Ci a ion: Benedic o, A.; A e a, B.;
Du an i, A.; Alonso-Alconada, D. The
Syn he ic Cannabinoid URB447
Exe s An i umo and An ime as a ic
E ec in Melanoma and Colon
Cance . Pha maceu icals 2022,15, 1166.
h ps://doi.o g/10.3390/
ph15101166
Academic Edi o s: Jean
Jacques Vanden Eynde and
Annie Mayence
Recei ed: 29 Augus 2022
Accep ed: 17 Sep embe 2022
Published: 20 Sep embe 2022
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pha maceu icals
A icle
The Syn he ic Cannabinoid URB447 Exe s An i umo and
An ime as a ic E ec in Melanoma and Colon Cance
Ai o Benedic o 1, Bea iz A e a 1, And ea Du an i 2and Daniel Alonso-Alconada 1,*
1Depa men o Cell Biology and His ology, Facul y o Medicine and Nu se y, Uni e si y o he Basque
Coun y, 48940 Leioa, Bizkaia, Spain
2Depa men o Biomolecula Sciences, Uni e si y o U bino Ca lo Bo, 61029 U bino, I aly
*Co espondence: [email p o ec ed]; Tel.: +34-946013294
Abs ac :
The endocannabinoid sys em is widesp ead h ough he body and ca ies ou a wide
a ie y o unc ions. Howe e , i s in ol emen in o he pa hologies, such as cance , s ill needs u he
a en ion. We aim o in es iga e he ole o CB2 ecep o du ing melanoma and colo ec al cance
(CRC) agg essi eness and me as a ic g ow h in he li e . We used he syn he ic cannabinoid URB447,
a known CB2 agonis and CB1 an agonis d ug, and s udied p ome as a ic abili y o mouse B16
melanoma and MCA38 CRC cells, by means o p oli e a ion, apop osis, cell cycle, mig a ion and
ma ix deg ada ion
in i o
upon URB447 ea men . We epo ed a dose-dependen iabili y dec ease
in bo h umo ypes. This esul is pa ly media ed by apop o ic cell dea h and cell cycle a es in
G1/G0 phase, as obse ed h ough low cy ome y. Melanoma and CRC cell mig a ion was a ec ed
in a dose-dependen ashion as obse ed h ough sc a ch assay, whe eas he sec e ion o ma ix
deg ading p o eins me allop o ease 2 (MMP2) and 9 (MMP9) in umo cells did no signi ican ly
change. Mo eo e , daily ea men o umo bea ing mice wi h URB447 dec eased he de elopmen o
li e me as asis in a melanoma model
in i o
. This p oo o concep s udy poin s ou o he syn he ic
cannabinoid URB447 as a po en ial candida e o deepe s udies o con i m i s po en ial as an i umo
he apy and li e me as asis ea men o CRC and melanoma.
Keywo ds: li e me as asis; cannabinoids; URB447; colon cance ; melanoma
1. In oduc ion
The endogenous cannabinoid sys em (ECS) ep esen s a ubiqui ous neu omodula o y
sys em in he body, wi h a wide spec um o di e en unc ions. I is composed by he
cannabinoid ecep o s and hei endogenous ligands and he enzymes ha syn hesize and
deg ade hese ligands [
1
]. Cannabinoid ecep o 1 (CB1) and cannabinoid ecep o 2 (CB2)
a e he main e ec o s o he ECS unc ions, along wi h o he ecep o s such as such as
PPAR’s and T ansien Recep o Po en ial (TRP) channels [
2
]. CB1 and CB2 ecep o s a e G
p o ein coupled ecep o s ha exe se e al in acellula changes and, he e o e, media e
cellula p ocesses such as gene ansc ip ion, cell mo ili y and angiogenesis [3,4].
ECS ecep o s a e widesp ead h oughou he body and in ol ed in di e en physi-
ological p ocesses. I is he omnip esence wha makes CB ecep o s a po en ial a ge o
se e al pa hologies [
5
,
6
]. In ac , CB ecep o s a e exp essed in a wide a ie y o cance s
wi h di e en o igin, such as glioma and melanoma, o name a ew [
7
,
8
]. Howe e , he ole
o hese ecep o s is no ye e y well unde s ood. While in some cance s, such as glioma o
melanoma, he ac i a ion o CB1 and CB2 lead o impai ed p o umo al ac i i ies [
9
,
10
], in
o he malignancies such as non-small cell lung ca cinoma, hese ecep o s ac as p o umo al
media o s [11].
