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Short communication
CB2 and TRPV1 receptors mediate cannabinoid
actions on MDR1 expression in multidrug
resistant cells
Jonathon C. Arnold1, Phoebe Hone1, Michelle L. Holland1, John D. Allen2
1School of Medical Sciences (Pharmacology) and Bosch Institute, The University of Sydney, NSW 2006, Sydney,
Australia
2The Centenary Institute of Cancer Medicine and Cell Biology, NSW 2006, Sydney, Australia
Correspondence: Jonathon C. Arnold, e-mail: [email protected]
Abstract:
Background: Cannabis is the most widely used illicit drug in the world that is often used by cancer patients in combination with con-
ventional anticancer drugs. Multidrug resistance (MDR) is a major obstacle in the treatment of cancer. An extensively characterized
mechanism of MDR involves overexpression of P-glycoprotein (P-gp), which reduces the cellular accumulation of cytotoxic drugs
in tumor cells.
Methods: Here we examined the role of cannabinoid receptors and transient receptor potential vanilloid type 1 (TRPV1) receptors in
the effects of plant-derived cannabinoids on MDR1 mRNA expression in MDR CEM/VLB100 cells which overexpress P-gp due to
MDR1 gene amplification.
Results: We showed that both cannabidiol (CBD) and D9-tetrahydrocannabinol (D9-THC) (10 µM) transiently induced the MDR1
transcript in P-gp overexpressing cells at 4 but not 8 or 48 h incubation durations. CBD and THC also concomitantly increased P-gp
activity as measured by reduced accumulation of the P-gp substrate Rhodamine 123 in these cells with a maximal inhibitory effect
observed at 4 h that slowly diminished by 48 h. CEM/VLB100 cell lines were shown to express CB2 and TRPV1 receptors. D9-THC
effects on MDR1 expression were mediated by CB2 receptors. The effects of CBD were not mediated by either CB2 or TRPV1 recep-
tors alone, however, required activation of both these receptors to modulate MDR1 mRNA expression.
Conclusion: This is the first evidence that CB2 and TRPV1 receptors cooperate to modulate MDR1 expression.
Key words:
cannabinoids, P-glycoprotein, CB2, TRPV1
Introduction
Multidrug resistance (MDR) is a major obstacle in the
treatment of cancer. MDR reduces the sensitivity of
tumor cells to an array of structurally and functionally
unrelated compounds, often through repeated expo-
sure to cytotoxic agents [3, 28]. An extensively char-
acterized mechanism of MDR involves a 170 kDa ef-
flux transporter termed P-glycoprotein (P-gp) [13],
which reduces the cellular accumulation of diverse
cytotoxic drugs [8]. P-gp belongs to the superfamily
of adenosine triphosphate (ATP)-binding cassette
(ABC) transporters [9] and is encoded by the MDR1
gene [17, 27]. P-gp was discovered due to its role in
mediating MDR in cancer, however, it is also ex-
pressed in many tissues throughout the body where its
Pharmacological Reports, 2012, 64, 751�757 751
Pharmacological Reports2012, 64, 751�757ISSN 1734-1140
Copyright © 2012by Institute of PharmacologyPolish Academy of Sciences
modulation affects the pharmacokinetics of substrate
drugs including cannabinoids [23, 24].
Cannabis is the most widely used illicit drug in the
world and it is being increasingly used for medicinal
purposes. The widespread use of this drug raises the
question of whether plant-derived cannabinoids affect
MDR1 expression. This might have implications for
MDR and also the pharmacokinetics of co-administered
therapeutic drugs. We have shown that the most abun-
dant cannabinoid constituents, D9-tetrahydrocannab-
inol (D9-THC) and cannabidiol (CBD), inhibit P-gp
expression in the acute T lymphoblastoid leukemic
line CEM/VLB100 which overexpresses P-gp due to
MDR1 gene amplification [2, 12]. Here we examined
the role of cannabinoid and transient receptor poten-
tial vanilloid type 1 (TRPV1) receptors in the effects
of cannabinoids on MDR1 mRNA expression.
Materials and Methods
Cell lines
The CEM/VLB100 cell line over-expresses P-gp, as
a result of MDR1 gene amplification, yielding an ap-
proximate 200- to 800-fold resistance to VLB [12].
