Amador Haro

Cannabinoids Inhibit the Vascular Endothelial Growth Factor Pathway in Gliomas

Cannabinoids inhibit tumor angiogenesis in mice, but the mechanism of their antiangiogenic action is still unknown. Because the vascular endothelial growth factor (VEGF) pathway plays a critical role in tumor angiogenesis, here we studied whether cannabinoids affect it. As a first approach, cDNA array analysis showed that cannabinoid administration to mice bearing s.c. gliomas lowered the expression of various VEGF pathway-related genes. The use of other methods (ELISA, Western blotting, and confocal microscopy) provided additional evidence that cannabinoids depressed the VEGF pathway by decreasing the production of VEGF and the activation of VEGF receptor (VEGFR)-2, the most prominent VEGF receptor, in cultured glioma cells and in mouse gliomas. Cannabinoid-induced inhibition of VEGF production and VEGFR-2 activation was abrogated both in vitro and in vivo by pharmacological blockade of ceramide biosynthesis. These changes in the VEGF pathway were paralleled by changes in tumor size. Moreover, intratumoral administration of the cannabinoid Δ9-tetrahydrocannabinol to two patients with glioblastoma multiforme (grade IV astrocytoma) decreased VEGF levels and VEGFR-2 activation in the tumors. Because blockade of the VEGF pathway constitutes one of the most promising antitumoral approaches currently available, the present findings provide a novel pharmacological target for cannabinoid-based therapies.

Cannabinoids Inhibit Glioma Cell Invasion by Down-regulating Matrix Metalloproteinase-2 Expression

Cannabinoids, the active components of Cannabis sativa L. and their derivatives, inhibit tumor growth in laboratory animals by inducing apoptosis of tumor cells and impairing tumor angiogenesis. It has also been reported that these compounds inhibit tumor cell spreading, but the molecular targets of this cannabinoid action remain elusive. Here, we evaluated the effect of cannabinoids on matrix metalloproteinase (MMP) expression and its effect on tumor cell invasion. Local administration of Delta(9)-tetrahydrocannabinol (THC), the major active ingredient of cannabis, down-regulated MMP-2 expression in gliomas generated in mice, as determined by Western blot, immunofluorescence, and real-time quantitative PCR analyses. This cannabinoid-induced inhibition of MMP-2 expression in gliomas (a) was MMP-2-selective, as levels of other MMP family members were unaffected; (b) was mimicked by JWH-133, a CB(2) cannabinoid receptor-selective agonist that is devoid of psychoactive side effects; (c) was abrogated by fumonisin B1, a selective inhibitor of ceramide biosynthesis; and (d) was also evident in two patients with recurrent glioblastoma multiforme. THC inhibited MMP-2 expression and cell invasion in cultured glioma cells. Manipulation of MMP-2 expression by RNA interference and cDNA overexpression experiments proved that down-regulation of this MMP plays a critical role in THC-mediated inhibition of cell invasion. Cannabinoid-induced inhibition of MMP-2 expression and cell invasion was prevented by blocking ceramide biosynthesis and by knocking-down the expression of the stress protein p8. As MMP-2 up-regulation is associated with high progression and poor prognosis of gliomas and many other tumors, MMP-2 down-regulation constitutes a new hallmark of cannabinoid antitumoral activity.

Cannabinoids and gliomas.

Cannabinoids, the active components of Cannabis sativa L., act in the body by mimicking endogenous substances—the endocannabinoids—that activate specific cell surface receptors. Cannabinoids exert various palliative effects in cancer patients. In addition, cannabinoids inhibit the growth of different types of tumor cells, including glioma cells, in laboratory animals. They do so by modulating key cell signaling pathways, mostly the endoplasmic reticulum stress response, thereby inducing antitumoral actions such as the apoptotic death of tumor cells and the inhibition of tumor angiogenesis. Of interest, cannabinoids seem to be selective antitumoral compounds, as they kill glioma cells, but not their non-transformed astroglial counterparts. On the basis of these preclinical findings, a pilot clinical study of Δ9-tetrahydrocannabinol (THC) in patients with recurrent glioblastoma multiforme has been recently run. The good safety profile of THC, together with its possible growth-inhibiting action on tumor cells, justifies the setting up of future trials aimed at evaluating the potential antitumoral activity of cannabinoids.

Ceramide-induced translocation of protein kinase C ζ in primary cultures of astrocytes

The present research was undertaken to study the possible involvement of the atypical protein kinase C (PKC) ζ in ceramide signal transduction in primary cultures of rat astrocytes. As shown by Western blot analysis, translocation of immunoreactive PKCζ to the particulate fraction occurred upon exposure of astrocytes to cell‐permeable ceramide analogs or to exogenous sphingomyelinase. The particulate fraction may correspond to a perinuclear area, as indicated by immunocytochemical techniques. Furthermore, treatment of cells with N‐octanoylsphingosine led to an increased phosphorylation of PKCζ. Results thus show that stimulation of PKCζ may be one of the intracellular events triggered by activation of the sphingomyelin pathway.

