Inhibition of skin tumor growth and angiogenesis in vivo by activation of cannabinoid receptors
Nonmelanoma skin cancer is one of the most common malignancies in humans. Different therapeutic strategies for the treatment of these tumors are currently being investigated. Given the growth-inhibiting effects of cannabinoids on gliomas and the wide tissue distribution of the two subtypes of cannabinoid receptors (CB1 and CB2), we studied the potential utility of these compounds in anti–skin tumor therapy. Here we show that the CB1 and the CB2 receptor are expressed in normal skin and skin tumors of mice and humans. In cell culture experiments pharmacological activation of cannabinoid receptors induced the apoptotic death of tumorigenic epidermal cells, whereas the viability of nontransformed epidermal cells remained unaffected. Local administration of the mixed CB1/CB2 agonist WIN-55,212-2 or the selective CB2 agonist JWH-133 induced a considerable growth inhibition of malignant tumors generated by inoculation of epidermal tumor cells into nude mice. Cannabinoid-treated tumors showed an increased number of apoptotic cells. This was accompanied by impairment of tumor vascularization, as determined by altered blood vessel morphology and decreased expression of proangiogenic factors (VEGF, placental growth factor, and angiopoietin 2). Abrogation of EGF-R function was also observed in cannabinoid-treated tumors. These results support a new therapeutic approach for the treatment of skin tumors.
The epidermis is a stratified squamous epithelium composed mainly of keratinocytes, whose proliferation and differentiation must be tightly regulated and coordinated. Basal keratinocytes, which are attached to the basement membrane, are undifferentiated and have proliferative potential. Before entering the differentiation program, they withdraw from the cell cycle and migrate toward the surface of the epidermis, leading to the formation of the outermost layer of the epidermis composed of enucleated dead squames, which are continuously shed from the surface of the skin (1). The incidence of both benign and malignant skin neoplasms has been rising at an alarming rate for the past several years. Thus, nonmelanoma skin cancer is one of the most common malignancies in humans: basal cell carcinomas and squamous cell carcinomas represent the vast majority of the malignant tumors diagnosed (2). These tumors are believed to arise mainly from stem cells of hair follicles (3), and their growth and development seems to rely on an early burst of neovascularization (4) in which VEGF (5–7) and EGF-R (8, 9) are essential components. The skin is also a major site for metastasis of internal disease (2, 10). Early recognition, biopsy confirmation, and treatment selection can reduce patient morbidity. Different types of strategies are currently being investigated as therapies for the treatment of these tumors, including cryotherapy, topical chemotherapeutic agents such as 5-fluorouracil, and photodynamics, the success of which is hampered by limitations such as the poor penetration of molecules into the skin and the difficulty to gain access to the whole tumor (10–12).
Cannabinoids, the active components of Cannabis sativa linnaeus (marijuana) and their derivatives, exert a wide array of effects on the CNS as well as on peripheral sites such as the immune, cardiovascular, digestive, reproductive, and ocular systems (13–15). Nowadays, it is widely accepted that most of these effects are mediated by the activation of specific G protein–coupled receptors that are normally bound by a family of endogenous ligands — the endocannabinoids (14, 16, 17). Two different cannabinoid receptors have been characterized and cloned from mammalian tissues: the “central” CB1 receptor, mostly expressed in brain and responsible for cannabinoid psychoactivity (18), and the “peripheral” CB2 receptor, mostly expressed in the immune system and unrelated to cannabinoid psychoactivity (19). Marijuana and its derivatives have been used in medicine for many centuries, and currently there is a renaissance in the study of the therapeutic effects of cannabinoids, which constitutes a widely debated issue with ample scientific and social relevance. Ongoing research is determining whether cannabinoid ligands may be effective agents in the treatment of, for example, pain and inflammation, neurodegenerative disorders such as multiple sclerosis and Parkinson’s disease, and the wasting and emesis associated with AIDS and cancer chemotherapy (13–15). In addition, cannabinoids may be potential antitumoral agents owing to their ability to induce the regression of various types of tumors, including lung adenocarcinoma (20), glioma (21, 22), and thyroid epithelioma (23) in animal models. Although cannabinoids directly induce apoptosis or cell cycle arrest in different transformed cells in vitro (24), the involvement of this and other potential mechanisms (e.g., inhibition of tumor angiogenesis) in their antitumoral action in vivo is as yet unknown.
This background prompted us to explore whether (a) the skin and skin tumors express cannabinoid receptors; (b) cannabinoid receptor activation exerts a growth-inhibiting action on skin tumors in vivo; and (c) inhibition of angiogenesis is implicated in the anti-tumoral effect of cannabinoids. Our data show that (a) CB1 and CB2 receptors are present in the skin and skin tumors; (b) local cannabinoid receptor activation induces the regression of skin tumors in vivo; and (c) at least two mechanisms may be involved in this action: direct apoptosis of tumor cells and inhibition of tumor angiogenesis.
