Jacob Bell
New Member
The FASEB Journal express article 10.1096/fj.02-0795fje. Published online January 2, 2003.
Inhibition of tumor angiogenesis by cannabinoids
Cristina Blázquez,* M. Llanos Casanova," Anna Planas,"¡ Teresa Gómez del Pulgar,* Concepción
Villanueva,§ MarÃa 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 (8). However, the mechanism of cannabinoid antitumoral action in vivo is as yet
unknown. Here we show that local administration of a nonpsychoactive cannabinoid to mice
inhibits angiogenesis of malignant gliomas as determined by immunohistochemical analyses and
vascular permeability assays. In vitro and in vivo experiments show that at least two mechanisms
may be involved in this cannabinoid action: the direct inhibition of vascular endothelial cell
migration and survival as well as the decrease of the expression of proangiogenic factors
(vascular endothelial growth factor and angiopoietin-2) and matrix metalloproteinase-2 in the
tumors. Inhibition of tumor angiogenesis may allow new strategies for the design of
cannabinoid-based antitumoral therapies.
Key words: cancer - vascular endothelium - angiogenic factor - apoptosis - experimental
therapeutics
C annabinoids, the active components of Cannabis sativa L. (marijuana) and their
derivatives, exert a wide array of effects on the central nervous system as well as on
peripheral sites such as the immune, cardiovascular, digestive, reproductive, and ocular
systems (1, 2). It is now widely accepted that most of these effects are mediated by the
activation of specific G protein-coupled receptors that are normally bound by a family of
endogenous ligands--the endocannabinoids (3—5). Marijuana and its derivatives have been used
in medicine for many centuries, and currently there is a renaissance in the study of the
therapeutic effects of cannabinoids, which constitutes a widely debated issue with ample
scientific and social relevance. Ongoing research is determining whether cannabinoid ligands
may be effective agents in the treatment of pain and inflammation, glaucoma, neurodegenerative
disorders such as multiple sclerosis and Parkinson's disease, and the wasting and emesis
associated with acquired immunodeficiency syndrome and cancer chemotherapy (1, 2, 6). In
addition, cannabinoids might be antitumoral agents owing to their ability to induce the regression
of various types of tumors in animal models (7—10). Although cannabinoids directly induce
apoptosis or cell cycle arrest in different transformed cells in vitro (11, 12), the involvement of
this and other mechanisms in their antitumoral action in vivo is as yet unknown.
To grow beyond minimal size, tumors must generate a new vascular supply for purposes of gas
exchange, cell nutrition, and waste disposal (13). They do so by secreting proangiogenic
cytokines that promote the formation of blood vessels. Vascular endothelial growth factor
(VEGF) 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
for endothelial cells, and ability to regulate most and perhaps all the steps in the angiogenic
cascade (13—15). Other cellular mediators such as the angiostatic factors angiopoietin-1 (Ang1)
and angiopoietin-2 (Ang2) also control the process of angiogenesis (13—15). Growing tumors
also produce matrix metalloproteinases (MMPs) that allow tissue breakdown and remodeling
during angiogenesis and invasiveness (16). Because overexpression of proangiogenic factors and
MMPs is causally involved in the progression of the majority of solid tumors, considerable effort
is being made in developing effective antiangiogenic drugs (17) and MMP inhibitors (18) to treat
cancer. This background prompted us to explore whether inhibition of angiogenesis is implicated
in the antitumoral effect of cannabinoids. Here, we report that cannabinoid administration
inhibits tumor angiogenesis in vivo and that at least two mechanisms may be involved in this
action: direct inhibition of vascular endothelial cell migration and survival as well as suppression
of proangiogenic factor and MMP expression in the tumors.
MATERIALS AND METHODS
Cannabinoids
JWH-133 was prepared in Dr. J.W. Huffman's laboratory (19). HU-210 was kindly given by Dr.
R. Mechoulam (Hebrew University, Jerusalem, Israel). WIN-55,212—2 and Δ9-
tetrahydrocannabinol were from Sigma (St. Louis, MO). SR141716 and SR144528 were kindly
given by Sanofi-Synthelabo (Montpellier, France). Stock solutions of cannabinoid ligands were
prepared in dimethyl sulfoxide (DMSO). For in vitro incubations, cannabinoids were directly
applied at a final DMSO concentration of 0.1—0.2% (v/v). For in vivo experiments, cannabinoids
were prepared at 1% (v/v) DMSO in 100 μl phosphate-buffered saline (PBS) supplemented with
5 mg/ml bovine serum albumin. No significant influence of the vehicle was observed on any of
the parameters determined.
Endothelial cell culture
Human umbilical vein endothelial cells (HUVECs) were isolated from umbilical veins (20) and
grown on gelatin-coated dishes in medium 199 supplemented with 20% fetal calf serum (FCS),
0.05% endothelial cell growth factor, and 100 μg/ml heparin. Cells were used between passages
4 and 7. Twenty-four hours before the experiments, cells were transferred to medium 199 plus
2% FCS. The ECV304 cell line was grown in Dulbecco's modified Eagle's medium (DMEM)
supplemented with 10% FCS. Twenty-four hours before the experiments, cells were transferred
to serum-free DMEM plus 0.5% FCS.
Endothelial cell migration
Cell migration was routinely monitored using the Boyden chamber. After exposure to
cannabinoids for 18 h, a time at which cannabinoid-induced apoptosis does not yet occur (data
not shown), cells were trypsinized, washed, and loaded into the upper well. Lysophosphatidic
acid (10 μM) was placed in the lower well as a cell migration stimulator. Cells were allowed to
migrate for 3 h at 37°C through a Falcon 5-μm filter. Then, cells from the upper side of the
membrane were removed, and the remaining cells on the bottom side of the membrane were
fixed with 50% ethanol, stained with eosin blue, and counted. In other experiments, cell
migration was assessed by counting the number of cells that had covered a scratch directly made
on the culture dish.
Endothelial cell death
Cell viability was determined by Trypan blue exclusion. Apoptosis was determined by TUNEL
staining (8) and oligonucleosomal DNA fragmentation (21) after treatment with cannabinoids
and other effectors for 48 h.
