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Cyclooxygenase enzymes (COX-1 and COX-2) catalyse the production of prostaglandins from arachidonic acid. Prostaglandins are important mediators in the inflammatory process and their production can be reduced by COX-inhibitors. Endocannabinoids, endogenous analogues of the plant derived cannabinoids, occur normally in the human body. The Endocannabinoids are structurally similar to arachidonic acid and have been suggested to interfere with the inflammatory process. They have also been shown to inhibit cancer cell proliferation. Anti-inflammatory effects of cannabinoids and endocannabinoids have been observed, however the mode of action is not yet clarified. Anti-inflammatory activity (i.e., inhibition of COX-2) is proposed to play an important role in the development of colon cancer, which makes this subject interesting to study further. In the present work, the six cannabinoids tetrahydrocannabinol (Δ⁹-THC), tetrahydrocannabinolic acid (Δ⁹-THC-A), cannabidiol (CBD), cannabidiolic acid (CBDA), cannabigerol (CBG) and cannabigerolic acid (CBGA), isolated from Cannabis sativa, were evaluated for their effects on prostaglandin production. For this purpose an in vitro enzyme based COX-1/COX-2 inhibition assay and a cell based prostaglandin production radioimmunoassay were used. Cannabinoids inhibited cyclooxygenase enzyme activity with IC₅₀ values ranging from 1.7·10⁻³ to 2.0·10⁻⁴ M.
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Cyclooxygenase enzymes (COX enzymes) catalyse the
production of prostaglandins, which are important mediators
in the inflammatory process. To date, two isoforms of COX
have been identified; a constitutively expressed enzyme,
COX-1 and an inducible enzyme, COX-2,1,2) of which the lat-
ter is induced by inflammatory stimuli. An important group
of anti-inflammatory drugs is the Non-Steroid Anti Inflam-
matory Drugs (NSAIDs), of which aspirin and indomethacin
are representatives. These compounds act by inhibiting the
COX enzymes. The substrate for the prostaglandin produc-
tion is arachidonic acid (Fig. 1),1) an eicosanoid, which is
produced on demand by phospholipase A2from arachido-
nate, which is stored in the lipid bilayers of the cell wall.3) In
recent years, COX-2 overexpression has been associated with
colon cancer development, and COX enzyme inhibition is
studied as a potential target for cancer chemoprevention.4,5)
Other compounds in the eicosanoid group are the endo-
cannabinoids. These endogenous compounds bind to cellular
receptors, including the cannabinoid receptors, which are the
molecular targets of the active principle in Cannabis sativa.
The biological function of the endocannabinoids involves
several regulatory agents, forming the endocannabinoid sys-
tem (ECS).6) It has been reported that endocannabinoids also
can function as substrates for the COX enzymes resulting in
production of prostaglandin ethanolamides and prostaglandin
glycerol esters.7,8) Recently, the endogenous cannabinoid
anandamide was shown to induce COX-2 dependent cell
death in colon cancer cells.9)
There are several structural (Fig. 1) and physiological sim-
ilarities between human endocannabinoids and cannabinoids
occurring in plant material. Structurally the 5-carbon side
chain in cannabinoids is present in the endocannabinoids as
the last five carbons of the fatty acid chain, and the C-3 OH
might correspond to the polar hydroxyl end of the endo-
cannabinoids. Furthermore, the relative distances between
774 Vol. 34, No. 5Note
Evaluation of the Cyclooxygenase Inhibiting Effects of Six Major
Cannabinoids Isolated from Cannabis sativa
Lucia Renee RUHAAK,*,a,b,† Jenny FELTH,a,# Pernilla Christina KARLSSON,cJoseph James RAFTER,c
Robert VERPOORTE,band Lars BOHLINa
aDivision of Pharmacognosy, Department of Medicinal Chemistry, Biomedical Centre, Uppsala University; Box 574, SE-
751 23 Uppsala, Sweden: bDivision of Pharmacognosy, Section Metabolomics, Institute of Biology, Leiden University;
2300 RA, Leiden, the Netherlands: and cDepartment of Biosciences and Nutrition, Karolinska Institutet; Novum, S-141 86
Huddinge, Sweden. Received December 22, 2010; accepted February 19, 2011; published online February 28, 2011
Cyclooxygenase enzymes (COX-1 and COX-2) catalyse the production of prostaglandins from arachidonic
acid. Prostaglandins are important mediators in the inflammatory process and their production can be reduced
by COX-inhibitors. Endocannabinoids, endogenous analogues of the plant derived cannabinoids, occur normally
in the human body. The Endocannabinoids are structurally similar to arachidonic acid and have been suggested
to interfere with the inflammatory process. They have also been shown to inhibit cancer cell proliferation. Anti-
inflammatory effects of cannabinoids and endocannabinoids have been observed, however the mode of action is
not yet clarified. Anti-inflammatory activity (i.e., inhibition of COX-2) is proposed to play an important role in
the development of colon cancer, which makes this subject interesting to study further. In the present work, the
six cannabinoids tetrahydrocannabinol (DD9-THC), tetrahydrocannabinolic acid (DD9-THC-A), cannabidiol (CBD),
cannabidiolic acid (CBDA), cannabigerol (CBG) and cannabigerolic acid (CBGA), isolated from Cannabis sativa,
were evaluated for their effects on prostaglandin production. For this purpose an in vitro enzyme based COX-
1/COX-2 inhibition assay and a cell based prostaglandin production radioimmunoassay were used. Cannabinoids
inhibited cyclooxygenase enzyme activity with IC50 values ranging from 1.7· 10
3to 2.0 · 104M.
