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Antitumor Activity of Plant Cannabinoids with Emphasis on the Effect of Cannabidiol on Human Breast Carcinoma

Authors:
  • Institute of Pharmacology Polish Academy of Sciences, Krakow, Poland

Abstract and Figures

Delta(9)-Tetrahydrocannabinol (THC) exhibits antitumor effects on various cancer cell types, but its use in chemotherapy is limited by its psychotropic activity. We investigated the antitumor activities of other plant cannabinoids, i.e., cannabidiol, cannabigerol, cannabichromene, cannabidiol acid and THC acid, and assessed whether there is any advantage in using Cannabis extracts (enriched in either cannabidiol or THC) over pure cannabinoids. Results obtained in a panel of tumor cell lines clearly indicate that, of the five natural compounds tested, cannabidiol is the most potent inhibitor of cancer cell growth (IC(50) between 6.0 and 10.6 microM), with significantly lower potency in noncancer cells. The cannabidiol-rich extract was equipotent to cannabidiol, whereas cannabigerol and cannabichromene followed in the rank of potency. Both cannabidiol and the cannabidiol-rich extract inhibited the growth of xenograft tumors obtained by s.c. injection into athymic mice of human MDA-MB-231 breast carcinoma or rat v-K-ras-transformed thyroid epithelial cells and reduced lung metastases deriving from intrapaw injection of MDA-MB-231 cells. Judging from several experiments on its possible cellular and molecular mechanisms of action, we propose that cannabidiol lacks a unique mode of action in the cell lines investigated. At least for MDA-MB-231 cells, however, our experiments indicate that cannabidiol effect is due to its capability of inducing apoptosis via: direct or indirect activation of cannabinoid CB(2) and vanilloid transient receptor potential vanilloid type-1 receptors and cannabinoid/vanilloid receptor-independent elevation of intracellular Ca(2+) and reactive oxygen species. Our data support the further testing of cannabidiol and cannabidiol-rich extracts for the potential treatment of cancer.
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Antitumor Activity of Plant Cannabinoids with Emphasis on the
Effect of Cannabidiol on Human Breast Carcinoma
Alessia Ligresti, Aniello Schiano Moriello, Katarzyna Starowicz, Isabel Matias,
Simona Pisanti, Luciano De Petrocellis, Chiara Laezza, Giuseppe Portella, Maurizio Bifulco,
and Vincenzo Di Marzo
Endocannabinoid Research Group, Istituto di Chimica Biomolecolare (A.L., A.S.M., K.S., I.M., V.D.M.), and Istituto di
Cibernetica (A.S.M., L.D.P.), Consiglio Nazionale delle Ricerche Pozzuoli, Italy; Dipartimento di Biologia e Patologia Cellulare e
Molecolare “L. Califano”, Universita` di Napoli “Federico II”, Napoli, Italy (S.P., C.L., G.P., M.B.); and Dipartimento di Scienze
Farmaceutiche, Universita` degli Studi di Salerno, Fisciano, Italy (S.P., M.B.)
Received March 25, 2006; accepted May 23, 2006
ABSTRACT
9
-Tetrahydrocannabinol (THC) exhibits antitumor effects on
various cancer cell types, but its use in chemotherapy is limited
by its psychotropic activity. We investigated the antitumor ac-
tivities of other plant cannabinoids, i.e., cannabidiol, cannabig-
erol, cannabichromene, cannabidiol acid and THC acid, and
assessed whether there is any advantage in using Cannabis
extracts (enriched in either cannabidiol or THC) over pure can-
nabinoids. Results obtained in a panel of tumor cell lines clearly
indicate that, of the five natural compounds tested, cannabidiol
is the most potent inhibitor of cancer cell growth (IC
50
between
6.0 and 10.6
M), with significantly lower potency in noncancer
cells. The cannabidiol-rich extract was equipotent to cannabi-
diol, whereas cannabigerol and cannabichromene followed in
the rank of potency. Both cannabidiol and the cannabidiol-rich
extract inhibited the growth of xenograft tumors obtained by
s.c. injection into athymic mice of human MDA-MB-231 breast
carcinoma or rat v-K-ras-transformed thyroid epithelial cells
and reduced lung metastases deriving from intrapaw injection
of MDA-MB-231 cells. Judging from several experiments on its
possible cellular and molecular mechanisms of action, we pro-
pose that cannabidiol lacks a unique mode of action in the cell
lines investigated. At least for MDA-MB-231 cells, however, our
experiments indicate that cannabidiol effect is due to its capa-
bility of inducing apoptosis via: direct or indirect activation of
cannabinoid CB
2
and vanilloid transient receptor potential va-
nilloid type-1 receptors and cannabinoid/vanilloid receptor-in-
dependent elevation of intracellular Ca
2
and reactive oxygen
species. Our data support the further testing of cannabidiol and
cannabidiol-rich extracts for the potential treatment of cancer.
The therapeutic properties of the hemp plant, Cannabis
sativa, have been known since antiquity, but the recreational
use of its euphoric and other psychoactive effects has re-
stricted for a long time research on its possible pharmaceu-
tical application. The isolation of
9
-tetrahydrocannabinol
(THC), the main psychoactive component of Cannabis (Gaoni
and Mechoulam, 1964), opened the way to further investiga-
tions. After the discovery of the two specific molecular targets
for THC, CB
1
, and CB
2
(for review, see Pertwee, 1997), it
became clear that most of the effects of marijuana in the
brain and peripheral tissues were due to activation of these
two G-protein-coupled cannabinoid receptors. However, evi-
dence is also accumulating that some pharmacological effects
of marijuana are due to Cannabis components different from
THC. Indeed, C. sativa contains at least 400 chemical com-
ponents, of which 66 have been identified to belong to the
class of the cannabinoids (Pertwee, 1997).
To date, cannabinoids have been successfully used in the
treatment of nausea and vomiting (for review, see Robson,
This study was supported by GW Pharmaceuticals (research grant to
V.D.M.).
Article, publication date, and citation information can be found at
http://jpet.aspetjournals.org.
doi:10.1124/jpet.106.105247.
ABBREVIATIONS: THC,
9
-tetrahydrocannabinol; CB
1
, cannabinoid receptor type-1; CB
2
, cannabinoid receptor type-2; TRPV1, transient
receptor potential vanilloid type-1; SR141716A, N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxa-
mide HCl; SR144528, N-[(1S)-endo-1,3,3-trimethylbicyclo[2.2.1]heptan-2-yl]-5-(4-chloro-3-methylphenyl)-1-(4-methylbenzyl)-1-
pyrazole-3-carboxamide; JWH-133, 1,1-dimethylbutyl-1-deoxy-
9
-tetrahydrocannabinol; AM251, N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-di-
chlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide; AM630, 6-iodo-2-methyl-1-[2-(4-morpholinyl)-ethyl]-1H-indol-3-yl](4-methoxyphenyl)-
methanone; ANOVA, analysis of variance; ROS, reactive oxygen species; RT, reverse transcription; PCR, polymerase chain reaction; GAPDH,
glyceraldehyde-3-phosphate dehydrogenase; nt, nucleotide; PBS, phosphate-buffered saline; I-RTX, 5-iodo-resiniferatoxin; HEK, human
embryonic kidney; CBD, cannabidiol; BAPTA-AM, 1,2-Bis(2-aminophenoxy)ethane-N,N,N,N-tetraacetic acid tetrakis (acetoxymethyl ester).
0022-3565/06/3183-1375–1387$20.00
T
HE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 318, No. 3
Copyright © 2006 by The American Society for Pharmacology and Experimental Therapeutics 105247/3130654
JPET 318:1375–1387, 2006 Printed in U.S.A.
1375
2005), two common side effects that accompany chemother-
apy in cancer patients. Nevertheless, the use of cannabinoids
in oncology might be somehow underestimated since increas-
ing evidence exist that plant, synthetic, and endogenous can-
nabinoids (endocannabinoids) are able to exert a growth-
inhibitory action on various cancer cell types. However, the
precise pathways through which these molecules produce an
antitumor effect has not been yet fully characterized, also
because their mechanism of action appears to be dependent
on the type of tumor cell under study. It has been reported
that cannabinoids can act through different cellular mecha-
nisms, e.g., by inducing apoptosis, cell-cycle arrest, or cell
growth inhibition, but also by targeting angiogenesis and cell
migration (for review, see Bifulco and Di Marzo, 2002; Guz-
man, 2003; Kogan, 2005). Furthermore, the antitumoral ef-
fects of plant, synthetic and endocannabinoids can be medi-
ated by activation of either CB
1
(Melck et al., 2000; Bifulco et
al., 2001; Ligresti et al., 2003; Mimeault et al., 2003) or CB
2
receptors or both (Sanchez et al., 2001; Casanova et al., 2003;
McKallip et al., 2005), and, at least in the case of the endo-
cannabinoid anandamide, by transient receptor potential va-
nilloid type-1 (TRPV1) receptors (Maccarrone et al., 2000;
Jacobsson et al., 2001; Contassot et al., 2004) as well as by
noncannabinoid, nonvanilloid receptors (Ruiz et al., 1999).
