Chemopreventive effect of the non-psychotropic phytocannabinoid cannabidiol on experimental colon cancer

Abstract

Colon cancer affects millions of individuals in Western countries. Cannabidiol, a safe and non-psychotropic ingredient of Cannabis sativa, exerts pharmacological actions (antioxidant and intestinal antinflammatory) and mechanisms (inhibition of endocannabinoid enzymatic degradation) potentially beneficial for colon carcinogenesis. Thus, we investigated its possible chemopreventive effect in the model of colon cancer induced by azoxymethane (AOM) in mice. AOM treatment was associated with aberrant crypt foci (ACF, preneoplastic lesions), polyps, and tumour formation, up-regulation of phospho-Akt, iNOS and COX-2 and down-regulation of caspase-3. Cannabidiol-reduced ACF, polyps and tumours and counteracted AOM-induced phospho-Akt and caspase-3 changes. In colorectal carcinoma cell lines, cannabidiol protected DNA from oxidative damage, increased endocannabinoid levels and reduced cell proliferation in a CB(1)-, TRPV1- and PPARγ-antagonists sensitive manner. It is concluded that cannabidiol exerts chemopreventive effect in vivo and reduces cell proliferation through multiple mechanisms.

Full text

ORIGINAL ARTICLE
Chemopreventive effect of the non-psychotropic
phytocannabinoid cannabidiol on experimental
colon cancer
Gabriella Aviello &Barbara Romano &Francesca Borrelli &Raffaele Capasso &
Laura Gallo &Fabiana Piscitelli &Vincenzo Di Marzo &Angelo A. Izzo
Received: 11 October 2011 / Revised: 23 November 2011 /Accepted: 14 December 2011
#Springer-Verlag 2012
Abstract Colon cancer affects millions of individuals in
Western countries. Cannabidiol, a safe and non-psychotropic
ingredient of Cannabis sativa, exerts pharmacological actions
(antioxidant and intestinal antinflammatory) and mechanisms
(inhibition of endocannabinoid enzymatic degradation)
potentially beneficial for colon carcinogenesis. Thus, we
investigated its possible chemopreventive effect in the model
of colon cancer induced by azoxymethane (AOM) in mice.
AOM treatment was associated with aberrant crypt foci (ACF,
preneoplastic lesions), polyps, and tumour formation, up-
regulation of phospho-Akt, iNOS and COX-2 and down-
regulation of caspase-3. Cannabidiol-reduced ACF, polyps
and tumours and counteracted AOM-induced phospho-Akt
and caspase-3 changes. In colorectal carcinoma cell lines,
cannabidiol protected DNA from oxidative damage, increased
endocannabinoid levels and reduced cell proliferation in a
CB
1
-, TRPV1- and PPARγ-antagonists sensitive manner. It
is concluded that cannabidiol exerts chemopreventive effect in
vivo and reduces cell proliferation through multiple
mechanisms.
Keywords Non-psychotropic cannabinoid .Colon cancer .
Adenocarcinoma cells .DNA damage
Introduction
Colon cancer is a major cause of morbidity and mortality in
Western countries. In 2011, an estimated 101,340 new cases
of colorectal cancer were diagnosed in the USA, with
49,380 estimated deaths [1]. Colon cancer is thought to
arise as the result of a series of histopathological and
molecular changes that transform normal colonic epithe-
lial cells into a colorectal carcinoma, with aberrant crypt
foci (ACF) and polyps as intermediate steps in this
process [2]. This multi-step process spans 10–15 years,
thereby providing an opportunity for prevention [3].