The cell line was grown in complete medium consist-
ing of RPMI-1640 supplemented with 10% (v/v) fetal
calf serum (FCS), 50 IU/ml penicillin and 50 mg/ml
streptomycin (all purchased from Invitrogen, Austra-
lia) in a humidified atmosphere of 5% CO2 at 37°C.
Cells were grown in 75 cm2 culture flasks maintained
at a density between 105 and 106 cells/ml, in exponen-
tial growth, and subcultured every 3–4 days. Viable
cells were counted on a hemocytometer using trypan
blue exclusion.
Drugs and drug treatment
D9-THC and CBD were purchased from Sigma-Aldrich
(Sydney, NSW) and Tocris Cookson Inc. (Sydney,
NSW), respectively, and prepared as described previ-
ously [10–12]. The selective CB2 receptor antagonist
SR 144528 (Sanofi-Aventis Pharmaceuticals, France)
and the selective TRPV1 receptor antagonist SB
366791 (Sapphire Biosciences, Australia) were stored
as stock solutions in ethanol and were diluted with
complete medium prior to each experiment. The final
ethanol concentration in the medium never exceeded
0.1%. Cells were treated in 25 cm2 culture flasks at
a cell density of 107 cells/ml with 10 µM CBD and/or
D9-THC or an equivalent volume of ethanol vehicle
(VEH), in each case diluted in complete medium and
incubated for 4, 8 or 48 h. In 4 h experiments where
cannabinoid receptor antagonists were also included,
they were added at the same time as the cannabinoids,
to a final concentration of 5 µM. For each drug treat-
ment and incubation time we repeated the experiment
at least 3 times in separate culture flasks. RNA was
extracted on each occasion and analyzed in triplicate
by real time reverse transcriptase polymerase chain
reaction (real time RT PCR).
RNA extraction and reverse transcription
Total RNA was isolated using the TRIzol reagent (In-
vitrogen, Australia). RNA was treated with DNase I to
remove any contaminating genomic DNA. The con-
centration of RNA was assessed using the Nanodrop
ND-1000 (NanoDrop Technologies Inc., USA). RNA
(2 µg) was reverse transcribed with moloney-murine
leukemia virus reverse transcriptase (Promega, Aus-
tralia), random hexamers and dNTPs.
Real time reverse transcriptase polymerase
chain reaction
The real-time reverse transcriptase polymerase chain
reaction (RT PCR) was performed in multiplex using
TaqMan Gene Expression Assay primer and probes
and TaqMan Universal PCR Master Mix (Applied
Biosystems, USA). Briefly, PCR was performed in
triplicate in standard 96-well plates containing 1.25 µl
of TaqMan MDR1 forward and reverse primers and
MGB FAM-labelled probe, 1.25 µl of the TaqMan en-
dogenous control 18s rRNA forward and reverse
primers and MGB VIC-labelled probe, 12.5 µl of
TaqMan universal PCR master mix, 1 µl of cDNA
template and 9 µl of diethyl pyrocarbonate (DEPC)-
treated water to give a total reaction volume of 25 µl
per well. The RT PCR products were amplified using
the ABI Prism 7000 Sequence Detection System (Ap-
plied Biosystems, USA). Thermal cycling conditions
were 50°C for 2 min, followed by 95°C for 10 min, 40
cycles of 95°C for 15 s, with a final annealing tem-
perature of 60°C for 60 s. TaqMan primers and probes
were designed to amplify a 67 bp region of the MDR1
mRNA sequence (NCBI Entrez nucleotide sequence
ID: NM_000927.3), and a 187 bp amplicon from the
752 Pharmacological Reports, 2012, 64, 751�757
18s rRNA mRNA sequence (NCBI Entrez nucleotide
sequence ID: X03205.1). A non-template control us-
ing DEPC-treated water instead of cDNA was used as
a negative control in each plate analyzed.
