ArticlePDF Available

Anti-cancer effects of selective cannabinoid agonists in pancreatic and breast cancer cells

Authors:

Abstract

Objective: Cancer ranks first among the causes of morbidity and mortality all over the world, and it is expected to continue to be the main cause of death in the coming years. Therefore, new molecular targets and therapeutic strategies are urgently needed. In many cases, some reports show increased levels of endocannabinoids and their receptors in cancer, a condition often associated with tumour aggressiveness. Recent studies have suggested that cannabinoid-1/2 receptors contribute to tumour growth in a variety of cancers, including pancreatic, colon, prostate, and breast cancer. Understanding how cannabinoids can regulate key cellular processes involved in tumorigenesis, such as: cell proliferation and cell death, is crucial to improving existing and new therapeutic approaches for the cancer patients. The present study was aimed to characterize the in-vitro effect of L-759633 (a selective CB2 receptor agonist), ACPA (a selective CB1 receptor agonist) and ACEA (a selective CB1 receptor agonist) on the cell proliferation, clonogenicity, and apoptosis in pancreatic (PANC1) and breast (MDA-MB-231) cancer cells. Methods: The viability and/or proliferation of cells were detected by MTS assay. A clonogenic survival assay was used to detect the ability of a single cell to grow into a colony. Apoptosis was determined with Annexin V staining (Annexin V-FITC/PI test) and by analyzing the expression of Bcl-2-associated X protein (Bax) and B-cell lymphoma 2 (Bcl-2). Results: We found that selective CB1/2 agonists suppressed cell proliferation, clonogenicity and induced proapoptotic function in human PANC1 pancreatic and MDA-MB-231 breast cancer cells. Based on our findings, these agonists led to the inhibition of both cell viability and clonogenic growth in a dose dependent manner. CB1/2 agonists were observed to induce intrinsic apoptotic pathway by upregulating Bax, while downregulating Bcl-2 expression levels. Conclusion: Our data suggests that CB1/2 agonists have the therapeutic potential through the inhibition of survival of human PANC1 pancreatic and MDA-MB-231 breast cancer cells and also might be linked with further cellular mechanisms for the prevention (Fig. 5, Ref. 49).
Indexed and abstracted in Science Citation Index Expanded and in Journal Citation Reports/Science Edition
Bratisl Med J 2022; 123 (11)
813821
DOI: 10.4149/BLL_2022_130
EXPERIMENTAL STUDY
Anti-cancer effects of selective cannabinoid agonists in
pancreatic and breast cancer cells
TURGUT Nergiz Hacer1, ARMAGAN Guliz2, KASAPLIGIL Gozde3, ERDOGAN Mumin Alper4
Izmir Katip Celebi University, Faculty of Medicine, Department of Physiology, Izmir, Turkey.
alpero86@gmail.com
ABSTRACT
OBJECTIVE: Cancer ranks rst among the causes of morbidity and mortality all over the world, and it is
expected to continue to be the main cause of death in the coming years. Therefore, new molecular targets
and therapeutic strategies are urgently needed. In many cases, some reports show increased levels of
endocannabinoids and their receptors in cancer, a condition often associated with tumour aggressiveness.
Recent studies have suggested that cannabinoid-1/2 receptors contribute to tumour growth in a variety of
cancers, including pancreatic, colon, prostate, and breast cancer. Understanding how cannabinoids can
regulate key cellular processes involved in tumorigenesis, such as: cell proliferation and cell death, is crucial
to improving existing and new therapeutic approaches for the cancer patients. The present study was aimed
to characterize the in-vitro effect of L-759633 (a selective CB2 receptor agonist), ACPA (a selective CB1
receptor agonist) and ACEA (a selective CB1 receptor agonist) on the cell proliferation, clonogenicity, and
apoptosis in pancreatic (PANC1) and breast (MDA-MB-231) cancer cells.
METHODS: The viability and/or proliferation of cells were detected by MTS assay. A clonogenic survival
assay was used to detect the ability of a single cell to grow into a colony. Apoptosis was determined with
Annexin V staining (Annexin V-FITC/PI test) and by analyzing the expression of Bcl-2-associated X protein
(Bax) and B-cell lymphoma 2 (Bcl-2).
RESULTS: We found that selective CB1/2 agonists suppressed cell proliferation, clonogenicity and induced
proapoptotic function in human PANC1 pancreatic and MDA-MB-231 breast cancer cells. Based on our
ndings, these agonists led to the inhibition of both cell viability and clonogenic growth in a dose dependent
manner. CB1/2 agonists were observed to induce intrinsic apoptotic pathway by upregulating Bax, while
downregulating Bcl-2 expression levels.
CONCLUSION: Our data suggests that CB1/2 agonists have the therapeutic potential through the inhibition
of survival of human PANC1 pancreatic and MDA-MB-231 breast cancer cells and also might be linked with
further cellular mechanisms for the prevention (Fig. 5, Ref. 49). Text in PDF www.elis.sk
KEY WORDS: L-759633, ACPA, ACEA, apoptosis, pancreatic cancer, breast cancer.
1Department of Pharmacology, Faculty of Pharmacy, Izmir Katip Celebi
University, Izmir, Turkey, 2Department of Biochemistry, Faculty of Phar-
macy, Ege University, Izmir, Turkey, 3Department of Biotechnology, Grad-
uate School of Natural and Applied Science, Ege University, Izmir, Turkey,
and 4Department of Physiology, Faculty of Medicine, Izmir Katip Celebi
University, Izmir, Turkey
Address for correspondence: Mumin Alper ERDOGAN, Assoc Prof,
DVM, PhD, Izmir Katip Celebi University, Faculty of Medicine, Depart-
ment of Physiology, Izmir, Turkey.
Phone: +905433818677
Introduction
Cancer is the second leading cause of death after cardiovas-
cular diseases in many countries (1). Over the past years, there
has been an increasing trend in cancer cases and cancer-related
deaths, particularly in low- and middle-income countries, depend-
ing on the diversity of lifestyle, diverse habits, and geographic and
environmental factors (2). Especially in recent years, there have
been great developments in treatment strategies targeting various
biomolecules in cancer cells for the treatment of cancer.
Cannabinoids are the group of chemicals known as marijuana
and obtained from the plant called “Cannabis sativa linnaeus”.
The major active ingredient in Cannabis sativa linnaeus is delta-
9-tetrahydrocannabinol (9-THC) with lipophilic properties which
acts by mimicking the effects of endogenous cannabinoids (3).
Cannabinoids have been used for therapeutic purposes with the
discovery of their mechanism of action, agonists and antagonists
of their receptors (4–6). First, in 1975, Munson et al. showed that
9-THC improved survival in the lung cancer xenograft model (7).
After this study, numerous studies have been carried out on many
biological effects of cannabinoids for cancer (8–12).
