Jacob Bell
New Member
Inhibitory effects of cannabinoid CB1 receptor stimulation
on tumor growth and metastatic spreading: actions on
signals involved in angiogenesis and metastasis
Giuseppe Portella,* Chiara Laezza,* Paolo Laccetti,* Luciano De Petrocellis,"
Vincenzo Di Marzo,"¡ and Maurizio Bifulco*,§
*," ,"¡Endocannabinoid Research Group: *Istituto di Endocrinologia ed Oncologia Sperimentale,
Consiglio Nazionale delle Ricerche, and Dipartimento di Biologia e Patologia Cellulare e
Molecolare "L. Califano," Università di Napoli "Federico II, " Istituto di Cibernetica and "¡Istituto
di Chimica Biomolecolare, C.N.R., Via Campi Flegrei 34, Comprensorio Olivetti, 80078,
Pozzuoli (NA), Italy; §Dipartimento di Scienze Farmaceutiche, Università degli Studi di Salerno,
via Ponte Don Melillo, 84084 Fisciano (SA), Italy
Corresponding authors: V. Di Marzo, E-mail: vdimarzo@icmib.na.cnr.it; M. Bifulco, E-mail:
maubiful@unina.it
G.P. and C.L. contributed equally to this work.
ABSTRACT
Stimulation of cannabinoid CB1 receptors by 2-methyl-arachidonyl-2'-fluoro-ethylamide (Met-FAEA)
inhibits the growth of a rat thyroid cancer cell-derived tumor in athymic mice by
inhibiting the activity of the oncogene product p21ras. Here we report that Met-F-AEA also
blocks the growth of tumors previously induced in nude mice by the s.c. injection of the same rat
thyroid carcinoma cells. Met-F-AEA significantly inhibited, in tumors as well as transformed
cells, the expression of the vascular endothelial growth factor, an angiogenetic factor known to
be up-regulated by p21ras, as well as of one of its receptors, flt-1/VEGFR-1. The levels of the
cyclin-dependent kinase inhibitor p27(kip1), which is down-regulated by p21ras, were instead
increased by Met-F-AEA. All these effects were antagonized by the selective CB1 receptor
antagonist SR141716A. Met-F-AEA inhibited in vitro the growth of a metastasis-derived thyroid
cancer cell line more potently than a primary cancer cell line. Therefore, the hypothesis that CB1
receptor stimulation interferes not only with angiogenesis but also with metastatic processes was
tested in a widely used model of metastatic infiltration in vivo, the Lewis lung carcinoma (3LL)
in C57Bl/6 mice. Three weeks from the paw injection of 3LL cells, Met-F-AEA reduced
significantly the number of metastatic nodes, in a way antagonized by SR141716A. Our findings
indicate that CB1 receptor agonists might be used therapeutically to retard tumor growth in vivo
by inhibiting at once tumor growth, angiogenesis, and metastasis.
Key words: thyroid - cancer - endocannabinoid - p27 - lung - VEGF
9-Tetrahydrocannabinol (Δ9-THC) is the major psychoactive component of Cannabis
sativa, but it also exerts profound effects on several biological systems other than the
central nervous system by binding to specific receptor sites, the CB1 and CB2 cannabinoid Δ
receptors, thereby eliciting a variety of pharmacological responses (1). Both cannabinoid
receptors are seven-transmembrane domain, Gi/o protein-coupled proteins whose various
biological responses are abolished following treatment of tissues and cells with pertussis toxin
(2). In 1992, Devane et al. isolated N-arachidonoylethanolamine (anandamide) from pig brain as
an endogenous cannabinoid receptor ligand (3). Anandamide seems to be ubiquitous in mammals
(4), and several studies have suggested that this as well as the other endogenous cannabinoid
receptor ligands (or endocannabinoids) discovered to date, that is, 2-arachidonoylglycerol (2-
AG) (5, 6), noladin ether (7), and N-arachidonoyldopamine (8), can modulate cell survival or
death (see ref 9 for review).
The endocannabinoids were shown to inhibit the growth of human breast and prostate cancer
cells (see refs 10—12 and ref 13 for review). Anandamide inhibits the proliferation of human
breast cancer cells by blocking the G0/G1-S phase transition of the cell cycle through interference
with CB1 receptor-coupled signal transducing events (10, 14). It has been demonstrated that an
analog of anandamide, 2-methyl-arachidonyl-2'-fluoro-ethylamide (Met-F-AEA), which is more
metabolically stable and more potent as a CB1 ligand, is able to greatly reduce the growth of
experimental tumors induced by s.c. injection in athymic mice of rat thyroid cells transformed by
the Kirsten murine sarcoma virus (KiMSV) and expressing high levels of v-K-ras oncogene (15).
We suggested that anandamide and CB1 receptors are part of an endogenous signaling system
that can be targeted pharmacologically for the inhibition of v-K-ras oncogene-dependent cancer
cell proliferation (15) and have hypothesized that activation of this system may interfere also
with other biological processes involved in tumor growth and spreading, such as angiogenesis
and metastasis (16). This hypothesis is based also on the finding that Δ9-THC and synthetic
agonists of CB1 and CB2 receptors inhibit glioma growth in vitro and in vivo by inducing the
apoptosis of glioma cells (17, 18).
In the present study, we wanted to assess whether cancer growth in vivo would be limited by
cannabinoid receptors when the tumor is already established and growing. Therefore, we studied
the possible tumor-growth inhibitory effect of intratumoral administrations of Met-F-AEA and
investigated the possibility that this compound, by acting at CB1 receptors, also interferes with
angiogenesis and metastatic processes. We studied the effect of Met-F-AEA on the expression of
molecules controlling angiogenesis and metastasis, such as the vascular endothelial growth factor
(VEGF) and one of its receptors, and the cyclin-dependent kinase inhibitor p27(kip1). We
examined whether Met-F-AEA affects in the same way the proliferation of v-K-ras-expressing
cell lines derived either from a rat thyroid carcinoma (TK-6 cells) or from its lung metastasis
(MPTK-6 cells). Finally, by using a different as well as widely used in vivo model for the study
of metastasis, we evaluated the direct effect of Met-F-AEA on the number of metastatic nodes in
mice. Our data indicate for the first time that substances capable of targeting the
endocannabinoid system can interfere at multiple levels with the growth and spreading of
established tumors in vivo.