Δ
9
-tetrahydrocannabinol (Δ
9
-THC), the main psychotro-
pic ingredient of the marijuana plant Cannabis sativa, binds
two G
i/o
coupled membrane receptors, named cannabinoid
receptors (CB
1
and CB
2
), which are also activated by en-
dogenous ligands [anandamide and 2-arachidonoylglycerol
(2-AG)] [4,5]. Endocannabinoids are biosynthesised ‘on
demand’from membrane phospholipids and are inactivated
through a reuptake process (facilitated by a putative endo-
cannabinoid membrane transporter), followed by enzymatic
degradation catalysed by the fatty acid amide hydrolase
(FAAH, in the case of anandamide and, to some extent,
2-AG) or monoacylglycerol lipase (MAGL, in the case
G. Aviello :B. Romano :F. Borrelli :R. Capasso :A. A. Izzo
Department of Experimental Pharmacology,
Endocannabinoid Research Group,
University of Naples Federico II,
Naples, Italy
L. Gallo :F. Piscitelli :V. Di Marzo
Institute of Biomolecular Chemistry,
Endocannabinoid Research Group,
Consiglio Nazionale delle Ricerche (CNR),
Pozzuoli, Italy
A. A. Izzo (*)
Department of Experimental Pharmacology,
University of Naples Federico II,
Via D. Montesano 49,
80131 Naples, Italy
e-mail: aaizzo@unina.it
V. Di Marzo (*)
Institute of Biomolecular Chemistry, CNR,
Via Campi Flegrei, 34, Comprensorio Olivetti,
80078 Pozzuoli, NA, Italy
e-mail: vdimarzo@icmib.na.cnr.it
J Mol Med
DOI 10.1007/s00109-011-0856-x

of 2-AG) [4]. Cannabinoids—via direct or indirect acti-
vation of CB
1
and/or CB
2
receptors—exert protective
effects in well-established models of colon cancer [6–8].
In addition to Δ
9
-THC, the plant Cannabis also contains
non-psychotropic cannabinoids with potential therapeutic
interest. The best studied among these compounds is canna-
bidiol, which, unlike Δ
9
-THC, has very low affinity for both
CB
1
and CB
2
receptors [5,9]. Cannabidiol has an extremely
safe profile in humans [9] and exerts a number of pharma-
cological actions (e.g. analgesic/anti-inflammatory, antioxi-
dant, neuroprotective) of potential clinical interest [9]. Few
studies have investigated the effect of cannabidiol in the gut.
Specifically, cannabidiol has been shown to reduce intesti-
nal contractility [10,11] and, more importantly, to exert
antinflammatory effects [12,13], a relevant observation in
the light of the well-known association existing between
intestinal inflammation and colorectal cancer [14]. In addi-
tion, cannabidiol may inhibit FAAH [15] and exerts antiox-
idant action in colorectal carcinoma cell lines [12]. Both
FAAH inhibition [6,16] and antioxidant effects [17] are
potentially beneficial for colon carcinogenesis.
Because cannabidiol exerts pharmacological effects
(e.g. antioxidant, intestinal antinflammatory) and mech-
anisms (e.g. inhibition of endocannabinoids enzymatic
degradation) potentially beneficial for colon carcinogen-
esis, we investigated the potential chemopreventive ef-
fect of this non-psychotropic marijuana component in an
experimental model of colon cancer. The possible mode of
action was evaluated in colorectal carcinoma cell lines.
Materials and methods
Animals
Male ICR mice (Harlan Italy, S. Pietro al Natisone UD,
Italy) weighting 26–30 g were used in conformity to the
Italian DL no. 116 of 27 January 1992 and associated guide-
lines in the European Communities Council (86/609/ECC
and 2010/63/UE).
Cell cultures
For in vitro experiments, two human colon adenocarcinoma
cell lines (i.e. Caco-2 and HCT116 cells, ATCC from LGC
Standards, Milan, Italy), with a different genetic profile
(APC gene mutated in Caco-2 cells, K-RAS mutated in
HCT116 cells) [18,19] were used. Both cell lines were
cultured in Dulbecco’s modified Eagle’s medium containing
10% foetal bovine serum, 100 U/ml penicillin and 100 μg/
ml streptomycin, 1% non-essential amino acids, and 2 mM
L-glutamine, in conformity with the manufacturer’s
protocols.
In vivo treatments and tumour evaluation
Mice were randomly divided into the following groups:
Group 1 (control) was treated with vehicles; group 2
was treated with azoxymethane (AOM) plus the vehicle
used to dissolve cannabidiol; group 3 was treated with
AOM plus cannabidiol (1 mg/kg); and group 4 was
treated with AOM plus cannabidiol (5 mg/kg). AOM
(40 mg/kg in total, IP) was administered in four single
doses of 10 mg/kg, at the beginning of the first, second,
third, and fourth week. Cannabidiol was given (IP) three
times per week for the whole duration of the experiment
starting 1 week before the first administration of AOM
[6]. All animals were euthanised by asphyxiation with
CO
2
3 months after the first injection of AOM. Based
on our laboratory experience, this time (at the dose of
AOM used) was associated with the occurrence of a
significant number of aberrant crypt foci (ACF), polyps
and tumours. Colons were examined as previously
reported [6] using a light microscope at 20× magnifica-
tion (Leica Microsystems, Milan, Italy). Only foci with
four or more crypts (which are best correlated with the
final tumour incidence) were evaluated. ACF were dis-
tinguished from surrounding normal crypts by greater
size, larger and elongated luminal opening, thicker lin-
ing, and compression of the surrounding epithelium. The
criterion to distinguish polyps from tumors was established
considering the main characteristic features of these two
lesions (i.e. crypt distortion around a central focus and in-
creased distance from luminal to basal surface of cells for
polyps and high grade of dysplasia with complete loss of crypt
morphology for tumors) [20].