Additionally, cannabidiol has been suggested to inhibit gli-
oma cell growth in vitro and in vivo independently from
cannabinoid and vanilloid receptors (Massi et al., 2004; Vac-
cani et al., 2005).
The main limitation of the possible future use of THC in
oncology might be represented by adverse effects principally
at the level of the central nervous system, consisting mostly
of perceptual abnormalities, occasionally hallucinations, dys-
phoria, abnormal thinking, depersonalization, and somno-
lence (Walsh et al., 2003). However, most non-THC plant
cannabinoids seem to be devoid of direct psychotropic propri-
eties. In particular, it has been ascertained that cannabidiol
is nonpsychotropic (for review, see Mechoulam et al., 2002;
Pertwee, 2004) and may even mitigate THC psychoactivity
by blocking its conversion to the more psychoactive 11-hy-
droxy-THC (Bornheim and Grillo, 1998; Russo and Guy,
2006). Moreover, it has been recently found that systematic
variations in its constituents (i.e., cannabidiol and canna-
bichromene) do not affect the behavioral or neurophysiolog-
ical responses to marijuana (Ilan et al., 2005). Finally, it has
been also shown that, unlike THC, systemic administration
to rats of cannabigerol does not provoke poly-spike dis-
charges in the cortical electroencephalogram during wake-
fulness and behavioral depression (Colasanti, 1990). These
and other observations reinforce the concept that at least
cannabidiol, cannabigerol, and cannabichromene lack psych-
otropic activity and indicate that for a promising medical
profile in cancer therapy, research should focus on these
compounds, which instead have been poorly studied with
regard to their potential antitumor effects. By keeping this
goal in mind, we decided to investigate the antitumor prop-
erties of cannabigerol and cannabichromene. We also
screened THC acid and cannabidiol acid and two distinct
Cannabis extracts (enriched in either cannabidiol or THC),
where the presence of nonpsychotropic cannabinoids along
with THC has been reported to mitigate the potential side
effects of the latter compound in clinical trials (Russo and
Guy, 2006).
Materials and Methods
Drugs. All plant cannabinoids, the two cannabinoid acids, and the
two Cannabis extracts were kindly provided by GW Pharmaceuticals
(Wiltshire, UK; Fig. 1). Cannabidiol- and THC-rich extracts con-
tained approximately 70% cannabidiol or THC, respectively, to-
gether with lesser amounts of other cannabinoids. The two cannabi-
noid receptor antagonists, SR141716A and SR144528, were a kind
gift from Sanofi-Aventis (Paris, France), whereas methyl-
-cyclodex-
trin, all of the antioxidant drugs (
-tocopherol, vitamin C, astaxan-
tine), N-Acetyl-Asp-Glu-Val-Asp-aldehyde, and BAPTA-AM were
purchased from Sigma-Aldrich (St. Louis, MO). The endocannabi-
noid uptake inhibitor (S)-1-(4-hydroxybenzyl)-N-ethyl-oleoylamide
was synthesized as previously described in Ortar et al. (2003). Fi-
nally, all of the TRPV1 or cannabinoid receptor agonists and antag-
onists (capsaicin, resiniferatoxin, arachidonoyl-2-chloro-ethylamide,
JWH-133, AM251, AM630) were obtained from Tocris Cookson (Bris-
tol, UK).
Cell Cultures. Cell lines from various origins (MCF-7 and MDA-
MB-231 human breast carcinoma cells, DU-145 human prostate
carcinoma cells, CaCo-2 human colorectal carcinoma cells, AGS hu-
man gastric adenocarcinoma cells, C
6
rat glioma cells, KiMol rat
thyroid cells transformed with the v-K-ras oncogene, and rat baso-
philic leukemia cells) were maintained at 37°C in a humidified
atmosphere containing 5% CO
2
. Media, sera, and subculturing pro
-
cedures differed from line to line and were according to the informa-
tion provided in each case by the supplier company (DSMZ, Braun-
schweig, Germany). Primary cells derived from normal human
mammary glands were purchased from Cell Applications, Inc. (San
Diego, CA) and cultured as described in the data sheet from the
supplier.
Cell Proliferation Assay. Six-well culture plates were incubated
at 37°C at a cell density of 5 10
4
cells/well in a humidified atmo
-
sphere containing 5% CO
2
. Three hours after seeding, vehicle or
cannabinoids at different concentrations were added to the medium
and then daily with each change of medium for 4 days, and the effect
Fig. 1. Chemical structures of the plant-derived cannabinoids used in
this study.
1376 Ligresti et al.
of compounds on cell growth was measured by Crystal Violet vital
staining. After staining, cells were lysated in 0.01% acetic acid and
analyzed by spectrophotometer analysis (PerkinElmer Lambda 12,
595 nm; PerkinElmer Life and Analytical Sciences, Boston, MA).
Optical density values from vehicle-treated cells were considered as
100% of proliferation. Statistical analysis was performed using
ANOVA followed by Bonferroni test.
Detection of Reactive Oxygen Species. Intracellular reactive
oxygen species (ROS) generation was determined by spectrofluoro-
metric analysis. MDA-MB-231 cells were plated (16 10
3
cells/well)
in Porvair PS-White Microplate 96-well (PerkinElmer Life and An-
alytical Sciences) for 12 h. The day of the experiment, cells were
rinsed once with Tyrode’s buffer, then loaded (1 h at 37°C in dark-
ness) with 10
M2,7-dichlorofluorescein diacetate (fluorescent
probe; Molecular Probes, Eugene, OR) in the presence of 0.05%
Pluronic F-127. Reactive oxygen species (ROS)-induced fluorescence
of intracellular 2,7-dichlorofluorescein diacetate was measured
with a microplate reader (PerkinElmer LS50B,
Ex
, 495 nm;
Em
, 521
nm). Fluorescence detections were carried out after the incubation of
100
MH
2
O
2
and/or increasing concentrations of cannabidiol at
room temperature in the darkness for different times (0–30-60–120
min). The fluorescence measured at time 0 was considered as basal
ROS production and subtracted from the fluorescence at different
times (
1
). Data are reported as mean S.E. of
2
, i.e., fluorescence
1
values at different doses subtracted of the
1
values of cells
incubated with vehicle. In some experiments, a buffer containing
MgCl
2
in amounts equivalent to CaCl
2
and 0.1 mM EGTA and cells
preloaded for 30 min with BAPTA-AM (40
M) were used instead.
Reverse Transcription-Polymerase Chain Reaction Analy-
sis. Total RNAs from these cells were extracted using the Trizol
reagent according to the manufacturer’s recommendations (Invitro-
gen, Carlsbad, CA). Following extraction, RNA was precipitated
using ice-cold isopropanol, resuspended in diethyl pyrocarbonate
(Sigma-Aldrich)-treated water, and its integrity was verified follow-
ing separation by electrophoresis on a 1% agarose gel containing
ethidium bromide. RNA was further treated with RNase-free DNase
I (Ambion DNA-free kit; Ambion, Austin, TX) according to the man-
ufacturer’s recommendations to digest contaminating genomic DNA
and to subsequently remove the DNase and divalent cations.
The expression of mRNAs for CB
1
,CB
2
, TRPV1, and GAPDH were
examined by semiquantitative RT-PCR. Total RNA was reverse-
transcribed using random primers. DNA amplifications were carried
out in PCR buffer (Invitrogen) containing 2
l of cDNA, 500
M
dNTP, 2 mM MgCl2, 0.8
M of each primer, and 0.5 U of Taq
polymerase platinum (Invitrogen). The thermal reaction profile con-
sisted of a denaturation step at 94°C for 1 min, annealing at 55°C
(GAPDH) or 57°C (CB
2
and TRPV1) or 60°C (CB
1
) for 1 min, and an
extension step at 72°C for 1 min. A final extension step of 10 min was
carried out at 72°C. The PCR cycles observed to be optimal and in the
linear portion of the amplification curve were 24 for GAPDH, 29 for
CB
1
and CB
2
, and 28 for TRPV1 (data not shown). Reaction was
performed in a PE Gene Amp PCR System 9600 (PerkinElmer Life
and Analytical Sciences). After reaction, the PCR products were
electrophoresed on a 2% agarose gel containing ethidium bromide for
UV visualization.
Specific rat and human oligonucleotides were synthesized on the
basis of cloned rat and human cDNA sequences of CB
1
(GenBank
accession nos. NM_012784.3 and X81120 for rat and human, respec-
tively), CB
2
(GenBank accession nos. NM_0205433 and X74328 for
rat and human, respectively), TRPV1 (GenBank accession nos.