To da e, se e al s udies ha e p o en he e ec i eness o some cannabinoids agains
cance . Cannabidiol (CBD) is he main non-psychoac i e cons i uen o Cannabis. CBD
has shown an icance p ope ies, a ec ing di e se umo al p ocesses. CBD blocked he
cell cycle o gas ic cance cells, leading o educed CDK2/Cyclin E p o ein le els [
12
] and
Pha maceu icals 2022,15, 1166. h ps://doi.o g/10.3390/ph15101166 h ps://www.mdpi.com/jou nal/pha maceu icals
Pha maceu icals 2022,15, 1166 2 o 11
exe ed p oapop o ic e ec in b eas cance cells h ough endoplasmic e iculum s ess,
d i ing o apop osis [
13
]. In iguingly, CBD showed an an agonis ic e ec o CB1 while
ha ing an agonis ic e ec o CB2 [14].
The
in i o
an icance p ope ies o cannabinoids include cell cycle a es , he in-
duc ion o cance cell apop osis, impai ed mig a ion and in asion, educ ion o ma ix
me allop o ease 2 and 9 (MMP-2,9), impai men o angiogenic esponse h ough VEGF
down egula ion, inhibi ion o epi helial o mesenchymal ansi ion (EMT) and lead o
a ec ed me as a ic g ow h [15,16].
Howe e , a sho numbe o s udies has alida ed his
in i o
e ec using animal
models. In ac , jus one s udy has es ed
in i o
he e ec o a cannabinoid, speci ically a
hexahyd ocannabinol analog, on colo ec al cance (CRC) me as asis in a xenog a model
o CRC [
17
]). Rega ding o melanoma, only six s udies ha e alida ed he e icacy o
cannabinoids du ing cance de elopmen
in i o
[
18
]. Despi e he animal models s udied,
none o hem analyzed he e ec o cannabinoids du ing he me as a ic p ocess in he li e .
This o gan ep esen s a common a ge o gan o se e al cance s, such as CRC, panc ea ic
cance and melanoma [
19
], which signi ican ly complica es he p ognosis and su i al
o pa ien s. I is in e es ing o no e ha se e al cannabinoids ha e a high impac on he
de elopmen o a wide a ay o li e pa hologies.
Based on he abo e-men ioned e idences, s imula ing CB2 ecep o while an agoniz-
ing CB1 ecep o may ep esen a p omising he apeu ic op ion o he ea men o CRC
and melanoma li e me as asis.
URB447 ({[4-amino-1-(4-chlo obenzyl)-2-me hyl-5-phenyl-1H-py ole-3-yl](phenyl)
me hanone}) (Figu e 1) is a syn he ic cannabinoid ligand able o ac as CB2 agonis and
CB1 an agonis . I s abili y o show ano exian ac i i y wi hou he ypical side e ec s o
d ugs ac ing on he cen al ne ous sys em was o iginally disco e ed, due o he ac he
molecule educes eeding and body-weigh gain in mice wi h a pe iphe ally es ic ed
ac ion [
20
]. Mo e ecen ly, i has been demons a ed ha URB447 educes b ain inju y and
he associa ed whi e ma e demyelina ion a e hypoxia-ischemia in neona al a s [21].
Pha maceu icals 2022, 15, x FOR PEER REVIEW 2 o 11
has shown an icance p ope ies, a ec ing di e se umo al p ocesses. CBD blocked he
cell cycle o gas ic cance cells, leading o educed CDK2/Cyclin E p o ein le els [12] and
exe ed p oapop o ic e ec in b eas cance cells h ough endoplasmic e iculum s ess,
d i ing o apop osis [13]. In iguingly, CBD showed an an agonis ic e ec o CB1 while
ha ing an agonis ic e ec o CB2 [14].
The in i o an icance p ope ies o cannabinoids include cell cycle a es , he induc-
ion o cance cell apop osis, impai ed mig a ion and in asion, educ ion o ma ix me al-
lop o ease 2 and 9 (MMP-2,9), impai men o angiogenic esponse h ough VEGF down-
egula ion, inhibi ion o epi helial o mesenchymal ansi ion (EMT) and lead o a ec ed
me as a ic g ow h [15,16].
