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| | Cannabis
Marijuana Reduces Skin Cancer
Inhibition of skin tumor growth and angiogenesis
in vivo by activation of
cannabinoid receptors
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Back To Main Medical Reports Page
M. Llanos Casanova1, Cristina Blázquez2,
Jesús Martínez-Palacio1, Concepción Villanueva3,
M. Jesús Fernández-Aceńero3, John W. Huffman4,
José L. Jorcano1 and Manuel Guzmán2
1 Project on Cellular and Molecular Biology and
Gene Therapy, Centro de Investigaciones Energéticas, Medioambientales y Tecnológicas,
Madrid, Spain 2 Department of Biochemistry and Molecular Biology I,
School of Biology, Complutense University, Madrid, Spain 3 Department
of Pathology, Hospital General de Móstoles, Madrid, Spain 4
Department of Chemistry, Clemson University, Clemson, South Carolina, USA
Address correspondence to: 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.
Received for publication June 7, 2002, and accepted in
revised form November 19, 2002.
Nonmelanoma skin cancer is one of the most common malignancies in
humans. Different therapeutic strategies for the treatment of these
tumors are currently being investigated. Given the growth-inhibiting
effects of cannabinoids on gliomas and the wide tissue distribution
of the two subtypes of cannabinoid receptors (CB1 and CB2),
we studied the potential utility of these compounds in anti–skin
tumor therapy. Here we show that the CB1 and the CB2
receptor are expressed in normal skin and skin tumors of mice and
humans. In cell culture experiments pharmacological activation of
cannabinoid receptors induced the apoptotic death of tumorigenic
epidermal cells, whereas the viability of nontransformed epidermal
cells remained unaffected. Local administration of the mixed CB1/CB2
agonist WIN-55,212-2 or the selective CB2 agonist JWH-133
induced a considerable growth inhibition of malignant tumors
generated by inoculation of epidermal tumor cells into nude mice.
Cannabinoid-treated tumors showed an increased number of apoptotic
cells. This was accompanied by impairment of tumor vascularization,
as determined by altered blood vessel morphology and decreased
expression of proangiogenic factors (VEGF, placental growth factor,
and angiopoietin 2). Abrogation of EGF-R function was also observed
in cannabinoid-treated tumors. These results support a new therapeutic
approach for the treatment of skin tumors.
The epidermis is a stratified squamous epithelium composed mainly of
keratinocytes, whose proliferation and differentiation must be
tightly regulated and coordinated. Basal keratinocytes, which are
attached to the basement membrane, are undifferentiated and have
proliferative potential. Before entering the differentiation program,
they withdraw from the cell cycle and migrate toward the surface of
the epidermis, leading to the formation of the outermost layer of the
epidermis composed of enucleated dead squames, which are continuously
shed from the surface of the skin
(1). The
incidence of both benign and malignant skin neoplasms has been rising
at an alarming rate for the past several years. Thus, nonmelanoma
skin cancer is one of the most common malignancies in humans: basal
cell carcinomas and squamous cell carcinomas represent the vast
majority of the malignant tumors diagnosed (2).
These tumors are believed to arise mainly from stem cells of hair
follicles (3), and their growth and development seems to
rely on an early burst of neovascularization (4) in which
VEGF (5-7) and EGF-R (8,
9) are essential components. The skin is also a
major site for metastasis of internal disease (2, 10).
Early recognition, biopsy confirmation, and treatment selection can
reduce patient morbidity. Different types of strategies are currently
being investigated as therapies for the treatment of these tumors,
including cryotherapy, topical chemotherapeutic agents such as
5-fluorouracil, and photodynamics, the success of which is hampered
by limitations such as the poor penetration of molecules into the
skin and the difficulty to gain access to the whole tumor (10-12).
Cannabinoids, the active components of Cannabis sativa linnaeus (marijuana)
and their derivatives, exert a wide array of effects on the CNS as
well as on peripheral sites such as the immune, cardiovascular,
digestive, reproductive, and ocular systems (13-15).
Nowadays, it is 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 (14, 16, 17).
Two different cannabinoid receptors have been characterized and
cloned from mammalian tissues: the "central" CB1
receptor, mostly expressed in brain and responsible for cannabinoid
psychoactivity (18), and the
"peripheral" CB2 receptor, mostly expressed in the immune
system and unrelated to cannabinoid psychoactivity (19).