Full Study can be found at: http://www.jci.org/articles/view/16116
Here we report that CB1 and CB2 cannabinoid receptors are expressed in normal epidermis and in skin tumors and that both receptors are functional in the induction of apoptosis of skin tumor cells and the regression of skin carcinomas. It is therefore plausible that apoptosis of tumor cells and tumor regression are two causally related events. Nonetheless, our data indicate that cannabinoid antitumoral action may also rely on the inhibition of tumor angiogenesis. It has been shown that mouse skin tumor growth and progression depends on critical events leading to epithelial and stromal changes, including the establishment of an active angiogenesis (4). Here, we report that blood vessels developed by cannabinoid-treated carcinomas are small, in line with the finding that blood vessel enlargement constitutes a prominent feature of skin tumor progression (4, 32). Moreover, we show that in cannabinoid-treated carcinomas the expression of proangiogenic factors is depressed and that of antiangiogenic factors is unchanged, which fits well with the observations that link skin carcinoma development with a clear imbalance toward positive angiogenic-factor action (6, 7, 9). Ha-ras activation seems to be a critical event in mouse skin tumor initiation as well as a major component of the angiogenic response (6) in which VEGF plays a pivotal role (5, 9). Ha-ras activation induces VEGF expression in mouse keratinocytes (6), as well as in other cell types (33, 34). Our data also show that cannabinoid treatment decreases the expression of PIGF (another VEGF family member) and Ang2, and these two proangiogenic factors may act in concert with VEGF because their expression is highly increased since the early stages of tumor development (9, 28, 29).
EGF-R participates in the regulation of key epidermal functions (35–38). Moreover, we have shown that in mouse skin carcinomas EGF-R–dependent Ha-ras activation plays a pivotal role in VEGF expression and tumor angiogenesis and growth (9). Carcinoma growth arising from subcutaneous injection of tumor epidermal cells is a biphasic process. The first phase of slow growth occurs independently of EGF-R function. Later, an angiogenic switch response mediated by the EGF-R seems to be an essential requirement for complete tumor growth, involving high VEGF levels. Other members of the EGF-R family such as HER2 may also exert their relevant anticarcinogenic role via modulation of angiogenesis (39). Here we show that cannabinoid treatment impairs EGF-R function, VEGF expression, and angiogenesis in skin tumors. It is of interest that inhibition of EGF-R function also occurred upon exposure of cultured skin tumor cells to cannabinoids, indicating that the changes observed in EGF-R activity in vivo reflect a direct impact of cannabinoids on tumor cells and are not a mere consequence of decreased tumor size. Although at present we cannot establish the mechanism for the decrease of EGF-R phosphorylation in cannabinoid-treated tumors, it is tempting to speculate that cannabinoid treatment interferes with the tumor angiogenic switch and that this, together with the direct induction of apoptosis on tumor cells, is a reason for the inhibition of tumor growth in our system.
Nonmelanoma skin cancer is one of the most common malignancies in humans. Different types of strategies are currently being investigated as therapies for the treatment of these tumors, including cryotherapy, topical chemotherapeutic agents such as 5-fluorouracil, and photodynamics, the success of which is hampered by limitations such as the poor penetration of molecules into the skin and the difficulty of gaining access to the whole tumor (10–12). The present data indicate that local cannabinoid administration may constitute an alternative therapeutic approach for the treatment of nonmelanoma skin cancer. Of further therapeutic interest, we show that skin cells express functional CB2 receptors. The synergy between CB1 and CB2 receptors in eliciting skin tumor cell apoptosis reported here is nonetheless intriguing because it is not observed in the case of cannabinoid-induced glioma cell apoptosis (21, 22). In any event, the present report, together with the implication of CB2– or CB2-like receptors in the control of peripheral pain (40–42) and inflammation (41), opens the attractive possibility of finding cannabinoid-based therapeutic strategies for diseases of the skin and other tissues devoid of nondesired CB1-mediated psychotropic side effects.
We are indebted to M.I. de los Santos for expert technical assistance and to F. Larcher for discussion and advice. This work was supported by grants from the Ministerio de Ciencia y Tecnología (PM 98-0079 to M. Guzmán, SAF 98-0047 to J.L. Jorcano, and BMC 2001-1018 to J.L. Jorcano); the Comunidad Autónoma de Madrid (08.1/0079/2000 to M. Guzmán); the Fundación Ramón Areces (to M. Guzmán); and the National Institute on Drug Abuse (DA03590 to J.W. Huffman).
Full Study can be found at: http://www.jci.org/articles/view/16116