Howe e , a sho numbe o s udies has alida ed his in i o e ec using animal
models. In ac , jus one s udy has es ed in i o he e ec o a cannabinoid, speci ically a
hexahyd ocannabinol analog, on colo ec al cance (CRC) me as asis in a xenog a model
o CRC [17]). Rega ding o melanoma, only six s udies ha e alida ed he e icacy o can-
nabinoids du ing cance de elopmen in i o [18]. Despi e he animal models s udied,
none o hem analyzed he e ec o cannabinoids du ing he me as a ic p ocess in he
li e . This o gan ep esen s a common a ge o gan o se e al cance s, such as CRC, pan-
c ea ic cance and melanoma [19], which signi ican ly complica es he p ognosis and su -
i al o pa ien s. I is in e es ing o no e ha se e al cannabinoids ha e a high impac on
he de elopmen o a wide a ay o li e pa hologies.
Based on he abo e-men ioned e idences, s imula ing CB2 ecep o while an agoniz-
ing CB1 ecep o may ep esen a p omising he apeu ic op ion o he ea men o CRC
and melanoma li e me as asis.
URB447 ({[4-amino-1-(4-chlo obenzyl)-2-me hyl-5-phenyl-1H-py ole-3-yl](phenyl)
me hanone}) (Figu e 1) is a syn he ic cannabinoid ligand able o ac as CB2 agonis and
CB1 an agonis . I s abili y o show ano exian ac i i y wi hou he ypical side e ec s o
d ugs ac ing on he cen al ne ous sys em was o iginally disco e ed, due o he ac he
molecule educes eeding and body-weigh gain in mice wi h a pe iphe ally es ic ed
ac ion [20]. Mo e ecen ly, i has been demons a ed ha URB447 educes b ain inju y and
he associa ed whi e ma e demyelina ion a e hypoxia-ischemia in neona al a s [21].
Figu e 1. Chemical s uc u e o he syn he ic cannabinoid URB447.
The e o e, we es ed he an icance e icacy o he syn he ic cannabinoid URB447 in
CRC and melanoma in i o and u he explo ed i s unc ion using a syngeneic model o
li e me as asis in i o. I is emp ing o hypo hesize ha he abili y o URB447 o exe a
s imula o y e ec in CB2 may educe he p o umo al ac i i y o bo h melanoma and CRC
in i o and p e en me as a ic g ow h in he li e .
2. Resul s
2.1. An i umo E ec o URB447 in Cance Cell Viabili y
We ha e checked he po en ial o URB447 o modula e melanoma and CRC cell ia-
bili y. Ou esul s show ha his syn he ic cannabinoid in e e es wi h umo cell iabili y
in bo h cance models. In de ail, URB447 10 µM did no a ec cell iabili y a e 24 h,
whe eas 25 µM and 50 µM exe ed an an i umo e ec a his ime poin . Howe e , 10 µM
signi ican ly educed cance cell iabili y abou 10% a e 48 h in B16-F10 melanoma cells,
N
H
2
N
O
Cl
Figu e 1. Chemical s uc u e o he syn he ic cannabinoid URB447.
The e o e, we es ed he an icance e icacy o he syn he ic cannabinoid URB447 in
CRC and melanoma
in i o
and u he explo ed i s unc ion using a syngeneic model o
li e me as asis
in i o
. I is emp ing o hypo hesize ha he abili y o URB447 o exe a
s imula o y e ec in CB2 may educe he p o umo al ac i i y o bo h melanoma and CRC
in i o and p e en me as a ic g ow h in he li e .
2. Resul s
2.1. An i umo E ec o URB447 in Cance Cell Viabili y
We ha e checked he po en ial o URB447 o modula e melanoma and CRC cell
iabili y. Ou esul s show ha his syn he ic cannabinoid in e e es wi h umo cell
iabili y in bo h cance models. In de ail, URB447 10
µ
M did no a ec cell iabili y a e
24 h
, whe eas 25
µ
M and 50
µ
M exe ed an an i umo e ec a his ime poin . Howe e ,
10 µM
signi ican ly educed cance cell iabili y abou 10% a e 48 h in B16-F10 melanoma
cells, whe eas 25
µ
M and 50
µ
M d o e o 40% and 60% cell dea h, espec i ely (Figu e 2A).
