Alzheimer's disease (AD) is characterized by enhanced ␤-amyloid peptide (␤A) deposition along with glial activation in senile plaques, selective neuronal loss, and cognitive deficits. Cannabinoids are neuroprotective agents against excitotoxicity in vitro and acute brain damage in vivo. This background prompted us to study the localization, expression, and function of cannabinoid receptors in AD and the possible protective role of cannabinoids after ␤A treatment, both in vivo and in vitro. Here, we show that senile plaques in AD patients express cannabinoid receptors CB 1 and CB 2 , together with markers of microglial activation, and that CB 1 -positive neurons, present in high numbers in control cases, are greatly reduced in areas of microglial activation. In pharmacological experiments, we found that G-protein coupling and CB 1 receptor protein expression are markedly decreased in AD brains. Additionally, in AD brains, protein nitration is increased, and, more specifically, CB 1 and CB 2 proteins show enhanced nitration. Intracerebroventricular administration of the synthetic cannabinoid WIN55,212-2 to rats prevent ␤A-induced microglial activation, cognitive impairment, and loss of neuronal markers. Cannabinoids (HU-210, WIN55,212-2, and JWH-133) block ␤A-induced activation of cultured microglial cells, as judged by mitochondrial activity, cell morphology, and tumor necrosis factor-␣ release; these effects are independent of the antioxidant action of cannabinoid compounds and are also exerted by a CB 2 -selective agonist. Moreover, cannabinoids abrogate microglia-mediated neurotoxicity after ␤A addition to rat cortical cocultures. Our results indicate that cannabinoid receptors are important in the pathology of AD and that cannabinoids succeed in preventing the neurodegenerative process occurring in the disease.

Cannabinoids, the active components of marijuana and their derivatives, induce tumor regression in rodents (8). However, the mechanism of cannabinoid antitumoral action in vivo is as yet unknown. Here we show that local administration of a nonpsychoactive cannabinoid to mice inhibits angiogenesis of malignant gliomas as determined by immunohistochemical analyses and vascular permeability assays. In vitro and in vivo experiments show that at least two mechanisms may be involved in this cannabinoid action: the direct inhibition of vascular endothelial cell migration and survival as well as the decrease of the expression of proangiogenic factors (vascular endothelial growth factor and angiopoietin-2) and matrix metalloproteinase-2 in the tumors. Inhibition of tumor angiogenesis may allow new strategies for the design of cannabinoid-based antitumoral therapies.

Rho proteins belong to the small GTPases superfamily. They function as molecular switches that, in response to diverse stimuli, control key signaling and structural aspects of the cell. Although early studies proposed a role for Rho GTPases in cellular transformation, this effect was underestimated due to the fact that no genetic mutations affecting Rho-encoding genes were found in tumors. Recently, it has become evident that Rho GTPases participate in the carcinogenic process by either overexpression of some of the members of the family with oncogenic activity, downmodulation of other members with suggested tumor suppressor activity, or by alteration of upstream modulators or downstream effectors. Thus, alteration of the levels of expression of different members of the family of Rho GTPases has been detected in many types of human tumors leading to a great interest in the cellular effects elicited by these oncoproteins. This essay reviews the current evidence of dysregulation of Rho signaling by overexpression in human tumors.

Delta(9)-Tetrahydrocannabinol (THC) and other cannabinoids have been shown to induce apoptosis of glioma cells via ceramide generation. In the present study, we investigated the metabolic origin of the ceramide responsible for this cannabinoid-induced apoptosis by using two subclones of C6 glioma cells: C6.9, which is sensitive to THC-induced apoptosis; and C6.4, which is resistant to THC-induced apoptosis. Pharmacological inhibition of ceramide synthesis de novo, but not of neutral and acid sphingomyelinases, prevented THC-induced apoptosis in C6.9 cells. The activity of serine palmitoyltransferase (SPT), which catalyses the rate-limiting step of ceramide synthesis de novo, was remarkably enhanced by THC in C6.9 cells, but not in C6.4 cells. However, no major changes in SPT mRNA and protein levels were evident. Changes in SPT activity paralleled changes in ceramide levels. Pharmacological inhibition of ceramide synthesis de novo also prevented the stimulation of extracellular-signal-regulated kinase and the inhibition of protein kinase B triggered by cannabinoids. These findings show that de novo-synthesized ceramide is involved in cannabinoid-induced apoptosis of glioma cells.