The effects of cannabinoids on signalling pathways in cancer
cells are mediated via G-protein coupled cannabinoid receptors
(CB receptors), CB1 receptor and CB2 receptor, as well as through
other receptors and in a receptor-independent manner. Both re-
ceptors are highly expressed in different cancer tissues. CB1/2
receptors belong to the seven transmembrane spanning receptor
superfamily. CB1 receptors are abundant in the brain areas relat-
ed to anxiety, memory, motor coordination, pain, and endocrine
functions. CB1 receptors are also expressed in other sites such as
Bratisl Med J 2022; 123 (11)
813821
814
uterus, testis, and spleen (13). CB1 receptor activation inhibits
forskolin stimulated adenylyl cyclase through stimulating cel-
lular signal transduction via Gi/o, to inhibit calcium channels (P,
Q, N type). CB2 receptors are expressed in the immune system
organs and cells. CB2 receptors are also found in the pancreas,
bones, heart, liver, and endothelium, at a lower level. CB2 recep-
tor activation strongly stimulates Gi, leading to adenylate cyclase
inhibition (14). The survival and growth of tumour cells often
depends on increased signalling through pathways that regulate
cell proliferation and survival. Activation of cannabinoid recep-
tors additionally leads to activation of phosphoinositide 3-kinase
(PI3K) and mitogen-activated protein kinase (MAPK) pathways
(15, 16). As both CB1/2 receptors are highly expressed in differ-
ent cancer tissues, these receptors are emerging targets for cancer
treatment, even though their exact role in cancer progression is
still not completely known. The general consensus in literature
suggests that cannabinoids have anticancer effects. It has been
indicated that different cannabinoids inhibit cell proliferation in
vitro and tumour growth in vivo and that in the mechanism of these
effects, apoptosis has a signi cant role (17-20).
Cannabinoid therapy promotes cancerous cell death, reduc-
tion of tumour angiogenesis, as well as blockade of invasion and
metastasis. Cannabinoid anticancer mechanism is substantially
related to inducing autophagy mediated apoptotic cancer cell
death (21, 22). In addition to the cancer cell death, treatment
with cannabinoids has been shown to normalize the tumour vas-
culature, which is considered to be based on the inhibition of
the endothelial growth factor (VEGF) pathway. Again, cannabi-
noids have been shown to reduce distant tumour mass formation
in animal models with metastasis. These compounds have been
shown to inhibit migration, and invasion in different types of
cancer cells (23–25). Regulation of extracellular proteases and
their inhibitors also contribute to the antimetastatic effects of can-
nabinoids (24, 26). These items also show an acceptable safety
pro le. However, there are still con icting results regarding the
anticancer effects of cannabinoids and new studies are needed on
the mechanism of this effect. In this study, we aimed to evalu-
ate the in-vitro impact of L-759633 (a selective CB2 receptor
agonist), ACPA (a selective CB1 receptor agonist) and ACEA (a
selective CB1 receptor agonist) on cell proliferation, clonogen-
ity and apoptosis against pancreatic (PANC1) and breast (MDA-
MB-231) cancer cells.
Material and methods
Cell culture and reagents
The human breast and pancreas cancer cell lines (MDA-
MB-231 and PANC1) employed were obtained from American
Type Culture Collection (ATCC, Manassas, VA). All the cell lines
were cultured in DMEM/F12 (Life Technologies, Gibco BRL,
Grand Island, NY) supplemented with 10 % FBS (FBS, Hyclone).
Media was supplemented with penicillin and streptomycin (100
U/mL, Invitrogen). The cells were cultured under standard condi-
tions at 37 °C in a humidi ed incubator containing 5 % CO2 and
were used between passages 4 and 15. CB2-agonist-L-759633,
CB1-agonist-ACPA and CB1-agonist-ACEA were purchased from
Tocris Bioscience (Wiesbaden-Nordenstadt, Germany).
Cell viability and proliferation assays
Cell viability and proliferation was evaluated by MTS assay
(Promega, Madison, WI, USA). A hemocytometer was used for
cell count and viable cells were identi ed by trypan blue exclu-
sion. Cells (1.5x103 cells/well) were seeded in 96-well plates
and treated with CB2-agonist -L-759633, CB1-agonist-ACPA
and CB1-agonist-ACEA at a dose range between 1-250 μM for
72 h. Following treatment, a solution which contained MTS (3-
(4, 5-dimethylthiazol-2-yl)-5-(3-carboxy-methoxyphenyl)-2-(4-
sulfophenyl)-2H-tetrazolium) and PMS (phenazine methosulfate)
(20:1 v/v) was applied to each cell at 37 °C during 3 h. For viable
growing cell estimation, the absorbance was read at 490 nm. Ex-
periments were carried out in triplicate and the results were pre-
sented as the mean absorption ± standard deviation.
Clonogenic survival assay
This assay is a cell survival and proliferation assay, which is
based on a single cell growing into a colony in vitro (27). Gently
mixed 500 cells were plated on a 6 well culture plate. Following
incubation for 24 hours, cells were treated with 50 and 100 μM
doses of CB2-agonist -L-759633, CB1-agonist-ACPA and CB1-
agonist-ACEA once a week and grown for 2–3 weeks. Then cells
were washed with phosphate-buffered saline and crystal violet
staining was performed. Colonies greater than 50 cells in diameter
were counted. Experiments were done in triplicate.
Western blot analysis
For analysis of Bcl-2-associated X protein (Bax) and B-cell
lymphoma 2 (Bcl-2) levels, cells were seeded in 5x105 cells /
culture asks. After 72 h treatment with control and test com-
pounds, cells were collected and centrifuged, then washed twice
in ice cold phosphate buffered saline (PBS). Cells were lysed in a
lysis buffer at 4 °C, lysates were centrifuged at 13,000x g for 10
min and collected supernatant fractions were used for Werstern
blot analysis. For each sample, a total protein concentration was
determined by detergent protein assay kit (Bio-Rad, Hercules,
CA). Quanti cation of proteins was performed on 40 μg protein/
lane on 4–15 % SDS-PAGE gels. After electrophoretic transfer to
PVDF membranes, the membranes were blocked for 60 min in a
blocking buffer (0.1 % Triton X-100 with 5 % non-fat dry milk
in TBS-Tween 20). Following wash with TBS-T (diluted in TBS-
Tween 20 containing 5 % non-fat dry milk, and incubated at 4 °C
overnight), membranes were probed with primary antibodies Bax
and Bcl-2(Cell Signalling Technology, Danvers, MA). Wash with
TBS-T was followed by incubation with horseradish peroxidase
(HRP)-conjugated anti-rabbit and anti-mouse secondary antibody
(Cell Signalling Technology, Danvers, MA). As a loading control,
the mouse anti-β-actin antibody (Sigma Chemical, St. Louis, MO)
was used to observe β-actin expression. For chemiluminescent
detection, chemi-glow detection reagents were used (Alpha In-
notech, San Leandro, CA). FluorChem 8900 imager was used
for visualizing blots and blots were quanti ed by densitometric
TURGUT Nergiz Hacer et al. Anti-cancer effects of selective cannabinoid agonists in pancreatic and breast cancer cells
xx
815
scanning using an image analysis program (ImageJ 1.48s process-
ing software, National Institutes of Health, Bethesda, MD, USA).
Experiments were performed at least twice.
