Jacob Bell
New Member
MAURIZIO BIFULCO,*," ,2 CHIARA LAEZZA,* GIUSEPPE PORTELLA,* MARIO VITALE,*
PIERANGELO ORLANDO,"¡ LUCIANO DE PETROCELLIS,§ AND VINCENZO DI MARZO" " ,2
*," ,"¡,§," " Endocannabinoid Research Group, *Centro di Endocrinologia ed Oncologia Sperimentale,
Consiglio Nazionale delle Ricerche, and Dipartimento di Biologia e Patologia Cellulare e Molecolare
'L. Califano', Universita` di Napoli Federico II, " Dipartimento di Medicina Sperimentale e Clinica 'G.
Salvatore', Universita` di Catanzaro, "¡Istituto di Biochimica delle Proteine, §Istituto di Cibernetica and
" " Istituto di Chimica Biomolecolare, Consiglio Nazionale delle Ricerche, Comprensorio Olivetti,
80078, Pozzuoli, Napoli, Italy
SPECIFIC AIMS
The active principal of Cannabis, (2)-D9-tetrahydrocannabinol
and the endogenous cannabinoid anandamide,
exert growth inhibitory effects on cancer cells.
Here we investigated the control by a metabolically
stable anandamide analog via the cannabinoid CB1
receptor, of ras oncogene-dependent growth of a thyroid
tumor in vivo and in vitro.
PRINCIPAL FINDINGS
1. Effect of CB1 receptor activation on thyroid tumor
growth in nude mice
Since anandamide is rapidly metabolized in vivo, we
used a metabolically stable analog, 2-methyl-arachidonyl-
29-fluoro-ethylamide (Met-F-AEA). The effect
of Met-F-AEA in vivo was evaluated in a nude mouse
xenograft model where K-ras-transformed FRTL-5
(Ki Mol) cells were implanted subcutaneously (s.c.).
Ki Mol cells are able to induce the growth of undifferentiated
carcinomas when injected s.c. into syngenic
animals or athymic mice. To evaluate the
efficacy of Met-F-AEA treatment in vivo on the growth
of thyroid Ki Mol cells, 30 athymic mice were inoculated
s.c. with 1 3 106 Ki Mol cells; after 2 days,
animals were injected with saline solution containing
Met-F-AEA. Saline solution was injected in the control
group. Met-F-AEA was injected s.c. in the peritumoral
area on days 2 and 5 of a 7 day cycle for three
cycles. Met-F-AEA treatment (0.5 mg/kg/dose) induced
a drastic reduction in tumor weight (;80%)
with respect to the vehicle-control treated mice,
with no detectable toxic or hypolocomotor effects on
the treated animals (Fig. 1A). This effect was significantly
inhibited by the CB1 receptor antagonist
SR141716A (0.7 mg/kg/dose, s.c. intratumor) (Fig.
1), thus suggesting the involvement of CB1 receptors
in the tumor growth inhibitory effect of Met-F-AEA.
This was also supported by the finding in
tumor tissue of a CB1 mRNA transcript, detected by
RT-PCR technique, and of a CB1 immunoreactive
receptor protein as detected by Western immunoblotting.
The size of both the mRNA transcript and
the immunoreactive protein were compatible with
what was expected for CB1 receptors. The anti-tumor
effect of Met-F-AEA was accompanied by a strong
decrease in of p21ras activity in tumors, which was
almost abolished by SR 141716A (Fig. 2, upper
panel).
2. Effect of CB1 receptor activation on normal and
K-ras-transformed FRTL-5 cell cycle and proliferation
We also looked at the effect of Met-F-AEA on Ki Mol
cells in vitro and found that the anandamide analog
(10 mM) inhibits their proliferation. After a 24 h
treatment, cells had proliferated ;35% less than
vehicle-treated cells (P,0.05 by ANOVA). By contrast,
proliferation of nontransformed FRTL-5 cells
after a 24 h treatment was not affected by 10 mM
Met-F-AEA (9% inhibition, not statistically significant
by ANOVA, see below). The effect of Met-F-AEA
was not due to toxicity or apoptosis of cells, but to
the dose-dependent (IC50 ;5 mM) arrest of the cell
cycle at the G0/G1 phase, associated with a significant
reduction of cells in the S phase, as shown by
FACScan cytometry. The anti-proliferative effect was
accompanied by a striking reduction of p21ras activity
(Fig. 2, lower panel). All these effects were abolished
by SR141716A (0.1 mM). In fact, Ki Mol cells also
expressed a CB1 mRNA transcript and a CB1 immunoreactive
protein.