Induction of nerve growth factor synthesis by sphingomyelinase and ceramide in primary astrocyte cultures.

Astrocytes synthesize nerve growth factor (NGF) in response to pro-inflammatory cytokines. To further study the signaling mechanism involved in this induction of NGF production, the sphingomyelin (SM) pathway was studied. Addition of exogenous neutral SMase (Staphylococcus aureus) or C2-ceramide to primary cultures of newborn rat cortical astrocytes elicited a dose-response increase of NGF synthesis, with maximal effect at 1 U/ml and 25 microM, respectively. Induction of NGF synthesis by SMase and ceramide was shown to be independent of classical PKC activity. Intracellular cAMP-raising agents, such as forskolin and 3-isobutyl-1-methylxanthine, partially prevented the SMase- and C2-ceramide-induced secretion of NGF to the cell supernatant. PD098059 and apigenin, inhibitors of the mitogen-activated protein (MAP) kinase pathway, produced a dose-response inhibition of the SMase- and C2-cer-induced release of NGF. This observation points to the possibility that regulation of NGF synthesis and secretion by the SMase pathway may be mediated downstream by the MAP kinase cascade. As a matter of fact, pre-treatment of astrocytes with SMase or C8-ceramide led to an increased phosphorylation of raf-1. Moreover, MAP kinase activity was enhanced in astrocytes treated with SMase or both ceramides. In conclusion, results suggest that the SMase pathway may control NGF synthesis in the central nervous system, and raise the possibility of an involvement of the MAP kinase cascade in this process.

Regulation of nerve growth factor secretion and mRNA expression by bacterial lipopolysaccharide in primary cultures of rat astrocytes

The present work was undertaken to study the effect of bacterial lipopolysaccharide (LPS), a potent activator of the host inflammatory response, on the synthesis of nerve growth factor (NGF) by newborn rat brain astrocytes. Treatment of primary rat astroglial cells cultured in chemically defined medium with LPS resulted in a dose‐dependent accumulation of NGF mRNA, and an increased release of NGF protein in the cell medium. NGF mRNA levels were maximal after 24 hr of stimulation (8‐fold increase), whereas extracellular NGF peaked after 72 hours of treatment (17‐fold increase). This dramatic increase of extracellular NGF was abrogated if cells were treated with actinomycin D or cycloheximide, a fact which implies that the accumulation of extracellular NGF by LPS‐treated cells requires DNA transcription and RNA translation. Stimulation of NGF synthesis and secretion was: (i) unaffected by treatment with the protein kinase C inhibitor bisindolylmaleimide, and (ii) prevented by forskolin and 3‐isobutyl‐1‐methylxanthine, two agents which increase cAMP levels. Inhibition of LPS effect was also obtained with apigenin, a proposed inhibitor of the mitogen‐activated protein kinase pathway. Results thus show that LPS stimulates NGF synthesis by astroglial cells through a mechanism that is independent of protein kinase C (PKC), antagonized by cAMP‐elevating agents, and probably mediated by the mitogen‐activated protein kinase cascade. The data raise the possibility that LPS exerts stimulatory effects on NGF synthesis that are independent of those elicited by astrocyte‐derived inflammatory lymphokines such as IL‐1β, TNFα or TGFβ1. J. Neurosci. Res. 49:569–575, 1997.

Down-regulation of tissue inhibitor of metalloproteinases-1 in gliomas: a new marker of cannabinoid antitumoral activity?

Cannabinoids, the active components of Cannabis sativa L. and their derivatives, inhibit tumor growth in laboratory animals by inducing apoptosis of tumor cells and inhibiting tumor angiogenesis. It has also been reported that cannabinoids inhibit tumor cell invasiveness, but the molecular targets of this cannabinoid action remain elusive. Here we evaluated the effects of cannabinoids on the expression of tissue inhibitors of metalloproteinases (TIMPs), which play critical roles in the acquisition of migrating and invasive capacities by tumor cells. Local administration of Delta(9)-tetrahydrocannabinol (THC), the major active ingredient of cannabis, down-regulated TIMP-1 expression in mice bearing subcutaneous gliomas, as determined by Western blot and immunofluorescence analyses. This cannabinoid-induced inhibition of TIMP-1 expression in gliomas (i) was mimicked by JWH-133, a selective CB(2) cannabinoid receptor agonist that is devoid of psychoactive side effects, (ii) was abrogated by fumonisin B1, a selective inhibitor of ceramide synthesis de novo, and (iii) was also evident in two patients with recurrent glioblastoma multiforme (grade IV astrocytoma). THC also depressed TIMP-1 expression in cultures of various human glioma cell lines as well as in primary tumor cells obtained from a glioblastoma multiforme patient. This action was prevented by pharmacological blockade of ceramide biosynthesis and by knocking-down the expression of the stress protein p8. As TIMP-1 up-regulation is associated with high malignancy and negative prognosis of numerous cancers, TIMP-1 down-regulation may be a hallmark of cannabinoid-induced inhibition of glioma progression.