Analysis of cell d eath
Apoptosis was evaluated by an Annexin V assay. Cells were
seeded in 25-cm2 culture asks (5x105 cells/ ask). The cells were
treated with indicated CB2-agonist -L-759633 at doses of 10
and 50 μM, CB1-agonist-ACPA and CB1-agonist-ACEA at dose
of 50 μM for 72 h, and then analyzed by Annexin V to identify
apoptotic cells and propidium iodide (PI) staining to distinguish
viable cells from the non-viable cells according to the manufac-
turer’s protocol (BD Pharmingen FITC–Annexin V kit, San Diego,
CA) using a benchtop ow cytometer (Accuri C6 ow cytometry,
Becton Dickinson). Positive cells were determined and quanti ed
by FACS analysis. The membrane phospholipid PS of apoptotic
cells is displaced internally for exposure to the outer lea et of the
membrane. This can be identi ed by using Annexin V, a PS bind-
ing protein (28).
Fig. 1. Effect of increasing concentrations of selective CB2-agonist (L-759633), CB1-agonist-ACPA and CB1-agonist-ACEA agonists on cell
proliferation (A) in human PANC1 pancreatic cancer cells and (B) in human MDA-MB-231 breast cancer cells measured by MTS assay. Cells
were treated with cannabinoid agonists at a dose range between 1-250 μM or cisplatin 5 ng/ml for 72 h. Data are expressed as the mean (± SD)
values. * p < 0.005; ** p < 0.0001 compared to the control group.
A
B
Bratisl Med J 2022; 123 (11)
813821
816
Statistical analysis
Data were expressed as the means ± standard deviation val-
ues, from at least three experiments. Statistical analysis was per-
formed by one-way analysis of variance (ANOVA) followed by
post hoc Tukey’s test. A p value < 0.05 were considered statisti-
cally signi cant.
Results
Effect on cell viability and proliferation
Considering the results obtained, selective CB2-agonist -L-
759633, CB1-agonist-ACPA and CB1-agonist-ACEA applications
signi cantly and dose-dependently decreased cell proliferation in
PANC1 cancer cells (* p < 0.005; ** p < 0.0001) (Fig. 1a). Again,
selective cannabinoid agonist administration signi cantly and
dose-dependently decreased cell proliferation in MDA-MB-231
breast cancer cells (* p < 0.005; ** p < 0.0001) (Fig. 1b). For
ongoing experiments, the dose that inhibits cell proliferation by
approximately 50 % and twice of this dose were chosen as 50
and 100 μM.
Effect on cell clonogenicity
The effect on viability of the cannabinoid agonists on human
PANC1 pancreatic and MDA-MB-231 breast cancer cells was
tested for their in uence on clonogenicity. Tests were conducted at
50 and 100 μM for L-759633, ACPA and ACEA in culture plates.
All the three cannabinoid agonists strongly inhibited colony for-
mation for human PANC1 pancreatic cell line (* p < 0.0001) (Fig.
A
B
Fig. 2. Effect of increasing concentrations of selective CB2-agonist (L-759633), CB1-agonist-ACPA and CB1-agonist-ACEA agonists on colony
formation capacity (A) in human PANC1 pancreatic cancer cells, (B) in human MDA-MB-231 breast cancer cells. Clonogenicity assays were
performed by incubating cells with 50 or 100 μM L-759633, ACPA, ACEA or cisplatin 5 ng/ml. Data are expressed as the mean (± SD) values
of three independent experiments. * p < 0.0001 compared to the control group.
TURGUT Nergiz Hacer et al. Anti-cancer effects of selective cannabinoid agonists in pancreatic and breast cancer cells
xx
817
2a). Again, selective cannabinoid agonist
administration signi cantly and dose-de-
pendently decreased cell clonogenicity in
MDA-MB-231 breast cancer cells (* p <
0.0001) (Fig. 2b). The colony counts were
graphed to better visualize (Fig. 2).
Western blot analysis
In Western blot analysis, a signifi-
cant and dose-dependent increase was ob-
served in pro-apoptotic protein Bax levels in
PANC1 pancreatic cancer cells with selec-
tive CB2-agonist -L-759633, CB1-agonist-
ACPA and CB1-agonist-ACEA application.
On the contrary, a signi cant decrease was
observed in anti-apoptotic protein Bcl-2
levels (Fig. 3). In Western blot analysis,
a signi cant and dose-dependent increase
in proapoptotic protein Bax levels was ob-
served in MDA-MB-231 breast cancer cells
with selective cannabinoid agonist applica-
tions, on the contrary, a signi cant decrease
was observed in anti-apoptotic protein Bcl-2
levels (Fig. 4).
Flow cytometry analysis
To investigate underlying molecular
mechanisms of growth inhibition observed
by treatments with CB2-agonist-L-759633,
CB1-agonist-ACPA and CB1-agonist-
ACEA in breast and pancreatic cancer cells,
after 72 h of treatment, we evaluated apop-
tosis induction through cannabinoid agents,
using double staining annexin V / propidium
iodide (Fig. 5). AnnexinV +/PI – stained
section de nes cells in early apoptosis; cells
which are stained with only PI (AnnexinV
-/PI +) are early necrotic cells, although
AnnexinV +/PI + stained part represents
the cells in primary necrosis and late apop-
tosis related with secondary necrosis (28).
As shown in Figure 5a, administration of
cannabinoid agents triggered apoptosis (as
evaluated by induction of the apoptotic cells
as positively stained in AnV +/PI - and AnV
+/PI + groups, as 17.2 %, 20.3 %, 31.3 %,
98.8 % and 99.5 % in cisplatin 5 ng/ml,
L-759 10 μM, L-759 50 μM, ACPA 50 μM
and ACEA 50 μM treated groups in PANC1
pancreatic cancer cells, respectively). And
as shown in Figure 5b, administration of
cannabinoid agents triggered apoptosis (as
evaluated by induction of the apoptotic cells
as positively stained in AnV +/PI - and AnV
+/PI + groups, as 16.7 %, 21.1 %, 32.1 %,
Fig. 3. Western blot analysis for human PANC1 pancreatic cancer cells. The changes in Bax
and Bcl-2 protein levels following CB2-agonist (L-759633) (1, 50 and 100 μM), CB1-agonist-
ACPA (50, 100 μM) and CB1-agonist-ACEA (50, 100 μM) agonist treatments. The graphs show
the relative densitometric values of the indicated proteins. Determination of the amount of
protein product was performed by densitometric scanning. Data are normalized using β-actin
signal and expressed in arbitrary densitometric units. Values are means SD, * p < 0.005 sig-
ni cant difference from untreated cells.
Bratisl Med J 2022; 123 (11)
813821
818
55.7 % and 94.6 % in Cisplatin 5ng/ml,
L-759 10 μM, L-759 50 μM, ACPA 50 μM
and ACEA 50 μM treated groups in MDA-
MB-231 cells, respectively.