Tumor induction
Gliomas were induced in mice deficient in recombination activating gene 2 by s.c. flank
inoculation of tumor cells (8). When tumors had reached a volume of 200—300 mm3, animals
were assigned randomly to two groups and injected intratumorally with vehicle or 50 μg d—1 of
JWH-133 for 8 days (rat C6 glioma cells) or 25 days (human grade IV astrocytoma cells
[glioblastoma multiforme] obtained from tumor biopsies as previously described [9]).
Histopathology and immunohistochemistry
Tumors were fixed in 10% buffered formalin and embedded in paraffin. Sections were stained
with hematoxylin/eosin for histopathological evaluation. For immunohistochemistry, tumor
sections were treated as previously described (22) and stained with antibodies against CD31
(Pharmigen, San Diego, CA) and SMA (Sigma, St. Louis, MO).
Vascular permeability assay
Vascular permeability was assessed using a modified Miles assay. Tumor-bearing animals were
anesthesized and Evans blue (1% in PBS, 100 μl/mouse) was injected into the tail vein. Dye
leakage was subsequently detected as blue spots on the tumor surface.
VEGF, Ang1, and Ang2 expression
Total RNA was extracted from the tumor samples by the acid-guanidinium method (22). The
anti-VEGF probe has been described previously (22). Probes for Ang1 and Ang2 detection were
kindly provided by Dr. G. D. Yancopoulos (Regeneron Pharmaceuticals, Tarrytown, NY).
Ribosomal 7S RNA was used as a loading control.
MMP-2 and TIMP-2 expression
Tumor extracts were incubated with gelatin-Sepharose 4B, and MMPs were eluted with buffer
containing 10% DMSO. Samples were subjected to zymography in gels containing 1 mg/ml
porcine gelatin, and gels were subsequently incubated and stained with amido black as
previously described (23). Western blotting was performed with antibodies against MMP-2 and
TIMP-2 (both from Chemicon, Temecula, CA) and subsequent luminography.
CB1 and CB2 receptor expression
Western blot analysis of cannabinoid receptors was performed in cell membrane fractions as
previously described (8). The anti-CB1 receptor antibody was kindly given by Dr. K. Mackie
(University of Washington, Seattle, WA). The anti-CB2 receptor antibody was from Cayman
Chemical (Ann Arbor, MI).
ERK activity
ERK activity was determined in cell extracts as the incorporation of [γ-32P]ATP into a specific
peptide substrate as previously described (8).
Statistics
Results shown represent means±SD. Differences among groups were evaluated by the unpaired
Student's t test or ANOVA, with a post hoc analysis by the Student-Neuman-Keuls test.
RESULTS
Two different cannabinoid receptors have been characterized and cloned from mammalian
tissues: the "central" CB1 receptor, which is responsible for cannabinoid psychoactivity (24), and
the "peripheral" CB2 receptor, which is unrelated to cannabinoid psychoactivity (25). Because
cannabinoid-based therapeutic strategies should be as devoid as possible of psychotropic side
effects, we administered to tumor-bearing mice the selective CB2 agonist JWH-133 (19). We
have recently provided pharmacological, biochemical, and behavioral evidence that JWH-133
activates selectively the CB2 receptor in the present experimental system (9).
Cannabinoid administration inhibits tumor angiogenesis
As reported before (9), JWH-133 administration inhibited the growth of C6-cell (Fig. 1A) and
human gliomas (Fig. 1B) in mice. In addition, whereas control tumors had a reddish color,
cannabinoid-treated tumors had a pale appearance (Fig. 1A and 1B), suggesting that a deficient
blood supply might be a cause for such growth inhibition. Analysis of the vasculature by
immunostaining of CD31, a marker of endothelial cells, revealed no significant effect of JWH-
133 administration on microvascular count (number of blood vessels per unit area) in the tumors
(data not shown). However, we observed a notable effect of cannabinoid administration on blood
vessel morphology. Thus, in control animals, C6-cell gliomas displayed microvascular
hyperplasia, in which proliferating blood vessels are lined by disorderly heaped up endothelial
cells that ultimately transform into glomeruloid tufts (Fig. 1C). Similarly, control human
astrocytomas showed a network of dilated immature vessels (Fig. 1E). In contrast, all JWH-133-
treated tumors showed a pattern of blood vessels characterized predominantly by very small and
narrow capillaries (Fig. 1D and 1F).
We subsequently analyzed the expression of smooth muscle α-actin (SMA), a marker of smooth
muscle cells and pericytes. In control tumors, SMA-positive cells were detached from the
endothelial cells that constitute the walls of the blood vessels, pointing to the disruption of the
normally stable link of endothelial and perivascular cells (Fig. 1G). This process is known to
occur during active tumor growth (26—28). In contrast, in JWH-133-treated tumors, SMApositive
cells had a mature appearance and remained closely around the endothelial wall (Fig.
1H). This high and well-defined coverage of smooth muscle cells and pericytes is indicative of a
differentiated vascular phenotype (26—28).
We next examined whether these changes in tumor vascularization in vivo lead to actual
differences in vascular functionality as assessed by a vascular permeability assay. Dye
accesibility to the tumors was much lower in cannabinoid-treated animals than in controls (Fig.
2A and 2B). Analysis of the in situ-tumor pictures showed that the dye extravasation area in the
tumor relative to total tumor area was 84±11% in control animals and 15±7% in JWH-133-
treated animals for C6-cell gliomas (n=4, P<0.01) and 88±19% in control animals and 21±11%
in JWH-133-treated animals for human astrocytomas (n=4, P<0.01). These observations,
together with the CD31/SMA staining data, indicate that the vascular network of actively
growing control tumors is large, plastic, and leaky, whereas that of slowly growing cannabinoidtreated
tumors is small, differentiated, and impermeable.
Cannabinoid administration inhibits vascular endothelial cell migration
We examined the direct impact of cannabinoids on vascular endothelial cell migration and
survival. Primary HUVECs expressed the two subtypes of cannabinoid receptors (Fig. 3A). The
mixed CB1/CB2 cannabinoid agonist WIN-55,212—2 (25 nM) inhibited HUVEC migration as
assessed by the Boyden chamber method and by scratch coverage, and this effect was prevented
by the CB1 selective antagonist SR141716 (0.5 μM) and the CB2 selective antagonist SR144528
(0.5 μM) (Fig. 3B and C), pointing to the involvement of cannabinoid receptors. Moreover,
selective activation of the CB2 receptor with JWH-133 (25 nM) also blocked cell migration (Fig.
