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In Vitro and In Vivo Efficacy of Non-Psychoactive Cannabidiol in Neuroblastoma

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Background: Neuroblastoma (nbl) is one of the most common solid cancers in children. Prognosis in advanced nbl is still poor despite aggressive multimodality therapy. Furthermore, survivors experience severe long-term multi-organ sequelae. Hence, the identification of new therapeutic strategies is of utmost importance. Cannabinoids and their derivatives have been used for years in folk medicine and later in the field of palliative care. Recently, they were found to show pharmacologic activity in cancer, including cytostatic, apoptotic, and antiangiogenic effects. Methods: We investigated, in vitro and in vivo, the anti-nbl effect of the most active compounds in Cannabis, Δ(9)-tetrahydrocannabinol (thc) and cannabidiol (cbd). We set out to experimentally determine the effects of those compounds on viability, invasiveness, cell cycle distribution, and programmed cell death in human nbl SK-N-SH cells. Results: Both compounds have antitumourigenic activity in vitro and impeded the growth of tumour xenografts in vivo. Of the two cannabinoids tested, cbd was the more active. Treatment with cbd reduced the viability and invasiveness of treated tumour cells in vitro and induced apoptosis (as demonstrated by morphology changes, sub-G1 cell accumulation, and annexin V assay). Moreover, cbd elicited an increase in activated caspase 3 in treated cells and tumour xenografts. Conclusions: Our results demonstrate the antitumourigenic action of cbd on nbl cells. Because cbd is a nonpsychoactive cannabinoid that appears to be devoid of side effects, our results support its exploitation as an effective anticancer drug in the management of nbl.
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EFFICACY OF NON-PSYCHOACTIVE CANNABIDIOL ON NEUROBLASTOMA, Fisher et al.
S15Current Oncology, Vol. 23, Supp. 2, March 2016
© 2016 Multimed Inc.
ORIGINAL ARTICLE
In vitro and in vivo efficacy of
non-psychoactive cannabidiol in neuroblastoma
T. Fisher phd,*a H. Golan md,*†a G. Schiby md, S. PriChen phd,§ R. Smoum phd,|| I. Moshe msc,*
N. Peshes-Yaloz phd,# A. Castiel phd,# D. Waldman md,* R. Gallily phd,** R. Mechoulam phd,||
and A. Toren md phd*†††
ABSTRACT
Background Neuroblastoma (nbl) is one of the most common solid cancers in children. Prognosis in advanced
nbl is still poor despite aggressive multimodality therapy. Furthermore, survivors experience severe long-term
multi-orga n sequelae. Hence, the identi fication of new ther apeutic str ategies is of ut most importa nce. Can nabinoids
and their derivatives have been used for years in folk medicine and later in the field of palliative care. Recently, they
were found to show pharmacologic activity in cancer, including cytostatic, apoptotic, and antiangiogenic effects.
Methods We investigated, in vitro and in vivo, the anti-nbl effect of the most active compounds in Cannabis,
Δ9-tetrahydrocannabinol (thc ) and cannabidiol (cbd). We set out to experimentally determine the effects of those
compound s on viabil ity, invasiven ess, cell cy cle distr ibution, and prog rammed c ell death i n human nbl SK-N-SH cells.
Results Both compounds have antitumourigenic activity in vitro and impeded the growth of tumour xenografts
in vivo. Of the two cannabinoids tested, cbd was the more active. Treatment with cbd reduced the viability and
inva siveness of tre ated tumour c ells in vitro a nd induced apopto sis (as demonst rated by morpholog y changes, su b-G1
cell accumulation, and annexin V assay). Moreover, cbd elicited an increase in activated caspase 3 in treated cells
and tumour xenografts.
Conclusions Our results demonstrate the antitumourigenic action of cbd on nbl cells. Because cbd is a non-
psychoa ctive cannabi noid that appea rs to be devoid of side eff ects, our results s upport its exploit ation as an ef fective
anticancer drug in the management of nbl.
Key Words Neuroblastoma, cannabidiol, Δ9-tetra hydrocannabinol, apoptosis, tumour xenograft models, non-
psychoactive cannabinoids
Curr Oncol. 2016 Mar;23(S2):S15-S22 www.current-oncology.com
INTRODUCTION
Neuroblastoma (nbl) is the most frequent extracranial
solid tumour in childhood. It accounts for approxi mately
8% of childhood ca ncers and is cha racterized by variable
clinica l behaviour, reflecting molec ular di fferences i n the
tumour1. Using cur rent risk str atificat ion cr iteria, approx-
imately 40% of n bl tumours are classified as high-risk.
Treatment for children with high-risk nbl involves mul-
timodal ity therapy, including chemotherapy, autologous
stem-cel l transplantation, su rgery, radiat ion therapy, and
immunotherapy using differentiation therapy. Despite
that aggressive approach, children with nbl have very
poor outcomes, and the sur vivors ex perience serious side
effects related to treatment toxicity2. Hence, the need for
new and less-toxic therapeutic strategies to treat the dis-
ease is urgent.