Discussion
In this study, we investigated the ef-
fects of three different selective canna-
binoid agonists, selective CB2-agonist
-L-759633, CB1-agonist-ACPA and CB1-
agonist-ACEA on the cell proliferation,
colonogenicity and apoptosis in human
PANC1 pancreatic and MDA-MB-231
breast cancer cells. Our results demonstrate
that these agents with increasing concen-
trations exert an intense antiproliferative
effect and supress colony formation capac-
ity in both human PANC1 pancreatic and
MDA-MB-231 breast cancer cells. Also,
our results suggest apoptosis as one of the
mechanisms of these agonists to reduce tu-
mour cell survival.
In various cancer types, endo-, phy-
to- and synthetic cannabinoids have been
shown to have antiproliferative, antiangio-
genic, proapoptotic and antimetastatic ef-
fects due to regulating cellular signalling
pathways, which are critical for cell survival
and growth (15, 18, 29, 30). More exten-
sive research is needed to determine the full
potential of synthetic cannabinoids in can-
cer. The anti-cancer effects of cannabinoids
have been reported in the case of pancreatic
cancer (31–34). It has been demonstrated
that cannabidiol and tetrahydrocannabinol
can suppress pancreatic cancer growth, and
that this may be partially through inhibition
of p-21 activated kinase 1 (PAK1) (34). Par-
allel to our study, it has been shown that both
CB1 and CB2 receptor agonists act through
a widely common mechanism that involves
cell growth regulation and apoptosis in pan-
creatic adenocarcinoma (35). Again, it has
been shown that cannabinoids induce apop-
tosis on pancreatic cancer cells by the acti-
vation of the p8-ATF-4-TRB3 proapoptotic
pathway (31). Dando et al stated that in
pancreatic adenocarcinoma cells, autophagy
induction dependent to cannabinoids was
related to ROS-dependent increase of the
AMP/ATP ratio (32).
Increased expression of CB1 and CB2
receptors has been reported in various breast
cancer cell lines and tissues (36). The rst
Fig. 4. Western blot analysis for human MDA-MB-231 breast cancer cells. The changes in
Bax and Bcl-2 protein levels following CB2-agonist (L-759633) (1, 50 and 100 μM), CB1-
agonist-ACPA (50, 100 μM) and CB1-agonist-ACEA (50, 100 μM) agonist treatments. The
graphs show the relative densitometric values of the indicated proteins. Determination of the
amount of protein product was performed by densitometric scanning. Data are normalized
using β-actin signal and expressed in arbitrary densitometric units. Values are means SD, *
p < 0.005 signi cant difference from untreated cells.
TURGUT Nergiz Hacer et al. Anti-cancer effects of selective cannabinoid agonists in pancreatic and breast cancer cells
xx
819
identi ed endogenous ligand for cannabinoid receptor, anadamide
has been shown to block breast cancer cell proliferation by CB1
like receptor mediated inhibition of prolactin action (37). Anan-
damide analogue, 2-methyl-2-F-anandamide (Met-F-AEA), has
been suggested to have the ability to inhibit the invasion of breast
cancer cells and that this effect is mediated through the inactivation
of the β-catenin (38). Again, CB1 receptor activation has been sug-
gested to be a target for therapeutic strategies to retard the growth
of breast carcinoma to inhibit metastatic spreading (30). Selective
CB1 receptor agonist, ACEA has been shown to decrease the inva-
sive potential of breast cancer stem cells, demonstrating that CB1
receptor contributes to stem cell properties (39). CB2 receptors are
over expressed in breast tumours, and this expression positively
correlates with the histological grade. It has been suggested that in
breast cancer cells, the apoptosis induced by CB2-selective agonist
JWH-015 may be mediated by MAPK/ERK activity (40). It has
been previously shown that through activation of CB2 receptors,
Delta (9)-tetrahydrocannabinol can reduce cell proliferation and
induce apoptosis (36). Again, it has been shown that CB2 activa-
tion under in vitro and in vivo conditions suppressed breast can-
cer through inhibiting EGFR/IGF-IR signalling pathways and it
has been stated that CB2 might be an important target in breast
cancer subtypes (41). According to the type of cannabinoid and
the model used, the proposed mechanisms for the regulation of
proliferation of cancer cells differ in literature. Consistent with our
study it has been observed that synthetic agonists JWH-133 (CB2
agonist) and WIN-55,212-2 (CB1 and CB2 agonist) inhibited cell
proliferation, migration and induced apoptosis in breast cancer
cell lines in vitro (42). Again, mixed CB1/CB2 agonist CP55 940
(43) have been shown to inhibit cell proliferation in breast cancer
cells. Tamoxifen has been shown to act as an inverse agonist for
CB1/2 receptors via modulating adenylate cyclase activity and to
A
B
Fig. 5. Flow cytometric analysis of selective CB2-agonist (L-759633) (10, 50 μM), CB1-agonist-ACPA (50 μM), and CB1-agonist-ACEA (50 μM),
agonists in human PANC1 pancreatic cancer cells and human MDA-MB-231 cancer cells. Histograms show cell cycle pro les and percentage
of apoptosis is shown in bar graphic. Values are means SD, * p < 0.0001 compared with the control group.
Bratisl Med J 2022; 123 (11)
813821
820
lead to increases in intracellular cAMP (44). In the current study,
we showed that CB2 agonist -L-759633, CB1 agonist-ACPA and
CB1agonist-ACEA exerted a marked cytotoxic effect against hu-
man PANC1 pancreatic and MDA-MB-231 breast cancer cells
with a potency comparable with that observed for cisplatin. The
results of MTS assay on human PANC1 pancreatic cancer cells in
the presence of selective cannabinoid agonists illustrated that these
agonists increased cell proliferation. In particular, we showed that
selective cannabinoid agonists, CB2 agonist -L-759633 (50, 100,
250 μM), CB1 agonist-ACPA (50, 100, 250 μM) and CB1agonist-
ACEA (100, 250 μM) were capable of eliciting cytotoxic effects
in MDA-MB-231 breast cancer cells. Performed MTS assay con-
rmed this data, clearly showing a signi cant anti-proliferative
effect in a dose dependent manner.
To further evaluate the effect of cannabinoid receptor agonism
on human PANC1 pancreatic cells and MDA-MB-231 breast can-
cer cell lines, clonogenicity assay was performed with treatment
with L-759633, ACPA and ACEA. All the three agonists caused
a decreased colony formation in both cell lines dose dependently.
Colony formation from cell assemblages may be associated with
cell-cell adhesion and cell motility (45) therefore, suggesting that
in the presence of CB1/2 agonist administration, both PANC1 pan-
creatic cells and MDA-MB-231 breast cancer cells are less mobile
and more adherent to each other.