3B). Similar inhibitory effects of WIN-55,212—2 and JWH-133 were observed when the cell
migration stimulator (lysophosphatidic acid) was added to both wells of the Boyden chamber
(data not shown), pointing to a real depression of cell locomotion (chemokinesis) rather than of
chemotaxis.
We also tested the possible involvement of the extracellular signal-regulated kinase (ERK)
cascade in cannabinoid action, because ERK seems to be involved in the control of cell function
by cannabinoids in other cell types (29). Thus, cannabinoid receptor activation increased ERK
activity in HUVECs (Fig. 3D), and pharmacological blockade of the ERK cascade with the
selective inhibitor PD98059 (25 μM) abrogated cannabinoid inhibition of cell migration (Fig.
3B).
Cannabinoid administration induces vascular endothelial cell apoptosis
We next studied whether cannabinoids affect vascular endothelial cell survival. WIN-55,212—2
(25 nM) induced HUVEC death, and this effect was prevented by SR141716 (0.5 μM) and by
SR144528 (0.5 μM) (Fig. 4A), pointing again to the involvement of cannabinoid receptors.
Cannabinoid-induced HUVEC death occurred by apoptosis, as determined by oligonucleosomal
DNA fragmentation (Fig. 4B) and TUNEL staining (Fig. 4C).
The HUVEC line ECV304 was used to further study the pharmacological profile of cannabinoidinduced
vascular endothelial cell death. Preliminary experiments had shown that, like HUVEC,
the ECV304 cell line expresses the two subtypes of cannabinoid receptors (Fig. 3A) and ensues
apoptosis upon activation of those receptors with WIN-55,212—2 (Fig. 4D and data not shown).
The apoptotic effect of WIN-55,212—2 on ECV304 cells was mimicked by HU-210 (25 nM),
another nonselective cannabinoid agonist, and by Δ9-tetrahydrocannabinol (1 μM), the main
active component of marijuana (Fig. 4D). Moreover, selective activation of the CB2 receptor
with JWH-133 (25 nM) also induced cell death. PD98059 (25 μM) (but not the protein kinase A
inhibitor H-7 [5 μM)], the protein kinase C inhibitor GF109203X [2 μM], or the p38 mitogenactivated
protein kinase inhibitor SB203580 [10 μM] [data not shown]) abrogated cannabinoidinduced
death of ECV304 cells (Fig. 4D).
Cannabinoid administration inhibits tumor expression of proangiogenic factors
Tumor cells produce proangiogenic factors that promote the migration and survival of vascular
cells (13—15). Therefore, we next tested whether cannabinoids inhibit angiogenesis not only by
targeting vascular endothelial cells directly, but also by interfering with proangiogenic factor
expression in the tumors. In both C6-cell and human gliomas, VEGF and Ang2 expression was
markedly reduced by JWH-133 treatment (Fig. 5A). Ang1 expression was not affected by
cannabinoid treatment (Fig. 5A), in line with previous reports showing that this cytokine is not
majorly involved in the pathogenesis of gliomas (26, 27).
Cannabinoid administration improves other markers of tumor malignancy
The occurrence of abnormal vascular proliferation, aggressive invasion into surrounding normal
tissue, and enhanced necrosis are general markers of progression of gliomas and other tumors
(26—28). In addition to its aforementioned action on angiogenesis, we found that cannabinoid
administration improves those other two parameters of tumor malignancy. First, we determined
MMP expression as a molecular marker for tissue breakdown and remodeling during malignant
tumor growth (16). In particular, we focused on MMP-2 because this protease plays a pivotal
role in glioma invasiveness (30—32) and angiogenesis (31—33). JWH-133 administration
decreased MMP-2 activity and expression in C6-cell and human gliomas, whereas tissue
inhibitor of metalloproteinase-2 (TIMP-2) expression was not affected (Fig. 5B). Second, we
observed that areas of necrosis with palisading nuclei, a factor that is considered predictive of a
poor prognosis (27, 28), appeared in control but not in JWH-133-treated tumors (Fig. 5C).
DISCUSSION
Cannabinoids induce tumor regression in vivo (7—10). However, the exact mechanism of
cannabinoid antitumoral action is still unknown. Here, we report that cannabinoids inhibit tumor
angiogenesis in vivo and that at least two mechanisms may be involved in this cannabinoid
action: direct inhibition of vascular endothelial cell migration and survival as well as suppression
of proangiogenic factor and MMP expression in the tumors. A parsimonious interpretation of
these and our previous findings (8, 9) is that cannabinoids inhibit tumor growth by activating
cannabinoid receptors in both vascular endothelial cells and tumor cells. By inhibiting vascular
endothelial cell migration and survival, cannabinoids would directly prevent blood vessel
formation. By targeting tumor cells, cannabinoids would induce their apoptosis (8, 9) and would
also suppress proangiogenic factor and MMP production, further blocking tumor growth and
angiogenesis.
The observation that cannabinoids act directly on vascular endothelial cells is in line with recent
reports showing that vascular endothelial cells express functional CB1 receptors (34) that may be
responsible for the hypotensive effects of cannabinoids in different pathophysiological situations
(35—37). Our data expand those findings by showing that activation of cannabinoid receptors
modulates essential endothelial cell functions such as migration and proliferation. Of further
therapeutic interest, here, we show that vascular endothelial cells--like glioma cells (9)--express
functional CB2 receptors. Hence, the present report--together with the possible implication of
CB2 or CB2-like receptors in the control of peripheral pain (38, 39) and multiple sclerosis-linked
spasticity (40), for example--opens the attractive possibility of finding cannabinoid-based
therapeutic strategies devoid of nondesired CB1-mediated psychotropic side effects.
Nevertheless, the existence in some blood vessels of non-CB1, non-CB2 endothelial receptors for
cannabinoids may not be ruled out (41).
