Manuel Guzmán – Nobel Prize 2005 Physiology and Medicine

These research articles have been published by 2005 Nobel Prize winner for Physiology and Medicine Dr.

Manuel Guzmán and his associates from the Department of Biochemistry and Molecular Biology I,

School of Biology, Complutense University, 28040 Madrid, Spain and are summarized here. The full

reports are in the Journals quoted.

Neuroscientist 2007 Apr; 13 (2):109-14.

The endocannabinoid system and neurogenesis in health and disease.

The endocannabinoid system exerts an important neuromodulatory function in different brain

areas and is also known to be involved in the regulation of neural cell fate. Thus, CB(1) cannabinoid

receptors are neuroprotective in different models of brain injury, and their expression is altered in various

neurodegenerative diseases. Recent findings have demonstrated the presence of a functional

endocannabinoid system in neural progenitor cells that participates in the regulation of cell proliferation

and differentiation. In this Research Update, the authors address the experimental evidence

regarding the regulatory role of cannabinoids in neurogenesis [nerve growth] and analyze them in

the context of those pathological disorders in which cannabinoid function and altered neuronal or

glial [fiberous cells between nerves] generation is most relevant, for example, stroke and multiple

sclerosis.

Journal Biological Chemistry 2007 Mar 2; 282(9):6854-62. Epub 2007 Jan 2.

Cannabinoids induce glioma stem-like cell differentiation and inhibit gliomagenesis.

Glioma stem-like cells constitute one of the potential origins of gliomas, and therefore, their elimination

is an essential factor for the development of efficient therapeutic strategies. Cannabinoids are known to

exert an antitumoral action on gliomas [tumours] that relies on at least two mechanisms: induction

of apoptosis [programmed cell death] of transformed cells and inhibition of tumor angiogenesis.

However, whether cannabinoids target human glioma stem cells and their potential impact in

gliomagenesis are unknown. Here, we show that glioma stem-like cells derived from glioblastoma

multiforme [malignant tumour] biopsies and the glioma cell lines U87MG and U373MG express

cannabinoid type 1 (CB(1)) and type 2 (CB(2)) receptors and other elements of the endocannabinoid

system. In gene array experiments, CB receptor activation altered the expression of genes involved in the

regulation of stem cell proliferation and differentiation. The cannabinoid agonists HU-210 and JWH-133

promoted glial differentiation in a CB receptor-dependent manner as shown by the increased number of

S-100beta- and glial fibrillary acidic protein-expressing cells. In parallel, cannabinoids decreased the cell

population expressing the neuroepithelial progenitor marker nestin. Moreover, cannabinoid challenge

decreased the efficiency of glioma stem-like cells to initiate glioma formation in vivo, a finding that

correlated with decreased neurosphere formation and cell proliferation in secondary xenografts. Gliomas

derived from cannabinoid-treated cancer stem-like cells were characterized with a panel of neural markers

and evidenced a more differentiated phenotype and a concomitant decrease in nestin expression. Overall,

our results demonstrate that cannabinoids target glioma stem-like cells, promote their

differentiation, and inhibit gliomagenesis, thus giving further support to their potential use in the

management of malignant gliomas. [tumours]

Trends Pharmacological Science 2007 Jan; 28 (1):39-45. Epub 2006 Dec 1.

Cannabinoid CB2 receptor: a new target for controlling neural cell survival?

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Two types of cannabinoid receptor have been cloned and characterized. Whereas CB1 receptors are

ubiquitously expressed in neurons of the CNS, CB2 receptors have been thought to be absent from the

CNS. Recent data now question this notion and support the expression of CB2 receptors in microglial

cells, astrocytes and even some neuron subpopulations. This discrete distribution makes CB2 receptors

interesting targets for treating neurological disorders because CB2-selective agonists lack psychoactivity.

Here, we review evidence supporting the idea that CB2 receptors are implicated in the control of

fundamental neural cell processes, such as proliferation and survival, and that their pharmacological

manipulation might be useful for both delaying the progression of neurodegenerative disorders and

inhibiting the growth of glial tumors.

FASEB Journal 2006 (Federation of European Biochemical Societies)Dec; 20 (14):2633-5. Epub 2006

Oct 25.

Cannabinoid receptors as novel targets for the treatment of melanoma.

Melanoma causes the greatest number of skin cancer-related deaths worldwide. Despite intensive

research, prevention and early detection are the only effective measures against melanoma, 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. Human melanomas and melanoma cell lines express CB1 and CB2 cannabinoid receptors.

Activation of these receptors decreased growth, proliferation, angiogenesis and metastasis, and increased

apoptosis, of melanomas in mice. Cannabinoid antimelanoma activity was independent of the immune

status of the animal, could be achieved without overt psychoactive effects and was selective for

melanoma cells vs. normal melanocytes. Cannabinoid antiproliferative action on melanoma cells was due,

at least in part, to cell cycle arrest at the G1-S transition via inhibition of the prosurvival protein Akt and

hypophosphorylation of the pRb retinoblastoma protein tumor suppressor. These findings may

contribute to the design of new chemotherapeutic strategies for the management of melanoma.

FASEB Journal 2006 (Federation of European Biochemical Societies) Nov; 20 (13):2405-7. Epub 2006

Oct 2.

Non-psychoactive CB2 cannabinoid agonists stimulate neural progenitor proliferation.