Preparation of cytosolic lysates
Lysates from full-thickness colons were obtained as
previously described [12]. Briefly, tissues were homoge-
nised using a buffer solution (1:2, w/v) containing
0.5 M β-glycerophosphate, 20 mM MgCl
2
,10mM
EGTA, 100 mM dithiothreitol, 100 mM dimethylsul-
fonyl fluoride, 2 mg/ml apronitin, 2 mM leupeptin and
10 mM Na
3
VO
4
.
For lysate preparations from Caco-2 cells, 8 ×10
5
cells were seeded in Petri dishes, brought to subconflu-
ence (∼70%) and, after 24-h exposure to cannabidiol,
collected using the following buffer: 50 mM Tris–HCl,
pH 7.4, 0.25% sodium deoxycholate, 150 mM NaCl,
1 mM EGTA, 1 mM NaF, 1% NP-40, 1 mM PMSF,
1mMNa
3
VO
4
plus a complete protease inhibitor cock-
tail (Roche Diagnostics, Mannheim, Germany). The pro-
tein concentration was determined on supernatant
(following centrifugation at 16,200×gfor 15 min) using
the Bradford method.
J Mol Med

Western blot analysis
Western blot analyses were performed ex vivo in full-
thickness colonic tissues of animals treated or not with
AOM (alone or with cannabidiol 1 mg/kg) to investigate
the expression of inducible nitric oxide synthase (iNOS),
cycloxygenase (COX-2), phospho-Akt and caspase-3. We
also investigated the expression of phospho-Akt in Caco-2
cells, treated with cannabidiol (10 μM). Protein lysates (50–
70 μg) were separated on sodium dodecyl sulfate polyacryl-
amide gels, and membranes were incubated with anti-iNOS,
anti-COX-2 (BD Biosciences from Becton Dickinson,
Buccinasco, Italy), anti-β-actin (Sigma, Milan, Italy), anti-
phosho-Akt or anti-Akt and anti-cleaved-caspase-3 (frag-
ment p17) or anti-uncleaved caspase-3 (fragment p30) (Cell
Signaling from Euroclone, Milan, Italy). Signals were
visualised using ImageQuant 400 equipped with Quantity
One Software 4.6.3 (GE Healthcare, Milan, Italy).
Cytotoxicity studies: neutral red (NR) uptake
Caco-2 and HCT116 cells were seeded in 96-well plates [1 ×
10
4
cells per well (Caco-2) and 2.5×10
3
cells per well
(HCT116)] and allowed to adhere for 48 h; after this period,
cells were incubated with cannabidiol (0.01–10 μM) for
24 h and subsequently with NR dye solution (50 μg/ml)
for 3 h. Cells were lysed with 1% acetic acid, and the
absorbance was read at 532 nm (iMark
TM
microplate absor-
bance reader, BioRad). Dimethyl sulphoxide (DMSO)
(20%) was used as a positive control. The results are
expressed as percentage of cell viability (n03 experiments
including 8–10 replicates for each treatment).
Proliferation studies: MTT assay and
3
H-thymidine
incorporation
Caco-2 and HCT116 cells were seeded, allowed to adhere
and starved by serum deprivation for 24 h. For the 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
(MTT) assay, cells were treated with cannabidiol (0.01–
10 μM) for 24 h and incubated with MTT (250 μg/ml) for
1 h at 37°C. The mitochondrial reduction of MTT to formazan
was then quantitated at 490 nm (iMarkTM microplate reader,
BioRad, Italy).