NM_031982 and NM_080706.2 for rat and human, respectively), and
GAPDH (GenBank accession nos. NM_017008.2 and BT006893.1 for
rat and human, respectively).
For rat and human CB
1
, the primers sequences were 5-GAT GTC
TTT GGG AAG ATG AAC AAG C-3 (nt 1250 –1274 for rat and nt
1187–1211 for human; sense) and 5-AGA CGT GTC TGT GGA CAC
AGA CAT GG-3 (nt 1558 –1534 for rat and nt 1495–1470 for human;
antisense). The rat CB
2
sense and antisense primers were 5-TA(C/T)
CC(G/A) CCT (A/T)CC TAC AAA GCT C-3 (nt 407– 428) and 5-C
(A/T)GG CAC CTG CCT GTC CTG GTG-3 (nt 698 676), respec-
tively. For human CB
2
, the primers sequences were 5-TTT CCC
ACT GAT CCC CAA TG-3 (nt 672– 691; sense) and 5-AGT TGA
TGA GGC ACA GCA-3 (nt 1000 –983; antisense). For rat TRPV1,
the primers sequences were 5-GAC ATG CCA CCC AGC AGG-3 (nt
2491–2508; sense) and 5-TCA ATT CCC ACA CAC CTC CC-3 (nt
2752–2733; antisense). The human TRPV1 sense and antisense
primers were 5-TGG ACG AGG TGA ACT GGA C-3 (nt 2761–2779)
and 5-ACT CTT GAA GAC CTC AGC GTC-3 (nt 3023–3003), re-
spectively. For rat and human GAPDH, the primers sequences were
5-CCC TTC ATT GAC CTC AAC TAC ATG GT-3 (nt 949 –974 for
rat and nt 106–131 for human; sense) and 5-GAG GGG CCA TCC
ACA GTC TTC TG-3 (nt 1418 –1396 for rat and nt 575–553 for
human; antisense).
The expected sizes of the amplicons were 309 bp for rat and human
CB
1
, 291 bp for rat CB
2
, 329 bp for human CB
2
, 263 bp for rat
TRPV1, 262 bp for human TRPV1, and 470 bp for rat and human
GAPDH. In the presence of contaminant genomic DNA, the expected
size of the amplicons would be 1062 bp for GAPDH (data not shown).
No PCR product was detected when the reverse transcriptase step
was omitted.
Western Immunoblotting Analysis for Caspase-3. Immuno-
blotting analysis was performed on the cytosolic fraction of cells
treated as described above and according to previous published work
(Iuvone et al., 2004). Cytosolic fraction proteins were mixed with gel
loading buffer (50 mM Tris/10% SDS/10% glycerol 2-mercaptoetha-
nol/2 mg of bromphenol/ml) in a ratio of 1:1, boiled for 5 min and
centrifuged at 10,000g for 10 min. Protein concentration was deter-
mined, and equivalent amounts (50
g) of each sample were sepa-
rated under reducing conditions in 12% SDS-polyacrylamide mini-
gel. The proteins were transferred onto nitrocellulose membrane,
according to the manufacturer’s instructions (Bio-Rad, Hercules,
CA). The membranes were blocked by incubation at 4°C overnight in
high-salt buffer (50 mM Trizma base, 500 mM NaCl, 0.05% Tween
20) containing 5% bovine serum albumin and then incubated for 2 h
with anticaspase 3 (1:2000, v/v) at room temperature, followed by
incubation for 2 h with horseradish peroxidase-conjugate secondary
antibody (Dako, Glostrup, Denmark). The immune complexes were
developed using enhanced chemiluminescence detection reagents
(GE Healthcare, Little Chalfont, Buckinghamshire, UK) according to
the manufacturer’s instructions and exposed to Kodak X-OMAT film
(Eastman Kodak, Rochester, NY). The bands of
protein on X-ray
film were scanned and densitometrically analyzed with a GS-700
imaging densitometer.
Immunofluorescence. For immunoreaction, MDA-MB-231 cells
were seeded on sterile coverslips (22 22 mm; Menzel, Braun-
schweig, Germany) in six-well culture plates and incubated under
standard conditions until they were at least 70% confluent. Cultured
cells were processed for immunofluorescence. After three washes
with PBS, cells were fixed by incubating them in 4% (v/v) parafor-
maldehyde in PBS for 20 min at room temperature, rinsed with PBS,
permeabilized for 15 min in 0.5% Triton X-100 in PBS, and incubated
overnight at 4°C with rabbit polyclonal rabbit anti-CB
1
or anti-CB
2
antibody (Cayman Chemical, Ann Arbor, MI), both diluted 1:50 in
0.5% Triton X-100 in PBS, or goat anti-TRPV1 antibody (Santa Cruz
Biochemicals, Santa Cruz, CA) diluted 1:100 in 0.5% Triton X-100 in
PBS. After three washes in PBS, fluorescence was revealed by incu-
bation for2hinanAlexa Fluor 488-labeled secondary anti-rabbit
antibody (Invitrogen) diluted 1:100 in 0.5% Triton X-100 in PBS or
Alexa Fluor 546-labeled secondary anti-goat antibody (Invitrogen)
diluted 1:200 in 0.5% Triton X-100 in PBS. The preabsorption of
antibodies with the respective blocking peptides as well as omission
of primary antibodies (control immunoreaction) resulted in much
weaker or negative staining, respectively. Sections processed for
immunofluorescence were studied with an epifluorescence micro-
scope equipped with the appropriate filter (Leica DM IRB; Leica,
Wetzlar, Germany). Images were acquired using a digital Leica DFC
Cannabidiol in Cancer
1377
320 camera connected to the microscope and the image analysis
software Leica IM500. Images were processed in Adobe Photoshop
(Adobe Systems, Mountain View, CA), with brightness and contrast
being the only adjustments made.
In Vivo Studies: Effect on Xenograft Models of Carcinoma.
All of the experiments were performed by using Charles River
6-week-old male athymic mice (Charles River, Margate, Kent, UK)
as described previously (Bifulco et al., 2001). Two different mouse
xenograft models of tumor growth were induced by s.c. injection (5
10
5
cells) of two distinct highly invasive tumoral cell lines (KiMol or
MBA-MD-231 cells) into the dorsal right side of athymic mice. Start-
ing from the appearance of tumoral mass, pure compounds or Can-
nabis extracts were injected intratumor in the same inoculation
region twice per week for 20 (KiMol cell-induced tumors) or 16
(MBA-MD-231 cell-induced tumors) days. THC and cannabidiol were
administered at the dose of 5 mg/kg, whereas THC-rich and canna-
bidiol-rich were administrated at the dose of 6.5 mg/kg, which con-
tains 5 mg/kg THC and cannabidiol, respectively. Tumor diameters
were measured with calipers every other day until the animals were
killed. Tumor volumes (V) were calculated by the formula of rota-
tional ellipsoid (V AxB2/2; A axial diameter, B rotational
diameter). Results were reported as means S.E. Statistical anal-
ysis was performed using ANOVA followed by the Bonferroni’s test.
In Vivo Analysis: Effect on Experimental Lung Metastasis.
Monocellular suspension of MDA-MB-231 cells containing 2.5 10
5
cells was injected into the left paw of 30-day-old BalB/c male mice.
Animals were divided into three groups: vehicle (n 11), cannabidiol
(5 mg/kg/dose, n 14), or cannabidiol-rich (6.5 mg/kg/dose, n 14).
The drugs were injected i.p. every 72 h. Experimental metastases
were evaluated 21 days after the injection. To contrast lung nodules,
lungs were fixed in Bouin’s fluid, and metastatic nodes were scored
on dissected lung under a stereoscopic microscope. All animal studies
were conducted in accordance with the Italian regulation for the
welfare of animals in experimental neoplasia. All data were pre-
sented as means S.D. Statistical analysis was performed using
one-way ANOVA.
Cell Cycle and Apoptosis Detection. Different cell lines were
exposed to 10
M of cannabidiol or cannabigerol for 48 h at 37°C in
a humidified atmosphere containing 5% CO
2
. The distribution of
cells among the different phases of the cell cycle and apoptosis rate
were evaluated by flow cytometric analysis of the DNA content. Cells
(5 10
5
) were collected, washed twice with PBS, fixed by ethanol
70%, and kept at 20°C for at least 4 h. Propidium iodide (10
g/ml)
in PBS containing 100 U/ml DNase-free RNase was added to the cells
for 15 min at room temperature. Cells were acquired by a FACScali-
bur flow cytometer (BD Biosciences, San Jose, CA), and then analy-
sis was performed using ModFit LT version 3.0 from Verity Software
House, Inc. (Topsham, ME); 10,000 events were collected and cor-
rected for debris and aggregate populations.