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, for example, pain and inflammation, neurodegenerative
disorders such as multiple sclerosis and Parkinson’s disease, and
the wasting and emesis associated with AIDS and cancer chemotherapy (13-15).
In addition, cannabinoids may be potential antitumoral agents owing
to their ability to induce the regression of various types of tumors,
including lung adenocarcinoma (20), glioma (21,
22), and thyroid epithelioma (23) in
animal models. Although cannabinoids directly induce apoptosis or
cell cycle arrest in different transformed cells in vitro (24),
the involvement of this and other potential mechanisms (e.g.,
inhibition of tumor angiogenesis) in their antitumoral action in vivo
is as yet unknown.
This background prompted us to explore whether (a) the skin and
skin tumors express cannabinoid receptors; (b) cannabinoid receptor
activation exerts a growth-inhibiting action on skin tumors in vivo;
and (c) inhibition of angiogenesis is implicated in the anti-tumoral
effect of cannabinoids. Our data show that (a) CB1 and CB2
receptors are present in the skin and skin tumors; (b) local
cannabinoid receptor activation induces the regression of skin tumors
in vivo; and (c) at least two mechanisms may be involved in this
action: direct apoptosis of tumor cells and inhibition of tumor
angiogenesis.
Cannabinoids. JWH-133 was prepared in J.W. Huffman’s
laboratory (25). WIN-55,212-2 was from
Sigma-Aldrich (St. Louis, Missouri, USA). SR141716 and SR144528 were
kindly given by Sanofi-Synthelabo (Montpellier, France).
Cell culture. The mouse tumorigenic epidermal cell lines
PDV.C57 and HaCa4, and the nontransformed epidermal cell lines MCA3D
(mouse) and HaCat (human), were routinely maintained in DMEM
supplemented with 10% FCS. Twenty-four hours before the experiments,
cells were transferred to low serum (0.5%) DMEM. Primary human
keratinocytes (Biowhittaker Europe SPRL, Vervier, Belgium) were grown
and cultured for the experiments in KGM-2 medium (Biowhittaker Europe
SPRL). Stock solutions of cannabinoid ligands were prepared in
DMSO. Control incubations had the corresponding DMSO content. No
significant influence of DMSO was observed on cell viability at the
final concentration used (0.1–0.2%, vol/vol).
Human tumor samples. Formalin-fixed, paraffin-embedded,
human tumor samples were obtained from the files of the Department of
Pathology, Hospital General de Móstoles (Madrid, Spain).
Six-micrometer tumor sections were stained with either hematoxylin-eosin
or used for immunohistochemistry (see below). The normal skin sample
(Figures 1 and 3, skin) came from the
face of a 70-year-old man. The slowly growing tumor sample (Figure 3,
BCC) came from the face of a 69-year-old woman. The histological
analysis revealed the growth of basaloid cell solid nests at the
dermoepidermal junction with diagnostic characteristics of basal cell
carcinoma. The highly malignant tumor sample (Figures 1
and 3, SCC) came from the retroauricular skin of a
73-year-old man. The histological analysis showed the neoplastic
growth of epithelial cells, with squamous differentiation and focal
areas of acantholysis.
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Figure 1. Western blot
analysis of cannabinoid receptor expression in normal skin and
skin tumors. (a) CB1 and CB2
receptor expression in murine and human epidermal cell lines,
normal skin, and skin tumors. (b) Controls with the
anti-CB1 or anti-CB2 Ab-blocking peptide
are shown (see Methods and Results for explanation). Mouse
papillomas were generated by chemical carcinogenesis (as
described in ref. 9), while mouse squamous
cell carcinomas (SCCs) were generated by inoculation of PDV.C57
epidermal tumor cells as described in Methods. The source of
human samples is described in Methods. Images of representative
samples are shown. Similar results were obtained in at least two
other blots. PB, immortalized mouse nontumorigenic cell line
derived from SENCAR mice papillomas; HK, human keratinocytes; m,
mouse; h, human.
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Figure 3. Immunohistochemical
analysis of cannabinoid receptor expression in human normal skin
and skin tumors. (a) Immunolocalization of CB1
and CB2 receptors. (b) Controls without
primary Ab (only with secondary biotinylated anti-rabbit Ab), as
well as controls with the Ab-blocking peptides, are shown (see
Methods and Results for explanation). The source of samples is
described in Methods. BCC, basal cell carcinoma; SCC, squamous
cell carcinoma; Hf, hair follicle.
