Aea Cb1 Cb2 Angiogenesis Invasion Migration

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Figure 18.1 Schematic diagram of the cannabinoid receptor-dependent mechanisms whereby AEA leads to tumor growth suppression and/or cell death. AEA may act via the Cb1 receptor to activate the cAMP/PKA/MAPK pathway or to increase the production of ceramide. These effects ultimately result in a decrease in the expression of various growth factor receptors and decrease cell cycle progression. Alternatively, AEA may activate either Cb2 or VR1 to elicit a similar response, although the mechanism by which this occurs is not clear.

Figure 18.1 Schematic diagram of the cannabinoid receptor-dependent mechanisms whereby AEA leads to tumor growth suppression and/or cell death. AEA may act via the Cb1 receptor to activate the cAMP/PKA/MAPK pathway or to increase the production of ceramide. These effects ultimately result in a decrease in the expression of various growth factor receptors and decrease cell cycle progression. Alternatively, AEA may activate either Cb2 or VR1 to elicit a similar response, although the mechanism by which this occurs is not clear.

(McKallip et al., 2002). In these types of cancers, AEA induced apoptosis in vitro and in vivo and it is suggested that because the Cb2 agonists lack psychotropic effects, targeting of the Cb2 receptor would be preferential to targeting the Cb1 receptor in cancers of immune origin (McKallip et al., 2002). Another study demonstrated that interleukin-12 treatment and/or overexpression in thyroid carcinoma cells leads to an increase in Cb2

expression and that either overexpression of interleukin-12 or Cb2 resulted in tumor regression and increased sensitivity to chemotherapy (Shi et al., 2008). Taken together, these data indicate that targeting Cb2 may be of therapeutic value in certain tumor types and warrants further investigation.

VR1: The actions of AEA on VR1 were shown to be antiproliferative in glioma cells (Contassot et al., 2004b) and in uterine cervix cancer cells (Contassot et al., 2004a). In both of these cell types, the VR1 was aberrantly expressed in the tumor cells and tissue compared to their nonmalignant counterparts (Contassot et al., 2004a,b). Furthermore, stimulation ofCb1 or Cb2 was protective against the VR1-mediated antiproliferative effects of AEA (Contassot et al., 2004a,b).

Combination of receptors: In addition to the above-mentioned findings, studies have shown an antiproliferative/growth suppressive effect of AEA that could not be attributable to just one receptor. Using rat C6 glioma cells, AEA was shown to have growth suppressing effects that were time and dose-dependent (Jacobsson et al., 2001). These effects could be partially blocked by the Cb1, Cb2, and VR1 antagonists alone, but was completely attenuated when the three receptor antagonists were added in combination (Jacobsson et al., 2001). In addition, in osteosarcoma cells, AEA induced apoptosis by increasing intracellular calcium levels, activation of p38 MAPK and subsequent activation of caspase 3 (Hsu et al., 2007). Unfortunately, the authors do not address the issue of receptor-dependence in this study, therefore for the purpose of this review we will assume potential involvement of all three receptors as none can be ruled out.

B. Receptor-independent effects

Cannabinoid receptor-independent actions of AEA have been described in several tumor cell types (DeMorrow et al., 2007; Hinz et al., 2004a,b; Patsos et al., 2005; Ramer et al., 2001). Furthermore, most of these effects are via the actions of AEA at the cell membrane (DeMorrow et al., 2007; Hinz et al., 2004a,b; Ramer et al., 2001). Treatment of human neuroglioma cells with the stable analogue of AEA (Met-AEA) resulted in the induction of apoptosis (Hinz et al., 2004a,b; Ramer et al., 2001). This effect could not be blocked by the coadministration of antagonists of the Cb1, Cb2, or VR1 receptors, nor the Gi/o protein inactivator pertussis toxin (Ramer et al., 2001). Coupled with the induction of apoptosis by Met-AEA, there was an increased synthesis of ceramide, expression of cyclooxygenase-2 (Cox-2) and subsequent prostaglandin E2 synthesis via a mechanism involving p38 and p42/44 MAPK activation (Ramer et al., 2001). Specific Cox-2 inhibitors, such as celecoxib and diclofenac, or the specific silencing ofCox-2 expression with small-interfering RNA blocked the Met-AEA-induced apoptosis (Hinz et al., 2004b). Furthermore, the lipid raft disruptor methyl-beta-cyclodextrin blocked the Met-AEA-induced effects of ceramide synthesis, phosphorylation ofp38 andp42/44 MAPK, expression ofCox-2, and subsequent prostaglandin E2 synthesis (Hinz et al., 2004a). Together, these data suggest that Met-AEA, via lipid raft-mediated events, induces apoptosis of neuroglioma cells. A similar effect of AEA was observed in colorectal cancer cells (Patsos et al., 2005). Treatment of these cells with AEA increased the expression ofCox-2 and induced a subsequent cell death pathway (Patsos et al., 2005). Interestingly, inhibition of FAAH potentiated this cell death, suggesting that AEA-induced cell death was mediated via the metabolism of AEA by Cox-2 rather than through the classical AEA degradation pathway (Patsos et al., 2005).