Anandamide Cellular Reuptake and Intracellular Hydroly-
sis. The effect of compounds on anandamide cellular reuptake was
analyzed on rat basophilic leukemia cells or MDA-MB-231 cells by
using 2.5
M (10,000 cpm) [
14
C]anandamide as described previously
(De Petrocellis et al., 2000). Briefly, cells were incubated with
[
14
C]anandamide for 5 min at 37°C, in the presence or absence of
varying concentrations of the compounds. Residual [
14
C]anandamide
in the incubation medium after extraction with CHCl
3
/CH
3
OH 2:1
(by volume), determined by scintillation counting of the lyophilized
organic phase, was used as a measure of the anandamide that was
taken up by cells (De Petrocellis et al., 2000). Nonspecific binding of
[
14
C]anandamide to cells and plastic dishes was determined in the
presence of 100
M anandamide and was never higher than 30%.
Data are expressed as the concentration exerting 50% inhibition of
anandamide uptake (IC
50
) calculated by GraphPad software (Graph
-
Pad Software Inc., San Diego, CA). The effect of compounds on the
enzymatic hydrolysis of anandamide was studied using membranes
prepared from N18TG2 cells, incubated with the test compounds and
[
14
C]anandamide (20,000 cpm; 5
M) in 50 mM Tris-HCl, pH 9, for
30 min at 37°C. [
14
C]Ethanolamine produced from [
14
C]anandamide
hydrolysis was measured by scintillation counting of the aqueous
phase after extraction of the incubation mixture with 2 volumes of
CHCl
3
/CH
3
OH 1:1 (by volume). Data are expressed as the concen
-
tration exerting 50% inhibition of [
14
C]anandamide hydrolysis
(IC
50
), calculated by GraphPad software.
Activity at Human Recombinant TRPV1. The effect of the
substances on [Ca
2
]
i
was determined by using Fluo-3 (Molecular
Probes), a selective intracellular fluorescent probe for Ca
2
(De Petro
-
cellis et al., 2000). Human embryonic kidney (HEK) 293 cells stably
overexpressing human TRPV1 receptor or MDA-MB-231 cells were
transferred into six-well dishes coated with poly-
L-lysine (Sigma-Al-
drich) 1 day prior to experiments and grown in the culture medium
mentioned above. On the day of the experiment, the cells (50 60,000
cells/well) were loaded for2hat25°C with 4
M Flu-3-methylester
(Invitrogen) in dimethyl sulfoxide containing 0.04% Pluronic F-127
(Invitrogen). After loading, cells were washed with Tyrode’s solution,
pH 7.4, trypsinized, resuspended in Tyrode’s solution, and transferred
to the cuvette of the fluorescence detector (PerkinElmer LS50B) under
continuous stirring. Experiments were carried out by measuring cell
fluorescence at 25°C (
EX
488 nm,
EM
540 nm) before and after the
addition of the test compounds at various concentrations. Data are
expressed as the concentration exerting a half-maximal effect (EC
50
).
The efficacy of the effect was determined by comparing it with the
analogous effect observed with 4
M ionomycin. In some experiments
with MDA-MB-231 cells, the effect of cannabidiol was measured also in
the absence of extracellular Ca
2
(i.e., in a Tyrode’s solution containing
Mg
2
instead of Ca
2
and 0.1 mM EGTA) and in cells preloaded with
BAPTA-AM (20
M).
Results
Effect on Cancer Cell Growth: In Vitro Studies. For in
vitro studies, the cannabinoids under investigation were
screened for their ability to reduce cell proliferation on a
collection of tumoral cell lines. Cannabidiol always exhibited
the highest potency with IC
50
values ranging between 6.0
TABLE 1
Effect of cannabinoids and Cannabis extracts on cancer cell growth
Various epithelial cell lines of various tumoral origin were treated with different concentrations of drugs, and after 4 days, the cell number was measured with Crystal Violet
Vital staining (see Materials and Methods). Data are reported as mean S.E. of IC
50
values (micromolar) calculated from three independent experiments. CBG, cannabigerol;
CBC, cannabichromene; CBD-A, cannabidiol-acid; THC-A, THC-acid; CBD-rich, cannabidiol-enriched cannabis extract; THC-rich, THC-enriched cannabis extract.
MCF-7 C
6
DU-145 KiMol CaCo-2 MDA-MB-231 RBL-2H3 AGS
9
-THC
14.2 2.1 23.0 4.2 25 23.2 1.5 16.5 0.2 24.3 4.2 15.8 3.7 19.3 1.5
THC-A 9.8 0.4 18.0 5.3 25 21.0 2.7 21.5 1.4 18.2 5.3 10.0 3.4 25
CBD 8.2 0.3 8.5 0.8 20.2 1.8 6.0 3.0 7.5 0.5 10.6 1.8 6.3 1.5 7.5 1.3
CBD-A 21.7 3.2 18.0 4.2 25 12.7 3.0 25 25 25 25
CBG 9.8 3.4 13.0 2.1 21.3 1.7 8.2 0.7 9.0 1.4 16.2 2.1 9.0 0.7 8.2 0.7
CBC 14.2 1.4 13.0 2.6 25 7.3 3.0 12.0 2.4 20.4 2.6 15.8 4.2 18.3 3.0
THC-rich 21.0 0.5 18.5 3.3 25 23.0 2.0 16.0 0.5 25.2 3.3 14.6 3.1 22.0 2.0
CBD-rich 6.0 1.0 4.7 0.6 20 4.6 6.2 2.9 12.3 1.2 14.1 1.6 7.0 0.6 10.0 1.9
1378 Ligresti et al.
3.0 and 10.6 1.8
M (Table 1). Cannabidiol acid was the
least potent compound. Among the other plant cannabinoids,
cannabigerol was almost always the second most potent com-
pound, followed by cannabichromene (Table 1). The effect of
the two Cannabis extracts (enriched in cannabidiol or THC)
was next investigated, and in some circumstances, the can-
nabidiol-rich extract appeared slightly more potent than
pure cannabidiol (Table 1). In the case of MCF-7 cells, both
compounds exhibited quite similar potency, as indicated by
the IC
50
values of 8.2 0.3 and 6.0 1.0
M, respectively,
for cannabidiol and cannabidiol-rich extract (Fig. 2A), on the
contrary, in the case of C
6
glioma cells, cannabidiol-rich
extract also exhibited significantly higher potency than pure
cannabidiol (IC
50
, 4.7 0.6 and 8.5 0.8
M, respectively,
p 0.05, Fig. 2B). Only in the case of human DU-145 pros-
tate carcinoma cells, plant cannabinoids induced a stimula-
tory effect on cancer growth at the lowest doses tested and an
inhibitory effect only at the highest concentration tested (25
M) (as also found by Sanchez et al., 2003 in another prostate
carcinoma cell line). In this case, however, the cannabidiol-
rich extract lacked the pro-proliferative effect even at the
lowest concentration tested of 2
M (Fig. 2, C and D).
For a comparison, we also tested cisplatinum on some cell
lines and found that this widely used anticancer compound
as compared with cannabidiol was only 2.5-, 8.8-, and 3.9-fold
more potent in MCF-7, MDA-MB-231, and AGS cells (IC
50
3.2 0.3, 1.2 0.2, and 1.9 0.2
M, respectively) and 17-
and 33.6-fold more potent in C
6
and DU-145 cells (IC
50
0.5 0.1 and 0.6 0.2
M, respectively).
The trypan blue dye exclusion method on the entire range
of cells was used to detect cytotoxicity and to assess cell
viability. All of the compounds under investigation showed a
statistically significant cytotoxic effect starting only from the
highest concentration tested (25
M) (data not shown).
Finally, to investigate the selectivity of cannabidiol effect
in tumoral versus nontumoral cells, various concentrations
(from 1–100
M) of cannabidiol on different stabilized non-
tumor cell lines such as HaCat (human keratinocyte), 3T3-
F442A (rat preadipocytes), and RAW 264.7 (mouse monocyte-
macrophages) were also tested. Cannabidiol, at a dose
similar to its IC
50
values in the various tumoral cell lines, did
not affect the vitality of nontumor cell lines (Fig. 2E). Only at
a concentration of 25
M, which exerts nearly 100% inhibi-
tion of cancer cell growth, cannabidiol exhibited a cytotoxic
effect in these nontumoral cell lines (Fig. 2E). Lastly, it was
examined the selectivity of cannabidiol versus a primary cell
line derived from mammary glands (human mammary epi-
thelial cells) since several experiments on the mechanism of
action of cannabidiol were performed using a human breast
carcinoma cell line (MBA-MD-231 cells). Cannabidiol af-
fected significantly the vitality of this cell line only at a 25
M concentration (Fig. 2F).