Cannabinoids, the active components of marijuana and their endogenous counterparts, act on the

brain and many other organs through the widely expressed CB1 cannabinoid receptor. In contrast,

the CB2 cannabinoid receptor is abundant in the immune system and shows a restricted expression pattern

in brain cells. CB2-selective agonists are, therefore, very attractive therapeutic agents as they do not cause

CB1-mediated psychoactive effects. CB2 receptor expression in brain has been partially examined in

differentiated cells, while its presence and function in neural progenitor cells remain unknown. Here we

show that the CB2 receptor is expressed, both in vitro and in vivo, in neural progenitors from late

embryonic stages to adult brain. Selective pharmacological activation of the CB2 receptor in vitro

promotes neural progenitor cell proliferation and neurosphere generation, an action that is impaired in

CB2-deficient cells. Accordingly, in vivo experiments evidence that hippocampal progenitor proliferation

is increased by administration of the CB2-selective agonist HU-308. Moreover, impaired progenitor

proliferation was observed in CB2-deficient mice both in normal conditions and on kainate-induced

excitotoxicity. These findings provide a novel physiological role for the CB2 cannabinoid receptor

and open a novel therapeutic avenue for manipulating neural progenitor cell fate.

Cancer Research 2006 Jul 1; 66(13):6748-55.

Cannabinoids induce apoptosis of pancreatic tumor cells via endoplasmic reticulum stress-related

genes.

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Pancreatic adenocarcinomas are among the most malignant forms of cancer and, therefore, it is of

especial interest to set new strategies aimed at improving the prognostic of this deadly disease. The

present study was undertaken to investigate the action of cannabinoids, a new family of potential

antitumoral agents, in pancreatic cancer. We show that cannabinoid receptors are expressed in human

pancreatic tumor cell lines and tumor biopsies at much higher levels than in normal pancreatic tissue.

Studies conducted with MiaPaCa2 and Panc1 cell lines showed that cannabinoid administration (a)

induced apoptosis, (b) increased ceramide levels, and (c) up-regulated mRNA levels of the stress protein

p8. These effects were prevented by blockade of the CB(2) cannabinoid receptor or by pharmacologic

inhibition of ceramide synthesis de novo. Knockdown experiments using selective small interfering

RNAs showed the involvement of p8 via its downstream endoplasmic reticulum stress-related targets

activating transcription factor 4 (ATF-4) and TRB3 in Delta(9)-tetrahydrocannabinol-induced apoptosis.

Cannabinoids also reduced the growth of tumor cells in two animal models of pancreatic cancer. In

addition, cannabinoid treatment inhibited the spreading of pancreatic tumor cells. Moreover,

cannabinoid administration selectively increased apoptosis and TRB3 expression in pancreatic

tumor cells but not in normal tissue. In conclusion, results presented here show that cannabinoids lead

to apoptosis of pancreatic tumor cells via a CB(2) receptor and de novo synthesized ceramide-dependent

up-regulation of p8 and the endoplasmic reticulum stress-related genes ATF-4 and TRB3. These findings

may contribute to set the basis for a new therapeutic approach for the treatment of pancreatic

cancer.

Cancer Research 2006 Jul 1;66(13):6615-21.

Delta9-tetrahydrocannabinol inhibits cell cycle progression in human breast cancer cells through

Cdc2 regulation.

It has been proposed that cannabinoids are involved in the control of cell fate. Thus, these compounds can

modulate proliferation, differentiation, and survival in different manners depending on the cell type and

its physiopathologic context. However, little is known about the effect of cannabinoids on the cell cycle,

the main process controlling cell fate. Here, we show that Delta(9)-tetrahydrocannabinol (THC), through

activation of CB(2) cannabinoid receptors, reduces human breast cancer cell proliferation by blocking the

progression of the cell cycle and by inducing apoptosis. In particular, THC arrests cells in G(2)-M via

down-regulation of Cdc2, as suggested by the decreased sensitivity to THC acquired by Cdc2-

overexpressing cells. Of interest, the proliferation pattern of normal human mammary epithelial cells was

much less affected by THC. We also analyzed by real-time quantitative PCR the expression of CB(1) and

CB(2) cannabinoid receptors in a series of human breast tumor and nontumor samples. We found a

correlation between CB(2) expression and histologic grade of the tumors. There was also an association

between CB(2) expression and other markers of prognostic and predictive value, such as estrogen

receptor, progesterone receptor, and ERBB2/HER-2 oncogene. Importantly, no significant CB(2)

expression was detected in nontumor breast tissue. Taken together, these data might set the basis

for a cannabinoid therapy for the management of breast cancer.

British Journal Cancer 2006 Jul 17; 95(2):197-203. Epub 2006 Jun 27.

A pilot clinical study of Delta9-tetrahydrocannabinol in patients with recurrent glioblastoma

multiforme.

Delta(9)-Tetrahydrocannabinol (THC) and other cannabinoids inhibit tumour growth and

angiogenesis in animal models, so their potential application as antitumoral drugs has been

suggested. However, the antitumoral effect of cannabinoids has never been tested in humans. Here

we report the first clinical study aimed at assessing cannabinoid antitumoral action, specifically a pilot

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phase I trial in which nine patients with recurrent glioblastoma multiforme were administered THC

intratumoraly. The patients had previously failed standard therapy (surgery and radiotherapy) and

had clear evidence of tumour progression. The primary end point of the study was to determine the

safety of intracranial THC administration. We also evaluated THC action on the length of survival and

various tumour-cell parameters. A dose escalation regimen for THC administration was assessed.

Cannabinoid delivery was safe and could be achieved without overt psychoactive effects. Median survival

of the cohort from the beginning of cannabinoid administration was 24 weeks (95% confidence interval:

15-33). Delta(9)-Tetrahydrocannabinol inhibited tumour-cell proliferation in vitro and decreased tumour-

cell Ki67 immunostaining when administered to two patients. The fair safety profile of THC, together

with its possible antiproliferative action on tumour cells reported here and in other studies, may set

the basis for future trials aimed at evaluating the potential antitumoral activity of cannabinoids.