Cannabinoid administration was associated with a notable decrease in the expression of the
major proangiogenic factors VEGF and Ang2, which are essential for the vascularization of
gliomas and many other types of tumors such as those from mammary gland, lung, and skin (13,
22, 26, 27). VEGF is likely the most important proangiogenic cytokine in health and disease, and
Ang2, at least in the presence of VEGF, may also allow robust tumor angiogenesis and growth
(13, 14, 26). Thus, Ang2 is not expressed by normal human brain but is strongly induced in
human gliomas (26, 27, 42). Coinciding with high VEGF and Ang2 expression, vascular profiles
are characterized by abnormal deposition of extracellular matrix and weak association with
perivascular cells (26, 27, 42). In addition, the failure of many solid tumors to generate a welldifferentiated
and stable vasculature may indeed be attributed to a continuous overexpression of
Ang2, which prevents vessel maturation and contributes to the generally plastic, tenuous, and
leaky state of tumor vessels (26, 27, 42). All this is precisely what we have observed in tumors
from control animals (i.e., high Ang2 expression associated with enhanced MMP-2 activity,
fractionated SMA staining, and increased vascular permeability), just the opposite occurring in
cannabinoid-treated tumors.
In conclusion, our data show that cannabinoids inhibit the growth of gliomas in vivo by targeting
both tumor cells and vascular endothelial cells. Gliomas are one of the most malignant forms of
cancer, resulting in the death of affected patients within months after diagnosis. Current
therapies for glioma treatment are usually ineffective or just palliative (43, 44). One of the
alternative therapeutic approaches might be based on the use of nonpsychoactive cannabinoid
ligands, because these compounds inhibit tumor growth and angiogenesis in vivo. In line with the
idea that antiangiogenic treatments constitute one of the most promising antitumoral approaches
currently available (13, 14), the present findings provide a novel pharmacological target for
cannabinoid-based therapies.
ACKNOWLEDGMENTS
We are indebted to I. De los Santos, S. Gozalo, P. Labordeta, S. Moreno, and J. Oliver for expert
technical assistance and to Dr. F. Larcher and Dr. M. Ortiz de Landázuri for critical comments
on the manuscript. This work was supported by grants from Ministerio de Ciencia y TecnologÃa
(PM 98—0079 to M.G.), Comunidad Autónoma de Madrid (08.1/0079/2000 to M.G.), Fundación
Ramón Areces (to M.G.), Fondo de Investigación Sanitaria (01/0099 to A.P.), and National
Institute on Drug Abuse (DA03590 to J.W.H.).
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Received September 25, 2002; accepted November 19, 2002.
Fig. 1
Figure 1. Cannabinoid administration inhibits tumor angiogenesis. A, B) General appearance of dissected tumors.
Animals bearing C6-cell gliomas (A) or human astrocytomas (B) were treated without (—) or with (+) JWH-133. Note the
small size and pale color of cannabinoid-treated tumors. C—F) CD31 staining showing the blood vessel pattern in the
tumors. Animals bearing C6-cell gliomas (C, D) or human astrocytomas (E, F) were treated without (C, E) or with (D, F)
JWH-133. Note the small size of the vessels in cannabinoid-treated tumors. G, H) Smooth muscle α-actin staining
showing the pattern of smooth muscle cells and pericytes in the tumor blood vessels. Animals bearing human
astrocytomas were treated without (G) or with (H) JWH-133. Note the high perivascular coverage in cannabinoid-treated
tumors. Representative tumors are shown in each panel. Similar data were obtained in 7—9 (A, B) or 3—4 (C—H) additional
animals.
Fig. 2
Figure 2. Cannabinoid administration decreases vascular permeability of the tumors. Animals bearing C6-cell
gliomas (A) or human astrocytomas (B) were treated without (top) or with (bottom) JWH-133. Examples of subcutaneous
tumors in situ before (—) and after (+) systemic Evans blue injection. Note the small size and restricted dye accesibility of
cannabinoid-treated tumors. Similar data were obtained in three additional animals.
Fig. 3
Figure 3. Cannabinoid administration inhibits vascular endothelial cell migration. A) Expression of CB1 and CB2
cannabinoid receptors in HUVECs and ECV304 cells as determined by Western blot. Similar data were obtained in two
additional cell extracts. B, C) HUVECs were cultured with WIN-55,212—2 (WIN), JWH-133 (JWH), SR141716 (SR1),
SR144528 (SR2), and/or PD98059 (PD), and cell migration was monitored by using the Boyden chamber (B, n=4—6) or
by making a scratch on the culture dish (C, representative pictures of one experiment out of four; note the decrease in
scratch coverage by WIN-55,212). (D) ERK activity in HUVECs cultured as in C (n=4). *Significantly different (P<0.01)
from control incubations.
Fig. 4
Figure 4. Cannabinoid administration induces vascular endothelial cell apoptosis. A—C) HUVECs were cultured
with WIN-55,212—2 (WIN), SR141716 (SR1), and/or SR144528 (SR2), and cell viability (A, n=6), oligonucleosomal
DNA fragmentation (B, n=4), and TUNEL staining (C, representative pictures of one experiment out of three) were
assessed. D) ECV304 cells were cultured with WIN, HU-210 (HU), Δ9-tetrahydrocannabinol (THC), JWH-133 (JWH),
SR1, SR2, and/or PD98059 (PD), and cell viability was determined (n=6). *Significantly different (P<0.01) from control
incubations.
Fig. 5
Figure 5. Cannabinoid administration inhibits expression of pro-angiogenic factors and improves other markers
of tumor malignancy. A, B) Animals bearing C6-cell gliomas (C6) or human astrocytomas (HA) were treated without (—)
or with (+) JWH-133. A) Expression of VEGF, Ang1, and Ang2 in the tumors as determined by Northern blot. 7S RNA
was used as loading control. B) MMP-2 activity (zymogram, top), MMP-2 expression (Western blot, middle), and TIMP-
2 expression (Western blot, bottom) in the tumors. C) Necrosis (N) with palisading nuclei (P) in C6-cell gliomas from
control (left) but not JWH-133-treated (right) animals. Representative tumors are shown in each panel. Similar data were
obtained in two to four additional animals.