More recently, we have shown that AEA can induce growth-suppressive/pro-apoptotic effects in cholangiocarcinoma cells which could not be blocked by any Cb1, Cb2, or VR1 antagonists nor the Gi/o protein inactivator pertussis toxin (DeMorrow et al., 2007). This is via the stabilization of the lipid raft structures within the plasma membrane, the increased production of ceramide, and the subsequent recruitment of the death receptor complex components into the lipid raft structures (DeMorrow et al., 2007). Interestingly, the other more prevalent endocannabinoid, 2-AG, had growth-promoting effects, which were shown to be via the complete disruption of the lipid raft structure (DeMorrow et al., 2007), an event which has previously been shown to result in growth-promoting effects in other cell types (Lambert et al., 2006; Mathay et al., 2008).

These receptor-independent mechanisms are summarized in Fig. 18.2. These so-called "receptor-independent" effects of AEA must be taken with a note of caution. Most of these studies were performed prior to the identification of GPR55 as a putative cannabinoid receptor. Therefore, the possibility that AEA is exerting its effects through GPR55 or some other, as yet unidentified cannabinoid receptor cannot be ruled out.

IV. Effects of AEA on Migration, Invasion, and Angiogenesis

The acquisition of metastatic abilities by cancer cells often leads to clinically incurable disease. Metastasis consists of a series of sequential steps including detachment of cells from the primary tumor, survival of the cells in circulation, arrest in a new organ, initiation and maintenance of growth in the new tissue, and vascularization of the metastatic tumor (Fidler, 2002). AEA has been shown to have a regulatory role at each of these stages in the metastatic process (Fig. 18.3). Firstly, AEA was shown to be an endogenous inhibitor of migration of both colon carcinoma cells and nonmalignant T lymphocytes in vitro (Joseph et al., 2004). There was a differential mechanism involved in the regulation of the tumor versus

Apoptosis

Figure 18.2 Schematic diagram of the cannabinoid receptor-independent mechanisms whereby AEA induces apoptosis. AEA stabilizes the lipid raft microdomains at the plasma membrane and increases ceramide production. This then has an effect on Cox-2 expression and prostaglandin E2 production with a subsequent increase in apoptosis. Alternatively, AEA-induced ceramide production facilitates the Fas/FasL death receptor complex within the lipid raft structure, which ultimately results in increased apoptosis.

Apoptosis

Figure 18.2 Schematic diagram of the cannabinoid receptor-independent mechanisms whereby AEA induces apoptosis. AEA stabilizes the lipid raft microdomains at the plasma membrane and increases ceramide production. This then has an effect on Cox-2 expression and prostaglandin E2 production with a subsequent increase in apoptosis. Alternatively, AEA-induced ceramide production facilitates the Fas/FasL death receptor complex within the lipid raft structure, which ultimately results in increased apoptosis.

immune cells, that is, tumor cell migration could be stimulated by specific agonists for Cb1 receptor, whereas the immune cell migration was inhibited by a Cb2-dependent mechanism (Joseph ei a/., 2004). Furthermore, in an m two model of metastatic spreading using breast cancer cell lines, the AEA analogue, met-AEA significantly reduced the number and dimension of metastatic nodes, an effect that was inhibited by specific Cb1 receptor antagonists (Grimaldi ei a/., 2006). Molecular changes in the organization and distribution of cytoskeleton proteins are necessary for focal adhesion; cell motility and cell invasion were then assessed. No changes in expression of any of the integrins were detected after Met-AEA treatment, however, there was a marked decrease in the phosphorylation of focal adhesion kinase and src, both of which are normally localized to the focal adhesions and are involved in the metastatic formation and development (Grimaldi ei a/., 2006).