MATERIALS AND METHODS
Cells and culture
Normal rat thyroid FRTL-5 cell line was cultured in Coon's modified Ham's F-12 medium
supplemented with 5% calf serum (Sigma, Milano, Italia) and six growth factors (TSH,
hydrocortisone, insulin, transferrin, somatostatin, and glycil-histidil-lysine) (Sigma, St. Louis,
MO), as described previously (19). This cell line retains in vitro the typical markers of thyroid
differentiation, that is, thyroglobulin (TG), thyrotropin-receptor (TSH-R), and thyroperoxidase
(TPO) gene expression; iodide uptake; and dependency on TSH for growth. v-K-ras-transformed
FRTL-5 cells (KiMol cells), derived from FRTL-5 cells by infection with a wild-type strain of
KiMSV-MolMuLV (20, 21), were kindly provided by G. Vecchio and A. Fusco (Napoli, Italy).
We grew KiMol cells at 37°C in Coon's modified Ham's F-12 medium, supplemented with 5%
calf serum. TK-6 and MPTK-6 cell lines were obtained from a rat thyroid carcinoma (TK-6
cells) or its lung metastasis (MPTK-6 cells). TK-6 and MPTK-6 cells were cultured in Coon's
modified Ham's F-12 medium, supplemented with 5% calf serum (22). Lewis lung carcinoma
(3LL) cells were kindly provided by Dr. G. Zupi (Regina Elena Cancer Institute, Roma, Italy),
and cultured as described previously (23).
Cell proliferation assays
Cell proliferation assays were carried out in six-well dishes containing subconfluent cells at a
density of ∼60,000 cells/well, and according to the method described previously (15). Met-FAEA,
at a concentration (10 μM) previously found to exert maximal antiproliferative effects
(10—15), or vehicle was added every day at each change of medium for 4 days, after which, cells
were counted by a hemocytometer.
Tumorigenicity assay
We performed all experiments in 6-wk-old male athymic mice. Animals were injected (day 0) on
the dorsal right side with a suspension of 0.2 ml containing 5 × 105 KiMol cells. KiMol cells are
able to induce the growth of undifferentiated carcinomas when injected s.c. into athymic mice
(15, 22), and the site of injection was marked. When tumors on the dorsal right side were clearly
detectable, mice were divided into three groups, and 2-methyl-arachidonyl-2'-fluoro-ethylamide
(Met-F-AEA, 0.5 mg/kg/dose; Calbiochem, Darnstadt, Germany) or Met-F-AEA or Met-F-AEA
+ SR141716A (0.7 mg/kg/dose, a kind gift by Sanofi-Synthelabo), from stock solutions were
diluted into 0.2 ml of sterile saline solution (0.9% NaCl) and injected s.c. at the previous
injection site. Met-F-AEA was diluted from a stock solution in water and ethanol, whose final
concentration in the injected saline solution was <0.05%. SR141716A was introduced from a
stock solution in dimethylsulfoxide (DMSO), whose final concentration in the injected saline
solution was <0.1%.
The doses of Met-F-AEA and SR141716A were the same as those previously shown to exert,
respectively, maximal agonistic and antagonistic effects in vivo on thyroid cancer cell growth
(15). SR141716A was not tested alone because on KiMol cells this compound (0.1 μM) exerted
no effect per se, or, at concentrations higher than 0.5 μM, even inhibited KiMol cell proliferation
(15) as well as the proliferation of breast and mammary carcinoma cells (10—12). The vehicle
solution contained the same amounts of saline, ethanol, and DMSO as in the other two solutions.
Before injection, the solutions were sonicated in order to facilitate the solution of lipophilic
components. Treatment was repeated at 72-h intervals. Tumor diameters were measured weekly
and blind, using calipers. Tumor volumes (V) were calculated by the formula of rotational
ellipsoid: V = A × B2/2 (A, axial diameter; B, rotational diameter). After 27 days in the control
group, tumor burden exceeded 10% of the host weight and, therefore, according to the regulation
for animal welfare, the experiment was stopped, the animals were killed, and the tumor weight
was evaluated. During the treatment, no mice showed signs of wasting or other visible
indications of toxicity. Furthermore, the dose of Met-F-AEA showed no detectable reduction of
the spontaneous activity, as we observed unimpaired locomotion of the treated mice. All mice
were maintained at the Dipartimento di Biologia e Patologia animal facility, and all animal
studies were conducted in accordance with the Italian regulation for the welfare of animals in
experimental neoplasia.
Experimental lung metastasis
Monocellular suspension of 3LL cells containing 2.5 × 105 cells was injected into the left paw of
30-day-old C57BL/6N male mice (23). Animals were divided into three groups (20 animals
each), and Met-F-AEA (0.5 mg/kg/dose) or Met-F-AEA + SR141716A (0.7 mg/kg/dose) were
prepared as described above and injected i.p. every 72 h. Experimental metastases were
evaluated 21 days after the injection. To contrast lung nodules, lungs were fixed in Bouin's fluid,
and metastatic nodes were scored on dissected lung lobes under a stereoscopic microscope, as
previously described (23).
Western immunoblot
We prepared cell extracts from subconfluent cells grown in 100-mm Petri dishes. Cells were
washed twice in phosphate-buffered saline (PBS), scraped in PBS, and pelleted by
centrifugation. Cell pellets were resuspended in lysis buffer (10 mM Tris-HCl, pH 7.2; 1 mM
phenylmethylsulphonylfluoride; 2 µM aprotinin; and 10 µM leupeptin) disrupted by sonication
and centrifuged at 3000 rpm for 10 min at 4°C. Proteins from tumor tissue were extracted
following the same procedure described above. Proteins (50 µg) were electrophoresed on 12%
sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to
nitrocellulose membrane, and blocked with 7.5% milk in Tris-buffered saline and Tween 20 for 1
h at room temperature. The filters were then probed with CB1 receptor polyclonal antibodies
(Cayman, East Lansing, MI), VEGF or VEGF-R Flt-1 rabbit policlonal antibodies (Santa Cruz
Biotechnology, Santa Cruz, CA), or p27(kip1) mouse monoclonal antibody (Oncogene Research
Products, San Diego, CA). Immunoreactive proteins were detected by incubation with
horseradish peroxidase-conjugated donkey anti-rabbit IgG (Bio-Rad, Hercules, CA) by using the
enhanced chemiluminescence system (ECL;Amersham, Buckinghamshire, UK). The coefficient
of analytical variations of Western blot experiments was 4%. When commercially available, the
blocking peptides corresponding to each antibody were used to assess the specificity of the
immunoblots.
