source: BBC NEWS

An ingredient in marijuana may be useful for treating brain cancers, say Spanish researchers from Madrid.

Chemicals called cannabinoids could starve tumors to death by halting the growth of blood vessels that feed it, the Complutense University team hope.

By studying mice, the team has shown for the first time how these chemicals block vessel growth.

Their study, published in Cancer Research, also shows the treatment appears to work in humans.

 Glioblastoma multiforme is the most common brain cancer and is notoriously difficult to treat.

It can evade destruction by radiotherapy, chemotherapy and surgery.

Dr Manuel Guzmán and colleagues set out to determine whether they could prevent the cancer from growing by destroying its blood supply.

Previous research has shown cannabinoids block the growth of blood vessels in mice, but little is known about how these chemicals do this and whether they might do the same in human tumors.

Starving tumors

The researchers first gave mice cancer resembling the human form of brain cancer they wanted to study.

They then treated the mice with cannabinoid and examined the genes of the mice.

The activity of genes associated with blood vessel growth in tumors through the production of a substance called vascular endothelial growth factor (VEGF) was reduced.

Cannabinoids appeared to stifle VEGF production by increasing the activity of a substance that controls cell death, called ceramide.

Lead researcher Dr Guzmán said: "As far as we know, this is the first report showing that ceramide depresses VEGF pathway by interfering with VEGF production."

Their next challenge was to see if cannabinoids had the same effect in humans.

They took samples from two patients with glioblastoma multiforme who had not responded to surgery, chemotherapy and radiotherapy treatment.

Treatment for humans

Samples were taken before and after the patients were treated with cannabinoid solution infused directly into the tumour.

In both patients, VEGF levels in the tumour were reduced following treatment with cannabinoids.

Although they only looked at two patients, the researchers hope their findings could lead to new treatments.

"The present findings provide a novel pharmacological target for cannabinoid-based therapies," said Dr Guzmán.

Dr Richard Sullivan, Head of Clinical Programmes at Cancer Research UK, said: "This research provides an important new lead compound for anti-cancer drugs targeting cancer's blood supply.

"Although this work is at an early stage of development, other research has already demonstrated that VEGF is an important drug target for a range of cancers.

"The key now will be to show further activity in pre-clinical cancer models, find out in which combinations cannabinoids show greatest activity and formulate a product that can be tested in man.

"It is important to note that cannabinoids would need to generate very strong data in the future as there are already a number of VEGF inhibitors in clinical development," he said.

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by Shaoni Bhattacharya

Cannabis extracts may shrink brain tumours and other cancers by blocking the growth of the blood vessels which feed them, suggests a new study.

An active component of the street drug has previously been shown to improve brain tumours in rats. But now Manuel Guzmán at Complutense University, Spain, and colleagues have demonstrated how the cannabis extracts block a key chemical needed for tumours to sprout blood vessels - a process called angiogenesis.

And for the first time, the team has shown the cannabinoids impede this chemical in people with the most aggressive form of brain cancer - glioblastoma multiforme.

Cristina Blázquez at Complutense University, and one of the team, stresses the results are preliminary. "But it's a good point to start and continue," she told New Scientist.

"The cannabinoid inhibits the angiogenesis response - if a tumour doesn't do angiogenesis, it doesn't grow," she explains. "So if you can improve angiogenesis on one side and kill the tumour cells on the other side, you can try for a therapy for cancer."

"This research provides an important new lead compound for anti-cancer drugs targeting cancer's blood supply," says Richard Sullivan, head of clinical programmes, at Cancer Research UK.

Fat molecule

The team tested the effects of delta-9-tetrahydrocannabinol in 30 mice. They found the marijuana extract inhibited the expression of several genes related to the production of a chemical called vascular endothelial growth factor (VEGF).

VEGF is critical for angiogenesis, which allows tumours to grow a network of blood vessels to supply their growth. The cannabinoid significantly lowered the activity of VEGF in the mice and two human brain cancer patients, the study showed.

The drug did this by increasing the activity of a fat molecule called ceramide, suggests the study, as adding a ceramide inhibitor stifled the ability of the cannabinoid to block VEGF.

Small and pallid

"We saw that the tumours [in mice] were smaller and a bit pallid," adds Blázquez. The paleness of the cancer reflected its lack of blood supply as a result of the treatment. In the human patients, she says: "It seems that it works, but it's very early."

Sullivan points out: "Although this work is at an early stage of development other research has already demonstrated that VEGF is an important drug target for a range of cancers."

He emphasises the need for further work on cannabinoid combinations. "Cannabinoids would need to generate very strong data in the future as there are already a number of VEGF inhibitors in clinical development," he says.

The two patients in the ongoing study are among 14 in a clinical trial of the drug. The patients are given one cycle of treatment, lasting a few days, and their survival and general health are being studied.

Journal reference: Cancer Research (vol 64, p 5617)

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Paola Massi, Angelo Vaccani, Stefania Ceruti, Arianna Colombo, Maria P. Abbracchio, and
Daniela Parolar

Department of Pharmacology, Chemotherapy and Toxicology (P.M., A.C.), and Department of Pharmacological Sciences, School of Pharmacy, and Center of Excellence for Neurodegenerative Diseases, University of Milan, Milan, Italy (S.C., M.P.A.); and Department of Structural and Functional Biology, Pharmacology Unit and Center of Neuroscience, University of Insubria,
Busto Arsizio (Varese), Italy (A.V., D.P.Received October 3, 2003; accepted November 7, 2003

ABSTRACT

Recently, cannabinoids (CBs) have been shown to possess antitumor properties. Because the psychoactivity of cannabinoid compounds limits their medicinal usage, we undertook the present study to evaluate the in vitro antiproliferative ability of cannabidiol (CBD), a nonpsychoactive cannabinoid compound, on U87 and U373 human glioma cell lines. The addition of CBD to the culture medium led to a dramatic drop of mitochondrial oxidativemetabolism [3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H tetrazolium bromide test] and viability in glioma cells, in a concentration- dependent manner that was already evident 24 h after CBD exposure, with an apparent IC50 of 25
M. The antiproliferative effect of CBD was partially prevented by the CB2 receptor antagonist N-[(1S)-endo-1,3,3-trimethylbicyclo[2,2,1]heptan-2-yl]- 5-(4-chloro-3-methylphenyl)-1-(4-methylbenzyl)-pyrazole-3-carboxamide (SR144528; SR2) and -tocopherol.

By contrast, the CB1 cannabinoid receptor antagonist N-(piperidin-1-yl)-5-(4 chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole- 3-carboximide hydrochloride (SR141716; SR1), capsazepine (vanilloid receptor antagonist), the inhibitors of ceramide
generation, or pertussis toxin did not counteract CBD effects.

We also show, for the first time, that the antiproliferative effect of CBD was correlated to induction of apoptosis, as determined by cytofluorimetric analysis and single-strand DNA staining, which was not reverted by cannabinoid antagonists.

Finally, CBD, administered s.c. to nude mice at the dose of 0.5 mg/mouse, significantly inhibited the growth of subcutaneously implanted U87 human glioma cells. In conclusion,
the nonpsychoactive CBD was able to produce a significant antitumor activity both in vitro and in vivo, thus suggesting a possible application of CBD as an antineoplastic
agent.

Marijuana and its derivatives have been used in medicine for many centuries, and currently there is a renewed interest in the study of the therapeutic effects of cannabinoids. Cannabinoids
produce their effects by binding to specific plasma membrane G protein-coupled receptors. To date, two cannabinoid receptors have been characterized: the CB1 receptor, expressed primarily in the brain and in some peripheral tissues, and CB2 receptors, expressed by cells of the immune
system (Howlett et al., 2002; Pertwee and Ross, 2002).

Ongoing research is determining whether cannabinoid ligands may be effective agents in the treatment of pain, glaucoma, the wasting and emesis associated with cancer chemotherapy
and AIDS, and neurodegenerative disorders such as multiple sclerosis (Goutopoulos and Makriyannis, 2002).

Among the potential therapeutic activities, one of the most exciting and promising areas of current cannabinoid research is the demonstrated ability of these compounds to affect a number of pathways involved in the cell survival/death decision (Bifulco and Di Marzo, 2002; Guzman et al., 2002).

Both natural and synthetic as well as endogenous cannabinoids have been found to affect the rate of cell proliferation in cell lines derived from the central nervous system.

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This work was supported by a grant from the Cannabinoid Research Institute,
affiliated with GW Pharmaceuticals, Oxford, UK, and by a grant from the
Italian Ministry for University and Scientific and Technological Research
(FIRST 2001).
Article, publication date, and citation information can be found at
http://jpet.aspetjournals.org.

DOI: 10.1124/jpet.103.061002.

ABBREVIATIONS: CB, cannabinoid; CBD, cannabidiol; THC,
9-tetrahydrocannabinol; VR, vanilloid receptor; AEA, N-arachidonoylethanolamide (anandamide); MTT, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H tetrazolium bromide; PMA, phorbol 12-myristate 13-acetate; WIN 55,212-2,R- ()-[2,3-dihydro-5-methyl-3-[(morpholinyl)methyl]pyrrolo[1,2,3-de]-1,4-benzoxazinyl]-(1-napthanlenyl) methanone mesylate; SR141716A (SR2), N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3 carboximide hydrochloride; SR144528 (SR1), N-[(1S)-endo- 1,3,3-trimethylbicyclo[2,2,1]heptan-2-yl]-5-(4-chloro-3-methylphenyl)-1-(4-methylbenzyl)-pyrazole-3-carboxamide; PBS, phosphate-buffered saline;
PI, propidium iodide; ssDNA, single-strand DNA; ELISA, enzyme-linked immunosorbent assay; CPZ, capsazepine; PTX, pertussis toxin.

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Inhibition of C6 glioma cell proliferation by anandamide, 1-arachidonoylglycerol, and by a water soluble phosphate ester of anandamide: variability in response and involvement of arachidonic acid.

The FASEB Journal express article 10.1096/fj.02-0795fje. Published online January 2, 2003.

Cristina Blázquez, M. Llanos Casanova,† Anna Planas,‡ Teresa Gómez del Pulgar, Concepción
Villanueva, María J. Fernández-Aceñero, Julián Aragonés, John W. Huffman, José L.
Jorcano,† and Manuel Guzmán

 Department of Biochemistry and Molecular Biology I, School of Biology, Complutense
University, Madrid, Spain;

†Department of Cellular and Molecular Biology and Gene Therapy,
CIEMAT, Madrid, Spain;

‡Department of Pharmacology and Toxicology, IIBB-CSIC, IDIBAPS,
Barcelona, Spain;

Department of Pathology, Hospital General de Móstoles, Madrid, Spain;

Department of Immunology, Hospital de la Princesa, Madrid, Spain; Department of
Chemistry, Clemson University, Clemson, South Carolina, USA

Cristina Blázquez and M. Llanos Casanova contributed equally to this work.

Corresponding author: Manuel Guzmán, Department of Biochemistry and Molecular Biology I,
School of Biology, Complutense University, 28040 Madrid, Spain. E-mail: mgp@bbm1.ucm.es

ABSTRACT
Cannabinoids, the active components of marijuana and their derivatives, induce tumor regression
in rodents.

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.

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Burkhard Hinz, Robert Ramer, Karin Eichele, Ulrike Weinzierl, and Kay Brune

Department of Experimental and Clinical Pharmacology and Toxicology, Friedrich Alexander University, Erlangen, Germany
Received May 24, 2004; accepted September 10, 2004

ABSTRACT
Cannabinoids have been implicated in the reduction of glioma growth. The present study investigated a possible relationship between the recently shown induction of cyclooxygenase
(COX)-2 expression by the endocannabinoid analog R(
)- methanandamide [R(
)-MA] and its effect on the viability of H4 human neuroglioma cells. Incubation with R(
)-MA for up to 72 h decreased the cellular viability and enhanced accumulation of cytoplasmic DNA fragments in a time-dependent manner.

Suppression of R(
)-MA-induced prostaglandin (PG) E2 synthesis with the selective COX-2 inhibitor celecoxib (0.01–1 M) or inhibition of COX-2 expression by COX-2-silencing small-interfering RNA was accompanied by inhibition of R(
)- MA-mediated DNA fragmentation and cell death. In contrast, the selective COX-1 inhibitor SC-560 was inactive in this respect.

Cells were also protected from apoptotic cell death by other COX-2 inhibitors (NS-398 {[N-[2-(cyclohexyloxy)-4-nitrophenyl]- methanesulfonamide]} and diclofenac) and by the ceramide synthase inhibitor fumonisin B1, which interferes with COX-2 expression by R(
)-MA. Moreover, the proapoptotic action of R(
)-MA was mimicked by the major COX-2 product
PGE2.

Apoptosis and cell death by R(
)-MA were not affected by antagonists of cannabinoid receptors (CB1, CB2) and vanilloid receptor 1. In further experiments, celecoxib was demonstrated to suppress apoptotic cell death elicited by anandamide, which is structurally similar to R(
)-MA. As a whole, this study defines COX-2 as a hitherto unknown target by which a cannabinoid induces apoptotic death of glioma cells. Furthermore, our data show that pharmacological concentrations of celecoxib may interfere with the proapoptotic action of R(
)-MA and anandamide, suggesting that cotreatment with COX-2 inhibitors could diminish glioma regression induced by
these compounds.

There is presently a renaissance in studying potential therapeutical effects of cannabinoids that exert a broad array of actions within the central nervous system as well as on immune, cardiovascular, respiratory, digestive, reproductive, and ocular functions. Although the antineoplastic activity of 9-tetrahydrocannabinol (9-THC), the principal psychoactive component of marijuana, has been known since the 1970s (Munson et al., 1975), cannabinoids have been no more than some years ago associated with the management of malignant brain tumors. In this context, cannabinoids have been shown to induce regression of malignant gliomas in rodents (Galve-Roperh et al., 2000).

Different mechanisms have been proposed to account for the proapoptotic and antiproliferative
effects of different cannabinoids on glioma cells (for review, see Guzman et al., 2001). In rat C6 glioma cells, 9-THC has been demonstrated to reduce cellular viability by a mechanism involving activation of cannabinoid receptors and sustained generation of ceramide (Galve-
Roperh et al., 2000).

On the other hand, a recent study suggests that the antiproliferative effects of the endocannabinoids anandamide and 2-arachidonoylglycerol in these cells are mediated by a mechanism involving combined activation of cannabinoid and vanilloid receptors (Jacobsson et al.,
2001).

All together, the results of these and other studies suggest that there is no universal mechanisms by which.

This study was supported by the Deutsche Forschungsgemeinschaft (HI
813/1-1 and SFB 539, BI.6). Article, publication date, and citation information can be found at
http://molpharm.aspetjournals.org. doi:10.1124/mol.104.002618.

ABBREVIATIONS: 9-THC, 9-tetrahydrocannabinol; PG, prostaglandin; COX, cyclooxygenase; R(
)-MA, R(
)-methanandamide, R(
)-arachidonyl-
1-hydroxy-2-propylamide); AM-251, N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide;
AM-630, (6-iodo-2-methyl-1-[2-(4-morpholinyl)ethyl]-1H-indol-3-yl) (4-methoxyphenyl)methanone; NS-398, N-[2-(cyclohexyloxy)-4-nitrophenyl]-
methanesulfonamide; RT-PCR, reverse transcriptase-polymerase chain reaction; WST-1, 4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-
1.6-benzene disulfonate; siRNA, small-interfering RNA; CB, cannabinoid; SC-560, 5-(4-chloro-phenyl)-1-(4-methoxyphenyl)-3-trifluoromethylpyrazole;
NSAID, nonsteroidal anti-inflammatory drug.

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Manuel Guzmán
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.
So, could cannabinoids be used to develop new anticancer therapies?

 Preparations from Cannabis sativa have been used for many centuries both medicinally and recreationally. However, the chemical structure of their unique active
components — the CANNABINOIDS — was not elucidated until the early 1960s.As they are highly hydrophobic, cannabinoids were initially believed to mediate their actions by inserting directly into biomembranes.

This scenario changed markedly in the early 1990s, when specific cannabinoid receptors were cloned and their endogenous ligands were characterized, therefore providing a mechanistic basis for cannabinoid action.This led not only to an impressive expansion of basic cannabinoid research, but also to a renaissance in the study of the therapeutic effects of cannabinoids, which now constitutes a widely debated issue with ample scientific, clinical and social relevance.

The scientific community has gained substantial knowledge of the palliative and antitumour actions of cannabinoids during the past few years.However, further basic research and more exhaustive clinical trials are still required before cannabinoids can be routinely used in cancer therapy.

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Copyright © 2001 American Association for Cancer Research

[CANCER RESEARCH 61, 5784–5789, August 1, 2001]

Cristina Sa´nchez,2 Marı´a L. de Ceballos,2 Teresa Go´mez del Pulgar,2 Daniel Rueda, Ce´sar Corbacho, Guillermo Velasco, Ismael Galve-Roperh, John W. Huffman, Santiago Ramo´n y Cajal, and Manuel Guzma´n3

Department of Biochemistry and Molecular Biology I, School of Biology, Complutense University, 28040 Madrid, Spain [C. S., T. G. d. P., D. R., G. V., I. G-R., M. G.];
Neurodegeneration Group, Cajal Institute, CSIC, 28002 Madrid, Spain [M. L. d. C.];

Department of Pathology, Clı´nica Puerta de Hierro, 28035 Madrid, Spain [C. C., S. R. y C.];
and Department of Chemistry, Clemson University, Clemson, South Carolina 29634-1905 [J. W. H.]

ABSTRACT
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 CB1 receptor. By contrast, the other cannabinoid receptor subtype, the CB2 receptor, shows a much more restricted distribution and is absent from normal brain. Here we show that local administration of the selective CB2 agonist JWH-133 at 50 mg/day to Rag-22/2 mice induced a considerable regression of malignant tumors generated by inoculation of C6 glioma cells.

The selective involvement of the CB2 receptor in this action was evidenced by: (a) the prevention
by the CB2 antagonist SR144528 but not the CB1 antagonist SR141716; (b) the down-regulation of the CB2 receptor but not the CB1 receptor in the tumors; and (c) the absence of typical CB1-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 CB2 receptor expression was directly related with tumor malignancy. In addition, the growth of grade IV human astrocytoma cells in Rag-22/2 mice was completely blocked by JWH-133 administration at 50 mg/day.

Experiments carried out with C6 glioma cells in culture evidenced the internalization of the CB2 but not the CB1 receptor upon JWH-133 challenge and showed that selective activation of the CB2 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.

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Source: Alternet

Source: WebMD

WebMD Health News

Feb. 28, 2000 (Atlanta) -- If results of a recent rat study hold true in human trials, marijuana could be the treatment of choice for patients with malignant glioma -- an especially aggressive and often fatal form of brain cancer.

No, rats haven't started smoking pot. But when researchers injected tumorous animals with cannabinoids -- the drug's active ingredient -- about a third of them went into remission, and another third lived significantly longer than untreated rats. The findings appear in the March issue of the journal Nature Medicine.

The study does not mean that smoking pot will cure cancer in humans, says Daniele Piomelli, PhD, author of an editorial accompanying the paper. "What it does show is that in about one-third of animals injected with a potent cannabis mimic, the cancer disappears and in another one third, it is reduced. Given the seriousness of malignant glioma, it's a very important observation that deserves to be followed up," he tells WebMD. Piomelli is professor of pharmacology at the University of California in Irvine.