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Tumor induction in mice. Malignant skin tumors were induced
in nude (NMRI nu) mice by subcutaneous flank inoculation of 106
PDV.C57 epidermal tumor cells. When tumors had reached an average
volume of 500 mm3 (range, 400–600 mm3), a continuous
flow pump (Alzet 2002; Alza Corporation, Palo Alto, California, USA)
was surgically implanted on the flank of every mouse near the site of
tumor inoculation. The pump was filled with either vehicle, 1,580
µg of WIN-55,212-2, or 1,580 µg of JWH-133 in 236 µl PBS
supplemented with 5 mg/ml BSA and operated at a flux of 0.52 µl/h
for 11 days. Tumors were measured with an external caliper and their
volume calculated as (4 /3)
x (width/2)2 x
(length/2). Cannabinoid treatment did not affect animal weight gain
(data not shown).
Viability and apoptosis in vitro. Cell viability in the
cultures was determined by the 3-4,5-dimethylthiazol-2,5-diphenyltetrazolium
bromide thiazol blue test. Apoptosis was determined by both TUNEL
staining (21) and oligonucleosomal DNA fragmentation (26)
according to kit manufacturer’s instructions (Boehringer Mannheim
GmbH, Mannheim, Germany).
Proliferation and apoptosis in vivo. For proliferation
assays, mice received an intraperitoneal injection of BrdU (120 mg/kg
body weight) (Boehringer Mannheim GmbH) 2 hours before tumor
harvesting. Detection of BrdU-positive cells was performed using an
anti-BrdU mouse mAb (Boehringer Mannheim GmbH) as described (9).
Apoptosis was determined by TUNEL staining (Boehringer Mannheim GmbH)
according to kit manufacturer’s instructions (21).
Western blot analysis. Particulate cell or tissue fractions
were subjected to SDS-PAGE, and proteins were transferred from the
gels onto polyvinylidene fluoride membranes. To determine cannabinoid
receptor expression the blots were incubated with polyclonal Ab’s
raised in rabbits against residues 1–14 of the human/mouse CB1
receptor (1:500; kindly given by A. Howlett, North Carolina Central
University, Durham, North Carolina, USA) or residues 20–33 of the
human/mouse CB2 receptor (1:2,000; Cayman Chemical, Ann Arbor,
Michigan, USA) as described (21). Antigen preabsorption
experiments were performed by preincubating (37°C, 1 hour) 1 µl
(= 0.5 µg) of the anti-CB1 or anti-CB2 Ab and 100
µl PBS with or without 20 µl (= 20 µg) of the corresponding immune
peptide (Cayman Chemical). Western blots were subsequently done with
the aforementioned Ab dilutions. To determine EGF-R phosphorylation
the blots were incubated with anti-phosphotyrosine (1:500; 4G10 mAb;
Upstate Biotechnology Inc., Lake Placid, New York, USA) or
anti-keratin 5 (1:1,000; Berkeley Antibody Co., Richmond, California,
USA) Ab’s, the latter used as a loading control. In all cases,
samples were subjected to luminography with an enhanced
chemiluminescence detection kit (Amersham Life Sciences, Arlington
Heights, Illinois, USA). Densitometric analysis of the blots was
performed with the Molecular Analyst software package (Bio-Rad
Laboratories Inc., Hercules, California, USA).
Immunohistochemistry. Tissues were fixed in 10% buffered
formalin and embedded in paraffin. Sections of mouse and human skin
and tumors were stained with the aforementioned Ab’s against CB1
(1:300) and CB2 (1:300) receptors. Control immunostainings
using the secondary Ab in the absence of the primary Ab were
routinely performed. In addition, antigen preabsorption experiments
were carried out with the corresponding blocking peptides as
described above. Immunodetection of blood vessels in cryosections of
mouse tumors was performed with an anti-CD31 Ab (1:40; PharMingen,
San Diego, California, USA). Sections were incubated with a
biotinylated anti-rabbit (CB1 and CB2) or
anti-rat Ab (CD31) and then with peroxidase-conjugated streptavidin (LSAB
Kit Peroxidase; DAKO A/S, Glostrup, Denmark). Ab localization was
determined using 3,3'-diaminobenzidine (Vector Laboratories,
Burlingame, California, USA). Morphometric values were obtained by
examination of six 0.11-mm2 sections per tumor with the
image analysis system Leica Qwin (Leica Microsystems Inc., Chantilly,
Virginia, USA).