Angiogenesis Migration Invasion

Figure 18.3 AEA inhibits other aspects of tumorigenesis such as angiogenesis, tumor cell migration, and tumor invasion. AEA inhibits angiogenesis via a decrease in VEGF expression, whereas the decrease in migration is thought to be via a decrease in the activation of focal adhesion kinases and src kinase, both of which are thought to be involved in cell migration and metastasis. Lastly, AEA inhibits tumor cell invasion by decreasing the expression of proteins responsible for breaking down the extracellular matrix of the target organ, such as MMPs and increasing the expression of the tissue inhibitors of MMPs.

Angiogenesis Migration Invasion

Figure 18.3 AEA inhibits other aspects of tumorigenesis such as angiogenesis, tumor cell migration, and tumor invasion. AEA inhibits angiogenesis via a decrease in VEGF expression, whereas the decrease in migration is thought to be via a decrease in the activation of focal adhesion kinases and src kinase, both of which are thought to be involved in cell migration and metastasis. Lastly, AEA inhibits tumor cell invasion by decreasing the expression of proteins responsible for breaking down the extracellular matrix of the target organ, such as MMPs and increasing the expression of the tissue inhibitors of MMPs.

In order for a migrating cancer cell to then invade another organ, the existing extracellular matrix components (e.g., collagens and proteoglycans) must be broken down and hence the rigid architecture of the target organ must be compromised. Matrix metalloproteinases (MMPs) are emerging as a family of enzymes that exerts important functions during tumor invasion (Curran and Murray, 2000; Stamenkovic, 2000). Tissue inhibitors of MMPs (TIMPs), and in particular TIMP-1, have also been shown to inhibit the proteolytic activity of MMPs and suppress vascular tumor growth and angiogenesis in xenograft animal models (Zacchigna ei a/., 2004). Furthermore, there appears to be a correlation between high cancer invasiveness and decreased TIMP-1 expression (Chan ei a/., 2005; Khokha ei a/., 1989). Recently, the effects of AEA on MMP and TIMP expression were evaluated in various cancer cell types. Using a cervical cancer cell line, Met-AEA as well as D9-THC, inhibited the invasive properties of these cells via the increased expression of TIMP-1 (Ramer and Hinz, 2008). The effects of the cannabinoids on invasion and TIMP-1 expression were inhibited by the pretreatment of the cells with Cb1 and Cb2 antagonists as well as specific inhibitors of the p38 and p42/44 MAPkinases (Ramer and Hinz, 2008). This effect of cannabinoids on TIMP-1 expression was mimicked by treatment of glioma cells with D9-THC (Blazquez ei a/., 2008a).

Conversely, in glioma cells, cannabinoid treatment selectively decreased the expression of MMP-2 via a Cb2-dependent mechanism and requiring the synthesis of ceramide (Blazquez ei a/., 2008b). By manipulating the levels of MMP-2 expression by siRNA and cDNA overexpression, the authors were able to show that the decrease in MMP-2 expression was critical for the cannabinoid-mediated inhibition of cell invasion (Blazquez ei a/., 2008b).

The last aspect of the metastatic process that is regulated by cannabinoids is angiogenesis. Met-AEA inhibited the basic fibroblast growth factor-stimulated endothelial cell proliferation and induced apoptosis in a Cb1-dependent manner (Pisanti ei a/., 2007). Furthermore, Met-AEA was able to inhibit bidimensional capillary-like tube formation and tumor-induced angiogenesis in a three-dimensional model of endothelial and thyroid tumor cell spheroid cocultures (Pisanti ei a/., 2007). In support of this, we have shown that AEA treatment of an m two xenograft model of cholan-giocarcinoma also markedly inhibits the expression of members of the vascular endothelial growth factor family (DeMorrow which are key regulators of both normal and abnormal angiogenesis (Ferrara, 2005; Ferrara and Kerbel, 2005).