RESULTS
Effect of Met-F-AEA on the growth of established tumors
We used a metabolically stable analog of anandamide, Met-F-AEA, because anandamide is
rapidly metabolizd in vivo (4). Because the effect of Met-F-AEA on incipient tumors has been
previously analyzed, in order to evaluate the effects of this compound on already established
tumors, we 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
(15, 22). Twenty days later, when tumors were clearly detectable, saline solution containing Met-
F-AEA (0.5 mg/kg/dose) was injected in the peritumoral area on days 2 and 5 of a 7-day cycle
for 4 wk. Met-F-AEA treatment induced a drastic reduction in tumor volume with respect to the
vehicle control-treated mice (Fig. 1). This effect was significantly inhibited by the CB1 receptor
antagonist SR141716A (0.7 mg/kg/dose, s.c. intratumor, P<0.01 by ANOVA, Fig. 1).
Effect of Met-F-AEA on the levels of endogenous pro- and antiangiogenic factors
Because we observed that the tumors from Met-F-AEA-treated mice were paler than the tumors
from untreated mice, and because we have described that Met-F-AEA is able to block p21ras
activity via CB1 receptors (15), we investigated whether this compound could inhibit
angiogenesis by affecting the expression of VEGF, which in turn depends on the activity of
p21ras (24). Western blot analysis of proteins from treated or untreated tumors (Fig. 2) showed
that VEGF levels were dramatically reduced by Met-F-AEA treatment. In addition, we observed
that Met-F-AEA treatment also reduced the expression of one of the VEGF receptors (Flt-
1/VEGFR-1) in tumors (Fig. 2), thus indicating that this treatment is very likely to result in a
strong inhibition of VEGF signaling and, hence, tumor angiogenesis. These inhibitory effects of
Met-F-AEA were attenuated by the selective CB1 receptor antagonist, SR141716A (Fig. 2), thus
strongly suggesting the involvement of CB1 receptors in the anti-VEGF action of the compound.
Note that Met-F-AEA addition to KiMol cells was also able to significantly decrease VEGF and
VEGF receptor (Flt-1/VEGFR-1) expression (Fig. 2).
The cyclin-dependent kinase inhibitor p27(kip1) is another protein suggested to play a role as a
proangiogenic factor (25), and is under the negative control of the ras oncogene in proliferating
human thyroid cells (26). We found that Met-F-AEA treatment of tumors and Ki-Mol cells
increased p27(kip1) levels, and that this effect was attenuated by the selective CB1 receptor
antagonist, SR141716A (Fig. 3).
Effect of Met-F-AEA on metastatic cells
Because underexpression of p27(kip1) has been correlated with increased spreading of thyroid
cancer cells to lymph nodes (27), and VEGF itself has also been implicated in cancer cell
metastasis (28), we investigated the effect of Met-F-AEA on metastatic processes. We compared
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). The metastasis-derived MPTK-6
cell line displays a highly more malignant phenotype with respect to TK-6 cells (22, 29). Fourday
treatment with Met-F-AEA was able to inhibit the proliferation of both neoplastic thyroid
cell lines (Fig. 4a). However, the growth of metastasis-derived cells was inhibited more
efficaciously than that of primary thyroid carcinoma-derived cells (P<0.01, Fig. 4a), and this was
accompanied by a stronger up-regulation of CB1 receptor levels in MPTK-6 cells than in TK-6
(Fig. 4b), together with a stronger down-regulation of VEGF receptor levels in MPTK-6 than in
TK-6 cells (data not shown).
Effect of Met-F-AEA on the formation of metastatic nodules in the 3LL model
The data just described prompted us to test 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. As
shown in Figure 5, a dramatic inhibitory effect of Met-F-AEA was observed against lung
metastatic nodules induced by 3LL cells. Note that even those few metastatic nodules observed
in Met-F-AEA-treated animals were smaller compared with those present in control animals
(data not shown). The metastatic growth inhibitory effect was blocked by the CB1 receptor
antagonist SR141716A (P<0.01 by ANOVA).
DISCUSSION
Previous data indicate that cannabinoid receptors are likely to represent a new endogenous
signaling system that can be targeted pharmacologically for the inhibition of cancer growth and
suggest the use of anandamide-based drugs as anticancer drugs (see ref 16 for review). In
particular, we recently reported that stimulation of cannabinoid CB1 receptors by the
metabolically stable endocannabinoid analog Met-F-AEA, by inhibiting p21ras activity, prevents
proliferation of v-K-ras-transformed rat thyroid cells both in vitro and in vivo, when injected
concomitantly to transformed cells into athymic mice (15). In the present study, we observed that
Met-F-AEA is also able to block the growth of already established tumors, thus indicating that
anandamide-based drugs may be efficacious therapeutic drugs for the inhibition of cancer cell
growth.
The major functional emphasis on oncogenes, such as mutant ras, as contributors to tumor
development has been on their impact on promoting aberrant cellular mitogenesis. However,
oncogenes may also have an impact on tumor formation and growth through indirect
mechanisms, such as tumor angiogenesis (30). Thus, oncogenic mutations or amplification of ras
are associated with up-regulation of the proangiogenic factor VEGF, whose enhanced expression
is in turn associated with a large number of human tumor types or with cell transformation events
(31). In the mouse skin cancerogenesis model, characterized by the activation of the Ha-ras
oncogene, the development of papillomas is preceded by a burst of angiogenesis, and Ha-ras
activation induces VEGF expression in mouse keratinocytes and other cell types (32).
Furthermore, it has been described that genetic disruption of the single mutant K-ras allele in
two different human colorectal carcinoma cell lines was associated with a loss of tumorigenic
competence and significant suppression of VEGF expression (33). VEGF up-regulation has also
been associated with malignancy in human thyroid tumors and cancer cells (34—36), and because
activation of CB1 receptors by Met-F-AEA is able to block p21ras activity (15), the effect of this
compound on VEGF signaling was analyzed here, showing a dramatic reduction of VEGF
expression.
