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Control by the endogenous cannabinoid system of ras oncogene-dependent tumor growth ¹

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’, Università di Napoli Federico II,
Dipartimento di Medicina Sperimentale e Clinica ‘G. Salvatore’, Università 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, (-)-⁹-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-2'-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 x 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. 1 A). 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 p21^ras activity in tumors, which was almost abolished by SR 141716A (Fig. 2 , upper panel).

View larger version (48K): [in this window] [in a new window]   Figure 1. Met-F-AEA inhibits growth of Ki Mol -induced tumors in athymic mice. A) Tumor volume at different days from inoculation. Data are means ± SE of n = 10. Differences in tumor volumes after 4 wk were significant (P<0.01 by ANOVA, followed by Bonferroni’s test) between the control and Met-F-AEA groups and between the Met-F-AEA and the Met-F-AEA + SR141716A groups. B) Size of typical tumors grown in the presence of vehicle (control), Met-F-AEA, or Met-F-AEA + SR141716A.

View larger version (35K): [in this window] [in a new window]   Figure 2. Met-F-AEA suppresses p21ras activity in Ki Mol cells in vitro and Ki Mol cell-derived tumors in vivo. Upper panel: tumors obtained by s.c. injection of Ki Mol cells from vehicle-treated, Met-F-AEA-treated, and Met-F-AEA plus SR141716A-treated mice were processed for the assay of p21ras activity. Lower panel: Ki Mol cells were incubated with 10 µM Met-F-AEA in the presence of 0.1 µM SR141617 or vehicle for 24 h. The same amount of proteins (50 µg) was loaded in each lane. Data are representative of three experiments yielding similar results.

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 µM) 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 µM 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 µM) 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 p21^ras activity (Fig. 2 , lower panel). All these effects were abolished by SR141716A (0.1 µM). In fact, Ki Mol cells also expressed a CB₁ mRNA transcript and a CB₁ immunoreactive protein.

3. Effect of chronic CB₁ receptor activation on CB₁ receptor expression in normal and K-ras-transformed FRTL-5 cells
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 µM). 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 agonists. In accordance with this opposite regulation of CB₁ receptor expression in transformed vs. healthy cells we found that after treatment of cells with 5 µM Met-F-AEA, the proliferation of Ki Mol cells was significantly more strongly inhibited by the cannabimimetic substance (up to 70% inhibition), whereas the response of FRTL-5 cells barely reached statistical significance after 3 days (up to 28% inhibition).

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 p21^ras 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 p21^ras 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 p21^ras 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 p21^ras and MAPKs, the CB₁ receptor may regulate the fate of cancer cells through mechanisms that may depend on the cell phenotype (Fig. 3 ).

View larger version (24K): [in this window] [in a new window]   Figure 3. Possible role of the endocannabinoid system in the control of cancer cell growth. As shown in this study with the exogenous anandamide analog Met-F-AEA, endogenous anandamide might down-regulate cancer cell proliferation by exerting a negative control on p21ras activity, leading (possibly via the MAPK cascade) to inhibition of mitosis. This effect is accompanied by up-regulation of the expression of CB1 cannabinoid receptors, which mediate anandamide inhibition of p21ras activity, with subsequent increasing suppression of proliferation. Conversely, anandamide does not induce inhibition of proliferation in nontransformed cells because of its inhibition of the expression of CB1 cannabinoid receptors in these cells.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.01-0320fje; to cite this article, use FASEB J. (October 29, 2001) 10.1096/fj.01-0320fje

² E-mail: maubiful@unina.it (M.B.); vdimarzo@icmib.na.cnr.it (V.D.M.). Present address (M.B.) Dipartimento di Scienze Farmaceutiche, Università degli Studi di Salerno, via Ponte Don Melillo, 84084 Fisciano, Salerno, Italy.

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