Key words cannabinoid; cyclooxygenase inhibition; prostaglandin production
Biol. Pharm. Bull. 34(5) 774—778 (2011)
© 2011 Pharmaceutical Society of Japan
To whom correspondence should be addressed. e-mail: Lruhaak@ucdavis.edu
#Equal contribution with the first author.
Present address: Department of Chemistry, University of California at Davis,
Davis, CA 95616, U.S.A.
Fig. 1. Structural Formulas of the Endocannabinoid Anandamide (1) and
the Endocannabinoid Precursor Arachidonic Acid (2) together with the Six
Cannabinoids; D9-THC (3), D9-THCA-A (4), CBD (5), CBDA (6), CBG (7),
CBGA (8)
the groups are comparable due to the ring system in cannabi-
noids, which can be mimicked by the U-shaped endocannabi-
noids and their four double bonds.10) Also physiologically
there are similarities, since both cannabinoids and endo-
cannabinoids bind to the cannabinoid receptors.11) Endo-
cannabinoids, such as anandamide, are derived from arachi-
donic acid and are structurally similar to this compound (Fig.
1).
Altogether, these similarities gave rise to the hypothesis
that cannabinoids can affect the COX enzyme activity.
Several studies have demonstrated anti-inflammatory activi-
ties in vivo and in vitro for various cannabinoid com-
pounds,12—18) which makes this hypothesis very plausible. In-
hibiting effects on COX enzyme activity have also previously
been observed for cannabidiol and cannabidiolic acid,17,19)
and cannabinoids have potential to affect the potency of
NSAIDs.20,21) Furthermore, in recent years, it has been shown
that the ECS can protect against colonic inflammation,6,22)
which is of interest in prevention of bowel disease and colo-
rectal cancer. The cannabinoid receptors are suggested to be
involved in the control of colonic inflammation,6,22) however,
the mode of action for the anti-inflammatory effects of
cannabinoids is not yet clarified.
In the present study we evaluated the COX-mediated anti-
inflammatory properties of six different naturally occuring
cannabinoids; tetrahydrocannabinol (D9-THC), tetrahydro-
cannabinolic acid-A (THCA-A), cannabidiol (CBD), canna-
bidiolic acid (CBDA), cannabigerol (CBG) and cannabigero-
lic acid (CBGA) (Fig. 1). An enzyme-based in vitro COX
inhibition assay was used to evaluate the effects on both
COX-1 and COX-2 on enzyme-level, while a cell-based
prostaglandin production assay was used to evaluate the
effects on COX-2 at cellular level.
MATERIALS AND METHODS
Materials All solvents were purchased from Lab-Scan,
Dublin, Ireland, and were of analytical grade. Scientific sam-
ples of cannabinoids (D9-THC, THCA-A, CBD, CBDA,
CBG and CBGA) were provided by Prof. Robert Verpoorte
and Dr. Arno Hazekamp, Leiden University, The Nether-
lands. The cannabinoids were isolated from Cannabis sativa
and characterized and quantified using the chromatography
and 1H-NMR methods as described by Hazekamp et al.23,24)
All cannabinoid samples were at least 92% pure.
COX-1 enzyme, purified from ram seminal vesicles and
COX-2 enzyme, purified from sheep placental cotyledons,
and the reference compound NS-398 (N-[2-(cyclo-hexyl-
oxy)-4-nitrophenyl]methanesulphonamide) were purchased
from Cayman Chemical Co., Ann Arbor, MI, U.S.A.
Hematin was obtained from ICN biomedicals Inc., Aurora,
Ohio, U.S.A. Adrenalin was purchased from Apoteket AB,
Göteborg, Sweden. Reduced gluthatione, indomethacine, un-
labeled arachidonic acid, anti-prostaglandin E2, prostaglandin
E2standard, Bovine Serum Albumin, tumor necrosis factor
(TNF)-
a
and charcoal were obtained from Sigma-Aldrich,
St. Louis, MO, U.S.A. 14C-Arachidonic acid, [5,6,8,11,12,-
14,15(n)-3H] Prostaglandin E2and dextran molecular weight
(mw) 70000 was purchased from Amersham Pharmacia,
Stockholm, Sweden, while silica gel 60, particle size 0.063—
2 mm was obtained from Merck, Darmstadt, Germany. Dul-
becco’s modified Eagle’s medium (DMEM)-high glucose and
trypsin-ethylenediaminetetraacetic acid (EDTA) were ob-
tained from Invitrogen, Taastrup, Denmark.
Cell Culture The human colon adenocarcinoma cell line
HT29, was cultured in monolayer in DMEM (Dulbecco’s
modified Eagle medium supplemented with 10% fetal bovine
serum (FBS), 2 mML-glutamine, and 1% penicillin/strepto-
mycin) at 37 °C and 5% CO2. All experiments were per-
formed with 60—80% confluent cells and 0.1% DMEM
(0.1% FBS). Pure compounds were dissolved in ethanol and
diluted in 0.1% DMEM (with the final concentration in the
cell cultures being maximum 0.25% ethanol).