In cancer treatment, as a general rule, controlling unwanted
cellular toxicity, weakening the proliferation of cancer cells and
overcoming intrinsic drug resistance is important. For this purpose,
induction of apoptosis is an ideal approach for a selective killing of
the cancer cells. Apoptosis, programmed cell death, is responsible
for the eradication of damaged cells and has an important role in
regulating cell proliferation, balancing cell survival and death. In-
trinsic (mitochondrial dependent) and extrinsic (receptor mediated)
pathways activate caspase 3 and result in apoptosis (46). Caspase 3
and 9 are activated when proapoptotic (i.e. Bak and Bax) and anti-
apoptotic (i.e. Mcl-1, Bcl-2 and Bcl-xL) proteins stabilize the mem-
brane of mitochondria (47). Cancer cells regulate this process and
reduce apoptosis resulting in elevated drug resistance. We evaluated
Bcl-2 and Bax, in order to investigate in more details the underly-
ing mechanism of selective agonists. Bax and Bcl-2 with opposite
effects are two members of the Bcl-2 family. Bcl-2, is found at high
levels of many human tumours and neutralizes the proapoptotic
effect of BAX by forming a heterodimer with Bax (48, 49). In our
study, after treatment of PANC1 pancreatic and MDA-MB-231
breast cancer cells with cannabinoid agonist the proapoptotic/an-
tiapoptotic balance drifted to the proapoptotic side. CB2 agonist
-L-759633, CB1 agonist-ACPA and CB1agonist-ACEA decreased
the expression of antiapoptotic BCL2 and increased the proapop-
totic Bax levels. The results were con rmed by ow cytometry. An
increase in apoptotic cells was seen after treatment with selective
cannabinoid agonists, CB2 agonist -L-759633 (10–50 μM), CB1
agonist-ACPA (50 μM) and CB1agonist-ACEA (50 μM) both in
human PANC1 pancreatic and MDA-MB-231 breast cancer cells.
The cells showed a clear positivity of the Annexin V test. As shown
by western blot and ow cytometric analysis, selective cannabi-
noid agonists, CB2 agonist -L-759633, CB1 agonist-ACPA and
CB1agonist-ACEA are good apoptosis inducers in human PANC1
pancreatic and MDA-MB-231 breast cancer cells.
Identifying effective treatments is critical for managing and
improving cancer treatment. Inhibiting cannabinoid receptors se-
lectively produces a potential for the therapy of various cancers,
including pancreatic and breast cancers. The results presented
here show that selective cannabinoid agonists, CB2 agonist -L-
759633, CB1 agonist-ACPA and CB1agonist-ACEA supress cell
proliferation, clonogenicity and induce apoptosis in human PANC1
pancreatic and MDA-MB-231 breast cancer cells in vitro. We ob-
served that both CB1 and CB2 agonists act through a mechanism
that involves up- and down regulations of proteins, which are
related to cell growth regulation and energetic metabolism. This
evidence suggests a link between the inhibition of cell survival
and proapoptotic activity of selective CB1/2 receptor agonists
considering as novel pharmacological anti-cancer agents. Over-
all, we think that our results may contribute to the development of
cannabinoid-based therapy in the prevention and management of
pancreatic and breast cancers. However, further studies investigat-
ing speci c mechanisms and the molecular pathways associated
with cannabinoid activities are needed.
References
1. Ma X, Yu H. Global burden of cancer. Yale J Biol Med 2006; 79: 85–94.
2. Adeloye D, David RA, Aderemi AV et al. An Estimate of the Incidence
of Prostate Cancer in Africa: A Systematic Review and Meta-Analysis. Plos
One 2016; 11: e0153496.
3. Kalant H. Medicinal use of cannabis: history and current status. Pain Res
Manag 2001; 6 (2): 80–91.
4. Croxford JL, Yamamura T. Cannabinoids and the immune system: po-
tential for the treatment of in ammatory diseases? J Neuroimmunol 2005;
166 (1–2): 3–18.
5. Ständer S, Reinhardt HW, Luger TA. Topical cannabinoid agonists.
An effective new possibility for treating chronic pruritus. Hautarzt 2006;
57 (9): 801–807.
6. Mach F, Steffens S. The role of the endocannabinoid system in athero-
sclerosis. J Neuroendocrinol 2008; 20 Suppl 1: 53–57.
7. Munson A, Harris L, Friedman M, Dewey W, Carchman R. Anti-
neoplastic activity of cannabinoids. J Nat Cancer Inst 1975; 55: 597–602.
8. Vecera L, Gabrhelik T, Prasil P, Stourac P. The role of cannabinoids in
the treatment of cancer. Bratisl Med J 2020; 121 (1): 79–95.
9. Donadelli M, Dando I, Zaniboni T et al. Gemcitabine/cannabinoid
combination triggers autophagy in pancreatic cancer cells through a ROS-
mediated mechanism. Cell Death Dis 2011; 2 (4): e152.
10. Müller L, Radtke A, Decker J, Koch M, Belge G. The Synthetic Can-
nabinoid WIN 55,212-2 Elicits Death in Human Cancer Cell Lines. Anti-
cancer Res 2017; 37 (11): 6341–6345.
11. Fonseca BM, Correia-da-Silva G, Teixeira NA. Cannabinoid-induced
cell death in endometrial cancer cells: involvement of TRPV1 receptors in
apoptosis. J Physiol Biochem 2018; 74 (2): 261–272.
12. Milian L, Mata M, Alcacer J et al. Cannabinoid receptor expression in
non-small cell lung cancer. Effectiveness of tetrahydrocannabinol and can-
nabidiol inhibiting cell proliferation and epithelial-mesenchymal transition
in vitro. PLoS One 2020; 15 (2): e0228909.
TURGUT Nergiz Hacer et al. Anti-cancer effects of selective cannabinoid agonists in pancreatic and breast cancer cells
xx
821
13. Ruhl T, Karthaus N, Kim BS, Beier JP. The endocannabinoid recep-
tors CB1 and CB2 affect the regenerative potential of adipose tissue MSCs.
Exp Cell Res 2020; 389 (1): 111881.
14. Howlett AC, Abood ME. CB (1) and CB (2) Receptor Pharmacology.
Adv Pharmacol 2017; 80: 169–206.
15. Greenhough A, Patsos HA, Williams AC, Paraskeva C. The canna-
binoid delta (9)-tetrahydrocannabinol inhibits RAS-MAPK and PI3K-AKT
survival signalling and induces BAD-mediated apoptosis in colorectal cancer
cells. Int J Cancer 2007; 121 (10): 2172–2180.
16. Ye L, Cao Z, Wang W, Zhou N. New Insights in Cannabinoid Recep-
tor Structure and Signaling. Curr Mol Pharmacol 2019; 12 (3): 239–248.
17. Singh UP, Singh NP, Singh B, Price RL, Nagarkatti M, Nagarkatti
PS. Cannabinoid receptor-2 (CB2) agonist ameliorates colitis in IL-10 (-/-)
mice by attenuating the activation of T cells and promoting their apoptosis.
Toxicol Appl Pharmacol 2012; 258 (2): 256–267.
18. Carpi S, Fogli S, Romanini A et al. AM251 induces apoptosis and
G2/M cell cycle arrest in A375 human melanoma cells. Anticancer Drugs
2015; 26 (7): 754–762.
19. Bachari A, Piva TJ, Salami SA, Jamshidi N, Mantri N. Roles of Can-
nabinoids in Melanoma: Evidence from In Vivo Studies. Int J Mol Sci 2020;
21 (17): 6040.