For millennia, Cannabis sativa has been used in folk
medic ine to alle viate pa in, depression, a menorrhea, i nflam-
mation, e pilepsy, and numerou s other medic al condition s3.
Correspondence to: Amos Toren, Department of Pediatric Hemato-Oncology, The Edmond and Lily Safra Children’s Hospital, The Chaim Sheba Medical Center,
Tel-Hashomer 52621 Israel.
E-mail: amost@post.tau.ac.il n DOI: http://dx.doi.org /10.3747/co.23.2893
a These authors contributed equally to the preparation of this
manuscript.
EFFICACY OF NON-PSYCHOACTIVE CANNABIDIOL ON NEUROBLASTOMA, Fisher et al.
S16 Current Oncology, Vol. 23, Supp. 2, March 2016
© 2016 Multimed Inc.
In ca ncer patients sp ecifica lly, canna binoids are we ll know n
to exert palliative effects; t heir best-established use is the
inh ibition of chemotherapy-induced nausea a nd vomiting,
but they a re also appl ied for pain a lleviat ion, appetite sti m-
ulation, and attenuation of wasting4.
Recently, increasing evidence suggests that Δ9-tetra-
hydrocanna binol (thc) a nd canna bidiol (cbd), major c om-
ponents of Cannabis sativa, and sy nthetic ca nnabinoids
and the endocannabinoid ananda mide have antitumour
activity5,6. Many adult cancer types (lung ca ncer, glioma,
thy roid cancer, ly mphoma, skin c ancer, pancre atic cancer,
uterine cancer, and breast and prostate carcinoma) have
been repor ted to be sensit ive to the ant iproliferat ive action
of can nabinoids in a w ide variety of experimental models,
including cancer cell lines in culture, xenograft mouse
models, and genetically engineered mice6.
Cannabinoids act chiefly by activating the specific
cannabinoid receptors cb1 and cb26. However, it is now
well-established that these molecules also have effects
that are cb receptor–independent; other receptors, such
as vanilloid receptor 17 and the peroxisome proliferator–
activated receptors8, could be responsible for thei r action.
The mech anisms i nvolved in t he antitu mour effec ts of
can nabinoids include proliferation inh ibition and growt h
arrest9, induction of apoptosis10,11, stimulation of auto-
phagy12,13, angiogenesis inhibition14, and anti-metastatic
effects15– 17. However, the a ntitumou rigenic mecha nism of
action of c bd is as yet unknown18.
At the mole cular lev el, canna binoids have been sho wn
to tr igger change s in var ious signa lling pa thways, i ncluding
Akt/mammalian target of rapamycin complex 119, erk,
upregulation of stress-associated transcription factor
p819,20, downregulation of matrix metalloproteinase 221,
and vascular endothelial growth factor signalling22. Nev-
ert heless, studies e xploring t he putative ant itumour igenic
properties of cannabinoids in pediatric tumours are st ill
limited, and the molecular mechanisms underly ing the
antitumourigenic effect are poorly understood. Recently
published data demonstr ated the antitumourigenic activ-
ity of cannabinoids—ma inly thc and synthetic cannabi-
noids— on alveola r rhabdomyosa rcoma and osteo sarcoma
by inducing apoptosis23 and triggering the endoplasmic
reticular stress and autophag y process24.
Our study aimed to characterize both the in vitro
and in vivo effects of ca nnabinoids on another pediatric
tumour, nbl, and to unravel the mechanism responsible
for those effects. Given our positive results, we suggest
that non-thc cannabinoids such as cbd might provide a
basis for the development of novel therapeutic strategies
in high-risk nbl, w ithout the typical psychotropic effects
of thc and without the strong side effects associated with
chemotherapeutic agents.
METHODS
Cannabinoids
Δ9-Tetrahydrocannabinol was supplied by Prof. Raphael
Mechoulam, In stitute for Dr ug Research, Medica l Faculty,
The Hebrew University, Ein Kerem Campus, Jerusalem,
Israel. Cannabidiol was supplied by THC Pharm GmbH,
Frankfurt, Germany.
Cell Cultures
The human nbl cell lines SK-N-SH25 and IMR-3226 were
purcha sed from ATCC ( Manassa s, VA, U.S.A .) and the Eu ro-
pean C ollection of Aut henticated Cell Cu ltures (Sa lisbur y,
U.K.) respectively. The NUB-627 and LA N-1 cell lines were
kindly prov ided by Dr. Shifra Ash, Schneider Children’s
Medica l Center of Israel28.
SK-N-SH cells were c ulture d in Eagle m ini mum essent ial
medium (ATCC), supplemented w ith 10% fetal bovine
ser um (fbs) and 100 U/mL pen icilli n–stre ptomycin (Gibco,
Paisley, U.K.). IMR-32 cells were cultured in Eagle basal
medium (Sigma–A ldrich, St. Louis, MO, U.S.A.) supple-
mented w ith 2 mmol/L g lutami ne, 1% non-essentia l amino
acids, 10% fbs, and 100 U/mL penicillin–streptomycin.
