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A new strategy to block tumor growth by inhibiting endocannabinoid inactivation

MAURIZIO BIFULCO^*,,1, CHIARA LAEZZA^*, MARTA VALENTI, ALESSIA LIGRESTI, GIUSEPPE PORTELLA^* and VINCENZO DI MARZO^,1

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",
Dipartimento di Scienze Farmaceutiche, Università degli Studi di Salerno, Fisciano, Italy; and
Istituto di Chimica Biomolecolare, C.N.R., Pozzuoli, Italy

¹ Correspondence: E-mail: V.D.M., vdimarzo{at}icmib.na.cnr.it and M.B., maubiful{at}unina.it

SPECIFIC AIMS

Endocannabinoid signaling has been shown to be enhanced in several cancer tissues and malignant cells, and studies in cell lines have suggested that this up-regulation might provide transformed cells with a further means to inhibit their proliferation via more than one molecular mechanism. To provide evidence to this hypothesis and to search for possible novel antitumor therapeutic strategies, we investigated the effect of inhibitors of endocannabinoid degradation on the growth of rat thyroid tumor xenografts.

PRINCIPAL FINDINGS

1. Inhibitors of endocannabinoid degradation inhibit rat thyroid tumor xenografts in vivo
Substances were administered the first time together with subcutaneous inoculation of Kras-transformed thyroid cells used to induce tumor xenografts in vivo in athymic mice, and then were administered intratumor twice a week for the 5 wk duration of the experiments. Mice that had been treated with vehicle alone developed subcutaneous tumors whose size was 5–6 mm³ after 5 wk. In agreement with previous studies, administration of metabolically stable anandamide analog Met-F-AEA (0.5 mg/kg/dose) significantly inhibited the growth and size of the tumors at the end of the experiment (Fig. 1 A, B). We also showed here for the first time that administration of the other endocannabinoid, 2-AG, at a dose higher than that of Met-F-AEA (5 mg/kg), causes a smaller but still statistically significant reduction of tumor size (Fig. 1A ). That this compound is less efficacious than Met-F-AEA is not surprising if one takes into consideration that 2-AG was shown to undergo degradation in vivo more rapidly than anandamide. Intratumor administration of selective endocannabinoid reuptake inhibitor, VDM11, and of selective anandamide hydrolysis inhibitor, AA-5-HT, both at the dose of 5 mg/kg, strongly and significantly inhibited tumor growth over the course of the experiments (Fig. 1B ). The two inhibitors were selected for their inactivity at cannabinoid receptors (K_i>10 µM) and their proven selectivity: 1) VDM11 is an inhibitor of the putative endocannabinoid membrane transporter, and as such it inhibits anandamide cellular uptake (IC₅₀=8-10 µM), but unlike other similar inhibitors, has little effect on anandamide hydrolysis or vanilloid TRPV1 receptors; 2) AA-5-HT is a very selective, metabolically stable inhibitor of fatty acid amide hydrolase (FAAH, IC₅₀=6-12µM), the enzyme mostly responsible for anandamide hydrolysis and which can catalyze 2-AG hydrolysis. No inhibitory effect of either VDM-11 or AA-5-HT on locomotion was observed during the course of the experiments, which is in agreement with previous data showing that these two compounds are inactive on this as well as other behavioral parameters of central cannabimimetic activity even when administered systemically. Arvanil, a metabolically stable, mixed inhibitor of endocannabinoid cellular reuptake (IC₅₀=3.5 µM) and a partial CB₁ agonist(K_i=0.5–1.9 µM) inactive on FAAH, significantly inhibited tumor growth (Fig. 1A ). This compound is a very potent (K_i=0.28 µM, EC₅₀ 0.5 nM) agonist of vanilloid receptors, which can be directly activated by anandamide, and have been implicated in inhibition of cancer cell growth through induction of apoptosis. The extent of the effect of arvanil as compared with VDM-11, which is chemically very similar to arvanil and hence is likely to have a similar pharmacokinetic profile, suggests that: 1) vanilloid receptors and induction of apoptosis may not be involved in anticancer effects of either endocannabinoids or of inhibitors of their inactivation, at least in the in vivo model used in this study (see below); and 2) no further enhancement of cancer growth-inhibitory actions of a compound is achieved when the capability to directly activate CB₁ receptors is added to its ability to activate this receptor indirectly via inhibition of endocannabinoid reuptake. A full assessment of the actual involvement of vanilloid receptors in the assay of tumorigenicity used here was not among the aims of the present study and should be investigated further by using specific agonists and antagonists for these receptors.