Effect on Cancer Cell Growth: In Vivo Studies. For
the in vivo studies, the efficacy of cannabidiol and its en-
riched extract at reducing tumor size and volume was eval-
uated. Mice treated with either pure cannabidiol or the can-
nabidiol-rich extract exhibited significantly smaller tumors
in comparison with control mice. A strong and statistically
significant antitumor effect was observed with both treat-
ments and with both in vivo xenograft tumor models used
(Fig. 3, A and B). The effect of cannabidiol and cannabidiol-
rich compounds on the formation of lung metastatic nodules
of MBA-MD-231 cells injected into the paw was also investi-
gated. Both cannabidiol and cannabidiol-rich exhibited a
strong and significant reduction of metastatic lung infiltra-
tion (Fig. 3C).
Study on the Cellular Mechanism of Action of Can-
nabidiol. With the intention to evaluate if the inhibitory
effect on cell growth of cannabidiol was associated with apo-
ptotic events or blockade of mitogenesis, the percentage of G
1
population cells was estimated by flow cytometry. In MCF-7
cells, a hormone-sensitive cell line, cannabidiol, exerted an-
tiproliferative effect by causing a cell cycle block at the G
1
/S
phase transition (Fig. 4A; Table 2). A similar result was
observed in another hormone-sensitive cell line KiMol cells,
where, however, the antiproliferative effect of cannabidiol
was also accompanied by a proapoptotic action (Fig. 4C;
Table 2). Finally, in C
6
glioma and MDA-MB-231 cells (two
nonhormone-sensitive cell lines), cannabidiol provoked a
pure proapoptotic effect (Fig. 4D; Table 2). The proapoptotic
effect of cannabidiol on MDA-MB-231 cells was also estab-
lished by evaluating the involvement of caspase-3. The pro-
apoptotic effect of cannabidiol was confirmed in this cell line
but not in DU-145 cells, as indicated by the procaspase-3
cleavage into caspase-3 by Western immunoblotting analysis
following a 48-h treatment of cells with the compound (Fig.
4E). In agreement with a role of apoptosis and caspase-3
in cannabidiol anticancer effect in MDA-MB-231 cells, N-
Acetyl-Asp-Glu-Val-Asp-aldehyde (10
M), an inhibitor of
caspase-3, significantly attenuated the growth-inhibitory ef-
fect of both 5 and 10
M cannabidiol as indicated by the
reduction of the percentage inhibition of cell proliferation
induced by these two doses of the cannabinoid (from 21.8
3.1 to 7.8 1.1% at 5
M and from 55.8 4.9 to 11.9 1.6%
at 10
M; means S.E.; n 3, p 0.01).
Study on the Molecular Mechanism of Action of
CBD. When using PCR, we found that both vanilloid TRPV1
receptors and cannabinoid CB
1
and CB
2
receptors are ex
-
pressed in most of the cell lines used in this study (Table 3).
To estimate the involvement of TRPV1 receptors in the an-
tiproliferative properties, all cannabinoids were screened for
their capability to generate TRPV1-mediated intracellular
calcium elevation in stably transfected TRPV1-HEK293
cells. Apart from cannabidiol, only cannabigerol and canna-
bidiol acid activated TRPV1 receptors, with a significantly
lower potency than cannabidiol, whereas cannabichromene,
THC, and THC acid were almost inactive (Fig. 5). The can-
nabidiol-rich extract was as efficacious and potent as canna-
bidiol, whereas the THC-rich extract was more efficacious
and potent than THC, possibly due to the presence of other
TRPV1-active cannabinoids, including cannabidiol and can-
nabigerol (Fig. 5).
To assess whether plant cannabinoids, which are very
weak agonists of CB
1
and CB
2
receptors, activate these
receptors indirectly, i.e., by elevating endocannabinoid lev-
els, we studied their effects on anandamide cellular uptake
and enzymatic hydrolysis (Bifulco et al., 2004). Although
most of the compounds tested did inhibit anandamide me-
tabolism (Table 4), particularly at the level of cellular
uptake, their rank of potency (cannabichromene canna-
bigerol cannabidiol THC) did not reflect their potency
at inhibiting cancer cell proliferation.
To conclusively investigate the role of vanilloid TRPV1
receptors and cannabinoid CB
1
and CB
2
receptors in the
Cannabidiol in Cancer 1379
Fig. 2. Effect of cannabinoids and Cannabis extracts on the proliferation of some of the cell lines investigated in this study. MCF-7 cells (A), C
6
cells
(B), and DU-145 cells (C and D) were treated with increasing concentrations of cannabidiol, cannabinoids (C), and cannabidiol-rich extracts (daily
added with each change of medium for 4 days). Effect on cell proliferation was measured by Crystal Violet vital staining. After staining, cells were
lysated in 0.01% acetic acid and analyzed by spectrophotometric analysis (PerkinElmer Lambda 12,
595 nm). Results are reported as percentage
of inhibition of proliferation where optical density value from vehicle-treated cells was considered as 100% of proliferation and represent the mean
S.E. of three different experiments. , p 0.05 versus cannabidiol pure by ANOVA followed by Bonferroni’s test. E and F, effect of cannabidiol on
nontumoral cell lines. Cells were treated with two different concentrations of cannabidiol for 4 (E) or 3 days (F), and vitality was evaluated by using
trypan blue dye exclusion assay (see Materials and Methods). In cells treated with vehicle, mortality was always lower than 2%. Data are expressed
as percentage of control and represent the mean S.E. of three different experiments. Statistical analysis was carried out by ANOVA followed by
Bonferroni’s test (ⴱⴱ, p 0.01; ⴱⴱⴱ, p 0.001 versus the same concentration of cannabidiol on MDA-MB-231 cells). CBG, cannabigerol; CBC,
cannabichromene; CBD-A, cannabidiol acid; THC-A, THC acid; CBD-rich, cannabidiol-enriched cannabis extract; THC-rich, THC-enriched cannabis
extract.
1380 Ligresti et al.
anticancer effects of plant cannabinoids, in all those cell lines
where cannabinoid or vanilloid receptors were expressed (Ta-
ble 3), we studied the effect of selective antagonists, alone or
in combination, on the inhibitory effect of 10
M cannabidiol.
Whereas 5-iodo-resiniferatoxin (I-RTX, 100 nM) was used as
a TRPV1-selective antagonist, SR141716A (0.5
M) and
SR144528 (0.5
M) were used as selective antagonists for
CB
1
and CB
2
receptors, respectively. A statistically signifi
-
cant effect of selective concentrations of the two antagonists,
I-RTX and SR144528, was found only in MDA-MB-231 cells;
however, these molecules were able to revert only partially
the effect of cannabidiol. Higher doses of the two compounds
inhibited cell number per se and were not used. When I-RTX
and SR144528 were administered together, cannabidiol ef-
fect was attenuated by about 40%, although this effect was
probably minimized by the fact that the mixture of antago-
nists significantly inhibited cell growth per se (Fig. 6). These
findings are in agreement with the results obtained by im-
munofluorescence and showing high levels of CB
2
and
TRPV1 receptors in the plasma membrane of intact MDA-
MB-231 cells (Fig. 7). Regarding the CB
1
antagonist, despite
the presence in MDA-MB-231 cells of CB
1
receptors (Fig. 7),
SR141716A (0.5
M) did not influence the effect of cannabi-
diol (data not shown). No effect was observed with any of the
three antagonists in the other cell lines, except for KiMol
cells, were the mixture of antagonists showed a slight inhi-
bition (15 2%), which was not statistically significant (data
not shown).
Role of Vanilloid and Cannabinoid Receptors in
MDA-MB-231 Cells. Starting from the experiments with
TRPV1 and CB
1
and CB
2
receptor antagonists, we further
investigated the role of direct or indirect activation of these
receptors in cannabidiol effect on MDA-MB-231 cell growth.
Cannabidiol and THC-A, tested at a 25
M concentration, did
inhibit the uptake of [
14
C]anandamide by MDA-MB-231 cells
(25.1 2.5% and 21.0 3.0% inhibition, respectively,
Fig. 3. In vivo actions of cannabidiol on tumor growth and metastasis. A and B, effect of cannabidiol (5 mg/kg) and cannabidiol-rich extract (6.5 mg/kg)
on two different xenograft tumor models in athymic mice. KiMol cells (A) or MBA-MD-231 cells (B) were injected s.c. (day 0 of treatment) into the dorsal
right side of athymic mice, and the intratumor treatments were administered twice per week. Results represent mean S.E. (, p 0.05 versus
vehicle; n 6 by ANOVA followed by Bonferroni’s test). C, effect of cannabidiol and cannabidiol-rich extract on breast cancer cell metastasis.