Curr Pharm Des 2006; 12 (18):2319-25.

Endocannabinoids: a new family of lipid mediators involved in the regulation of neural cell

development.

The endocannabinoids (eCBs) anandamide and 2-arachidonoylglycerol are important retrograde

messengers that inhibit neurotransmitter release via presynaptic CB1 receptors. In addition,

cannabinoids are known to modulate the cell death/survival decision of different neural cell types,

leading to different outcomes that depend on the nature of the target cell and its

proliferative/differentiation status. Thus, cannabinoids protect primary neurons, astrocytes and

oligodendrocytes from apoptosis, whereas transformed glial cells are prone to apoptosis by

cannabinoid challenge. Moreover, a potential role of the eCB system in neurogenesis and neural

differentiation has been proposed. Recent research shows that eCBs stimulate neural progenitor

proliferation and inhibit hippocampal neurogenesis in normal adult brain. Cannabinoids inhibit cortical

neuron differentiation and promote glial differentiation. On the other hand, experiments with

differentiated neurons have shown that cannabinoids also regulate neuritogenesis, axonal growth and

synaptogenesis. These new observations support that eCBs constitute a new family of lipid signaling cues

responsible for the regulation of neural progenitor proliferation and differentiation, acting as instructive

proliferative signals through the CB1 receptor.

Exp Cell Res 2006 Jul 1; 312(11):2121-31. Epub 2006 Apr 19

The CB2 cannabinoid receptor signals apoptosis via ceramide-dependent activation of the

mitochondrial intrinsic pathway.

Delta9-tetrahydrocannabinol and other cannabinoids exert pro-apoptotic actions in tumor cells via

the CB2 cannabinoid receptor. However, the molecular mechanism involved in this effect has

remained elusive. Here we used the human leukemia cell line Jurkat-that expresses CB2 as the unique

CB receptor-to investigate this mechanism. Our results show that incubation with the selective CB2

antagonist SR144528 abrogated the pro-apoptotic effect of Delta9-tetrahydrocannabinol. Cannabinoid

treatment led to a CB2 receptor-dependent stimulation of ceramide biosynthesis and inhibition of this

pathway prevented Delta9-tetrahydrocannabinol-induced mitochondrial hypopolarization and cytochrome

c release, indicating that ceramide acts at a pre-mitochondrial level. Inhibition of ceramide synthesis de

novo also prevented caspase activation and apoptosis. Caspase 8 activation-an event typically related with

the extrinsic apoptotic pathway-was also evident in this model. However, activation of this protease was

post-mitochondrial since (i) a pan-caspase inhibitor as well as a selective caspase 8 inhibitor were unable

to prevent Delta9-tetrahydrocannabinol-induced loss of mitochondrial-membrane transmembrane

potential, and (ii) cannabinoid-induced caspase 8 activation was not observed in Bcl-xL over-expressing

cells. In summary, results presented here show that CB2 receptor activation signals apoptosis via a

ceramide-dependent stimulation of the mitochondrial intrinsic pathway.

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Cancer Cell 2006 Apr; 9 (4):301-12.

The stress-regulated protein p8 mediates cannabinoid-induced apoptosis of tumor cells.

One of the most exciting areas of current research in the cannabinoid field is the study of the

potential application of these compounds as antitumoral drugs. Here, we describe the signaling

pathway that mediates cannabinoid-induced apoptosis of tumor cells. By using a wide array of

experimental approaches, we identify the stress-regulated protein p8 (also designated as candidate of

metastasis 1) as an essential mediator of cannabinoid antitumoral action and show that p8 upregulation is

dependent on de novo-synthesized ceramide. We also observe that p8 mediates its apoptotic effect via

upregulation of the endoplasmic reticulum stress-related genes ATF-4, CHOP, and TRB3. Activation of

this pathway may constitute a potential therapeutic strategy for inhibiting tumor growth.

Molecular Pharmacology 2006 Jul; 70(1):51-9. Epub 2006 Mar 29

A cannabinoid quinone inhibits angiogenesis by targeting vascular endothelial cells.

Recent findings on the inhibition of angiogenesis and vascular endothelial cell proliferation by

anthracycline antibiotics, which contain a quinone moiety, make this type of compound a very promising

lead in cancer research/therapy. We have reported that a new cannabinoid anticancer quinone, cannabidiol

hydroxyquinone (HU-331), is highly effective against tumor xenografts in nude mice. For evaluation of

the antiangiogenic action of cannabinoid quinones, collagen-embedded rat aortic ring assay was used. The

ability of cannabinoids to cause endothelial cell apoptosis was assayed by TUNEL staining and flow

cytometry analysis. To examine the genes and pathways targeted by HU-331 in vascular endothelial cells,

human cDNA microarrays and polymerase chain reaction were used. Immunostaining with anti-CD31 of

tumors grown in nude mice served to indicate inhibition of tumor angiogenesis. HU-331 was found to be

strongly antiangiogenic, significantly inhibiting angiogenesis at concentrations as low as 300 nM. HU-

331 inhibited angiogenesis by directly inducing apoptosis of vascular endothelial cells without changing

the expression of pro- and antiangiogenic cytokines and their receptors. A significant decrease in the total

area occupied by vessels in HU-331-treated tumors was also observed. These data lead us to consider

HU-331 to have high potential as a new antiangiogenic and anticancer drug.

Journal Neuroscience 2006 Feb 1; 26(5):1551-61

The endocannabinoid system promotes astroglial differentiation by acting on neural progenitor

cells.