Cannabinoids, the active components of marijuana and their endogenous counterparts, exert many of their actions in brain through the seven-transmembrane receptor CB 1 . This receptor is coupled to the activation of the extracellular signal-regulated kinase (ERK) cascade. However, the precise molecular mechanism for CB 1 -mediated ERK activation is still unknown. Here, we show that in U373 MG human astrocytoma cells, CB 1 receptor activation with the cannabinoid agonist ⌬ 8 -tetrahydrocannabinol dimethyl heptyl (HU-210) was coupled to ERK activation and protection from ceramide-induced apoptosis. HU-210-induced ERK activation was inhibited by tyrphostin AG1478 and PP2, widely employed inhibitors of the epidermal growth factor receptor (EGF R ) and the Src family of cytosolic tyrosine kinases, respectively. However, HU-210 stimulation resulted in neither EGF R phosphorylation, Src tyrosine phosphorylation, nor increased Src activity. In addition, dominantnegative forms of both proteins were unable to prevent cannabinoid-induced ERK activation, thus excluding the existence of CB 1 -mediated EGF R transactivation or Src activation. Wortmannin and 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294,002), inhibitors of the phosphatidylinositol 3-kinase (PI3K) signaling pathway, blocked cannabinoid-induced ERK activation. Likewise, HU-210 stimulated the PI3K downstream targets protein kinase B (PKB), as shown by its phosphorylation in Thr 308 and Ser 473 residues, and Raf-1. Moreover, ␤␥ subunit release mimicked ERK and PI3K/PKB activation, suggesting that activation of class IB PI3K mediates cannabinoid action. Pro-survival HU-210 action also required activation of both PI3K and ERK signaling pathways. In conclusion, CB 1 -induced ERK activation was mediated by PI3K IB and this effect may have important consequences in the control of cell death/ survival decision.

Cannabinoids, the active components of marijuana and their endogenous counterparts, exert many of their actions on the central nervous system by binding to the CB 1 cannabinoid receptor. Different studies have shown that cannabinoids can protect neural cells from different insults. However, those studies have been performed in neurons, whereas no attention has been focused on glial cells. Here we used the pro-apoptotic lipid ceramide to induce apoptosis in astrocytes, and we studied the protective effect exerted by cannabinoids. Results show the following: (i) cannabinoids rescue primary astrocytes from C 2 -ceramide-induced apoptosis in a doseand time-dependent manner; (ii) triggering of this antiapoptotic signal depends on the phosphatidylinositol 3-kinase/protein kinase B pathway; (iii) ERK and its downstream target p90 ribosomal S6 kinase might be also involved in the protective effect of cannabinoids; and (iv) cannabinoids protect astrocytes from the cytotoxic effects of focal C 2 -ceramide administration in vivo. In summary, results show that cannabinoids protect astrocytes from ceramide-induced apoptosis via stimulation of the phosphatidylinositol 3-kinase/protein kinase B pathway. These findings constitute the first evidence for an "astroprotective" role of cannabinoids.

Cannabinoids exert most of their effects in the central nervous system through the CB(1) cannabinoid receptor. This G-protein-coupled receptor has been shown to be functionally coupled to inhibition of adenylate cyclase, modulation of ion channels and activation of extracellular-signal-regulated kinase. Using Chinese hamster ovary cells stably transfected with the CB(1) receptor cDNA we show here that Delta(9)-tetrahydrocannabinol (THC), the major active component of marijuana, induces the activation of protein kinase B/Akt (PKB). This effect of THC was also exerted by the endogenous cannabinoid anandamide and the synthetic cannabinoids CP-55940 and HU-210, and was prevented by the selective CB(1) antagonist SR141716. Pertussis toxin and wortmannin blocked the CB(1) receptor-evoked activation of PKB, pointing to the sequential involvement of a G(i)/G(o) protein and phosphoinositide 3'-kinase. The functionality of the cannabinoid-induced stimulation of PKB was proved by the increased phosphorylation of glycogen synthase kinase-3 serine 21 observed in cannabinoid-treated cells and its prevention by SR141716 and wortmannin. Cannabinoids activated PKB in the human astrocytoma cell line U373 MG, which expresses the CB(1) receptor, but not in the human promyelocytic cell line HL-60, which expresses the CB(2) receptor. Data indicate that activation of PKB may be responsible for some of the effects of cannabinoids in cells expressing the CB(1) receptor.

Abstract. Cdc42, a member of Rho GTPases family, is involved in the regulation of several cellular functions, such as rearrangement of actin cytoskeleton, membrane trafficking, cell-cycle progression, and transcriptional regulation. Aberrant expression or activity of Cdc42 has been reported in several tumours. Here, the specific role of Cdc42 in development and progression of colorectal cancer was analyzed through microarrays technology. A comparative analysis of Cdc42 overexpressing cells versus cells with decreased Cdc42 levels through siRNA revealed that Cdc42 overexpression downregulated the potential tumour suppressor gene ID4. Results were validated by quantitative RT-PCR and the methylation status of the specific promoter, analyzed. Methylation-specific PCR and bisulfite sequencing PCR analysis revealed that Cdc42 induced the methylation of the CpG island of the ID4 promoter. Colorectal adenocarcinoma samples were compared with the corresponding adjacent normal tissue of the same patient in order to determine specific gene expression levels. The downregulation of ID4 by Cdc42 was also found of relevance in colorectal adenocarcinoma biopsies. Cdc42 was found to be overexpressed with high incidence (60%) in colorectal cancer samples, and this expression was associated with silencing of ID4 with statistical significance (p<0.05). Cdc42 may have a role in the development of colon cancer. Furthermore, inhibition of Cdc42 activity may have a direct impact in the management of colorectal cancer.