20. Mazuz M, Tiroler A, Moyal L et al. Synergistic cytotoxic activity
of cannabinoids from cannabis sativa against cutaneous T-cell lymphoma
(CTCL) in-vitro and ex-vivo. Oncotarget 2020; 11 (13): 1141–1156.
21. Zhang G, Bi H, Gao J, Lu X, Zheng Y. Inhibition of autophagy and
enhancement of endoplasmic reticulum stress increase sensitivity of osteo-
sarcoma Saos-2 cells to cannabinoid receptor agonist WIN55,212-2. Cell
Biochem Funct 2016; 34 (5): 351–358.
22. Semlali A, Beji S, Ajala I, Rouabhia M. Effects of tetrahydrocannabi-
nols on human oral cancer cell proliferation, apoptosis, autophagy, oxidative
stress, and DNA damage. Arch Oral Biol 2021; 129: 105200.
23. McAllister SD, Christian RT, Horowitz MP, Garcia A, Desprez
PY. Cannabidiol as a novel inhibitor of Id-1 gene expression in aggres-
sive breast cancer cells. Mol Cancer Ther 2007; 6 (11): 2921–2927.
24. Preet A, Qamri Z, Nasser MW et al. Cannabinoid receptors, CB1 and
CB2, as novel targets for inhibition of non-small cell lung cancer growth
and metastasis. Cancer Prev Res (Phila) 2011; 4 (1): 65–75.
25. Tegeder I. Endocannabinoids as Guardians of Metastasis. Int J Mol
Sci 2016; 17 (2): 230.
26. Ramer R, Hinz B. Inhibition of cancer cell invasion by cannabinoids
via increased expression of tissue inhibitor of matrix metalloproteinases-1.
J Natl Cancer Inst 2008; 100 (1): 59–69.
27. Plumb JA. Cell sensitivity assays: clonogenic assay. Methods Mol Med
1999; 28: 17–23.
28. Vermes I, Haanen C, Steffens-Nakken H, Reutelingsperger C. A
novel assay for apoptosis. Flow cytometric detection of phosphatidylserine
expression on early apoptotic cells using uorescein labelled Annexin V. J
Immunol Methods 1995; 184: 39–51.
29. Blázquez C, Salazar M, Carracedo A et al. Cannabinoids inhibit glioma
cell invasion by down-regulating matrix metalloproteinase-2 expression.
Cancer Res 2008; 68 (6): 1945–1952.
30. Grimaldi C, Pisanti S, Laezza C et al. Anandamide inhibits adhesion
and migration of breast cancer cells. Exp Cell Res 2006; 312 (4): 363–373.
31. Carracedo A, Gironella M, Lorente M et al. Cannabinoids induce
apoptosis of pancreatic tumor cells via endoplasmic reticulum stress-related
genes. Cancer Res 2006; 66 (13): 6748–6755.
32. Dando I, Donadelli M, Costanzo C et al. Cannabinoids inhibit energetic
metabolism and induce AMPK-dependent autophagy in pancreatic cancer
cells. Cell Death Dis 2013; 4 (6): e664.
33. Aizikovich A. Anticancer Effect of New Cannabinoids Derived from
Tetrahydrocannabinolic Acid on PANC-1 and AsPC-1 Human Pancreas Tu-
mor Cells. J Pancreat Cancer 2020; 6 (1): 40–44.
34. Yang Y, Huynh N, Dumesny C, Wang K, He H, Nikfarjam M. Can-
nabinoids Inhibited Pancreatic Cancer via P-21 Activated Kinase 1 Mediated
Pathway. Int J Mol Sci 2020; 21 (21): 8035.
35. Brandi J, Dando I, Palmieri M, Donadelli M, Cecconi D. Compara-
tive proteomic and phosphoproteomic pro ling of pancreatic adenocarci-
noma cells treated with CB1 or CB2 agonists. Electrophoresis 2013; 34
(9–10): 1359–1368.
36. Caffarel MM, Sarrió D, Palacios J, Guzmán M, Sánchez C. Delta9-
tetrahydrocannabinol inhibits cell cycle progression in human breast can-
cer cells through Cdc2 regulation. Cancer Res 2006; 66 (13): 6615–6621.
37. De Petrocellis L, Melck D, Palmisano A et al. The endogenous can-
nabinoid anandamide inhibits human breast cancer cell proliferation. Proc
Natl Acad Sci USA 1998; 95 (14): 8375–8380.
38. Laezza C, d’Alessandro A, Mal tano AM, Bifulco M. Anandamide
inhibits the Wnt/beta-catenin signalling pathway in human breast cancer
MDA MB 231 cells. Eur J Cancer 2012; 48 (16): 3112–3122.
39. Mohammadpour F, Ostad SN, Aliebrahimi S, Daman Z. Anti-invasion
Effects of Cannabinoids Agonist and Antagonist on Human Breast Cancer
Stem Cells. Iran J Pharm Res 2017; 16 (4): 1479–1486.
40. Hanlon KE, Lozano-Ondoua AN, Umaretiya PJ et al. Modulation of
breast cancer cell viability by a cannabinoid receptor 2 agonist, JWH-015,
is calcium dependent. Breast Cancer (Dove Med Press) 2016; 8: 59–71.
41. Elbaz M, Ahirwar D, Ravi J, Nasser MW, Ganju RK. Novel role of
cannabinoid receptor 2 in inhibiting EGF/EGFR and IGF-I/IGF-IR pathways
in breast cancer. Oncotarget 2017; 8 (18): 29668–29678.
42. Qamri Z, Preet A, Nasser MW et al. Synthetic cannabinoid receptor
agonists inhibit tumor growth and metastasis of breast cancer. Mol Cancer
Ther 2009; 8 (11): 3117–3129.
43. McAllister SD, Christian RT, Horowitz MP, Garcia A, Desprez
PY. Cannabidiol as a novel inhibitor of Id-1 gene expression in aggres-
sive breast cancer cells. Mol Cancer Ther 2007; 6 (11): 2921–2927.
44. Prather PL, Francis Devaraj F, Dates CR et al. CB1 and CB2 receptors
are novel molecular targets for Tamoxifen and 4OH-Tamoxifen. Biochem
Biophys Res Commun 2013; 441 (2): 339–343.
45. Sungkaworn T, Triampo W, Nalakarn P et al. The effects of TiO2
nanoparticles on tumor cell colonies: fractal dimension and morphological
properties. Int J Medical Health Biomed Bioeng Pharm Eng 2008; 2 (1): 20–27.
46. Wang W, Zhu M, Xu Z et al. Ropivacaine promotes apoptosis of he-
patocellular carcinoma cells through damaging mitochondria and activating
caspase-3 activity. Biol Res 2019; 52 (1): 36.
47. Zhang X, Qin Y, Pan Z et al. Cannabidiol Induces Cell Cycle Arrest
and Cell Apoptosis in Human Gastric Cancer SGC-7901 Cells. Biomol-
ecules 2019; 9 (8): 302.