Endocannabinoids exert an important neuromodulatory role via presynaptic cannabinoid CB1 receptors

and may also participate in the control of neural cell death and survival. The function of the

endocannabinoid system has been extensively studied in differentiated neurons, but its potential role in

neural progenitor cells remains to be elucidated. Here we show that the CB1 receptor and the

endocannabinoid-inactivating enzyme fatty acid amide hydrolase are expressed, both in vitro and in vivo,

in postnatal radial glia (RC2+ cells) and in adult nestin type I (nestin(+)GFAP+) neural progenitor cells.

Cell culture experiments show that CB1 receptor activation increases progenitor proliferation and

differentiation into astroglial cells in vitro. In vivo analysis evidences that, in postnatal CB1(-/-) mouse

brain, progenitor proliferation and astrogliogenesis are impaired. Likewise, in adult CB1-deficient mice,

neural progenitor proliferation is decreased but is increased in fatty acid amide hydrolase-deficient mice.

In addition, endocannabinoid signaling controls neural progenitor differentiation in the adult brain

by promoting astroglial differentiation of newly born cells. These results show a novel physiological

role of endocannabinoids, which constitute a new family of signaling cues involved in the regulation

of neural progenitor cell function.

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Life Science 2005 Aug 19; 77(14):1723-31.

Cannabinoids and ceramide: two lipids acting hand-by-hand.

Cannabinoids, the active components of Cannabis sativa (marijuana) and their endogenous

counterparts, exert their effects by binding to specific G-protein-coupled receptors that modulate

adenylyl cyclase and ion channels. Recent research has shown that the CB1 cannabinoid receptor is also

coupled to the generation of the lipid second messenger ceramide via two different pathways:

sphingomyelin hydrolysis and ceramide synthesis de novo. Sustained ceramide accumulation in tumor

cells mediates cannabinoid-induced apoptosis, as evidenced by in vitro and in vivo studies. This effect

seems to be due to the impact of ceramide on key cell signalling systems such as the extracellular signal-

regulated kinase cascade and the Akt pathway. These findings provide a new conceptual view on how

cannabinoids act, and raise interesting physiological and therapeutic questions.

Journal Neuroscience 2005 Feb 23; 25(8):1904-13.

Prevention of Alzheimer's disease pathology by cannabinoids: neuroprotection mediated by

blockade of microglial activation.

Alzheimer's disease (AD) is characterized by enhanced beta-amyloid peptide (betaA) 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 betaA treatment, both in vivo and in

vitro. Here, we show that senile plaques in AD patients express cannabinoid receptors CB1 and CB2,

together with markers of microglial activation, and that CB1-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 CB1 receptor protein expression are markedly decreased in AD brains.

Additionally, in AD brains, protein nitration is increased, and, more specifically, CB1 and CB2 proteins

show enhanced nitration. Intracerebroventricular administration of the synthetic cannabinoid WIN55,212-

2 to rats prevent betaA-induced microglial activation, cognitive impairment, and loss of neuronal markers.

Cannabinoids (HU-210, WIN55,212-2, and JWH-133) block betaA-induced activation of cultured

microglial cells, as judged by mitochondrial activity, cell morphology, and tumor necrosis factor-alpha

release; these effects are independent of the antioxidant action of cannabinoid compounds and are also

exerted by a CB2-selective agonist. Moreover, cannabinoids abrogate microglia-mediated neurotoxicity

after betaA 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.

Cancer Research 2004 Aug 15; 64(16):5617-23.

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

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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 Delta9-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.

Neuropharmacology 2004 Sep; 47(3):315-23.

Hypothesis: cannabinoid therapy for the treatment of gliomas?

Gliomas, in particular glioblastoma multiforme or grade IV astrocytoma, are the most frequent class of

malignant primary brain tumours and one of the most aggressive forms of cancer. Current therapeutic

strategies for the treatment of glioblastoma multiforme are usually ineffective or just palliative. During

the last few years, several studies have shown that cannabinoids-the active components of the plant

Cannabis sativa and their derivatives--slow the growth of different types of tumours, including gliomas,

in laboratory animals. Cannabinoids induce apoptosis of glioma cells in culture via sustained ceramide

accumulation, extracellular signal-regulated kinase activation and Akt inhibition. In addition, cannabinoid

treatment inhibits angiogenesis of gliomas in vivo. Remarkably, cannabinoids kill glioma cells

selectively and can protect non-transformed glial cells from death. These and other findings

reviewed here might set the basis for a potential use of cannabinoids in the management of gliomas.

Nature Reviews Cancer 2003 Oct; 3(10):745-55.

Cannabinoids: potential anticancer agents.

Cannabinoids - the active components of Cannabis sativa and their derivatives - exert palliative

effects in cancer patients by preventing nausea, vomiting and pain and by stimulating appetite. In

addition, these compounds have been shown to inhibit the growth of tumour cells in culture and

animal models by modulating key cell-signalling pathways. Cannabinoids are usually well tolerated, and

do not produce the generalized toxic effects of conventional chemotherapies. Some people consider

cannabinoids as addictive drugs. A withdrawal syndrome, which consists of irritability, insomnia,

restlessness and a sudden, temporary sensation of heat — ‘hot flashes’— has been occasionally observed

in chronic cannabis smokers after abrupt cessation of drug use. However, this occurs rarely, and

symptoms are mild and usually dissipate after a few days. Similarly, after chronic tetrahydrocannabinol

(THC) treatment, no somatic signs of spontaneous withdrawal appear in different animal species, even at

extremely high doses. So, could cannabinoids be used to develop new anticancer therapies?

Molecular Pharmacology 2002 Dec; 62(6):1385-92.

Mechanism of extracellular signal-regulated kinase activation by the CB(1) cannabinoid receptor.