Finally, we assessed whether cannabinoid CB1 receptors
are regulated during endocannabinoid inhibition of
tumor development. We were surprised to find that the
decrease in tumor volume induced by Met-F-AEA was
accompanied by a strong up-regulation of CB1 receptor
mRNA and protein vs. vehicle-treated tumors. Likewise,
Ki Mol cells treated with Met-F-AEA expressed significantly
more CB1 receptors, and this effect was
abolished by SR141716A (0.1 mM). Cell immunofluorescence
studies with permeabilized and nonpermeabilized
cells showed that Met-F-AEA increased the levels
of CB1 receptors on both the cell membrane and
cytosol. By contrast, nontransformed FRTL-5 cells
treated with Met-F-AEA exhibited fewer CB1 receptors
than vehicle-treated cells, as assessed by Western immunoblot
and immunofluorescence analysis, in agreement
with the down-regulation of the expression of CB1
receptors generally observed in several healthy cell
types and tissues after chronic exposure to CB1 receptor
CONCLUSIONS AND SIGNIFICANCE
Our findings indicate that two fundamental components
of the endocannabinoid system, anandamide and
the cannabinoid CB1 receptor, represent a potential
target for the development of therapeutic agents controlling
ras oncogene-dependent tumor growth. Previously
it was shown that THC reduces the growth of
glioma tumors in mice by inducing apoptosis of glioma
cancer cells. This effect was blocked by a combination
of CB1 and CB2 cannabinoid receptor antagonists but
not by each antagonist alone, leaving the role of each
cannabinoid receptor subtype in this effect to be clarified.
By contrast, we have reported previously that the
cytostatic effect of anandamide on human breast and
prostate cancer cells is due to activation of CB1 receptors
and inhibition of cAMP-mediated signaling and/or
activation of p42/p44 ERK. Therefore, the data presented
here are important since they indicate for the
first time that 1) anandamide analogs, which have a
lower potential for physical dependence than THC and
synthetic cannabinoids, inhibit tumor growth in vivo at
nonpsychotropic doses; 2) the anti-tumor effects of
these substances in vivo can be exerted through CB1
cannabinoid receptors and inhibition of p21ras activity;
and 3) anandamide analogs inhibit the growth of
epithelial tumors, particularly those derived from K-ras
transformed thyroid cells. Indeed, epithelial cell-derived
tumor models have particular importance since
the large majority of human neoplasias are of epithelial
origin.
Another important finding described here is that
CB1 receptors may be regulated by endocannabinoids
in K-ras transformed cells in a manner entirely different
from that observed with nontransformed cells, thus
allowing cancer cells (but not healthy cells) to respond
ever more efficaciously to the anti-proliferative effect of
CB1 agonists. We speculate that this phenomenon may
contribute to the putative tumor suppressing role proposed
for endocannabinoids in previous studies. Furthermore,
this finding indicates that Met-F-AEA can
inhibit the growth of cancer thyroid cells much more
effectively than healthy, nontransformed cells and
strengthens our proposal that endocannabinoid-based
drugs should be taken into account as novel anti-tumor
agents.
Additional work is necessary to establish the exact
molecular mechanism of Met-F-AEA-induced suppression
of p21ras activity. It has been shown recently that
endocannabinoids stimulate ERK, JNK, and p38 mitogen-
activated protein kinases (MAPKs) in several cell
lines via CB1/CB2 receptor-dependent and independent
mechanisms, whereas an inhibitor of p21ras farnesyl
transferase was shown to attenuate the CB1-mediated
activation of JNK. Hence, on the basis of data reported here and previously we speculate that by
modulating the activity of both p21ras and MAPKs, the
CB1 receptor may regulate the fate of cancer cells
through mechanisms that may depend on the cell
phenotype (Fig. 3).