According to lead researcher Manuel Guzmán, PhD, his team's previous studies showed that cannabinoids could stop growth and kill cancer cells but did not harm normal cells. The current work examined the action behind this effect and whether it would also work in living animals. Guzmán is a biochemistry lecturer at Complutense University, Madrid.

The researchers first caused tumors in the brains of 18 rats. They then injected the animals over the course of seven days with either a natural or artificial cannabinoid, or a placebo for comparison. Additional groups of healthy, tumor-free rats also received the various treatments.

All of the untreated animals with tumors died between days 12 and 18, but those treated with the cannabinoids lived much longer, and had significantly smaller tumors. Approximately one-third of treated animals showed no response at all to the cannabinoids, indicating that the treatment might not work for all patients. There were no negative side effects at all in the healthy animals receiving treatment.

According to Guzmán, in the body there are two kinds of cannabinoid receptors, or parts of a cell that the cannabinoid connects with like a key fits into a lock. Once connected, the receptor is activated or "turned on." In the brain these receptors are called CB₁, and in the rest of the body they are called CB₂. In another set of experiments, the researchers tested exactly which of these receptors had to be activated in order to cause cancer cell death. They found that the cannabinoid was activating both receptors. Guzmán says activating either of the receptors is enough to induce cell death, while blocking both completely eliminates the effect.

It is only CB₁ activation that induces marijuana's euphoric or "high" effects, says Guzmán, so if we could "specifically activate only CB₂ receptors, we could kill the cancer cells without producing any kind of psychotropic effect." Unfortunately, however, the cannabinoids that would only activate the CB₂ receptor are not yet available for experimentation.

Both Guzmán and Piomelli express concern that ethical debate over medical marijuana use will hinder future investigation.

"It is stupid," says Guzmán, "because if these compounds were present in pine leaves or lettuce, then most likely things would be different. But they are present in marijuana, so it's controversial ... which is nonsense. Hospitalized patients are given morphine and other drugs, but for some reason, it's considered immoral to give them cannabis."

In Piomelli's opinion, placing restrictions on clinical use and testing of marijuana-based therapies is "not only silly, it can be criminal. When patients are dying, there should be no consideration to such matters," he tells WebMD.

Malignant glioma is "fairly common and very deadly," Piomelli says. "I believe it would be ethically acceptable to offer [cannabinoids] to patients, especially in light of the fact that the toxicity is likely to be very, very small."

ISMAEL GALVE-ROPERH1, CRISTINA SÁNCHEZ1, MARÍA LUISA CORTÉS2,
TERESA GÓMEZ DEL PULGAR1, MARTA IZQUIERDO2 & MANUEL GUZMÁN1

Department of Biochemistry and Molecular Biology I, School of Biology, Complutense University,
28040-Madrid, Spain

Department of Molecular Biology, Severo Ochoa Molecular Biology Center, School of Sciences,
Autónoma University, 28049-Madrid, Spain

I.G.-R. and C.S. contributed equally to this work

Correspondence should be addressed to M.G.; email: mgp@solea.quim.ucm.es

Δ9-Tetrahydrocannabinol, the main active component of marijuana, induces apoptosis of transformed neural cells in culture.

Here, we show that intratumoral administration of Δ9-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

Read full article (PDF) via Nature America INC

Source: Journal of Biological Chemistry

1. Mauro Maccarrone,
2. Tatiana Lorenzon,
3. Monica Bari,
4. Gerry Melino and
5. Alessandro Finazzi-Agrò

Abstract

The endocannabinoid anandamide (AEA) is shown to induce apoptotic bodies formation and DNA fragmentation, hallmarks of programmed cell death, in human neuroblastoma CHP100 and lymphoma U937 cells. RNA and protein synthesis inhibitors like actinomycin D and cycloheximide reduced to one-fifth the number of apoptotic bodies induced by AEA, whereas the AEA transporter inhibitor AM404 or the AEA hydrolase inhibitor ATFMK significantly increased the number of dying cells.

Furthermore, specific antagonists of cannabinoid or vanilloid receptors potentiated or inhibited cell death induced by AEA, respectively. Other endocannabinoids such as 2-arachidonoylglycerol, linoleoylethanolamide, oleoylethanolamide, and palmitoylethanolamide did not promote cell death under the same experimental conditions.

The formation of apoptotic bodies induced by AEA was paralleled by increases in intracellular calcium (3-fold over the controls), mitochondrial uncoupling (6-fold), and cytochrome c release (3-fold). The intracellular calcium chelator EGTA-AM reduced the number of apoptotic bodies to 40% of the controls, and electrotransferred anti-cytochrome c monoclonal antibodies fully prevented apoptosis induced by AEA.

Moreover, 5-lipoxygenase inhibitors 5,8,11,14-eicosatetraynoic acid and MK886, cyclooxygenase inhibitor indomethacin, caspase-3 and caspase-9 inhibitors Z-DEVD-FMK and Z-LEHD-FMK, but not nitric oxide synthase inhibitorNω-nitro-l-arginine methyl ester, significantly reduced the cell death-inducing effect of AEA. The data presented indicate a protective role of cannabinoid receptors against apoptosis induced by AEA via vanilloid receptors.

Anandamide (arachidonoylethanolamide, AEA)¹ belongs to an emerging class of endogenous lipids including amides and esters of long chain polyunsaturated fatty acids and collectively indicated as “endocannabinoids”.

In fact, AEA has been isolated and characterized as an endogenous ligand for cannabinoid receptors in the central nervous system (CB1 subtype) and peripheral immune cells (CB2 subtype). AEA is released from depolarized neurons, endothelial cells and macrophages, and mimics the pharmacological effects of Δ⁹-tetrahydrocannabinol, the active principle of hashish and marijuana.

Recently, attention has been focused on the possible role of AEA and other endocannabinoids in regulating cell growth and differentiation, which might account for some pathophysiological effects of these lipids. An anti-proliferative action of AEA has been reported in human breast carcinoma cells, due to a CB1-like receptor-mediated inhibition of the action of endogenous prolactin at its receptor.

An activation of cell proliferation by AEA has been reported instead in hematopoietic cell lines. Moreover, preliminary evidence that the immunosuppressive effects of AEA might be associated with inhibition of lymphocyte proliferation and induction of programmed cell death (PCD or apoptosis) has been reported, and growing evidence is being collected that suggests that AEA might have pro-apoptotic activity, both in vitro and in vivo.

This would extend to endocannabinoids previous observations on Δ⁹-tetrahydrocannabinol, shown to induce PCD in glioma tumors, glioma cells, primary neurons, hippocampal slices, and prostate cells . However, the mechanism of AEA-induced PCD is unknown.

The various effects of AEA in the central nervous system and in immune system, as well as its ability to reduce the emerging pain signals at sites of tissue injury, are terminated by a rapid and selective carrier-mediated uptake of AEA into cells, followed by its degradation to ethanolamine and arachidonic acid by the enzyme fatty acid amide hydrolase (FAAH). Recently, we showed that human neuroblastoma CHP100 cells and human lymphoma U937 cells do have these tools to eliminate AEA.

Therefore, these cell lines were chosen to investigate how AEA and related endocannabinoids induce apoptosis and how the removal and degradation of AEA are related to this process. The existence of a neuroimmune axis appears to be confirmed by the finding that endocannabinoids elicit common responses in these two cell types.

RESULTS

AEA-induced PCD Is Not Mediated by CB1 or CB2 Receptors and Is Potentiated by Inhibitors of AEA Degradation

Treatment of human neuroblastoma CHP100 cells and human lymphoma U937 cells with AEA led to apoptotic body formation in a dose- (Fig.1 A) and time- (Fig.1 B) dependent manner. Detection by ELISA of DNA fragments in the cell cytosols under the same experimental conditions confirmed the cytofluorimetric data (Fig. 1 C).

Apoptosis at 48 h was significant in both cell lines already at 0.25 μm AEA (approximately 2.5-fold over the control) and reached a level of 6.0-fold over the control at 1 μm AEA (Fig.1 A). These concentrations are in the physiological range.

Time course experiments showed that 1 μm AEA induced a significant increase in apoptotic bodies 24 h after treatment (2.5-fold over the control) and a maximum of 6-fold the control after 48 h (Fig. 1 B). Unlike AEA, 2-AG and other endocannabinoids failed to induce significant cell death in CHP100 or U937 cells (Table I).

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Induction of apoptosis by AEA in different cell lines. AEA induced apoptotic body formation and DNA fragmentation in human neuroblastoma CHP100 cells (white bars) and human lymphoma U937 cells (gray bars).A, the number of apoptotic bodies was measured at 48 h, and B, it was measured in cells treated with 1 μm AEA. C, DNA fragmentation was measured in the same samples as in B. Rat glioma C6 cells (light gray bars) and human leukemia DAUDI cells (black bars) did not show apoptotic body formation or DNA fragmentation under the same experimental conditions. *, p > 0.05 compared with control; §, p < 0.01 compared with control.

Effect of AEA analogues on apoptotic body formation, mitochondrial uncoupling and intracellular calcium concentration in CHP100 cells

 In order to investigate the possible role of cannabinoid receptors on PCD induced in CHP100 or U937 cells by AEA, two specific CBR antagonists were used as follows: SR141716 and SR144528, which bind CB1R and CB2R, respectively.

Neither SR141716 nor SR144528, even if used at an excess concentration of 5 μm, could significantly prevent the AEA-induced toxicity, suggesting that the effect was not mediated by “classical” CB1 or CB2 receptors (TableII).

In line with these data, the synthetic cannabinoid [³H]CP55.940, a high affinity ligand for both CB1 and CB2 receptors, did not bind to human neuroblastoma or lymphoma cells, suggesting that they had no functional cannabinoid receptors on their surface (Fig.2).

Instead rat brain, used as a positive control, did bind [³H]CP55.940, which was displaced by AEA (Fig. 2, inset) with an inhibition constant (K _i = 30 ± 4 nm) close to that previously reported.

Either the inhibitor of AEA transporter AM404 or the FAAH inhibitor ATFMK, each used at 10 μm, significantly increased (up to approximately 180 or 165% of the control, respectively) the AEA toxicity (Table II).

On the other hand, actinomycin D and cycloheximide (both at 10 μg/ml) reduced AEA-induced PCD in CHP100 cells down to approximately 20 or 30% of the control, respectively (Table II). Superimposable results were observed with the human lymphoma U937 cell line (Table II).

Effect of various compounds on AEA-induced cell death in human CHP100 and U937 cells

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Analysis of functional cannabinoid receptors in different cell lines. Binding of the synthetic cannabinoid [³H]CP55.940 (400 pm) to CHP100 (white bars), U937 (gray bars), C6 (light gray bars), and DAUDI (black bars) cells and displacement by 0.1 μm SR141716, 0.1 μm SR144528, or 1 μm AEA. Unspecific binding was measured in the presence of 10 μm AEA.

Values were expressed as percentage of the maximum (100% = 1000 ± 100 cpm). Inset, binding of [³H]CP55.940 (400 pm) by rat brain membranes and displacement by various concentrations of AEA. Values were expressed as percentage of the maximum (100% = 1000 ± 100 cpm).CTR, control. *, p < 0.01 compared with control C6 cells; **, p > 0.05 compared with control C6 cells; ***, p > 0.05 compared with control DAUDI cells; §, p < 0.01 compared with control DAUDI cells.

AEA-induced PCD Is Mediated by Vanilloid Receptors

CHP100 and U937 cells were able to bind [³H]AEA, according to a saturable process with an apparent affinity constant of 75 ± 10 nm (Fig. 3 A). Cold AEA, but not 2-AG, SR141716 or SR144528 (each used at 1 μm), displaced 200 nm [³H]AEA from the binding site (Fig. 3 B). Also, 1 μm cannabidiol (CBD), a selective antagonist of a newly discovered type of cannabinoid receptor, failed to affect the binding of 200 nm [³H]AEA to CHP100 or U937 cells, whereas 1 μm capsazepine (Caps), a selective antagonist of vanilloid receptors, fully displaced it (Fig.3 B). Interestingly, 10 μm CBD did not protect CHP100 or U937 cells against PCD induced by 1 μm AEA, whereas 10 μm Caps led to a 30% reduction of apoptotic bodies in both cell lines (Table II).

Moreover, when we treated CHP100 or U937 cells with 1 μm capsaicin, the physiological agonist of vanilloid receptors, we found a 5-fold increase in apoptotic bodies after 48 h, which resembled the effect of 1 μm AEA on these cells (Fig. 1 A).

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Binding of AEA to CHP100 and U937 cells. A, represents the binding of [³H]AEA to CHP100 (circles) or U937 (triangles) cells.B, the effect of (cold) AEA, 2-AG, SR141716, SR144528, CBD, or Caps (each used at 1 μm) on the binding of 200 nm [³H]AEA to CHP100 (white bars) or U937 (gray bars) is shown, and values were expressed as percentage of the untreated controls (100% = 170 ± 20 (CHP100) or 160 ± 20 (U937) cpm, respectively). CTR, control. *, p < 0.01 compared with control cells.

Cannabinoid Receptors Prevent AEA-induced PCD

Rat glioma C6 cells and human leukemia DAUDI cells were found to have a specific AEA transporter, with apparent K _m andV _max values for AEA of 0.15 ± 0.02 μm and 40 ± 4 pmol·min^{− 1}·mg protein^{− 1} (C6 cells) and 0.10 ± 0.01 μm and 150 ± 15 pmol·min^{− 1}·mg protein^{− 1} (DAUDI cells). FAAH activity, which has been already reported in C6 cells, was found to depend on AEA concentration according to a Michaelis-Menten kinetics in these cells and in DAUDI cells (not shown), with apparent K _m of 5.0 ± 0.5 μm and V _max of 135 ± 15 pmol·min^{− 1}·mg protein^{− 1} (C6), and 5.0 ± 0.5 μm and 450 ± 50 pmol·min^{− 1}·mg protein^{− 1} (DAUDI). These kinetic parameters of AEA transporter and FAAH in C6 and DAUDI cells closely resembled those measured in CHP100 or U937 cells, respectively.

Moreover, C6 cells have been reported to express CB1R on their surface, whereas in DAUDI cells the mRNA for the CB2 receptor subtype has been found.

Consistently, these cells were able to bind [³H]CP55.940, which was displaced by 0.1 μmSR141716 in C6 cells or 0.1 μm SR144528 in DAUDI cells and by 1 μm AEA in both cell types (Fig. 2). Quite interestingly, neither cell line showed apoptotic body formation or DNA fragmentation when treated with 1 μm AEA under the experimental conditions previously tested with CHP100 or U937 cells (Fig. 1).

However, at 10 μm AEA concentration a 3.5–4.0-fold increase in PCD after 48 h (TableIII) was observed, an effect that was enhanced in C6 cells by 1 μm SR141716, but not by SR144528, whereas in DAUDI cells the opposite was found (Table III). Inhibition of the AEA transporter by 10 μm AM404 almost doubled apoptotic body formation induced by AEA in both cell lines, and this effect was additive to that of SR141716 in C6 cells and of SR144528 in DAUDI cells (Table III).

Apoptosis induced by 10 μm AEA in C6 or DAUDI cells was reduced to approximately 40% by 10 μm Caps, whereas CBD at the same concentration was ineffective (Table III). Remarkably, C6 and DAUDI cells were able to bind [³H]AEA (400 ± 40 or 350 ± 40 cpm respectively, upon incubation with 200 nm[³H]AEA), which was fully displaced by 1 μmSR141716 + 1 μm Caps (C6 cells) or 1 μmSR144528 + 1 μm Caps (DAUDI cells). Caps alone displaced approximately 30% [³H]AEA from each cell type, under the same experimental conditions.

Effect of various compounds on AEA-induced cell death in rat C6 and human DAUDI cells.

AEA Induces Mitochondrial Uncoupling, Intracellular Calcium Rise, and Cytochrome c Release

AEA led to a dose-dependent mitochondrial uncoupling, which was most evident 6 h after treatment of CHP100 or U937 cells (TableIV). At this time interval, the increase in mitochondrial uncoupling was already significant with 0.25 μm AEA, a concentration that also induced significant apoptotic body formation (Fig. 1). Treatment with AEA also caused a dose-dependent and rapid (within 6 min) increase of intracellular calcium concentration (Table IV). Again, AM404 or ATFMK (both at 10 μm) significantly potentiated, whereas 10 μm Caps inhibited, the effect of AEA on both mitochondrial uncoupling and calcium rise (Table IV). Instead, 2-AG and the other endocannabinoids did not affect mitochondrial integrity or intracellular calcium concentration (Table I).

Interestingly, 50 μm EGTA-AM, a permeant calcium chelator, reduced the number of apoptotic bodies induced by AEA to approximately 40 or 35% of the control, in CHP100 or U937 cells, respectively (Table II).

Also 10 μm ETYA or 10 μm MK886, specific 5-lipoxygenase inhibitors, reduced to approximately 40–50% the pro-apoptotic activity of AEA in both cell lines (Table II). Similar results were observed by treating the cells with 10 μm indomethacin, a cyclooxygenase inhibitor.

Instead, treatment with 10 μm cyclosporin A, a mitochondrial permeability transition pore inhibitor, or 50 μm l-NAME, a nitric oxide synthase inhibitor, were ineffective (Table II).

Effect of AEA on mitochondrial uncoupling and intracellular calcium concentration in human CHP100 and U937 cells.

Western blot showed that anti-cytochrome c monoclonal antibodies specifically recognized a single immunoreactive band in CHP100 and U937 cell saps, corresponding to a molecular mass of approximately 15 kDa (Fig.4 A).

These antibodies were used to quantify cytochrome c release into cell cytosol by ELISA. Treatment of CHP100 or U937 cells with 1 μm AEA led to a 3–3.5-fold increase in cytochrome c release after 8 h, an effect that was not prevented by 5 μmSR141716, 5 μm SR144528, or 10 μm CBD (Fig.4 B). 10 μm AM404 or 10 μm ATFMK enhanced cytochrome c release up to approximately 5-fold over the controls, whereas 10 μm Caps fully inhibited cytochrome c release induced by 1 μm AEA in both cell lines (Fig. 4 B). 2-AG and the other endocannabinoids did not stimulate cytochrome c release in CHP100 or U937 cells, when used at 10 μm (Fig.4 B and data not shown).

Electrotransfer of anti-cytochromec monoclonal antibodies into U937 cells reduced the number of apoptotic bodies induced by 1 μm AEA after 24 h, from 2.8-fold (Fig. 1 B) to 1.3-fold over the controls, whereas non-immune mouse serum under the same experimental conditions was ineffective. Since the release of cytochrome c can trigger an apoptotic caspase cascade, we tested the effect of inhibitors of caspase-3 and caspase-9 on AEA-induced PCD. Table II shows that the caspase-3 inhibitor Z-DEVD-FMK or the caspase-9 inhibitor Z-LEHD-FMK, each used at 50 μm, reduced PCD induced by 1 μm AEA in CHP100 or U937 cells to 20–30% of the controls.

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Figure 4

Effect of AEA on cytochrome crelease from CHP100 and U937 cells. A shows Western blot analysis of cytochrome c in cell homogenates (25 μg/lane).