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 delta(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

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cytosolic tyrosine kinases, respectively. However, HU-210 stimulation resulted in neither EGF(R)

phosphorylation, Src tyrosine phosphorylation, nor increased Src activity. In addition, dominant-negative

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, betagamma 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.

British Journal Pharmacology 2003 Oct; 140(3):547-57.

Neurons on cannabinoids: dead or alive?

Preparations from Cannabis sativa (marijuana) have been used for many centuries both

medicinally and recreationally. However, the chemical structure of their active components – the

cannabinoids – was not elucidated until the early 1960s. Among the 60 cannabinoids produced by

marijuana, 9-tetrahydrocannabinol (THC) is the most relevant one owing to its high potency and

abundance. Since the early 1990s, it is widely accepted that THC acts in the organism by mimicking

endogenous substances – the so-called endocannabinoids – that bind to and activate specific cell surface

cannabinoid receptors, two of which have been cloned and characterized so far from mammalian tissues:

CB1 and CB2. These recent findings have led to a remarkable expansion of basic cannabinoid research as

well as to a renaissance in the study of the therapeutic value of cannabinoids.

The CB1 cannabinoid receptor is highly abundant in discrete areas of the brain, and its activation lowers

the release of neurotransmitters such as dopamine and GABA, thereby affecting processes such as

movement and memory. Besides these well-established neuromodulatory events, cannabinoids may

control the survival/death decision of neurons. Thus, cannabinoids have been shown to protect neurons

from toxic insults such as excitotoxicity, traumatic injury and ischaemia both in vitro and in vivo. This

neuroprotective action may rely on different mechanisms, including (i) inhibition of glutamatergic

neurotransmission, (ii) antioxidant capacity, (iii) modulation of glial cell function and (iv) control of the

microvasculature, and is supported by the observation that the brain overproduces endocannabinoids upon

damage. In contrast, a few studies have shown that cannabinoids induce apoptosis of cultured neurons,

but the mechanism of this neurotoxic action is largely unknown.

In this issue, Downer et al. (2003) show that THC-induced apoptosis of primary rat cortical neurons relies

on the activation of the c-Jun N-terminal kinase (JNK) cascade. The JNK protein kinases participate in

the blockade of the cell cycle and the induction of cell death triggered by stress signals such as irradiation,

heat shock, osmotic shock and proinflammatory cytokines. As selective JNK inhibitors are not available,

Downer used selective antisense oligonucleotides to target JNK mRNAs and therefore deplete neurons of

JNKs. The results of these elegant experiments were rather clear-cut, except perhaps for the relative role

of the different JNK isoforms in the apoptotic process. Cannabinoids may therefore lead to opposite

effects on neuron survival/death. It is conceivable that different experimental factors account for this

'ying-yang' action, for example:

(i) cannabinoid neuroprotection is usually more evident in whole-animal than in cultured-neuron

models, which may result from their aforementioned impact on various brain cell types (neurons, glia,

vascular endothelium);

(ii) cannabinoids may exert dual effects on neural cell fate depending on signal input (e.g. agonist dose

and time of exposure), high inputs usually exerting growth inhibition or death;

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(iii) endocannabinoids and exogenous cannabinoids may display distinct pharmacological behaviour (e.g.

agonistic potency and stability); and

(iv) the origin of the neural cell and its stage of differentiation may affect sensitivity to death.

Modulation of neural cell fate by cannabinoids is therefore a complex and still obscure issue, and

although the bulk of the experimental evidence supports that these compounds protect the brain

from damage, the paper by Downer adds a cautionary note to the field.

Journal Biological Chemistry 2002 Nov 29; 277(48):46645-50. Epub 2002 Sep 16.

The endocannabinoid anandamide inhibits neuronal progenitor cell differentiation through

attenuation of the Rap1/B-Raf/ERK pathway.

Endocannabinoids are neuromodulators that act as retrograde synaptic messengers inhibiting the release

of different neurotransmitters in cerebral areas such as hippocampus, cortex, and striatum. However, little

is known about other roles of the endocannabinoid system in brain. In the present work we provide

substantial evidence that the endocannabinoid anandamide (AEA) regulates neuronal differentiation both

in culture and in vivo. Thus AEA, through the CB(1) receptor, inhibited cortical neuron progenitor

differentiation to mature neuronal phenotype. In addition, human neural stem cell differentiation and

nerve growth factor-induced PC12 cell differentiation were also inhibited by cannabinoid challenge. AEA

decreased PC12 neuronal-like generation via CB(1)-mediated inhibition of sustained extracellular signal-

regulated kinase (ERK) activation, which is responsible for nerve growth factor action. AEA thus

inhibited TrkA-induced Rap1/B-Raf/ERK activation. Finally, immunohistochemical analyses by confocal

microscopy revealed that adult neurogenesis in dentate gyrus was significantly decreased by the AEA

analogue methanandamide and increased by the CB(1) antagonist SR141716. These data indicate that

endocannabinoids inhibit neuronal progenitor cell differentiation through attenuation of the ERK

pathway and suggest that they constitute a new physiological system involved in the regulation of

neurogenesis.

Pharmacol Ther 2002 (pharmacologica et therapeutica latinoamericana) Aug; 95(2):175-84.

Cannabinoids and cell fate.

Cannabinoids recently have been shown to control the cell survival/death decision. Thus,

cannabinoids induce growth arrest or apoptosis in a number of transformed neural and non-neural cells in

culture. In addition, cannabinoid administration induces regression of malignant gliomas in rodents by a

mechanism that may involve sustained ceramide generation and extracellular signal-regulated kinase

activation. In contrast, most of the experimental evidence indicates that cannabinoids may protect normal

neurons from toxic insults, such as glutamatergic overstimulation, ischaemia, and oxidative damage.