This finding suggests that because Met-F-AEA is not capable of inducing apoptosis of thyroid
cancer cells in our rat thyroid cancer model (15), a likely means by which that CB1 stimulation
leads to blockade of the growth of established tumors observed here might be the inhibition of
angiogenesis. This hypothesis was strengthened by our finding of a suppressing effect by Met-FAEA
on the expression of the VEGF receptor Flt-1 (also known as fms-like tyrosine kinase, or
VEGFR-1), one of the two receptors that play a crucial role in mediating VEGF-induced
neoangiogenesis and endothelial cell proliferation (34, 37, 38). Importantly, we found that the
expression of both VEGF and Flt-1 was suppressed not only in the tumor in vivo, but also in
KiMol, TK-6, and MPTK-6 cells in vitro. This finding is in agreement with the recent
observation that VEGF receptor-neutralizing antibodies inhibit the proliferation of VEGF
receptor-containing tumor cells, indicating that blocking VEGF signaling may have a direct
effect on tumor cell growth by disrupting a VEGF-VEGF receptor autocrine pathway (28).
Moreover, VEGF released from cancer cells can also exert a role as a paracrine factor able to
stimulate the proliferation of endothelial cells, the formation of new vessels, and, possibly, the
migration and spreading of cancer cells (28, 39). Therefore, the down-regulation of both VEGF
and Flt-1 induced in vivo by Met-F-AEA may also lead to a direct effect not only on tumor
neoangiogenesis but also on growth and metastasis.
Another molecule involved in cancer cell proliferation, by governing cyclin-dependent kinase-2
activity during the transition from G1 to S phase, and whose levels are regulated by degradation
via the ubiquitin-proteasome pathway and whose overexpression can block cell cycle
progression in the G1 phase, is p27(kip1). This protein was recently shown to inhibit endothelial
cell proliferation and migration, and its overexpression also was found to inhibit angiogenesis
(25). Furthermore, p27(kip1) is under the negative control of the ras oncogene in proliferating
human epithelial thyroid cells (26). These previous observations, and our finding of the
inhibition of p21ras activity and VEGF signaling by Met-F-AEA, provided the rationale to also
test this compound on p27(kip1) levels, which were found to be sensibly increased in both
tumors and KiMol cells. This suggests that overexpression of p27(kip1) might contribute to the
effect of Met-F-AEA on tumor growth and neoangiogenesis.
Because both p27(kip1) and VEGF have also been implicated in the processes of cancer cell
migration and/or metastasis (27, 28), and their expression was shown here to be blocked by Met-
F-AEA, we investigated the effects of this compound on metastatic cells. We found that 4-day
treatment with Met-F-AEA, which was previously shown to inhibit the proliferation of ras-
transformed KiMol cells much more efficaciously than nontransformed FRTL-5 cells (15),
inhibited the proliferation of the metastasis-derived thyroid cancer MPTK-6 cell line more
efficaciously than in the primary thyroid TK-6 cancer line. This was probably due to the fact that
4-day treatment with Met-F-AEA led to a stronger up-regulation of CB1 receptors in MPTK-6
cells than in TK-6 cells, much in the same way the compound was previously shown (15) to upregulate
or slightly down-regulate CB1 expression in Ki-Mol and FRTL-5 cells, respectively.
In summary, Met-F-AEA, probably via differential effects on CB1 receptor expression, appears
to be the more efficacious as an antiproliferative agent against rat thyroid cells the more these
cells become malignant and invasive. This observation is in agreement with the previous finding
of higher levels of cannabinoid CB2 receptors in astrocytomas with a higher degree of
malignancy (18) and with the finding of little responsiveness to endocannabinoids of colorectal
carcinoma CaCo-2 cells in culture when they are differentiated into noninvasive cells (A.
Ligresti, L. De Petrocellis, G. D'Argenio, M. Bifulco, I. Sorrentini, and V. Di Marzo,
unpublished observations). More importantly, these findings strengthen our hypothesis that Met-
F-AEA might also interfere efficaciously with the spread of cancer cells and with the formation
of metastases.
We decided to gain direct evidence for this hypothesis by testing the effect of Met-F-AEA on a
widely used model of metastatic spreading in vivo, the formation of lung nodules after
inoculation of 3LL cells. We found that the compound produced a strong reduction in both the
number and size of metastatic nodes in this model, in a way that was again counteracted by the
CB1 receptor antagonist. Therefore, our findings indicate that stimulation of CB1 receptors might
be a therapeutically useful strategy not only to retard the growth, but also to inhibit the metastatic
spreading, of cancer cells in vivo.
In conclusion, we have shown here that local administration of the stable anandamide analog and
cannabinoid CB1 receptor agonist, Met-F-AEA, blocks the growth of an already established rat
thyroid carcinoma in athymic mice. We have also provided evidence that this strong anticancer
effect might be due at least in part to inhibition of angiogenesis, because it was accompanied by
blockade of VEGF signaling and overexpression of p21(kip1). Furthermore, we have shown that
Met-F-AEA, by acting at CB1 receptors, more efficaciously inhibits the proliferation of
metastasis-derived than primary tumor-derived rat thyroid cancer cells and counteracts the
formation of metastatic loci in an in vivo model of metastasis. These actions were exerted after
local administration of a Met-F-AEA dose (0.5 mg/kg) at least 10-fold lower than those
previously shown to be necessary, when administered systemically, to exert a 50% decrease of
motor behavior, nociception, and body temperature in mice (40). Hence, it is very likely that this
type of treatment produces no important "central" side-effects.
Clearly, further studies are required to analyze in detail the mechanism(s), and the extent in vivo,
of the antiangiogenetic and antimetastatic effects of Met-F-AEA. During the submission of this
manuscript, two studies carried out by another laboratory independently from ours, have indeed
reported that cannabinoid receptor activation inhibits angiogenesis via inhibition of
proangiogenic factor expression, interference with endothelial cell migration, and induction of
endothelial cell apoptosis, thereby retarding skin cancer and glioma growth in vivo (41, 42). Our
findings go beyond these latest observations by demonstrating for the first time that CB1 receptor
stimulation a) may inhibit tumor growth by also targeting VEGF receptor and p27(kip1)
expression and b) interferes with metastatic processes in vitro and in vivo, and indicate
unequivocally that the endocannabinoid system might be targeted pharmacologically to block the
processes intervening in cancer growth and spreading at multiple levels.
ACKNOWLEDGMENTS
We thank M. Berardone for the art work. This study was supported by the Associazione Italiana
per la Ricerca sul Cancro (AIRC) (grant to M.B.).