Using this assay, the antiproliferative effect of cannabidiol
(10 μM) was evaluated (in Caco-2 cells) in the presence of
rimonabant (0.1 μM, CB
1
receptor antagonist), AM251
(10 μM, CB
1
receptor antagonist), SR144528 (0.1 μM, CB
2
receptor antagonist), AM630 (0.1 μM, CB
2
receptor antago-
nist), capsazepine [1 μM, transient receptor potential vanilloid
1 (TRPV1) antagonist] and GW9662 [10 μM, peroxisome
proliferator-activated receptor γ(PPARγ) antagonist], all in-
cubated 30 min before cannabidiol.
For
3
H-thymidine incorporation assay [21], cells were
incubated with cannabidiol (0.01–10 μM) in the presence of
[methyl-
3
H]-thymidine (1 μCi/well) for 24 h and collected
with 1 N NaOH for β-counting (PerkinElmer, Milan, Italy).
Identification and quantification of endocannabinoids
(anandamide and 2-AG), PEA and OEA
Endocannabinoids (anandamide and 2-AG), palmitoylethano-
lamide (PEA) and oleoylethanolamide (OEA) levels were
measured in Caco-2 cells. Cells were exposed to cannabidiol
(10 μM) for 24 h and harvested in 70 % methanol before cell
processing, subsequently extracted, purified and analysed by
isotope dilution liquid chromatography-atmospheric pressure-
chemical ionisation mass spectrometry [6,12].
Genotoxicity studies: comet assay
Genotoxicity studies were performed by single cell electro-
phoresis assay (Comet assay) [21] Following 24-h exposure to
cannabidiol (10 μM), Caco-2 cells were incubated with
75 μMH
2
O
2
(damaging stimulus) or phosphate-buffered
saline PBS (undamaging stimulus) for 5 min. After centrifu-
gation at 1,000×gfor 5 min, pellets were mixed with 0.85%
low melting point agarose and added to 1% normal melting
point agarose gels. Gels were then suspended in 2.5 M NaCl,
100 mM Na
2
EDTA, 10 mM Tris and 1% Triton X-100, pH 10
at 4°C for 1 h and electrophoresed in alkaline buffer (300 mM
NaOH, 1 mM Na
2
EDTA, pH 12) at 26 V, 300 mA for 20 min.
After neutralisation in 0.4 M Tris–HCl (pH 7.5), gels were
stained with 2 μg/ml ethidium bromide. Images were analysed
using a Leica microscope equipped with a Casp software.
Drugs
AOM, MTT, 3-amino-7-dimethylamino-2-methylphenazine
hydrochloride (NR) were purchase from Sigma (Milan,
Italy); AM251, AM630, capsazepine and GW9662 were
obtained from Tocris Cookson (Bristol, UK). Rimonabant
and SR144528 (N-[-1S-endo-1,3,3-trimethyl bicyclo [2.2.1]
heptan-2-yl]-5-(4-chloro-3-methylphenyl)-1-(4-methylben-
zyl)-pyrazole-3-carboxamide) were from Sanofi-Aventis
(Montpellier, France). Methyl-[
3
H]-thymidine was pur-
chased from PerkinElmer (Monza, Italy). Cannabidiol (pu-
rity by high-performance liquid chromatography, 99.76%)
was kindly supplied by GW Pharmaceuticals (Porton Down,
Wiltshire, UK). All reagents for Western blot analyses, and
cell cultures were obtained from Sigma (Milan, Italy), Bio-
Rad Laboratories (Milan, Italy) and Microtech Srl (Naples,
Italy). The vehicles for in vivo experiments (10% ethanol,
10% Tween-20, 80% saline and 0.2 ml/kg) and in vitro
experiments (0.1% DMSO v/vin cell media) had no effect
on the response under study.
J Mol Med

Statistics
Data are expressed as the mean± standard error (SE mean) of n
experiments. To determine statistical significance, Student’st
test was used for comparing a single treatment mean with a
control mean, and an one-way analysis of variance followed
by a Tukey–Kramer multiple comparisons test was used for
analysis of multiple treatment means. The chi-square test was
used to evaluate the significance between the number of mice
with or without ACF, polyps or tumours. P’s< 0.05 were
considered significant. The IC
50
value, i.e. the concentration
of cannabidiol able to produce 50% of maximal inhibition of
cell proliferation (geometric mean±95% CL), was calculated
with the aid of a computer programme (GraphPad Prism 4).