RT-PCR analysis of receptor expression
All receptor PCR reactions were performed using
0.05 × the final volume of template in reactions con-
sisting of 1 × HotStarTaq PCR buffer, 125 nM of each
primer, 100 nM dNTPs and 0.1 U/µl of HotStarTaq
DNA Polymerase. Thermal cycling conditions con-
sisted of an initial activation step of 95°C for 15 min,
followed by n cycles of 94°C for 45 s, annealing
temperature (Tm) °C for 20 s and 72°C for 30 s, with
a final cycle of 72°C for 5 min. Both the number of
amplification cycles (n), and the Tm were determined
for each primer set. Primers were designed based on
sequences available from the National Centre for Bio-
technology Information databases, using Primer 3
software [22]. Where alternatively spliced transcripts
of the gene exist, primers were designed across a re-
gion that is common to all variants. Specific reaction
conditions, primer sequences and amplicon sizes are
described in Table 1. We utilized a negative RT con-
trol which showed no signal upon amplification high-
lighting that our product derived from cDNA rather
than genomic DNA.
Rhodamine 123 accumulation assay of P-gp
activity
Cells were plated at 2 × 105/ml and 1/10th volume of
11X cannabinoids added to wells to a final concentra-
tion of 10 µM, 48, 8 or 4 h prior to harvest. Untreated
controls received 1/10th volume complete medium 4 h
prior to harvest. At harvest, cells were pelleted,
washed free of cannabinoids, and resuspended in
complete medium. Rhodamine 123 accumulation was
performed in triplicate. Aliquots of 0.5 ml (~4 × 105)
cells were transferred to 5 ml serology tubes pre-
loaded with 0.5 ml 1 µM Rhodamine 123 in complete
medium. The racked tubes were placed in a humidi-
fied CO2 incubator at 37°C for 60 min, with quick
mixing every 20 min, and then cooled in ice-water
slurry. Cells were pelleted by centrifugation at 2°C,
washed once with PBS containing 1% FCS and 1 mM
EDTA, resuspended in 0.3 ml of the same, and kept
on ice until analysis. Rhodamine 123 accumulation
was assayed by flow cytometry, using 488 nm excita-
tion and 530 nm fluorescence.
Data analysis
The changes in gene expression levels were evaluated
by relative quantification between cDNA templates
from drug-treated cells and their respective vehicle-
treated controls at each time point. The expression of
the housekeeping gene, 18s rRNA, was used as an en-
Pharmacological Reports, 2012, 64, 751�757 753
CB2 and TRPV1 mediate cannabinoid action on MDR1 expressionJonathon C. Arnold et al.
Tab. 1. National Centre for Biotechnology Information database accession number, primer sequences, annealing temperature (Tm), amplifica-tion cycle number (n) and amplicon sizes of CB1, CB2 and TRPV1 receptor mRNA
Accession (mRNA) Forward primer (5’-3’) Reverse primer (5’-3’) Tm (°C) n Amplicon size (bp)
cDNA DNA
Cannabinoid receptor (CB1)
NM_016083.3 aagaccctggtcctgatcct cgcaggtccttactcctcag 52 35 188 188
Cannabinoid receptor (CB2)
NM_001841 tagacacggacccctttttg ttctcccaagtccctcattg 57 35 243 243
Transient Receptor Potential Vanilloid (subtype 1) (TRPV1)
NM_080704 gcctggagctgttcaagttc tctcctgtgcgatcttgttg 56 35 177 177
b actin (ACTB)
NM_001101 ggacttcgagcaagagatgg agcactgtgttggcgtacag 55 30 234 329
dogenous control, to normalize the data between sam-
ples. Data were analyzed according to the mathemati-
cal model for relative quantification modified in ac-
cordance with ‘delta-delta method’ used by Applied
Biosystems [20]. Real-time RT PCR CT results were
analyzed using Sequence detection software version
1.2.3 (Applied Biosystems, USA) where baseline and
threshold parameters were kept constant between
plates. The relative expression ratios between treated
and vehicle control groups were statistically analyzed
using the non-parametric Mann-Whitney U test as an
equality of variance F-test showed that the variance
between experimental groups were heterogenous.
Rhodamine 123 accumulation was analyzed by one-
way ANOVA with the difference between untreated
cells and the 4, 8 and 48 h time-points tested explic-
itly by Tukey’s post-test (here, variances were homo-
geneous).
