ArticlePDF Available

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


Abstract and Figures

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.
Content may be subject to copyright.
S15Current Oncology, Vol. 23, Supp. 2, March 2016
© 2016 Multimed Inc.
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*†††
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
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: n DOI: /10.3747/co.23.2893
a These authors contributed equally to the preparation of this
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.
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.).
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.
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. ).
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
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
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.
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.
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.
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.
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.
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.
We have read a nd understood Current Oncology ’s policy on dis-
closing conflicts of interest, and we declare t hat we have none.
*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.
1. Mari s JM, Hogar ty MD, Bagat ell R, Cohn SL . Neurobla stoma.
Lancet 2007;369:2106–20.
2. London W B, Castel V, Monclai r T, et al. Cli nical and biologic
featu res pred ictive of s urv ival a fter rela pse of neurobl astoma:
a report from the Internat ional Neuroblastoma Risk Group
project. J Clin Oncol 2011;29:3286–92.
3. Mechoula m R, ed. The Pharmacohistory of Cannabis sativa
Cannabinoids as Therapeutic Agents. Boca Raton, FL: CRC
Pre ss; 1986:1–19.
4. Pertwee RG. Emerging st rategies for exploiting canna-
binoid receptor agonists as medici nes. Br J Pharmacol
200 9;156 :397– 411.
5 Galve-Roperh I, Sa nchez C, Cortes ML, Gomez del Pulgar
T, Izquierdo M, Guzman M. Anti-tumoral action of can na-
binoids : involvement of sustained cera mide accumulation
and extracellula r signal-regulated kinase activation. Nat
Med 2000;6:313–19.
6 Velasco G, S anchez C, Gu zman M. Towa rds the use of c anna-
binoids a s antitu mour agent s. Nat Rev Cancer 2012;12 :436– 44.
7 Zygmunt PM, Petersson J, Andersson DA, et al. Van illoid
receptors on sensory nerves mediate the vasodilator action
of anandamide. Nature 1999;400:452–7.
8. O’Sullivan SE. Cannabinoids go nuclear: evidence for acti-
vation of peroxisome proliferator-activated receptors. Br J
Pharmacol 2007;152: 576–82.
9. Ga lanti G, Fisher T, Kventsel I, et al. Delta9tetrahydro-
can nabinol i nhibit s cell cy cle progre ssion by down regu lation
of e2f1 in human glioblastoma mu ltiforme cells. Acta Oncol
2008 ; 47:106 2–70.
10. Car racedo A, Lorente M, Eg ia A, et al. The stress-regulated
protein p 8 mediate s cann abinoid-induc ed apoptosi s of tumor
cells. Cancer Cell 2 006 ;9:301–12.
11. Ca lvar uso G, Pell erito O, Notar o A, Giuli ano M. Can nabinoid-
asso ciated cel l death mec hanis ms in tu mor models (rev iew).
Int J Oncol 2012 ;41:407–13.
12. Sala zar M, Carracedo A, Sala nueva IJ, et al. Cannabinoid
action induces autophagy-mediated cell death through
stimulat ion of er stress in human glioma cel ls. J Clin Invest
S22 Current Oncology, Vol. 23, Supp. 2, March 2016
© 2016 Multimed Inc.
13. Vara D, Sala zar M, Olea-Herrero N, Gu zma n M, Velasco G,
Diaz-Laviada I. A nti-tumora l action of cannabinoids on he-
patocellular carcinoma: role of ampk-dependent activation
of autophag y. Cell Death Differ 2011;18 :1099 –111.
14. Portella G, L aezza C, Laccetti P, De Petrocellis L , Di Marzo
V, Bifulco M. Inhibitory ef fects of ca nnabinoid c b1 receptor
stimulation on tumor growth and metastatic spreading:
actions on signals involved in angiogenesis and metastasis.
FASEB J 2003 ;17:17 71–3.
15. Qamri Z , Preet A, Nasser MW, et al. Synt hetic cannabinoid
receptor agonists inhibit tu mor growt h and metastasis of
breast cancer. Mol Cancer T her 2009;8:3117–29.
16. Ra mer R, Hin z B. Inhibi tion of canc er cell in vasion by ca nna-
binoids v ia incr eased ex pression of ti ssue inh ibitor of matr ix
metalloproteinases-1. J Natl Cancer Inst 2008;100:59–69.
17. McAllister SD, Mura se R, Christian RT, et al. Pathways me-
diating t he effects of cannabidiol on the reduct ion of breast
cancer cell proli ferat ion, invasion, and metastasis. Breast
Cancer Res Treat 2011;129:37–47.
18. M assi P, Solina s M, Cinqui na V, Parolar o D. Cannabi diol as po-
tential anti-cancer drug. Br J Clin Pharmacol 2012;75:303–12.
19. Gomez del Pulgar T, Velasco G, Sanchez C, Haro A, Guzman
M. De novo synt hesi zed ceramide is involved in cannabi-
noid-induced apoptosis. Biochem J 20 02;363:18 3–8.