Source: Inhibition of tumor angiogenesis by cannabinoids
Inhibition of tumor angiogenesis by cannabinoids
Cristina Blázquez,* M. Llanos Casanova," Anna Planas,"¡ Teresa Gómez del Pulgar,* Concepción
Villanueva,§ MarÃa 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 (8). However, the mechanism of cannabinoid antitumoral action in vivo is as yet
unknown. Here we show that local administration of a nonpsychoactive cannabinoid to mice
inhibits angiogenesis of malignant gliomas as determined by immunohistochemical analyses and
vascular permeability assays. In vitro and in vivo experiments show that at least two mechanisms
may be involved in this cannabinoid action: the direct inhibition of vascular endothelial cell
migration and survival as well as the decrease of the expression of proangiogenic factors
(vascular endothelial growth factor and angiopoietin-2) and matrix metalloproteinase-2 in the
tumors. Inhibition of tumor angiogenesis may allow new strategies for the design of
cannabinoid-based antitumoral therapies.
Key words: cancer - vascular endothelium - angiogenic factor - apoptosis - experimental
therapeutics
C annabinoids, the active components of Cannabis sativa L. (marijuana) and their
derivatives, exert a wide array of effects on the central nervous system as well as on
peripheral sites such as the immune, cardiovascular, digestive, reproductive, and ocular
systems (1, 2). It is now widely accepted that most of these effects are mediated by the
activation of specific G protein-coupled receptors that are normally bound by a family of
endogenous ligands--the endocannabinoids (3—5). Marijuana and its derivatives have been used
in medicine for many centuries, and currently there is a renaissance in the study of the
therapeutic effects of cannabinoids, which constitutes a widely debated issue with ample
scientific and social relevance. Ongoing research is determining whether cannabinoid ligands
may be effective agents in the treatment of pain and inflammation, glaucoma, neurodegenerative
disorders such as multiple sclerosis and Parkinson's disease, and the wasting and emesis
associated with acquired immunodeficiency syndrome and cancer chemotherapy (1, 2, 6). In
addition, cannabinoids might be antitumoral agents owing to their ability to induce the regression
of various types of tumors in animal models (7—10). Although cannabinoids directly induce
apoptosis or cell cycle arrest in different transformed cells in vitro (11, 12), the involvement of
this and other mechanisms in their antitumoral action in vivo is as yet unknown.
To grow beyond minimal size, tumors must generate a new vascular supply for purposes of gas
exchange, cell nutrition, and waste disposal (13). They do so by secreting proangiogenic
cytokines that promote the formation of blood vessels. Vascular endothelial growth factor
(VEGF) 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
for endothelial cells, and ability to regulate most and perhaps all the steps in the angiogenic
cascade (13—15). Other cellular mediators such as the angiostatic factors angiopoietin-1 (Ang1)
and angiopoietin-2 (Ang2) also control the process of angiogenesis (13—15). Growing tumors
also produce matrix metalloproteinases (MMPs) that allow tissue breakdown and remodeling
during angiogenesis and invasiveness (16). Because overexpression of proangiogenic factors and
MMPs is causally involved in the progression of the majority of solid tumors, considerable effort
is being made in developing effective antiangiogenic drugs (17) and MMP inhibitors (18) to treat
cancer. This background prompted us to explore whether inhibition of angiogenesis is implicated
in the antitumoral effect of cannabinoids. Here, we report that cannabinoid administration
inhibits tumor angiogenesis in vivo and that at least two mechanisms may be involved in this
action: direct inhibition of vascular endothelial cell migration and survival as well as suppression
of proangiogenic factor and MMP expression in the tumors.
MATERIALS AND METHODS
Cannabinoids
JWH-133 was prepared in Dr. J.W. Huffman's laboratory (19). HU-210 was kindly given by Dr.
R. Mechoulam (Hebrew University, Jerusalem, Israel). WIN-55,212—2 and Δ9-
tetrahydrocannabinol were from Sigma (St. Louis, MO). SR141716 and SR144528 were kindly
given by Sanofi-Synthelabo (Montpellier, France). Stock solutions of cannabinoid ligands were
prepared in dimethyl sulfoxide (DMSO). For in vitro incubations, cannabinoids were directly
applied at a final DMSO concentration of 0.1—0.2% (v/v). For in vivo experiments, cannabinoids
were prepared at 1% (v/v) DMSO in 100 μl phosphate-buffered saline (PBS) supplemented with
5 mg/ml bovine serum albumin. No significant influence of the vehicle was observed on any of
the parameters determined.
Endothelial cell culture
Human umbilical vein endothelial cells (HUVECs) were isolated from umbilical veins (20) and
grown on gelatin-coated dishes in medium 199 supplemented with 20% fetal calf serum (FCS),
0.05% endothelial cell growth factor, and 100 μg/ml heparin. Cells were used between passages
4 and 7. Twenty-four hours before the experiments, cells were transferred to medium 199 plus
2% FCS. The ECV304 cell line was grown in Dulbecco's modified Eagle's medium (DMEM)
supplemented with 10% FCS. Twenty-four hours before the experiments, cells were transferred
to serum-free DMEM plus 0.5% FCS.
Endothelial cell migration
Cell migration was routinely monitored using the Boyden chamber. After exposure to
cannabinoids for 18 h, a time at which cannabinoid-induced apoptosis does not yet occur (data
not shown), cells were trypsinized, washed, and loaded into the upper well. Lysophosphatidic
acid (10 μM) was placed in the lower well as a cell migration stimulator. Cells were allowed to
migrate for 3 h at 37°C through a Falcon 5-μm filter. Then, cells from the upper side of the
membrane were removed, and the remaining cells on the bottom side of the membrane were
fixed with 50% ethanol, stained with eosin blue, and counted. In other experiments, cell
migration was assessed by counting the number of cells that had covered a scratch directly made
on the culture dish.
Endothelial cell death
Cell viability was determined by Trypan blue exclusion. Apoptosis was determined by TUNEL
staining (8) and oligonucleosomal DNA fragmentation (21) after treatment with cannabinoids
and other effectors for 48 h.