LA N-1 and NUB-6 cells were cultured in RPMI-1640 (Gibco)
supplemented with 10% fbs and 100 U/mL penicillin–
streptomycin. All the cell lines were cultured at 37°C in a
humidified atmosphere containing 5% CO2.
MTT Test
An mtt assay (Biological Industries, Kibbutz Beit-Haemek,
Israel) was used to eva luate the effect of c bd and t hc on
nbl cell viability. SK-N-SH, LAN-1, IMR-32, and NUB-6
cells (5×103 cells/mL) were plated (200 μL) in triplicate in
flat-bottom 96-well plates in the appropriate medium. The
cells were allowed to adhere to the plate surface overnight
and were t hen cultured with increasing doses of thc or c bd
(0–50 μg/mL) for 24, 48, a nd 72 hours. Cel l viabil ity was t hen
determined by mtt assay, which measures t he reduction of
mtt to formazan by the mitochondria of viable cells29. For-
maz an was mea sured spe ctrophotomet rical ly by absorpt ion
at 560 nm i n a PowerWaveX plate reader (BioTek, Winooski,
VT, U.S.A.). All experiments were repeated at least 3 times.
Cell mor phologies were assessed dai ly by light microscopy.
Microscopy Analysis
One day before treatment, SK-N-SH cells were plated
(1×106 cells per 9-cm plate). A fter 48 hours of incubation
with cbd (10 μg/mL), cell morphology changes were as-
sessed by light microscopy (Olympus CKX41: Olympus,
Tokyo, Japan).
Cell-Cycle Analysis
One day before thc or cbd treatment, SK-N-SH cells were
plate d (1×106 c ells per 9-c m plate). Af ter 24, 48, and 72 hou rs
of treat ment, the cel ls were washed i n phosphate-buf fered
saline (pbs: Biological Industries), detached using a solu-
tion of 0.1% trypsin (Biological Industries), and spun at
1100 rpm. The resulting pellet was resuspended in 250 μL
cold pbs, and the cells were fixed over night with 5 mL cold
75% ethanol (Sigma–Aldrich) and pbs at –20°C. The pellet
was then washed twice with cold pbs (followed by centri-
fuga tion at 1100 rpm f or 7 minutes). Di stribut ion of the cells
in G1, S, and G2/M phases of the cell c ycle were monitored
after nuclei had been stained with 50 μg/mL propidium
iodide ( Sigma–A ldrich) cont aini ng 125 U/mL protease-f ree
rnase (Sig ma–Ald rich) in 0.5 % Triton ( Bio-Lab, Jerusa lem,
Israel) and had been pbs-buffered for 30 minutes in the
dark. The cells were ana lyzed usi ng an Epics XL-MCL flow
cytometer and the FlowJo soft ware application (Beckman
Coulter, Brea, CA, U.S.A.).
EFFICACY OF NON-PSYCHOACTIVE CANNABIDIOL ON NEUROBLASTOMA, Fisher et al.
S17Current Oncology, Vol. 23, Supp. 2, March 2016
© 2016 Multimed Inc.
Apoptotic Cell Death
Annexin V Assay
One day before cbd treatment (7.5 μg/mL and 10 μg/mL for
24 hours and 48 hours), cells were plated (1×106 cells per
9-cm plate). Cells treated w ith 1–10 μmol/L staurosporine
(Sigma–Aldrich) for 24 hours ser ved as a positive control;
untreated cells served as a negative control. Treated cells,
untreated cells, a nd positive control cells were har vested,
and after the annexin V assay [human recombinant an-
nexin V (apc conjugate, catalogue no. AL X-209–252: Enzo
Life Sciences, Ann Arbor, MI, U.S.A.); annexin V binding
buf fer, no. 55645 4 (BD Pharm ingen, San Die go, CA, U.S.A. );
and 7-ami noactinomyc in D, no. 559925 (BD Phar mingen)]
were analy zed using an Epics X L-MCL flow cytometer a nd
the FlowJo software application.
Caspase Assay
One day before cbd treatment (7.5 μg/mL and 10 μg/mL
for 24 hours), cells were plated (1×106 cells per 9-cm plate).
Cells were harvested, and proteins were extracted with
radioimmunoprecipitation assay buffer (Sigma–Aldrich).
Protein concentrations were ca librated using the BCA
Protein Assay Reagent Kit (Pierce, Rockford, IL, U.S.A.).
Samples were separated on 12% sds-pag e (Bio-Rad,
Rishon LeZion, Israel) and transferred onto nitro filters
(Sch leicher and Schuel l Bioscience, Das sel, Germa ny). The
blots were re acted using c aspase 3 ( 8G10) rabbit monoclon al
ant ibody (Cel l Signal ling Technolog y, Danvers, M A, U.S.A.) a s
the primary a ntibody. The secondary antibody, horseradish
peroxidase conjugated goat anti-rabbit antibody (Jackson
ImmunoResearch Laboratories, Farmington, CT, U.S.A.),
was dete cted by chemi lumine scence. Sign als were dete cted
using a n ECL Kit (Amersham Pharmacia, Little Chalfont,
U.K.) and visualized by exposure to radiography film.
