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Cannabinoid receptors as novel targets for the treatment of melanoma

Cristina Blázquez^*, Arkaitz Carracedo^*, Lucía Barrado, Pedro José Real, José Luis Fernández-Luna, Guillermo Velasco^*, Marcos Malumbres and Manuel Guzmán^*,1

^* Department of Biochemistry and Molecular Biology I, School of Biology, Complutense University, Madrid, Spain;

Cell Division and Cancer Group, Centro Nacional de Investigaciones Oncológicas, Madrid, Spain; and

Unit of Molecular Genetics, Marqués de Valdecilla University Hospital, Santander, Spain

¹Correspondence: Department of Biochemistry and Molecular Biology I, School of Biology, Complutense University, 28040 Madrid, Spain. E-mail: mgp{at}bbm1.ucm.es

SPECIFIC AIMS

Melanoma is the leading cause of death from cutaneous malignancies, so new therapeutic strategies are necessary for the management of this devastating disease. Here, we evaluated the efficacy of cannabinoid receptor agonists, a new family of potential antitumoral compounds, at skin melanoma.

PRINCIPAL FINDINGS

1. Melanoma cells express cannabinoid receptors
CB₁ and CB₂ cannabinoid receptor expression was analyzed by confocal microscopy in a series of human cutaneous melanoma biopsies. Of the 61 tumors examined, 36 expressed significant immunoreactivity for CB₁ and CB₂ receptors, 10 just for the CB₁ receptor, 10 just for the CB₂ receptor, and only 5 were negative for the two CB receptor types. In line with these observations, Western blot analysis and RT-polymerase chain reaction (RT-PCR) experiments showed that CB₁ and CB₂ receptor protein and mRNA, respectively, were expressed in the melanoma cell lines B16 (mouse) and A375 (human).

2. Cannabinoids inhibit the growth of melanoma cells but not of normal melanocytes
We tested the functionality of cannabinoid receptors in the control of melanoma cell growth by using two mixed CB₁/CB₂ agonists: the plant-derived cannabinoid ⁹-tetrahydrocannabinol (THC) and the synthetic cannabinoid WIN-55,212–2. These compounds decreased the number of viable B16 and A375 melanoma cells in the cultures in a dose- and time-dependent manner. For example, values of A375 cell density (as a percentage of the corresponding vehicle incubations) after 48 h of cannabinoid challenge were 71 ± 6 (1 µM THC), 61 ± 6 (2 µM THC), 39 ± 4 (2.5 µM THC), and 22 ± 7 (3 µM THC). Incubation with the CB₁ antagonist SR141716 (0.5 µM) or the CB₂ antagonist SR144528 (0.5 µM) prevented THC and WIN-55,212–2 action in both cell lines, pointing to the involvement of cannabinoid receptors. In addition, cannabinoid antiproliferative activity seemed to be selective for tumor cells, as neither THC (1 µM, up to 72 h) nor WIN-55,212–2 (100 nM, up to 72 h) induced a significant change in the number of viable mouse melan-c and human Hermes 2b cells (two nontumorigenic lines of melanocytes) in the cultures.

3. Cannabinoid administration inhibits melanoma progression and metastatic spreading in mice
We evaluated the effect of cannabinoid treatment in vivo. We induced malignant tumors in C57BL/6 mice by subcutaneous (s.c.) flank inoculation of B16 melanoma cells line and injected them peritumorally with vehicle or WIN-55,212–2. We found that tumors from cannabinoid-treated animals were notably smaller than controls (Fig. 1 A). This antitumoral action of WIN-55,212–2 was studied in further detail.