20. El lert-Miklaszewska A, Kaminska B, Kona rska L. Ca nnabi-
noids down-regulate pi3k/Akt and erk signalling pathways
and ac tivate pro apoptotic f unction of B ad protein. C ell Signal
20 0 5 ;17 : 25 – 3 7.
21. Blazquez C, Sa lazar M, Car racedo A, et al. Can nabinoids
inh ibit glioma cell invasion by down-regulat ing matrix
meta lloprotei nase-2 expr ession. Canc er Res 2008;68:1945–52.
22. Blazquez C, Gonza lez-Feria L, A lvarez L, Haro A, Casa-
nova ML, Guzman M. Can nabinoids inhibit the vascula r
endothelial growth factor pathway in gliomas. Cancer Res
2004; 64:5617–23.
23. Oesc h S, Walter D, Wachtel M, et al . Canna binoid recept or 1 is
a potential drug target for treatment of translocation-positive
rhabdomyosarcoma. Mol Cancer Ther 20 09; 8:1838–45.
24. Notaro A , Sabella S, P ellerito O, et al. I nvolvement of pa r-4 i n
can nabinoid-de pendent sensit ization of o steosar coma cell s
to trail-induced apoptosis. Int J Biol Sci 2014 ;10:46 6–78 .
25. Gi lbert LC, Wachsman JT. Characterization and pa rt ial
puri fication of the plasm inogen activator from human neu-
roblastoma cel l line, SK-N-SH. A compa rison with human
urokinase. Biochim Biophys Acta 1982;704 :450 –60.
26. Tumilow icz JJ, Nichol s WW, Cholon JJ, Gre ene AE. Defi nition
of a conti nuous huma n cell li ne derive d from neur oblastoma .
Cancer Res 1970; 30 :2110–18.
27. Yeger H, Bau mal R, Paw lin G, et al. Phe notypic a nd molecul ar
characterization of inducible human neuroblastoma cell
lines. Differentiation 19 88 ;39 : 21627.
28. Yaari S, Jacob-Hirsch J, Amar iglio N, Haklai R, Rechavi G,
Kloog Y. Disruption of cooperat ion bet ween Ras and MycN
in hum an neurobl astoma cel ls promotes gr owth ar rest. Clin
Cancer Res 2005;11:4321–3 0.
29. Mosm ann T. Rapid color imetr ic assay fo r cellul ar grow th and
sur viva l: applic ation to prol iferation a nd cyt otoxicit y assays .
J Immunol Methods 1983;65:55–63.
30. Sar fara z S, Adha mi VM, Sy ed DN, Afaq F, Mukh tar H. Ca nna-
binoids for cancer treatment: prog ress and promise. Cancer
Res 2008;68:339–42.
31. Roma no B, Borrell i F, Paga no E, Casc io MG, Pertw ee RG, Izzo
AA. Inhibition of colon carci nogenesis by a standardized
Cannabis sativa ex tract with high content of ca nnabidiol.
Phytomedicine 2014; 21:631–9.
32. Nabissi M, Morelli MB, A mantini C, et al. Cannabidiol
stimulates Aml-1a–dependent glial differentiation and
inhibits glioma stem-like cells proliferation by inducing
autophag y in a trpv2-dependent manner. Int J Cancer
2015;137:1855 69.
33. Morelli MB, Offida ni M, A lesiani F, et al. The effect s of
cannabidiol and its sy nerg ism w ith bortezomib in multiple
myeloma cell lines. A role for transient receptor potentia l
vanilloid type-2. Int J Cancer 2014;134:2534–46.
34. Elba z M, Nasser MW, Ravi J, et al. Modulation of the tumor
micr oenviron ment and in hibitio n of egf/egfr pathway : nov-
el anti-tumor mecha nisms of ca nnabidiol in breast cancer.
Mol Oncol 2015;9:906–19.
35. Ligre sti A, Moriel lo AS, Sta rowicz K, et al . Antitu mor activ ity
of plant cannabinoids with emphasis on the ef fect of can-
nabidiol on huma n breast carcinoma. J Phar macol Exp Ther
20 0 6 ; 318 : 13 75 – 8 7.
36. Ramer R, Merkord J, Rohde H, Hin z B. Cannabidiol inhib-
its cancer cell invasion via upregu lation of tissue inhib-
itor of matrix metalloproteina ses-1. Biochem Pharmacol
2010 ;7 9 :95566 .
37. Ra mer R, Rohde A, Merkord J, Rohde H, Hin z B. Decrease
of plasm inogen activator i nh ibitor-1 may contr ibute to t he
anti-invasive action of cannabidiol on huma n lung cancer
cells. Pharm Res 2010 ;27:216 2–74.
38. Aviel lo G, Romano B, Bor relli F, et al. Che mopreventive ef fect
of the non-psychotropic phy tocannabinoid can nabidiol on
expe rimenta l colon canc er. J Mol Med (Berl) 2012;90:925–34.
39. Singer E, Judkins J, Salomon is N, et al. React iv e ox ygen
species-mediated therapeutic response and resistance in
glioblastoma. Cell Death Dis 2 015 ;6:e1601.