MDA-MB-231 cells were injected into the left paw of 30-day-old BalB/c male mice. Animals were divided into three groups (n 11 for vehicle; n 14
for treated) and treated with vehicle (CTR), cannabidiol (5 mg/kg/dose), or cannabidiol-rich extract (6.5 mg/kg/dose). The drugs were injected i.p. every
72 h. Lung metastatic nodules were evaluated 21 days after the injection. Data represent mean S.E. of number of nodules (, p 0.05; ⴱⴱ, p 0.01
versus CTR). Statistical analysis was performed by ANOVA followed by Bonferroni’s test. CBD-rich, cannabidiol-enriched cannabis extract.
Cannabidiol in Cancer 1381
mean S.E.; n 4), but cannabigerol and a selective inhib-
itor of anandamide cellular uptake, (S)-1-(4-hydroxybenzyl)-
N-ethyl-oleoylamide, were significantly more efficacious at
exerting this effect (82.0 3.5% and 77.0 3.1% inhibition,
respectively), even though they were significantly less effica-
cious than cannabidiol at inhibiting cell growth (Table 1;
data not shown). Furthermore, direct agonists of CB
1
and
CB
2
receptors, i.e., arachidonoylchloroethanolamide and
JWH-133, were also less potent and efficacious than canna-
bidiol at inhibiting MDA-MB-231 cell growth (data not
shown). We also studied in MDA-MB-231 cells the effect of
Fig. 4. A to D, representative fluorescence-activated cell sorter analyses
showing the effect of 2 days of treatment of 10
M cannabidiol (CBD) on
apoptosis rate in various cell lines calculated as the percentage of cells
showing a subdiploid DNA peak (subG1). Graphs are representative of
three independent experiments with similar results. Graphs on the left
are from cells treated with vehicle and those on the right from cells
treated with cannabidiol. Line bar shows where the subdiploid DNA peak
is calculated. CTR, vehicle-treated cells. E, effect of cannabidiol on
caspase 3 release from procaspase. Western immunoblotting analysis was
performed to detect the levels of caspase-3 expression. Proteins were
extracted from DU-145 cells (lanes 1 and 2) or MDA-MB-231 cells (lanes
3 and 4) treated with vehicle (CTR, lanes 1 and 3) or 10
M cannabidiol
(cannabidiol, lanes 2 and 4) for 48 h. Determination of relative band
intensity was carried out using a GS700 densitometer, and the results are
presented in arbitrary scanning units. DU-145, CTR 5.7 0.81; can-
nabidiol 4.2 0.74; MDA-MB-231, CTR 3.11 0.67; cannabidiol
2.64 0.26 (Procaspase), 2.89 0.51 (Caspase), mean S.E. of n 3
separate experiments.
Fig. 5. Effect of plant cannabinoids and Cannabis extracts on vanilloid
TRPV1 receptor activation. HEK293 cells overexpressing the human
recombinant TRPV1 receptor were loaded with a selective fluorescent
probe (see Materials and Methods). The TRPV1-mediated effect on
[Ca
2
]
i
was determined by measuring cell fluorescence before and after
the addition of the test compounds at increasing concentrations. Data are
reported as percentage of the maximal effect obtained with Ionomycin 4
M and are means of n 3 separate experiments. Error bars are not
shown for the sake of clarity and were never higher than 5% of the means.
CBG, cannabigerol; CBC, cannabichromene; CBD-A, cannabidiol-acid;
THC-A, THC acid; CBD-rich, cannabidiol-enriched cannabis extract;
THC-rich, THC-enriched cannabis extract.
TABLE 2
Determination of cell cycle arrest, apoptosis, and mortality in the
various cell lines exposed for 48 h to 10
M of cannabidiol before flow
cytometry analysis (see Fig. 4 and Materials and Methods); each
experiment was repeated three times
Cell Type Cell Cycle Arrest Apoptosis Mortality
DU-145 Absent 10 Absent
MCF-7 G
1
/S
Absent Absent
C
6
Absent 9–10% 25–27%
KiMol G
1
/S
12–15% 20–22%
MDA-MB-231 Absent 15% Absent
TABLE 3
Schematic and qualitative representation of the results of the RT-PCR
analyses of mRNAs for cannabinoid and vanilloid receptors in the cell
lines under study
Total RNA from cells was extracted, and its integrity was verified. RNA was further
treated with RNase-free DNase I (Ambion DNA-free kit) to digest contaminating
genomic DNA and to subsequently remove the DNase and divalent cations. The
expression of mRNAs was examined by RT-PCR. Transcripts for fatty acid amide
hydrolase (FAAH) and CB
1
and CB
2
receptors were analyzed and are classified as: a,
abundant; m, medium; w, weak; and nd, not detected, based on the intensity of the
band normalized to the band corresponding to glyceraldehyde-3-phosphate dehydro-
genase as the housekeeping gene and on the number of cycles necessary to obtain a
visible band. Results are based on n 3 separate determinations.
Cell Type CB
1
CB
2
TRPV1
AGS nd nd a
DU-145 a w a
MCF-7 w w a
C
6
mw m
KiMol w a m
CaCo-2 w a a
RBL-2H3 nd a nd
MDA-MB-231 w m a
1382 Ligresti et al.
cannabidiol (5
M) after a 10-min exposure to methyl-
-
cyclodextrin (0.5 mM), a potent membrane cholesterol depl-
eter that is able to destroy the lipid raft microdomains and to
block the clustering of CB
1
at the plasma membrane in MDA-
MB-231 cells (Sarnataro et al., 2005). We found no significant
effect on the inhibitory action of cannabidiol (from 29.9
3.5% to 30.2 3.6% inhibition, mean S.E.; n 3; p 0.05).
Regarding TRPV1 receptors, we investigated whether can-
nabidiol induces intracellular Ca
2
elevation also in MDA-
MB-231 cells. Cannabidiol did induce a rapid and sustained
elevation of intracellular Ca
2
in MDA-MB-231 cells (EC
50
0.7 0.1
M, maximal effect at 10
M cannabidiol 24.5
0.3% of the effect of 4
M ionomycin, Fig. 8, A and B) but in
a way that was not blocked by I-RTX, nor by CB
1
or CB
2
receptor antagonists (Fig. 8D). In agreement with these data,
we also found that potent selective agonists of TRPV1 recep-
tors, such as capsaicin and RTX, respectively, exerted little
effect on MDA-MB-231 cell growth (data not shown). More-
over, cannabidiol effect on intracellular Ca
2
did not require
the presence of extracellular Ca
2
(EC
50
0.14 0.01
M,
Fig. 8C), indicating that it occurs mostly at the level of
intracellular stores and was in fact blocked after loading the
cells with the Ca
2
chelating agent BAPTA-M (Fig. 8D).
Involvement of Oxidative Stress in CBD Actions on
MDA-MB-231 Cells. MDA-MB-231 cells were selected also
to investigate the implications of cannabidiol effects on oxi-
dative stress phenomena. The effects of antioxidant agents
on the antiproliferative action of 10
M cannabidiol were
evaluated. Already at 0.1
M concentration,
-tocopherol
significantly prevented, although in a partial manner, the
antiproliferative effects of cannabidiol on these cells (Fig.
9A); also, vitamin C and astaxantine, at 25
M concentra-
tion, were able to counteract the inhibitory effect of cannabi-
diol by 30% (data not shown). Further experiments were
performed to measure the intracellular ROS generation.
Cannabidiol in a dose- and time-dependent manner induced
ROS formation in MDA-MB-231 cells in a Ca
2
-containing
buffer (Fig. 9B). Importantly, the effect of cannabidiol (10
M) on ROS production (60 min) was Ca
2
-dependent be
-
cause it was erased when cells were preloaded with
BAPTA-AM (40
M) and incubated in an isotonic buffer with
the same ionic strength but with Mg
2
instead of Ca
2
(Fig.
9B, inset). Next, we carried out different incubations under
both standard and severe growth cell culturing conditions
that lead to a strong production of ROS, i.e., with 12-h serum
deprivation; subsequently, cells were treated either with low
or high concentration of cannabidiol only for 24 h, as opposed
to the 96-h incubation used in most of the experiments pre-
sented here. In nonserum-deprived cells, cannabidiol exerted
Fig. 6. Influence of selective receptor antagonists on CBD antiprolifera-
tive action. MDA-MB-231 cells were treated with 10
M cannabidiol in
presence or in absence of selective antagonist for CB
2
receptors [0.5
M
SR144528 (SR
2
)], TRPV1 receptors [100 nM 5-I-resiniferatoxin (I-RTX)],
or a mixture of both compounds (mix). Data are shown as percent inhi-
bition of proliferation. Cells vehicle-treated were used as 100% of prolif-
eration. , p 0.05 versus cannabidiol only, by ANOVA followed by
Bonferroni’s test.