Results
Effect of cannabidiol on the formation of ACF, polyps
and tumours
The carcinogenic agent AOM given alone induced the
expected appearance of ACF (Fig. 1a), polyps (Fig. 1b)
and tumours (Fig. 1c)after3monthsoftreatment.The
percentage of mice bearing ACF containing four or more
crypts, polyps and tumours observed after the treatment with
AOM is shown in Table 1.
Cannabidiol (1 mg/kg) significantly reduced AOM-
induced ACF (67% inhibition) (Fig. 1a), polyps (57% inhi-
bition) (Fig. 1b) and tumours (66% inhibition) (Fig. 1c)as
well as the percentage of mice bearing polyps (Table 1). In
addition, 1 mg cannabidiol showed a strong trend towards
the inhibition of the percentage of mice bearing tumours
(Table 1), although a conventional statistical significance
was not fully achieved (P00.06). Cannabidiol (5 mg/kg)
significantly reduced the formation of polyps (Fig. 1b) and
the percentage of mice bearing polyps only (Table 1).
COX-2, iNOS, phospho-Akt and caspase-3 expression
in colonic tissues
Western blot analysis revealed the expression of COX-2,
iNOS, phospho-Akt and caspase-3 (Fig. 2a–d) in colonic
tissues of both healthy and AOM-treated animals. The den-
sitometric analysis indicated a significant increase in the
expression of COX-2 (Fig. 2a), iNOS (Fig. 2b)and
phospho-Akt (Fig. 2c) in the colons of AOM-treated mice.
Cannabidiol (1 mg/kg) did not cause significant changes in
the expression of COX-2 and iNOS in AOM-treated animals
(Fig. 2a, b) but significantly reduced AOM-induced Akt
protein phosphorylation (Fig. 2c). AOM treatment caused
a significant down-regulation of cleaved caspase-3 expres-
sion, which was restored by cannabidiol (Fig. 2d).
Cytotoxicity (neutral red) assays in human colon
adenocarcinoma cells
The effect of cannabidiol on cell viability, using the
neutral red assay, was evaluated in both Caco-2 and
HCT116 cell lines (Table 2). Cannabidiol, at the con-
centration ranging from 0.01 to 10 μM, did not affect
Fig. 1 Aberrant crypt foci with four or more crypts (ACF ≥4/mouse)
(a), polyps (b) and tumours (c) induced in the mouse colon by AOM:
effect of cannabidiol (CBD, non-psychotropic cannabinoid, 1 and
5 mg/kg). AOM (40 mg/kg in total, IP) was administered, at the single
dose of 10 mg/kg, at the beginning of the first, second, third and fourth
week. CBD was given (IP) three times a week for the whole duration of
the experiment starting 1 week before the first administration of AOM.
Measurements were performed 3 months after the first injection of
AOM. Insets: representative images of an ACF (a), polyp (b) and
tumour (c) captured at ×20, ×10 and ×5 magnifications, respectively.
Each bar represents the mean±SE mean of 9–11 mice. *P<0.05 and
**P<0.01 vs vehicle
J Mol Med

Caco-2 or HCT116 cell viability after 24-h exposure.
DMSO (20% v/v), used as positive control, significantly
(P<0.001) reduced both Caco-2 and HCT116 cell
viability (Table 2).
Antiproliferative effect of cannabidiol
The effect of non-cytotoxic concentrations of cannabidiol
(CBD, 0.01–10 μM) was evaluated on cell proliferation in
both Caco-2and HCT116 cells using two different techniques.
In both cell lines and using MTT assay and
3
H-thymidine
incorporation, cannabidiol exerted a significant antiprolifera-
tive effect (Fig. 3)[IC
50
(95% CL) in the
3
H-thymidine
incorporation assay: 0.67 (0.0145–31.4)μM]. Using the
MTT assay, we found that the effect of CBD (10 μM) on
Caco-2 cell proliferation was counteracted by rimonabant
(0.1 μM) and AM251 (1 μM) (two CB
1
receptor antagonists),
capsazepine (1 μM, a TRPV1 receptor antagonist) and
GW9662 (10 μM, a PPARγreceptor antagonist) (Fig. 4).
By contrast, the effect of cannabidiol was not signifi-
cantly changed by SR144528 (10 μM) and AM630
(1 μM) (CB
2
receptor antagonists) (Fig. 4). All receptor
antagonists employed in this set of experiments were not
cytotoxic and did not affect, per se, cell proliferation
(data not shown).