Northern blot analysis. Total RNA was extracted from the
tumor samples by the acid guanidinium method (27).
The VEGF probe has been described previously (7). The
placental growth factor (PIGF) probe was kindly given by G. Persico (Istituto
Internazionale di Genetica e Biofisica, Naples, Italy). Probes for
angiopoietin 1 (Ang1) and angiopoietin 2 (Ang2) detection were kindly
provided by G.D. Yancopoulos (Regeneron Pharmaceuticals, Tarrytown,
New York, USA). A 1-kb fragment of the 5' region of the hEGF-R cDNA
was used as probe for EGF-R detection (9).
Ribosomal 7S RNA was used as a loading control. Densitometric
analysis of the blots was performed with a PhosphorImager using
Quantity One software (Bio-Rad Laboratories Inc.).
Statistics. Results shown represent means ± SD. Statistical
analysis was performed by ANOVA with a post hoc analysis by the
Student-Neuman-Keuls test. Data in Table 1, Figure 5a,
and Figure 6c were analyzed by the Mann-Whitney (Wilcoxon)
W test to compare medians for nonparametric data.
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Figure 5. Cannabinoids
inhibit skin tumor growth in vivo. PDV.C57 cells were injected
subcutaneously in mice. When tumors had reached the desired
size, animals were treated with either vehicle (Co),
WIN-55,212-2 (WIN), or JWH-133 (JWH), for 11 days. (a)
Tumor size (n = 8 for each experimental group).
*Significantly different (P < 0.01) from control mice.
(b) Examples of subcutaneous tumors in the flank of mice
after the indicated treatments. (c) Appearance of tumors
dissected from mice after the indicated treatments.
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Figure 6. Cannabinoids
inhibit angiogenesis in skin tumors in vivo. PDV.C57 cells were
injected subcutaneously in mice. When tumors had reached the
desired size, animals were treated with either vehicle (Co),
WIN-55,212-2 (WIN,) or JWH-133 (JWH) for 11 days. (a)
Northern blot of the proangiogenic factors VEGF, PIGF, and Ang2.
C1, C2; J1, J2; W1, W2 designate tumors from two different
animals of each experimental group, that is, treated with
vehicle (Co), JWH-133, or WIN-55,212-2, respectively. OD values
relative to those of loading controls are given in arbitrary
units. 7S, Ribosomal 7S RNA. (b) CD31 immunostaining.
Note that control carcinomas show dilated blood vessels, while
vessels of cannabinoid-treated tumors are narrow. (c)
Morphometric analysis of tumor vasculature (n = 4–6 for
each experimental group). *Significantly different (P
< 0.05) from control mice.
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Cannabinoid receptors are expressed in skin and skin tumors.
The expression of cannabinoid receptors in epidermal cell lines, normal
skin, and skin tumors of mice and humans was examined by Western blot
analysis and immunohistochemistry. Western blot experiments showed
that CB1 and CB2 receptors were expressed in a
number of tumorigenic and nontransformed epidermal cell lines of
murine and human origin (Figure 1a). In addition, the two
receptors were present in normal mouse skin as well as in benign (papillomas)
and malign (squamous cell carcinomas) mouse skin tumors. Likewise, CB1
and CB2 receptors were expressed in human skin,
keratinocytes, and carcinomas (Figure 1a). To ascertain
the specificity of the cannabinoid receptor Ab’s used in the
blotting experiments, antigen preabsorption experiments were carried
out with the corresponding blocking peptides. As shown in Figure 1b,
the peptides blocked anti-CB1 and anti-CB2 Ab
binding, not only in skin-derived samples but also in other cell
types used as well-established controls for the presence of CB1
(rat cortical neurons) (18), CB2 (human
promyelocytic HL60 cells) (19), and CB1
plus CB2 (rat C6 glioma cells) (21).
Immunocytochemical analyses showed that in mouse (Figure 2a)
and human (Figure 3a) normal skin CB1 and CB2
receptors were mostly present in suprabasal layers of the epidermis
and hair follicles. Basal staining was also observed in some sporadic
regions. CB1 and CB2 receptor immunoreactivity was
also evident in both papillomas and squamous cell carcinomas of mouse
origin (Figure 2a), as well as in human basal cell
carcinomas and squamous cell carcinomas (Figure 3a).