Together, these data suggest that cannabinoids, and in particular anan-damide, may be key endogenous inhibitors of various stages in metastatic processes, including migration, invasion, and angiogenesis. This further supports the notion that drugs directed at regulating the endocannabinoid system may prove to be valuable tools in the fight against various cancers.

V. Targeting Degradation Enzymes of Cannabinoids as an Anticancer Therapy

As mentioned previously, the degradation of endocannabinoids is an active and rapid process. Therefore, blocking the degradation pathway may enhance the antiproliferative effects of AEA and have beneficial effects in cancer treatment. Indeed, treatment of human breast cancer cells m Wire with palmitoylethanolamide enhances the antiproliferative effects of AEA (Di Marzo ei a/., 2001). This agent was shown to reduce the expression of FAAH up to 30—40% thereby allowing the accumulation of AEA and increasing its antiproliferative effects (Di Marzo ei a/., 2001). In addition, treatment of athymic mice with thyroid tumor xenografts with VDM-11 (a selective inhibitor of endocannabinoid cellular reuptake) or arachidonyl-serotonin (a selective blocker of endocannabinoid hydrolysis) increased the intratumoral levels ofanandamide and significantly decreased tumor volume (Bifulco ei a/., 2004). The antiproliferative actions of these agents could be only partly inhibited by the pretreatment of the Cb1 receptor antagonist, suggesting that endocannabinoids tonically control tumor growth in vivo by both Cb1-mediated and non-Cb1-mediated mechanisms (Bifulco et al., 2004). Regardless of the molecular mechanism by which anandamide and other endocannbinoids regulate tumor growth, inhibitors of their inactiva-tion might be useful for the development of novel anticancer drugs (Bifulco et al., 2004).

VI. Tumor Promoting Effects of Anandamide

The evidence supporting growth-promoting effect of AEA in tumors is pallid in comparison to the antitumoral effects described above. There is a greater volume of research indicating that the structurally similar, plant-derived cannabinoid, D9-THC stimulates growth in a number of cancer cell lines via Cb1 and Cb2 receptor-independent mechanisms (McKallip et al., 2005; Takeda et al., 2008). However, several cannabinoids, including AEA, have been shown to accelerate proliferation via the transactivation of the EGFR in a TACE/ADAM17 metalloprotease-dependent manner (Hart et al., 2004). This effect was observed in several cell lines from various origins including lung cancer, squamous cell carcinoma, bladder carcinoma, glioblastoma, astrocytoma, and kidney cancer (Hart et al., 2004). The cannabinoid-induced activation of the EGFR leads to the subsequent phosphorylation and activation of the adaptor protein Src homology 2 domain-containing (shc), and downstream activation of the ERK1/2 and Akt/PKB pathways (Hart et al., 2004). Thus, the cross-communication of cannabinoid receptors and EGFR may provide an explanation as to how cannabinoids may stimulate cancer cell proliferation (Hart et al., 2004).

In addition, the stable analogue of AEA, methanandamide, has a mito-genic effect on an androgen-dependent prostate cancer cell line that could be blocked by antagonists for either the Cb1 or Cb2 receptors as well as by the PI-3kinase inhibitor (Sanchez et al., 2003). The downstream consequence of activation of the endocannabinoid system was an increase in the expression of the androgen receptor, which is directly linked to the growth of these cells (Sanchez et al., 2003).

VII. Conclusions

In conclusion, the endocannabinoid system exerts a myriad of effects on tumor cell growth, progression, angiogenesis, and migration. With a notable few exceptions, targeting the endocannabinoid system with agents that activate cannabinoid receptors or increase the endogenous levels of

AEA may prove to have therapeutic benefit in the treatment of various cancers. Further studies into the downstream consequences of AEA treatment are required and may illuminate other potential therapeutic targets.

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