A possible new class of octopamine receptors coupled to adenylate cyclase in the brain of the dipterous Ceratitis capitata. Pharmacological characterization and regulation of 3H-octopamine binding

Octopamine exerts its effects in insects through interaction with at least two classes of receptors, designated octopamine-1 and octopamine-2. Octopamine-2 receptors are positively coupled to adenylate cyclase, while octopamine-1 receptors are not coupled to this enzyme system. Ceratitis capitata brain appears to have octopamine receptors as unique aminergic receptors coupled to adenylate cyclase. These receptors show some pharmacological analogies with respect to octopamine-2 receptors, however they should constitute a new class of octopamine receptors. C. capitata brain octopamine receptors have also been characterized by [3H]octopamine-binding studies, exhibiting similar regulatory mechanisms to other receptors coupled to adenylate cyclase activation.

Adenylyl cyclase system is affected differently by endurance physical training in heart and adipose tissue

Adaptive changes in the beta-adrenergic adenylyl cyclase (AC) system in response to endurance training were studied in heart and adipose tissue. Training was performed by making male Wistar rats run on a motor-driven treadmill. The changes following exercise training were opposite in the two tissue studied. The density of beta-adrenergic receptors in left ventricular membranes of trained rats showed a marked decrease. Comparison of AC activities in cardiac membranes prepared from trained and sedentary rats revealed a depressing effect of endurance training on: 1. the beta-adrenergic stimulatory pathway and the inhibition of AC via receptor; 2. the Gs component and the Gs-adenylyl cyclase coupling, as shown by the response of adenylyl cyclase to GppNHp and NaF; and 3. the enzyme catalytic activity in the presence of Mn2+ or forskolin. The levels of Gsalpha subunits in the left ventricle, as measured in terms of ADP-ribosylated and immunologically reactive proteins, were decreased by endurance exercise, whereas immunodetectable levels of Gialpha2 increased in the membranes of trained myocardium. In contrast to the diminished sensitivity that characterizes the behavior of the cardiac beta-adrenergic-AC system, endurance physical training increased sensitivity of this signal transduction system in adipose tissue. Thus, the density of beta-ARs as well as AC activity and the beta-adrenergic stimulatory pathway were increased in adipose membranes of trained rats compared with the corresponding sedentary controls. In addition, the levels of Gsalpha subunits were higher in the adipose plasma membranes of trained rats. However, immunodetectable levels of Gi1alpha and Gi3alpha increased with training, whereas the amount of Gi2alpha decreased in membranes of trained rats. In conclusion, the present study shows that chronic exercise is associated with a tissue-specific adaptation of the beta-adrenergic AC system.

Cardiac β-adrenoceptors, G-proteins and adenylate cyclase regulation during myocardial hypertrophy

The role of the beta-adrenoceptor-G-protein-adenylate cyclase system in the pathogenesis of cardiac hypertrophy was studied. We have used a minipig model of pressure-overload cardiac hypertrophy secondary to aortic banding. Four groups of five animals were used: minipigs made hypertrophic were evaluated 2 months (CH2 group) and 9 months (CH9 group) later and compared to controls (C2 and C9 groups, respectively). A decrease in beta-adrenergic receptor density and an increase in antagonist affinity were shown in left ventricular membranes of hypertrophied animals compared with controls. In both groups, CH2 and CH9, an increase in EC50 for isoproterenol-stimulated adenylate cyclase activity, an increase in forskolin-stimulated adenylate cyclase activity and a diminished inhibition by carbachol of isoproterenol-stimulated adenylate cyclase were observed. In contrast, fluoride-stimulated adenylate cyclase activity was markedly increased only in the end stage of hypertrophy. alpha s-cholera toxin-catalysed ADP-ribosylation is increased in early hypertrophy and then decreases with late hypertrophy and a similar pattern is observed with alpha o pertussis toxin-catalysed ADP-ribosylation, whereas alpha i-ADP-ribosylation remains unchanged. Tissue content of Gs-, Gi- and Go-proteins, as assessed by specific antibodies, was found unchanged in CH9 and CH2 groups when compared with that in C9 and C2 control groups, respectively. Modifications in Gs functional activity in later hypertrophic stages, expressed as alterations in cholera toxin ADP-ribosylation and adenylate cyclase fluoride responsiveness, may be important in the pathogenesis of decompensation from compensated hypertrophy to cardiac failure.