View larger version (13K): [in this window] [in a new window]   Figure 1. Inhibitors of endocannabinoid inactivation inhibit rat thyroid carcinoma growth in vivo. A) Effect of intratumor administration of 2-methyl-2’-F-anandamide (AEA, 0.5 mg/kg/dose), 2-arachidonoyl-glycerol (2-AG, 5 mg/kg/dose), and arvanil (1 mg/kg/dose) on the growth of thyroid tumor xenografts induced in athymic mice. B) Effect of intratumor administration of 2-methyl-2’-F-anandamide (AEA, 0.5 mg/kg/dose), VDM-11 (VDM, 5 mg/kg/dose) and arachidonoyl-serotonin (AA-5-HT, 5 mg/kg/dose) on the growth of thyroid tumor xenografts induced in athymic mice. Data are means ± SE of n = 10 mice per data point. Effects of all compounds at all time-points were statistically different (P<0.05 by ANOVA followed by Bonferroni’s test) from vehicle (control).

2. Inhibitors of endocannabinoid degradation enhance intratumoral endocannabinoid levels
We next needed to demonstrate that VDM11 and AA-5-HT were indeed acting through enhancement of endocannabinoid tissue concentrations. Therefore, we compared levels of anandamide and 2-AG, as measured by isotope-dilution liquid chromatography mass spectrometry, in tumors excised from treated and untreated mice on the last day of the in vivo experiments described above. AA-5-HT produced a significant elevation of the concentrations of both anandamide and 2-AG, whereas VDM-11 only enhanced 2-AG levels (Fig. 2 ). In agreement with its mechanism of action via inhibition of FAAH, AA-5-HT elevated levels of the FAAH substrate and anandamide congener, palmitoylethanolamide, although to a lesser extent, possibly because this compound can be hydrolyzed by another amidase. These findings suggest that elevation of levels of endocannabinoids, and 2-AG in particular, might be sufficient alone to inhibit growth of thyroid tumor xenografts "from inside." The observation that VDM-11, which enhanced significantly only 2-AG levels, was more efficacious than 2-AG at blocking tumor growth, albeit based solely on the comparison between just one dose of the two compounds, suggests that enhancing local levels of an endocannabinoid may achieve better results in terms of tumor growth inhibition in vivo than administering exogenously the metabolically unstable compound.

View larger version (20K): [in this window] [in a new window]   Figure 2. Inhibitors of endocannabinoid inactivation enhance endocannabinoid levels in rat thyroid carcinomas. Effect of VDM-11 (VDM, 5 mg/kg/dose) and arachidonoyl-serotonin (AA-5-HT, 5 mg/kg/dose) on levels of anandamide, palmitoylethanolamide (PEA) and 2-arachidonoylglycerol (2-AG) in tumors excised from athymic mice after a 5 wk intratumor treatment with the two substances or vehicle. Anandamide, PEA, and 2-AG levels were measured by isotope dilution-liquid chromatography-mass spectrometry in tissue lipid extracts. Basal tumor levels from two sets of vehicle-treated mice are shown. Data are means ± SE of n = 5 mice per data point. *P< 0.05; **P< 0.01 vs. control (vehicle), by ANOVA followed by Bonferroni’s test.