Invasion Assay
Tumour cell invasion was assayed in Transwel l chambers
(Transwell 3422: Corning, Corning, NY, U.S.A.) pre-coated
with Cultrex Basement Membrane Extract (Trevigen,
Gaithersburg, MD, U.S.A.). Membrane filters were placed
in 24-wel l tissue-c ulture pl ates accordi ng to manufac turer
guidelines. After 24 hours of treatment with cbd (15 μg/
mL and 20 μg/mL), cells were harvested, and 2×105 cells
suspende d in 200 μL seru m-free mediu m were added to th e
upper sur face of each cha mber. The bottom of t he chamber
was fi lled wit h 750 μL medium w ith 10% fbs. Af ter 24 hours
in which cells were a llowed to migrate to the underside of
the membrane, the invaded cells were fi xed w ith parafor-
maldehyde (Electron Microscopy Sciences, Hatfield, PA,
U.S.A.) and sta ined with crystal violet (Sig ma–Aldrich).
In Vivo Studies
All experiments involving mice were approved and per-
formed according to the guidelines issued by the Sheba
Medica l Center Research Committee for the Ca re and Use
of Laboratory Animals (permit no. 803/12).
To study the in vivo antitumour activity of cannabi-
noids, nbl tumours were induced in nonobese diabetic
immunodeficient (nod/scid) mice by subcuta neous in-
jection. Briefly, 1×107 SK-N-SH cells suspended in 100 μL
serum-free medium and Cultrex (1:1) were injected sub-
cutaneously into the rear flank of 5- to 8-week-old nod/
scid mice. Mice were maintained in a pathogen-free
environment and monitored week ly for tumour grow th.
Secondary tumours were detected by palpation and were
measured with external callipers. Volume was calculated
as (width)2 × (length) × 0.52. When tumours had reached
an average size of 400 mm3, the mice were randomly as-
signed to treatment a nd control groups (each n = 12). T hey
were then injected intraperitonea lly for 14 days wit h thc
(20 mg/kg da ily), cbd (20 m g/kg daily), or v ehicle (etha nol)
or were left untreated. At the end of the treatment period,
the mice were euthanized, and the tumours were excised
and processed for further ana lyses.
Histology
Formalin-fixed tissues were dehydrated, embedded in
paraffin, and sectioned at 4 μm. The slides were warmed
to 60°C for 1 hour and then processed by a f ully auto-
mated protocol. Immunostainings were ca librated on a
Benchmark XT staining module (Ventana Medical Sys-
tems, Tucson, AZ, U.S.A.). Briefly, after sections had been
dewaxed and rehydrated, a CC1 Standard Benchmark XT
pre-treatment for antigen retrieval (Ventana Medical Sys-
tems) was selected for active caspase 3. Active caspase 3
antibody (Epitomics, Burlingame, CA, U.S.A.) was diluted
1:10 with Antibody Diluent (Ventana Medical Systems)
and in cubated for 1 hour at 37° C. Detection w as performe d
using a n ultraVie w detection k it (Ventana Med ical System s)
and counterstained with hematoxylin ( Ventana Medical
Systems). After the run on the automated stainer was
completed, t he slides were dehy drated in 70 % ethanol, 95 %
etha nol, and 100% et hanol (10 s each). Before c over-slippi ng,
the sections were cleared in xylene (10 s) and mounted
with Entellan (EMD Millipore, Billerica, MA, U.S.A.). The
stai ned sect ions were revie wed under lig ht microscopy a nd
analyzed by a pathologist.
Statistical Analysis
Unless otherwise specified, results are shown as means
or medians ± standa rd dev iation. A Kruskal–Wallis test,
followed by a post hoc Mann–Whitney test, was used to
eva luate signi ficant dif ferences in t he viabi lity of cell l ines,
the growth rate of xenografts, and the counts of positive
cleaved c aspase 3 cel ls for the va rious tre atment groups . A p
value l ess than 0.0 5 was considered s tatistic ally sig nifica nt.
All ana lyses were per formed using t he IBM SPSS Statistics
soft ware appl ication (version 21: IBM, A rmonk, N Y, U.S.A. ).
RESULTS
Viability of NBL Cell Lines In Vitro
We used an mtt assay to a ssess the ef fect of thc a nd cbd on
the v iabilit y of the SK-N-SH, NUB-6, I MR-32, and LA N-1 nbl
cell l ines [Figure 1(A)]. In v itro, af ter 24 hours of tr eatment,
cbd and t hc had a lready ef fectivel y reduced the v iabilit y of
nbl cel l lines in a dose- (0 –50 μg/mL) and time-dependent
man ner, wit h cbd havi ng the better ef fect. Bet ter response
to treatment wa s observed in t he SK-N-SH and NUB-6 cel l
line s, as demonstr ated by a 50% reduc tion in cel l viabil ity at
lower cbd or thc conc entration s (5 μg/mL and 15 μg/mL for
EFFICACY OF NON-PSYCHOACTIVE CANNABIDIOL ON NEUROBLASTOMA, Fisher et al.
