells were implanted subcutaneously.
The treatment with Met-F-AEA induced a
drastic reduction in the tumour volume. This effect was
inhibited by the CB1 receptor antagonist SR141716 and
was accompanied by a strong reduction in the K-ras
activity. The decrease in tumour volume induced by
Met-F-AEA was accompanied by a strong upregulation
of CB1 receptor mRNA and protein when compared
with vehicle-treated tumours. Similarly, KiMol cells
treated with Met-F-AEA expressed significantly more
CB1 receptors, and this effect was abolished by
SR141716. Cell immunofluorescence studies with both
permeabilised and non-permeabilised cells showed that
Met-F-AEA increased the levels of CB1 receptors on
both the cellmembrane and the cytosol.By contrast, nontransformed
FRTL-5 cells treated with Met-F-AEA
exhibited less CB1 receptors than vehicle-treated cells
in both the cellmembrane and the cytosol. In accordance
with this opposite regulation of CB1 receptor expression
in transformed versus healthy cells, the proliferation of
KiMol cells treated with Met-F-AEA was significantly
more strongly inhibited by the cannabimimetic substance
(up to 70%inhibition) than the response of FRTL-5 cells,
which barely reached statistical significance after 3 days
(up to 28% inhibition). Afterwards, Portella et al. (2003)
studied whether cancer growth in vivo would be limited
by CBs when the tumour is already established and
growing. Therefore, they investigated the possible
tumour growth inhibitory effect of intratumoural
administrations of Met-F-AEA and the possibility that
this compound, by acting at CB1 receptors, also
interferes with angiogenesis and metastatic processes.
In order to evaluate the effects of this compound on
already established tumours, the authors s.c. injected 45
nude mice with K-ras-transformed FRTL-5 cells
(KiMol), which are able to induce the growth of
undifferentiated carcinomas when injected s.c. into
athymic mice. Twenty days later, when tumours were
clearly detectable, saline solution containing Met-FAEA
was injected in the peritumoural area on days 2 and
5 of a 7-day cycle for 4weeks.TheMet-F-AEAtreatment
induced a drastic reduction in the tumour volume with
respect to the vehicle control-treated mice. Subsequently,
it has been observed that this compound inhibited
angiogenesis by affecting the expression of VEGF. In
addition, Met-F-AEA treatment also reduced the
expression of one of the VEGF receptors (Flt-1/
VEGFR-1) in tumours, thus indicating that this treatment
was very likely to result in a strong inhibition of VEGF
signalling and, hence, tumour angiogenesis (Fig. 3).
These inhibitory effects of Met-F-AEA were attenuated
by the selectiveCB1 receptor antagonist,SR141716, thus
strongly suggesting the involvement of CB1 receptors in
the anti-VEGF action of the compound. The addition of
Met-F-AEA to KiMol cells was also able to significantly
decrease VEGF and VEGF receptor (Flt-1/VEGFR-1)
expression. They also founded that Met-F-AEA treatment
of tumours and KiMol cells increased p27(kip1)
levels (Fig. 3), and that this effect was attenuated by the
selective CB1 receptor antagonist, SR141716. The
cyclin-dependent kinase inhibitor p27(kip1) is another
protein suggested to play a role as a proangiogenic factor
and is under the negative control of the ras oncogene in
proliferating human thyroid cells. Moreover, it has been
described the effect of Met-F-AEA on metastatic
processes, comparing the antiproliferative action of this
compound on two other cell lines derived from a rat
thyroid carcinoma (TK-6 cells) or its lung metastasis
(MPTK-6 cells). A 4-day treatment with Met-F-AEA
was able to inhibit the proliferation of both neoplastic
thyroid cell lines. The growth of metastasis-derived cells
was inhibited more efficaciously than that of primary
thyroid carcinoma-derived cells, and this was accompanied
by a stronger upregulation of CB1 receptor levels
in MPTK-6 cells than in TK-6, together with a stronger
downregulation of VEGF receptor levels in MPTK-6
than in TK-6 cells. Finally, the authors tested the effects
of Met-F-AEA in vivo on the induction of metastatic foci
in mice lungs after intra-paw injection of the highly
metastatic 3LL cells.Adramatic inhibitory effect ofMet-
F-AEA was observed against lung nodules induced by
3LL cells. The metastatic growth inhibitory effect was
blocked by the CB1 receptor antagonist SR141716. In
conclusion, the local administration of the stable
anandamide analogue and cannabinoid CB1 receptor
agonist, Met-F-AEA, blocked the growth of an already
established rat thyroid carcinoma in athymic mice,
underlining that this strong anticancer effect might be
due at least in part to inhibition of angiogenesis, because
it was accompanied by blockade ofVEGF signalling and
overexpression of p27(kip1; Fig. 3). Furthermore, Met-
F-AEA, by acting at CB1 receptors, more efficaciously
inhibited the proliferation of metastasis-derived than
primary tumour-derived rat thyroid cancer cells and
counteracts the formation of metastatic loci in an in vivo
model of metastasis. Anandamide-based drugs may be
efficacious for the inhibition of K-ras-induced epithelial
cancer cell growth in vivo through the activation of CB1
receptors, inhibition of p21ras activity (Fig. 3) and
blockade of the cell cycle. This strong anticancer effect
might be due at least in part to inhibition of angiogenesis
because it was accompanied by the blockade of VEGF
signalling (Fig. 3).