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Source: Inhibitory effects of cannabinoid CB1 receptor stimulation on tumor growth and metastatic spreading: actions on signals involved in angiogenesis and metastasis
on tumor growth and metastatic spreading: actions on
signals involved in angiogenesis and metastasis
Giuseppe Portella,* Chiara Laezza,* Paolo Laccetti,* Luciano De Petrocellis,"
Vincenzo Di Marzo,"¡ and Maurizio Bifulco*,§
*," ,"¡Endocannabinoid Research Group: *Istituto di Endocrinologia ed Oncologia Sperimentale,
Consiglio Nazionale delle Ricerche, and Dipartimento di Biologia e Patologia Cellulare e
Molecolare "L. Califano," Università di Napoli "Federico II, " Istituto di Cibernetica and "¡Istituto
di Chimica Biomolecolare, C.N.R., Via Campi Flegrei 34, Comprensorio Olivetti, 80078,
Pozzuoli (NA), Italy; §Dipartimento di Scienze Farmaceutiche, Università degli Studi di Salerno,
via Ponte Don Melillo, 84084 Fisciano (SA), Italy
Corresponding authors: V. Di Marzo, E-mail: vdimarzo@icmib.na.cnr.it; M. Bifulco, E-mail:
maubiful@unina.it
G.P. and C.L. contributed equally to this work.
ABSTRACT
Stimulation of cannabinoid CB1 receptors by 2-methyl-arachidonyl-2'-fluoro-ethylamide (Met-FAEA)
inhibits the growth of a rat thyroid cancer cell-derived tumor in athymic mice by
inhibiting the activity of the oncogene product p21ras. Here we report that Met-F-AEA also
blocks the growth of tumors previously induced in nude mice by the s.c. injection of the same rat
thyroid carcinoma cells. Met-F-AEA significantly inhibited, in tumors as well as transformed
cells, the expression of the vascular endothelial growth factor, an angiogenetic factor known to
be up-regulated by p21ras, as well as of one of its receptors, flt-1/VEGFR-1. The levels of the
cyclin-dependent kinase inhibitor p27(kip1), which is down-regulated by p21ras, were instead
increased by Met-F-AEA. All these effects were antagonized by the selective CB1 receptor
antagonist SR141716A. Met-F-AEA inhibited in vitro the growth of a metastasis-derived thyroid
cancer cell line more potently than a primary cancer cell line. Therefore, the hypothesis that CB1
receptor stimulation interferes not only with angiogenesis but also with metastatic processes was
tested in a widely used model of metastatic infiltration in vivo, the Lewis lung carcinoma (3LL)
in C57Bl/6 mice. Three weeks from the paw injection of 3LL cells, Met-F-AEA reduced
significantly the number of metastatic nodes, in a way antagonized by SR141716A. Our findings
indicate that CB1 receptor agonists might be used therapeutically to retard tumor growth in vivo
by inhibiting at once tumor growth, angiogenesis, and metastasis.
Key words: thyroid - cancer - endocannabinoid - p27 - lung - VEGF
9-Tetrahydrocannabinol (Δ9-THC) is the major psychoactive component of Cannabis
sativa, but it also exerts profound effects on several biological systems other than the
central nervous system by binding to specific receptor sites, the CB1 and CB2 cannabinoid Δ
receptors, thereby eliciting a variety of pharmacological responses (1). Both cannabinoid
receptors are seven-transmembrane domain, Gi/o protein-coupled proteins whose various
biological responses are abolished following treatment of tissues and cells with pertussis toxin
(2). In 1992, Devane et al. isolated N-arachidonoylethanolamine (anandamide) from pig brain as
an endogenous cannabinoid receptor ligand (3). Anandamide seems to be ubiquitous in mammals
(4), and several studies have suggested that this as well as the other endogenous cannabinoid
receptor ligands (or endocannabinoids) discovered to date, that is, 2-arachidonoylglycerol (2-
AG) (5, 6), noladin ether (7), and N-arachidonoyldopamine (8), can modulate cell survival or
death (see ref 9 for review).
The endocannabinoids were shown to inhibit the growth of human breast and prostate cancer
cells (see refs 10—12 and ref 13 for review). Anandamide inhibits the proliferation of human
breast cancer cells by blocking the G0/G1-S phase transition of the cell cycle through interference
with CB1 receptor-coupled signal transducing events (10, 14). It has been demonstrated that an
analog of anandamide, 2-methyl-arachidonyl-2'-fluoro-ethylamide (Met-F-AEA), which is more
metabolically stable and more potent as a CB1 ligand, is able to greatly reduce the growth of
experimental tumors induced by s.c. injection in athymic mice of rat thyroid cells transformed by
the Kirsten murine sarcoma virus (KiMSV) and expressing high levels of v-K-ras oncogene (15).
We suggested that anandamide and CB1 receptors are part of an endogenous signaling system
that can be targeted pharmacologically for the inhibition of v-K-ras oncogene-dependent cancer
cell proliferation (15) and have hypothesized that activation of this system may interfere also
with other biological processes involved in tumor growth and spreading, such as angiogenesis
and metastasis (16). This hypothesis is based also on the finding that Δ9-THC and synthetic
agonists of CB1 and CB2 receptors inhibit glioma growth in vitro and in vivo by inducing the
apoptosis of glioma cells (17, 18).
In the present study, we wanted to assess whether cancer growth in vivo would be limited by
cannabinoid receptors when the tumor is already established and growing. Therefore, we studied
the possible tumor-growth inhibitory effect of intratumoral administrations of Met-F-AEA and
investigated the possibility that this compound, by acting at CB1 receptors, also interferes with
angiogenesis and metastatic processes. We studied the effect of Met-F-AEA on the expression of
molecules controlling angiogenesis and metastasis, such as the vascular endothelial growth factor
(VEGF) and one of its receptors, and the cyclin-dependent kinase inhibitor p27(kip1). We
examined whether Met-F-AEA affects in the same way the proliferation of v-K-ras-expressing
cell lines derived either from a rat thyroid carcinoma (TK-6 cells) or from its lung metastasis
(MPTK-6 cells). Finally, by using a different as well as widely used in vivo model for the study
of metastasis, we evaluated the direct effect of Met-F-AEA on the number of metastatic nodes in
mice. Our data indicate for the first time that substances capable of targeting the
endocannabinoid system can interfere at multiple levels with the growth and spreading of
established tumors in vivo.