S18 Current Oncology, Vol. 23, Supp. 2, March 2016
© 2016 Multimed Inc.
SK-N-SH and NUB-6 respectively, compared with >20 μg/
mL for IMR-32 and LAN-1). The same trend was found,
and even enhanced, after treatment with t hc and c bd for
48 hours [Figure 1(A)]. More importantly, the response
after treatment of SK-N-SH cells with cbd (10 μg/mL) was
better than the response after treatment with the same
concentration of thc [Figure 1(B), p = 0.0004 for 24 and 48
hours of treatment].
The foregoing data indicate that anti-nbl activity is
better w ith cbd than with t hc in all n bl cell lines tested.
Because cell lines showed varying sensitiv ity to cbd, we
chose the most sensitive SK-N-SH cell line to confirm the
antiproli ferative ef fect of cbd in fu rther in vit ro and in vivo
experiments.
Cell-Cycle Analysis
We next studied the effect of 48 hours of treatment with
increasing doses of cbd (5–20 μg/mL) on cell-cycle pro-
gression [Figure 2(A)]. During treatment w ith cbd (5 μg/
mL), the percentage of SK-N-SH cells sequestered in the
G1 compartment rose to 82.4% from 65.8% in untreated
control cells [Figure 2(B)]. Accordingly, the percentages
of cells in G2 and S phase were found to be decreased,
indicating that those cell populations had undergone G1
phase arrest (similar results were obtained when NUB-6
cells were treated with 10 μg/mL cbd; data not shown).
Further more, an accumulat ion of SK-N-SH cells i n sub-G1
phase [Figure 2(B)] was detected when that line was incu-
bated w ith 10 μg/mL cbd (4.27%) a nd 20 μg/mL cbd (25.3%),
indic ating the p ossibilit y that trea tment with c bd induce d
apoptosis in a dose-dependent manner.
Apoptotic Cell Death
To verify our hypothesis that the reduction in nbl cell v ia-
bilit y associated with cbd treatment was indeed attribut-
able to apoptot ic cell death, w e first exa mined morpholo gy
chan ges after cbd treat ment. Micros copic anal ysis showed
that treatment with 10 μg/mL cbd affected cell morphol-
ogy; the number of cells that had lost their norma l shape,
becoming rounded and swollen, and that floated in the
medium increased [Figure 3(A)]. Those results confirmed
that cbd t reatment mig ht induce the appe arance of t ypica l
features of apoptosis.
FIGURE 1 Δ9-Tetrahydrocannabinol (THC) and cannabidiol (CBD)
reduce viability of neuroblastoma (NBL) cell lines in vitro, with CBD
having a better effect. (A) Cell lines SK-N-SH (open squares), NUB-6
(open circles), IMR-32 (open triangles), and LAN-1 (crosses) were
incubated with increasing concentrations (0–50 μg/mL) of THC or
CBD for 24 hours and 48 hours. Viability was measured by MTT assay.
(B) Mean ± standard deviation of SK-N-SH cell viability after incubation
with 10 μg/mL THC or CBD for 24 and 48 hours. *** Denotes a
significant change relative to control (p = 0.0004 ). Data are expressed as
a percentage of the vehicle control and are the mean of pooled results
from experiments performed in triplicate.
FIGURE 2 Alteration of SK-N-SH cell cycle progression induced by
cannabidiol (CBD). (A) Cell cycle analysis in untreated SK-N-SH cells
and in cells treated with increasing concentrations of CBD for 48
hours. (B) Change in cell accumulation percentages during cell cycle
progression after incubation with CBD for 48 hours. UT = untreated.
EFFICACY OF NON-PSYCHOACTIVE CANNABIDIOL ON NEUROBLASTOMA, Fisher et al.
S19Current Oncology, Vol. 23, Supp. 2, March 2016
© 2016 Multimed Inc.
Next, we used an annexin V assay to measure the per-
centage of cells undergoing apoptosis after cbd treatment
[Figure 3(B)]. Staurosporine-treated cells were used as
a positive control. Treatment of SK-N-SH cells wit h cbd
(7.5 μg/mL and 10 μg/mL) for 24 hours and for 48 hours re-
sulted in an increase i n the early apoptotic cel l population
(an nexin V–po sitive and 7-ami noactinomy cin D–neg ative)
in a ti me-dependent man ner. A dose- and ti me-dependent
increase in the late apoptotic cell population was also
demonstrated (annexin V–positive and 7-aminoactino-
mycin D –positive). Early apoptosis was demonstrated in
24% of cells incubated for 24 hours with cbd (7. 5 μg/mL),
but in only 0.7% of untreated cells. As Figure 3 shows, the
proport ion of late apoptotic c ells incr eased to 63 % from 35%
af ter 24 hours of tre atment wit h increasi ng concentrat ions
of cbd (10 μg/mL and 7.5 μg/mL respectively)
Fina lly, to fur ther confir m the apoptotic ef fects of cbd
on SK-N-SH cells, we measured apoptosis by caspase 3
assay [Figure 3(C)]. After 24 hours of treatment with in-
creasing doses of cbd ( 7. 5 μg/mL and 10 μg/mL), a dose-
dependent cleavage of caspase 3 was found as evaluated
by the appearance of activated p17 and p19 fragments on
Western blot analysis.