Tumor induction
Gliomas were induced in mice deficient in recombination activating gene 2 by s.c. flank
inoculation of tumor cells (8). When tumors had reached a volume of 200—300 mm3, animals
were assigned randomly to two groups and injected intratumorally with vehicle or 50 μg d—1 of
JWH-133 for 8 days (rat C6 glioma cells) or 25 days (human grade IV astrocytoma cells
[glioblastoma multiforme] obtained from tumor biopsies as previously described [9]).
Histopathology and immunohistochemistry
Tumors were fixed in 10% buffered formalin and embedded in paraffin. Sections were stained
with hematoxylin/eosin for histopathological evaluation. For immunohistochemistry, tumor
sections were treated as previously described (22) and stained with antibodies against CD31
(Pharmigen, San Diego, CA) and SMA (Sigma, St. Louis, MO).
Vascular permeability assay
Vascular permeability was assessed using a modified Miles assay. Tumor-bearing animals were
anesthesized and Evans blue (1% in PBS, 100 μl/mouse) was injected into the tail vein. Dye
leakage was subsequently detected as blue spots on the tumor surface.
VEGF, Ang1, and Ang2 expression
Total RNA was extracted from the tumor samples by the acid-guanidinium method (22). The
anti-VEGF probe has been described previously (22). Probes for Ang1 and Ang2 detection were
kindly provided by Dr. G. D. Yancopoulos (Regeneron Pharmaceuticals, Tarrytown, NY).
Ribosomal 7S RNA was used as a loading control.
MMP-2 and TIMP-2 expression
Tumor extracts were incubated with gelatin-Sepharose 4B, and MMPs were eluted with buffer
containing 10% DMSO. Samples were subjected to zymography in gels containing 1 mg/ml
porcine gelatin, and gels were subsequently incubated and stained with amido black as
previously described (23). Western blotting was performed with antibodies against MMP-2 and
TIMP-2 (both from Chemicon, Temecula, CA) and subsequent luminography.
CB1 and CB2 receptor expression
Western blot analysis of cannabinoid receptors was performed in cell membrane fractions as
previously described (8). The anti-CB1 receptor antibody was kindly given by Dr. K. Mackie
(University of Washington, Seattle, WA). The anti-CB2 receptor antibody was from Cayman
Chemical (Ann Arbor, MI).
ERK activity
ERK activity was determined in cell extracts as the incorporation of [γ-32P]ATP into a specific
peptide substrate as previously described (8).
Statistics
Results shown represent means±SD. Differences among groups were evaluated by the unpaired
Student's t test or ANOVA, with a post hoc analysis by the Student-Neuman-Keuls test.
RESULTS
Two different cannabinoid receptors have been characterized and cloned from mammalian
tissues: the "central" CB1 receptor, which is responsible for cannabinoid psychoactivity (24), and
the "peripheral" CB2 receptor, which is unrelated to cannabinoid psychoactivity (25). Because
cannabinoid-based therapeutic strategies should be as devoid as possible of psychotropic side
effects, we administered to tumor-bearing mice the selective CB2 agonist JWH-133 (19). We
have recently provided pharmacological, biochemical, and behavioral evidence that JWH-133
activates selectively the CB2 receptor in the present experimental system (9).
Cannabinoid administration inhibits tumor angiogenesis
As reported before (9), JWH-133 administration inhibited the growth of C6-cell (Fig. 1A) and
human gliomas (Fig. 1B) in mice. In addition, whereas control tumors had a reddish color,
cannabinoid-treated tumors had a pale appearance (Fig. 1A and 1B), suggesting that a deficient
blood supply might be a cause for such growth inhibition. Analysis of the vasculature by
immunostaining of CD31, a marker of endothelial cells, revealed no significant effect of JWH-
133 administration on microvascular count (number of blood vessels per unit area) in the tumors
(data not shown). However, we observed a notable effect of cannabinoid administration on blood
vessel morphology. Thus, in control animals, C6-cell gliomas displayed microvascular
hyperplasia, in which proliferating blood vessels are lined by disorderly heaped up endothelial
cells that ultimately transform into glomeruloid tufts (Fig. 1C). Similarly, control human
astrocytomas showed a network of dilated immature vessels (Fig. 1E). In contrast, all JWH-133-
treated tumors showed a pattern of blood vessels characterized predominantly by very small and
narrow capillaries (Fig. 1D and 1F).
We subsequently analyzed the expression of smooth muscle α-actin (SMA), a marker of smooth
muscle cells and pericytes. In control tumors, SMA-positive cells were detached from the
endothelial cells that constitute the walls of the blood vessels, pointing to the disruption of the
normally stable link of endothelial and perivascular cells (Fig. 1G). This process is known to
occur during active tumor growth (26—28). In contrast, in JWH-133-treated tumors, SMApositive
cells had a mature appearance and remained closely around the endothelial wall (Fig.
1H). This high and well-defined coverage of smooth muscle cells and pericytes is indicative of a
differentiated vascular phenotype (26—28).
We next examined whether these changes in tumor vascularization in vivo lead to actual
differences in vascular functionality as assessed by a vascular permeability assay. Dye
accesibility to the tumors was much lower in cannabinoid-treated animals than in controls (Fig.
2A and 2B). Analysis of the in situ-tumor pictures showed that the dye extravasation area in the
tumor relative to total tumor area was 84±11% in control animals and 15±7% in JWH-133-
treated animals for C6-cell gliomas (n=4, P<0.01) and 88±19% in control animals and 21±11%
in JWH-133-treated animals for human astrocytomas (n=4, P<0.01). These observations,
together with the CD31/SMA staining data, indicate that the vascular network of actively
growing control tumors is large, plastic, and leaky, whereas that of slowly growing cannabinoidtreated
tumors is small, differentiated, and impermeable.
Cannabinoid administration inhibits vascular endothelial cell migration
We examined the direct impact of cannabinoids on vascular endothelial cell migration and
survival. Primary HUVECs expressed the two subtypes of cannabinoid receptors (Fig. 3A). The
mixed CB1/CB2 cannabinoid agonist WIN-55,212—2 (25 nM) inhibited HUVEC migration as
assessed by the Boyden chamber method and by scratch coverage, and this effect was prevented
by the CB1 selective antagonist SR141716 (0.5 μM) and the CB2 selective antagonist SR144528
(0.5 μM) (Fig. 3B and C), pointing to the involvement of cannabinoid receptors. Moreover,
selective activation of the CB2 receptor with JWH-133 (25 nM) also blocked cell migration (Fig.