Fig. 7. Representative photomicro-
graphs demonstrating localization of
CB
1
,CB
2
, and TRPV1 receptors in hu
-
man breast adenocarcinoma (MDA-
MB-231) cells as determined by the im-
munofluorescence technique described
under Materials and Methods.A,CB
1
receptor immunoreactivity. B, CB
2
re
-
ceptor immunoreactivity. C, TRPV1-
immunoreactivity was performed using
rabbit polyclonal anti-CB
1
, anti-CB
2
(both diluted 1:50), and Alexa Fluor
488-conjugated secondary antibody (1:
100) and goat polyclonal anti-TRPV1
diluted 1:100 and Alexa Fluor 546-con-
jugated secondary antibody (1:200).
Magnification, 63. Scale bar, 40
m.
Immunofluorescence was almost unde-
tectable when preincubating antibodies
with the corresponding blocking pep-
tides (data not shown).
TABLE 4
Effect of plant cannabinoids on anandamide inactivation
Membranes from N18TG2 cells were incubated with
14
Canandamide in the pres
-
ence of compounds for 30 min at 37°C (see Material and Methods) to determine the
effect on the enzymatic hydrolysis by fatty acid amide hydrolase (FAAH). Intact
RBL-2H3 cells were incubated with
14
Canandamide in the presence of compounds
for 5 min at 37°C (see Material and Methods) to determine the effect on anandamide
cellular uptake (ACU). Data represent mean S.E. of three different experiments.
FAAH Assay IC
50
ACU Assay IC
50
M
THC 50 22 5
CBD 28 3
a
22 2
a
CBG 50 15 3
CBC 50 13 2
THC-A 50 25
CBD-A 50 25
CBG, cannabigerol; CBC, cannabichromene; CBD-A, cannabidiol-acid; THC-A,
THC-acid.
a
Data from Bisogno et al., 2001.
Cannabidiol in Cancer 1383
a pro-proliferative effect at low doses (0.5
M), whereas it
was ineffective after serum deprivation (Fig. 9, A and B). At
the highest concentration tested (10
M), the growth-inhib-
itory effect was much stronger than that caused by the same
dose without serum deprivation (Fig. 10, A and B). The effect
of cannabidiol on ROS formation induced by 100
MH
2
O
2
was also measured. In conformity with the results obtained
in the short-term cell proliferation assays, cannabidiol, de-
spite its stimulatory activity on ROS formation when admin-
istered per se (Fig. 9B), was able to reduce ROS production
induced by 100
MH
2
O
2
, but only at the lowest concentra
-
tion tested (Fig. 10C).
Discussion
The aim of this study was to identify natural cannabinoids
with antitumor activities at least similar to those of THC and
devoid of the potential central effects of this compound.
Given that the efficacy of cannabinoids as antitumoral agents
appears to be strictly correlated to the cell type under inves-
tigation, we screened a panel of plant cannabinoids in a wide
range of tumoral cell lines distinct in origin and typology. We
found that, surprisingly, cannabidiol acted as a more potent
inhibitor of cancer cell growth than THC and that cannabig-
erol and cannabichromene usually followed cannabidiol in
the rank of potency. The cell growth-inhibitory effect of can-
nabidiol depended on its chemical structure since the addi-
tion of a carboxylic acid group (as in cannabidiol acid) dra-
matically reduced its activity. This is unlikely due to simple
modification of the lipophilicity of the compound and subse-
quent decrease of its capability to penetrate the cell mem-
brane since THC-A was often more efficacious than THC. We
also found that the cannabidiol-rich Cannabis extract was as
potent as pure cannabidiol in most cases or even more potent
in some cell lines. These results suggest the use in cancer
therapy for cannabidiol, a compound lacking the psychotropic
effects typical of THC. Indeed, the efficacy of cannabidiol and
of the cannabidiol-rich extract were confirmed in vivo in two
different models of xenograft tumors obtained by inoculation
in athymic mice of either v-K-ras-transformed thyroid epi-
thelial cells or of the highly invasive MDA-MB-231 breast
Fig. 8. Effect of CBD on intracellular Ca
2
in MDA-MB-231 cells. A, dose-related effect of cannabidiol in the presence of extracellular Ca
2
,as
determined with Fluo-4. Data are mean S.E. of n 4 experiments and are expressed as percentage of the effect obtained with 4
M ionomycin. B,
time-related effect of cannabidiol (10
M) in the presence of extracellular Ca
2
. Trace is representative of n 4 experiments. C, dose-related effect
of cannabidiol in the absence of extracellular Ca
2
, as determined with Fura-2. Data are mean S.E. of n 4 experiments. Maximal
fluorescence
was
0.235 0.031 at 10
M cannabidiol and was usually attained after 200 s (D). Effect of various antagonists (the CB
1
antagonist AM251, 1
M; the CB
2
antagonist AM630, 1
M; the TRPV1 antagonist I-RTX, 0.1
M; 5-min pretreatment) and the intracellular calcium chelating agent BAPTA-AM (20
M, loaded onto the cells before stimulation) on cannabidiol (1
M) action on intracellular Ca
2
. Similar results were obtained with SR141716A and
SR144528.
1384 Ligresti et al.
cancer cells. Furthermore, cannabidiol and the cannabidiol-
rich extract also inhibited the formation of lung metastases
subsequent to inoculation of MDA-MB-231 cells, in agree-
ment with the inhibitory actions on cancer cell migration
previously described for this compound (Vaccani et al., 2005).
The weak effects observed here with THC might be re-
garded as surprising. In fact, THC was reported to induce
apoptosis in both C
6
glioma and human prostate PC-3 cells
(Sanchez et al., 1998; Ruiz et al., 1999; Sarfaraz et al., 2005),
although it may even enhance breast cancer growth and
metastasis (McKallip et al., 2005). The low potency found
here for this compound, at least in glioma and prostate can-
cer, could be explained by the different experimental condi-
tions used and supports the notion that the efficacy of can-
Fig. 9. Study of the involvement of oxidative stress in the effect of CBD. A, antiproliferative effect of 10
M cannabidiol on MDA-MB-231 cells was
measured after 4 days of treatment in absence or in presence of increasing concentrations of
-tocopherol. Data represent mean S.E. of percent
inhibition of proliferation (, p 0.05 by ANOVA followed by Bonferroni’s test). B, time course of ROS production by MDA-MB-231 cells (16 10
3
cells/well) as measured by spectrofluorometric analysis. Cells were loaded 1 h with 10
M fluorescent probe in the presence of 0.05% Pluronic;
fluorescence was measured in a 96-well microplate reader (PerkinElmer LS50B,
Ex
, 495 nm;
Em
, 521 nm). Fluorescence detection was carried out
after the incubation of either 100
MH
2
O
2
or increasing concentrations of cannabidiol at different times (0–30-60–120 min). 100
MH
2
O
2
was used
as a positive control in these experiments. The fluorescence measured at time 0 was considered as basal ROS production and subtracted from the
fluorescence at different times (
1
). Data are reported as
2
, i.e.,
1
values at different doses subtracted of the
1
values of cells incubated with vehicle,
and are mean S.E. of n 3 experiments. The effects of H
2
O
2
and of all doses of cannabidiol were significantly different from control values as
determined by ANOVA followed by Bonferroni’s test. Inset, lack of effect of cannabidiol 10
M on ROS production (after 60 min) in the absence of both
extracellular and intracellular Ca
2
is shown. ⴱⴱⴱ, p 0.005 by ANOVA, n 5.
Fig. 10. Study of the involvement of oxidative stress in the effect of CBD. A and B, antiproliferative effect of cannabidiol (24-h incubation) was
evaluated in standard growth conditions or after 12 h of serum deprivation to induce oxidative stress. Poststarvation, cells were treated with 0.5 or
10
M cannabidiol for 24 h, and the effect on proliferation was evaluated by Crystal Violet staining. Data are reported as mean S.E. of percent
inhibition of proliferation, n 3. C, ROS production after2hofincubations with cannabidiol or H
2
O
2
was measured in MDA-MB-231 cells (16 10
3
cells/well) by spectrofluorometric analysis. The effect of cannabidiol per se (0.5 and 10
M) is reported in Fig. 9. Cells were loaded 1 h with 10
M
fluorescent probe in presence of 0.05% Pluronic, and fluorescence was measured in a 96-well microplate reader (PerkinElmer LS50B,
Ex
, 495 nm;
Em
,
521 nm). Fluorescence was measured at t 0 and after2hofincubation with H
2
O
2
in the presence or absence of increasing concentration of
cannabidiol. Data are expressed as explained in the legend to Fig. 9. Cannabidiol inhibited ROS production by H
2
O
2
only at the lowest concentration
(0.5
M, p 0.05 by ANOVA followed by Bonferroni’s test).
Cannabidiol in Cancer 1385
nabinoids is strongly dependent on the cell type utilized. In
fact, regarding glioma cells, THC induction of apoptosis was
reported not in C
6
cells, but in a THC-sensitive subclone
(C
6.9
). Furthermore, Ruiz et al. (1999) used a human prostate
cancer cell line different from the one used here. Melck et al.