Altoge ther, the foregoi ng results c onfirm t hat treat ment
with cbd induces apoptosis in the SK-N-SH nbl cell line.
Cell Invasiveness
As shown in Fig ure 4, cell invasion in Transwell chambers
was dramatically decreased for SK-N-SH nbl cells treated
for 24 hours w ith cbd (15 μg/mL and 20 μg/mL) than for
untreated cells.
Tumour Growth Rate in Mouse Xenograft Model
Because tumour reg ression in an animal xenograft model
represent s an import ant endpoint of cli nical rele vance, we
eva luated the abi lity of ca nnabinoids t o reduce nbl tumour
growth in vivo. Tumour xenograf ts were first generated by
subcutaneous injection of SK-N-SH cells into nod/scid
mice. T he mice were t hen treated w ith dai ly intrap eritoneal
injec tions of 20 mg/kg thc, 20 mg/k g cbd, or ethanol v ehicle
(control), or were left untreated for 14 days.
Tumour grow th was significantly reduced in thc- and
cbd-treated mice than in the vehicle-treated or untreated
mice [Figure 5(A)]. Interestingly, response to treatment was
obser ved to be bette r in the gr oup treated w ith cbd t han in t he
group t reated wit h thc: Median xenog raft volume at t he end
of treat ment was 2. 31 cm3 in t he cbd-treated group compared
wit h 4.28 cm3 in t he untreated g roup (p = 0.029) and 4. 31 cm3
in the vehicle-treated group (p = 0.036). In the thc-treated
group, median volume was 3.46 cm3, which was significant
only compared with the untreated group (p = 0.039).
FIGURE 3 Apoptotic effects of cannabidiol (CBD) on SK-N-SH cells. (A) Changes in SK-N-SH cell morphology: untreated cells compared with
cells treated with 10 μg/mL CBD for 48 hours. (B) Apoptotic effects of CBD on SK-N-SH cells analyzed by annexin-V assay. Cells were treated
with CBD in a dose- and time-dependent manner (7.5 μg/mL, 10 μg/mL; 24 hours, 48 hours) and were stained with annexin-V and 7-amino
actinomycin D (7AAD). Q1 = percentage of dead cells; Q2 = percentage of cells in late apoptosis; Q3 = percentage of cells in early apoptosis;
Q4 = percentage of live cells. (C) Apoptotic effects of CBD on SK-N-SH cells analyzed by caspase-3 assay. Cells were treated with increasing
doses of CBD (7.5 μg/mL, 10 μg/mL) for 24 hours.
EFFICACY OF NON-PSYCHOACTIVE CANNABIDIOL ON NEUROBLASTOMA, Fisher et al.
S20 Current Oncology, Vol. 23, Supp. 2, March 2016
© 2016 Multimed Inc.
To further define the in vivo effect of cbd treatment
with respect to apoptosis induction, we ana lyzed tissue
obtained from tumour xenografts. Tumours were excised
after the last day of treatment, and paraffin-embedded
sections were analyzed immunohistochemica lly with the
apoptosis indicator cleaved caspase 3. Cells positive for
cleaved caspase 3 were detected w ith significantly greater
frequency i n sections of xenog raft s from cbd-tre ated mice
[Figure 5(B)] than in sect ions from ethanol-treated mice
[p < 0.001, Figure 5(C)].
To summarize, thc and cbd both suppressed the SK-
N-SH tumour xenograft growt h rate, with cbd treatment
demonst rating a bet ter effect . Moreover, the better e fficacy
of cbd and its ef fect on the indu ction of activ ated caspa se 3
are consistent w ith the results obtained in vitro.
DISCUSSION
In rece nt years, inter est in the role of c annabinoid s, main ly
thc, in cancer therapy has been renewed because of the
ability of these molecules to limit tumour cell prolifera-
tion and to induce selective cell death5,6,30. The response
to treatment with cannabinoids has been investigated
and demonstrated in a wide variety of adult tumours30;
however, the effect has been studied in only a few pedi-
atric tumours23,24. We therefore investigated the role of
can nabinoids in a p ediatr ic tumour, nbl, w hich is the mo st
frequ ent extrac rania l solid tumou r of childho od and which
still carries a ver y poor prognosis1.