The arrow indicates the expected molecular size for cytochrome c. Molecular mass markers are shown on the right-hand side. B shows the effect of 1 μm AEA, in the absence or in the presence of 5 μm SR141716, 5 μm SR144528, 10 μm AM404, 10 μm ATFMK, 10 μmCBD, or 10 μm Caps, on cytochrome c release from CHP100 (white bars) or U937 (gray bars) cells, as determined by ELISA at 405 nm (see “Experimental Procedures”).

Treatment of either cell line with any of the compounds listed, in the absence of AEA, was ineffective under the same experimental conditions. CTR, control. *, p< 0.01 compared with control cells; **, p > 0.05 compared with control cells; #, p > 0.05 compared with AEA-treated cells; §, p < 0.05 compared with AEA-treated cells; @, p < 0.01 compared with AEA-treated cells.

DISCUSSION

We have shown that AEA can induce apoptotic body formation and DNA fragmentation, hallmarks of PCD, in human neuronal and immune cells through a pathway involving rise in intracellular calcium, mitochondrial uncoupling, and cytochrome c release.

Activation of the arachidonate cascade and of the caspase cascade are critical steps in the death program. The pro-apoptotic activity of AEA was observed at physiological concentrations of this compound. Unlike AEA, other structurally related and biologically active endocannabinoids, such as 2-AG, LEA, OEA, and PEA (1-3), were unable to force cells into PCD under the same experimental conditions (Table I), ruling out the possibility that the observed effects of AEA were due to unspecific cell poisoning. Since 2-AG may release arachidonate through FAAH activity faster than AEA, the lack of pro-apoptotic activity of this compound rules out the possibility that AEA-induced PCD might be due to arachidonate, as reported for U937 cells.

Consistently, inhibition of FAAH by ATFMK potentiated, instead of reducing, the apoptotic activity of AEA (Table II). Also inhibition of AEA degradation by blocking its uptake enhanced AEA-induced PCD (Table II).

Since a slower degradation leads to an increased concentration of AEA in the extracellular matrix, these findings suggest that the pro-apoptotic activity of AEA is mediated by a target molecule on the cell surface. Indeed, [³H]AEA binds to CHP100 and U937 cell membranes (Fig. 3 A). However, in these cell lines a different binding site must be involved because the “classical” CB1 or CB2 receptors are not present (Fig. 2).

Previous reports have shown that AEA can bind and modulate receptors other than CB1R and CB2R, and recently a CB receptor for AEA, distinct from type 1 or type 2, has been described in endothelial cells.

However, this new CB receptor was not expressed in CHP100 or U937 cells, because its selective antagonist CBD was ineffective on [³H]AEA binding (Fig. 3 B) and on AEA-induced PCD (Table II).

On the other hand, it is becoming increasingly evident that AEA behaves as a full agonist at human vanilloid receptors, whose activation can induce apoptosis in neuronal and immune cells.

Therefore, the possibility that the pro-apoptotic activity of AEA might occur through this receptor was investigated. Indeed, it was found that capsazepine, a selective antagonist of VR, prevented [³H]AEA binding to CHP100 or U937 cells (Fig. 3 B) and inhibited AEA-induced PCD (Table II), whereas the VR agonist capsaicin  mimicked the pro-apoptotic activity of AEA in these cells. Altogether, these findings suggest that AEA-induced PCD was mediated by vanilloid receptors.

It should be stressed that this hypothesis is consistent with the observation that 2-AG and the other endocannabinoids did not promote PCD (Table I), because these compounds do not activate vanilloid receptors or have a much lower potency than AEA. In this context, it seems noteworthy that AM404 alone was ineffective on PCD, mitochondrial uncoupling, intracellular calcium concentration, or cytochrome c release from cells, although it did potentiate the effect of AEA (Tables II-IV and Fig. 4 B).

These findings suggest that AM404 was unable to activate directly human VR, at variance with a previous report suggesting that it is an agonist for rat VR.

A major finding of this investigation is that CB1R or CB2R antagonists, SR141716 or SR144528, were ineffective in CHP100 or U937 cells, which lack cannabinoid receptors (Fig. 2), but they did potentiate AEA-induced PCD in C6 or DAUDI cells (Table III).

In fact, these cells express functional CB1 or CB2 receptors, respectively (Fig. 2), and were able to bind larger amounts of [³H]AEA than CHP100 or U937 cells. Capsazepine displaced approximately 30% [³H]AEA from C6 or DAUDI cells, suggesting that the remaining 70% was bound to CB receptors.

Remarkably, capsazepine prevented AEA-induced PCD in these cells in a way fully analogous to that observed in CHP100 or U937 cells (Table III), suggesting that vanilloid receptors mediate the pro-apoptotic activity of AEA also in C6 and DAUDI cells. As a matter of fact, specific vanilloid responses have been described in C6 cells.

Like in CHP100 or U937 cells, cannabidiol was ineffective on the pro-apoptotic activity of AEA in C6 or DAUDI cells, ruling out that the new “endothelial” CBR might be involved.

On the other hand, it seems noteworthy that the ability of C6 or DAUDI cells to degrade AEA through intracellular uptake and degradation by FAAH was similar to that of CHP100 or U937 cells, respectively. Therefore, it is tempting to speculate that cells bearing functional CB1 or CB2 receptors on their surface are protected against the toxic effects of physiological concentrations of AEA.

In C6 or DAUDI cells, the effects on PCD of co-administration of the transporter inhibitor AM404, which increases extracellular concentration of AEA, or of CBR antagonists SR141716 and SR144528, which prevent CBR activation (Table III), support this concept. These findings can be interpreted by suggesting a regulatory loop between CB receptors and the AEA transporter, which has been recently demonstrated in human endothelial cells.

In this loop, the binding of AEA to CB receptors triggers the activation of AEA uptake by cells, followed by intracellular degradation of AEA by FAAH. Elimination of AEA from the extracellular space might terminate its activity at vanilloid receptors, thus inhibiting the induction of apoptosis. Scheme FSI summarizes the main features of this model.

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Figure FSI

Role of vanilloid receptor and cannabinoid receptor in AEA-induced programmed cell death. Binding of extracellular AEA to VR triggers a sequence of events starting with a rise in intracellular calcium and followed by activation of cyclooxygenase and lipoxygenase, drop in mitochondrial membrane potential (Δψ), release of cytochrome c, and activation of caspases, ultimately leading to programmed cell death (apoptosis).

Binding of AEA to cannabinoid receptors (CBR) activates transporter (T)-mediated uptake of AEA and its subsequent cleavage to arachidonic acid and ethanolamine by membrane-bound FAAH. These latter events inhibit the pro-apoptotic activity of AEA.

PCD of CHP100 or U937 cells induced by AEA was executed through a series of events common to several types of unrelated apoptotic stimuli. It involved the following: (i) rise in cytosolic calcium concentration (within 6 min), (ii) uncoupling of mitochondria (within 6 h), and (iii) release of cytochrome c (within 8 h).

These events required gene expression of proteins necessary for apoptosis, as shown by the protective effect of actinomycin D and cycloheximide (Table II). Consistently with the data on apoptotic body formation and [³H]AEA binding to cell membranes, (i) capsazepine inhibited the events triggered by AEA, (ii) AM404 or ATFMK potentiated them, and (iii) SR141716, SR144528, or CBD were ineffective (Table IV and Fig. 4 B). At variance with other types of PCD, calcium rise induced by AEA was not acting through activation of nitric-oxide synthase, because the nitric-oxide synthase inhibitorl-NAME was ineffective in protecting cells against AEA.

Instead, arachidonate degradation by 5-lipoxygenase and cyclooxygenase activities, which might be enhanced as a consequence of a rise in intracellular Ca²⁺, had a role in the process, because the inhibitors ETYA and MK886 significantly inhibited AEA-induced PCD (Table II).

It must be mentioned that MK886 can exert lipoxygenase-unrelated effects on mammalian cells .

However, the observation that ETYA and MK886 yielded the same inhibition of apoptosis seems to rule out the involvement of lipoxygenase-independent pathways.

This seems interesting, because formation of arachidonate products unbalances the intracellular redox level and has been implicated in apoptotic death of several cell types.

In particular, it should be stressed that a function for lipoxygenase in programmed organelle degradation has been recently demonstrated, showing that the enzyme can make pore-like structures in the lipid bilayer.

This activity might contribute to uncouple directly the mitochondria (Table IV). However, opening of the mitochondrial permeability transition pore did not contribute to AEA-induced PCD, as suggested by the lack of effect of cyclosporin A (Table II).

On the other hand, an unbalanced redox level in the cell has been associated to release of cytochrome c, a converging point in apoptosis induced by different stimuli in various cell types. Cytochrome c release was observed also in AEA-induced PCD (Fig. 4 B), and it was essential for apoptosis, because sequestering cytochrome c within intact U937 cells by electrotransferred anti-cytochrome cmonoclonal antibodies was able to prevent AEA-induced PCD.

Cytochrome c release in the cell cytosol is usually followed by activation of a caspase cascade, initiated by caspase-3 and caspase-9 which are the most proximal members of the proteolytic chain.

Caspases are thought to form a proteolytic machinery within the cell, resulting in the breakdown of key enzymes and cellular structures, and to activate DNases responsible for chromatin degradation seen in apoptosis.

Also AEA-induced PCD seemed to be executed through this series of events, because caspase-3 or caspase-9 inhibitors reduced apoptotic body formation to approximately 20–30% of the controls (Table II).

Altogether, these results suggest that PCD induced by AEA occurs through an apoptotic pathway based on calcium rise, mitochondrial uncoupling, and cytochrome c release. Upstream activation of the arachidonate cascade leads to redox unbalance and organelle disruption, which both favor cytochromec release, then caspases act as downstream executioners of the death program.

In this context, it seems noteworthy that also capsaicin-induced PCD occurs through intracellular calcium rise, imbalance of the redox level, and drop in mitochondrial membrane potential, further strengthening the hypothesis that AEA is acting through vanilloid receptors.

Scheme FSI summarizes the series of events responsible for AEA-induced cell death. It seems noteworthy that these findings might be relevant also for neuronal apoptosis induced by alcohols, where an increase in AEA concentration has been reported.

Moreover, they demonstrate major differences in the cytotoxicity of the different endocannabinoids, which might be relevant for understanding their pathophysiological roles. Finally, this study shows that endocannabinoids exert similar actions in neuronal and immune cells, perhaps (and significantly) through common signals.

U.S. War on Drugs Stalling Mind-Blowing Research into Pot's Cancer-Healing Properties By Paul Armentano

Clinical research published in a journal of the American Association for Cancer Research showing that marijuana's components can inhibit the growth of cancerous brain tumours is the latest in a long line of studies demonstrating the drug's potential as an anti-cancer agent.

This latest study, performed by researchers at Madrid's Complutense University, found that cannabis restricts the blood supply to glioblastoma multiforme tumours, an aggressive brain tumour that kills some 7,000 people in the United States every year.

But despite the value of such findings both in terms of the treatment of life-threatening illnesses and as news, U.S. media coverage has been almost non-existent.

Why the blackout? Not one such study has been acknowledged by the U.S. government.

This wasn't always the case. In fact, the first experiment documenting pot's anti-tumour effects took place in 1974 at the Medical College of Virginia at the behest of the U.S. government.

It showed that marijuana's psychoactive component, THC, "slowed the growth of lung cancers, breast cancers and a virus-induced leukemia in laboratory mice and prolonged their lives by as much as 36 per cent."

Despite these favourable preliminary findings, U.S. government officials refused to fund any follow-up research for two decades, until it conducted a similar - though secret - clinical trial in the mid-1990s.

That study, carried out by the U.S. National Toxicology Program, concluded that mice and rats administered high doses of THC over long periods had greater protection against malignant tumours than untreated controls.

Rather than publicize these findings, government researchers shelved the results, which only became public after a draft copy of the findings were leaked in 1997 to a medical journal that in turn forwarded the story to the national media.

However, in the eight years since then, the U.S. government has yet to fund a single additional study examining the drug's potential anti-cancer properties.

Is this a case of federal bureaucrats valuing politics more than the health and safety of patients? You be the judge.

Fortunately, scientists overseas have generously picked up where U.S. researchers so abruptly left off.

This month, researchers at the University of Milan in Italy, reported that marijuana's constituents inhibit the spread of brain cancer in human tumour biopsies from patients failed by standard cancer therapies.

Last year, the same researchers reported in the Journal of Pharmacology and Experimental Therapeutics that non-psychoactive compounds in marijuana inhibited the growth of glioma cells in a dose-dependent manner and selectively targeted and killed malignant cells, stimulating them to "commit suicide" in a natural process called apoptosis.

In 2000, a research team at Complutense's department of biochemistry and molecular biology reported in the journal Nature Medicine that injections of synthetic THC eradicated malignant gliomas (brain tumours) in one-third of treated rats.

The study was undertaken after the discovery in 1998 that THC can selectively induce apoptosis in brain tumour cells without negatively affecting the surrounding healthy cells.

Nevertheless, federal officials in the U.S. continue to refuse to express any interest in funding - or even acknowledging - this clinical research.

1. Cristina Blázquez
2. Luis González-Feria
3. Luis Álvarez
4. Amador Haro
5. M. Llanos Casanova
6. Manuel Guzmán
   

7. Abstract

   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.

8. 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.

9. 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.

   
   

   INTRODUCTION

   To grow beyond minimal size, tumors must generate a new vascular supply for purposes of gas exchange, cell nutrition, and waste disposal. They do so by secreting proangiogenic cytokines that promote the formation of blood vessels.

10. Vascular endothelial growth factor (VEGF; also known as VEGF-A) is considered the most important proangiogenic molecule because it is expressed abundantly by a wide variety of animal and human tumors and because of its potency, selectivity, and ability to regulate most and perhaps all of the steps in the angiogenic cascade. The best characterized VEGF receptors are two related receptor tyrosine kinases termed VEGF receptor (VEGFR)-1 (also known as Flt-1) and VEGFR-2 (also known as kinase domain region or Flk-1).

11. Although VEGF binds to VEGFR-1 with higher affinity, numerous studies in cultured cells and laboratory animals have provided evidence that VEGFR-2 is the major mediator of the mitogenic, antiapoptotic, angiogenic, and permeability-enhancing effects of VEGF.

12. Because overexpression of VEGF and VEGFR-2 is causally involved in the progression of many solid tumors, several strategies to inhibit VEGF signaling have been translated into clinical trials in cancer patients, including anti-VEGF and anti-VEGFR-2 antibodies, small VEGFR-2 inhibitors, and a soluble decoy VEGFR. In addition, clinical trials are being performed with a number of promising anticancer compounds such as Iressa and Herceptin that block proteins involved in the induction of the VEGF pathway.

    Cannabinoids, the active components of Cannabis sativa L. (marijuana), and their derivatives exert a wide array of effects by activating their specific G protein-coupled receptors CB₁ and CB₂, which are normally engaged by a family of endogenous ligands–the endocannabinoids.

13. Marijuana and its derivatives have been used in medicine for many centuries, and there is currently a renaissance in the study of the therapeutic effects of cannabinoids. Today, cannabinoids are approved to palliate the wasting and emesis associated with cancer and AIDS chemotherapy, and ongoing clinical trials are determining whether cannabinoids are effective agents in the treatment of pain, neurodegenerative disorders such as multiple sclerosis, and traumatic brain injury.

14. In addition, cannabinoid administration to mice and/or rats induces the regression of lung adenocarcinomas, gliomas, thyroid epitheliomas, lymphomas  , and skin carcinomas. These studies have also evidenced that cannabinoids display a fair drug safety profile and do not produce the generalized cytotoxic effects of conventional chemotherapies, making them potential antitumoral agents.

    Little is known, however, about the mechanism of cannabinoid antitumoral action in vivo. By modulating key cell signaling pathways, cannabinoids directly induce apoptosis or cell cycle arrest in different transformed cells in vitro .

15. However, the involvement of these events in their antitumoral action in vivo is as yet unknown. More recently, immunohistochemical and functional analyses of the vasculature of gliomas and skin carcinomas have shown that cannabinoid administration to mice inhibits tumor angiogenesis.

16. These findings prompted us to explore the mechanism by which cannabinoids impair angiogenesis of gliomas and, particularly, the possible impact of cannabinoids on the VEGF pathway. Here, we report that cannabinoid administration inhibits the VEGF pathway in cultured glioma cells, in glioma-bearing mice, and in two patients with glioblastoma multiforme. In addition, this effect may be mediated by ceramide, a sphingolipid second messenger implicated previously in cannabinoid signaling in glioma cells.

    
    

    MATERIALS AND METHODS

    Cannabinoids.

    The Δ⁹-tetrahydrocannabinol was kindly given by Alfredo Dupetit (The Health Concept, Richelbach, Germany). JWH-133 was kindly given by Dr. John Huffman (Department of Chemistry, Clemson University, Clemson, SC; Ref. 24 ). WIN-55,212-2 and anandamide were from Sigma (St. Louis, MO). SR141716 and SR144528 were kindly given by Sanofi-Synthelabo (Montpellier, France). For in vitro incubations, cannabinoid agonists and antagonists were directly applied at a final DMSO concentration of 0.1–0.2% (v/v).

17.  For in vivo experiments, ligands were prepared at 1% (v/v) DMSO in 100 μl PBS supplemented with 5 mg/ml BSA. No significant influence of the vehicle was observed on any of the parameters determined.

    Cell Culture.

    The rat C6 glioma , the human U373 MG astrocytoma, the mouse PDV.C57 epidermal carcinoma, and the human ECV304 bladder cancer epithelioma  were cultured as described previously. Human glioma cells were prepared from a glioblastoma multiforme (grade IV astrocytoma; Ref. 26 ).

18. The biopsy was digested with collagenase (type Ia; Sigma) in DMEM at 37°C for 90 min, the supernatant was seeded in DMEM containing 15% FCS and 1 mm glutamine, cells were grown for 2 passages, and 24 h before the experiments, cells were transferred to 0.5%-serum DMEM. Cell viability was determined by trypan blue exclusion. Rat recombinant VEGF and N-acetylsphingosine (C₂-ceramide) were from Sigma.

    Tumor Induction in Mice.

    Tumors were induced in mice deficient in recombination activating gene 2 by s.c. flank inoculation of 5 × 10⁶ C6 glioma cells in 100 μl PBS supplemented with 0.1% glucose. When tumors had reached a volume of 350–450 mm³, animals were assigned randomly to the various groups and injected intratumorally for up to 8 days with 50 μg/day JWH-133 and/or 60 μg/day fumonisin B1 (Alexis, San Diego, CA).

19. Control animals were injected with vehicle. Tumors were measured with external caliper, and volume was calculated as (4π/3) × (width/2)² × (length/2).

    Human Tumor Samples.

    Tumor biopsies were obtained from two of the patients enrolled in an ongoing Phase I/II clinical trial (at the Neurosurgery Department of Tenerife University Hospital, Spain) aimed at investigating the effect of Δ⁹-tetrahydrocannabinol administration on the growth of recurrent glioblastoma multiforme. The patients had failed standard therapy, which included surgery, radiotherapy (60 Gy), and temozolomide chemotherapy (4 cycles). Patients had clear evidence of tumor progression on sequential magnetic resonance scanning before enrollment in the study, had received no anticancer therapy for ∼1 year, and had a fair health status (Karnofski performance score = 90). The patients provided written informed consent. The protocol was approved by the Clinical Trials Committee of Tenerife University Hospital and by the Spanish Ministry of Health.