Results
We determined whether changes in MDR1 mRNA
levels occurred in the CEM/VLB100 cells in response
to treatment with cannabinoids for 4, 8 or 48 h. At 4 h,
both D9-THC and CBD (10 µM) significantly in-
creased MDR1 mRNA expression, by approximately
2.5 and 2.0 fold, respectively, over VEH-treated cells
(p < 0.05, Fig. 1A, B). The induction of the MDR1
transcript was short-lived as no significant effects
were observed at 8 or 48 h for either cannabinoid. The
functional consequences of MDR1 induction were
tested by cellular Rhodamine 123 accumulation; the
dye is a good P-gp substrate and its accumulation is
inversely related to P-gp efflux activity. The increased
MDR1 transcript levels in the cannabinoid-treated
cells were accompanied by reduced Rhodamine 123
754 Pharmacological Reports, 2012, 64, 751�757
Fig. 1. Effects of cannabinoids onMDR1 mRNA levels and P-gp function.The mean (± SEM) MDR1 mRNA ex-pression relative to vehicle-treatedCEM/VLB100 cells following treatmentfor 4, 8 and 48 h with (A) 10 µM D9-THCand (B) 10 µM CBD. (C, D) Conse-quent changes in cellular Rhodamine123 accumulation; relative to un-treated controls. The levels of MDR1
mRNA were normalized to the 18srRNA housekeeping gene. Statisticalsignificance is denoted by * p < 0.05,** p < 0.01 or *** p < 0.001 (n = 3–4 pergroup)
accumulation (Fig. 1C, D). The increased P-gp activ-
ity persisted longer than the elevated transcript level
as reduced Rhodamine 123 accumulation was ob-
served for all THC and CBD incubation times (that is
4, 8 and 48 h) compared to untreated controls. Such
an effect is consistent with P-gp protein having
a longer half-life than MDR1 transcript [1, 5, 21].
However, a maximal inhibitory effect on Rhodamine
123 accumulation was observed at 4 h which slowly
dissipated as Rhodamine 123 levels were significantly
higher at 48 h than the 4 h time-point for both THC
and CBD (p < 0.001 and 0.05, respectively).
The ability of D9-THC or CBD to modulate MDR1
mRNA levels raises the important question of whether
such effects are mediated by receptors. We assessed
the expression in CEM/VLB100 cells of some common
targets of cannabinoid drugs, namely CB1, CB2 and
TRPV1 receptors, using RT-PCR [19]. Both CB2 and
TRPV1 receptor mRNAs were found to be present in
these cells, but not CB1 receptor mRNA (Fig. 2). We
then attempted to block the induction of MDR1 by
CBD or D9-THC with receptor antagonists. The CB2receptor antagonist, SR 144528, effectively blocked
D9-THC-induced upregulation of MDR1 mRNA (p <
0.05, Fig. 3) whereas the TRPV1 receptor antagonist
SB 366791 had little, if any, effect. In contrast, the
combination of these antagonists was required to re-
verse CBD-mediated induction of MDR1 mRNA (p <
0.05, Fig. 3). Neither SR 144528, SB 366791 or their
combination significantly modulated MDR1 mRNA
in the absence of cannabinoid exposure compared to
VEH-treated CEM/VLB100 cells (83.33 ± 10.40,
184.30 ± 72.12, 79.67 ± 20.33; the mean ± SEM, re-
spectively).
Discussion
D9-THC and CBD were shown to modulate MDR1
gene expression by inducing the levels of transcript in
CEM/VLB100 cells. In the present study, 10 µM
D9-THC significantly increased the level of MDR1
mRNA at 4 h in CEM/VLB100 cells. The effect on
MDR1 mRNA expression was only short-lived as it
was not sustained at 8 or 48 h. The consequent in-
crease in P-gp activity, reflected in reduced Rhodam-
ine 123 accumulation, persisted longer, as expected
from the half-life of the protein [1, 5, 21]. Further, we
Pharmacological Reports, 2012, 64, 751�757 755
CB2 and TRPV1 mediate cannabinoid action on MDR1 expressionJonathon C. Arnold et al.