Source: Control by the endogenous cannabinoid system of ras oncogene-dependent tumor growth
PIERANGELO ORLANDO,"¡ LUCIANO DE PETROCELLIS,§ AND VINCENZO DI MARZO" " ,2
*," ,"¡,§," " Endocannabinoid Research Group, *Centro di Endocrinologia ed Oncologia Sperimentale,
Consiglio Nazionale delle Ricerche, and Dipartimento di Biologia e Patologia Cellulare e Molecolare
'L. Califano', Universita` di Napoli Federico II, " Dipartimento di Medicina Sperimentale e Clinica 'G.
Salvatore', Universita` di Catanzaro, "¡Istituto di Biochimica delle Proteine, §Istituto di Cibernetica and
" " Istituto di Chimica Biomolecolare, Consiglio Nazionale delle Ricerche, Comprensorio Olivetti,
80078, Pozzuoli, Napoli, Italy
SPECIFIC AIMS
The active principal of Cannabis, (2)-D9-tetrahydrocannabinol
and the endogenous cannabinoid anandamide,
exert growth inhibitory effects on cancer cells.
Here we investigated the control by a metabolically
stable anandamide analog via the cannabinoid CB1
receptor, of ras oncogene-dependent growth of a thyroid
tumor in vivo and in vitro.
PRINCIPAL FINDINGS
1. Effect of CB1 receptor activation on thyroid tumor
growth in nude mice
Since anandamide is rapidly metabolized in vivo, we
used a metabolically stable analog, 2-methyl-arachidonyl-
29-fluoro-ethylamide (Met-F-AEA). The effect
of Met-F-AEA in vivo was evaluated in a nude mouse
xenograft model where K-ras-transformed FRTL-5
(Ki Mol) cells were implanted subcutaneously (s.c.).
Ki Mol cells are able to induce the growth of undifferentiated
carcinomas when injected s.c. into syngenic
animals or athymic mice. To evaluate the
efficacy of Met-F-AEA treatment in vivo on the growth
of thyroid Ki Mol cells, 30 athymic mice were inoculated
s.c. with 1 3 106 Ki Mol cells; after 2 days,
animals were injected with saline solution containing
Met-F-AEA. Saline solution was injected in the control
group. Met-F-AEA was injected s.c. in the peritumoral
area on days 2 and 5 of a 7 day cycle for three
cycles. Met-F-AEA treatment (0.5 mg/kg/dose) induced
a drastic reduction in tumor weight (;80%)
with respect to the vehicle-control treated mice,
with no detectable toxic or hypolocomotor effects on
the treated animals (Fig. 1A). This effect was significantly
inhibited by the CB1 receptor antagonist
SR141716A (0.7 mg/kg/dose, s.c. intratumor) (Fig.
1), thus suggesting the involvement of CB1 receptors
in the tumor growth inhibitory effect of Met-F-AEA.
This was also supported by the finding in
tumor tissue of a CB1 mRNA transcript, detected by
RT-PCR technique, and of a CB1 immunoreactive
receptor protein as detected by Western immunoblotting.
The size of both the mRNA transcript and
the immunoreactive protein were compatible with
what was expected for CB1 receptors. The anti-tumor
effect of Met-F-AEA was accompanied by a strong
decrease in of p21ras activity in tumors, which was
almost abolished by SR 141716A (Fig. 2, upper
panel).
2. Effect of CB1 receptor activation on normal and
K-ras-transformed FRTL-5 cell cycle and proliferation
We also looked at the effect of Met-F-AEA on Ki Mol
cells in vitro and found that the anandamide analog
(10 mM) inhibits their proliferation. After a 24 h
treatment, cells had proliferated ;35% less than
vehicle-treated cells (P,0.05 by ANOVA). By contrast,
proliferation of nontransformed FRTL-5 cells
after a 24 h treatment was not affected by 10 mM
Met-F-AEA (9% inhibition, not statistically significant
by ANOVA, see below). The effect of Met-F-AEA
was not due to toxicity or apoptosis of cells, but to
the dose-dependent (IC50 ;5 mM) arrest of the cell
cycle at the G0/G1 phase, associated with a significant
reduction of cells in the S phase, as shown by
FACScan cytometry. The anti-proliferative effect was
accompanied by a striking reduction of p21ras activity
(Fig. 2, lower panel). All these effects were abolished
by SR141716A (0.1 mM). In fact, Ki Mol cells also
expressed a CB1 mRNA transcript and a CB1 immunoreactive
protein.