Enzyme-Based Inhibition Assay The assay followed
the original method described by White and Glassman,25)
with modifications as described by Noreen et al.26) The assay
described below was used for both COX-1 and COX-2 en-
zymes. In short, 20
m
l of each sample was dispensed in a 96-
well plate. All samples were dissolved in 20% dimethyl sul-
foxide (DMSO) in TRIS buffer. To determine minimal and
maximal activity of the enzyme, 20% DMSO in TRIS buffer
was used as the sample. Total inhibition of the enzyme in the
minimum wells was reached by addition of 10
m
l of 2 MHCl
to the wells before the enzyme was added. Cofactors were
dissolved in TRIS buffer to concentrations of 1.27 mg/ml
hematin, 6.50 mg/ml adrenalin and 1.50 mg/ml gluthatione,
giving final concentrations in the wells of 1.3
m
g/ml, 1.3
mg/ml and 0.3 mg/ml respectively. COX enzyme was mixed
with the co-factors, pre-incubated and activated on ice for
5 min. Sixty microliters of enzyme-cofactor solution was
added to the sample in the wells, and the plate was incubated
for 10 min on ice. The activity of the enzyme in the wells was
6U (COX-1) or 3U (COX-2). Twenty microliters of 14C-
arachidonic acid (14C-AA) solution was dispensed in each
well and to start the enzymatic reaction, the plate was incu-
bated in a 37 °C waterbath for 15 min (COX-1) or 3min
(COX-2). The reaction was stopped by addition of 10
m
l
of HCl (2 M). To separate the non-converted 14C-AA from
the 14C-labeled prostaglandins, column chromatography
(Silica gel 60, particle size 0.063—2 mm) was used. The
columns were equilibrated using 2 ml of eluent, consisting of
heptane : ethyl acetate : acetic acid (70 : 30 : 1), thereafter the
samples were applied, and the non-converted AA was eluted
using 4 ml of the same eluent. The prostaglandins were
then eluted using 3 ml of a second eluent, consisting of
dioxane : methanol (85 : 15). Scintillation fluid was added
to the samples, and the amount of radioactively labeled
prostaglandin in the samples was determined using a Packard
scintillation spectrometer. Percent inhibition values were cal-
culated and IC50-values were obtained by applying the non-
linear regression analysis tool of Graph Pad Prism (Graph-
Pad Software Inc., CA, U.S.A.).
Prostaglandin E2(PGE2) Production in HT29 Cells
PGE2is a major product produced by COX from arachidonic
acid and is often used to estimate COX activity in cells. The
method used is a standard procedure for measuring PGE2
production in cells, and has previously been described in de-
tail.27—29) In brief, HT29 cells were seeded out at a concen-
tration of 3.30105cells/well. At day 2, 100
m
Maspirin was
added to the wells to prevent activation of COX-1. At day 3,
the cells were incubated with TNF-
a
(50 ng/ml) and cannabi-
noid samples (12.5 or 25
m
M) for 5 h, thereafter the test solu-
May 2011 775
tion was replaced with medium containing 100
m
mol/l
arachidonic acid (Sigma) and the cells were incubated for 1h.
The concentration of released prostaglandin E2(PGE2) was
quantified using radio immuno-assay (RIA), according to the
protocol supplied by Sigma Chemical Co., using [3H]PGE2
and polyclonal antiserum to PGE2(Sigma). The amount of
prostaglandins in each sample was detected using a scintilla-
tion counter, and expressed as the percentage inhibition of
the TNF-
a
treated cells. Each cannabinoid was tested at least
twice in the cell system and later analyzed in duplicate in the
RIA. The results were expressed as the percentage inhibition
of the TNF-
a
treated cells. In all experiments untreated cells
were included as controls, and the selective COX-2 inhibitor
NS398 was used as a reference compound for comparison of
inhibiting activity.
Prior to the PGE2 experiments, all cannabinoid samples
were tested for cytotoxicity in the AlamarBlueTM assay to en-
sure that potential COX-2 inhibitory effects were not due to
cell death.30,31) A cell survival of approximately 70% was
considered as acceptable for studying the prostaglandin pro-
duction. Cannabinoid concentrations causing cell death (i.e.,
cell survival 70%) were excluded from the PGE2produc-
tion experiments.
RESULTS
Enzyme-Based Inhibition Assay The inhibitory effects
of six cannabinoids on the cyclooxygenase enzyme activity
was evaluated by an in vitro COX enzyme inhibition assay.
D9-THC, D9-THCA-A, CBD, CBDA, CBG and CBGA were
screened for their ability to inhibit COX-1 and COX-2 at a
concentration of 100 mg/ml (approximately 3·104M), since
higher concentrations were assumed to be irrelevant. In this
screening, an enzyme inhibition of 30% was considered as
sufficient to be relevant, and was set as a cutoff limit for
compounds to investigate further. D9-THCA-A, CBDA, CBG
and CBGA showed more than 30% inhibition on COX-1
(Fig. 2). The concentration-dependent activity (i.e. inhibition
of COX-1) for these compounds was further evaluated at
concentrations ranging from 3.18 · 103to 2.78 · 105M, as
presented by concentration–effect graphs (Fig. 3A). The
IC50-values are presented in Table 1. The IC50-value of the
reference compound indomethacin was within acceptable
limits of the value reported previously for this COX-1 assay
(1.4 · 106M),26) confirming that the assay was successful.
When screened for COX-2 enzyme inhibiting activity D9-
THCA-A, CBG and CBGA showed more than 30% inhibi-
tion. Interestingly, CBDA, which was recently reported to se-
lectively inhibit COX-2,19) did not reach the 30% inhibition
threshold (Fig. 2), and was therefore not considered in our
further COX-2 inhibition studies. The inhibition of D9-
THCA-A, CBG and CBGA was measured at concentrations
ranging from 3.18 · 103to 2.78 · 105M, as represented by
the concentration–effect graphs (Fig. 3B) with IC50-values
presented in Table 1. The IC50 value of the reference com-
pound indomethacin was within acceptable limits of the
value previously reported for this COX-2 assay (1.64·106
M),26) confirming that the assay results were reliable.