Fig. 3. Effect of cannabinoid receptor antagonists on cannabinoid in-duction of MDR1 mRNA. Graphs show the mean (± SEM) MDR1
mRNA expression, relative to vehicle-treated controls (VEH), ofCEM/VLB100 cells treated with (A) 10 µM D9-THC and (B) 10 µMCBD, co-administered with either VEH or 5 µM of the CB2 receptorantagonist SR 144528 and/or the TRPV1 receptor antagonist SB366791 for 4 h. The levels of MDR1 mRNA were normalized to the 18srRNA housekeeping gene. Statistical significance was determinedby comparing cannabinoid-antagonist relative to the cannabinoid-VEH control group (* p < 0.05, n = 3–4 per condition)
Fig. 2. The expression of CB1, CB2 and TRPV1 mRNA in MCF-7(+ve) and CEM/VLB100 (VLB) cells as determined by RT-PCR.b-Actin (ACTB) was used as a control for cDNA quality. Water wasused as a negative control (–ve). n = 2, single experiment shown
showed that CEM/VLB100 express CB2 and TRPV1
receptors but not CB1 receptors. The ability of the
cannabinoids to modulate MDR1 mRNA was depend-
ent on slightly different mechanisms, with the actions
of D9-THC mediated by the CB2 receptor alone,
whereas CBD’s effects required both CB2 and TRPV1
activation, as neither a CB2 or a TRPV1 antagonist
alone were effective in reversing CBD-induced
MDR1 mRNA upregulation.
In the present study, 10 µM D9-THC and CBD tran-
siently increased the level of MDR1 mRNA that cor-
related with increased functional P-gp activity as sup-
ported by reduced Rhodamine 123 accumulation in
CEM/VLB100 cells at 4 h but not 8 or 48 h. We have
previously demonstrated that CBD and D9-THC re-
duce the protein expression of P-gp in CEM/VLB100cells after 72 h exposure, which correlated with in-
creased Rhodamine 123 accumulation and enhanced
sensitivity of the cells to the cytotoxic actions of the
P-gp substrate, vinblastine [12]. Taken together with
the present results this suggests that THC and CBD
have opposing effects on P-gp expression and conse-
quent transport capacity that is dependent on incuba-
tion duration – that is short-term cannabinoid expo-
sure (4 h) transiently increases P-gp transport whereas
longer-term exposure (72 h) decreases P-gp activity.
The literature is replete with examples of cannabi-
noids having biphasic effects on various biological
measures e.g., [6, 18, 25, 26]. The mechanism respon-
sible for this biphasic action of cannabinoids on P-gp
expression and activity requires further investigation.
D9-THC binds to both CB1 and CB2 with similar af-
finities and behaves as a partial agonist at these recep-
tors [7, 14, 16, 19, 29], however, it does not bind
TRPV1 receptors [15]. Thus, given that only CB2 re-
ceptor mRNA was found expressed in CEM/VLB100
cells, it is not surprising that this receptor accounted
for all of D9-THC’s ability to upregulate MDR1
mRNA. Interestingly, CBD exhibited a more complex
profile, as only combination blockade of CB2 and
TRPV1 significantly inhibited CBD-induced MDR1
mRNA expression, and neither CB2 nor TRPV1 an-
tagonists were effective when tested alone. CBD, in
its (–)-enantiomeric form utilized here, lacks affinity
for CB1 and CB2 receptors, unlike its (+)-enantiomer
which displays significant affinity for both receptor
subtypes [4]. This raises the possibility that CBD’s ef-
fectiveness here is explained by CBD’s propensity to
inhibit endogenous fatty acid amide hydrolase and
elevate levels of the endocannabinoid anandamide.
This suggests that anandamide’s simultaneous activa-
tion of both CB2 and TRPV1 receptors [4] is necessary
for increased MDR1 mRNA expression following
CBD exposure. Here we provide the first evidence
showing that TRPV1 and CB2 receptors may cooper-
ate to affect MDR1 expression.
Acknowledgments:
This work was supported by a National Health and Medical
Research Council (NH&MRC) Project Grant and a NARSAD Young
Investigator Award awarded to JCA.
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Received: April 18, 2011; in the revised form: January 17, 2012;
accepted: February 2, 2012.
Pharmacological Reports, 2012, 64, 751�757 757
CB2 and TRPV1 mediate cannabinoid action on MDR1 expressionJonathon C. Arnold et al.