Finally, we assessed whether cannabinoid CB1 receptors
are regulated during endocannabinoid inhibition of
tumor development. We were surprised to find that the
decrease in tumor volume induced by Met-F-AEA was
accompanied by a strong up-regulation of CB1 receptor
mRNA and protein vs. vehicle-treated tumors. Likewise,
Ki Mol cells treated with Met-F-AEA expressed significantly
more CB1 receptors, and this effect was
abolished by SR141716A (0.1 mM). Cell immunofluorescence
studies with permeabilized and nonpermeabilized
cells showed that Met-F-AEA increased the levels
of CB1 receptors on both the cell membrane and
cytosol. By contrast, nontransformed FRTL-5 cells
treated with Met-F-AEA exhibited fewer CB1 receptors
than vehicle-treated cells, as assessed by Western immunoblot
and immunofluorescence analysis, in agreement
with the down-regulation of the expression of CB1
receptors generally observed in several healthy cell
types and tissues after chronic exposure to CB1 receptor
CONCLUSIONS AND SIGNIFICANCE
Our findings indicate that two fundamental components
of the endocannabinoid system, anandamide and
the cannabinoid CB1 receptor, represent a potential
target for the development of therapeutic agents controlling
ras oncogene-dependent tumor growth. Previously
it was shown that THC reduces the growth of
glioma tumors in mice by inducing apoptosis of glioma
cancer cells. This effect was blocked by a combination
of CB1 and CB2 cannabinoid receptor antagonists but
not by each antagonist alone, leaving the role of each
cannabinoid receptor subtype in this effect to be clarified.
By contrast, we have reported previously that the
cytostatic effect of anandamide on human breast and
prostate cancer cells is due to activation of CB1 receptors
and inhibition of cAMP-mediated signaling and/or
activation of p42/p44 ERK. Therefore, the data presented
here are important since they indicate for the
first time that 1) anandamide analogs, which have a
lower potential for physical dependence than THC and
synthetic cannabinoids, inhibit tumor growth in vivo at
nonpsychotropic doses; 2) the anti-tumor effects of
these substances in vivo can be exerted through CB1
cannabinoid receptors and inhibition of p21ras activity;
and 3) anandamide analogs inhibit the growth of
epithelial tumors, particularly those derived from K-ras
transformed thyroid cells. Indeed, epithelial cell-derived
tumor models have particular importance since
the large majority of human neoplasias are of epithelial
origin.
Another important finding described here is that
CB1 receptors may be regulated by endocannabinoids
in K-ras transformed cells in a manner entirely different
from that observed with nontransformed cells, thus
allowing cancer cells (but not healthy cells) to respond
ever more efficaciously to the anti-proliferative effect of
CB1 agonists. We speculate that this phenomenon may
contribute to the putative tumor suppressing role proposed
for endocannabinoids in previous studies. Furthermore,
this finding indicates that Met-F-AEA can
inhibit the growth of cancer thyroid cells much more
effectively than healthy, nontransformed cells and
strengthens our proposal that endocannabinoid-based
drugs should be taken into account as novel anti-tumor
agents.
Additional work is necessary to establish the exact
molecular mechanism of Met-F-AEA-induced suppression
of p21ras activity. It has been shown recently that
endocannabinoids stimulate ERK, JNK, and p38 mitogen-
activated protein kinases (MAPKs) in several cell
lines via CB1/CB2 receptor-dependent and independent
mechanisms, whereas an inhibitor of p21ras farnesyl
transferase was shown to attenuate the CB1-mediated
activation of JNK. Hence, on the basis of data reported here and previously we speculate that by
modulating the activity of both p21ras and MAPKs, the
CB1 receptor may regulate the fate of cancer cells
through mechanisms that may depend on the cell
phenotype (Fig. 3).
Source: Control by the endogenous cannabinoid system of ras oncogene-dependent tumor growth