48. Changizi Z, Moslehi A, Rohani AH, Eidi A. Chlorogenic acid induces
4T1 breast cancer tumor’s apoptosis via p53, Bax, Bcl-2, and caspase-3 signal-
ing pathways in BALB/c mice. J Biochem Mol Toxicol 2021; 35 (2): e22642.
49. Liu X, Dong J, Cai W, Pan Y, Li R, Li B. The Effect of Thymoquinone
on Apoptosis of SK-OV-3 Ovarian Cancer Cell by Regulation of Bcl-2 and
Bax. Int J Gynecol Cancer 2017; 27 (8): 1596–1601.
Received April 18, 2022.
Accepted July 6, 2022.
ResearchGate has not been able to resolve any citations for this publication.
Article
Full-text available
The anti-cancer effects of cannabinoids including CBD (Cannabidiol) and THC ((−)-trans-∆9-tetrahydrocannabinol) have been reported in the case of pancreatic cancer (PC). The connection of these cannabinoids to KRas oncogenes that mutate in more than 90% of PC, and their effects on PD-L1, a key target of immune checkpoint blockade, have not been thoroughly investigated. Using cell lines and mouse models of PC, the effects of CBD and THC on cancer growth, the interaction between PC cells and a stromal cell, namely pancreatic stellate cells (PSCs), and the mechanism(s) involved were determined by cell-based assays and mouse study in vivo. CBD and THC inhibited the proliferation of PC, PSC, and PSC-stimulated PC cells. They also suppressed pancreatic tumour growth in mice. Furthermore, CBD and/or THC reduced the expression of PD-L1 by either PC or PSC cells. Knockout of p-21 activated kinase 1 (PAK1, activated by KRas) in PC and PSC cells and, in mice, dramatically decreased or blocked these inhibitory effects of CBD and/or THC. These results indicated that CBD and THC exerted their inhibitions on PC and PSC via a p-21 activated kinase 1 (PAK1)-dependent pathway, suggesting that CBD and THC suppress Kras activated pathway by targeting PAK1. The inhibition by CBD and THC of PD-L1 expression will enhance the immune checkpoint blockade of PC.
Article
Full-text available
Melanoma is the fourth most common type of cancer diagnosed in Australians after breast, prostate, and colorectal cancers. While there has been substantial progress in the treatment of cancer in general, malignant melanoma, in particular, is resistant to existing medical therapies requiring an urgent need to develop effective treatments with lesser side effects. Several studies have shown that “cannabinoids”, the major compounds of the Cannabis sativaL. plant, can reduce cell proliferation and induce apoptosis in melanoma cells. Despite prohibited use of Cannabis in most parts of the world, in recent years there have been renewed interests in exploiting the beneficial health effects of the Cannabis plant-derived compounds. Therefore, the aim of this study was in the first instance to review the evidence from in vivo studies on the effects of cannabinoids on melanoma. Systematic searches were carried out in PubMed, Embase, Scopus, and ProQuest Central databases for relevant articles published from inception. From a total of 622 potential studies, six in vivo studies assessing the use of cannabinoids for treatment of melanoma were deemed eligible for the final analysis. The findings revealed cannabinoids, individually or combined, reduced tumor growth and promoted apoptosis and autophagy in melanoma cells. Further preclinical and animal studies are required to determine the underlying mechanisms of cannabinoids-mediated inhibition of cancer-signaling pathways. Well-structured, randomized clinical studies on cannabinoid use in melanoma patients would also be required prior to cannabinoids becoming a viable and recognized therapeutic option for melanoma treatment in patients.
Article
Full-text available
Purpose: New tetrahydrocannabinolic acid (THCA) derivatives ALAM027 and ALAM108 were proposed for the treatment of the pancreatic cancer disease. Methods: The in vitro effect of new cannabinoids ALAM027 and ALAM108 was tested against PANC-1 and AsPC-1 cell lines by CellTiter Glo assay. Pancreatic cancer xenograft model was used for the in vivo anticancer activity study of these compounds on PANC-1 cells. Results: The in vitro study of new cannabinoids showed greater activity of ALAM108 than ALAM027 both for PANC-1 and AsPC-1 cells. The in vivo study of new cannabinoids on PANC-1 cells showed that their oral administration was effective in reducing tumor volume and tumor weight, and did not lead to any discomfort and weight loss of mice. Conclusion: The cannabinoids ALAM108 and ALAM027 inhibited the tumor growing 1.6-2 times in mice with human PANC-1 cells.
Article
Full-text available
The main chemical component of cannabis, cannabidiol (CBD), has been shown to have antitumor properties. The present study examined the in vitro effects of CBD on human gastric cancer SGC-7901 cells. We found that CBD significantly inhibited the proliferation and colony formation of SGC-7901 cells. Further investigation showed that CBD significantly upregulated ataxia telangiectasia-mutated gene (ATM) and p53 protein expression and downregulated p21 protein expression in SGC-7901 cells, which subsequently inhibited the levels of CDK2 and cyclin E, thereby resulting in cell cycle arrest at the G0–G1 phase. In addition, CBD significantly increased Bax expression levels, decreased Bcl-2 expression levels and mitochondrial membrane potential, and then upregulated the levels of cleaved caspase-3 and cleaved caspase-9, thereby inducing apoptosis in SGC-7901 cells. Finally, we found that intracellular reactive oxygen species (ROS) increased after CBD treatment. These results indicated that CBD could induce G0–G1 phase cell cycle arrest and apoptosis by increasing ROS production, leading to the inhibition of SGC-7901 cell proliferation, thereby suggesting that CBD may have therapeutic effects on gastric cancer.
Article
Full-text available
Abstract Background Recent evidences indicated that some local anaesthetic agents played a role in inhibiting the proliferation of cancer cells; Whether ropivacaine is able to promote apoptosis of hepatocellular carcinoma (HCC) cells is still unclear. The aim of this study was to investigate the effect of ropivacaine on the apoptosis of HCC cells. Methods In the present study, we treated the HCC cell lines, Bel7402 and HLE with ropivacaine. MTT, DAPI stain, trypan blue exclusion dye assay, flow cytometry, electron microscopy, computational simulation, laser confocal microscope, Western blotting, and enzyme activity analysis of caspase-3 were applied to detect the growth and apoptosis of HCC cells and to explore the role mechanism of ropivacaine. Results Ropivacaine was able to inhibit proliferation and promote apoptosis of HCC cells in a dose- and time-dependent manner. Ropivacaine also has a trait to inhibit the migration of HCC cells; ropivacaine damaged the mitochondria of HCC cells. The results also indicated that ropivacaine was able to interact with caspase-3, promote cytoplasmic caspase-3 migration into the nucleus, stimulate cleavage of caspase-3 and PARP-1, caspase-9 proteins, inhibit the expression of Bcl-2, promote expression of Apaf-1 and mitochondria release cytochrome C, and activate the activity of caspase-3. Conclusions Ropivacaine has a novel role in promoting apoptosis of HCC cells; The role mechanism of ropivacaine maybe involve in damaging the function of mitochondria and activating the caspase-3 signalling pathway in HCC cells. Our findings provide novel insights into the local anaesthetic agents in the therapy of HCC patients.