40. McA lliste r SD, Christ ian RT, Horowit z MP, Garcia A, D esprez
PY. Can nabidiol a s a novel inh ibitor of Id-1 gene expres sion in
agg ressive br east ca ncer cell s. Mol Cancer Th er 2007;6:2921–7.
41. Massi P, Solinas M, Cinquina V, Parolaro D. Cannabidiol as
potent ial ant icance r drug. B r J Clin Pharmacol 2013;75:303–12.
42. El An daloussi-L ilja J, Lundqv ist J, Forsby A . trpv1 expression
and ac tivit y duri ng retinoic a cid–indu ced neurona l dif feren-
tiation. Neurochem Int 2009;55:768–74.
43. Caba llero FJ, So ler-Torron teras R, La ra-Chi ca M, et al. AM4 04
inh ibits nfat and nf-κb signaling pathways and impairs
migration and invasiveness of neuroblastoma cells. Eur J
Pharmacol 2015 ;746 :221– 32.
44. L ouhiv uori LM, Bar t G, Lars son KP, et al. Di fferentiation de-
pendent ex pression of tr pa 1 and trpm8 channels in IMR-32
human neuroblastoma cells. J Cell Physiol 20 09 ;221: 67–74.
45. Cellai I, Benvenuti S, Luciani P, et al. Ant ineoplastic effect s
of rosig litaz one and ppa rγ t ransac tivation i n neurobla stoma
cells. Br J Cancer 2006;95:879–88.
46. Baek Y M, Hwang HJ, K im SW, et al. A compa rative prot eomic
analysis for capsaicin-induced apoptosis between huma n
hepatocarcinoma (HepG2) a nd human neuroblastoma (SK-
N-SH) cel ls. Proteomics 2008;8:4748–67.
47. Wiskerke J, Pattij T, Schoffelmeer AN, De Vries TJ. The role
of cb1 receptors in psychosti mulant add iction. Addict Biol
48. Hollister LE, Gillespie H. Interactions in man of delta-9-tet-
rahydrocannabinol. ii. Can nabinol and cannabidiol. Clin
Pharmacol Ther 1975;18:80–3.
49. Gu zma n M, Duarte MJ, Blazquez C, et al. A pilot cli nical
study o f Δ9-tetrah ydroca nnabinol i n patients w ith rec urrent
glioblastoma mult ifor me. Br J Cancer 2006;95:197–203.
50. Holland ML, Lau DT, Allen JD, Arnold JC. The multidrug
transporter abcg2 (bcrp) is inhibited by plant-der ived can-
nabinoids. Br J Pharmacol 20 07;152:815–24.
... A recent comprehensive review summarizes the biological effects of CBD in different tumor types and is highly recommendable for interested readers [6]. The biological effects of CBD have been tested in a broad range of tumor cells in vitro and in vivo (Table A1 in Appendix A), including glioma/glioblastoma [9,[11][12][13][14][15][16][17][18][19], breast 38], lung cancer [39][40][41], cervical cancer [25,39,42]), neuroblastoma [43,44], medulloblastoma [45], ependymoma [45], pancreatic cancer [46,47], ovarian cancer [28], endometrial cancer [48], bladder urothelial carcinoma [49], and head and neck squamous cell carcinoma [50]. ...
... Since mitochondrial damage causes reduced ATP production, the energy-consuming basic cellular functions such as migration are affected by CBD. Decreased migration capacities were reported from the first hour of CBD treatment in glioblastoma [13] and leukemia [25] and during 24-48 h of observation in multiple cancer types such as bladder carcinoma [49], neuro-and glioblastoma [17,41,43], breast [20,21], cervical [39], lung [39], and endometrial cancer [48], and squamous cell carcinoma [50]. ...
... The anticancer properties of CBD were confirmed in experiments in vivo. CBD reduced tumor growth and metastasis in animal models such as human xenografts of squamous carcinoma [50], colorectal and gastric cancer [35,37], lung cancer [39,40], prostate carcinoma [29], glioma [12], and neuroblastoma [43], as well as orthotopic implants in mice, such as medulloblastoma/ependymoma [45], breast cancer [21,23], and leukemia [31]. Working CBD doses were within 1-100 mg/kg body weight, typically 5 mg/kg [9,39,40,50], which is roughly equivalent to a low micromolar range. ...
Full-text available
Cannabidiol (CBD), a major non-psychotropic component of cannabis, is receiving growing attention as a potential anticancer agent. CBD suppresses the development of cancer in both in vitro (cancer cell culture) and in vivo (xenografts in immunodeficient mice) models. For critical evaluation of the advances of CBD on its path from laboratory research to practical application, in this review, we wish to call the attention of scientists and clinicians to the following issues: (a) the biological effects of CBD in cancer and healthy cells; (b) the anticancer effects of CBD in animal models and clinical case reports; (c) CBD’s interaction with conventional anticancer drugs; (d) CBD’s potential in palliative care for cancer patients; (e) CBD’s tolerability and reported side effects; (f) CBD delivery for anticancer treatment.