Complementary to the enzyme-inhibition assay, the effects
776 Vol. 34, No. 5
Fig. 2. Screening of Six Cannabinoids for Their Potential to Inhibit COX-
1 and COX-2 Enzymes
All cannabinoids were screened at concentrations of 100
m
g/ml. To justify further
analysis, a cut off value of at least 30% inhibition was used, represented by the black
dotted line.
Fig. 3. (A) Graphs Representing the COX-1 Inhibition of D9-THCA-A,
CBDA, CBGA and Indomethacin in the Enzyme Based Assay
For each datapoint n3.
(B) Graphs Representing the COX-2 Inhibiton of D9-THCA-A, CBG,
CBGA and Indomethacin in the Enzyme Based Assay
For each datapoint n3.
Table 1. COX Inhibition IC50-Values Determined for D9-THCA-A, CBG,
CBGA and Indomethacin Using an Enzyme Based in Vitro Assay
IC50 (M)
Compound
COX-1 COX-2
D9-THCA-A 1.7 · 1036.3·10
4
CBDA 4.7 · 104N.D.a)
CBG N.D.a)2.7·10
4
CBGA 4.6 · 1042.0·10
4
Indomethacin 3.1 · 1069.3·10
5
a) N.D., not determined.
of cannabinoids on prostaglandin production were examined
in a cell based assay. Six different cannabinoids were tested
for their ability to decrease prostaglandin production in TNF-
a
stimulated HT29 cells. Prior to measuring the prosta-
glandin production, the effects of cannabinoids on cell sur-
vival were investigated, to make sure that the effects were not
due to cell death. A cell survival of approximately 70% was
considered as acceptable for studying the prostaglandin pro-
duction, and the observed effects on the PGE2production are
very unlikely to be explained by cell death. Both apoptosis
and necrosis make the cells detach from the plate surface. No
such signs were observed. D9-THC, CBD, CBDA and CBG
were tested at concentrations of 2.5·105M, whereas D9-
THCA-A and CBGA were tested at a concentration of
6.25 · 105M. However, higher concentrations of cannabi-
noids caused a high cytotoxicity and could not be used in the
experiments. The results, as presented in Fig. 4, showed that
D9-THC, D9-THCA-A, CBD, CBG and CBGA inhibited
prostaglandin production, however the level of inhibition was
low (10%). CBDA, on the other hand seemed to stimulate
the prostaglandin production (Fig. 4).
DISCUSSION
Cannabinoids have been shown to possess anti-inflamma-
tory effects,12—18) but the mechanism of action is not yet
known. COX enzyme inhibiting activity has previously been
observed for CBD and CBDA.17,19) Overexpression of COX-
2 has in recent years also been associated with colon cancer
development,5) and COX-2 enzyme inhibition is regarded as
a potential target for cancer chemoprevention.4) Interestingly,
endocannabinoid levels are elevated in colon cancer tissue,
and they also inhibit cancer cell proliferation by acting at
cannabinoid receptors.32) Recently, it has also been shown
that the ECS can protect against colonic inflammation,6,22)
which is of interest in prevention of bowel disease and col-
orectal cancer. Additionally, cannabinoids have been shown
to affect the potency of NSAIDs,20,21) potentially via modula-
tion of the COX pathway.
In the present study, six major cannabinoids isolated from
plant material modulated the activity of COX enzymes, with
IC50 values ranging from 1.7·103to 2.0 · 104M. None of
the cannabinoids showed high COX selectivity except from
CBDA, which only inhibited COX-1. This finding is contra-
dictory to previously reported results by Takeda et al., where
CBDA was found to be a selective COX-2 inhibitor in an en-
zyme inhibition assay using purified COX enzymes.19) These
inconsistencies might be caused by differences in the detec-
tion method. In the present study radioactively labeled
prostaglandin was measured, while Takeda et al. measured
the oxidation of TMPD spectrophotometrically. Alternatively,
as the cannabinoids used in the studies were purified from
plant material, different impurities in the samples could
cause different results. Further studies, preferably in human
cell lines, are needed to validate the COX inhibition by
cannabinoids.
In the screening, it was observed that D9-THC showed
stimulation in a dose-related matter (between 3.18·104and
3.18 · 105M) both in the COX-1 inhibition assay and the
COX-2 inhibition assay (data not shown). However, the
COX-inhibition assay is not designed to quantify COX
enzyme activation, and hence no definitive conclusions can
be drawn from these findings.
Interestingly, CBD and D9-THC showed low activity in the
in vitro assay of the COX-enzymes in comparison with the
other cannabinoids tested. The COX inhibition assay is an in
vitro assay where purified COX enzyme (from ram seminal
vesicles and sheep placental cotyledons, respectively) is
used. This assay is far from the human in vivo conditions.
Therefore, we complementarily used human colon cancer
cells to investigate if the prostaglandin production would be
inhibited also in living cells. The inhibition of prostaglandin
production in cancer cells is of great interest, since the in-
flammatory process is believed to be of importance for colon
carcinogenesis.33) As shown in Fig. 4, the results (e.g. inhibi-
tion of PGE2production) from the cell-based assay were sim-
ilar for all cannabinoids. All compounds tested inhibited the
production of PGE2only slightly. An experiment with higher
concentrations might give more clear results. However,
higher concentrations of the cannabinoids were cytotoxic,
causing detachment of cells and signs of cell death, and such
experiments were not possible to perform using this cell-
based assay.