Article
Objective Cannabinoids, including delta-8- and delta-9-tetrahydrocannabinol (THC) have a palliative care impact and may therefore be beneficial against cancer. The aim of this study was to investigate the effect of Δ⁹-THC and Δ⁸-THC on oral cancer cell behaviors. Design The Ca9-22 oral cancer cells were cultured in the presence or not of various concentrations of Δ⁹-THC and Δ⁸-THC for different times. The cultures were then used to measure cell viability/proliferation, apoptosis, autophagy, oxidative stress, antioxidant activity, and inhibition of signaling pathways MAP-Kinase, NF-κB, and β−catenin. Results Both cannabinoids were found to decrease cell viability/proliferation by blocking the cell cycle progression from the S to the G2/M phase and enhancing their apoptosis and autophagy. Δ⁹-THC and Δ⁸-THC also suppressed the migration/invasion by inhibiting epithelial-mesenchymal transition markers, such as E-cadherin, in addition to decreasing reactive oxygen species (ROS) production and increasing glutathione (GSH) and the expression of mtMP. Δ⁹-THC and Δ⁸-THC also downregulated cyclin D1, p53, NOXA, PUMAα, and DRAM expressions but increased p21 and H2AX expression. Conclusion We demonstrated that cannabinoids (Δ⁹-THC and Δ⁸-THC) were able to decrease oral cancer cell growth through various mechanisms, including apoptosis, autophagy, and oxidative stress. These results suggest a potential use of these molecules as a therapy against oral cancer.
Article
Despite all the new treatments, metastatic breast cancer (BC) causes many deaths. Chlorogenic acid (CGA) is a polyphenol compound with various pharmacological traits, such as anticancer properties. Targeting apoptotic death pathways has been propounded as the most effective therapeutic method in various cancers. In the current study, apoptotic agents such as p53, Bax, Bcl‐2, and caspase‐3 have been investigated. The experimental groups included saline, BC, CGA, protective (PR), and treatment (TM) groups. First, 4T1 mouse BC was established and then the effects of treatment with CGA were investigated through measurement of tumor weight and volume, metastatic nodules, liver biochemical tests, hematoxylin and eosin (H&E), immunohistochemistry (IHC) staining, and real‐time reverse transcription‐polymerase chain reaction (RT‐PCR) in experimental groups. The findings showed that CGA reduced tumor weight and volume in the PR group (P < .05) and in the TM group (P < .001). Surprisingly, it eliminated the tumors in the TM group. Metastatic nodules in the PR and TM groups were significantly reduced as compared with the BC group (P < .001). The evaluation by H&E staining showed cell apoptosis in both the PR and TM groups. The results of real‐time RT‐PCR showed that CGA therapy increased the expression ratio of Bax/Bcl‐2 (P < .001 and P < .05, respectively) and the expression of p53 (P < .001 and P < .05, respectively) and caspase‐3 genes (P < .01) in the PR and TM groups. The IHC data regarding the Bax/Bcl‐2 ratio confirmed the other results (P < .001). The findings demonstrate that CGA plays a significant role in the induction of apoptosis and the treatment of 4T1 BC tumors in BALB/c mice.
Article
Cannabis sativa produces hundreds of phytocannabinoids and terpenes. Mycosis fungoides (MF) is the most common type of cutaneous T-cell lymphoma (CTCL), characterized by patches, plaques and tumors. Sézary is a leukemic stage of CTCL presenting with erythroderma and the presence of neoplastic Sézary T-cells in peripheral blood. This study aimed to identify active compounds from whole cannabis extracts and their synergistic mixtures, and to assess respective cytotoxic activity against CTCL cells. Ethanol extracts of C. sativa were analyzed by high-performance liquid chromatography (HPLC) and gas chromatography/mass spectrometry (GC/MS). Cytotoxic activity was determined using the XTT assay on My-La and HuT-78 cell lines as well as peripheral blood lymphocytes from Sézary patients (SPBL). Annexin V assay and fluorescence-activated cell sorting (FACS) were used to determine apoptosis and cell cycle. RNA sequencing and quantitative PCR were used to determine gene expression. Active cannabis compounds presenting high cytotoxic activity on My-La and HuT-78 cell lines were identified in crude extract fractions designated S4 and S5, and their synergistic mixture was specified. This mixture induced cell cycle arrest and cell apoptosis; a relatively selective apoptosis was also recorded on the malignant CD4+CD26- SPBL cells. Significant cytotoxic activity of the corresponding mixture of pure phytocannabinoids further verified genuine interaction between S4 and S5. The gene expression profile was distinct in My-La and HuT-78 cells treated with the S4 and S5 synergistic mixture. We suggest that specifying formulations of synergistic active cannabis compounds and unraveling their modes of action may lead to new cannabis-based therapies.
Article
Human adipose tissue includes large quantities of mesenchymal stromal cells (atMSCs), which represent an abundant cell source for therapeutic applications in the field of regenerative medicine. Adipose tissue secrets various soluble factors including endocannabinoids, and atMSCs express the cannabinoid receptors CB1 and CB2. This indicates that adipose tissue possesses an endocannabinoid system (ECS). The ECS is also ascribed great significance for wound repair, e.g. by modulating inflammation. However, the exact effects of CB1/CB2 activation in human atMSCs have not been investigated, yet. In the present study, we stimulated human atMSCs with increasing concentrations (1-30 μM) of the unspecific cannabinoid receptor ligand WIN55,212-2 and the specific CB2 agonist JWH-133, either alone or co-applied with the receptor antagonist Rimonabant (CB1) or AM 630 (CB2). We investigated the effects on metabolic activity, cell number, differentiation and cytokine release, which are important processes during tissue regeneration. WIN decreased metabolic activity and cell number, which was reversed by Rimonabant. This suggests a CB1 dependent mechanism, whereas the number of atMSCs was increased after CB2 ligation. WIN and JWH increased the release of VEGF, TGF-β1 and HGF. Adipogenesis was enhanced by WIN, which could be reversed by blocking CB1. There was no effect on osteogenesis, and only WIN increased chondrogenic differentiation. Our results indicate that definite activation of the cannabinoid receptors exerted different effects in atMSCs, which could be of specific value in cell-based therapy for wound regeneration.
Article
Background: Cannabinoid has long been used for medicinal purposes. Cannabinoid signaling has been considered the therapeutic target for treating pain, addiction, obesity, inflammation, and other diseases. Recent studies have suggested that in addition to CB1 and CB2, there are non-CB1 and non-CB2 cannabinoid-related orphan GPCRs including GPR18, GPR55, and GPR119. In addition, CB1 and CB2 display allosteric binding and biased signaling, revealing correlations between biased signaling and functional outcomes. Interestingly, new investigations have indicated that CB1 is functionally present within the mitochondria of striated and heart muscles directly regulating intramitochondrial signaling and respiration. Conclusion: In this review, we summarize the recent progress in cannabinoid-related orphan GPCRs, CB1/CB2 structure, Gi/Gs coupling, allosteric ligands and biased signaling, and mitochondria-localized CB1, and discuss the future promise of this research.