... Andradas [111] points out that most of the conducted studies concern acute lymphoblastic leukemia, which indicates that cannabinoids destroy cancer cells both in vivo and in vitro and that cannabinoids THC and CBD interact with vincristine, cytarabine, and doxorubicin in vitro [123,124]. It has also been shown that synthetic cannabinoids inhibit rhabdomyosarcoma growth [125] and reduce the viability and invasiveness of neuroblastoma cells [126]. Furthermore, synthetic cannabinoids induced cell cycle arrest of osteosarcoma cells [127]. ...
... 2016 [126] neuroblastoma AM404 (ECS modulator) SK-N-SH AM404 inhibits NFAT and NF-κB transcriptional activity by CB1-and TRPV1-independent mechanism; AM404 inhibits MMP-1, -3, and -7 expression and cell migration; ...
Full-text available
Neoplastic diseases in children are the second most frequent cause of death among the young. It is estimated that 400,000 children worldwide will be diagnosed with cancer each year. The nutritional status at diagnosis is a prognostic indicator and influences the treatment tolerance. Both malnutrition and obesity increase the risk of mortality and complications during treatment. It is necessary to constantly search for new factors that impair the nutritional status. The endocannabinoid system (ECS) is a signaling system whose best-known function is regulating energy balance and food intake, but it also plays a role in pain control, embryogenesis, neurogenesis, learning, and the regulation of lipid and glucose metabolism. Its action is multidirectional, and its role is being discovered in an increasing number of diseases. In adults, cannabinoids have been shown to have anti-cancer properties against breast and pancreatic cancer, melanoma, lymphoma, and brain tumors. Data on the importance of both the endocannabinoid system and synthetic cannabinoids are lacking in children with cancer. This review highlights the role of nutritional status in the oncological treatment process, and describes the role of ECS and gastrointestinal peptides in regulating appetite. We also point to the need for research to evaluate the role of the endocannabinoid system in children with cancer, together with a prospective assessment of nutritional status during oncological treatment.
... The highest number of in vitro studies have been performed on tumour cells of the nervous system, followed by tests on breast cancer cell lines. IC 50 values of pure CBD varied widely from about 0.6 µM to more than 22 µM for tumours of the nervous system, and were more effective than CBD-E, THC or CBG [15][16][17][18][19][20]. In ependymoma cell lines, CBD and THC were similarly effective, whereas CBD was more potent in medulloblastoma [20]. ...
... Three studies compared, head-to-head, the antitumour effects between CBD and THC. CBD was equally potent to THC in glioma [57], and superior in neuroblastoma cell xenografts [19]. In the third study on medulloblastoma xenografts, neither CBD nor THC could demonstrate a clear superiority compared with controls [20]. ...
Full-text available
Preclinical models provided ample evidence that cannabinoids are cytotoxic against cancer cells. Among the best studied phytocannabinoids, cannabidiol (CBD) is most promising for the treatment of cancer as it lacks the psychotomimetic properties of delta-9-tetrahydrocannabinol (THC). In vitro studies and animal experiments point to a concentration- (dose-)dependent anticancer effect. The effectiveness of pure compounds versus extracts is the subject of an ongoing debate. Actual results demonstrate that CBD-rich hemp extracts must be distinguished from THC-rich cannabis preparations. Whereas pure CBD was superior to CBD-rich extracts in most in vitro experiments, the opposite was observed for pure THC and THC-rich extracts, although exceptions were noted. The cytotoxic effects of CBD, THC and extracts seem to depend not only on the nature of cannabinoids and the presence of other phytochemicals but also largely on the nature of cell lines and test conditions. Neither CBD nor THC are universally efficacious in reducing cancer cell viability. The combination of pure cannabinoids may have advantages over single agents, although the optimal ratio seems to depend on the nature of cancer cells; the existence of a 'one size fits all' ratio is very unlikely. As cannabinoids interfere with the endocannabinoid system (ECS), a better understanding of the circadian rhythmicity of the ECS, particularly endocannabinoids and receptors, as well as of the rhythmicity of biological processes related to the growth of cancer cells, could enhance the efficacy of a therapy with cannabinoids by optimization of the timing of the administration, as has already been reported for some of the canonical chemotherapeutics. Theoretically, a CBD dose administered at noon could increase the peak of anandamide and therefore the effects triggered by this agent. Despite the abundance of preclinical articles published over the last 2 decades, well-designed controlled clinical trials on CBD in cancer are still missing. The number of observations in cancer patients, paired with the anticancer activity repeatedly reported in preclinical in vitro and in vivo studies warrants serious scientific exploration moving forward.