MATERIALS AND METHODS
Cells and culture
Normal rat thyroid FRTL-5 cell line was cultured in Coon's modified Ham's F-12 medium
supplemented with 5% calf serum (Sigma, Milano, Italia) and six growth factors (TSH,
hydrocortisone, insulin, transferrin, somatostatin, and glycil-histidil-lysine) (Sigma, St. Louis,
MO), as described previously (19). This cell line retains in vitro the typical markers of thyroid
differentiation, that is, thyroglobulin (TG), thyrotropin-receptor (TSH-R), and thyroperoxidase
(TPO) gene expression; iodide uptake; and dependency on TSH for growth. v-K-ras-transformed
FRTL-5 cells (KiMol cells), derived from FRTL-5 cells by infection with a wild-type strain of
KiMSV-MolMuLV (20, 21), were kindly provided by G. Vecchio and A. Fusco (Napoli, Italy).
We grew KiMol cells at 37°C in Coon's modified Ham's F-12 medium, supplemented with 5%
calf serum. TK-6 and MPTK-6 cell lines were obtained from a rat thyroid carcinoma (TK-6
cells) or its lung metastasis (MPTK-6 cells). TK-6 and MPTK-6 cells were cultured in Coon's
modified Ham's F-12 medium, supplemented with 5% calf serum (22). Lewis lung carcinoma
(3LL) cells were kindly provided by Dr. G. Zupi (Regina Elena Cancer Institute, Roma, Italy),
and cultured as described previously (23).
Cell proliferation assays
Cell proliferation assays were carried out in six-well dishes containing subconfluent cells at a
density of ∼60,000 cells/well, and according to the method described previously (15). Met-FAEA,
at a concentration (10 μM) previously found to exert maximal antiproliferative effects
(10—15), or vehicle was added every day at each change of medium for 4 days, after which, cells
were counted by a hemocytometer.
Tumorigenicity assay
We performed all experiments in 6-wk-old male athymic mice. Animals were injected (day 0) on
the dorsal right side with a suspension of 0.2 ml containing 5 × 105 KiMol cells. KiMol cells are
able to induce the growth of undifferentiated carcinomas when injected s.c. into athymic mice
(15, 22), and the site of injection was marked. When tumors on the dorsal right side were clearly
detectable, mice were divided into three groups, and 2-methyl-arachidonyl-2'-fluoro-ethylamide
(Met-F-AEA, 0.5 mg/kg/dose; Calbiochem, Darnstadt, Germany) or Met-F-AEA or Met-F-AEA
+ SR141716A (0.7 mg/kg/dose, a kind gift by Sanofi-Synthelabo), from stock solutions were
diluted into 0.2 ml of sterile saline solution (0.9% NaCl) and injected s.c. at the previous
injection site. Met-F-AEA was diluted from a stock solution in water and ethanol, whose final
concentration in the injected saline solution was <0.05%. SR141716A was introduced from a
stock solution in dimethylsulfoxide (DMSO), whose final concentration in the injected saline
solution was <0.1%.
The doses of Met-F-AEA and SR141716A were the same as those previously shown to exert,
respectively, maximal agonistic and antagonistic effects in vivo on thyroid cancer cell growth
(15). SR141716A was not tested alone because on KiMol cells this compound (0.1 μM) exerted
no effect per se, or, at concentrations higher than 0.5 μM, even inhibited KiMol cell proliferation
(15) as well as the proliferation of breast and mammary carcinoma cells (10—12). The vehicle
solution contained the same amounts of saline, ethanol, and DMSO as in the other two solutions.
Before injection, the solutions were sonicated in order to facilitate the solution of lipophilic
components. Treatment was repeated at 72-h intervals. Tumor diameters were measured weekly
and blind, using calipers. Tumor volumes (V) were calculated by the formula of rotational
ellipsoid: V = A × B2/2 (A, axial diameter; B, rotational diameter). After 27 days in the control
group, tumor burden exceeded 10% of the host weight and, therefore, according to the regulation
for animal welfare, the experiment was stopped, the animals were killed, and the tumor weight
was evaluated. During the treatment, no mice showed signs of wasting or other visible
indications of toxicity. Furthermore, the dose of Met-F-AEA showed no detectable reduction of
the spontaneous activity, as we observed unimpaired locomotion of the treated mice. All mice
were maintained at the Dipartimento di Biologia e Patologia animal facility, and all animal
studies were conducted in accordance with the Italian regulation for the welfare of animals in
experimental neoplasia.
Experimental lung metastasis
Monocellular suspension of 3LL cells containing 2.5 × 105 cells was injected into the left paw of
30-day-old C57BL/6N male mice (23). Animals were divided into three groups (20 animals
each), and Met-F-AEA (0.5 mg/kg/dose) or Met-F-AEA + SR141716A (0.7 mg/kg/dose) were
prepared as described above and injected i.p. every 72 h. Experimental metastases were
evaluated 21 days after the injection. To contrast lung nodules, lungs were fixed in Bouin's fluid,
and metastatic nodes were scored on dissected lung lobes under a stereoscopic microscope, as
previously described (23).
Western immunoblot
We prepared cell extracts from subconfluent cells grown in 100-mm Petri dishes. Cells were
washed twice in phosphate-buffered saline (PBS), scraped in PBS, and pelleted by
centrifugation. Cell pellets were resuspended in lysis buffer (10 mM Tris-HCl, pH 7.2; 1 mM
phenylmethylsulphonylfluoride; 2 µM aprotinin; and 10 µM leupeptin) disrupted by sonication
and centrifuged at 3000 rpm for 10 min at 4°C. Proteins from tumor tissue were extracted
following the same procedure described above. Proteins (50 µg) were electrophoresed on 12%
sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to
nitrocellulose membrane, and blocked with 7.5% milk in Tris-buffered saline and Tween 20 for 1
h at room temperature. The filters were then probed with CB1 receptor polyclonal antibodies
(Cayman, East Lansing, MI), VEGF or VEGF-R Flt-1 rabbit policlonal antibodies (Santa Cruz
Biotechnology, Santa Cruz, CA), or p27(kip1) mouse monoclonal antibody (Oncogene Research
Products, San Diego, CA). Immunoreactive proteins were detected by incubation with
horseradish peroxidase-conjugated donkey anti-rabbit IgG (Bio-Rad, Hercules, CA) by using the
enhanced chemiluminescence system (ECL;Amersham, Buckinghamshire, UK). The coefficient
of analytical variations of Western blot experiments was 4%. When commercially available, the
blocking peptides corresponding to each antibody were used to assess the specificity of the
immunoblots.