View larger version (22K): [in this window] [in a new window]   Figure 1. Cannabinoid administration inhibits melanoma progression and metastatic spreading in mice. A) Tumors were generated by s.c. injection of B16 cells in C57BL/6 mice, and animals were treated with either vehicle or cannabinoid (either WIN-55,212–2 or JWH-133 at 50 µg/day, daily) for up to 8 days (n=8 for each experimental group). Tumor size was monitored during the treatment. Examples of tumors in the flank of mice and after dissection are shown. *Significantly different (P<0.01) from vehicle administration. B) Tumors were generated by s.c. injection of B16 cells in nude mice, and animals were treated with either vehicle or WIN-55,212–2 (50 µg/day, daily) for 8 days (n=6 for each experimental group). *Significantly different (P<0.01) from vehicle administration. C) B16 cells were injected intraplantarly in C57BL/6 and nude mice, and animals were treated with either vehicle or WIN-55,212–2 (50 µg/day, every 3 days) for 21 days (C57BL/6 mice, n=6) or 12 days (nude mice, n=5). The number of metastatic nodules in the lungs and the liver were subsequently counted. Significantly different (#P<0.05, *P<0.01) from vehicle administration.

1) Because cannabinoid-based therapeutic strategies should be as devoid as possible of psychotropic effects, which are mediated by brain CB₁ receptors, and melanoma cells express CB₂ receptors (see above), which lack cannabinoid psychoactivity, we administered to mice JWH-133, a nonpsychoactive, CB₂-selective agonist. As shown in Fig. 1A , JWH-133 was as effective as the mixed CB₁/CB₂ agonist WIN-55,212–2 in preventing tumor growth.

2) To distinguish between direct cannabinoid antitumoral activity on melanoma cells and potential immune-related responses induced by cannabinoid treatment, parallel experiments were conducted in immune-deficient (nude) mice. As shown in Fig. 1B , WIN-55,212–2 significantly inhibited melanoma growth in these animals.

3) To test whether the antitumoral effect of WIN-55,212–2 also targets melanoma cell spreading, we examined cannabinoid action in a model of metastatic-nodule formation. For this purpose, melanoma cells were injected into the paw of C57BL/6 and nude mice, and animals were administered vehicle or WIN-55,212–2 intraperitoneally (i.p.). As shown in Fig. 1C , the cannabinoid decreased the number of lung and liver metastases in both strains of mice.

We next tested whether cannabinoids inhibit proliferation of tumor cells in vivo. Quantification of proliferative (=5-bromo-2'-deoxyuridine-labeled) cells in tumor sections revealed that treatment with WIN-55,212–2 or JWH-133 decreased tumor cell proliferation. This was accompanied by an increase in the number of apoptotic cells, as determined by TUNEL staining, and by a decrease in tumor vascular density, as determined by CD31 immunostaining.

4. Akt is involved in cannabinoid-induced inhibition of melanoma cell proliferation
We investigated the mechanism by which cannabinoids inhibit melanoma cell proliferation. Flow cytometry analysis showed that cannabinoid treatment (100 nM WIN-55,212–2 or 1 µM THC; 48 h) inhibited B16 and A375 melanoma cell cycle, most likely at the G1-S transition, as inferred from the increase of cells in the G0/G1 compartment and the decrease of cells in the S compartment. Likewise, cannabinoids decreased the phosphorylation state of the retinoblastoma protein family member pRb, a master regulator of the G1-S transition, both in cultured melanoma cells and in tumor xenografts. At higher concentrations (2 µM for THC, 0.5 µM for WIN-55–212-2) both cannabinoids were also able to induce apoptosis as determined by 1) appearance of a hypodiploid (sub-G0/G1) population, 2) TUNEL staining, and 3) caspase 3 activation.

We next examined the possible contribution of various signaling pathways to the inhibition of melanoma cell proliferation. Cannabinoid incubation induced a rapid inhibition of the prosurvival protein Akt (Fig. 2 A), whereas extracellular signal-regulated kinase, c-Jun N-terminal kinase and p38 mitogen-activated protein kinase were not significantly affected. To test the involvement of Akt in cannabinoid action, we overexpressed the kinase in B16 melanoma cells with an adenoviral vector. Akt overexpression abrogated cannabinoid-induced antiproliferative effect (Fig. 2B ) and pRb hypophosphorylation (Fig. 2C ), suggesting that Akt inhibition is necessary for cannabinoid action.