Regarding immune cells, low doses of cannabinoids may enhance proliferation, whereas high doses of

cannabinoids usually induce growth arrest or apoptosis. The potential therapeutic applications of

these findings are discussed.

Journal Biological Chemistry 2002 Sep 27; 277(39):36527-33. Epub 2002 Jul 19.

Cannabinoids protect astrocytes from ceramide-induced apoptosis through the phosphatidylinositol

3-kinase/protein kinase B pathway.

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

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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 dose- and time-dependent manner; (ii) triggering of this anti-

apoptotic 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.

Cancer Research 2001 Aug 1; 61(15):5784-9.

Inhibition of glioma growth in vivo by selective activation of the CB(2) cannabinoid receptor.

The development of new therapeutic strategies is essential for the management of gliomas, one of

the most malignant forms of cancer. We have shown previously that the growth of the rat glioma C6

cell line is inhibited by psychoactive cannabinoids (I. Galve-Roperh et al., Nat. Med., 6: 313-319, 2000).

These compounds act on the brain and some other organs through the widely expressed CB(1) receptor.

By contrast, the other cannabinoid receptor subtype, the CB(2) receptor, shows a much more restricted

distribution and is absent from normal brain. Here we show that local administration of the selective

CB(2) agonist JWH-133 at 50 microg/day to Rag-2(-/-) mice induced a considerable regression of

malignant tumors generated by inoculation of C6 glioma cells. The selective involvement of the CB(2)

receptor in this action was evidenced by: (a) the prevention by the CB(2) antagonist SR144528 but not the

CB(1) antagonist SR141716; (b) the down-regulation of the CB(2) receptor but not the CB(1) receptor in

the tumors; and (c) the absence of typical CB(1)-mediated psychotropic side effects. Cannabinoid

receptor expression was subsequently examined in biopsies from human astrocytomas. A full 70% (26 of

37) of the human astrocytomas analyzed expressed significant levels of cannabinoid receptors. Of

interest, the extent of CB(2) receptor expression was directly related with tumor malignancy. In addition,

the growth of grade IV human astrocytoma cells in Rag-2(-/-) mice was completely blocked by JWH-133

administration at 50 microg/day. Experiments carried out with C6 glioma cells in culture evidenced the

internalization of the CB(2) but not the CB(1) receptor upon JWH-133 challenge and showed that

selective activation of the CB(2) receptor signaled apoptosis via enhanced ceramide synthesis de novo.

These results support a therapeutic approach for the treatment of malignant gliomas devoid of

psychotropic side effects.

Molecular Pharmacology 2001 May; 59(5):955-9.

The CB(1) cannabinoid receptor of astrocytes is coupled to sphingomyelin hydrolysis through the

adaptor protein fan.

Cannabinoids exert most of their effects through the CB(1) receptor. This G protein-coupled receptor

signals inhibition of adenylyl cyclase, modulation of ion channels, and stimulation of mitogen- and stress-

activated protein kinases. In this article, we report that Delta(9)-tetrahydrocannabinol (THC), the

major active component of marijuana, induces sphingomyelin hydrolysis in primary astrocytes but

not in other cells expressing the CB(1) receptor, such as primary neurons, U373 MG astrocytoma cells,

and Chinese hamster ovary cells transfected with the CB(1) receptor cDNA. THC-evoked sphingomyelin

breakdown in astrocytes was also exerted by the endogenous cannabinoid anandamide and the synthetic

cannabinoid HU-210 and was prevented by the selective CB(1) antagonist SR141716. By contrast, the

effect of THC was not blocked by pertussis toxin, pointing to a lack of involvement of G(i/o) proteins. A

role for the adaptor protein FAN in CB(1) receptor-coupled sphingomyelin breakdown is supported by

two observations: 1) coimmunoprecipitation experiments show that the binding of FAN to the CB(1)

receptor is enhanced by THC and prevented by SR141716; 2) cells expressing a dominant-negative form

of FAN are refractory to THC-induced sphingomyelin breakdown. This is the first report showing that a

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G-protein-coupled receptor induces sphingomyelin hydrolysis through FAN and that the CB(1)

cannabinoid receptor may signal independently of G(i/o) proteins.

Journal Molecular Medicine 2001; 78(11):613-25.

Control of the cell survival/death decision by cannabinoids.

Cannabinoids, the active components of Cannabis sativa (marijuana), and their derivatives produce

a wide spectrum of central and peripheral effects, some of which may have clinical application. The

discovery of specific cannabinoid receptors and a family of endogenous ligands of those receptors has

attracted much attention to cannabinoids in recent years. One of the most exciting and promising areas

of current cannabinoid research is the ability of these compounds to control the cell survival/death

decision. Thus cannabinoids may induce proliferation, growth arrest, or apoptosis in a number of cells,

including neurons, lymphocytes, and various transformed neural and nonneural cells. The variation in

drug effects may depend on experimental factors such as drug concentration, timing of drug delivery, and

type of cell examined. Regarding the central nervous system, most of the experimental evidence indicates

that cannabinoids may protect neurons from toxic insults such as glutamaergic overstimulation, ischemia

and oxidative damage. In contrast, cannabinoids induce apoptosis of glioma cells in culture and regression

of malignant gliomas in vivo. Breast and prostate cancer cells are also sensitive to cannabinoid-

induced antiproliferation. Regarding the immune system, low doses of cannabinoids may enhance cell

proliferation, whereas high doses of cannabinoids usually induce growth arrest or apoptosis. The

neuroprotective effect of cannabinoids may have potential clinical relevance for the treatment of

neurodegenerative disorders such as multiple sclerosis, Parkinson's disease, and ischemia/stroke,

whereas their growth-inhibiting action on transformed cells might be useful for the management of

malignant brain tumors. Ongoing investigation is in search for cannabinoid-based therapeutic strategies

devoid of non desired psychotropic effects.