3B). Similar inhibitory effects of WIN-55,212—2 and JWH-133 were observed when the cell
migration stimulator (lysophosphatidic acid) was added to both wells of the Boyden chamber
(data not shown), pointing to a real depression of cell locomotion (chemokinesis) rather than of
chemotaxis.
We also tested the possible involvement of the extracellular signal-regulated kinase (ERK)
cascade in cannabinoid action, because ERK seems to be involved in the control of cell function
by cannabinoids in other cell types (29). Thus, cannabinoid receptor activation increased ERK
activity in HUVECs (Fig. 3D), and pharmacological blockade of the ERK cascade with the
selective inhibitor PD98059 (25 μM) abrogated cannabinoid inhibition of cell migration (Fig.
3B).
Cannabinoid administration induces vascular endothelial cell apoptosis
We next studied whether cannabinoids affect vascular endothelial cell survival. WIN-55,212—2
(25 nM) induced HUVEC death, and this effect was prevented by SR141716 (0.5 μM) and by
SR144528 (0.5 μM) (Fig. 4A), pointing again to the involvement of cannabinoid receptors.
Cannabinoid-induced HUVEC death occurred by apoptosis, as determined by oligonucleosomal
DNA fragmentation (Fig. 4B) and TUNEL staining (Fig. 4C).
The HUVEC line ECV304 was used to further study the pharmacological profile of cannabinoidinduced
vascular endothelial cell death. Preliminary experiments had shown that, like HUVEC,
the ECV304 cell line expresses the two subtypes of cannabinoid receptors (Fig. 3A) and ensues
apoptosis upon activation of those receptors with WIN-55,212—2 (Fig. 4D and data not shown).
The apoptotic effect of WIN-55,212—2 on ECV304 cells was mimicked by HU-210 (25 nM),
another nonselective cannabinoid agonist, and by Δ9-tetrahydrocannabinol (1 μM), the main
active component of marijuana (Fig. 4D). Moreover, selective activation of the CB2 receptor
with JWH-133 (25 nM) also induced cell death. PD98059 (25 μM) (but not the protein kinase A
inhibitor H-7 [5 μM)], the protein kinase C inhibitor GF109203X [2 μM], or the p38 mitogenactivated
protein kinase inhibitor SB203580 [10 μM] [data not shown]) abrogated cannabinoidinduced
death of ECV304 cells (Fig. 4D).
Cannabinoid administration inhibits tumor expression of proangiogenic factors
Tumor cells produce proangiogenic factors that promote the migration and survival of vascular
cells (13—15). Therefore, we next tested whether cannabinoids inhibit angiogenesis not only by
targeting vascular endothelial cells directly, but also by interfering with proangiogenic factor
expression in the tumors. In both C6-cell and human gliomas, VEGF and Ang2 expression was
markedly reduced by JWH-133 treatment (Fig. 5A). Ang1 expression was not affected by
cannabinoid treatment (Fig. 5A), in line with previous reports showing that this cytokine is not
majorly involved in the pathogenesis of gliomas (26, 27).
Cannabinoid administration improves other markers of tumor malignancy
The occurrence of abnormal vascular proliferation, aggressive invasion into surrounding normal
tissue, and enhanced necrosis are general markers of progression of gliomas and other tumors
(26—28). In addition to its aforementioned action on angiogenesis, we found that cannabinoid
administration improves those other two parameters of tumor malignancy. First, we determined
MMP expression as a molecular marker for tissue breakdown and remodeling during malignant
tumor growth (16). In particular, we focused on MMP-2 because this protease plays a pivotal
role in glioma invasiveness (30—32) and angiogenesis (31—33). JWH-133 administration
decreased MMP-2 activity and expression in C6-cell and human gliomas, whereas tissue
inhibitor of metalloproteinase-2 (TIMP-2) expression was not affected (Fig. 5B). Second, we
observed that areas of necrosis with palisading nuclei, a factor that is considered predictive of a
poor prognosis (27, 28), appeared in control but not in JWH-133-treated tumors (Fig. 5C).
DISCUSSION
Cannabinoids induce tumor regression in vivo (7—10). However, the exact mechanism of
cannabinoid antitumoral action is still unknown. Here, we report that cannabinoids inhibit tumor
angiogenesis in vivo and that at least two mechanisms may be involved in this cannabinoid
action: direct inhibition of vascular endothelial cell migration and survival as well as suppression
of proangiogenic factor and MMP expression in the tumors. A parsimonious interpretation of
these and our previous findings (8, 9) is that cannabinoids inhibit tumor growth by activating
cannabinoid receptors in both vascular endothelial cells and tumor cells. By inhibiting vascular
endothelial cell migration and survival, cannabinoids would directly prevent blood vessel
formation. By targeting tumor cells, cannabinoids would induce their apoptosis (8, 9) and would
also suppress proangiogenic factor and MMP production, further blocking tumor growth and
angiogenesis.
The observation that cannabinoids act directly on vascular endothelial cells is in line with recent
reports showing that vascular endothelial cells express functional CB1 receptors (34) that may be
responsible for the hypotensive effects of cannabinoids in different pathophysiological situations
(35—37). Our data expand those findings by showing that activation of cannabinoid receptors
modulates essential endothelial cell functions such as migration and proliferation. Of further
therapeutic interest, here, we show that vascular endothelial cells--like glioma cells (9)--express
functional CB2 receptors. Hence, the present report--together with the possible implication of
CB2 or CB2-like receptors in the control of peripheral pain (38, 39) and multiple sclerosis-linked
spasticity (40), for example--opens the attractive possibility of finding cannabinoid-based
therapeutic strategies devoid of nondesired CB1-mediated psychotropic side effects.
Nevertheless, the existence in some blood vessels of non-CB1, non-CB2 endothelial receptors for
cannabinoids may not be ruled out (41).