The cannabinoids are known to be involved in the immune
system via the CB2receptor. The binding constants Kifor
D9-THC interacting with the CB1and CB2receptors are
8.0·10
5Mand 3.2 · 105Mrespectively.34) These binding
constants are in the same range as the IC50 values we found
for the COX-inhibition by cannabinoids. This might indicate
a possibility of physiologically important effects of the COX-
inhibiting cannabinoids via interaction with the COX-en-
zymes. Further in vitro studies are required to prove such ef-
fects, but the present study shows that several of the major
cannabinoids may also affect other receptors than CB1and
CB2. Interestingly, a recent report, linking COX-2 inhibition
to increased endocannabinoid levels, suggests the ECS and
the COX-mediated prostaglandin pathway to be closely con-
nected.35)
In conclusion, it is clear that cannabinoids inhibit COX-
enzymes, but in a higher concentration range, as compared
to anti-inflammatory drugs (i.e. indomethacin). The obvious
contradiction regarding the selectivity for CBDA, as com-
pared to the previous report by Takeda et al.,19) is interesting
May 2011 777
Fig. 4. Decrease in Prostaglandin Production in TNF-
a
Stimulated HT29
Cells
The prostaglandin production inhibitor NS398 was used as a reference compound.
Error bars represent S.D.
and should be object for further investigation. Additional
studies will also be needed to conclude the relevance of the
COX-inhibitory effects in relation to other anti-inflammatory
activities mediated by cannabinoids. As evident from recent
reports, the ECS plays an important role in the human body.
Interestingly, colonic inflammation can be controlled via the
ECS, and plant-derived cannabinoids may have a potential to
be used as future therapeutic agents.
Acknowledgements This work was financially support-
ed by Grants from the Agricultural Sciences and Spatial
Planning (FORMAS) and European University Consortium
for Pharmaceutical Research (ULLA). The authors also want
to thank Dr. Ulrika Huss Melin for helpful discussion and Dr.
Arno Hazekamp for providing purified cannabinoid samples.
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778 Vol. 34, No. 5
... While the effects were only numerical in the first experiment, PTGS2 was upregulated when REC were exposed to both LPS and CBD in experiment 2. Eicosanoids are intertwined with the ECS, with endocannabinoids being substrates for prostaglandin synthesis [56]. The immunomodulatory effects of phytocannabinoids on the arachidonic acid-associated pathways (mainly prostaglandin and leukotriene pathways) are varied, and are dependent on type of compound, dose, cell type and physiological status of the cells or tissue [57,58]. In lung fibroblasts, administration of cannabinoids, including THC, CBN and CBD, into the cell media resulted in stimulation of PGE2 synthesis [59]. ...
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Background Ruminant livestock experience a number of challenges, including high concentrate diets, weaning and transport, which can increase their risk of disorders such as ruminal acidosis, and the associated inflammation of the ruminal epithelium. Cannabidiol (CBD), a phytochemical from hemp ( Cannabis sativa ), is a promising target as a therapy for gastrointestinal inflammation, and may be extremely valuable as either a treatment or prophylactic. However, the effects of CBD in the the ruminant gastrointestinal tract have not been explored, in part due to the restrictions on feeding hemp to livestock. Therefore, the objective of this study was to investigate the immunomodulatory properties of CBD using a model of inflammation in primary ruminal epithelial cells (REC). In addition, CBD dose was evaluated for possible cytotoxic effects. Results Negative effects on cell viability were not observed when REC were exposed to 10 μM CBD. However, when the dose was increased to 50 μM for 24 h, there was a significant cytotoxic effect. When 10 μM CBD was added to culture media as treatment for inflammation induced with lipopolysaccharide (LPS), expression of genes encoding for pro-inflammatory cytokine IL1B was less compared to LPS exposure alone, and CBD resulted in a down-regulation of IL6 . As a pre-treatment, prior to LPS exposure, REC had decreased expression of IL6 and CXCL10 while CBD was present in the media, but not when it was removed prior to addition of LPS. Conclusions Results suggest that CBD may reduce cytokine transcription both during LPS-induced inflammation and when used preventatively, although these effects were dependent on its continued presence in the culture media. Overall, these experiments provide evidence of an immunomodulatory effect by CBD during a pro-inflammatory response in primary REC in culture.
... CBG may exert therapeutic effects through modulation of transient receptor potential (TRP) channels, cyclooxygenase (COX-1 and COX-2) enzymes, and cannabinoid 5-HT1A and α2 adrenergic receptors [84,[87][88][89]. Non-cytotoxic concentrations of CBG (0.05-20 µM) did not reduce TNFα secretion ( Figure 2G). ...