RESULTS
Effect of Met-F-AEA on the growth of established tumors
We used a metabolically stable analog of anandamide, Met-F-AEA, because anandamide is
rapidly metabolizd in vivo (4). Because the effect of Met-F-AEA on incipient tumors has been
previously analyzed, in order to evaluate the effects of this compound on already established
tumors, we 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
(15, 22). Twenty days later, when tumors were clearly detectable, saline solution containing Met-
F-AEA (0.5 mg/kg/dose) was injected in the peritumoral area on days 2 and 5 of a 7-day cycle
for 4 wk. Met-F-AEA treatment induced a drastic reduction in tumor volume with respect to the
vehicle control-treated mice (Fig. 1). This effect was significantly inhibited by the CB1 receptor
antagonist SR141716A (0.7 mg/kg/dose, s.c. intratumor, P<0.01 by ANOVA, Fig. 1).
Effect of Met-F-AEA on the levels of endogenous pro- and antiangiogenic factors
Because we observed that the tumors from Met-F-AEA-treated mice were paler than the tumors
from untreated mice, and because we have described that Met-F-AEA is able to block p21ras
activity via CB1 receptors (15), we investigated whether this compound could inhibit
angiogenesis by affecting the expression of VEGF, which in turn depends on the activity of
p21ras (24). Western blot analysis of proteins from treated or untreated tumors (Fig. 2) showed
that VEGF levels were dramatically reduced by Met-F-AEA treatment. In addition, we observed
that Met-F-AEA treatment also reduced the expression of one of the VEGF receptors (Flt-
1/VEGFR-1) in tumors (Fig. 2), thus indicating that this treatment is very likely to result in a
strong inhibition of VEGF signaling and, hence, tumor angiogenesis. These inhibitory effects of
Met-F-AEA were attenuated by the selective CB1 receptor antagonist, SR141716A (Fig. 2), thus
strongly suggesting the involvement of CB1 receptors in the anti-VEGF action of the compound.
Note that Met-F-AEA addition to KiMol cells was also able to significantly decrease VEGF and
VEGF receptor (Flt-1/VEGFR-1) expression (Fig. 2).
The cyclin-dependent kinase inhibitor p27(kip1) is another protein suggested to play a role as a
proangiogenic factor (25), and is under the negative control of the ras oncogene in proliferating
human thyroid cells (26). We found that Met-F-AEA treatment of tumors and Ki-Mol cells
increased p27(kip1) levels, and that this effect was attenuated by the selective CB1 receptor
antagonist, SR141716A (Fig. 3).
Effect of Met-F-AEA on metastatic cells
Because underexpression of p27(kip1) has been correlated with increased spreading of thyroid
cancer cells to lymph nodes (27), and VEGF itself has also been implicated in cancer cell
metastasis (28), we investigated the effect of Met-F-AEA on metastatic processes. We compared
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). The metastasis-derived MPTK-6
cell line displays a highly more malignant phenotype with respect to TK-6 cells (22, 29). Fourday
treatment with Met-F-AEA was able to inhibit the proliferation of both neoplastic thyroid
cell lines (Fig. 4a). However, the growth of metastasis-derived cells was inhibited more
efficaciously than that of primary thyroid carcinoma-derived cells (P<0.01, Fig. 4a), and this was
accompanied by a stronger up-regulation of CB1 receptor levels in MPTK-6 cells than in TK-6
(Fig. 4b), together with a stronger down-regulation of VEGF receptor levels in MPTK-6 than in
TK-6 cells (data not shown).
Effect of Met-F-AEA on the formation of metastatic nodules in the 3LL model
The data just described prompted us to test 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. As
shown in Figure 5, a dramatic inhibitory effect of Met-F-AEA was observed against lung
metastatic nodules induced by 3LL cells. Note that even those few metastatic nodules observed
in Met-F-AEA-treated animals were smaller compared with those present in control animals
(data not shown). The metastatic growth inhibitory effect was blocked by the CB1 receptor
antagonist SR141716A (P<0.01 by ANOVA).
DISCUSSION
Previous data indicate that cannabinoid receptors are likely to represent a new endogenous
signaling system that can be targeted pharmacologically for the inhibition of cancer growth and
suggest the use of anandamide-based drugs as anticancer drugs (see ref 16 for review). In
particular, we recently reported that stimulation of cannabinoid CB1 receptors by the
metabolically stable endocannabinoid analog Met-F-AEA, by inhibiting p21ras activity, prevents
proliferation of v-K-ras-transformed rat thyroid cells both in vitro and in vivo, when injected
concomitantly to transformed cells into athymic mice (15). In the present study, we observed that
Met-F-AEA is also able to block the growth of already established tumors, thus indicating that
anandamide-based drugs may be efficacious therapeutic drugs for the inhibition of cancer cell
growth.
The major functional emphasis on oncogenes, such as mutant ras, as contributors to tumor
development has been on their impact on promoting aberrant cellular mitogenesis. However,
oncogenes may also have an impact on tumor formation and growth through indirect
mechanisms, such as tumor angiogenesis (30). Thus, oncogenic mutations or amplification of ras
are associated with up-regulation of the proangiogenic factor VEGF, whose enhanced expression
is in turn associated with a large number of human tumor types or with cell transformation events
(31). In the mouse skin cancerogenesis model, characterized by the activation of the Ha-ras
oncogene, the development of papillomas is preceded by a burst of angiogenesis, and Ha-ras
activation induces VEGF expression in mouse keratinocytes and other cell types (32).
Furthermore, it has been described that genetic disruption of the single mutant K-ras allele in
two different human colorectal carcinoma cell lines was associated with a loss of tumorigenic
competence and significant suppression of VEGF expression (33). VEGF up-regulation has also
been associated with malignancy in human thyroid tumors and cancer cells (34—36), and because
activation of CB1 receptors by Met-F-AEA is able to block p21ras activity (15), the effect of this
compound on VEGF signaling was analyzed here, showing a dramatic reduction of VEGF
expression.