3. Endocannabinoids and inhibitors of their degradation inhibit cell proliferation via CB₁ cannabinoid receptors and possible additional mechanisms
We investigated the mechanism of action of VDM11 and AA-5-HT by studying their effects in vitro on the same transformed rat thyroid transformed cells used to induce tumors in vivo. The two compounds, as well as 2-AG, Met-F-AEA, and arvanil, dose-dependently inhibited cell proliferation with IC₅₀ below 10 µM in all cases, and with no effect on apoptosis. CB₁ receptor antagonist, SR141716A (0.2 µM), but not CB₂ receptor antagonist SR144528 (0.5 µM), counteracted significantly the effects of Met-F-AEA and VDM-11, thus suggesting that CB₁ receptors are uniquely involved in the in vitro antiproliferative effects of these compounds. To a smaller extent, effects of 2-AG, AA-5-HT, and arvanil were inhibited by SR141716A but not SR144528, suggesting that these compounds might also act through other non-CB₁-mediated mechanisms. SR141716A, but not SR144528, exerted a significant, albeit small, antiproliferative action per se. This effect is likely to have masked in part the antagonism of 2-AG, AA-5-HT and arvanil actions, and was also observed in vivo, where SR141716A, at a dose previously shown to be sufficient to attenuate Met-F-AEA inhibitory action in the same model (0.7 mg/kg, intratumor, twice a wk for 5 wk), exerted a small (27%) but significant antitumor effect. These findings suggest that endocannabinoids might exert tonic antiproliferative effects through non-CB₁ receptor-mediated mechanisms, particularly when CB₁ receptors are blocked. These mechanisms are not likely to involve cannabinoid CB₂ receptors, because we have shown previously that these receptors are not involved in endocannabinoid antiproliferative effects, and found here that a CB₂ antagonist has no effect on the antiproliferative actions of the compounds tested. However, apart from vanilloid receptors (see above), several other membrane proteins and ion channels are being proposed as endocannabinoid molecular targets under both physiological and pathological conditions. The mechanism underlying the anticancer effect of SR141716A will require separate investigations.

CONCLUSIONS AND SIGNIFICANCE

Irrespective of the mechanism of antiproliferative actions of endocannabinoids, and in view of the possibility that they may inhibit cancer cell growth and spreading via non-cannabinoid receptor-mediated pathways, our data suggest that an alternative and possibly more efficacious and safer strategy to block tumor growth in vivo may be the use of substances that selectively inhibit endocannabinoid degradation (Fig. 3 ). These compounds should be more specific than traditional CB₁ receptor agonists since they are likely to affect endocannabinoid levels, and hence the state of activation of CB₁ receptors, only in those tissues where these compounds are produced "on demand" to contribute exerting protective actions, and not, as in the case of cannabinoid receptor agonists, in all tissues expressing functionally active CB₁ receptors. Many examples of the successful use of endocannabinoid uptake and hydrolysis inhibitors in animal models of other disorders (e.g., for anxiety, cholera toxin-induced intestinal hypersecretion, kainate-induced excitotoxicity, and glutamate hyperactivity of corticostriatal neurons in Parkinson’s disease) have been reported recently. This is the first report that such substances, by prolonging an antiproliferative tone of endogenous cannabinoids (Fig. 3) , can inhibit cancer growth in vivo. Further experiments will tell whether the present findings will result in development of new anticancer drugs against endocannabinoid-sensitive tumors.

View larger version (30K): [in this window] [in a new window]   Figure 3. Schematic representation of findings. Transformed rat thyroid carcinoma cells produce endocannabinoids which inhibit proliferation by acting at least in part via cannabinoid CB1 receptors. This antiproliferative activity is tonically limited by endocannabinoid inactivation which occurs via cellular reuptake facilitated by an endocannabinoid membrane transporter and subsequent intracellular enzymatic hydrolysis via fatty acid amide hydrolase (FAAH). Inhibition of the transporter or of FAAH retards endocannabinoid inactivation, thereby prolonging endocannabinoid inhibition of cell proliferation with subsequent tumor growth inhibitory action. Blunted arrows denote pharmacological blockade, broken arrows denote inhibition, and normal arrows denote participation to enzymatic reactions.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-1754fje;

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