    Patient 1 (a 48-year-old man) had a right-occipital-lobe tumor (7.5 × 6 cm maximum diameters), and Patient 2 (a 57-year-old-man) had a right-temporal-lobe tumor (6 × 5 cm maximum diameters).

20. Both tumors were diagnosed by the Pathology Department of Tenerife University Hospital as glioblastoma multiforme and showed the hallmarks of this type of tumor (high vascularization, necrotic areas, abundant palisading and mitotic cells, and so on).

21. The tumors were removed extensively by surgery, biopsies were taken, and the tip (∼5 cm) of a silastic infusion cathether (9.6 French; 3.2 mm diameter) was placed into the resection cavity. The infusion cathether was connected to a Nuport subclavicular s.c. reservoir. Each day 0.5–1.5 (median 1.0) mg of Δ⁹-tetrahydrocannabinol (100 μg/μl in ethanol solution) were dissolved in 30 ml of physiological saline solution supplemented with 0.5% (w/v) human serum albumin, and the resulting solution was filtered and subsequently administered at a rate of 0.3 ml/min with a syringe pump connected to the s.c. reservoir. Patient 1 started the treatment 4 days after the surgery and received a total amount of 24.5 mg of Δ⁹-tetrahydrocannabinol for 19 days.

22. The posttreatment biopsy was taken 19 days after the cessation of Δ⁹-tetrahydrocannabinol administration. Patient 2 started the treatment 4 days after the surgery and received a total amount of 13.5 mg of Δ⁹-tetrahydrocannabinol for 16 days. The posttreatment biopsy was taken 43 days after the cessation of Δ⁹-tetrahydrocannabinol administration. Samples were either transferred to DMEM containing 15% FCS and 1 mm glutamine (for tumor-cell isolation, see above; Fig. 2B ⇓ ) and frozen (for VEGF determination, Patients 1 and 2; and for VEGFR-2 Western blotting, Patient 1; Fig. 6, A and C ⇓ ) or fixed in formalin and embedded in paraffin (for VEGFR-2 confocal microscopy, Patients 1 and 2; Fig. 6B ⇓ ).

    The cDNA Arrays.

    Total RNA was extracted  from tumors of vehicle- or JWH-133-treated mice (see above), and poly(A)⁺ RNA was isolated with oligotex resin (Qiagen Inc., Valencia, CA) and reverse-transcribed with Moloney murine leukemia virus reverse transcriptase in the presence of 50 μCi [α-³³P]dATP for the generation of radiolabeled cDNA probes.

23. Purified radiolabeled probes were hybridized to angiogenesis, hypoxia, and metastasis gene array membranes (GEArray Q Series; Superarray Bioscience Corporation, Frederick, MD) according to the manufacturer’s instructions. Hybridization signals were detected by phosphorimager and analyzed by Phoretix housekeeping genes in the blots as internal controls for normalization. The selection criteria were set conservatively throughout the process, and the genes selected were required to exhibit at least a 2-fold change of expression and a P < 0.01.

    ELISA.

    VEGF levels were determined in cell culture media and in tumor extracts, obtained by homogenization as described previously, by solid-phase ELISA using the Quantikine mouse VEGF Immunoassay (R&D Systems, Abingdon, United Kingdom; 70% cross-reactivity with rat VEGF) for rat and mouse samples and the Quantikine human VEGF Immunoassay (R&D Systems) for human samples.

    Western Blot.

    Particulate cell or tissue fractions were subjected to SDS-PAGE, and proteins were transferred from the gels onto polyvinylidene fluoride membranes.

24. Blots were incubated with antibodies against total VEGFR-2 (1:1000; Santa Cruz Biotechnology, Santa Cruz, CA), VEGFR-2 phosphotyrosine 996 (1:250; Cell Signaling, Beverly, MA), VEGFR-2 phosphotyrosine 1214 (1:250; kindly given by Dr. Francesco Pezzella, Nuffield Department of Clinical Laboratory Science, University of Oxford, United Kingdom), and α-tubulin (1:4000, Sigma). The latter was used as a loading control. In all of the cases, samples were subjected to luminography with an enhanced chemiluminescence detection kit (Amersham Life Sciences, Arlington Heights, IL).

25. Densitometric analysis of the blots was performed with the Multianalyst software (Bio-Rad Laboratories, Hercules, CA).

    Confocal Microscopy.

    Glioma cells were cultured in coverslips and fixed in acetone for 10 min. Mouse tumors were dissected and frozen, and 5-μm sections were fixed in acetone for 10 min. Human tumors were fixed in 10% buffered formalin and then paraffin-embedded, 5-μm sections were deparaffinized and rehydrated, and antigen retrieval was carried out by immersing the slides in 10 mm citrate (pH 6.0) and boiling for 3 min. All of the samples were incubated with 10% goat serum in PBS for 30 min at room temperature to block nonspecific binding. Slices were incubated for 1.5 h with the aforementioned primary antibodies against total VEGFR-2 (1:50) and VEGFR-2 phosphotyrosine 1214 (1:20).

26. After washing with PBS, slices were additionally incubated (1 h, room temperature, darkness) with a mixture of the secondary goat antimouse antibodies Alexa Fluor 488 and Alexa Fluor 546 (both at 1:400; Molecular Probes, Leyden, The Netherlands).

27.  After washing with PBS, sections were fixed in 1% paraformaldehyde for 10 min and mounted with DAKO fluorescence mounting medium containing TOTO-3 iodide (1:1000; Molecular Probes) to stain cell nuclei. Confocal fluorescence images were acquired using a Laser Sharp 2000 software (Bio-Rad) and a Confocal Radiance 2000 coupled to Axiovert S100 TV microscope (Carl Zeiss, Oberkochen, Germany). Pixel quantification and colocalization were determined with Metamorph-Offline software (Universal Imaging, Downingtown, PA).

    Ceramide Synthesis.

    C6 glioma cells were cultured for 48 h in serum-free medium with the additions indicated together with 1 μCi of l-U-[¹⁴C]serine/well, lipids were extracted, and ceramide resolved by thin-layer chromatography as described previously.

    Statistics.

    Results shown represent mean ± SD. Statistical analysis was performed by ANOVA with a post hoc analysis by the Student-Neuman-Keuls test or by unpaired Student’s t test.

    RESULTS

    Changes in Gene Expression Profile in Mouse Gliomas.

    The cDNA array analysis was used as a first approach to test whether cannabinoid administration affects the VEGF pathway in mouse gliomas. Because cannabinoid-based therapeutic strategies should be as devoid as possible of psychotropic side effects and glioma cells express functional CB₂ receptors, which do not mediate psychoactivity, mice bearing s.c. gliomas were injected with the selective CB₂ agonist JWH-133 .

    A total of 267 genes related to angiogenesis, hypoxia (perhaps the most potent stimulus for the onset of tumor angiogenesis), and metastasis (a characteristic of actively growing tumors related closely to angiogenesis) were analyzed, of which 126 were considered to be expressed in reliable amounts. JWH-133 administration altered the expression of 10 genes, all of which are directly or indirectly related to the VEGF pathway (Fig. 1) ⇓ . Thus, cannabinoid treatment lowered the expression of the following: (a) VEGF-A [confirming our previous

28. Northern blot data and its relative VEGF-B ; (b) hypoxia-inducible factor-1α [one of the subunits of hypoxia-inducible factor-1, the major transcription factor involved in VEGF gene expression ; (c) two genes known to be under the control of VEGF, namely those encoding connective tissue growth factor [a mitogen involved in extracellular matrix production and angiogenesis , and heme oxygenase-1 [an enzyme highly expressed during hypoxia and inflammation ; and (d) four genes known to encode proteins functionally related to VEGF, namely Id3 [a transcription factor inhibitor involved in angiogenesis and tumor progression  , midkine [a proangiogenic and tumorigenic growth factor , angiopoietin-2 [a prominent proangiogenic factor that cooperates with VEGF , and Tie-1 [an angiopoietin receptor .

29.  In addition, cannabinoid treatment increased the expression of the gene encoding type I procollagen α1 chain (a metalloproteinase substrate related to matrix remodeling during angiogenesis; Ref. 35 ).

    

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    Fig. 1.

    Changes in gene expression profile in mouse gliomas after cannabinoid treatment. Animals bearing gliomas were treated with either vehicle (Control) or JWH-133 (JWH) for 8 days as described in “Materials and Methods.” Equal amounts of poly(A)⁺ RNA from tumors of 2 animals/group were pooled and hybridized to angiogenesis, hypoxia, and metastasis cDNA array membranes. Genes affected by cannabinoid treatment are listed.

30. Examples of affected genes are pointed with arrows. Angiogenesis membrane, angiopoietin-2 (top), midkine (middle), and VEGF-A (bottom); Hypoxia membrane, procollagen Iα1 (top), heme oxygenase-1 (middle), and VEGF-A (bottom); and Metastasis membrane, VEGF-A.

    Inhibition of VEGF Production in Cultured Glioma Cells and in Mouse Gliomas.

    We focused next on the two main components of the VEGF pathway, namely VEGF and VEGFR-2, in both cultured glioma cells and gliomas in vivo. Incubation of C6 glioma cells with the synthetic cannabinoid WIN-55,212-2 (100 nm), a mixed CB₁/CB₂ receptor agonist, inhibited VEGF release into the medium in a time-dependent manner (Fig. 2A)  . The cannabinoid did not affect cell viability throughout the time interval in which VEGF determinations were performed (up to 48 h; data not shown). Cannabinoid-induced attenuation of VEGF production was evident in another glioma cell line (the human astrocytoma U373 MG) and, more importantly, in tumor cells obtained directly from a human glioblastoma multiforme biopsy (Fig. 2B) . The cannabinoid effect was also observed in the mouse skin carcinoma PDV.C57 and in the human bladder cancer epithelioma ECV304 (Fig. 2B).

    

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    Fig. 2.

    Inhibition of VEGF production by cannabinoids in cultured glioma cells and in mouse gliomas. A, C6 glioma cells were cultured for the times indicated with vehicle (□) or 100 nm WIN-55,212-2 (▪), and VEGF levels in the medium were determined (n = 4). B, U373 MG astrocytoma cells, tumor cells obtained from a patient with glioblastoma multiforme (GBM), PDV.C57 epidermal carcinoma cells, and ECV304 bladder cancer epithelioma cells were cultured for 48 h with vehicle (□) or 100 nm WIN-55,212-2 (▪), and VEGF levels in the medium were determined. Data represent the percentage of VEGF in cannabinoid incubations versus the respective controls (n = 3–4). C, C6 glioma cells were cultured for 48 h with vehicle (Control), 100 nm WIN-55,212-2 (WIN), 100 nm JWH-133 (JWH), 2 μm anandamide (AEA), 0.5 μm SR141716 (SR1), and/or 0.5 μm SR144528 (SR2), and VEGF levels in the medium were determined (n = 4–6). D, C6 glioma cells were cultured for 48 h with vehicle (Control), 100 μm WIN-55, 212-2 (WIN), 1 μm C₂-ceramide (CER), and/or 0.5 μm fumonisin B1 (FB1), and VEGF levels in the medium were determined (n = 4). E, animals bearing gliomas were treated with either vehicle (Control), JWH-133 (JWH), fumonisin B1 (FB1), or JWH-133 plus fumonisin B1 for 8 days as described in “Materials and Methods,” and VEGF levels in the tumors were determined (n = 4–6 for each experimental group).

31. Significantly different (∗, P < 0.01; ∗ ∗, P < 0.05) from control incubations or control animals. Bars, ±SD.

    To prove the specificity of WIN-55,212-2 action on VEGF release, we used other cannabinoid receptor agonists as well as selective cannabinoid receptor antagonists (Fig. 2C) ⇓ . The inhibitory effect of WIN-55,212-2 was mimicked by the endocannabinoid anandamide (2 μm), another mixed CB₁/CB₂ agonist, and by the synthetic cannabinoid JWH-133 (100 nm), a selective CB₂ agonist. In addition, the CB₁ antagonist SR141716 (0.5 μm) and the CB₂ antagonist SR144528 (0.5 μm) prevented WIN-55,212-2 action, pointing to the involvement of CB receptors in cannabinoid-induced inhibition of VEGF production.

    The sphingolipid messenger ceramide has been implicated in the regulation of tumor cell function by cannabinoids. The involvement of ceramide in cannabinoid-induced inhibition of VEGF production was tested by the use of N-acetylsphingosine (C₂-ceramide), a cell-permeable ceramide analog, and fumonisin B1, a selective inhibitor of ceramide synthesis de novo. In line with our previous data in primary cultures of rat astrocytes, fumonisin B1 was able to prevent cannabinoid-induced ceramide biosynthesis (relative values of [¹⁴C]serine incorporation into ceramide, n = 3: vehicle, 100; 100 nm WIN-55,212-2, 140 ± 1; 100 nm WIN-55,212-2 plus 0.5 μm fumonisin B1, 86 ± 9). C₂-ceramide (1 μm) depressed VEGF production, whereas pharmacological blockade of ceramide synthesis de novo with fumonisin B1 (0.5 μm) prevented the inhibitory effect of WIN-55,212-2 (Fig. 2D) ⇓ . We subsequently evaluated whether fumonisin B1 action was also evident in vivo. The decrease in tumor VEGF levels induced by cannabinoid administration  was prevented by cotreatment of the animals with fumonisin B1 (Fig. 2E) ⇓ .

    Inhibition of VEGFR-2 in Cultured Glioma Cells and in Mouse Gliomas.

    VEGFR-2 activation was determined by measuring the extent of phosphorylation of two of its essential tyrosine autophosphorylation residues, namely 996 and 1214. Western blot experiments showed that C6 glioma cells express highly phosphorylated VEGFR-2 in the absence of ligand, indicating that the receptor may be constitutively active. Incubation of C6 glioma cells with WIN-55,212-2 or JWH-133 decreased VEGFR-2 activation without affecting total VEGFR-2 levels (Fig. 3A) ⇓ .

32. Confocal microscopy experiments confirmed the decrease in VEGFR-2 immunoreactivity by cannabinoid challenge when fluorescence was expressed per cell nucleus (Fig. 3B) ⇓ or per total-VEGFR-2 fluorescence (data not shown). Moreover, fumonisin B1 prevented cannabinoid inhibitory action, and C₂-ceramide reduced VEGFR-2 activation (Fig. 3, A and B) ⇓ . Interestingly, on cannabinoid exposure the receptor seemed to be preferentially condensed in the perinuclear region, and this relocalization was prevented by fumonisin B1 (Fig. 3B) ⇓ . The functional impact of VEGF on C6 glioma cells was supported by the finding that VEGF induced a prosurvival action by preventing the loss of cell viability on prolonged (72 h) cannabinoid or C₂-ceramide challenge (Fig. 3C) ⇓ .

    

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    Fig. 3.

    Inhibition of VEGFR-2 by cannabinoids in cultured glioma cells. A, C6 glioma cells were cultured for 4 h with vehicle (Control), 100 nm WIN-55,212-2 (WIN), 100 nm JWH-133 (JWH), 10 μm C₂-ceramide (CER), and/or 0.5 μm fumonisin B1 (FB1), and VEGFR-2 activation (anti-VEGFR-2 PY996 and anti-VEGR2 PY1214 antibodies) and expression (antitotal VEGFR-2 antibody) were determined by Western blot. Absorbance values relative to those of total VEGFR-2 are given in arbitrary units.

33. Significantly different (∗, P < 0.01) from control incubations (n = 3). B, C6 glioma cells were cultured as in panel A, and VEGFR-2 activation (anti-VEGFR-2 PY1214 antibody, green) and expression (antitotal VEGFR-2 antibody, red) were determined by confocal microscopy. Cell nuclei are stained in blue. One representative experiment of 3 is shown. Relative values of activated-VEGFR-2 pixels/cell nucleus are given in parentheses. C, C6 glioma cells were cultured for 72 h with vehicle (Control), 100 nm WIN-55,212-2 (WIN), 100 nm JWH-133 (JWH), or 1 μm C₂-ceramide (CER) with (▪) or without (□) 50 ng/ml VEGF, and the number of viable cells was determined. Significantly different (∗, P < 0.01) from control incubations (n = 3–4). Bars, ±SD.

    The effect of cannabinoid administration on VEGFR-2 activation was subsequently tested in tumor-bearing mice. The ceramide-dependent cannabinoid-induced inhibition of VEGFR-2 activation found in cultured cells was also observed by Western blot (Fig. 4A) ⇓ and confocal microscopy (Fig. 4B) ⇓ in mouse gliomas. Like in the cultured-cell experiments and in line with the cDNA array experiments (data not shown), total VEGFR-2 expression in the tumors was unaffected by cannabinoid treatment (Fig. 4, A and B) ⇓ .

    

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    Fig. 4.

    Inhibition of VEGFR-2 by cannabinoids in mouse gliomas. A, animals bearing gliomas were treated with either vehicle (Control), JWH-133 (JWH), fumonisin B1 (FB1), or JWH-133 plus fumonisin B1 for 8 days as described in “Materials and Methods,” and VEGFR-2 activation (anti-VEGFR-2 PY996 and anti-VEGR2 PY1214 antibodies) and expression (antitotal VEGFR-2 antibody) were determined by Western blot. Absorbance values relative to those of total VEGFR-2 (phosphorylated VEGFR-2 blots) or of α-tubulin (total VEGFR-2 blots) are given in arbitrary units. Significantly different (∗, P < 0.01) from control animals (n = 3–4 for each experimental group). B, animals bearing gliomas were treated as in panel A, and VEGFR-2 activation (anti-VEGFR-2 1214 antibody, green) and expression (antitotal VEGFR-2 antibody, red) were determined by confocal microscopy. Cell nuclei are stained in blue. Low- and high-magnification pictures are shown. One representative tumor of 3–4 for each experimental group is shown. Relative values of activated-VEGFR-2 pixels/cell nucleus are given in parentheses.

    Phosphorylated VEGFR-2 has been found previously in the cell nucleus, and it has been postulated that this translocation process might play a role in VEGFR-2 signaling. However, by confocal microscopy, we found a rather variable fraction of phosphorylated VEGFR-2 in the nuclei of C6 glioma cells in culture and on inoculation in mice, and this fraction of nuclear VEGFR-2 was unaltered after treatment with cannabinoids and/or fumonisin B1 in vitro and in vivo (data not shown).

    Changes in the Size of Mouse Gliomas.

    To test whether the aforementioned ceramide-dependent changes in the VEGF pathway are functionally relevant, we measured tumor size along cannabinoid and fumonisin B1 treatment. In agreement with previous observations, JWH-133 administration blocked the growth of s.c. gliomas in mice. Of importance, cotreatment of the animals with fumonisin B1 prevented cannabinoid antitumoral action (Fig. 5) ⇓ .

    

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    Fig. 5.

    Changes in the size of mouse gliomas after cannabinoid and fumonisin B1 treatment. Animals bearing gliomas (n = 4–6 for each experimental group) were treated with either vehicle (Control, ○), JWH-133 (JWH, •), fumonisin B1 (FB1, □), or JWH-133 plus fumonisin B1 (▪) for up to 8 days as described in “Materials and Methods.” Examples of formaldehyde-fixed dissected tumors after 8 days of treatment are shown. Bars, ±SD.