This finding suggests that because Met-F-AEA is not capable of inducing apoptosis of thyroid
cancer cells in our rat thyroid cancer model (15), a likely means by which that CB1 stimulation
leads to blockade of the growth of established tumors observed here might be the inhibition of
angiogenesis. This hypothesis was strengthened by our finding of a suppressing effect by Met-FAEA
on the expression of the VEGF receptor Flt-1 (also known as fms-like tyrosine kinase, or
VEGFR-1), one of the two receptors that play a crucial role in mediating VEGF-induced
neoangiogenesis and endothelial cell proliferation (34, 37, 38). Importantly, we found that the
expression of both VEGF and Flt-1 was suppressed not only in the tumor in vivo, but also in
KiMol, TK-6, and MPTK-6 cells in vitro. This finding is in agreement with the recent
observation that VEGF receptor-neutralizing antibodies inhibit the proliferation of VEGF
receptor-containing tumor cells, indicating that blocking VEGF signaling may have a direct
effect on tumor cell growth by disrupting a VEGF-VEGF receptor autocrine pathway (28).
Moreover, VEGF released from cancer cells can also exert a role as a paracrine factor able to
stimulate the proliferation of endothelial cells, the formation of new vessels, and, possibly, the
migration and spreading of cancer cells (28, 39). Therefore, the down-regulation of both VEGF
and Flt-1 induced in vivo by Met-F-AEA may also lead to a direct effect not only on tumor
neoangiogenesis but also on growth and metastasis.
Another molecule involved in cancer cell proliferation, by governing cyclin-dependent kinase-2
activity during the transition from G1 to S phase, and whose levels are regulated by degradation
via the ubiquitin-proteasome pathway and whose overexpression can block cell cycle
progression in the G1 phase, is p27(kip1). This protein was recently shown to inhibit endothelial
cell proliferation and migration, and its overexpression also was found to inhibit angiogenesis
(25). Furthermore, p27(kip1) is under the negative control of the ras oncogene in proliferating
human epithelial thyroid cells (26). These previous observations, and our finding of the
inhibition of p21ras activity and VEGF signaling by Met-F-AEA, provided the rationale to also
test this compound on p27(kip1) levels, which were found to be sensibly increased in both
tumors and KiMol cells. This suggests that overexpression of p27(kip1) might contribute to the
effect of Met-F-AEA on tumor growth and neoangiogenesis.
Because both p27(kip1) and VEGF have also been implicated in the processes of cancer cell
migration and/or metastasis (27, 28), and their expression was shown here to be blocked by Met-
F-AEA, we investigated the effects of this compound on metastatic cells. We found that 4-day
treatment with Met-F-AEA, which was previously shown to inhibit the proliferation of ras-
transformed KiMol cells much more efficaciously than nontransformed FRTL-5 cells (15),
inhibited the proliferation of the metastasis-derived thyroid cancer MPTK-6 cell line more
efficaciously than in the primary thyroid TK-6 cancer line. This was probably due to the fact that
4-day treatment with Met-F-AEA led to a stronger up-regulation of CB1 receptors in MPTK-6
cells than in TK-6 cells, much in the same way the compound was previously shown (15) to upregulate
or slightly down-regulate CB1 expression in Ki-Mol and FRTL-5 cells, respectively.
In summary, Met-F-AEA, probably via differential effects on CB1 receptor expression, appears
to be the more efficacious as an antiproliferative agent against rat thyroid cells the more these
cells become malignant and invasive. This observation is in agreement with the previous finding
of higher levels of cannabinoid CB2 receptors in astrocytomas with a higher degree of
malignancy (18) and with the finding of little responsiveness to endocannabinoids of colorectal
carcinoma CaCo-2 cells in culture when they are differentiated into noninvasive cells (A.
Ligresti, L. De Petrocellis, G. D'Argenio, M. Bifulco, I. Sorrentini, and V. Di Marzo,
unpublished observations). More importantly, these findings strengthen our hypothesis that Met-
F-AEA might also interfere efficaciously with the spread of cancer cells and with the formation
of metastases.
We decided to gain direct evidence for this hypothesis by testing the effect of Met-F-AEA on a
widely used model of metastatic spreading in vivo, the formation of lung nodules after
inoculation of 3LL cells. We found that the compound produced a strong reduction in both the
number and size of metastatic nodes in this model, in a way that was again counteracted by the
CB1 receptor antagonist. Therefore, our findings indicate that stimulation of CB1 receptors might
be a therapeutically useful strategy not only to retard the growth, but also to inhibit the metastatic
spreading, of cancer cells in vivo.
In conclusion, we have shown here that local administration of the stable anandamide analog and
cannabinoid CB1 receptor agonist, Met-F-AEA, blocks the growth of an already established rat
thyroid carcinoma in athymic mice. We have also provided evidence that this strong anticancer
effect might be due at least in part to inhibition of angiogenesis, because it was accompanied by
blockade of VEGF signaling and overexpression of p21(kip1). Furthermore, we have shown that
Met-F-AEA, by acting at CB1 receptors, more efficaciously inhibits the proliferation of
metastasis-derived than primary tumor-derived rat thyroid cancer cells and counteracts the
formation of metastatic loci in an in vivo model of metastasis. These actions were exerted after
local administration of a Met-F-AEA dose (0.5 mg/kg) at least 10-fold lower than those
previously shown to be necessary, when administered systemically, to exert a 50% decrease of
motor behavior, nociception, and body temperature in mice (40). Hence, it is very likely that this
type of treatment produces no important "central" side-effects.
Clearly, further studies are required to analyze in detail the mechanism(s), and the extent in vivo,
of the antiangiogenetic and antimetastatic effects of Met-F-AEA. During the submission of this
manuscript, two studies carried out by another laboratory independently from ours, have indeed
reported that cannabinoid receptor activation inhibits angiogenesis via inhibition of
proangiogenic factor expression, interference with endothelial cell migration, and induction of
endothelial cell apoptosis, thereby retarding skin cancer and glioma growth in vivo (41, 42). Our
findings go beyond these latest observations by demonstrating for the first time that CB1 receptor
stimulation a) may inhibit tumor growth by also targeting VEGF receptor and p27(kip1)
expression and b) interferes with metastatic processes in vitro and in vivo, and indicate
unequivocally that the endocannabinoid system might be targeted pharmacologically to block the
processes intervening in cancer growth and spreading at multiple levels.
ACKNOWLEDGMENTS
We thank M. Berardone for the art work. This study was supported by the Associazione Italiana
per la Ricerca sul Cancro (AIRC) (grant to M.B.).
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Source: Inhibitory effects of cannabinoid CB1 receptor stimulation on tumor growth and metastatic spreading: actions on signals involved in angiogenesis and metastasis