Cannabinoid administration was associated with a notable decrease in the expression of the
major proangiogenic factors VEGF and Ang2, which are essential for the vascularization of
gliomas and many other types of tumors such as those from mammary gland, lung, and skin (13,
22, 26, 27). VEGF is likely the most important proangiogenic cytokine in health and disease, and
Ang2, at least in the presence of VEGF, may also allow robust tumor angiogenesis and growth
(13, 14, 26). Thus, Ang2 is not expressed by normal human brain but is strongly induced in
human gliomas (26, 27, 42). Coinciding with high VEGF and Ang2 expression, vascular profiles
are characterized by abnormal deposition of extracellular matrix and weak association with
perivascular cells (26, 27, 42). In addition, the failure of many solid tumors to generate a welldifferentiated
and stable vasculature may indeed be attributed to a continuous overexpression of
Ang2, which prevents vessel maturation and contributes to the generally plastic, tenuous, and
leaky state of tumor vessels (26, 27, 42). All this is precisely what we have observed in tumors
from control animals (i.e., high Ang2 expression associated with enhanced MMP-2 activity,
fractionated SMA staining, and increased vascular permeability), just the opposite occurring in
cannabinoid-treated tumors.
In conclusion, our data show that cannabinoids inhibit the growth of gliomas in vivo by targeting
both tumor cells and vascular endothelial cells. Gliomas are one of the most malignant forms of
cancer, resulting in the death of affected patients within months after diagnosis. Current
therapies for glioma treatment are usually ineffective or just palliative (43, 44). One of the
alternative therapeutic approaches might be based on the use of nonpsychoactive cannabinoid
ligands, because these compounds inhibit tumor growth and angiogenesis in vivo. In line with the
idea that antiangiogenic treatments constitute one of the most promising antitumoral approaches
currently available (13, 14), the present findings provide a novel pharmacological target for
cannabinoid-based therapies.
ACKNOWLEDGMENTS
We are indebted to I. De los Santos, S. Gozalo, P. Labordeta, S. Moreno, and J. Oliver for expert
technical assistance and to Dr. F. Larcher and Dr. M. Ortiz de Landázuri for critical comments
on the manuscript. This work was supported by grants from Ministerio de Ciencia y TecnologÃa
(PM 98—0079 to M.G.), Comunidad Autónoma de Madrid (08.1/0079/2000 to M.G.), Fundación
Ramón Areces (to M.G.), Fondo de Investigación Sanitaria (01/0099 to A.P.), and National
Institute on Drug Abuse (DA03590 to J.W.H.).
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Received September 25, 2002; accepted November 19, 2002.
Fig. 1
Figure 1. Cannabinoid administration inhibits tumor angiogenesis. A, B) General appearance of dissected tumors.
Animals bearing C6-cell gliomas (A) or human astrocytomas (B) were treated without (—) or with (+) JWH-133. Note the
small size and pale color of cannabinoid-treated tumors. C—F) CD31 staining showing the blood vessel pattern in the
tumors. Animals bearing C6-cell gliomas (C, D) or human astrocytomas (E, F) were treated without (C, E) or with (D, F)
JWH-133. Note the small size of the vessels in cannabinoid-treated tumors. G, H) Smooth muscle α-actin staining
showing the pattern of smooth muscle cells and pericytes in the tumor blood vessels. Animals bearing human
astrocytomas were treated without (G) or with (H) JWH-133. Note the high perivascular coverage in cannabinoid-treated
tumors. Representative tumors are shown in each panel. Similar data were obtained in 7—9 (A, B) or 3—4 (C—H) additional
animals.
Fig. 2
Figure 2. Cannabinoid administration decreases vascular permeability of the tumors. Animals bearing C6-cell
gliomas (A) or human astrocytomas (B) were treated without (top) or with (bottom) JWH-133. Examples of subcutaneous
tumors in situ before (—) and after (+) systemic Evans blue injection. Note the small size and restricted dye accesibility of
cannabinoid-treated tumors. Similar data were obtained in three additional animals.
Fig. 3
Figure 3. Cannabinoid administration inhibits vascular endothelial cell migration. A) Expression of CB1 and CB2
cannabinoid receptors in HUVECs and ECV304 cells as determined by Western blot. Similar data were obtained in two
additional cell extracts. B, C) HUVECs were cultured with WIN-55,212—2 (WIN), JWH-133 (JWH), SR141716 (SR1),
SR144528 (SR2), and/or PD98059 (PD), and cell migration was monitored by using the Boyden chamber (B, n=4—6) or
by making a scratch on the culture dish (C, representative pictures of one experiment out of four; note the decrease in
scratch coverage by WIN-55,212). (D) ERK activity in HUVECs cultured as in C (n=4). *Significantly different (P<0.01)
from control incubations.
Fig. 4
Figure 4. Cannabinoid administration induces vascular endothelial cell apoptosis. A—C) HUVECs were cultured
with WIN-55,212—2 (WIN), SR141716 (SR1), and/or SR144528 (SR2), and cell viability (A, n=6), oligonucleosomal
DNA fragmentation (B, n=4), and TUNEL staining (C, representative pictures of one experiment out of three) were
assessed. D) ECV304 cells were cultured with WIN, HU-210 (HU), Δ9-tetrahydrocannabinol (THC), JWH-133 (JWH),
SR1, SR2, and/or PD98059 (PD), and cell viability was determined (n=6). *Significantly different (P<0.01) from control
incubations.
Fig. 5
Figure 5. Cannabinoid administration inhibits expression of pro-angiogenic factors and improves other markers
of tumor malignancy. A, B) Animals bearing C6-cell gliomas (C6) or human astrocytomas (HA) were treated without (—)
or with (+) JWH-133. A) Expression of VEGF, Ang1, and Ang2 in the tumors as determined by Northern blot. 7S RNA
was used as loading control. B) MMP-2 activity (zymogram, top), MMP-2 expression (Western blot, middle), and TIMP-
2 expression (Western blot, bottom) in the tumors. C) Necrosis (N) with palisading nuclei (P) in C6-cell gliomas from
control (left) but not JWH-133-treated (right) animals. Representative tumors are shown in each panel. Similar data were
obtained in two to four additional animals.
Source: Inhibition of tumor angiogenesis by cannabinoids