    Inhibition of the VEGF Pathway in Two Patients with Glioblastoma Multiforme.

    To obtain additional support for the potential therapeutic implication of cannabinoid-induced inhibition of the VEGF pathway, we analyzed the tumors of two patients enrolled in a clinical trial aimed at investigating the effect of Δ⁹-tetrahydrocannabinol, a mixed CB₁/CB₂ agonist, on recurrent glioblastoma multiforme. The patients were subjected to local Δ⁹-tetrahydrocannabinol administration, and biopsies were taken before and after the treatment. In both patients, VEGF levels in tumor extracts were lower after cannabinoid inoculation (Fig. 6A) ⇓ . The Δ⁹-tetrahydrocannabinol also lowered the expression of phosphorylated VEGFR-2 in the tumors of the two patients, and this was accompanied (in contrast to the mouse glioma experiments shown above) by a decrease in total VEGFR-2 levels (Fig. 6B) ⇓ . This was confirmed by Western blot analysis in Patient 1 (Fig. 6C) ⇓ . Unfortunately, we were unable to obtain appropriate samples for Western blot from Patient 2.

    

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    Fig. 6.

    Inhibition of the VEGF pathway in two patients with glioblastoma multiforme after cannabinoid treatment. The patients were subjected to Δ⁹-tetrahydrocannabinol (THC) administration as described in “Materials and Methods.” A, VEGF levels in the tumors before (□) and after (▪) THC treatment. B, VEGFR-2 activation (anti-VEGFR-2 PY1214 antibody, green) and expression (antitotal VEGFR-2 antibody, red) in the tumors before and after THC treatment as determined by confocal microscopy. Cell nuclei are stained in blue. Relative values of activated-VEGFR-2 pixels (parentheses) and of total-VEGFR-2 pixels (square brackets) per cell nucleus are given for the two patients. C, VEGFR-2 activation (anti-VEGFR-2 PY996 and anti-VEGR2 PY1214 antibodies) and expression (antitotal VEGFR-2 antibody) in the tumor of Patient 1 before and after THC treatment, as determined by Western blot. Absorbance values relative to those of loading controls (α-tubulin) are given in arbitrary units.

    DISCUSSION

    Angiogenesis is a prerequisite for the progression of most solid tumors. In particular, gliomas first acquire their blood supply by co-opting existing normal brain vessels to form a well-vascularized tumor mass without the necessity to initiate angiogenesis. When gliomas progress, they become hypoxic as the co-opted vasculature regresses and malignant cells rapidly proliferate. These hypoxic conditions, in turn, induce robust angiogenesis via the VEGF pathway and angiopoietin-2, and in fact, this angiogenic sprouting distinguishes a grade IV astrocytoma (glioblastoma multiforme) from lower-grade astrocytomas.

34. Here, we show that cannabinoid treatment impairs the VEGF pathway in mouse gliomas by blunting VEGF production and signaling. Cannabinoid-induced inhibition of VEGF expression and VEGFR-2 activation also occurred in cultured glioma cells, indicating that the changes observed in vivo may reflect the direct impact of cannabinoids on tumor cells. Moreover, a depression of the VEGF pathway was also evident in two patients with glioblastoma multiforme. Although the changes in VEGFR-2 expression observed in these two patients do not fully mirror the cultured-cell and mouse data, they clearly follow the same direction. The molecular basis of this discrepancy is, however, unknown.

    Our observations do not exclude that cannabinoids may also blunt tumor VEGF signaling indirectly by targeting other receptor-mediated processes that stimulate the VEGF pathway. For example, it is known that engagement of epidermal growh factor and nerve growth factor receptors induces the VEGF pathway, and cannabinoids have been reported to inhibit the epidermal growth factor receptor in skin carcinoma and prostate carcinoma cells as well as the TrkA neurotrophin receptor in breast carcinoma and pheochromocytoma cells. However, the molecular mechanisms by which cannabinoid receptor activation impact these growth factor receptors remain obscure.

    Recent work has shown that cannabinoids can modulate sphingolipid-metabolizing pathways by increasing the intracellular levels of ceramide, a lipid second messenger that controls cell fate in different systems . After cannabinoid receptor activation, two peaks of ceramide generation are observed in glioma cells that have different mechanistic origin: (a) the first peak comes from sphingomyelin hydrolysis; and (b) the second peak originates from ceramide synthesis de novo.

35. The findings reported here expand the role of de novo-synthesized ceramide in cannabinoid action. Moreover, as far as we know, this is also the first report showing that ceramide depresses the VEGF pathway by interfering with VEGF production and VEGFR-2 activation, a notion that is in line with the observation that ceramide analogs prevent VEGF-induced cell survival. In the context of the “sphingolipid rheostat” theory, the mitogenic sphingolipid sphingosine 1-phosphate would shift the balance toward angiogenesis and tumorigenesis, whereas the antiproliferative sphingolipid ceramide would blunt angiogenesis and tumorigenesis (present study).

    The use of cannabinoids in medicine is limited by their psychoactive effects mediated by neuronal CB₁ receptors. Although these adverse effects are within the range of those accepted for other medications, especially in cancer treatment, and tend to disappear with tolerance on continuous use, it is obvious that cannabinoid-based therapies devoid of side-effects would be desirable.

36. As glioma cells express functional CB₂ receptors, we used a selective CB₂ ligand to target the VEGF pathway. Selective CB₂ receptor activation in mice also inhibits the growth and angiogenesis of skin carcinomas. Unfortunately, very little is known about the pharmacokinetics and toxicology of the selective CB₂ ligands synthesized to date, making them as yet unavailable for clinical trials.

    Gliomas are one of the most malignant forms of cancer, resulting in the death of affected patients within 1–2 two years after diagnosis. Current therapies for glioma treatment are usually ineffective or just palliative.

37. Therefore, it is essential to develop new therapeutic strategies for the management of glioblastoma multiforme, which will most likely require a combination of therapies to obtain significant clinical results. In line with the idea that anti-VEGF treatments constitute one of the most promising antitumoral approaches currently available, the present laboratory and clinical findings provide a novel pharmacological target for cannabinoid-based therapies.

    Acknowledgments

    We are indebted to M. A. Muñoz and C. Sánchez for expert technical assistance in the confocal microscopy experiments, Dr. L. García for personal support, and Drs. G. Velasco and I. Galve-Roperh for discussion and advice.

    Footnotes

    • Grant support: Fundación Científica de la Asociación Española Contra el Cáncer and Ministerio de Ciencia y Tecnología Grant SAF2003-00745.

    • The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

    • Requests for reprints: Manuel Guzmán, Department of Biochemistry and Molecular Biology I, School of Biology, Complutense University, 28040 Madrid, Spain. Phone: 34-913944668; Fax: 34-913944672; E-mail: mgp@bbm1.ucm.es.

    •  See Internet address http://www.superarray.com for a detailed list of the genes analyzed.

    • Received December 16, 2003.
    • Revision received April 1, 2004.
    • Accepted June 10, 2004.
38. ©2004 American Association for Cancer Research.

 ABSTRACT
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 CB1 receptor. By contrast, the other cannabinoid receptor subtype, the CB2 receptor, shows a much more restricted distribution and is absent from normal brain. Here we show
that local administration of the selective CB2 agonist JWH-133 at 50 mg/day to Rag-22/2 mice induced a considerable regression of malignant tumors generated by inoculation of C6 glioma cells.

The selective involvement of the CB2 receptor in this action was evidenced by: (a) the prevention
by the CB2 antagonist SR144528 but not the CB1 antagonist SR141716; (b) the down-regulation of the CB2 receptor but not the CB1 receptor in the tumors; and (c) the absence of typical CB1-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 CB2 receptor expression was directly related with tumor malignancy. In addition, the growth of grade IV human astrocytoma cells in Rag-22/2 mice was completely blocked by JWH-133 administration at 50 mg/day.

Experiments carried out with C6 glioma cells in culture evidenced the internalization of the CB2 but not the CB1 receptor upon JWH-133 challenge and showed that selective activation of the CB2 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.

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Guzman M, Duarte MJ, Blazquez C, Ravina J, Rosa MC, Galve-Roperh I, Sanchez C, Velasco G, Gonzalez-Feria L.

Br J Cancer 2006;95(2):197-203.

THC was well tolerated in this pilot study of intrakranial cannabinoid administration.

 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 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.British Journal of Cancer advance online publication, 27 June 2006; doi:10.1038/sj.bjc.6603236 www.bjcancer.com

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Massi P, Vaccani A, Bianchessi S, Costa B, Macchi P, Parolaro D
Department of Pharmacology, Chemotherapy and Medical Toxicology, University of Milan, via Vanvitelli 32, 20129 Milan, Italy.

Recently, we have shown that the non-psychoactive cannabinoid compound cannabidiol  (CBD) induces apoptosis of glioma cells in vitro and tumor regression in vivo. The present study investigated a possible involvement of caspase activation and reactive oxygen species (ROS) induction in the apoptotic effect of CBD. CBD produced a gradual, time-dependent activation of caspase-3, which preceded the appearance of apoptotic death. In addiction, release of cytochrome c and caspase-9 and caspase-8 activation were detected. The exposure to CBD caused in glioma cells an early production of ROS, depletion of intracellular glutathione  and increase activity of glutathione  reductase and glutathione  peroxidase enzymes. Under the same experimental condition, CBD did not impair primary glia. Thus, we found a different sensitivity to the anti-proliferative effect of CBD in human glioma cells and non-transformed cells that appears closely related to a selective ability of CBD in inducing ROS production and caspase activation in tumor cells.

Cell. Mol. Life Sci. (2006)

Article Date: 15 Aug 2004

 Researchers in Spain have discovered that a cannabis extract makes brain tumors shrink by halting the growth of blood vessels that supply the tumors with life. Cannabis has chemicals called cannabinoids, these are the chemicals that could effectively starve tumors to death, say the researchers.

The study was carried out at the Complutense University, Madrid, Spain.

The team used mice to demonstrate that the cannabinoids block vessel growth.

You can read about this latest research in the journal Cancer Research.

Apparently, the procedure is also effective in humans.

The Spanish team, led by Dr Manuel Guzmn, wanted to see whether they could prevent glioblastoma multiforme cancer from growing by cutting off its blood supply. Glioblastoma multiforme is one of the most difficult cancers to treat - it seldom responds to any medical intervention, such as radiotherapy, chemotherapy and surgery.

The scientists knew that cannabinoids will block the growth of blood vessels (to tumors) in mice - they wanted to find out whether the same thing would happen with humans.

The mice were given a cancer similar to the human brain cancer (glioblastoma multiforme). The mice were then given cannabinoids and the genes examined.

The genes associated with blood vessel growth in tumors through the production of a chemical called vascular endothelial growth factor (VEGF) had their activity reduced.

Cannabinoids halt VEGF production by producing Ceramide. Ceramide controls cell death.

Dr Guzmn said: "As far as we know, this is the first report showing that ceramide depresses VEGF pathway by interfering with VEGF production."

They then wanted to see if this would also happen with humans.

They selected two patients who had glioblastoma multiforme and had not responded to chemotherapy, radiotherapy or surgery. The scientists took samples from them before and after treating them with a cannabinoids solution - this was administered directly into the tumor.

Amazingly, both patients experienced reduced VEGF levels in the tumor as a result of treatment with cannabinoids.

The researchers said that the results were encouraging. In order to be sure about their findings they need to carry out a larger study, they said.

Dr Guzmn said "The present findings provide a novel pharmacological target for cannabinoid-based therapies."

IACM-Bulletin of 09 July 2006

Science: THC tested against brain tumour in pilot clinical study

Results of a clinical study with THC in nine brain cancer patients conducted in a hospital on Tenerife, Spain, were published in the British Journal of Cancer. Patients suffered from a gioblastoma, a very aggressive brain tumour, and had previously failed standard therapy (surgery and radiotherapy). Median survival was 24 weeks. Two patients survived nearly one year.

THC was administered directly into the tumour by a small catheter, whose tip was placed into the tumour during a surgery. The initial THC dose was 20-40 micrograms, which was increased to 80-180 micrograms daily. Patients were treated for 10-64 days. The treatment was well tolerated by all patients.

The tumours of the nine patients expressed different amounts of CB1 and CB2 receptors, but there was no correlation between receptor expression and survival. Due to the study design it was not possible to determine the effect of THC on survival. This would have required a control group with no or with a different treatment. A comparison with survival in pilot studies with other drugs suggests that THC may have been beneficial to the patients in this study. Researchers noted that THC at least did "not facilitate tumour growth nor decrease patient survival." They suggest further trials with cannabinoids on this and other types of tumours either alone or in combination with other anti-tumoural drugs.

(Source: Guzman M, Duarte MJ, Blazquez C, Ravina J, Rosa MC, Galve-Roperh I, Sanchez C, Velasco G, Gonzalez-Feria L. A pilot clinical study of Delta(9)-tetrahydrocannabinol in patients with recurrent glioblastoma multiforme. Br J Cancer, 2006 Jun 27; [electronic publication ahead of print])

Germany: Federal Institute for Pharmaceuticals and Medical Products makes unrealistically high demands of patients for the approval of their medical use of cannabis

On 5 July, the Federal Institute for Pharmaceuticals and Medical Products sent identical letters to applicants for permission to use cannabis for medicinal purposes, in which the institute requests additional information and documents. Referencing provisions of the Federal Narcotics Control Law patients are also asked to comply with requirements, which at best can be met by pharmacies or pharmaceutical companies.

For example, they demand the storage of cannabis in a safe or in rooms made of reinforced concrete and a certificate documenting the patient’s experience in the handling of narcotics. If the cannabis were to be imported from abroad, the Institute would require importation permits for each purchase. The Institute asks applicants to respond by 31 August or to request an extension.

The chairman of the German Association for Cannabis as Medicine (ACM), Dr. Franjo Grotenhermen, points out that in Canada and in those states in the USA, where the medical use of cannabis is allowed, no such demands are made of patients. In addition, patients who are prescribed opiates in Germany are allowed to store a ration that suffices three months. "Even after the 2005 ruling by the Federal Administrative Court the Institute has been trying by all means to prevent patients from gaining legal access to the medical use of cannabis," he said. "The demands of the Federal Institute for Pharmaceuticals and Medical Products contradict the spirit of the court ruling." The Federal Administrative Court stated in its ruling of 19 May 2005 that "in particular in the case of cannabis" the federal institute needs to consider granting a permission for cultivation by patients.

The letter sent to patients by the Federal Institute for Pharmaceuticals and Medical Products is available for download at www.cannabis-med.org/german/bfarm2006.pdf

Galanti G, Fisher T, Kventsel I, Shoham J, Gallily R, Mechoulam R, Lavie G, Amariglio N, Rechavi G, Toren A

The Mina and Everard Goodman Faculty of Life Science, Bar-Ilan University, Ramat-Gan, Israel.

Background. The active components of Cannabis sativa L., Cannabinoids, traditionally used in the field of cancer for alleviation of pain, nausea, wasting and improvement of well-being have received renewed interest in recent years due to their diverse pharmacologic activities such as cell growth inhibition, anti-inflammatory activity and induction of tumor regression.

Here we used several experimental approaches, which identified delta-9-tetrahydrocannabinol (Delta(9)-THC) as an essential mediator of cannabinoid antitumoral action. Methods and results. Administration of Delta(9)-THC to glioblastoma multiforme (GBM) cell lines results in a significant decrease in cell viability. Cell cycle analysis showed G(0/1) arrest and did not reveal occurrence of apoptosis in the absence of any sub-G(1) populations.

Western blot analyses revealed a THC altered cellular content of proteins that regulate cell progression through the cell cycle. The cell content of E2F1 and Cyclin A, two proteins that promote cell cycle progression, were suppressed in both U251-MG and U87-MG human glioblastoma cell lines, whereas the level of p16(INK4A), a cell cycle inhibitor was upregulated. Transcription of thymidylate synthase (TS) mRNA, which is promoted by E2F1, also declined as evident by QRT-PCR. The decrease in E2F1 levels resulted from proteasome mediated degradation and was prevented by proteasome inhibitors. Conclusions.

Delta(9)-THC is shown to significantly affect viability of GBM cells via a mechanism that appears to elicit G(1) arrest due to downregulation of E2F1 and Cyclin A. Hence, it is suggested that Delta(9)-THC and other cannabinoids be implemented in future clinical evaluation as a therapeutic modality for brain tumors.

Published 15 October 2007 in Acta Oncol.
Full-text of this article is available online (may require subscription)

Blázquez C, Carracedo A, Salazar M, Lorente M, Egia A, González-Feria L, Haro A, Velasco G, Guzmán M

Department of Biochemistry and Molecular Biology I, School of Biology, Complutense University, 28040 Madrid, Spain.

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 was mimicked by JWH-133, a selective CB(2) cannabinoid receptor agonist that is devoid of psychoactive side effects, 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.

Blázquez C, Salazar M, Carracedo A, Lorente M, Egia A, González-Feria L, Haro A, Velasco G, Guzmán M

Department of Biochemistry and Molecular Biology I, School of Biology, Complutense University, Madrid, Spain.

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.

Published 14 March 2008 in Cancer Res, 68(6): 1945-52.
Full-text of this article is available online (may require subscription)

by GW Pharmaceuticals

Emerging research indicates that cannabinoids may help protect against the development of certain types of tumors. Most recently, a Spanish research team reported in Nature that injections of synthetic THC eradicated malignant brain tumors - so-called gliomas - in one-third of treated rats, and prolonged life in another third by as much as six weeks. 

Team leader Manuel Guzman called the results "remarkable" and speculated that they "may provide a new therapeutic approach for the treatment of malignant gliomas. An accompanying commentary remarked that this was the first convincing study to demonstrate that cannabis-based treatment may combat cancer. Other journals have also recently reported on cannabinoids' antitumoral potential. 

Earlier studies also indicate that cannabinoids may successfully stave certain types of tumors. One study examined the effects of delta-9-tetrahydrocannabinol (THC), delta-8-THC, and cannabinol (CBN) on cancer cells in mice lungs. Researchers reported that cannabinoids reduced the size of the tumors by 25 to 82 percent, depending on dose and duration of treatment, with a corresponding increase in survival time. 

A two-year federal study by the U.S. National Toxicology Program found that mice and rats given high doses of THC over long periods of time appeared to have greater protection against malignancies than untreated controls.

Researchers concluded that in both mice and rats, "the incidence of benign and malignant neoplasms was decreased in a dose dependent manner." Dr. Lester Grinspoon writes in Marihuana: The Forbidden Medicine (with James Bakalar) that other animal studies also suggest that some cannabinoids have tumor-reducing properties

An Italian research team reported in 1998 that the endocannabinoid anandamide, which binds to the same brain receptors as cannabis, "potently and selectively inhibits the proliferation of human breast cancer cells in vitro" by interfering with their DNA production cycle.  Non-mammary tumor cells were not affected by anandamide. Clearly, further research is necessary and appropriate.