(2000) found that stimulation of CB
1
receptors causes inhi
-
bition of DU-145 cell proliferation only when this is induced
by nerve growth factor. Using similar culturing conditions as
those used here, we previously showed that CB
1
, but not CB
2
,
stimulation inhibits the proliferation of MCF-7, KiMol, and
CaCo-2 cells more potently than what observed here with
THC (De Petrocellis et al., 1998; Bifulco et al., 2001; Ligresti
et al., 2003). This might be due to the use in those studies of
CB
1
agonists with higher potency or efficacy than THC or of
cells clones with a higher expression of CB
1
receptors than
that observed here. Indeed, McKallip et al. (2005) proposed
for human breast cancer cells that resistance to THC toxicity
is correlated to low expression of CB
1
receptors and high
expression of vanilloid receptors.
To date, the receptor-mediated anticancer effects of canna-
binoids and endocannabinoids have been ascribed to either
CB
1
-mediated inhibition of mitosis, as in the case of some
hormone-sensitive cells, or to the induction of apoptosis fol-
lowing activation of TRPV1 and/or CB
2
receptors. Starting
from the hypothesis that cannabidiol decreased cell number
by induction of apoptosis at least for human glioma cell lines
(Massi et al., 2004), we decided to evaluate the percentage of
apoptotic cells after exposure to cannabidiol and found that
the effect of the compound was due to an arrest of the cell
cycle in the case of MCF-7 (hormone-sensitive) cells, to both
cell cycle arrest and apoptosis in KiMol cells (which are also
hormone-sensitive to some extent), and only to induction of
apoptosis in C
6
and MDA-MB-231 (nonhormone-sensitive)
cells. It was, therefore, clear that cannabidiol lacks a unique
mode of action for its anticancer effect on the cell lines under
investigation. Based on previous evidence that cannabidiol,
although inactive as a direct agonist at cannabinoid CB
1
and
CB
2
receptors, activates directly the vanilloid TRPV1 recep
-
tor and is capable of increasing endocannabinoid levels by
inhibiting their inactivation (Bisogno et al., 2001), we first
investigated the capability of plant cannabinoids to interact
with the key components of the endocannabinoid or endova-
nilloid systems. Indeed, cannabidiol, cannabigerol, and can-
nabichromene were found here to activate TRPV1 receptors
and/or inhibit anandamide inactivation to some extent. How-
ever, despite the presence of either cannabinoid or vanilloid
receptors (or both) in all cell lines under study, in none but
one of these cell lines the direct or indirect stimulation of
these receptors seemed to be entirely or even partially re-
sponsible for the anticancer effects of cannabidiol. The only
important exception was represented by MDA-MB-231 cells,
where a partial, although significant, reversion of the effect
of cannabidiol was observed in the presence of selective an-
tagonists for TRPV1 and CB
2
receptors, thus pointing to the
partial involvement of these receptors in the anticancer ac-
tion of this cannabinoid in breast carcinoma cells. This find-
ing is important in view of the fact that these cells were the
ones used in the present study to investigate the anticancer
and antimetastatic effects of cannabidiol in vivo. However,
the present observation that “pure” agonists of CB
2
and
TRPV1 receptors, or a selective inhibitor of anandamide up-
take, were less efficacious than cannabidiol at inhibiting
MDA-MB-231 cell growth strongly suggests that the two
receptors act cooperatively with other mechanisms at induc-
ing apoptosis and that other unique effects of cannabidiol
also contribute to its anticancer actions.
It has been reported that plant and endogenous cannabi-
noids can induce apoptosis through several molecular mech-
anisms (Galve-Roperh et al., 2000; Jacobsson et al., 2001).
When TRPV1 is involved, apoptosis is induced by mitochon-
drial events triggered by TRPV1-mediated calcium influx
(Maccarrone et al., 2000), whereas when CB
2
receptors are
involved, ceramide accumulation seems to be the most im-
portant intracellular event causing programmed cell death
(Galve-Roperh et al., 2000). Our data indicate that a part of
the proapoptotic effect of cannabidiol in MDA-MB-231 cells
might be due to these mechanisms. However, a TRPV1- and
CB
2
-independent mechanism known to induce apoptosis is
the rise of intracellular ROS levels, as demonstrated by the
fact that nonvanilloid-, noncannabinoid receptor-mediated
anandamide-induced apoptosis is prevented by antioxidant
agents (Sarker et al., 2000). Hence, the effect of cannabidiol
might also be attributed to ROS production. For this reason
we investigated the involvement of oxidative stress in can-
nabidiol effects in MDA-MB-231 cells. We found that antioxi-
dants attenuated the proapoptotic effects of cannabidiol in
these cells, suggesting that this compound is indeed capable
of exerting pro-oxidative properties, at least in tumor cell
lines. Importantly, the extent of the effect of the antioxidants
accounted for that part of cannabidiol action that was not
blocked by CB
2
and TRPV1 receptor antagonists. Accord
-
ingly, cannabidiol, at the concentrations exerting antiprolif-
erative effects, also induces a significant enhancement of
ROS levels in MDA-MB-231 cells. The capability of cannabi-
diol to induce ROS might be surprising in view of its phenolic
chemical structure, which would rather favor an inhibitory
effect on oxidative stress. However, we provided here data
suggesting that cannabidiol might cause ROS elevation indi-
rectly, i.e., by elevating intracellular Ca
2
. Cannabidiol-in
-
duced intracellular Ca
2
elevation occurred in the same
range of concentrations as those necessary to cause growth
inhibition, was independent of TRPV1 and CB
1
and CB
2
receptor activation, and might be related to the analogous
effect recently observed with THC, cannabinol and cannabi-
diol in T cells (Rao and Kaminski, 2006). Finally, at a sub-
micromolar concentration, cannabidiol was also capable of
inhibiting H
2
O
2
-induced ROS formation, similar to what ob
-
served previously in nontumor cells (Hampson et al., 1998;
Iuvone et al., 2004), thus possibly explaining why also in the
present study, in certain cells and at low concentrations, or
with short incubation times and in cell culturing conditions
in which not so many ROS are present (i.e., in the presence of
serum), this compound can also produce pro-proliferative
effects.
In conclusion, our data indicate that cannabidiol, and pos-
sibly Cannabis extracts enriched in this natural cannabinoid,
represent a promising nonpsychoactive antineoplastic strat-
egy. In particular, for a highly malignant human breast
carcinoma cell line, we have shown here that cannabidiol and
a cannabidiol-rich extract counteract cell growth both in vivo
and in vitro as well as tumor metastasis in vivo. Cannabidiol
exerts its effects on these cells through a combination of
mechanisms that include either direct or indirect activation
of CB
2
and TRPV1 receptors and induction of oxidative
1386 Ligresti et al.
stress, all contributing to induce apoptosis. Additional inves-
tigations are required to understand the mechanism of the
growth-inhibitory action of cannabidiol in the other cancer
cell lines studied here.
Acknowledgments
We thank Teresa Iuvone and Daniele De Filippis for valuable help
with caspase-3 data and Tiziana Bisogno for critical comments and
general contribution to the work.
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Cannabidiol in Cancer 1387
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... However, there is contradictory evidence; it can stimulate tumour cell proliferation, angiogenesis and immunosuppression or inhibit tumour cell growth, angiogenesis, invasion and metastasis [84]. Noteworthy, some of these molecules have been recently proposed as possible therapeutic targets [44,83] since there is evidence that some nonpsychoactive cannabinoids can have antimetastatic, anti-invasive effects [85,86] and antiproliferative capabilities [87] by inducing apoptosis [88] and autophagy [89,90]. ...
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Both delta 9-tetrahydrocannabinol (delta 9-THC) and cannabigerol, two naturally occurring marihuana cannabinoids, produced only a modest fall in intraocular pressure after acute topical application to the eyes of cats. After chronic administration unilaterally to the cornea via Alzet osmotic minipumps and connecting extraocular cannulas, however, a considerable fall in ocular tension amounting to 4 to 7 mm Hg occurred. After systemic administration of delta 9-THC to rats, polyspike discharges appeared in the cortical electroencephalogram initially during wakefulness and behavioral depression. These polyspikes subsequently became evident within rapid eye movement sleep episodes. Cannabigerol was devoid of this effect. After removal of either sympathetic or parasympathetic input to the eyes of cats, the intraocular pressure lowering effect of delta 9-THC was not changed. Neither delta 9-THC nor cannabigerol altered the rate of formation of aqueous humor. On the other hand, both cannabinoids produced a two-to three-fold increase in aqueous outflow facility. These results suggest that cannabigerol and related cannabinoids may have therapeutic potential for the treatment of glaucoma.
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