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Intestinal inflammation is mediated by a subset of cells populating the intestine, such as enteric glial cells (EGC) and macrophages. Different studies indicate that phytocannabinoids could play a possible role in the treatment of inflammatory bowel disease (IBD) by relieving the symptoms involved in the disease. Phytocannabinoids act through the endocannabinoid system, which is distributed throughout the mammalian body in the cells of the immune system and in the intestinal cells. Our in vitro study analyzed the putative anti-inflammatory effect of nine selected pure cannabinoids in J774A1 macrophage cells and EGCs triggered to undergo inflammation with lipopolysaccharide (LPS). The anti-inflammatory effect of several phytocannabinoids was measured by their ability to reduce TNFα transcription and translation in J774A1 macrophages and to diminish S100B and GFAP secretion and transcription in EGCs. Our results demonstrate that THC at the lower concentrations tested exerted the most effective anti-inflammatory effect in both J774A1 macrophages and EGCs compared to the other phytocannabinoids tested herein. We then performed RNA-seq analysis of EGCs exposed to LPS in the presence or absence of THC or THC-COOH. Transcriptomic analysis of these EGCs revealed 23 differentially expressed genes (DEG) compared to the treatment with only LPS. Pretreatment with THC resulted in 26 DEG, and pretreatment with THC-COOH resulted in 25 DEG. To evaluate which biological pathways were affected by the different phytocannabinoid treatments, we used the Ingenuity platform. We show that THC treatment affects the mTOR and RAR signaling pathway, while THC-COOH mainly affects the IL6 signaling pathway.
... CBG may exert thrapeutic effect through modulation of transient receptor potential (TRP) channels, cyclooxygenase (COX-1 and COX-2) enzymes and cannabinoid 5-HT1A and α2 adrenergic receptors [106,[109][110][111]. Non-cytotoxic concentrations of CBG (0.05-20µM) did not reduce TNFɑ secretion ( Figure 2G). ...
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Inflammatory bowel diseases (IBD) includes Crohn's disease and ulcerative colitis, are idiopathic chronic relapsing inflammatory disorders of the intestinal tract. Different studies indicate that phytocanna-binoids, could play a possible role in the treatment of IBD by relieving the symptoms involved in the dis-ease. Phytocannabinoids act through the endocannabinoid system, which is distributed throughout the mammalian body in the cells of the immune system and in the intestinal cells. Our in vitro study analyzed the putative-anti-inflammatory effect of nine-selected pure cannabinoids in J774A1 macrophages cells and enteric glial cells (EGC’s) triggered to undergo inflammation with lipopolysaccharide (LPS). The an-ti-inflammatory effect of several phytocannabinoids was measured by their ability to reduce TNF tran-scription and translation in J774A1 macrophages and to diminish S100B and GFAP secretion and tran-scription in EGC’s. Our results demonstrate that THC at the lower concentrations tested exerted the most effective anti- inflammatory effect in both J774A1 macrophages and EGC’s compared to the other phy-tocannabinoids tested herein. We then performed RNA-seq analysis of EGC’s exposed to LPS in the presence or absence of THC or THC-COOH. Transcriptomic analysis of these EGC’s revealed 23 differ-entially expressed genes (DEG) compared to treatment with only LPS. Pretreatment with THC resulted in 26 DEG and pretreatment with THC-COOH resulted in 25 DEG. To evaluate which biological pathways were affected by the different phytocannabinoid treatments we used the Ingenuity platform. We show that THC treatment affected the mTOR and RAR signaling pathway while THC-COOH affected mainly the IL6 signaling pathway.
... COX2 is a well-known inflammatory mediator and the enzyme that catalyses the production of prostaglandins from arachidonic acid. Accumulating evidence has demonstrated that cannabinoids CBD, THC, THCV, and cannabis oil extract suppressed activity/ production/release of COX2 and cytokines [66][67][68][69]. Interestingly, CB2R agonists and GPR55 antagonists also showed anti-inflammatory properties by inhibiting the expression of COX2 and cytokines [70][71][72], supporting a crucial role of cannabinoid receptors in mediating the anti-inflammatory effects induced by cannabinoids and cannabis extracts. ...
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The incidence of chronic inflammatory disorders and autoimmune diseases is rapidly growing. To date, the COVID-19 pandemic caused by SARS-CoV-2 has killed over 6,209,000 people globally, while no drug has been proven effective for the disease. Screening natural anti-inflammatory compounds for clinical application has drawn much attention. In this study, we showed that high-CBD cannabis extracts #1, #5, #7, #169, and #317 suppressed the levels of expression of proinflammatory cyclooxygenase 2 (COX2) and increased the expression of the anti-inflammatory suppressor of cytokine signaling 3 (SOCS3) in human small intestinal epithelial cells (HSIEC) in TNFα/IFNγ-triggered inflammation. We revealed that these extracts, with the exception of extract #169, also profoundly attenuated induction of proinflammatory cytokines interleukin-6 (IL-6) and/or IL-8 proteins through miR-760- and miR-302c-3p-mediated silencing. The prevalent components in extracts #1 and #7 influenced the levels of IL-8 both individually as well as in combination with each other. However, the high-dose cannabis extracts displayed an inhibitory effect in the growth of HSIEC cells. These results show that our high-CBD cannabis extracts decrease the levels of proinflammatory molecules COX2, IL-6, and IL-8 via transcriptional suppression or miRNA-mediated silencing, highlighting their potential against COVID-19-associated cytokine storm syndrome.