The specificity of the immunolabeling was shown by experiments in
which the primary Ab was omitted and—as mentioned above for Western
blots—by antigen preabsorption experiments carried out with the
corresponding blocking peptides (Figures 2b and 3b).
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Figure 2. Immunohistochemical
analysis of cannabinoid receptor expression in mouse normal skin
and skin tumors. (a) Immunolocalization of CB1
and CB2 receptors. (b) Controls without
primary Ab (only with secondary biotinylated anti-rabbit Ab), as
well as controls with the Ab-blocking peptides, are shown (see
Methods and Results for explanation). Normal skin came from a
5-day-old mouse. Papillomas were generated by chemical
carcinogenesis (as described in ref. 9), while
SCCs were generated by inoculation of PDV.C57 epidermal tumor
cells as described in Methods. Images of representative samples
are shown. Similar results were obtained in at least two other
samples. Hf, hair follicle; bl, basal layer; sbl, suprabasal
layers.
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Cannabinoid receptor activation induces skin tumor cell apoptosis.
We tested the functionality of cannabinoid receptors in the induction
of apoptosis in skin tumor cells. The mixed CB1/CB2 agonist
WIN-55,212-2 decreased the viability of the tumorigenic epidermal
cell lines PDV.C57 and HaCa4 (Figure 4a). Values of cell
viability after WIN-55,212-2 treatment (as percentage of the
corresponding incubations with no additions) were 74 ± 10 (day 3)
and 62 ± 7 (day 4) for PDV.C57 cells, and 75 ± 7 (day 3) and 71 ±
4 (day 4) for HaCa4 cells. Of interest, the cannabinoid was unable to
induce any statistically significant change in the viability of MCA3D
and HaCat cells, two nontransformed epidermal cell lines, and of
primary human keratinocytes (Figure 4b). The mixed
CB1/CB2 agonist HU-210 and the selective CB2
agonist JWH-133 (Ki = 677 nM for CB1 and 3.4
nM for CB2; ref, 25) (both at 25 nM) also induced PDV.C57 death
to an extent similar to that of WIN-55,212-2 (data not shown).
WIN-55,212-2–induced death of PDV.C57 cells occurred by a process
of apoptosis, as determined by oligonucleosomal DNA fragmentation
(Figure 4c) and TUNEL staining (Figure 4d).
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Figure 4. Cannabinoid
receptor activation induces skin tumor cell apoptosis. (a)
The tumorigenic epidermal cell lines PDV.C57 (open symbols) and
HaCa4 (closed symbols) were cultured with 25 nM WIN-55,212-2
alone (circles), or in combination with 0.2 µM SR141716 (SR1)
(squares), or 0.2 µM SR144528 (SR2) (triangles), and cell
viability was determined (n = 5). (b) The
nontransformed epidermal cell lines MCA3D (open circles) and
HaCat (closed circles), as well as primary human keratinocytes
(closed squares), were cultured with 25 nM WIN-55,212-2, and
cell viability was determined (n = 4). (c and d)
PDV.C57 cells were cultured as described before, and
oligonucleosomal DNA fragmentation (c) (n = 6),
and TUNEL staining (d) (one representative experiment of
four) were assessed. *Significantly different (P <
0.01) from control incubations.
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To evaluate the possible implication of the CB1 and CB2
receptors in cannabinoid-induced apoptosis, the effect of selective
receptor antagonists was studied. Thus, the CB1 antagonist
SR141716 and the CB2 antagonist SR144528 prevented
WIN-55,212-2–induced apoptosis of PDV.C57 cells (Figures 4,
a, c, and d), pointing to the involvement of both receptors in the
apoptotic action of cannabinoids.
Cannabinoids inhibit skin tumor growth in vivo. Given the
inhibition of tumorigenic epidermal cell survival in culture by
cannabinoids, we evaluated the effect of cannabinoid treatment on
skin tumor growth in vivo. Tumors generated by inoculation of the
highly malignant PDV.C57 cell line were treated with vehicle or
WIN-55,212-2. As shown in Figure 5, a–c, cannabinoid
administration blocked the growth of tumor cells in vivo (in 75%
of the mice treated).