Pha maceu icals 2022,15, 1166 3 o 11
Rega ding MCA38, he same end was obse ed a e 48 h, educing cell iabili y 10%,
40% and 67% when ea ed wi h 10 µM, 25 µM and 50 µM, espec i ely (Figu e 2B).
Pha maceu icals 2022, 15, x FOR PEER REVIEW 3 o 11
whe eas 25 µM and 50 µM d o e o 40% and 60% cell dea h, espec i ely (Figu e 2A).
Rega ding MCA38, he same end was obse ed a e 48 h, educing cell iabili y 10%,
40% and 67% when ea ed wi h 10 µM, 25 µM and 50 µM, espec i ely (Figu e 2B).
Figu e 2. Tumo cell iabili y upon ea men wi h URB447. Melanoma (A) and CRC cance cells (B)
we e incuba ed in he p esence o URB447 o 24 and 48 h. Cell iabili y was measu ed using P es-
oblue™ iabili y assay. Images show ep esen a i e cell popula ion a he ime o he measu emen
(n = 3). Di e ences we e conside ed s a is ically signi ican o * p < 0.05, ** < 0.01 using S uden
es .
2.2. Apop o ic E ec o URB447 in Cance Cells
To unco e he mechanisms o educed iabili y, we analyzed he e ec o URB447
in he apop o ic cell dea h in bo h melanoma and CRC models. As shown in Figu e 3,
URB447 p omo es apop osis in bo h models. URB447 p omo ed apop o ic cell dea h in
B16-F10 melanoma cells in a dose-dependen manne a e 24 h. In de ail, whe eas 10 µM
and 25 µM sligh ly inc eased apop o ic cells, 50 µM ende ed melanoma cells o apop osis
media ed cell dea h, inc easing 3- old ea ly apop o ic cell coun s (Figu e 3A). Rega ding
MCA38, he same p oapop o ic pa e n was obse ed in cells ea ed wi h 10 µM, 25 µM
and 50 µM URB447. In his ega d, 10 µM inc eased 2,5- old he pe cen age o apop o ic
cells, whe eas 25 and 50 µM led o 4- old augmen a ion in ea ly apop o ic cell numbe s
(Figu e 3B).
Figu e 2.
Tumo cell iabili y upon ea men wi h URB447. Melanoma (
A
) and CRC cance cells
(
B
) we e incuba ed in he p esence o URB447 o 24 and 48 h. Cell iabili y was measu ed using
P es oblue
™
iabili y assay. Images show ep esen a i e cell popula ion a he ime o he measu emen
(n= 3). Di e ences we e conside ed s a is ically signi ican o * p< 0.05, ** p< 0.01 using S uden es .
2.2. Apop o ic E ec o URB447 in Cance Cells
To unco e he mechanisms o educed iabili y, we analyzed he e ec o URB447 in
he apop o ic cell dea h in bo h melanoma and CRC models. As shown in Figu e 3, URB447
p omo es apop osis in bo h models. URB447 p omo ed apop o ic cell dea h in B16-F10
melanoma cells in a dose-dependen manne a e 24 h. In de ail, whe eas 10
µ
M and 25
µ
M
sligh ly inc eased apop o ic cells, 50
µ
M ende ed melanoma cells o apop osis media ed cell
dea h, inc easing 3- old ea ly apop o ic cell coun s (Figu e 3A). Rega ding MCA38, he same
p oapop o ic pa e n was obse ed in cells ea ed wi h 10
µ
M, 25
µ
M and 50
µ
M URB447. In
his ega d, 10
µ
M inc eased 2,5- old he pe cen age o apop o ic cells, whe eas 25 and
50 µM
led o 4- old augmen a ion in ea ly apop o ic cell numbe s (Figu e 3B).
Pha maceu icals 2022,15, 1166 4 o 11
Pha maceu icals 2022, 15, x FOR PEER REVIEW 4 o 11
Figu e 3. Apop o ic e ec o URB447. Melanoma (A) and CRC cance cells (B) we e incuba ed in he
p esence o URB447 o 24 h. Flow cy ome y was ca ied ou o analyze ea ly and la e apop o ic
cells. Images show ep esen a i e FACS esul (n = 3). Di e ences we e conside ed s a is ically sig-
ni ican o * p < 0.05, ** p < 0.01 using S uden es .