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1. I. Galve-Roperph et al. "Antitumoral action of cannabinoids: involvement of sustained ceramide accumulation of ERK activation." Nature Medicine 6 (2000): 313-319.
2. ACM Bulletin. "THC destroys brain cancer in animal research," March 5, 2000.
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3. D. Piomelli. "Pot of gold for glioma therapy." Nature Medicine 6 (2000): 255-256.
4. J. Benard. "Cannabinoids, among others, send malignant tumors to nirvana." Bull Cancer 87 (2000): 299-300.
5. J. Molnar et al. "Membrane associated with antitumor effects of crocine-ginsenoside and cannabinoid derivatives." Anticancer Res 20 (2000): 861-867.
6. L. Ruiz et al. "Delta-9-tetrahydrocannabinol induces apoptosis in human prostate PC-3 cells via a receptor-independent mechanism." FEBS Letter 458 (1999): 400-404.
7. S. Baek et al. "Antitumor activity of cannabigerol against human oral epitheloid carcinoma cells." Arch Pharm Res 21 (1998): 353-356.
8. L. Harris et al., "Anti-tumoral Properties of Cannabinoids," The Pharmacology of Marihuana, ed. M. Braude et al., 2 vols., New York: Raven Press (1976) 2: 773-776 as cited by L. Grinspoon et al., Marihuana: The Forbidden Medicine (second edition), New Haven, CT: Yale University Press (1997), 173.
9. J. James, "Unpublished Federal Study Found THC-Treated Rats Lived Longer, Had Less Cancer," AIDS Treatment News 263 (1997).
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10. "Toxicology and Carcinogenesis Studies of 1 trans-delta-9-tetrahydrocannabinol in F344N/N Rats and BC63F1 Mice," National Institutes of Health National Toxicology Program, NIH Publication No. 97-3362 (November 1996).
11. L. Grinspoon et al., Marihuana: "The Forbidden Medicine" (second edition), 173.
12. L. De Petrocellis et al., The endogenous cannabinoid anandamide inhibits human breast cancer cell proliferation, Proceedings of the National Academy of Sciences 95 (1998): 8375-8380.
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13. "Pot Chemicals Might Inhibit Breast Tumors, Stroke Damage," Dallas Morning News, July 13, 1998.
    http://www.mapinc.org/drugnews

The Journal of Pharmacology And Experimental Therapeutics, First published on November 14, 2003

Anti-tumor effects of cannabidiol, a non-psychotropic cannabinoid, on human glioma cell lines
Paola Massi , Angelo Vaccani , Stefania Ceruti , Arianna Colombo , Maria Pia Abbracchio , Daniela Parolaro 

1 Dept. of Pharm. Chem. and Toxicol. University of Milan, Milan, Italy 2 Dept. of Sruct. & Funct. Biol. Center of Neuroscience, University of Insubria Busto Arsizio (VA) Ita 3 Dept. Pharmacol. Sci., Center of Excellence for neurodeg. diseases, Univ. of Milan, Milan Italy 4 University of Insubria

Address correspondence to: E-mail: daniela.parolaro@uninsubria.it

Abstract: Recently, cannabinoids have been shown to possess antitumor properties. Because the psycho-activity of cannabinoid compounds limits their medicinal usage, we undertook the present study to evaluate the in vitro antiproliferative ability of CBD, a non- psychoactive cannabinoid compound, on U87 and U373 human glioma cell lines.

The addition of CBD to the culture medium led to a dramatic drop of mitochondrial oxidative metabolism (MTT test) and viability in glioma cells, in a concentration-dependent manner, already evident 24 h after CBD exposure with an apparent IC50 of 25 µM.

The antiproliferative effect of CBD was partially prevented by the CB2 receptor antagonist SR144528 and -tocopherol. By contrast, the CB1 cannabinoid receptor antagonist SR141716, capsazepine (vanilloid receptor antagonist), the inhibitors of ceramide generation or PTX did not counteract CBD effects. We also show, for the first time, that the antiproliferative effect of CBD was correlated to induction of apoptosis, as determined by cytofluorimetric analysis and ssDNA staining, which was not reverted by cannabinoid antagonists.

Finally, CBD administered s.c. to nude mice at the dose of 0.5 mg/mouse, significantly inhibited the growth of subcutaneously implanted U87 human glioma cells. Concluding, the non-psychoactive CBD was able to produce a significant antitumor activity both in vitro and in vivo, thus suggesting a possible application of CBD as an antineoplastic agent.

Autophagy can promote cell survival or cell death, but the molecular basis underlying its dual role in cancer remains obscure. Here we demonstrate that Δ⁹-tetrahydrocannabinol (THC), the main active component of marijuana, induces human glioma cell death through stimulation of autophagy. Our data indicate that THC induced ceramide accumulation and eukaryotic translation initiation factor 2α (eIF2α) phosphorylation and thereby activated an ER stress response that promoted autophagy via tribbles homolog 3–dependent (TRB3-dependent) inhibition of the Akt/mammalian target of rapamycin complex 1 (mTORC1) axis.

We also showed that autophagy is upstream of apoptosis in cannabinoid-induced human and mouse cancer cell death and that activation of this pathway was necessary for the antitumor action of cannabinoids in vivo. These findings describe a mechanism by which THC can promote the autophagic death of human and mouse cancer cells and provide evidence that cannabinoid administration may be an effective therapeutic strategy for targeting human cancers.... .......read more

We evaluated the ability of cannabidiol (CBD) to impair the migration of tumor cells stimulated by conditioned medium. CBD caused concentration-dependent inhibition of the migration of U87 glioma cells, quantified in a Boyden chamber. Since these cells express both cannabinoid CB1 and CB2 receptors in the membrane, we also evaluated their engagement in the antimigratory effect of CBD.

The inhibition of cell was not antagonized either by the selective cannabinoid receptor antagonists SR141716 (CB1) and SR144528 (CB2) or by pretreatment with pertussis toxin, indicating no involvement of classical cannabinoid receptors and/or receptors coupled to Gi/o proteins. These results reinforce the evidence of antitumoral properties of CBD, demonstrating its ability to limit tumor invasion, although the mechanism of its pharmacological effects remains to be clarified....read more

Cudaback E, Stella N

 The last quarter century has borne witness to great advances in both the detection and treatment of numerous cancers. Even so, malignancies of the central nervous system, especially high-grade astrocytomas, continue to thwart our best efforts toward effective chemotherapeutic strategies. With prognosis remaining bleak, the time for serious consideration of alternative therapies has arrived. Various preparations of the marijuana plant, Cannabis sativa, and related synthetic and endogenous compounds, may constitute just such an alternative.

Cannabinoids, although much maligned historically for their psychotropic effects and clear abuse potential, have long been used medicinally and are now staging an impressive comeback, as recent studies have begun to explore their powerful anti-tumoral properties. In this study, we review in vitro and in vivo evidence supporting the use of cannabinoids for treatment of brain tumors.

We further propose the continued intense investigation of cannabinoid efficacies as novel anti-cancer agents, especially in models recapitulating such properties within the unique environment of the brain.

Salazar M, Carracedo A, Salanueva IJ, Hernández-Tiedra S, Lorente M, Egia A, Vázquez P,

Blázquez C, Torres S, García S, Nowak J, Fimia GM, Piacentini M, Cecconi F, Pandolfi PP, González-Feria L, Iovanna JL, Guzmán M, Boya P, Velasco G

 J Clin Invest 2009 May

 Autophagy can promote cell survival or cell death, but the molecular basis underlying its dual role in cancer remains obscure. Here we demonstrate that delta(9)-tetrahydrocannabinol (THC), the main active component of marijuana, induces human glioma cell death through stimulation of autophagy.

Our data indicate that THC induced ceramide accumulation and eukaryotic translation initiation factor 2alpha (eIF2alpha) phosphorylation and thereby activated an ER stress response that promoted autophagy via tribbles homolog 3-dependent (TRB3-dependent) inhibition of the Akt/mammalian target of rapamycin complex 1 (mTORC1) axis.

We also showed that autophagy is upstream of apoptosis in cannabinoid-induced human and mouse cancer cell death and that activation of this pathway was necessary for the antitumor action of cannabinoids in vivo. These findings describe a mechanism by which THC can promote the autophagic death of human and mouse cancer cells and provide evidence that cannabinoid administration may be an effective therapeutic strategy for targeting human cancers.

More from this journal

The Journal of clinical investigation[J Clin Invest]

• Sarah Aldington, Matire Harwood,  Brian Cox,  Mark Weatherall,  Lutz Beckert,  Anna Hansell, Alison Pritchard,Geoffrey Robinson, Richard Beasley, and Cannabis and Respiratory Disease Research Group
• Medical Research Institute of New Zealand, Wellington, New Zealand
• Department of Preventive and Social Medicine, University of Otago, Dunedin, New Zealand
• University of Otago, Wellington, New Zealand
• Imperial College London, London, United Kingdom
• University of Southampton, Southampton, United Kingdom.

Corresponding author: Dr Richard Beasley, Medical Research Institute of New Zealand, PO Box 10055, Wellington 6143, New Zealand. Email: Richard.Beasley@mrinz.ac.nz

 

Objective

To investigate whether cannabis smoking increases the risk of head and neck cancer.

Design

Case-control study.

Subjects and Methods

Cases of head and neck cancer ≤55 years identified from hospital databases and the Cancer Registry, and controls randomly selected from the electoral roll completed interviewer-administered questionnaires. Logistic regression was used to estimate the relative risk of head and neck cancer.

Results

There were 75 cases and 319 controls. An increased risk of cancer was found with increasing tobacco use, alcohol consumption, and decreased income but not increasing cannabis use. The highest tertile of cannabis use (>8.3 joint years) was associated with a nonsignificant increased risk of cancer (relative risk = 1.6, 95% confidence interval, 0.5-5.2) after adjustment for confounding variables.

Conclusions

Cannabis use did not increase the risk of head and neck cancer; however, because of the limited power and duration of use studied, a small or longer-term effect cannot be excluded.

 

Head and neck cancers represent a group of diverse cancers with varied etiology.The two main risk factors are tobacco smoking and alcohol, which have synergistic effects.Tobacco is a particularly strong risk factor for laryngeal, lip, and tongue cancers but also contributes to the risk at other upper-airway sites. Dietary factors modify risk with salted fish a risk factor for nasopharyngeal cancer, and diets rich in vitamin A and C are protective because of their antioxidative effects. A range of occupational risk factors has been identified such as the hardwood furniture industry and nasal fossa carcinoma.

 

Viruses play an important role with DNA from Epstein-Barr virus found in nasopharyngeal cancers of all types and human papilloma virus being implicated in other cancers of the head and neck. At most sites, cancer occurs more commonly in men except postcricoid carcinoma, which predominates in women because of its association with Plummer-Vinson syndrome.

 

Cannabis smoking also has the potential to contribute to the risk of head and neck cancer. Cannabis smoke is qualitatively similar to tobacco smoke, although it contains up to twice the concentration of the carcinogenic polyaromatic hydrocarbons.

 

Cannabis is less densely packed than tobacco cigarettes and tends to be smoked without filters to a smaller butt size, leading to higher concentrations of smoke inhaled.

 

Several studies have shown precancerous histologic and genetic abnormalities in the respiratory tracts of cannabis smokers, and carcinogenic effects of cannabis smoke have been shown in vitro and in different in vivo animal models. An increased risk of lung cancer with cannabis smoking has been reported in most but not all case-control studies that have investigated the association.

 

Epidemiologic evidence for an association between cannabis and head and neck cancer is limited and conflicting. Case reports and case series have suggested a causative role for cannabis in cancers at different sites including lip, tongue, nasopharynx, pyriform fossa, tonsillar fossa, pharynx, and larynx. Some of the cases have been striking for their young age and lack of other risk factors, suggesting that cannabis may be an early initiator of head and neck cancers.

 

There have been 3 case-control studies of cannabis and cancers of the oral cavity and 2 case-control studies of head and neck cancer. In only one of these five case-control studies was there a statistically significant association reported between cannabis use and cancer; however, the interpretation of these studies has been limited by the choice of controls, inability to quantify use,low response rates, and low power with the possibility of type II error.

 

To investigate the association between cannabis use and head and neck cancer, we undertook a case-control study in young adults in New Zealand.

• 
  

Methods

Study Participants

Cases were patients with confirmed head and neck cancer aged 55 years and under at the time of diagnosis, diagnosed between January 2001 and July 2005, and identified from hospital databases and the New Zealand Cancer Registry. Cases were a mixture of prevalent and incident cases, in which the diagnosis was made within the previous five years.

 

Subjects were excluded if they had metastasis from a distant primary other than head and neck or a histologic diagnosis of carcinoid, melanoma, or adenocystic carcinomas. Age at diagnosis, anatomic location of their malignancy, and histologic type were collected for cases.

 

Controls (without respiratory tract cancer, head and neck cancer, or lung cancer) were randomly selected from the electoral roll and frequency matched in five-year age groups to the expected national incidence of head and neck cancer and district health boards to increase the study efficiency. The expected prevalence and regional variation of head and neck cancer were not available for this purpose.

 

Subjects came from eight district health board regions, serving both urban and rural populations. The study was approved by the regional ethics committees, and each participant gave written informed consent.

 

Data Collection

Questionnaires were administered face to face by trained interviewers, usually at the home of the participant. Information on demographics (including ethnic group), smoking history, passive smoking exposure, recreational drug use, diet, occupation, income, education, alcohol consumption, and family history of malignancy was collected.

 

A family history of upper respiratory tract cancer was defined as having a sibling or parent reported to have head and neck cancer. Cannabis smokers were asked about the amount, frequency, age of onset and duration of use, and the characteristics of their smoking. Subjects were asked to express their cannabis use in terms of frequency of joint use. If they smoked cannabis in another way (eg, pipes or bongs), they were asked to estimate the number of joints that quantity of cannabis would equate to.

 

This conversion allowed cannabis use for all participants to be quantified in terms of the total number of joints smoked. The lifetime amount of cannabis use was expressed as joint years of use, with one joint year being equivalent to one joint per day for one year. Pack years of cigarette smoking were calculated, with one pack year equivalent to 20 cigarettes per day for one year. The questionnaire was piloted on reformed cannabis smokers before use. Patients with lung cancer were also interviewed, with the findings published separately.

 

Statistical Analysis

Standard methods for analysis of case-control studies were used. The mean delay from diagnosis to interview was subtracted from the date of interview to calculate a reference date for duration of exposure for each control. Relative risks were estimated by calculating odds ratios by logistic regression using SPSS version 11.0 for Mac OSX (SPSS Inc, Chicago, IL) and adjusted for confounding variables. Tests for trend in relative risks for ordered categories were also performed.

 

Adjustment for age, joint years of cannabis smoking, and pack years of cigarette smoking was made by using them as continuous variables in the regression models. Level of income and a semiquantitative measure of total alcohol consumption were each categorized into four groups and also included in the regression models.

 

The effects of pack years of cigarette smoking (quintiles of smoking for all subjects included) and joint years of cannabis smoking (tertiles of use for all subjects included) were assessed. The relative risks were also calculated based on cannabis use up to five years before diagnosis on the basis that exposure after that time was unlikely to have caused the malignancy. The age at which cannabis smoking started was categorized, and the relative risk associated with starting cannabis smoking under 16 years of age was compared with starting over 21 years of age.

• 
  

Results

A total of 106 cases were contacted and invited for interview, of whom 81 (76%) agreed to participate. After exclusion of six in whom the diagnosis of cancer had been made more than five years before interview, there were 75 cases of head and neck cancer (Table 1). There were 493 controls contacted and invited for interview, of whom 324 (66%) agreed to participate. Five control subjects declined to provide their income and were excluded, leaving 319 control subjects for the analysis. None of these five controls reported using cannabis. The characteristics of the cases and controls are shown in Table 2.

Table 1 Anatomic site of malignancy by ICD code

Table 2 The frequency distribution of cases and controls for selected variables

The risk of head and neck cancer did not vary with age because of the controls being frequency matched on the age of cases in five-year age groups to improve the efficiency of the study. A family history of upper respiratory tract cancer was not significantly associated with an increased risk of head and neck cancer (relative ratio [RR] = 0.6; 95% confidence interval [CI], 0.2-1.9) (Table 3). Males had a significantly increased risk of cancers of the head and neck compared with females (RR = 3.4, 95% CI, 1.7-6.7).

 

In the age group studied, the increased relative risk of head and neck cancer for Maori and Pacific Island people compared with all other ethnicities was not statistically significant when adjusted for age, sex, pack years of tobacco smoking, joint years of marijuana use, alcohol consumption, and income level (RR = 1.9; 95% CI, 0.8-4.4) (data not shown).

 

When ethnic groups were further divided into New Zealand European, Chinese or Indian, Maori, and other ethnic groups, the relative risks for Maori and Chinese or Indian ethnic groups compared with New Zealand Europeans were 2.2 (95% CI, 1.0-5.2) and 3.9 (95% CI, 0.6-24.8), respectively (Table 3).

Table 3 Tobacco use, cannabis use, and alcohol consumption and risk of head and neck cancer

A low level of income was strongly associated with an increased risk of cancer of the head and neck, after adjustment for potential confounders, with the risk for those earning more than $70,000 per annum about one fifth of those earning less than or equal to $25,000 per annum (RR = 0.2; 95% CI, 0.1-0.4).

 

The relative risk from ever smoking cigarettes was statistically significant (RR = 2.1; 95% CI, 1.1-4.1) (Table 3). The relative risk of head and neck cancer increased with successive increases in cigarette smoking, with the highest quintile of pack years of cigarette smoking having a relative risk of 4.9 (95% CI, 1.9-12.4) after adjusting for confounding variables.

 

This increased risk was 4% for each pack year of exposure (95% CI, 2%-6%) after adjustment for confounding variables (Table 4). A significant increase in the relative risk with increasing alcohol consumption was observed (test for trend, P < 0.01), and the relative risk of head and neck cancer from heavy alcohol consumption compared with nondrinkers was 5.7 (95% CI, 1.2-25.9).

Table 4 Cannabis use and tobacco use as continuous variables and relative risk of head and neck cancer

 

The median duration of cannabis use was 10.5 years among controls (range, 0.25 to 29 years) and 25 years among cases (range, two to 32 years). Ever use of cannabis was not associated with a significantly increased risk of head and neck cancer (Table 3).

 

The risk associated with the highest tertile of cannabis use (>8.3 joint-years of exposure) was not statistically significant, RR=1.6 (95% CI, 0.5 to 5.2) after adjustment for confounding variables including tobacco smoking, alcohol consumption, and level of income.

 

When cannabis use was fitted as a continuous variable, the estimated 4% increase in the risk of head and neck cancer for each joint year of exposure (RR = 1.04; 95% CI, 0.97-1.11) was not statistically significant, after adjustment for confounding variables (Table 4).

 

When cannabis use in the five years before diagnosis or reference date was excluded, the risk from each joint year of exposure increased but again was not statistically significant (RR = 1.08; 95% CI, 0.77-1.53). Adjustment for whether cannabis use was solely from joints or combined with other methods of use did not appreciably alter these results.

 

The age at which subjects started smoking cannabis was not associated with the risk of head and neck cancer (data not shown). Adjustment for occupational risk of respiratory tract cancer or the consumption of different food groups did not appreciably alter the estimate obtained for the relative risk of head and neck cancer from cannabis use.

 

Discussion

This population-based study did not find a statistically significant increase in the risk of head and neck cancer in young adults from cannabis use. However, the median duration of cannabis use in cases and controls may have been too short for longer-term risks to be observed.