Endocannabinoids, palmitoylethanolamide
and oleoylethanolamide levels in Caco-2 cells
The exposure to CBD (0.1–10 μM) for 24 h induced an
increase in 2-AG levels (Fig. 5b) in subconfluent Caco-2 cells.
The effect was significant for the 0.1 μM concentration.
No significant differences were observed in anandamide,
palmitoylethanolamide and oleoylethanolamide levels
following cannabidiol (0.1–10 μM) incubation for 24 h
(Fig. 5a, c and d).
Phospho-Akt expression in Caco-2 cells
Figure 6shows the effect of cannabidiol on Akt phosphor-
ylation in Caco-2 cells. Cannabidiol (10 μM for 24 h) was
able to significantly reduce the expression of phospho-Akt
in proliferating Caco-2 cells.
Genotoxicity assay
Compared to the control cells (a), cannabidiol (10 μM)
alone did not significantly affect DNA damage after 24-
h exposure (c), suggesting the absence of a genotoxic effect
(Fig. 7). Exposure of Caco-2 cells to hydrogen peroxide
(75 μM) produced a significant increase in the percentage
Table 1 Effect of cannabidiol (CBD) on the percentage of mice bearing ACF (with four or more crypts), polyps or tumours induced by the
carcinogen agent azoxymethane (AOM)
Treatments NMice bearing ACF(%) Mice bearing polyps (%) Mice bearing tumours (%)
AOM+ vehicle 10 90 100 90
AOM+ CBD 1 mg/kg 10 60 50* 40***
AOM+ CBD 5 mg/kg 10 60 40** 70
*P00.038; **P00.014; ***P00.060 (chi-square test)
Fig. 2 Cyclooxygenase-2 (COX-2) (a), inducible nitric oxide synthase
(iNOS) (b), phospho-Akt (c) and cleaved caspase-3 (active fragment
p17) (d) expression in colonic tissues of mice treated or not with AOM:
effect of cannabidiol (CBD, 1 mg/kg). AOM (40 mg/kg in total, IP)
was administered, at the single dose of 10 mg/kg, at the beginning of
the first, second, third and fourth week. CBD was administered (IP)
three times a week for the whole duration of the experiment starting
1 week before the first administration of AOM. Each bar represents the
mean± SE mean of four to five independent experiments. *P<0.05 and
**P<0.01 vs control;
#
P<0.05 vs AOM
J Mol Med

of DNA in comet tails (b), whereas a pre-treatment with
cannabidiol (10 μM) (D) for 24 h significantly reduced the
H
2
O
2
-induced DNA damage (Fig. 7).
Discussion
Although cannabidiol has been shown to kill glioma cells
[22], to inhibit cancer cell invasion [23] and to reduce the
growth of breast carcinoma and lung metastases in rodents
[24,25], its effect on colon carcinogenesis has not been
evaluated to date. This is an important omission, since colon
cancer affects millions of individuals in Western countries
[1–3]. In the present study, we have shown that cannabidiol
exerts (1) protective effects in an experimental model of
colon cancer and (2) antiproliferative actions in colorectal
carcinoma cells.
Effect of cannabidiol in the AOM model of colon cancer
We have shown that cannabidiol exerted beneficial effects in
AOM-treated mice. More specifically, we found that canna-
bidiol, at the dose of 1 mg/kg, exerted an optimal chemo-
preventive effect, by being able to significantly reduce ACF,
polyps and tumours. At the higher 5 mg/kg dose, it prevented
the formation of polyps only. The lack of a dose-related effect
of cannabidiol has been alsopreviously documented in several
in vivo pharmacological assays [9]. Because an optimal phar-
macological effect was observed at the 1 mg/kg, ex vivo
intestinal biochemical changes (i.e. caspase-3, phospho-Akt,
iNOS, COX-2 evaluations) were evaluated in animals treated
with this dose of cannabidiol. We found that the protective
effect of cannabidiol on colon carcinogenesis was associated
with up-regulation of the active fragment of caspase-3, i.e. one
of the major final effectors of the apoptotic process [26]. Pro-
apoptotic mechanisms induced by cannabidiol have been
previously documented in human breast carcinoma and glio-
ma cells [22,24].