2.3. Cell Cycle In e e ence upon URB447 T ea men
Since cell cycle dys egula ion is a hallma k o cance , we aimed o analyze whe he
URB447 may dis up he cell cycle and, he e o e, educe umo cell iabili y, as p e i-
ously obse ed. We epo ed ha URB447 sligh ly impai s he B16 melanoma cell cycle
h ough G0/G1 phase a es . In de ail, he pe cen age o cells in G0/G1 phase inc eased 9%
when ea ed wi h 10 µM URB447 o 24 h compa ed o he con ol cells. Howe e , 25 µM
and 50 µM blocked he cell cycle in G0/G1 phase. Rega ding o MCA38, no changes we e
de ec ed a e 24 h o ea men wi h 10 µM URB447. Howe e , 25 µM and 50 µM signi i-
can ly inc eased G0/G1 phase a es . Bo h cell lines exhibi ed educed cell numbe s in S
phase unde 50 µM URB447 ea men , which u he impai s he cell cycle. (Figu e 4).
Figu e 4. Cell cycle analysis in cells ea ed wi h URB447. Melanoma (A) and CRC cance cells (B)
we e incuba ed in he p esence o di e en concen a ions o URB447 o 24 h. P opidium iodide
RNAse Ki was used o low cy ome y and he numbe o cells in each cell cycle phase we e quan-
i ied (n = 3). Di e ences we e conside ed s a is ically signi ican o * p < 0.05 using S uden es .
2.4. Tumo Cell Mig a ion Is Comp omised by URB447
To inc ease abili y o s ep up om he p ima y lesion and mig a e o gene a e dis an
me as asis is a equi ed s ep o disease p og ession. To u he explo e he an i umo ac-
ion o URB447, we ca ied ou a wound healing assay using di e en URB447 concen a-
ions. As obse ed in Figu e 5, he highes es ed concen a ion, 50 µM, led o a p o-
nounced impai men o cell mig a ion in bo h models. In de ail, URB447 educed 50% he
mig a ion abili y in melanoma and almos 60% in colon cance cells. Mo eo e , 25 µM
exe ed an an i-mig a o y e ec in bo h cell ypes, dec easing he mig a ion po en ial by
Figu e 3.
Apop o ic e ec o URB447. Melanoma (
A
) and CRC cance cells (
B
) we e incuba ed in he
p esence o URB447 o 24 h. Flow cy ome y was ca ied ou o analyze ea ly and la e apop o ic cells.
Images show ep esen a i e FACS esul (n= 3). Di e ences we e conside ed s a is ically signi ican
o * p< 0.05, ** p< 0.01 using S uden es .
2.3. Cell Cycle In e e ence upon URB447 T ea men
Since cell cycle dys egula ion is a hallma k o cance , we aimed o analyze whe he
URB447 may dis up he cell cycle and, he e o e, educe umo cell iabili y, as p e iously
obse ed. We epo ed ha URB447 sligh ly impai s he B16 melanoma cell cycle h ough
G0/G1 phase a es . In de ail, he pe cen age o cells in G0/G1 phase inc eased 9% when
ea ed wi h 10
µ
M URB447 o 24 h compa ed o he con ol cells. Howe e , 25
µ
M
and
50 µM
blocked he cell cycle in G0/G1 phase. Rega ding o MCA38, no changes
we e de ec ed a e 24 h o ea men wi h 10
µ
M URB447. Howe e , 25
µ
M and 50
µ
M
signi ican ly inc eased G0/G1 phase a es . Bo h cell lines exhibi ed educed cell numbe s
in S phase unde 50
µ
M URB447 ea men , which u he impai s he cell cycle. (Figu e 4).
Pha maceu icals 2022, 15, x FOR PEER REVIEW 4 o 11
Figu e 3. Apop o ic e ec o URB447. Melanoma (A) and CRC cance cells (B) we e incuba ed in he
p esence o URB447 o 24 h. Flow cy ome y was ca ied ou o analyze ea ly and la e apop o ic
cells. Images show ep esen a i e FACS esul (n = 3). Di e ences we e conside ed s a is ically sig-
ni ican o * p < 0.05, ** p < 0.01 using S uden es .