 

The major limitation of this study was the small sample size, with 75 cases of head and neck cancer and 319 controls being included in the analysis. For example, it was not possible to assess the RR associated with the different types of cancer that make up the group of head and neck cancers. Although tobacco smoking dominates the etiology of head and neck cancers as a group, other diverse factors, including viral infection, alcohol, diet, and occupation, contribute to specific cancers within the group, and there may be a differential risk associated with long-term cannabis use for the different cancers within this group.

 

The strong association of low income with increased risk suggests that unadjusted residual confounding may have influenced the associations observed. Based on the frequency of cannabis use of 12% in our population, it is estimated that about 150 subjects with cancer at a specific site would be required to provide 80% statistical power to identify a two-fold increase in risk (alpha = 0.05) using a 2:1 ratio of controls to cases.

 

Cases were identified from both the National Cancer Registry and from hospital outpatient and discharge databases to ensure case ascertainment was as complete as possible. We used a population-based control group randomly selected from the electoral roll rather than hospital-based controls because the latter is susceptible to significant bias because of the many medical conditions associated with cannabis use.

 

In New Zealand, about 93% of adults are listed on the electoral roll, and this approach was used to avoid the potential sources of bias inherent in the selection of the control groups in previous case-control studies of head and neck cancer.

 

The study of Zhang et al selected a control group from healthy blood donors who were less likely to have a history of substance abuse, resulting in a prevalence of cannabis use considerably less that that reported from a comparative United States population. It has been suggested that these characteristics of the control group may have contributed to the increased risk of head and neck cancer with cannabis smoking reported in that study.

 

The two British case-control studies of oral cancer and cannabis use  recruited controls through the case’s general practitioner, although the method of selection was not stated and on occasions other local general practitioners had to be used. The most recent case-control study from the United States recruited controls by canvassing the neighborhood of each enrolled case.

 

This approach may have made detection of an association less likely as cannabis use is likely to be similar within distinct neighborhoods. This may explain the higher rate of cannabis use among the control group than would have been expected from the United States population data, limiting the potential to find an association.

 

In our study, no mention was made of the primary risk factor of interest to avoid recruitment bias with either the cases or the controls. To minimize response bias, the interviewer did not state the specific research hypothesis and took a detailed history of all well known risk factors. Recall of the amount of cannabis smoked over a long period of time may have been difficult for some subjects; however, this is likely to have been similar for both cases and controls.

 

The exposure assessed was joint years of cannabis use, which combines both the intensity (amount and frequency) and duration of use. This approach follows the current convention for quantifying lifelong cannabis consumption and recognizes the evidence that the risk of lung cancer with cigarette smoking is related to both intensity and duration of use.

 

The administration of the questionnaire by trained interviewers in this way overcame difficulties of incomplete data from self-completed questionnaires, which has occurred previously, and allowed quantification of cannabis use, which may have varied considerably during the smoker’s lifetime.

 

The study was limited to subjects 55 years and under because of case series that have suggested that the role of cannabis smoking in head and neck cancer may be particularly strong in this age group. Furthermore, time trends indicate a progressively increased use of cannabis in New Zealand since the 1950s, resulting in this age group being predominantly exposed to cannabis compared with older adults.

 

This study was undertaken in parallel with a case-control study of the role of cannabis and lung cancer, and it is informative to contrast and compare the findings relating to cancers of the upper and lower respiratory tract. The relative risks of cannabis use for head and neck cancer were lower than those found for lung cancer and did not reach statistical significance.

 

For example, when the tertiles of cannabis use in the analysis of lung cancer were used, the relative risk for lung cancer in those with the highest tertile of cannabis use compared with nonusers was 5.7 (95% CI, 1.5-21.6) but only 1.6 (95% CI, 0.5-7.0) for head and neck cancer.

 

When joint years of use were fitted as a continuous variable, the risk for head and neck cancer was not significant and was lower than the significant risk for lung cancer (4% vs 8% per joint year, respectively). When the relationship was assessed for cannabis use up to 5 years before diagnosis, we found the magnitude of the risk was increased for both head and neck cancers and lung cancer to 8% and 10%, respectively, but with the former estimate not being statistically significant. This observation suggests a biological plausibility in that it is the exposure several years before the diagnosis of the malignancy that is relevant to its causation.

 

The risk of head and neck cancer did not increase with early onset of cannabis use or whether joints alone or mixed methods of cannabis use were included.

 

Conclusion

 

No significantly increased risk of head and neck cancer from cannabis use was found in young adults; however, because of the limited power and duration of use studied, a small or longer-term effect cannot be excluded. Likewise, because head and neck cancer is a heterogeneous group, the negative overall finding does not preclude an effect of cannabis on specific histological subtypes. Cancers of the head and neck are the fourth most common cause of cancer worldwide, with approximately 500,000 new cases diagnosed each year.² Larger studies are required to assess the risk of cancers of the head and neck associated with cannabis use for the different cancer sites of the head and neck.

 

Author Contributions

Sarah Aldington, study design, data collection, analysis, writing; Matire Harwood, data collection; Brian Cox, study design, analysis, writing; Mark Weatherall, study design, writing; Lutz Beckert, data collection; Anna Hansell, analysis, writing; Alison Pritchard, database development; Geoffrey Robinson, study design, writing; Richard Beasley, study design, writing.

 

Financial Disclosure

Supported by The New Zealand Ministry of Health, The Hawke’s Bay Medical Research Foundation, and GlaxoSmithKline (UK). Associate Professor Brian Cox was funded by the Director’s Cancer Research Trust. Dr Anna Hansell is a Wellcome Trust Intermediate Clinical Fellow supported by Grant Number 075883.

 

Acknowledgments

The Cannabis and Respiratory Disease Research Group: Executive Steering Committee: S. Aldington, M. Harwood, B. Cox, M. Weatherall, A. Pritchard, G. Robinson, L. Beckert, A. Hansell, and R. Beasley. Regional Coordinators: M. Tweed, B. Mahon (Wellington), L. Beckert, M. Campbell (Christchurch), M.J. Sneyd (Dunedin), R. Armstrong, C. Crawford (Hastings), A. Watson, A. Lobhan (Palmerston North), N. Graham, S. Holt (Tauranga), J. McLachlan, P. Swann (Waikato), S.T. Tan (Hutt), G. Lear, and N. Sheikh (Gisborne).

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J Neurooncol. 2005 Aug;

McAllister SD, Chan C, Taft RJ, Luu T, Abood ME, Moore DH, Aldape K, Yount G.

California Pacific Medical Center Research Institute, 475 Brannan St., Suite 220, San Francisco, CA 94107, USA. mcallis@sutterhealth.org

1. Stefan Hart,
2. Oliver M. Fischer
3. Axel Ullrich

4. Abstract

   Cannabinoids, the active components of marijuana and their endogenous counterparts were reported as useful analgetic agents to accompany primary cancer treatment by preventing nausea, vomiting, and pain and by stimulating appetite.

5. Moreover, they have been shown to inhibit cell growth and to induce apoptosis in tumor cells. Here, we demonstrate that anandamide, Δ⁹-tetrahydrocannabinol (THC), HU-210, and Win55,212-2 promote mitogenic kinase signaling in cancer cells.

6. Treatment of the glioblastoma cell line U373-MG and the lung carcinoma cell line NCI-H292 with nanomolar concentrations of THC led to accelerated cell proliferation that was completely dependent on metalloprotease and epidermal growth factor receptor (EGFR) activity. EGFR signal transactivation was identified as the mechanistic link between cannabinoid receptors and the activation of the mitogen-activated protein kinases extracellular signal-regulated kinase 1/2 as well as prosurvival protein kinase B (Akt/PKB) signaling. Depending on the cellular context, signal cross-communication was mediated by shedding of proAmphiregulin (proAR) and/or proHeparin-binding epidermal growth factor-like growth factor (proHB-EGF) by tumor necrosis factor α converting enzyme (TACE/ADAM17). Taken together, our data show that concentrations of THC comparable with those detected in the serum of patients after THC administration accelerate proliferation of cancer cells instead of apoptosis and thereby contribute to cancer progression in patients.

7. Introduction

   Cannabinoids have been used in medicine for more than a century. Recently interest in their therapeutic value has been fuelled by suggestions to apply these drugs in cancer treatment to improve analgesia and to relieve insomnia. Because of their neuroprotective properties, cannabinoids have also been proposed to be useful drugs for the therapy of neurodegenerative diseases like Parkinson‘s disease, Huntington disease, and multiple sclerosis. Orally applicable Δ⁹-tetrahydrocannabinol (THC; Dronabinol, Marinol) and its synthetic derivative Nabilone (Cesamet) have been approved by the United States Food and Drug Administration to stimulate the appetite of patients with AIDS and to reduce the nausea of cancer patients undergoing chemotherapy.

   Moreover, recent investigations propose that drugs activating the endogenous cannabinoid system might be used in cancer therapy to slow down or block cancer growth. The endogenous cannabinoid anandamide (AEA) acts antiproliferatively in MCF-7, EFM-19, T47D, and DU145 cells. Interestingly, cannabinoid-induced inhibition of proliferation in breast cancer cells results from cycle arrest at the G₁-S phase transition and is independent of apoptosis. Furthermore, depending on drug concentration, the timing of drug delivery, and cellular context, cannabinoids may either inhibit or stimulate the function of immune cells. Although high concentrations of cannabinoids block immune cells, Derocq et al.demonstrated proliferation in human B cells after cannabinoid stimulation at nanomolar concentrations. In addition, murine hematopoietic cells depend on AEA for normal growth in serum-free medium.

   THC, the endogenous cannabinoid AEA and synthetic cannabinoids like HU-210 and Win55,212-2 interact with specific G protein-coupled receptors (GPCRs). Two subtypes of the cannabinoid receptors, CB1 and CB2, have been cloned and characterized. The CB1 receptor, which is responsible for the well-known psychotropic effects of cannabinoids, is highly expressed in the central nervous system, but lower levels are also present in immune cells and peripheral tissues including testis, whereas the CB₂ receptor is predominantly expressed in immune cells . Both cannabinoid receptors are coupled to heterotrimeric G_i/o-proteins and activate the mitogen-activated protein kinases (MAPK) extracellular signal-regulated kinase (ERK)1/2 and p38 as well as the Akt/PKB survival pathway. Extensive research efforts have addressed the question how cannabinoids induce MAPK activation. Thus far, the accumulation of ceramides after cannabinoid stimulation has been implicated in the induction of the ERK/MAPK signal, whereas other reports suggested intracellular ceramide levels not to be required for cannabinoid-induced MAPK activation. Previously we and others have shown that a wide variety of GPCR agonists leads to the activation of MAPK via transactivation of the epidermal growth factor receptor (EGFR) . This mechanistic concept involves the proteolytic processing of a membrane-spanning proEGF-like growth factor by a zinc-dependent metalloprotease of the ADAM family .

   The aim of this study was to identify critical elements that link the cannabinoid receptors to activation of the ERK/MAPK and the Akt/PKB pathway. Hence, we tested whether cannabinoid receptors transactivate the EGFR in cancer cell lines, thereby activating downstream mitogenic signaling events.

   Our results demonstrate that treatment of NCI-H292 (lung cancer), SCC-9 (squamous cell carcinoma), 5637 (bladder carcinoma), U373-MG (glioblastoma), 1321N1 (astrocytoma), and A498 (kidney cancer) cells with cannabinoids such as THC, AEA, HU-210, and Win55,212-2 leads to rapid EGFR tyrosine phosphorylation, phosphorylation of the adaptor protein Src homology 2 domain-containing (SHC), and downstream activation of ERK1/2 and Akt/PKB. EGFR transactivation is specifically mediated by cannabinoid-induced cleavage of proAmphiregulin (proAR) and/or proHeparin-binding epidermal growth factor-like growth factor (proHB-EGF) at the cell surface by tumor necrosis factor α-converting enzyme (TACE/ADAM17). Importantly, THC induced EGFR- and metalloprotease-dependent cancer cell proliferation. Thus, this cross-communication of CB1/CB2 receptors and the EGFR provides a molecular explanation of how cannabinoid receptors are linked to MAPK and Akt/PKB activation in a wide variety of human cancer cell lines.

   In the light of these results, the use of cannabinoids in cancer therapy has to be reconsidered, because relatively high concentrations of THC induce apoptosis in cancer cells, whereas nanomolar concentrations enhance tumor cell proliferation and may, therefore, accelerate cancer progression in patients.

   Materials and Methods

   Cell Culture.

   All of the cell lines (American Type Culture Collection, Manassas, VA) were routinely grown according to the supplier’s instructions. Heparin (Sigma, St. Louis, MO), Crm197 (Quadratech Ltd., Epsom Surrey, United Kingdom), batimastat (BB94, British Biotech, Oxford, United Kingdom), TNF-α protease inhibitor (TAPI; Calbiochem), and AG1478 (Alexis Biochemicals) were added to serum-starved cells 20 min before the respective growth factor. Arachidonylethanolamide [also called anandamide (AEA)] and THC were obtained from Sigma, and WIN 55,212-2 mesylate and HU-210 from TOCRIS (Bristol, United Kingdom).

   Protein Analysis.

   Cells were lysed and proteins immunoprecipitated as described previously. After SDS-PAGE, proteins were transferred to nitrocellulose membrane. Western blots were performed according to standard methods. The antibodies against human EGFR (108.1), SHC, and HER2/neu have been characterized before. Phosphotyrosine was detected with the 4G10 monoclonal antibody (UBI, Lake Placid, NY). Polyclonal anti-phospho-p44/p42 (Thr202/Tyr204) MAPK antibody and anti-phospho-Akt (Ser473) antibody were purchased from New England Biolabs (Beverly, MA). Polyclonal anti-Akt1/2 and anti-ERK2 antibody was from Santa Cruz Biotechnology (Santa Cruz, CA), and anti-TACE antibody was from Chemicon (Harrow, United Kingdom).

8. Apoptosis Assay.

   NCI-H292 lung cancer cells were seeded and grown for 20 h. On serum starvation for 24 h, cells were treated with THC as indicated in Fig. 3C  for 6 h. Cells were collected in assay buffer (1% sodium citrate, 0.1% Triton X-100) containing propidium iodine and were incubated at 4°C for 3 h. Nuclear DNA staining was analyzed on a Becton Dickinson FACScalibur flow cytometer.

   For statistical analysis, Student’s t test was used to compare data between two groups. Values are expressed as mean ± SD of at least triplicate samples. P < 0.05 was considered statistically significant.

   RNA Interference and Reverse Transcription-PCR Analysis.

   Transfection of 21-nucleotide siRNA duplexes (Dharmacon Research, Lafayette, CO) for targeting endogenous genes was carried out using OligofectAMINE (Invitrogen) and 4.2 μg small interfering RNA (siRNA) duplex per 6-well plate as described previously. Transfected SCC-9 cells and NCI-H292 cells were serum starved and assayed 4 days and 3 days after transfection, respectively. Highest efficiencies in silencing target genes were obtained by using mixtures of siRNA duplexes targeting different regions of the gene of interest. The sequences of siRNA used were described previously. Specific silencing of targeted genes was confirmed by Western blot (TACE) and reverse transcription-PCR analysis. RNA, isolated using RNeasy Mini kit (Qiagen, Hilden, Germany), was reverse transcribed using AMV Reverse Transcriptase (Roche, Mannheim, Germany). PuReTaq Ready-To-Go PCR Beads (Amersham Biosciences, Piscataway, NJ) were used for PCR amplification. Primers (Sigma Ark, Steinheim, Germany) were described previously. PCR products were subjected to electrophoresis on a 2.5% agarose gel, and DNA was visualized by ethidium bromide staining.

   [³H]Thymidine Incorporation Assay.

   For the [³H]thymidine incorporation assay, U373-MG cells were seeded into 12-well plates at 1,5 × 10⁴ cells/well. On serum deprivation for 48 h, cells were subjected to preincubation and stimulation as indicated in Fig. 3A ⇓ . After 18 h, cells were pulse labeled with [³H]thymidine (1 mCi/ml) for 4 h, and thymidine incorporation was measured by trichloroacetic acid precipitation and subsequent liquid scintillation counting.

   MTT Assay.

   In a 96-well flat-bottomed plate (Nunc, Naperville, IL), ∼2000 cells/100 μl of cell suspension were seeded. On serum starvation for 24 h, cells were incubated with inhibitors and growth factors as indicated for another 24 h. MTT, a tetrazolium dye [3-(4,5-dimethylthiazol-2-y1)-2,5-diphenyltetrazolium bromide; thiazolyl blue, SIGMA, St. Louis, MO] was added to each well to a final concentration of 1 mg/ml MTT. Plates were incubated in the presence of MTT for 4 h. Mitochondrial dehydrogenase activity reduces the yellow MTT dye to a purple formazan, which is solubilized (DMSO, acidic acid, SDS), and absorbance was read at 570 nm on a microplate reader.

   Results

   Cannabinoid-Induced EGFR Signal Transactivation in Human Carcinoma Cells Depends on Metalloprotease Activity.

   To address the question whether cannabinoids lead to transactivation of the EGFR in human cancer cell lines, we treated NCI-H292 (lung cancer), SCC-9 (squamous cell carcinoma), 5637 (bladder carcinoma), U373-MG (glioblastoma), 1321N1 (astrocytoma), and A498 (kidney cancer) cells with the synthetic cannabinoids Win55,212-2 and HU210, the endogenous cannabinoid AEA, and the naturally occurring THC. Resulting EGFR tyrosine phosphorylation levels were monitored by immunoblot analysis. As shown in Fig. 1, A–E ⇓ , cannabinoids rapidly induced EGFR activation within 3 min.

   

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   Fig. 1.

   EGFR signal transactivation requires EGFR tyrosine kinase activity and a metalloprotease activity. A, serum-starved NCI-H292 cells, SCC9 cells, and 5637 cells were preincubated with EGFR-specific tyrphostin AG1478 (250 nm, 20 min) or vehicle (DMSO) and were treated with Win55,212-2 (Win; 10 μm), Δ9-tetrahydrocannabinol (THC; 1 μm), HU210 (50 nm), anandamide [AEA (AN); 10 μm], and epidermal growth factor (EGF) (5 ng/ml) for 3 min.

9. After lysis, EGFR was immunoprecipitated (IP) using anti-EGFR antibody. Tyrosine-phosphorylated EGFR was detected by (IB) with anti-phosphotyrosine (PY) antibody, followed by reprobing of the same filter with anti-EGFR antibody. B, serum-starved NCI-H292 cells, SCC9 cells, and 5637 cells were preincubated with metalloprotease inhibitor batimastat (BB94; 5 μm, 20 min) or vehicle (DMSO), stimulated for 3 min, and analyzed as described in A. C, D, and E, Serum-starved U373-MG, 1321N1, and A498 cells were preincubated with inhibitors and were stimulated for 3 min as indicated, and were analyzed as described in A. F, serum-starved NCI-H292 cells were preincubated with BB94 (5 μm, 20 min) and were stimulated with arachidonyl-2′-chloroethylamide (ACEA) (200 nm) or BML-190 (5 μm) for 3 min, and were analyzed as described in A. G, serum-starved NCI-H292 cells were preincubated with BB94 (5 μm, 20 min), tyrphostin AG1478 (250 nm, 20 min), or vehicle (DMSO) and stimulated with anandamide (An; 10 μm) or THC (1 μm) for 3 min. After lysis, HER2 was immunoprecipitated and assayed for HER2 tyrosine phosphorylation content.