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Melanoma is one of the leading fatal forms of cancer, yet from a treatment perspective, we have minimal control over its reoccurrence and resistance to current pharmacotherapies. The endocannabinoid system (ECS) has recently been accepted as a multifaceted homeostatic regulator, influencing various physiological processes across different biological compartments, including the skin. This review presents an overview of the pathophysiology of melanoma, current pharmacotherapy used for treatment, and the challenges associated with the different pharmacological approaches. Furthermore, it highlights the utility of cannabinoids as an additive remedy for melanoma by restoring the balance between downregulated immunomodulatory pathways and elevated inflammatory cytokines during chronic skin conditions as one of the suggested critical approaches in treating this immunogenic tumor. This article is categorized under: Cancer > Molecular and Cellular Physiology
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Cannabis and cannabis products are becoming increasingly popular options for symptom management of inflammatory bowel diseases, particularly abdominal pain. While anecdotal and patient reports suggest efficacy of these compounds for these conditions, clinical research has shown mixed results. To date, clinical research has focused primarily on delta-9-tetrahydrocannabinol (THC) and cannabidiol (CBD). THC is a ligand of classical cannabinoid receptors (CBRs). CBD is one of a large group of nonintoxicating cannabinoids (niCBs) that mediate their effects on both CBRs and through non-CBR mechanisms of action. Because they are not psychotropic, there is increasing interest and availability of niCBs. The numerous niCBs show potential to rectify abnormal intestinal motility as well as have anti-inflammatory and analgesic effects. The effects of niCBs are frequently not mediated by CBRs, but rather through actions on other targets, including transient receptor potential channels and voltage-gated ion channels. Additionally, evidence suggests that niCBs can be combined to increase their potency through what is termed the entourage effect. This review examines the pre-clinical data available surrounding these niCBs in treatment of abdominal pain with a focus on non-CBR mechanisms.
Chapter
Multiple sclerosis (MS) is a chronic demyelinating disease of the central and peripheral nervous systems. The symptoms, such as vision problems, muscle weakness, pain, balance problems, and paralysis, are due to the uncontrolled or inappropriate neural transmission that gradually worsens as the disease progresses. During the early stage of the disease, patients may experience long symptom-free periods, and the periods are interrupted by flare-ups that last days to weeks. It has been indicated that Cannabis, delta-9-tetrahydrocannabinol (THC), nabiximols, and oral Cannabis extract (OCE) may diminish spasticity associated with MS [19]. Nabiximols is a mucosal spray containing delta-9-THC and cannabidiol (CBD) in the 1:1 ratio and has been developed for treating MS. The US FDA has approved nabiximols and has also been approved in several European countries, Canada, and New Zealand for treating spasticity associated with MS.
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A simple method is presented for the preparative isolation of seven major cannabinoids from Cannabis sativa plant material. Separation was performed by centrifugal partition chromatography (CPC), a technique that permits large‐scale preparative isolations. Using only two different solvent systems, it was possible to obtain pure samples of the cannabinoids; (−)‐Δ‐(trans)‐tetrahydrocannabinol (Δ‐THC), cannabidiol (CBD), cannabinol (CBN), cannabigerol (CBG), (−)‐Δ‐(trans)‐tetrahydrocannabinolic acid‐A (THCA), cannabigerolic acid (CBGA), and cannabidiolic acid (CBDA). A drug‐type and a fiber‐type cannabis cultivar were used for the isolation. All isolates were shown to be more than 90% pure by gas chromatography. This method makes acidic cannabinoids available on a large scale for biological testing. The method described in this report can also be used to isolate additional cannabinoids from cannabis plant material.
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A radiochemical enzyme assay for studying cyclooxygenase (COX)-catalyzed prostaglandin biosynthesis in vitro was optimized with respect to both COX-1 and COX-2 activity. The assay can be used to assess the relative selectivity of plant-derived inhibitors on COX-1 and COX-2 Assay conditions were optimized for both enzymes with respect to concentration of cofactors (l-epinephrine, reduced glutathione, and hematin), activation time (enzyme and cofactors), reaction time, and pH. Moreover, the kinetic parameters, Km and Kcat, of both enzymes were estimated. Five COX inhibitors were used to validate the assay, indomethacin, aspirin, naproxen, ibuprofen, and the arylsulfonamide NS-398, all with different COX selectivity and time dependency. Time-dependent inhibition was determined by comparing the inhibition, with and without preincubation of enzyme and inhibitor. Two flavonoids, (+)-catechin and quercitrin, were examined with respect to inhibition of COX-catalyzed prostaglandin biosynthesis. (+)-Catechin showed equal inhibitory effects on the two enzymes. Quercitrin was found to be inactive toward both COX-1- and COX-2-catalyzed prostaglandin biosynthesis. The optimization procedure resulted in a considerable reduction of the amount of enzyme required for adequate prostglandin biosynthesis and a reliable method suited to evaluate natural products on inhibition of COX-2-catalyzed prostaglandin biosynthesis, as well as on COX-1.
Article
Cannabinoids have been shown to increase the release of arachadonic acid, whereas nonsteroidal anti-inflammatory drugs (NSAIDs) have been shown to decrease the analgesic effects of cannabinoids. We evaluated the antinociceptive effects of chronic administration of Δ9-tetrahydrocannabinol (Δ9-THC), anandamide (an endogenous cannabinoid), arachadonic acid, ethanolamine, and methanandamide on several NSAIDs via p.o. and/or i.p. routes of administration using the mouse p -phenylquinone (PPQ) test, a test for visceral nociception. Our studies with a cannabinoid receptor (CB1) antagonist [ N -(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1 H -pyrazole-3-carboximide hydrochloride (SR141716A)], a CB2 antagonist [ N -((1 S )-endo-1,3,3-trimethyl-bicyclo-heptan-2-yl]-5-(4-chloro-3-methylphenyl)-1-(4-methylbenzyl)-pyrazole-3-carboxamide) (SR144528)], and an another CB2 agonist [1,1-dimethylbutyl-1-deoxy-Δ9-THC (JWH-133)] were performed to better characterize PPQ interactions with cannabinoid receptors. The acute affects of Δ9-THC were blocked by SR141716A (i.p.) and partially blocked by SR144528 (i.p.). When NSAIDs (p.o.) were administered, the ED50 values were as follows: 23 mg/kg aspirin, 3 mg/kg indomethacin, 5 mg/kg celecoxib, 3 mg/kg ketorolac, 57 mg/kg acetaminophen (32.3–99.8), and 0.8 mg/kg diclofenac (0.1–4.9). In animals given chronic Δ9-THC, only diclofenac and acetaminophen were active. Conversely, chronic methanandamide (i.p.) did not alter the antinociceptive effects of the NSAIDs. Neither the CB1 or CB2 antagonist blocked the effects of the NSAIDs. The effects of chronic arachadonic acid, ethanolamine, and anandamide could not be evaluated. In summary, our data indicate that chronic Δ9-THC alters the cyclooxygenase system. Alternatively, the data suggest that this alteration is not due to chronic endogenous cannabinoid release. Based upon these data, we hypothesize that human subjects who are chronic users of Δ9-THC may not respond to analgesic treatment with the above NSAIDs.