Effect of cannabinoids on other endocrine
tumours
Studies on the effects of cannabinoids on other types of
endocrine tumour have been performed.
Recently, it has been demonstrated that agonists of
cannabinoids receptors modulate insulin release in RIN
m5F rat insulinoma b cells (De Petrocellis et al. 2007).
In particular, the CB1 agonist arachidonoyl-chloroethanolamide
(ACEA) and the CB2 agonist JWH133,
elevated Ca(2C), in a way sensitive to the inhibitor of
phosphoinositide-specific phospholipase C (PI-PLC),
U73122, andindependentlyfrom extracellular Ca(2C).
ACEA, but not JWH133, significantly inhibited
the effect on Ca(2C) of bombesin, which acts via
G(q/11)- and PI-PLC-coupled receptors in insulinoma
cells. Anandamide and N-arachidonoyldopamine,
which also activate TRPV1 receptors expressed in
RIN m5F cells, elevated Ca(2C) in the presence of
extracellular Ca(2C) in a way sensitive to both CB1
and TRPV1 antagonists. These results suggest that, in
RIN m5F cells, CB(1) receptors are coupled to
PI-PLC-mediated mobilisation of Ca(2C).
Pheochromocytoma is a rare catecholamine-secreting
tumour derived from chromaffin cells. It has
been found that rat adrenal pheochromocytoma PC-
12 cells contain the endocannabinoids anandamide
and oleamide, together with the enzyme responsible
for their degradation, FAAH and the proposed
biosynthetic precursors for arachidonoylethanolamide
and related acylethanolamides, the N-acyl-phosphatidylethanolamines
(Bisogno et al. 1998). Moreover,
several studies have reported that anandamide
induces apoptosis in PC-12 cells triggering JNK
and p38 MAPK pathways. The activation of p38
MAPK/JNK was accompanied by the release of
cytochrome c from the mitochondria and caspase
activation, suggesting that anandamide triggers a
mitochondrial-dependent apoptotic pathway (Sarker
et al. 2000, 2003). Also, the synthetic cannabinoid
recptor CB1 agonist CP55,940 induced apoptosis in
PC12 cells, also inducing NF-kB. However,
the elevation in NF-kB activity was not demonstrated
an integral part of the apoptotic signalling cascade
in PC12 cells, because its inhibition was not related
to the reduction of TUNEL-positive cells (Erlandsson
et al. 2002).
A potentially important role of the endocannabinoid
system in pituitary pathophysiology has been studied
extensively (Pagotto et al. 2006). Normal human
pituitary gland and pituitary adenomas have been
reported to express CB type 1 and synthesise
endogenous cannabinoids. CB1 was present in adrenocorticotrophin
(ACTH)-, PRL- and growth hormone
(GH)-producing cells, whereas no immunoreactivity
was found in luteinizing hormone-, follicle-stimulating
hormone- and thyrotrophin-positive cells. CB1 was
detected in acromegaly-associated pituitary adenomas,
Cushing's adenomas and prolactinomas, whereas a faint
or no expression was found in non-functioning pituitary
adenomas. The content of endocannabinoids in
pituitary tumours was higher than that in normal
human pituitary. In particular, prolactinomas showed
the highest level of AEA, followed by acromegalyassociated
pituitary tumours and corticotropinomas,
where the 2-AG content was also increased. Moreover,
the endocannabinoid content in pituitary adenomas was
shown to be correlated with the presence of CB1, by
being elevated in the acromegaly-associated pituitary
adenomas, Cushing's adenomas and prolactinomas,
which were the tumours positive for CB1, and lower in
non-functioning adenomas, which are characterised by
a low or absent CB1 expression (Pagotto et al. 2001).
The existence of an auto/paracrine cannabinoid loop in
pituitary adenomas that may have an important role in
modulating hormone overproduction could be postulated.
Natural or synthetic cannabinoids have been
shown to affect hormonal pituitary release in several
in vivo and in vitro rodent models (Fernandez-Ruiz
et al. 1997, Jackson & Murphy 1997). To attribute a
functional significance to CB1, primary tumour cell
cultures were stimulated with cannabinoids in the
presence and absence of physiological stimulants. The
cannabinoid agonist WIN-55,212-2 (1 mM) inhibited
GH secretion in most of the acromegaly-associated
pituitary tumours tested, and this effect was generally
reversed by the specific CB1 antagonist SR 141716,
suggesting that cannabinoids are able to directly
influence basal GH secretion through CB1 activation.