   Preincubation of the cells with the metalloprotease inhibitor batimastat (BB94) or the EGFR kinase-specific inhibitor AG1478 prevented EGFR tyrosine phosphorylation in response to cannabinoid stimulation (Fig. 1, A–E) ⇓ . Stimulation of NCI-H292 cells with receptor subtype-specific agonists arachidonyl-2′-chloroethylamide (ACEA) and BML-190 for CB1 and CB2 receptor, respectively, demonstrated that both cannabinoid receptors are capable of transactivating the EGFR (Fig. 1F) ⇓ . Expression of both the CB1 and the CB2 receptor was detected by cDNA microarray and Northern blot analysis in all six cancer cell lines (data not shown). Interestingly, the EGFR relative HER2/neu, which serves as a prognostic marker in many different cancer types, was likewise activated in response to cannabinoid stimulation (Fig. 1G) ⇓ . Both AEA- and THC-induced tyrosine phosphorylation of HER2/neu in NCI-H292 cells depended on metalloprotease and EGFR activity.

10. Therefore, phosphorylation of Her2/neu appears to result from EGFR transphosphorylation. Taken together, these experiments demonstrate that cannabinoids rapidly induce EGFR and Her2/neu signal transactivation in a metalloprotease-dependent manner in different human cancer cell lines.

    Cannabinoid-Induced Activation of ERK and the Akt/PKB Survival Pathway Depends on EGFR Function.

    To assess whether the EGFR links cannabinoids to ERK1/2 activation, we first analyzed the tyrosine phosphorylation content of the adaptor protein SHC. Fig. 2A ⇓ demonstrates that treating cells with Win55,212-2, THC, HU210, and AEA resulted in EGFR-dependent tyrosine phosphorylation of SHC. We further analyzed ERK1/2 activation by immunoblot using activation state-specific MAPK antibodies. As shown in Fig. 2, B–D ⇓ , treating NCI-H292, SCC9, and U373-MG cells with cannabinoids potently activated ERK1/2 to a similar extent as EGF (5 ng/ml) stimulation. Preincubation with AG1478 or BB94 completely abrogated cannabinoid-induced MAPK activation, indicating that, in these human carcinoma cells, cannabinoid-induced ERK1/2 activation completely depends on EGFR signal transactivation (Fig. 2, B–D) ⇓ .

    

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    Fig. 2.

    Cannabinoid-induced SHC tyrosine phosphorylation and activation of extracellular signal-regulated kinase 1/2 (ERK1/2) and Akt/PKB pathways is EGFR- and metalloprotease-dependent. A, serum-starved SCC9 cells were treated as described in Fig. 1 ⇓ A, precipitated SHC was immunoblotted with αPY antibody followed by reprobing of the same filters with anti-SHC antibody. B and C, serum-starved SCC9 cells and NCI-H292 cells were preincubated with AG1478 (250 nm, 20 min) or BB94 (5 μm, 20 min), respectively, and were stimulated for 7 min as indicated. Phosphorylated ERK1/2 was detected by immunoblotting with phospho-specific ERK1/2 (P-ERK) antibody. The same filters were reprobed with anti-ERK antibody. D, serum-starved U373-MG cells were preincubated with inhibitors and stimulated for 7 min as indicated and were analyzed as described in B. E and F, serum-starved SCC9 cells and NCI-H292 cells were preincubated with AG1478 (250 nm, 20 min) or BB94 (5 μm, 20 min), respectively, and stimulated for 7 min as indicated. Activated Akt/PKB was detected by immunoblotting with phospho-specific Akt/PKB (P-Akt) antibody. The same filters were reprobed with anti-Akt/PKB antibody. Win, Win55,212-2; THC, Δ⁹-tetrahydrocannabinol; AEA, anandamide; EGF, epidermal growth factor; IB, immunoblotting; PY, anti-phosphotyrosine; P-ERK, phospho-specific ERK1/2; kDa, M_r in thousands; P-Akt, phospho-specific Akt/PKB.

    In addition to ERK stimulation, a variety of GPCR agonists were shown to activate the survival mediator Akt/PKB . Because cannabinoids were known to activate Akt/PKB in astrocytoma cells, we addressed the question as to whether cannabinoids activate Akt/PKB in human cancer cells depending on EGFR function. Indeed, activation of Akt/PKB by all four cannabinoids was completely blocked by the selective EGFR inhibitor AG1478 and by the metalloprotease inhibitor BB94 (Fig. 2, E and F) ⇓ .

    Taken together, tyrosine phosphorylation of SHC and activation of the Akt/PKB and the ERK/MAPK pathway after cannabinoid treatment of different human cancer cell lines critically depend on metalloprotease-mediated EGFR signal transactivation.

    EGFR Mediates Cannabinoid-Induced Proliferation.

    Because ERK1/2 are generally known to mediate proliferation in a variety of cells, we addressed the question as to whether cannabinoids induce proliferation of cancer cells. To determine the proliferation rate in response to THC stimulation, we measured DNA synthesis and the turnover of MTT. As shown in Fig. 3A ⇓ , THC at concentrations as low as 100 nm was capable of increasing the DNA synthesis of U373-MG cells. To investigate the involvement of the EGFR signal transactivation pathway for cannabinoid-induced DNA synthesis, we preincubated the cells with AG1478 and the metalloprotease inhibitor BB94. The increased DNA synthesis induced by THC was blocked in the presence of either AG1478 or BB94 (Fig. 3A) ⇓ .

11.  Furthermore, in the lung cancer cell line NCI-H292, THC (at 300 nm) was as potent as EGF (at 5 ng/ml) in increasing proliferation (Fig. 3B) ⇓ . THC-induced cell proliferation of NCI-H292 cells was completely blocked by preincubation with AG1478 and the metalloprotease inhibitor TAPI. In contrast to these observations, several earlier studies reported the induction of apoptosis on treatment of cells with higher concentrations of THC. Therefore, we tested whether, and in particular at what concentrations, THC would be able to trigger cell death of NCI-H292 cells. As shown in Fig. 3C ⇓ , micromolar concentrations of THC were capable of inducing apoptosis, whereas submicromolar concentrations did not affect cell survival.

    

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    Fig. 3.

    Δ⁹-Tetrahydrocannabinol (THC)-induced proliferation of U373-MG and NCI-H292 cells depends on EGFR transactivation. A, U373-MG cells were treated with inhibitors as indicated and incubated in the presence or absence of ligands (THC, 100 nm/300 nm; EGF, 2 ng/ml) for 18 h. Cells were then pulse labeled with [³H]thymidine, and thymidine incorporation was measured by liquid scintillation counting.

12. Quantitative analysis is from four independent experiments. ∗, P < 0.01 for the difference between DMSO versus THC and EGF; ∗∗, P < 0.01 for the difference between agonists versus inhibitors + agonists. B, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Serum-starved NCI-H292 cells were treated with AG1478 (250 nm) and the metalloprotease inhibitor TAPI (5 μm) and were incubated for 24 h in the presence or absence of ligands (THC 300 nm, EGF 5 ng/ml). Bars, ± SD of absorbance at 570 nm of three independent experiments. ∗, P < 0.01 for the difference between DMSO versus THC and EGF; ∗∗, P < 0.01 for the difference between agonists versus inhibitors + agonists. C, serum-starved NCI-H292 cells were treated with different concentrations of THC as indicated for 6 h.

13. After collection of the cells in assay buffer, nuclei were stained with propidium iodide and were analyzed by flow cytometric analysis. Quantification of experiments was performed in quadruplicate; bars, ±SD. ∗, P < 0.001 for the difference between DMSO versus THC; FL2-H, fluorescence channel 2-height.

    These results demonstrate that pharmacologically active concentrations of THC are capable of promoting the cell proliferation of human carcinoma cells and identify EGFR signal transactivation as the underlying molecular mechanism.

    TACE/ADAM17 Mediates Cannabinoid-Induced EGFR Activation.

    Our observations clearly showed that cannabinoid receptors transactivate the EGFR in a wide variety of human cancer cell lines involving metalloprotease activity (Fig. 1, A–E) ⇓ . We have previously shown the critical involvement of the metalloprotease-disintegrin TACE in proAmphiregulin shedding after GPCR stimulation.Therefore, we raised the question as to whether TACE is required for cannabinoid-induced EGFR signal transactivation.

14. We blocked endogenous TACE expression using a siRNA approach. TACE mRNA and protein was effectively and specifically reduced by transfecting siRNAs into NCI-H292 cells (Fig. 4, A and B) ⇓ . Inhibition of TACE expression but not of the related ADAM12 completely suppressed cannabinoid-induced EGFR tyrosine phosphorylation in NCI-H292 and SCC9 cells (Fig. 4C) ⇓ . Likewise, the cannabinoid-induced phosphorylation and activation of ERK1/2 and Akt/PKB was abolished in the absence of TACE but was unaffected by the down-regulation of ADAM12 (Fig. 4D) ⇓ . As expected, suppression of neither protease had an affect on signaling events induced by direct EGF stimulation of the EGFR.

    

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    Fig. 4.

    Tumor necrosis factor α-converting enzyme (TACE) mediates cannabinoid-induced EGFR transactivation and extracellular signal-regulated kinase 1/2 (ERK1/2) and Akt/PKB activation. A and B, NCI-H292 cells were transfected with TACE or ADAM12 siRNA. Gene expression was analyzed by reverse transcription-PCR or immunoblotting (IB) with TACE antibody. C, NCI-H292 cells and SCC9 cells were transfected with siRNAs raised against TACE and ADAM12, were serum starved, and were stimulated for 3 min with Δ⁹-tetrahydrocannabinol (THC; 1 μm), anandamide (An; 10 μm), and EGF (5 ng/ml), and were assayed for EGFR tyrosine phosphorylation content. D, NCI-H292 cells and SCC9 cells were transfected with siRNAs and stimulated for 7 min with agonists as indicated. Phosphorylated ERK1/2 and activated Akt/PKB were detected by immunoblotting with phospho-specific ERK1/2 (P-ERK) and Akt/PKB (P-Akt) antibodies, respectively.

15. The same filters were reprobed with anti-ERK1/2 antibody and anti-Akt/PKB, respectively. P-ERK, phospho-specific ERK1/2; P-Akt, phospho-specific Akt/PKB. E, NCI-H292 cells were transfected with siRNAs raised against proAR, proHB-EGF, and proTGFα.

16. Gene expression was analyzed by reverse transcription-PCR (RT-PCR). F, SCC9 cells were transfected with siRNAs raised against proAR, proHB-EGF, and proTGFα; stimulated with THC (1 μm) and AEA (10 μm) for 3 min; and assayed for EGFR tyrosine phosphorylation content. G, serum-starved SCC9 cells were preincubated with heparin (100 ng/ml, 15 min), stimulated with THC (1 μm) and AEA (10 μm), and assayed for EGFR tyrosine phosphorylation. H, NCI-H292 cells were transfected with siRNAs as indicated, stimulated with THC (1 μm) and AEA (10 μm), and assayed for EGFR tyrosine phosphorylation. I, serum-starved NCI-H292 cells were preincubated with CRM197 (10 μg/ml, 20 min), stimulated with THC (1 μm) and AEA (10 μm) for 3 min, and assayed for EGFR tyrosine phosphorylation. IB, immunoblotting; PY, anti-phosphotyrosine; IP, immunoprecipitated.

    Ectodomain Shedding of proHB-EGF and proAR Mediates Cannabinoid-Induced EGFR Activation.

    Among the different EGF-like precursors, proAR, proHB-EGF, and proTGFα are predominantly expressed in NCI-H292 and SCC9 cells as indicated by cDNA microarray analysis (data not shown; Ref. To investigate which ligand is involved in the EGFR signal transactivation pathway after cannabinoid stimulation, we transiently transfected siRNAs, and efficient and specific silencing of the endogenous expression of proAR, proHB-EGF, and proTGFα was monitored by reverse transcription-PCR (Fig. 4E ⇓ ; Ref. 19 ). AEA- and THC-induced tyrosine phosphorylation of the EGFR in SCC9 cells required proAR as well as proHB-EGF expression (Fig. 4F) ⇓ .

17. Suppression of either ligand resulted in partial reduction of cannabinoid-induced EGFR phosphorylation, whereas proTGFα inhibition did not affect EGFR phosphorylation at all. Furthermore, preincubation with heparin, which abrogates both proAR and proHB-EGF function, also interfered with cannabinoid receptor-EGFR cross-talk (Fig. 4G) ⇓ . In contrast, in NCI-H292 cells, cannabinoid-induced transactivation of the EGFR did solely depend on proHB-EGF (Fig. 4H) ⇓ . The ability of the diphtheria toxin mutant Crm197, a specific inhibitor of proHB-EGF function, to block EGFR phosphorylation in response to THC stimulation substantiated this observation (Fig. 4I) ⇓ .

    Together, these results show that cannabinoid-induced EGFR signal transactivation is mediated by specific proteolytic processing of the two heparin-binding EGFR ligands, proAR and proHB-EGF, by one and the same zinc-dependent metalloprotease TACE.

    
    

    Discussion

    Cannabinoids were shown to induce apoptosis in cells of the neuronal system including neurons, astrocytes, human grade IV astrocytoma, glioma C6, astrocytoma U373-MG, neuroblastoma N18 TG2, and pheochromocytoma PC12 cells and to inhibit proliferation of MCF-7, EFM-19, T47D, and DU145 cells. On the basis of these findings and their analgesic properties, cannabinoids were suggested as useful drugs to support cancer therapy. Here we show that various cannabinoids potently induce mitogenic kinase signaling in different cancer cell lines.

18. Moreover, we demonstrate, in contrast to other studies that used cannabinoids such as THC at micromolar concentrations, that nanomolar concentrations of THC induce proliferation of cancer cells (Fig. 3, A and B) ⇓ . Importantly, the concentration of THC that was used here is more likely to reflect the therapeutically relevant situation detected in serum after drug treatment.

    The binding of cannabinoids to their cognate receptors has been shown to enhance the activity of the MAPKs ERK1/2. The activation of the MAPK pathway in glioma cells on cannabinoid treatment was suggested to involve the activation of Raf1 by increased ceramide levels. However, our results identified signal transactivation of the EGFR as the key mechanism linking cannabinoid receptors to MAPK signaling cascades in a wide variety of human cancer cell lines. Activation of ERK1/2 by four different cannabinoids coincides with the phosphorylation of the EGFR and was blocked by a specific inhibitor of the EGFR, AG1478 (Fig. 2, B–D) ⇓ .

19. Although the ability of AG1478 to block cannabinoid-induced MAPK activation was noted before in U373-MG cells by Galve-Roperh et al, they excluded the existence of cannabinoid receptor-mediated EGFR transactivation because of the inability to detect tyrosine-phosphorylated EGFR, which was probably caused by a lack of sensitivity of detection.

    Interestingly, in addition to ERK1/2 activation, Akt/PKB phosphorylation was detected after cannabinoid treatment in an EGFR-dependent manner (Fig. 2E) ⇓ . Such a parallel contiguous activation of ERK1/2 and of Akt/PKB was observed before, e.g., in glioblastoma cells treated with cannabinoid receptor agonists, and was suggested to protect astrocytes from ceramide-induced apoptosis in a dose- and time-dependent manner .

    Our experimental finding that cannabinoid-induced EGFR cross-talk is established in a variety of human cancer cell lines (Fig. 1, A–E) ⇓ implicates the EGFR as a central integrator of cannabinoid signaling.

20. The cross-communication between GPCRs and the EGFR involves the proteolytic processing of different membrane-spanning proEGF-like growth factor ligands like proAR, proHB-EGF, and proTGFα by zinc-dependent metalloproteases like ADAM10, ADAM12, and TACE, depending on the cellular contex . In human cancer cell lines, we demonstrate that TACE mediates transactivation of the EGFR after cannabinoid stimulation via proteolytic processing of proHB-EGF and/or proAmphiregulin (Fig. 4, F–I) ⇓ . Abrogation of either TACE or the respective proEGF-like growth-factor function completely blocked cannabinoid-induced EGFR tyrosine phosphorylation and subsequent activation of the mitogenic ERK pathway and the prosurvival Akt/PKB pathway (Fig. 4) ⇓ . We previously described the involvement of TACE in EGFR signal transactivation after lysophosphatidic acid (LPA) and carbachol stimulation.

21. Moreover, TACE was found to mediate EGFR activation by cigarette smoke via proAR shedding in NCI-H292 cells. However, this is the first report demonstrating a TACE- and HB-EGF-dependent EGFR signal transactivation after GPCR stimulation. Interestingly, knockout experiments by Jackson et al. show that newborn mice lacking TACE, HB-EGF, and the EGFR have similar defective valvulogenesis and suggest EGFR activation by TACE-processed proHB-EGF . Moreover, our data substantiate the concept that, depending on the cell type and the stimulated GPCR, different ADAM proteases and proEGF-like growth factor ligands are capable of activating the EGFR.

    Cannabis-based drugs are in phase three clinical trials for treating pain associated with cancer. Furthermore, THC is currently used to treat nausea in cancer patients undergoing extensive chemotherapy. In contrast, Grand and Gandhi recently presented a case study of acute pancreatitis induced by cannabis smoking, indicating that cannabinoids may be a risk factor for pancreatic cancer.

22. Smoking of THC is the most effective route of delivery, as THC is rapidly absorbed after inhalation, and the effects become fully apparent within minutes. Pharmacological activity of smoked THC depends on the depth and length of inhalation. Maximum serum concentrations up to 267 ng/ml (850 nm) are measured after smoking THC, whereas maximum serum concentrations of oral or rectal administered THC or its derivatives as a drug are lower (35–350 nm;.

23. Here we observed a proliferative response of glioblastoma and lung cancer cells at concentrations of 100–300 nm THC, whereas THC at micromolar concentrations induced cell death in agreement with previous observations with neuronal cell types and immune cells (Fig. 3, A–C ⇓ These findings indicate that the biological responses to cannabinoids critically depend on drug concentration and cellular context. Taken together, these results have to be taken into account when considering therapeutic applications of cannabinoids. The risk in the medical use of THC or cannabis for the treatment of patients with established tumors is the further acceleration of tumor growth due to the proliferative potential of cannabinoids.

     

     

     

    Acknowledgments

    We thank U. Eichelsbacher, R. Gautsch, and R. Hornberger for their help with cell culture; T. Knyazeva for cDNA; P. Knyazeva for help with cDNA arrays and Northern blot analysis; and N. Prenzel and M. Buschbeck for critically reading the manuscript.

    Footnotes

    • Grant support: O. Fischer has been supported by a Boehringer Ingelheim Fonds Ph.D. scholarship.

    • The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

    • Requests for reprints: Axel Ullrich, Department of Molecular Biology, Max-Planck-Institute of Biochemistry, Am Klopferspitz 18A, D-82152 Martinsried, Germany. E-mail: ullrich@biochem.mpg.de.

    • Received November 28, 2003.
    • Revision received January 27, 2004.
    • Accepted February 4, 2004.
24. ©2004 American Association for Cancer Research.

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