When we investigated the potential role of the phosphoi-
nositide 3-kinase (PI3K)/Akt pathway, which is crucial for
the regulation of cell growth, migration, differentiation, and
apoptosis [27,28], we found that cannabidiol counteracted
AOM-induced up-regulation of the phosphorylated form of
Akt protein (and it also down-regulated Akt phorsphorylation
in Caco-2 cells). These data are suggestive of an involvement
of the PI3K-Akt survival signalling cascade in cannabidiol-
induced protective effect. Interestingly, Greenbough and col-
leagues found that the psychotropic cannabinoid Δ
9
-THC, via
CB
1
activation, induced apoptosis in colorectal cancer cells
and that its protective effect also involved inhibition of the
PI3K-Akt survival signalling cascade [29].
Table 2 Effect of 24-h exposure to cannabidiol on cell viability evaluated by neutral red uptake assay in Caco-2 and HCT116 cell lines
Cell lines Cannabidiol (μM) DMSO (20% v/v)
0 0.01 0.1 1 10
Caco-2 100.2± 6.1 98.0 ± 8.6 100.5± 2.0 97.0 ± 2.47 99.25 ± 4.5 45.0 ± 4.1***
HCT116 100.1± 2.5 105.3 ± 2.5 102.0 ± 5.5 101.7 ± 2.5 106.1 ±1.7 36.1±1.9***
DMSO (20% v/v) was used as positive control
Data are expressed as percentage of cell viability ± SE mean. ***P< 0.001 vs control (n03 independent experiments)
Fig. 3 Antiproliferative effects of cannabidiol (0.01–10 μM, 24-
h exposure) in Caco-2 (a,b) and HCT116 (c,d) cells. Proliferation
rate was studied using two different techniques: the MTT assay (a,c)
and the
3
H-thymidine incorporation (b,c). Each bar represents the
mean± SE mean of three independent experiments. *P<0.05, **P<
0.01 and ***P<0.001 vs control
J Mol Med

Finally, we found that cannabidiol did not change the over-
expressionof COX-2 and iNOS, two key enzymes involved in
colon carcinogenesis [30,31]. Likewise, the protective effect
of cannabidiol against glioma in vivo was not associated with
changes in COX-2 activity in glioma tumour tissues [26]. We
have previously shown that the antinflammatory effect of
cannabidiol in the gut is associated with down-regulation of
iNOS, but not COX-2, expression [12].
Effect of cannabidiol on colorectal carcinoma cell lines
In order to identify the potential receptor(s) underlying the
antitumoural action of cannabidiol in the gut, we investigated
the effect of this non-psychotropic cannabinoid on colorectal
carcinoma cell lines. Cannabidiol is known to exert antiproli-
ferative effects in different tumour cell lines [21,24]. In the
present study, we have shown that this compound, at non-
cytotoxic concentrations, exerts antiproliferative effects in two
different colorectal carcinoma cell lines, i.e. Caco-2 and
HCT116 cells. A complete concentration–response effect
was observed using the
3
H-thymidine incorporation—but
not the MTT—method. This difference probably reflects
the diverse sensitivity of the two methods, being the
3
H-thymidine incorporation assay more sensitive than
the MTT assay [32].
To evaluate the target(s) downstream the in vitro effect of
cannabidiol, we investigated, in Caco-2 cells, the potential
involvement of: (1) cannabinoid receptors because cannabi-
diol may increase endocannabinoid levels [33,15], which,
in turn, may exert antiproliferative effects in vitro via CB
1
and, only in part, CB
2
receptor activation [34]; (2) TRPV1
because cannabidiol may directly activate this receptor [15];
in addition, anandamide, an endogenous TRPV1 ligand
[15], is elevated in the AOM model of colon cancer [6], as
well as in biopsies of patients with colon cancer [34]; (3)
PPARγbecause cannabidiol may activate PPARγ[35] and
PPARγagonists exert protective effect in colon carcinogen-
esis [36]. Our data show that the antiproliferative effect of
Fig. 4 Antiproliferative effects,
evaluated by MTT assay,
of cannabidiol (CBD, 10 μM,
24 h-exposure) alone or in the
presence of rimonabant
(0.1 μM) and AM251 (1 μM)
(two selective CB
1
receptor
antagonists), SR144528
(10 μM) and AM630
(1 μM) (two selective CB
2
receptor antagonists),
capsazepine (1 μM) (a TRPV1
antagonist) and GW9662
(10 μM) (a PPARγantagonist).