Because cannabinoid-based therapeutic strategies should be as devoid
as possible of psychotropic side effects and PDV.C57 express
functional CB2 receptors, we administered to mice the selective
CB2 agonist JWH-133. Previously, we have provided pharmacological,
biochemical, and behavioral evidence that JWH-133 activates
selectively the CB2 receptor and does not elicit psychotropic effects
in mice (22). As shown in Figure 5, a–c,
tumors from JWH-133–treated animals were significantly smaller
than those from vehicle-treated controls (in 70%
of the mice treated).
We next examined whether, as occurs in cultured skin tumor cell lines,
cannabinoids induce apoptosis of malignant cells in vivo. As shown in
Table 1, quantification of apoptotic cells in tumor sections
revealed that treatment with WIN-55,212-2 or JWH-133 increased the
number of apoptotic cells. In contrast, the proliferation index did
not significantly differ between control and cannabinoid-treated carcinomas.
Cannabinoids inhibit skin tumor angiogenesis in vivo. Tumors
require an adequate supply of oxygen and nutrients to grow more than
a few millimeters. For that purpose they produce proangiogenic
factors that promote the formation of new blood vessels (28,
29). We therefore analyzed whether the vascularization of
growth-arrested cannabinoid-treated tumors was affected. Northern
blot analyses showed that the expression of major proangiogenic factors,
namely VEGF, PIGF, and Ang2, was strongly depressed by treatment with
WIN-55,212-2 or JWH-133 (Figure 6a). The mRNA expression
of Ang1 and the two antiangiogenic factors, thrombospondin 1 and
thrombospondin 2, was not significantly affected by cannabinoid administration
(data not shown). Furthermore, although immunostaining of CD31, a
marker of endothelial cells, revealed no significant differences in
vascular density (number of blood vessels per unit area) between
control and WIN-55,212-2– or JWH-133–treated tumors (Figures 6, b
and c), important differences were observed when vessel morphology
was examined: while control carcinomas showed a network of dilated
vessels, cannabinoid-treated tumors displayed a pattern of blood
vessels characterized predominantly by narrow capillaries (Figure 6b).
Morphometric analyses confirmed that cannabinoid treatment induced a
statistically significant decrease in blood vessel size, as
determined by the total area occupied by vessels, the area per
vessel, and the vessel larger diameter length (Figure 6c).
Cannabinoids decrease EGF-R activation in skin tumors in vivo.
We have recently found that in skin carcinomas EGF-R plays an important
role in triggering the angiogenic switch necessary for skin tumor
growth (9). Thus, we measured the expression levels
and activation state of EGF-R in control and cannabinoid-treated skin
tumors. While EGF-R mRNA was highly expressed in vehicle-treated tumors,
in line with its known overexpression in skin carcinomas (30,
31), the levels of EGF-R mRNA in cannabinoid-treated tumors
were very low (Figure 7a). In addition, the degree of
EGF-R activation (autophosphorylation) was markedly reduced in
cannabinoid-treated tumors (Figure 7b). Moreover,
exposure of cultured PDV.C57 cells to WIN-55,212-2 or JWH-133 blunted
EGF-R phosphorylation (Figure 7c), supporting the
direct impact of cannabinoids on skin tumor cells.
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Figure 7. Cannabinoids
inhibit EGF-R activation in skin tumors in vivo. (a and b)
PDV.C57 cells were injected subcutaneously in mice. When tumors
had reached the desired size, animals were treated with either
vehicle (Co), WIN-55,212-2, or JWH-133 for 11 days. Northern (a)
and Western blot (b) analyses show that EGF-R mRNA
expression and EGF-R activation (autophosphorylation),
respectively, are severely diminished in cannabinoid-treated
tumors. One representative experiment of three is shown in each
panel. (c) PDV.C57 cells were cultured for 24 hours with
either vehicle, 25 nM WIN-55,212-2, or 25 nM JWH-133, and EGF-R
phosphorylation was determined by Western blot analysis. One
representative experiment of three is shown. OD values relative
to those of loading controls are given in arbitrary units.