We focuse d only on the major c ompounds in ca nnabis,
thc a nd cbd. The result s obtained i n the in vitro studies can
be summarized as follows:
nBoth molecules—a nd cbd in particu lar—reduced t he
viabilit y of nbl cells.
nThe effect of cbd seemed to be mediated by apoptotic
cell death, as demonstrated by morpholog y changes,
accumulation of sub-G1 cells, annexin V assay, and
increased expression of cleaved caspase 3.
nThe invasiveness of nbl cells was also reduced with
cbd treatment.
Based on t hat first set of r esults, we st udied the ef fect of
cbd and t hc on xenograft tumours generated in nod/scid
mice from SK-N-SH cells that had already demonstrated
the greatest sensitivity to the effect of those molecules. In
accord w ith the find ings from t he in vitro ex periments, thc
and cbd both reduced the xenogra ft growth rate, wit h cbd
showing a superior effect.
FIGURE 4 Anti-invasiveness effect of cannabidiol (CBD) on
SK-N-SH cells. The invasion assays were performed using cell
cultures (2x105 cells/well) treated with CBD (15 μg/mL, 20 μg/mL)
for 24 hours; results were compared with those for untreated cells
(2x105 cells/well). For each well (treated or untreated cells), 10 fields
were examined by light microscopy.
FIGURE 5 Cannabidiol (CBD) suppresses tumour growth in a mouse
xenograft model and increases cleaved caspase-3 staining in treated
xenografts. (A) Growth rate of SK-N-SH cell–derived tumour xenografts
treated for 14 days with intraperitoneal injections of ethanol-vehicle
(n = 12, closed triangles), 20 mg/kg Δ9-tetrahydrocannabinol (n = 12,
closed squares), 20 mg/kg CBD (n = 12, closed circles) and untreated
controls (n = 12, open squares). Data represents tumour volume during
14 days of treatment. a p < 0.05 and b p < 0.01 for CBD compared with
ethanol treatment (Mann–Whitney U-test). (B) Activated caspase-3 im-
munostaining in SK-NS-H cell–derived tumour xenografts treated with
CBD 20 mg/kg or ethanol vehicle for 14 days. (C) Counts of cleaved
caspase-3 immunoreactive cells in 18×10 lens fields from xenografts of
CBD- and ethanol-treated mice. a p < 0.0001 compared with ethanol.
EFFICACY OF NON-PSYCHOACTIVE CANNABIDIOL ON NEUROBLASTOMA, Fisher et al.
S21Current Oncology, Vol. 23, Supp. 2, March 2016
© 2016 Multimed Inc.
Our in vitro data suggesting that cbd inhibits the pro-
liferation of, a nd induces apoptosis in, nbl cells —together
with its remarkable effect on nbl xenografts—are, to the
best of our k nowledge, the first to show an antitumour
effect of cbd on nbl cells. Moreover, the results obtained
in our study indicate that, of the two cannabinoids tested,
cbd was more effective on the SK-N-SH cell line a nd on
xenografts than was the more-studied t hc. Those results
accord with recently emerging data showing an effect of
cbd on other tumours such as glioblastoma and breast,
lung, prostate, and colon ca ncer18, 31– 34 .
Δ9-Tetra hydrocan nabinol, the se cond most abundant
can nabinoid in C annabis sativa, has be en shown to induc e
apoptosis a nd to inh ibit tumour cel l viabil ity and i nvasive-
ness in various tumours34–38, as our study also demon-
strated. Recently, cbd was also reported to enhance the
production of react ive ox ygen species in cancer cells39, to
downregulate the metastat ic factor Id1, and to upregulate
the pro-dif ferentiation factor Id216,40.
The mechanism by which cbd produces the observed
effects has not yet been completely clarified, but seems
to be independent of the cb1 and cb2 receptors. Various
studies have demonstrated that cbd acts as an agonist
for vanilloid receptor 1 and for the trpv2, trpa 1 , ppar g,
and 5-ht1a receptors, a nd as an antagonist for the trpm8
and gpr55 receptors41. However, cbd’s antitumourigenic
molecular mechanisms of action have not been studied in
nbl. A hint ca n be found in several reports showing that
various nbl cel l lines ex press the fore going receptor s shown
to be involved in t he action of cbd42– 44. Several studies
have demonstrated that activation of those receptors wit h
agonists other than cbd mediates cell death in a variety of
nbl cell lines, including SK-N-SH45,46, the cbd-responsive
cell line in our study.
As a potent ial ther apeutic agent, cbd c ould have many
advantages, especially compared with psychoactive thc.
Because most—if not all—of the psychoactive ef fects of
cannabinoids are produced by activation of the central
cb1 receptors47, cbd, which has been shown to act inde-
pendentl y of cb1, is devoid of psycho active ef fects48 and c an
serve as a more suitable treatment, especially in children.
Additionally, it shares the palliative properties and low
toxic ity profile desc ribed for other c annabinoid s, has none
of the strong side effects associated with chemotherapeu-
tic agents10,18,49, and might have synergistic activity w ith
well-established antineoplastic substances10,5 0.