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Several derivatives of cannabinol and the 1,1-dimethylheptyl homolog (DMH) of cannabinol were prepared and assayed for binding to the brain and the peripheral cannabinoid receptors (CB1 and CB2), as well as for activation of CB1- and CB2-mediated inhibition of adenylylcyclase. The DMH derivatives were much more potent than the pentyl (i.e., cannabinol) derivatives. 11-Hydroxycannabinol (4a) was found to bind potently to both CB1 and CB2 (Ki values of 38.0 ± 7.2 and 26.6 ± 5.5 nM, respectively) and to inhibit CB1-mediated adenylylcyclase with an EC50 of 58.1 ± 6.2 nM but to cause only 20% inhibition of CB2-mediated adenylylcyclase at 10 μM. It behaves as a specific, though not potent, CB2 antagonist. 11-Hydroxycannabinol-DMH (4b) is a very potent agonist for both CB1 and CB2 (Ki values of 100 ± 50 and 200 ± 40 pM; EC50 of adenylylcyclase inhibition 56.2 ± 4.2 and 207.5 ± 27.8 pM, respectively).
Article
We show here the identity of Alamar Blue as resazurin. The ‘resazurin reduction test’ has been used for about 50 years to monitor bacterial and yeast contamination of milk, and also for assessing semen quality. Resazurin (blue and nonfluorescent) is reduced to resorufin (pink and highly fluorescent) which is further reduced to hydroresorufin (uncoloured and nonfluorescent). It is still not known how this reduction occurs, intracellularly via enzyme activity or in the medium as a chemical reaction, although the reduced fluorescent form of Alamar Blue was found in the cytoplasm and of living cells nucleus of dead cells. Recently, the dye has gained popularity as a very simple and versatile way of measuring cell proliferation and cytotoxicity. This dye presents numerous advantages over other cytotoxicity or proliferation tests but we observed several drawbacks to the routine use of Alamar Blue. Tests with several toxicants in different cell lines and rat primary hepatocytes have shown accumulation of the fluorescent product of Alamar Blue in the medium which could lead to an overestimation of cell population. Also, the extensive reduction of Alamar Blue by metabolically active cells led to a final nonfluorescent product, and hence an underestimation of cellular activity.
Article
Despite recent advances in understanding colorectal tumour biology, there is still a need to improve the 5-year survival rate of patients with colorectal cancer as approximately 40% of patients presenting with advanced disease will remain resistant to therapy. One of the major contributing factors in resistance to therapy is the failure of colorectal tumour cells to undergo apoptosis. Hence there is an urgent need to develop novel therapeutic approaches that can target apoptosis-resistant cells. To this end, we investigated the potential efficacy of the endogenous cannabinoid anandamide to induce cell death in apoptosis-resistant colon cancer cells. Here, for the first time, we show that anandamide can induce cell death in the apoptosis-resistant HCT116 Bax-/- colorectal cell line. Importantly, we provide direct genetic evidence that this induction of cell death is dependent on COX-2 expression. Interestingly, increased COX-2 expression also sensitised the SW480 colorectal cancer cell line (low endogenous COX-2) to anandamide-induced death, whereas COX-2 suppression by RNAi inhibited anandamide-induced cell death in the HCA7 colorectal cancer cell line (high endogenous COX-2 expression). This COX-2-dependent death was independent of cannabinoid receptor engagement (CB1 or CB2), and not a direct consequence of reactive oxygen species (ROS) formation. This study demonstrates a novel utilisation for COX-2 expression, targeting apoptotic defective colorectal cancer cells for destruction by anandamide. As COX-2 is not expressed in the normal colorectal epithelium, but highly expressed in colorectal tumours and apoptosis resistance contributes to treatment failure, these data suggest that anandamide has the potential to be an effective therapeutic in colorectal cancer.
Article
The connection between inflammation and tumorigenesis is well-established and in the last decade has received a great deal of supporting evidence from genetic, pharmacological, and epidemiological data. Inflammatory bowel disease is an important risk factor for the development of colon cancer. Inflammation is also likely to be involved with other forms of sporadic as well as heritable colon cancer. The molecular mechanisms by which inflammation promotes cancer development are still being uncovered and could differ between colitis-associated and other forms of colorectal cancer. Recent work has elucidated the role of distinct immune cells, cytokines, and other immune mediators in virtually all steps of colon tumorigenesis, including initiation, promotion, progression, and metastasis. These mechanisms, as well as new approaches to prevention and therapy, are discussed in this review.