Trends Pharmacological Science 2001 Jan; 22(1):19-22.

Ceramide: a new second messenger of cannabinoid action.

Cannabinoids, the active components of Cannabis sativa (marijuana), and their endogenous counterparts

exert their effects by binding to specific G(i/o)-protein-coupled receptors that modulate adenylyl cyclase,

ion channels and extracellular signal-regulated kinases. Recent research has shown that the CB(1)

cannabinoid receptor is coupled to the generation of the lipid second messenger ceramide via two

different pathways: sphingomyelin hydrolysis, and ceramide synthesis de novo. Ceramide in turn

mediates cannabinoid-induced apoptosis, as shown by in vitro and in vivo studies. These findings

provide a new perspective on how cannabinoids act, and raise exciting physiological and

therapeutic questions.

Molecular Pharmacology 2000 Oct; 58(4):814-20.

The CB(1) cannabinoid receptor is coupled to the activation of c-Jun N-terminal kinase.

Cannabinoids exert most of their effects through the CB(1) receptor. This G-protein-coupled receptor has

been shown to be functionally coupled to inhibition of adenylyl 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 c-Jun N-terminal kinase (JNK). Western blot analysis

showed that both JNK-1 and JNK-2 were stimulated by THC. The effect of THC was also exerted by

endogenous cannabinoids (anandamide and 2-arachidonoylglycerol) and synthetic cannabinoids (CP-

55,940, HU-210, and methanandamide), and was prevented by the selective CB(1) antagonist SR141716.

Pertussis toxin, wortmannin, and a Ras farnesyltransferase inhibitor peptide blocked, whereas mastoparan

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mimicked, the CB(1) receptor-evoked activation of JNK, supporting the involvement of a G(i)/G(o)-

protein, phosphoinositide 3'-kinase and Ras. THC-induced JNK stimulation was prevented by tyrphostin

AG1296, pointing to the implication of platelet-derived growth factor receptor transactivation, and was

independent of ceramide generation. Experiments performed with several types of neural cells that

endogenously express the CB(1) receptor suggested that long-term JNK activation may be involved in

THC-induced cell death. The CB(1) cannabinoid receptor was also shown to be coupled to the activation

of p38 mitogen-activated protein kinase. Data indicate that activation of JNK and p38 mitogen-activated

protein kinase may be responsible for some of the cellular responses elicited by the CB(1) cannabinoid

receptor.

Biochemistry Journal 2000 Apr 15; 347(Pt 2):369-73.

The CB1 cannabinoid receptor is coupled to the activation of protein kinase B/Akt.

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.

Nature Medicine 2000 Mar; 6 (3):313-9.

Anti-tumoral action of cannabinoids: involvement of sustained ceramide accumulation and

extracellular signal-regulated kinase activation.

Delta9-Tetrahydrocannabinol, the main active component of marijuana, induces apoptosis of transformed

neural cells in culture. Here, we show that intratumoral administration of Delta9-tetrahydrocannabinol

and the synthetic cannabinoid agonist WIN-55,212-2 induced a considerable regression of malignant

gliomas in Wistar rats and in mice deficient in recombination activating gene 2. Cannabinoid treatment

did not produce any substantial neurotoxic effect in the conditions used. Experiments with two subclones

of C6 glioma cells in culture showed that cannabinoids signal apoptosis by a pathway involving

cannabinoid receptors, sustained ceramide accumulation and Raf1/extracellular signal-regulated kinase

activation. These results may provide the basis for a new therapeutic approach for the treatment of

malignant gliomas.

Life Science 1999 65(6-7):657-64.

Effects of cannabinoids on energy metabolism.

The present review summarizes the recent work carried out by our group on the link between signal

transduction pathways and metabolic regulation systems as affected by cannabinoids. In cells such as

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astrocytes and lymphocytes, which express cannabinoid receptors, physiologically relevant doses of

cannabinoids induce a remarkable metabolic stimulation as determined e.g. by enhanced glucose

utilization. Studies performed in astrocytes show that the cannabinoid-evoked stimulation of glucose

metabolism is independent of adenylyl cyclase inhibition, and seems to rely on the cascade CB1

cannabinoid receptor --> Sphingomyelin breakdown --> Ceramide --> Raf-1 --> Mitogen-activated

protein kinase (MAPK) --> Glucose utilization. A role for phosphoinositide 3'-kinase in the stimulation of

glucose utilization by cannabinoids is also put forward. In addition, ceramide generated upon CB1

cannabinoid receptor activation may enhance ketone body production by astrocytes independently of

MAPK. Anandamide has also been shown to exert metabolic effects in hepatocytes, cells that do not

express cannabinoid receptors. The biological role of cannabinoids as modulators of metabolism is as yet

unclear.

Molecular Pharmacology 1998 Nov; 54(5):834-43.

Involvement of sphingomyelin hydrolysis and the mitogen-activated protein kinase cascade in the

Delta9-tetrahydrocannabinol-induced stimulation of glucose metabolism in primary astrocytes.

