Inhibitory Effects Of Cannabinoid CB1 Receptor Stimulation On Tumor Growth And Metastatic Spreading: Actions On Signals Involved In Angiogenesis And MetastasisPRINCIPAL FINDINGS
1. Met-F-AEA inhibits the growth of an established tumor in vivo
Since the effect of Met-F-AEA on incipient tumors has been analyzed, to evaluate the effects of this compound on already established tumors, 45 nude mice were injected subcutaneously with K-ras-transformed FRTL-5 cells (Ki-Mol cells). Twenty days later, when tumors were clearly detectable, saline solution containing MetF-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 controltreated mice (not shown). This effect was signiﬁcantly inhibited by the CB1 receptor antagonist SR141716A (0.7 mg/kg/dose, s.c. intratumor, coadministered with Met-F-AEA; P0.01 by ANOVA).
2. Effect of Met-F-AEA on the expression of endogenous pro- and anti-angiogenic factors
Since we observed that the tumors from Met-F-AEAtreated mice were paler than the tumors from untreated mice and because we noted previously that Met-F-AEA is able to block p21ras activity via CB1 receptors, we investigated whether this compound could inhibit angiogenesis by affecting the expression of the vascular endothelial growth factor (VEGF), which in turn depends on the p21ras activity. Western blot analysis of proteins from treated or untreated tumors (Fig. 1) showed that VEGF levels were dramatically reduced by Met-F-AEA treatment. In addition, Met-F-AEA treatment reduced the expression of a VEGF receptor (Flt-1 or VEGFR-1) (Fig. 1). These inhibitory effects of Met-F-AEA were attenuated by the selective CB1 receptor antagonist, SR141716A (Fig. 1), strongly suggesting the involvement of CB1 receptors in the anti-VEGF action of the compound, and were observed in Ki-Mol cells in vitro. The cyclin-dependent kinase inhibitor p27(kip1) is another protein suggested to play a role as a proangiogenic factor and is under the negative control of the ras oncogene in proliferating human thyroid cells. 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 (not shown).
3. Effect of Met-F-AEA on metastatic cells
Since underexpression of p27(kip1) has been correlated with increased spreading of thyroid cancer cells to lymph nodes and VEGF itself has been implicated in cancer cell metastasis, we investigated the effect of Met-F-AEA on metastatic processes. We compared the anti-proliferative 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, which display a highly more malignant phenotype with respect to TK-6 cells). Four day treatment with Met-F-AEA was able to inhibit the proliferation of both neoplastic thyroid cell lines. However, the growth of metastasis-derived cells was inhibited more efﬁcaciously than that of primary thyroid carcinomaderived cells (62.07.0 vs. 38.08.0% inhibition, meansse, n3; P0.05 by ANOVA), and this effect was accompanied by a stronger up-regulation of CB1 receptor levels in MPTK-6 cells than in TK-6 (not shown).
4. Effect of Met-F-AEA on the formation of metastatic nodules in the Lewis lung carcinoma model
The above data 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 Lewis lung carcinoma (3LL) cells. A dramatic inhibitory effect of Met-F-AEA was observed against lung metastatic nodules induced by 3LL cells (Fig. 2). Even those few metastatic nodules observed in Met-FAEA treated animals were smaller than those present in control animals. The metastatic growth inhibitory effect was blocked by the CB1 receptor antagonist SR141716A (Fig. 2, P0.01 by ANOVA).
CONCLUSIONS AND SIGNIFICANCE
We recently reported that stimulation of cannabinoid CB1 receptors by the metabolically stable endocannabinoid analog Met-F-AEA, by inhibiting p21 ras activity, prevents the growth of v-K-ras-transformed rat thyroid cells both in vitro and in vivo when injected concomitantly to transformed cells into athymic mice. In the present study we found that Met F-AEA is 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 cells growth. As we observed earlier that Met-F-AEA is not capable of inducing apoptosis of our neoplastic cell line model, a likely means by which CB1 stimulation leads to blockade of the growth of established tumors might be the inhibition of tumor neo-angiogenesis. VEGF is an important proangiogenic factor of human thyroid tumors and is known to be up-regulated by p21ras. Since activation of CB1 receptors by Met-F-AEA blocks p21ras activity, the effect of this compound on VEGF signaling was analyzed, showing a dramatic reduction of VEGF expression. We also found that Met-F-AEA suppressed the expression of the VEGF receptor Flt-1 (also known as VEGFR-1), which plays a crucial role in mediating VEGF-induced neo-angiogenesis and endothelial cell proliferation. Finally, Met-F-AEA up regulated the levels of the cyclin-dependent kinase inhibitor p27(kip1), another signal that has been recognized as a possible endogenous suppressor of angiogenesis and whose expression is inhibited by p21ras. These effects, observed both in Met-F-AEA-treated tumors in vivo and in rat thyroid cancer cells in vitro and signiﬁcantly atten-uated by the CB1 receptor antagonist SR141716A, strongly suggest that stimulation of CB1 receptors might lead to inhibition of tumor angiogenesis and metastatic spreading in vivo (Fig. 3). While a direct effect of cannabinoids on new vessel formation and endothelial cell migration and apoptosis was reported during this study, we gained direct evidence for the effect of Met-F-AEA on tumor metastasis by ﬁnding that 4 day treatment with Met-F-AEA inhibited the proliferation of the metastasis derived thyroid cancer MPTK-6 cell line more efﬁcaciously than in the primary thyroid cancer-derived TK-6 cell line. This effect was probably because 4 day treatment with MetF-AEA led to a much stronger up-regulation of CB1 receptors in MPTK-6 cells than in TK-6 cells, and suggests that activation of these receptors might cause inhibition of metastasis in vivo. This hypothesis was supported by using an in vivo model of metastatic spreading, that is the formation of lung nodules after inoculation of Lewis lung carcinoma cells. We found that Met-F-AEA produced a reduction in the number and size of metastatic nodes in this model in a way that was again counteracted by the CB1 receptor antagonist. In conclusion, we have demonstrated for the ﬁrst time that CB1 receptor stimulation 1) may inhibit tumor angiogenesis and metastasis by both suppressing the expression of VEGF and of its Flt-1 receptor and enhancing p27(kip1) levels; and 2) does interfere with metastatic processes in vitro and in vivo. Our data indicate that the endocannabinoid system might be targeted pharmacologically to block at multiple levels the molecular and cellular processes intervening in cancer growth and spreading (Fig. 3).
Source: Inhibitory Effects Of Cannabinoid CB1 Receptor Stimulation On Tumor Growth And Metast