The antagonists were incubated
30 min before CBD. Each bar
represents the mean± SE mean
of three independent
experiments. *P<0.05,
**P<0.01 and ***P<0.001
vs control;
#
P<0.05 and
##
P<0.01 vs CBD
J Mol Med

cannabidiol was counteracted by rimonabant and AM251
(two CB
1
receptor antagonists), capsazepine (a TRPV1 re-
ceptor antagonist) and GW9662 (a PPARγreceptor
antagonist), thus suggesting that this non-psychotropic phy-
tocannabinoid may exert anti-cancer effects in vitro through
multiple mechanisms. In line with our results, it has been
recently demonstrated that cannabidiol reduces intestinal
permeability in Caco-2 cells in a CB
1
and TRPV1 antago-
nist sensitive manner [37]. Interestingly, it has been previ-
ously demonstrated that the TRPV1 agonist capsaicin
induces apoptosis in colorectal carcinoma cell lines by acti-
vating PPARγ[38]. Because cannabidiol does not bind CB
1
receptors with high affinity, the reversal by rimonabant
could be explained by indirect activation of such receptors,
e.g. via enhancement of endocannabinoid(s) in colorectal
carcinoma cell lines. Indeed, we have here demonstrated
that cannabidiol was able to increase 2-AG levels in Caco-
2 cells. In addition, anandamide levels appeared to be
increased with this concentration of cannabidiol although
in a non-statistically significant manner. Although FAAH
is not the primary enzyme involved in 2-AG metabolism
[4], we have previously demonstrated, in both Caco-2
cells and colon of AOM-treated mice [6,33], that arach-
idonoyl serotonin, another FAAH inhibitor, increases the
content of both anandamide and 2-AG.
Fig. 6 Effect of cannabidiol (CBD, 10 μM for 24 h) on phospho-Akt
expression in proliferating Caco-2 cells. Each bar represents the mean
±SE mean of three independent experiments. ***P< 0.001 vs control
Fig. 7 Effect of cannabidiol (CBD, 10 μM for 24 h) on hydrogen
peroxide (H
2
O
2
)-induced DNA damage evaluated by the comet assay.
The DNA damage was induced in Caco-2 cells by 75 μMH
2
O
2
(b) and
compared with PBS-treated (undamaged) cells (a). The effect of CBD
was studied in presence (d) or absence (c)ofH
2
O
2
.a–dRepresentative
comets. Each bar represents the mean±SE mean of three independent
experiments where at least 75 cells per gel in triplicate were scored.
###
P<0.001 vs undamaged cells (a, PBS) and ***P< 0.001 vs dam-
aged cells (b,H
2
O
2
). DNA damage, expressed as percentage of fluo-
rescence in the comet tail (% DNA tail) was quantified using at least 75
cells per gel were scored and each sample was evaluated in triplicate
(n03 independent experiments)
Fig. 5 Levels of anandamide (a), 2-arachidonoylglycerol (2-AG, b),
palmitoylethanolamide (PEA, c) and oleoylethanolamide (OEA, d)in
Caco-2 cells exposed to cannabidiol (0.1–10 μM, 24 h). Each bar
represents the mean± SE mean of three independent experiments.
*P<0.05 vs control
J Mol Med

Finally, using the single cell electrophoretic assay (Comet
assay), a widely accepted tool for investigating DNA dam-
age, we have demonstrated that cannabidiol was unable to
induce DNA damage and, more importantly, whereas it
exerted protective effects against hydrogen peroxide-
induced DNA damage. These results are of interest because
DNA mutation is a crucial step in carcinogenesis and oxi-
datively derived DNA lesions have been observed in many
tumours, where they are strongly implicated in the aetiology
of colon cancer.
Conclusions
In conclusion, we have here demonstrated here that the non-
psychotropic phytocannabinoid cannabidiol exerts chemo-
preventive effects in an experimental model of colon cancer,
an effect that is associated with down-regulation of
phospho-Akt and up-regulation of caspase-3. Studies on
colorectal carcinoma cells suggest that cannabidiol protects
DNA damage caused by an oxidative insult and exerts
antiproliferative effects through multiple mechanisms, in-
cluding involvement of CB
1
receptors, TRPV1 and PPARγ.
In the light of its safety records and considering that can-
nabidiol is already currently used in patients with multiple
sclerosis [9], our findings suggest that cannabidiol might be
worthy of clinical consideration in colon cancer prevention.
Acknowledgements GA is grateful to Nexus award “Marcello
Tonini”.
Conflict of interest This work was partly supported by GW Phar-
maceuticals (UK).
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