... Table 2 shows the latest studies on various cannabinoids used in cancer models [88]. AEA, AM251 0-0.5 µM Decrease in the invasiveness of CD44 + /CD24 −/low /ESA + cancer stem cell [92] CBDA (11), ST-247, GSK0660, GW501516 1-50 µM CBDA (11) prevents transcriptional activation of PPARβ/δ [93] CBD (10) 1-50 µM A synergistic effect observed after coadministration of CBDsol and paclitaxel or docetaxel [94] Human glioblastoma Δ 9 -THC (1), CBD (10) 0-5 µM The substantial apoptotic induction and GIC population reduction [95] CBD (10) 0-5 µM Downregulation of key stem cell regulators including Sox2 and p-STAT3 and activation of p-p38 pathway [96] CBD (10), SR141716, SR144528 5-40 µM Effects on apoptosis induction and antiproliferative activity [96] Human neuroblastoma Δ 9 -THC (1), CBD (10) 0-50 µg/mL Cell viability reduction and apoptosis [97] Human glioblastoma multiforme, Human GBM cultures Δ 9 -THC (1), WIN 55,212-2 0.1 nM-2 µM Increase in apoptosis and antiproliferative effects [98] Pancreatic cancer CBD (10) 0-10 µM GPR55-mediated antiproliferative effects [99] Human (10) 0.01-10 µM Decreased in viability of cell and cell cycle arrest [110] Human melanoma Δ 9 -THC (1), CBD (10) 0-10 µM Decreased in viability of cell [111] Murine squamous, non-melanoma skin cancer AEA, AMG9810, AM251, AM630 2.5-40 µM Reduction in viability of cell due to apoptosis [112] Human renal carcinoma WIN 55, 212-2, JWH-133, SR141716A, AM630 0-25 µM Induction of apoptosis and reduction in cell proliferation [113] Human ovarian cancer CBD (10) 10-50 µM Inhibition of proliferation of cell [94] Rat adrenal gland DHA-DA, AEA 0-80 µM NOS activation, enhanced Ca 2+ signalling, and GPR55 activation cause apoptosis [114] AEA (anandamide); MET-AEA (methanandamide, non-hydrolysable analogue of AEA); AM251 (CB1 antagonist); DHA-DA (N-docosahexaenoyl dopamine); HU-210 (CB1 agonist); JWH-133 (CB2 agonist); JWH-015 (CB2 agonist); SR141716 (CB1 inverse agonist); WIN 55,212-2 (CB1 agonist); Noleoylethanolamine (NOE) (acidic ceramidase inhibitor); SR144528 (CB2 inverse agonist); PD98059 (ERK inhibitor); LY294002 (PI3K inhibitor); PBMCs (peripheral blood mononuclear cells); GW9662 (PPAR-γ antagonist); AM630 (CB2 antagonist); GSK066 (PPARβ/δ antagonist); AMG9810 (TRPV1 antagonist); GSK501516 (PPARδ antagonist); NOS (nitric oxide synthases); EMT (epithelialmesenchymal transition); CSCs (cancer stem cells). ...
Full-text available
The development of new antibiotics is urgently needed to combat the threat of bacterial resistance. New classes of compounds that have novel properties are urgently needed for the development of effective antimicrobial agents. The extract of Cannabis sativa L. has been used to treat multiple ailments since ancient times. Its bioactivity is largely attributed to the cannabinoids found in its plant. Researchers are currently searching for new anti-infective agents that can treat various infections. Although its phytocannabinoid ingredients have a wide range of medical benefits beyond the treatment of infections, they are primarily associated to psychotropic effects. Different cannabinoids have been demonstrated to be helpful against harmful bacteria, including Gram-positive bacteria. Moreover, combination therapy involving the use of different antibiotics has shown synergism and broad-spectrum activity. The purpose of this review is to gather current data on the actions of Cannabis sativa (C. sativa) extracts and its primary constituents such as terpenes and cannabinoids towards pathogens in order to determine their antimicrobial properties and cytotoxic effects together with current challenges and future perspectives in biomedical application.
... (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. ...
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.
... Senescence-associated β-galactosidase (SA-β-gal) activity was measured with a βgalactosidase staining kit (Senescence B-Galactosidase Staining KIT, Cell Signaling Technology, #9860) according to the manufacturer's instructions. Briefly, SK-N-SH cells (5 × 10 4 cells\well) were plated in 6-well plates (3 mL) and treated according to the previously described treatment regimen [19] with 50, 75 and 100 µM of HU-600 or HU-585 for 48 h and then fixed and incubated overnight at 37 • C in CO 2 free environment. Accumulation of a distinctive blue color in senescent cells was then observed by microscope (Olympus Scientific Solutions, Waltham, MA, USA). ...
Full-text available
Modulation of the endogenous cannabinoid system has been suggested as a potential anticancer strategy. In the search for novel and less toxic therapeutic options, structural modifications of the endocannabinoid anandamide and the synthetic derivative of oleic acid, Minerval (HU-600), were done to obtain 2-hydroxy oleic acid ethanolamide (HU-585), which is an HU-600 derivative with the anandamide side chain. We showed that treatment of SK-N-SH neuroblastoma cells with HU-585 induced a better anti-tumorigenic effect in comparison to HU-600 as evidenced by 3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide assay, colony-forming assay, and migration assay. Moreover, HU-585 demonstrated pro-apoptotic properties shown by increased levels of activated caspase-3 following treatment and a better senescence induction effect in comparison to HU-600, as demonstrated by increased activity of lysosomal β-galactosidase. Finally, we observed that combined treatment of HU-585 with the senolytic drugs ABT-263 in vitro, and ABT-737 in vivo resulted in enhanced anti-proliferative effects and reduced neuroblastoma xenograft growth in comparison to treatment with HU-585 alone. Based on these results, we suggest that HU-585 is a pro-apoptotic and senescence-inducing compound, better than HU-600. Hence, it may be a beneficial option for the treatment of resistant neuroblastoma especially when combined with senolytic drugs that enhance its anti-tumorigenic effects.