2.3. Cell Cycle In e e ence upon URB447 T ea men
Since cell cycle dys egula ion is a hallma k o cance , we aimed o analyze whe he
URB447 may dis up he cell cycle and, he e o e, educe umo cell iabili y, as p e i-
ously obse ed. We epo ed ha URB447 sligh ly impai s he B16 melanoma cell cycle
h ough G0/G1 phase a es . In de ail, he pe cen age o cells in G0/G1 phase inc eased 9%
when ea ed wi h 10 µM URB447 o 24 h compa ed o he con ol cells. Howe e , 25 µM
and 50 µM blocked he cell cycle in G0/G1 phase. Rega ding o MCA38, no changes we e
de ec ed a e 24 h o ea men wi h 10 µM URB447. Howe e , 25 µM and 50 µM signi i-
can ly inc eased G0/G1 phase a es . Bo h cell lines exhibi ed educed cell numbe s in S
phase unde 50 µM URB447 ea men , which u he impai s he cell cycle. (Figu e 4).
Figu e 4. Cell cycle analysis in cells ea ed wi h URB447. Melanoma (A) and CRC cance cells (B)
we e incuba ed in he p esence o di e en concen a ions o URB447 o 24 h. P opidium iodide
RNAse Ki was used o low cy ome y and he numbe o cells in each cell cycle phase we e quan-
i ied (n = 3). Di e ences we e conside ed s a is ically signi ican o * p < 0.05 using S uden es .
2.4. Tumo Cell Mig a ion Is Comp omised by URB447
To inc ease abili y o s ep up om he p ima y lesion and mig a e o gene a e dis an
me as asis is a equi ed s ep o disease p og ession. To u he explo e he an i umo ac-
ion o URB447, we ca ied ou a wound healing assay using di e en URB447 concen a-
ions. As obse ed in Figu e 5, he highes es ed concen a ion, 50 µM, led o a p o-
nounced impai men o cell mig a ion in bo h models. In de ail, URB447 educed 50% he
mig a ion abili y in melanoma and almos 60% in colon cance cells. Mo eo e , 25 µM
exe ed an an i-mig a o y e ec in bo h cell ypes, dec easing he mig a ion po en ial by
Figu e 4.
Cell cycle analysis in cells ea ed wi h URB447. Melanoma (
A
) and CRC cance cells (
B
) we e
incuba ed in he p esence o di e en concen a ions o URB447 o 24 h. P opidium iodide RNAse Ki
was used o low cy ome y and he numbe o cells in each cell cycle phase we e quan i ied (n= 3).
Di e ences we e conside ed s a is ically signi ican o * p< 0.05 using S uden es .
2.4. Tumo Cell Mig a ion Is Comp omised by URB447
To inc ease abili y o s ep up om he p ima y lesion and mig a e o gene a e dis an
me as asis is a equi ed s ep o disease p og ession. To u he explo e he an i umo ac ion
o URB447, we ca ied ou a wound healing assay using di e en URB447 concen a ions.
As obse ed in Figu e 5, he highes es ed concen a ion, 50
µ
M, led o a p onounced
impai men o cell mig a ion in bo h models. In de ail, URB447 educed 50% he mig a ion
abili y in melanoma and almos 60% in colon cance cells. Mo eo e , 25
µ
M exe ed an
an i-mig a o y e ec in bo h cell ypes, dec easing he mig a ion po en ial by 30%. Finally,
Pha maceu icals 2022,15, 1166 5 o 11
al hough a educ ion was obse ed, 10
µ
M did no signi ican ly a ec cell mig a ion a e
24 h in melanoma, bu i educed ha o CRC in 20%.
Pha maceu icals 2022, 15, x FOR PEER REVIEW 5 o 11
30%. Finally, al hough a educ ion was obse ed, 10 µM did no signi ican ly a ec cell
mig a ion a e 24 h in melanoma, bu i educed ha o CRC in 20%.
Figu e 5. The mig a o y abili y o umo cells upon URB447 ea men . Wound healing assay was
ca ied ou upon s imula ion o melanoma (A) and CRC cance cells (B) in he p esence o inc easing
concen a ions o URB447 o 24 h. The closed wound a ea was calcula ed. Images show ep esen a-
i e esul (n = 3). Di e ences we e conside ed s a is ically signi ican o * p < 0.05, ** p < 0.01 using
S uden es .