The effects of cannabinoids on metabolic pathways and signal transduction systems were studied in

primary cultures of rat astrocytes. Delta9-Tetrahydrocannabinol (THC), the major active component of

marijuana, increased the rate of glucose oxidation to CO2 as well as the rate of glucose incorporation into

phospholipids and glycogen. These effects of THC were mimicked by the synthetic cannabinoid HU-210,

and prevented by forskolin, pertussis toxin, and the CB1 receptor antagonist SR 141716. THC did not

affect basal cAMP levels but partially antagonized the forskolin-induced elevation of intracellular cAMP

concentration. THC stimulated p42/p44 mitogen-activated protein kinase (MAPK) activity, Raf-1

phosphorylation, and Raf-1 translocation to the particulate cell fraction. In addition, the MAPK inhibitor

PD 098095 and the phosphoinositide 3-kinase inhibitors wortmannin and LY 294002 were able to

antagonize the THC-induced stimulation of glucose oxidation to CO2, phospholipid synthesis and

glycogen synthesis. The possible involvement of sphingomyelin breakdown in the metabolic effects of

THC was studied subsequently. THC produced a rapid stimulation of sphingomyelin hydrolysis that was

concomitant to an elevation of intracellular ceramide levels. This effect was prevented by SR 141716.

Moreover, the cell-permeable ceramide analog D-erythro-N-octanoylsphingosine, as well as exogenous

sphingomyelinase, were able in turn to stimulate MAPK activity, to increase the amount of Raf-1 bound

to the particulate cell fraction, and to stimulate glucose metabolism. The latter effect was prevented by PD

098059 and was not additive to that exerted by THC. Results thus indicate that THC produces a

cannabinoid receptor-mediated stimulation of astrocyte metabolism that seems to rely on sphingomyelin

hydrolysis and MAPK stimulation.

FEBS Lett. 1998 Sep 25; (Federation of European Biochemical Societies) 436(1):6-10.

Delta9-tetrahydrocannabinol induces apoptosis in C6 glioma cells.

Delta9-Tetrahydrocannabinol (THC), the major active component of marijuana, induced apoptosis

in C6.9 glioma cells, as determined by DNA fragmentation and loss of plasma membrane

asymmetry. THC stimulated sphingomyelin hydrolysis in C6.9 glioma cells. THC and N-

acetylsphingosine, a cell-permeable ceramide analog, induced apoptosis in several transformed neural

cells but not in primary astrocytes or neurons. Although glioma C6.9 cells expressed the CBI cannabinoid

receptor, neither THC-induced apoptosis nor THC-induced sphingomyelin breakdown were prevented by

SR141716, a specific antagonist of that receptor. Results thus show that THC-induced apoptosis in

glioma C6.9 cells may rely on a CBI receptor-independent stimulation of sphingomyelin breakdown.

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Brain Research 1997 Aug 29; 767(1):64-71.

Delta9-tetrahydrocannabinol stimulates glucose utilization in C6 glioma cells.

The present work was undertaken to study the metabolic response of C6 glioma cells to physiologically

relevant doses of delta9-tetrahydrocannabinol (THC), the major active component of marijuana. At those

concentrations (i.e. nanomolar range), THC produced a dose-dependent increase in the rates of glucose

oxidation to CO2 and glucose incorporation into phospholipids and glycogen. The THC-induced

stimulation of glucose utilization was (i) dose-dependent up to 100 nM THC, (ii) mimicked by the

synthetic cannabinoid HU-210, and (iii) prevented by pertussis toxin and the CB1 receptor antagonist

SR141716A. In contrast to THC, forskolin markedly depressed CO2 production, phospholipid synthesis

and glycogen synthesis from glucose. The forskolin-induced inhibition of glucose utilization was (i)

mimicked by dibutyryl-cAMP, and (ii) prevented by THC, HU-210 and H-7, an inhibitor of the cAMP-

dependent protein kinase. Likewise, THC was able to antagonize in part the forskolin-induced elevation

of intracellular cAMP concentration, and this antagonistic effect was prevented by SR141716A.

However, THC per se did not affect basal cAMP concentration. Results thus indicate that physiologically

relevant doses of THC stimulate glucose metabolism in C6 glioma cells through a cannabinoid receptor-

mediated process. Although cannabinoid receptors may be coupled to inhibition of adenylyl cyclase in C6

glioma cells, this does not seem to be the mechanism involved in the THC-induced stimulation of glucose

metabolism.

Life Science 1997; 60(19):1709-17.

Metabolic stimulation of mouse spleen lymphocytes by low doses of delta9-tetrahydrocannabinol.

The present work was undertaken to study the metabolic response of mouse spleen lymphocytes to

physiologically relevant doses of delta9-tetrahydrocannabinol (THC), the major active component of

marijuana. At those concentrations (i.e. nanomolar range), THC induced a 2-2.5-fold stimulation of both

glucose oxidation to CO2 and phospholipid synthesis from glucose. This stimulation was (i) dose-

dependent up to 1 microM THC, (ii) mimicked by the synthetic cannabinoid HU-210, (iii) prevented by

forskolin and pertussis toxin, and (iv) unaffected by the CB1 receptor antagonist SR141716A. THC was

also able to antagonize the forskolin-induced elevation of intracellular cAMP concentration. In contrast,

at non-physiological, cytotoxic doses (i.e. micromolar range) THC markedly depressed glucose

metabolism in lymphocytes by a cannabinoid receptor-independent pathway. Results thus indicate that

physiologically relevant doses of THC induce a metabolic stimulation of lymphocytes that seems to

be mediated by a cannabinoid receptor-dependent pathway.

Dr. Guzmán and his researchers try to determine why and how THC cures cancers. Rick Simpson of

Phoenix Tears and the people he has treated have proved that the topical application of THC cures skin

cancer (see FASEB Journal 2006 Dec., p2) (and other skin conditions) and that ingestion or aspiration

(via a vapourizer) shrinks and/or destroys other cancerous tumours in the body. His evidence also shows

that THC cures or controls diabetes, depression, hypertension and other diseases. Rick Simpson’s

preliminary results were forwarded to Dr. Guzmán.

Information gathered and presented by Phoenix Tears

www.PhoenixTears.ca

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