Ribosomal 7S RNA (7S) and keratin 5 (K5) were used as loading
controls in Northern and Western blots, respectively. PY,
phosphotyrosine.
|
|
Here we report that CB1 and CB2 cannabinoid receptors are
expressed in normal epidermis and in skin tumors and that both
receptors are functional in the induction of apoptosis of skin tumor
cells and the regression of skin carcinomas. It is therefore
plausible that apoptosis of tumor cells and tumor regression are two
causally related events. Nonetheless, our data indicate that
cannabinoid antitumoral action may also rely on the inhibition of
tumor angiogenesis. It has been shown that mouse skin tumor growth
and progression depends on critical events leading to epithelial and
stromal changes, including the establishment of an active angiogenesis
(4). Here, we report that blood vessels developed by
cannabinoid-treated carcinomas are small, in line with the finding
that blood vessel enlargement constitutes a prominent feature of skin
tumor progression (4, 32). Moreover, we
show that in cannabinoid-treated carcinomas the expression of
proangiogenic factors is depressed and that of antiangiogenic factors
is unchanged, which fits well with the observations that link skin
carcinoma development with a clear imbalance toward positive
angiogenic-factor action (6, 7, 9).
Ha-ras activation seems to be a critical event in mouse skin
tumor initiation as well as a major component of the angiogenic
response (6) in which VEGF plays a pivotal role (5,
9). Ha-ras activation induces VEGF expression in mouse
keratinocytes (6), as well as in other cell types (33,
34). Our data also show that cannabinoid treatment
decreases the expression of PIGF (another VEGF family member) and
Ang2, and these two proangiogenic factors may act in concert with
VEGF because their expression is highly increased since the early
stages of tumor development (9, 28, 29).
EGF-R participates in the regulation of key epidermal functions (35-38).
Moreover, we have shown that in mouse skin carcinomas EGF-R–dependent
Ha-ras activation plays a pivotal role in VEGF expression and
tumor angiogenesis and growth (9). Carcinoma growth
arising from subcutaneous injection of tumor epidermal cells is a
biphasic process. The first phase of slow growth occurs independently
of EGF-R function. Later, an angiogenic switch response mediated by
the EGF-R seems to be an essential requirement for complete tumor
growth, involving high VEGF levels. Other members of the EGF-R family
such as HER2 may also exert their relevant anticarcinogenic role via
modulation of angiogenesis (39). Here we show that
cannabinoid treatment impairs EGF-R function, VEGF expression, and
angiogenesis in skin tumors. It is of interest that inhibition of EGF-R
function also occurred upon exposure of cultured skin tumor cells to
cannabinoids, indicating that the changes observed in EGF-R activity
in vivo reflect a direct impact of cannabinoids on tumor cells and
are not a mere consequence of decreased tumor size. Although at present
we cannot establish the mechanism for the decrease of EGF-R
phosphorylation in cannabinoid-treated tumors, it is tempting to
speculate that cannabinoid treatment interferes with the tumor
angiogenic switch and that this, together with the direct induction
of apoptosis on tumor cells, is a reason for the inhibition of tumor
growth in our system.
Nonmelanoma skin cancer is one of the most common malignancies in
humans. Different types of strategies are currently being investigated
as therapies for the treatment of these tumors, including cryotherapy,
topical chemotherapeutic agents such as 5-fluorouracil, and
photodynamics, the success of which is hampered by limitations such
as the poor penetration of molecules into the skin and the difficulty
of gaining access to the whole tumor (10-12).
The present data indicate that local cannabinoid administration may
constitute an alternative therapeutic approach for the treatment of
nonmelanoma skin cancer. Of further therapeutic interest, we show
that skin cells express functional CB2 receptors. The
synergy between CB1 and CB2 receptors in eliciting skin
tumor cell apoptosis reported here is nonetheless intriguing because
it is not observed in the case of cannabinoid-induced glioma cell
apoptosis (21, 22). In any event, the
present report, together with the implication of CB2- or
CB2-like receptors in the control of peripheral pain (40-42)
and inflammation (41), opens the attractive
possibility of finding cannabinoid-based therapeutic strategies for
diseases of the skin and other tissues devoid of nondesired CB1-mediated
psychotropic side effects.
We are indebted to M.I. de los Santos for expert technical assistance and
to F. Larcher for discussion and advice. This work was supported by
grants from the Ministerio de Ciencia y Tecnología (PM 98-0079 to M.
Guzmán, SAF 98-0047 to J.L. Jorcano, and BMC 2001-1018 to J.L.
Jorcano); the Comunidad Autónoma de Madrid (08.1/0079/2000 to M.
Guzmán); the Fundación Ramón Areces (to M. Guzmán); and the
National Institute on Drug Abuse (DA03590 to J.W. Huffman).
Conflict of interest: The authors have declared that no conflict
of interest exists.
Nonstandard abbreviations used: placental growth factor (PIGF); angiopoietin
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