2.5. Ma ix Deg ading MMP-2 and MMP-9 Sec e ion Is No Al e ed by URB447
One o he main ea u es o me as a ic cance cell is he abili y o deg ade he ECM
p esen in bo h he p ima y issue and he seconda y o gan upon me as a ic coloniza ion.
He e, we show ha e en hough URB447 sligh ly in luences he sec e ion o MMP-2 and
MMP-9 in bo h umo models, he e is no signi ican change a e 24 h. Al hough a educ-
ion end can be obse ed, URB447 did no dis u b he sec e ion o ma ix deg ading
p o eins (Figu e 6).
Figu e 5.
The mig a o y abili y o umo cells upon URB447 ea men . Wound healing assay
was ca ied ou upon s imula ion o melanoma (
A
) and CRC cance cells (
B
) in he p esence o
inc easing concen a ions o URB447 o 24 h. The closed wound a ea was calcula ed. Images
show ep esen a i e esul (n= 3). Di e ences we e conside ed s a is ically signi ican o * p< 0.05,
** p< 0.01 using S uden es .
2.5. Ma ix Deg ading MMP-2 and MMP-9 Sec e ion Is No Al e ed by URB447
One o he main ea u es o me as a ic cance cell is he abili y o deg ade he ECM
p esen in bo h he p ima y issue and he seconda y o gan upon me as a ic coloniza ion.
He e, we show ha e en hough URB447 sligh ly in luences he sec e ion o MMP-2
and MMP-9 in bo h umo models, he e is no signi ican change a e 24 h. Al hough a
educ ion end can be obse ed, URB447 did no dis u b he sec e ion o ma ix deg ading
p o eins (Figu e 6).
2.6. Daily T ea men wi h URB447 Reduced he Me as a ic Bu den in he Li e
The ob ained esul s may unco e a bene icial e ec o URB447 du ing li e coloniza-
ion and umo g ow h. Li e me as asis o ho opic model e ealed ha daily ea men o
umo bea ing mice wi h URB447 led o educed umo bu den in he li e in a melanoma
model. In de ail, he me as a ic a ea was educed in 25% in melanoma compa ed o ehicle
ea ed g oup, a e a daily i.p. injec ion o 1 mg/kg URB447 (Figu e 7). Mo eo e , he
numbe o isible melanoma oci in he URB447 ea ed g oup was educed, al hough he
di e ences we e no s a is ically signi ican .
Pha maceu icals 2022,15, 1166 6 o 11
Pha maceu icals 2022, 15, x FOR PEER REVIEW 6 o 11
(A) (B)
Figu e 6. Sec e ion o MMP-2 and MMP-9 in URB447 ea ed umo cells. The sec e ion o MMP-2
was measu ed in melanoma (A) and CRC cance cells (B) in he p esence o inc easing concen a-
ions o URB447 o 24 h. (n = 3). No signi ican di e ences we e obse ed.
2.6. Daily T ea men wi h URB447 Reduced he Me as a ic Bu den in he Li e
The ob ained esul s may unco e a bene icial e ec o URB447 du ing li e coloni-
za ion and umo g ow h. Li e me as asis o ho opic model e ealed ha daily ea men
o umo bea ing mice wi h URB447 led o educed umo bu den in he li e in a mela-
noma model. In de ail, he me as a ic a ea was educed in 25% in melanoma compa ed o
ehicle ea ed g oup, a e a daily i.p. injec ion o 1 mg/kg URB447 (Figu e 7). Mo eo e ,
he numbe o isible melanoma oci in he URB447 ea ed g oup was educed, al hough
he di e ences we e no s a is ically signi ican .
Figu e 6.
Sec e ion o MMP-2 and MMP-9 in URB447 ea ed umo cells. The sec e ion o MMP-2
was measu ed in melanoma (
A
) and CRC cance cells (
B
) in he p esence o inc easing concen a ions
o URB447 o 24 h. (n= 3). No signi ican di e ences we e obse ed.