The most widely used route of cannabinoid admin-
istration is smoking—an unattractive clinical option,
particularly in children. Our work indicates that systemic
(intr aperitonea l) admin istration of cbd effec tively reduces
tumour growth, and use in a clinical sett ing can therefore
be based on other routes of administration, such as in oral
or oromucosa l treatments.
CONCLUSIONS
Our findings about the activity of cbd in nbl support and
extend previous findings about the a ntitumour activities
of cbd in other tumours and suggest that cannabis ex-
trac ts enriched i n cbd and not in thc c ould be suitable for
the development of novel non-psychotropic therapeutic
strategies in nbl . Use of cbd either as single agent or in
combination with ex ist ing compounds and chemother-
apy agents is a possibility. Combination therapy might
improve t he antitu mourigenic effects of ot her treatment s
and a llow for a reduction i n the chemotherapy dose, m in-
imizing toxicity and long-term sequelae. Future studies
are needed to highlight the pat hways involved in the
antitumourigenic effects of cbd in nbl as demonstrated
in the present work.
CONFLICT OF INTEREST DISCLOSURES
We have read a nd understood Current Oncology ’s policy on dis-
closing conflicts of interest, and we declare t hat we have none.
AUTHOR AFFILIATIONS
*Pedi atric Hemat o-Oncolog y Resear ch Laborator y, Sheba Cance r
Resea rch Center, Depar tment of Pediatric Hemato-Oncolog y, The
Edmond a nd Li ly Sa fra Chi ldren’s Hospital, a nd Depa rt ment of
Pathol ogy and §Pe diatr ic Stem Cell Re search I nstitut e, The Cha im
Sheba Med ical Cent er, Tel-Hashome r, Israel; ||In stitute f or Drug Re-
sear ch, Hebrew Univ ersity of Jer usalem , Jerusa lem, Israel ; #Cancer
Resea rch Center, The Chaim Sheba Medical Center, Tel-Hasho-
mer, Israel; **The Lautenberg Center for General and Tumour
Immu nology, Hebrew Universit y of Jerusalem, Jerusalem, Israel;
††Sack ler School of Medic ine, Tel-Aviv Univer sity, Tel-Aviv, Isra el.
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... (Table 7) 46 47 Fisher et al., found that THC and CBD reduced NBL cell viability in a dose and time dependant manor through induction of apoptosis and cell cycle arrest. 47 Alharris et al. expanded on this work, investigating precise CBD induced cell death mechanisms. Pretreatment of cells with caspase 2 and 3 inhibitors caused a significant reduction in apoptosis compared to CBD positive vehicle controls. ...
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Legislative change to cannabis use has generated significant interest into the therapeutic utility of cannabis-derived medical products, particularly in the field of oncology. However, much of this research has focused on adults, leaving physicians and caregivers uncertain as to the safety and efficacy of cannabinoids amongst the pediatric demographic. To this end, the aim of this review is to examine the scope of pharmaceutical cannabis in treatment of pediatric cancer, evaluating its utility as an anti-cancer therapeutic as well as symptom relief agent. This systematic review was conducted following the PRISMA guidelines. 30 included articles comprised of 16 clinical and 14 preclinical studies. There is reasonable evidence to support the use of cannabis in CINV, with plausible utility for other facets of symptomatic relief. Preclinical pediatric cancer models, investigating anti-cancer cannabinoid effect, have provided evidence that may warrant first phase clinical trials.
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TRPA1 and TRPM8 are transient receptor potential (TRP) channels involved in sensory perception. TRPA1 is a non-selective calcium permeable channel activated by irritants and proalgesic agents. TRPM8 reacts to chemical cooling agents such as menthol. The human neuroblastoma cell line IMR-32 undergoes a remarkable differentiation in response to treatment with 5-bromo-2-deoxyuridine. The cells acquire a neuronal morphology with increased expression of N-type voltage gated calcium channels and neurotransmitters. Here we show using RT-PCR, that mRNA for TRPA1 and TRPM8 are strongly upregulated in differentiating IMR-32 cells. Using whole cell patch clamp recordings, we demonstrate that activators of these channels, wasabi, allyl-isothiocyanate (AITC) and menthol activate membrane currents in differentiated cells. Calcium imaging experiments demonstrated that AITC mediated elevation of intracellular calcium levels were attenuated by ruthenium red, spermine, and HC-030031 as well as by siRNA directed against the channel. This indicates that the detected mRNA level correlate with the presence of functional channels of both types in the membrane of differentiated cells. Although the differentiated IMR-32 cells responded to cooling many of the cells showing this response did not respond to TRPA1/TRPM8 channel activators (60% and 90% for AITC and menthol respectively). Conversely many of the cells responding to these activators did not respond to cooling (30%). This suggests that these channels have also other functions than cold perception in these cells. Furthermore, our results suggest that IMR-32 cells have sensory characteristics and can be used to study native TRPA1 and TRPM8 channel function as well as developmental expression. J. Cell. Physiol. 221: 67–74, 2009. © 2009 Wiley-Liss, Inc