Interestingly, WIN-55,212-2 was able to suppress the
stimulatory effect on GH release produced by GH-releasing
hormone, but not that caused by growth
hormone-releasing peptide (GHRP). In all corticotropinomas
tested, WIN-55,212-2 alone was not able to
influence basal ACTH secretion, but together with CRF
had an additive effect on ACTH release that was
specifically blocked by SR 141716A, thereby indicating
a CB1-mediated effect (Pagotto et al. 2001). Cannabinoids
can modulate PRL secretion (Pagotto et al. 2006),
but it is still controversial whether this is a direct
pituitary action or an indirect activation of central
neurotransmitters. Nevertheless, in the single case
studied by Pagotto et al. (2001), WIN-55,212-2 was
able to inhibit basal PRL secretion.
Conclusion
It is extremely important today to identify newtargets for
drug development, either for cancers that are insensitive
to the present therapies, as substitutes for common toxic
chemotherapeutic regimens or to adjuvant other treatments
improving efficacy and avoiding recurrence and
resistance. In this frame, the case of endocannabinoidrelated
drugs appears intrinsically interesting and not
sufficiently explored, especially with regard to the
mechanistic insights into the triggered cellular events.
A summary of the main trophic actions of endo/cannabinoids
via their receptor in modulating tumour cell
proliferation is shown in Table 1. During the last few
years, it has become evident thatmultiple mechanisms of
action, not solely limited to the CNS, are involved in the
endocannabinoid-mediated control of cell proliferation.
In this review,we have tried to summarise the importance
of endo/CB expression and modulation to interfere with
tumour growth. There is compelling evidence that endo/
cannabinoids may regulate the growth and spread of
normal and neoplastic tissues. An accepted notion is that
endocannabinoid system very often induces opposite
effects in normal versus neoplastic cells in important
physiological processes, such as proliferation and
migration (Guzman et al. 2001). This apparent paradox
could be explained on the basis of CB receptors coupling
efficiency to different subsets of G-proteins, able to
activate different downstreampathways. However, there
is sill much to learn about this topic. In this review, we
focused our attention on endocrine and related cancers,
first because the endocannabinoid system seems to be
directly involved in the control of neuroendocrine
function, also through a direct effect on peripheral target
endocrine organs and second because initial studies of
endocannabinoid control of cell proliferation were
performed on endocrine cancer cells. Agonists of endo/
CBs seem to be effective drugs with antiproliferative
activity in breast, prostate and thyroid cancers in vitro and
in vivo, simultaneously affecting multiple signalling
pathways and biological processes that have been
implicated in the development of the malignant
phenotype and are downstream endocrine receptors
stimulation. An increasingly detailed knowledge of
those cell signalling pathways involved in malignancy
provides a sound basis for the development of drugs
aimed at selected components of the pathways.
Obviously, the modulation of a single target that
simultaneously inhibits multiple critical pathways is an
intriguing anticancer strategy. The inhibitory effect of
endo/cannabinoids on tumour growth could be dependent
on the differential localisation and expression of
different receptor subtypes and on the signal transduction
mechanisms activated following the binding of specific
agonists. Further studies may clarify whether CBs
stimulation could uncouple endocrine receptors from
their downstream signalling, thereby providing a useful
perturbation of hormonal-dependent cancers. Collected
evidence suggests a strong connection between endocannabinoid
system biology and lipid rafts (Sarnataro
et al. 2005, 2006). In this context, it should be very
interesting characterise the role of lipid rafts/caveolae in
CB receptors signalling and interplay with endocrine
receptors, since these compartments could represent a
cellular device for intracellular trafficking, as well as a
favourable platform to regulate intracellular signalling.
Furthermore, in view of the recent evidence that
endocannabinoid-induced cell arrest may occur via
both receptor-dependent and -independent mechanisms,
we venture to suggest that the clarification of the role of
endocannabinoid and its receptors in cancer development
may hold great promise for the treatment of patients
affected by endocrine and related malignancies. In sum,
CB1 receptors represent a promising endocrine tumour
drug target for several reasons: 1) this is due to the
ubiquity of these receptors expressed in a large variety of
endocrine cells; 2) cannabinoids selectively affect
tumour cells more than their non-transformed counterparts
thatmight even be protected fromcell death and 3) a
large number of ligands have been generated by
introducing several modifications in the structure of the
lead compounds, some of them with high affinity and
selectivity and lack of adverse psychotropic effects. It
appears clear that the documented antitumour activity of
the endo/cannabinoids, intrinsically interesting but not
sufficiently explored, needs a deeper knowledge
especially in regard to the mechanistic insights into the
triggered cellular events and to their safe translation into
the clinical setting.
Acknowledgements
This study was supported by the Associazione Educazione
e Ricerca Medica Salernitana, ERMES. The
authors declare that there is no conflict of interest that
would prejudice the impartiality of this scientific work.
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