... [101] Regarding the mechanism by which cannabis may act on pediatric brain tumors, there are four studies. A 2016 study found that CBD was able to decrease the viability and induce apoptosis of neuroblastoma cells, in addition to decreasing tumor growth with in vivo xenografts [92]. As CBD does not have a particularly high affinity for CB1 [27], this further suggests that anti-cancer effects may be triggered by making use of receptors other than CB1. ...
Full-text available
The antineoplastic effects of cannabis have been known since 1975. Since the identification of the components of the endogenous cannabinoid system (ECS) in the 1990s, research into the potential of cannabinoids as medicine has exploded, including in anti-cancer research. However, nearly all of this research has been on adults. Physicians and governing bodies remain cautious in recommending the use of cannabis in children, since the ECS develops early in life and data about cannabis exposure in utero show negative outcomes. However, there exist many published cases of use of cannabis in children to treat pediatric epilepsy and chemotherapy-induced nausea and vomiting (CINV) that show both the safety and efficacy of cannabis in pediatric populations. Additionally, promising preclinical evidence showing that cannabis has anti-cancer effects on pediatric cancer warrants further investigation of cannabis’ use in pediatric cancer patients, as well as other populations of pediatric patients. This review aims to examine the evidence regarding the potential clinical utility of cannabis as an anti-cancer treatment in children by summarizing what is currently known about uses of medical cannabis in children, particularly regarding its anti-cancer potential.
Full-text available
Tetrahydrocannabinols (THCs) antagonize the CB1 and CB2 cannabinoid receptors, whose signaling to the endocannabinoid system is essential for controlling cell survival and proliferation as well as psychoactive effects. Most tumor cells express a much higher level of CB1 and CB2; THCs have been investigated as potential cancer therapeutic due to their cannabimimetic properties. To date, THCs have been prescribed as palliative medicine to cancer patients but not as an anticancer modality. Growing evidence of preclinical research demonstrates that THCs reduce tumor progression by stimulating apoptosis and autophagy and inhibiting two significant hallmarks of cancer pathogenesis: metastasis and angiogenesis. However, the degree of their anticancer effects depends on the origin of the tumor site, the expression of cannabinoid receptors on tumor cells, and the dosages and types of THC. This review summarizes the current state of knowledge on the molecular processes that THCs target for their anticancer effects. It also emphasizes the substantial knowledge gaps that should be of concern in future studies. We also discuss the therapeutic effects of THCs and the problems that will need to be addressed in the future. Clarifying unanswered queries is a prerequisite to translating the THCs into an effective anticancer regime.
Endogenous and exogenous cannabinoids modulate many physiological and pathological processes by binding classical cannabinoid receptors 1 (CB1) or 2 (CB2) or non-cannabinoid receptors. Cannabinoids are known to exert antiproliferative, apoptotic, anti-migratory and anti-invasive effect on cancer cells by inducing or inhibiting various signaling cascades. In this chapter, we specifically emphasize the latest research works about the alterations in endocannabinoid system (ECS) components in malignancies and cancer cell proliferation, migration, invasion, angiogenesis, autophagy, and death by cannabinoid administration, emphasizing their mechanism of action, and give a future perspective for clinical use.
Colorectal cancer is the third leading cause of cancer incidence and mortality in the United States. Cannabidiol (CBD), the second most abundant phytocannabinoid in Cannabis sativa, has potential use in cancer treatment on the basis of many studies showing its anti-cancer activity in diverse types of cancer, including colon cancer. However, its mechanism of action is not yet fully understood. In the current study, we observed CBD to repress viability of different human colorectal cancer cells in a dose-dependent manner. CBD treatment led to G1-phase cell cycle arrest and an increased sub-G1 population (apoptotic cells); it also downregulated protein expression of cyclin D1, cyclin D3, cyclin-dependent kinase 2 (CDK2), CDK4, and CDK6. CBD further increased caspase 3/7 activity and cleaved poly(ADP-ribose) polymerase, and elevated expression of endoplasmic reticulum (ER) stress proteins including binding immunoglobulin protein (BiP), inositol-requiring enzyme 1α (IRE1α), phosphorylated eukaryotic initiation factor 2α (eIF2α), activating transcription factor 3 (ATF3), and ATF4. We found that CBD repressed cell viability and induced apoptotic cell death through a mechanism dependent on cannabinoid receptor type 2 (CB2), but not on CB1, transient receptor potential vanilloid, or peroxisome proliferator-activated receptor gamma. Anti-proliferative activity was also observed for other non-psychoactive cannabinoid derivatives including cannabidivarin (CBDV), cannabigerol (CBG), cannabicyclol (CBL), and cannabigerovarin (CBGV). Our data indicate that CBD and its derivatives could be promising agents for the prevention of human colorectal cancer.