View larger version (19K): [in this window] [in a new window]   Figure 2. Akt is involved in cannabinoid-induced inhibition of melanoma cell proliferation. A) B16 cells were cultured for the times indicated with vehicle, 100 nM WIN-55,212–2, 1 µM THC, or 100 nM JWH-133; extracts were obtained, and Western blot analysis was performed. One representative experiment is shown. OD values (in percentage of the respective vehicle incubations; n=4) are given for phospho-Akt relative to total Akt in short-term incubations. *Significantly different (P<0.01) from vehicle incubations. B, C) B16 cells were infected with HA-Akt or empty vector and further cultured for 48 h with vehicle or 100 nM WIN-55,212–2. The number of viable cells (B) and pRb phosphorylation status (C) were determined. Western blot controls of Akt expression in the different infection conditions and OD values for pRb are shown. Results correspond to four different experiments. *Significantly different (P<0.01) from vehicle incubations.

CONCLUSIONS AND SIGNIFICANCE

Melanoma remains a management challenge. Despite many years of intensive research, currently approved therapies—high-dose IFN -2b and dacarbazine—are only palliative or even ineffective, and ongoing therapeutic approaches, such as vaccine-based immunotherapy and targeted chemotherapy, are as yet far from the clinics. Here, we studied the potential efficacy of cannabinoids as antitumoral agents against melanoma, and show that these compounds exert a remarkable growth-inhibiting effect on melanoma cells in vivo that is evident under various experimental settings (animals with different immune statuses, melanoma cells inoculated at different sites, cannabinoids injected by different routes). In addition, this is associated with an improvement of various tumor-progression parameters (decreased proliferation and vascularization, increased apoptosis), as well as with an inhibition of tumor-cell metastatic spreading, one of the clinical hallmarks of advanced melanoma. Moreover, cannabinoid action seems to be selective for tumor vs. nontumor cells.

Activation of cannabinoid receptors decreases melanoma cell proliferation at least in part via inhibition of Akt (Fig. 3 ), a key element of a major prosurvival pathway that is deregulated in many types of tumors, including melanoma. Thus, Akt is constitutively activated in more than 60% of melanomas, with higher frequency of activation at later stages of the disease. Our findings also support that Akt inhibition arrests the cell cycle at the G1-S transition via pRb retinoblastoma protein hypophosphorylation (Fig. 3) . However, we cannot rule out that cannabinoid antiproliferative action in our system also relies on additional mechanisms.

View larger version (15K): [in this window] [in a new window]   Figure 3. Schematic diagram depicting the possible mechanism involved in cannabinoid-induced inhibition of melanoma cell proliferation. Activation of cannabinoid receptors on melanoma cells inhibits Akt, which decreases pRb retinoblastoma protein phosphorylation, leading, in turn, to cell cycle arrest at the G1-S transition and decreased cell proliferation. It cannot be ruled out that additional pathways contribute as well to cannabinoid antitumoral action in melanoma cells.

Although the use of cannabinoids in medicine is limited by their psychotropic effects, these compounds display a fair drug safety profile, their potential adverse effects are within the range of those accepted for other medications—especially in cancer treatment—and their psychoactive effects tend to disappear with tolerance on continuous use. Nonetheless, it is obvious that cannabinoid-based therapies devoid of side effects would be desirable. By showing that CB₂ receptor activation is functional in curbing melanoma cell growth in vitro and in vivo, the present report may therefore set the basis for a new psychoactivity-devoid, cannabinoid-based therapeutic approach for the management of malignant melanoma.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.06-6638fje

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This Article Abstract Full Text Full Text (PDF) Supplemental Data All Versions of this Article: fj.06-6638fjev1 20/14/2633    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Blázquez, C. Articles by Guzmán, M. Search for Related Content PubMed PubMed Citation Articles by Blázquez, C. Articles by Guzmán, M.