Full-text available
Glioblastoma (GBM) resistance to therapy is the most common cause of tumor recurrence, which is ultimately fatal in 90% of the patients 5 years after initial diagnosis. A sub-population of tumor cells with stem-like properties, glioma stem cells (GSCs), is specifically endowed to resist or adapt to the standard therapies, leading to therapeutic resistance. Several anticancer agents, collectively termed redox therapeutics, act by increasing intracellular levels of reactive oxygen species (ROS). In this study, we investigated mechanisms underlying GSC response and resistance to cannabidiol (CBD), a non-toxic, non-psychoactive cannabinoid and redox modulator. Using primary GSCs, we showed that CBD induced a robust increase in ROS, which led to the inhibition of cell survival, phosphorylated (p)-AKT, self-renewal and a significant increase in the survival of GSC-bearing mice. Inhibition of self-renewal was mediated by the activation of the p-p38 pathway and downregulation of key stem cell regulators Sox2, Id1 and p-STAT3. Following CBD treatment, a subset of GSC successfully adapted, leading to tumor regrowth. Microarray, Taqman and functional assays revealed that therapeutic resistance was mediated by enhanced expression of the antioxidant response system Xc catalytic subunit xCT (SLC7A11 (solute carrier family 7 (anionic amino-acid transporter light chain), member 11)) and ROS-dependent upregulation of mesenchymal (MES) markers with concomitant downregulation of proneural (PN) markers, also known as PN-MES transition. This 'reprogramming' of GSCs occurred in culture and in vivo and was partially due to activation of the NFE2L2 (NRF2 (nuclear factor, erythroid 2-like)) transcriptional network. Using genetic knockdown and pharmacological inhibitors of SLC7A11, we demonstrated that combining CBD treatment with the inhibition of system Xc resulted in synergistic ROS increase leading to robust antitumor effects, that is, decreased GSC survival, self-renewal, and invasion. Our investigation provides novel mechanistic insights into the antitumor activity of redox therapeutics and suggests that combinatorial approaches using small molecule modulators of ROS offer therapeutic benefits in GBM.
Full-text available
The synthetic cannabinoid WIN 55,212-2 is a potent cannabinoid receptor agonist with anticancer potential. Experiments were performed to determine the effects of WIN on proliferation, cell cycle distribution, and programmed cell death in human osteosarcoma MG63 and Saos-2 cells. Results show that WIN induced G2/M cell cycle arrest, which was associated with the induction of the main markers of ER stress (GRP78, CHOP and TRB3). In treated cells we also observed the conversion of the cytosolic form of the autophagosome marker LC3-I into LC3-II (the lipidated form located on the autophagosome membrane) and the enhanced incorporation of monodansylcadaverine and acridine orange, two markers of the autophagic compartments such as autolysosomes. WIN also induced morphological effects in MG63 cells consisting in an increase in cell size and a marked cytoplasmic vacuolization. However, WIN effects were not associated with a canonical apoptotic pathway, as demonstrated by the absence of specific features, and only the addition of TRAIL to WIN-treated cells led to apoptotic death probably mediated by up-regulation of the tumor suppressor factor PAR-4, whose levels increased after WIN treatment, and by the translocation of GRP78 on cell surface.
Full-text available
In recent years, cannabinoids (the active components of Cannabis sativa) and their derivatives have received considerable interest due to findings that they can affect the viability and invasiveness of a variety of different cancer cells. Moreover, in addition to their inhibitory effects on tumor growth and migration, angiogenesis and metastasis, the ability of these compounds to induce different pathways of cell death has been highlighted. Here, we review the most recent results generating interest in the field of death mechanisms induced by cannabinoids in cancer cells. In particular, we analyze the pathways triggered by cannabinoids to induce apoptosis or autophagy and investigate the interplay between the two processes. Overall, the results reported here suggest that the exploration of molecular mechanisms induced by cannabinoids in cancer cells can contribute to the development of safe and effective treatments in cancer therapy.
Glioma stem-like cells (GSCs) correspond to a tumor cell subpopulation, involved in glioblastoma multiforme (GBM) tumor initiation and acquired chemoresistance. Currently, drug-induced differentiation is considered as a promising approach to eradicate this tumor-driving cell population. Recently, the effect of cannabinoids (CBs) in promoting glial differentiation and inhibiting gliomagenesis has been evidenced. Herein, we demonstrated that cannabidiol (CBD) by activating Transient Receptor Potential Vanilloid-2 (TRPV2) triggers GSCs differentiation activating the autophagic process and inhibits GSCs proliferation and clonogenic capability. Above all, CBD and carmustine (BCNU) in combination overcome the high resistance of GSCs to BCNU treatment, by inducing apoptotic cell death. Acute myeloid leukemia (Aml-1) transcription factors play a pivotal role in GBM proliferation and differentiation and it is known that Aml-1 control the expression of several nociceptive receptors. So, we evaluated the expression levels of Aml-1 spliced variants (Aml-1a, b and c) in GSCs and during their differentiation. We found that Aml-1a is up-regulated during GSCs differentiation, and its down-regulation restores a stem cell phenotype in differentiated GSCs. Since it was demonstrated that CBD induces also TRPV2 expression and that TRPV2 is involved in GSCs differentiation, we evaluated if Aml-1a interacted directly with TRPV2 promoters. Herein, we found that Aml-1a binds TRPV2 promoters and that Aml-1a expression is up-regulated by CBD treatment., in a TRPV2 and PI3K/AKT dependent manner. Altogether, these results support a novel mechanism by which CBD inducing TRPV2-dependent autophagic process stimulates Aml-1a-dependent GSCs differentiation, abrogating the BCNU chemoresistance in GSCs. This article is protected by copyright. All rights reserved. © 2015 UICC.
The anti-tumor role and mechanisms of Cannabidiol (CBD), a non-psychotropic cannabinoid compound, are not well studied especially in triple-negative breast cancer (TNBC). In the present study, we analyzed CBD's anti-tumorigenic activity against highly aggressive breast cancer cell lines including TNBC subtype. We show here -for the first time-that CBD significantly inhibits epidermal growth factor (EGF)-induced proliferation and chemotaxis of breast cancer cells. Further studies revealed that CBD inhibits EGF-induced activation of EGFR, ERK, AKT and NF-kB signaling pathways as well as MMP2 and MMP9 secretion. In addition, we demonstrated that CBD inhibits tumor growth and metastasis in different mouse model systems. Analysis of molecular mechanisms revealed that CBD significantly inhibits the recruitment of tumor-associated macrophages in primary tumor stroma and secondary lung metastases. Similarly, our in vitro studies showed a significant reduction in the number of migrated RAW 264.7 cells towards the conditioned medium of CBD-treated cancer cells. The conditioned medium of CBD-treated cancer cells also showed lower levels of GM-CSF and CCL3 cytokines which are important for macrophage recruitment and activation. In summary, our study shows -for the first time-that CBD inhibits breast cancer growth and metastasis through novel mechanisms by inhibiting EGF/EGFR signaling and modulating the tumor microenvironment. These results also indicate that CBD can be used as a novel therapeutic option to inhibit growth and metastasis of highly aggressive breast cancer subtypes including TNBC, which currently have limited therapeutic options and are associated with poor prognosis and low survival rates. Copyright © 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
N-arachidonoylphenolamine (AM404), a paracetamol lipid metabolite, is a modulator of the endocannabinoid system endowed with pleiotropic activities. AM404 is a dual agonist of the Transient Receptor Potential Vanilloid type 1 (TRPV-1) and the Cannabinoid Receptor type 1 (CB1) and inhibits anandamide (AEA) transport and degradation. In addition, it has been shown that AM404 also exerts biological activities through TRPV-1- and CB1-independent pathways. In the present study we have investigated the effect of AM404 in the NFAT and NF-κB signaling pathways in SK-N-SH neuroblastoma cells. AM404 inhibited NFAT transcriptional activity through a CB1- and TRPV-1-independent mechanism. Moreover, AM404 inhibited both the expression of COX-2 at transcriptional and post-transcriptional levels and the synthesis of PGE2. AM404 also inhibited NF-κB activation induced by PMA/Ionomycin in SK-N-SH cells by targeting IKKβ phosphorylation and activation. We found that Cot/Tlp-2 induced NFAT and COX-2 transcriptional activities were inhibited by AM404. NFAT inhibition paralleled with the ability of AM404 to inhibit MMP-1 and MMP2 expression, cell migration and invasion in a cell-type specific dependent manner. Taken together, these data reveal that paracetamol, the precursor of AM404, can be explored not only as an antipyretic and painkiller drug but also as a co-adjuvant therapy in inflammatory and cancer diseases. Copyright © 2014. Published by Elsevier B.V.
Multiple myeloma (MM) is a plasma cell (PC) malignancy characterised by the accumulation of a monoclonal PC population in the bone marrow (BM). Cannabidiol (CBD) is a non-psychoactive cannabinoid with antitumoural activities, and the transient receptor potential vanilloid type-2 (TRPV2) channel has been reported as a potential CBD receptor. TRPV2 activation by CBD decreases proliferation and increases susceptibility to drug-induced cell death in human cancer cells. However, no functional role has been ascribed to CBD and TRPV2 in MM. In this study, we identified the presence of heterogeneous CD138+TRPV2+ and CD138+TRPV2- PC populations in MM patients, whereas only the CD138+ TRPV2- population was present in RPMI8226 and U266 MM cell lines. Because bortezomib (BORT) is commonly used in MM treatment, we investigated the effects of CBD and BORT in CD138+TRPV2- MM cells and in MM cell lines transfected with TRPV2 (CD138+TRPV2+). These results showed that CBD by itself or in synergy with BORT strongly inhibited growth, arrested cell cycle progression and induced MM cells death by regulating the ERK, AKT and NF-κB pathways with major effects in TRPV2+ cells. These data provide a rationale for using CBD to increase the activity of proteasome inhibitors in MM.
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