Pha maceu icals 2022, 15, x FOR PEER REVIEW 7 o 11
Figu e 7. Me as a ic g ow h o melanoma in con ol and URB447 ea ed mice li e . Mice we e in-
asplenically injec ed wi h 2 × 10
5
B16-F10 melanoma cells and ea ed wi h Vehicle o URB447 1
mg/kg o 10 days. The me as a ic a ea and isible oci numbe we e quan i ied. (n = 4). Di e ences
we e conside ed s a is ically signi ican o * p < 0.05 using S uden es .
3. Discussion
Cannabinoid ecep o s a e gaining ele ance no only in ne ous sys em linked pa-
hologies, bu also in a wide a ay o pa hologies wi h di e en o igins [22–24]. Among
o he s, hei ole du ing cance p og ession has been unco e ed, poin ing ou canna-
binoids as a po en ial a ge o disease managemen . He e, we show ha a CB2 ecep o
agonis syn he ic cannabinoid wi h no psychoac i e e ec s, URB447, educes se e al
p ome as a ic p ope ies o cance cells. Finally, daily ea men wi h URB447 a ec s u-
mo ea u es o melanoma, leading o educed me as a ic g ow h in he li e .
We ha e epo ed a dose-dependen educ ion in melanoma and CRC cell iabili y
when exposed o URB447. These indings a e in line wi h ecen s udies using a new CB2
agonis epo ing he same end [25]. We obse ed ha his dec eased cell iabili y is
pa ly media ed by umo cell apop osis. P e ious s udies om o he g oups ha e linked
CB2 ecep o agonis s wi h his phenomenon, poin ing ou o Caspase 3 and 7 inc ease as
esponsible o he obse ed pheno ype, along wi h PARP exp ession s imula ion [26],
which may accoun o he epo ed obse a ions. Mo eo e , CB2 s imula ion leads o
an iapop o ic p o ein Bcl-2 educ ion [27], he e o e, acili a ing cell dea h. A second
mechanism ha seems o be in ol ed in cance cell educed iabili y. URB447 ea men
al e ed cell cycle in bo h models, leading o G0/G1 cell cycle a es , accompanied wi h
dec ease in S phase cell coun s. This inding goes along a ecen s udy epo ing G1 phase
a es in CB2 s imula ed CRC cance cells. Fu he mo e, hey ound educed le els o cy-
clins in CB2 s imula ed colo ec al CRC cells [26].Mo eo e , CDK4 exp ession was also
educed when agonizing a CB2 ecep o in a glioblas oma cance model [28], which may
explain he esul s ob ained using URB447.
The agg essi eness o cance cells inc eases when hey unde go EMT and, he e o e,
acqui e inc eased mo ili y o lea e he p ima y lesion and colonize dis an o gans. In e -
es ingly, URB447 impai ed cance cell mig a ion in a dose-dependen end. Thus, he in-
ol emen o CB2 ecep o in he egula ion o umo cell mig a ion needs u he a en-
ion. In his ega d, he pa ial CB2 ecep o agonis , CBD, showed an i-mig a o y e ec s
on panc ea ic cance [29], in line wi h ou obse a ions. In e es ingly, CBD blocked epi-
helial g ow h ac o media ed lung cance cell mig a ion in i o con olling he le els o
EMT genes, such as imen in [30], suppo ing ou obse a ions. Mo eo e , he CB2 ecep-
o agonis educed MMP-9 sec e ion in dend i ic cells [31], impai ing hei mig a ion,
which may also occu in cance cells. Rega ding he exp ession o MMPs, URB447 did no
al e he sec e ion o me allop o eases a e 24 h incuba ion. Howe e , se um s a a ion
Figu e 7.
Me as a ic g ow h o melanoma in con ol and URB447 ea ed mice li e . Mice we e
in asplenically injec ed wi h 2
×
10
5
B16-F10 melanoma cells and ea ed wi h Vehicle o URB447
1 mg/kg
o 10 days. The me as a ic a ea and isible oci numbe we e quan i ied. (n= 4). Di e ences
we e conside ed s a is ically signi ican o * p< 0.05 using S uden es .
Pha maceu icals 2022,15, 1166 7 o 11
3. Discussion
Cannabinoid ecep o s a e gaining ele ance no only in ne ous sys em linked
pa hologies, bu also in a wide a ay o pa hologies wi h di e en o igins [
22
–
24
]. Among
o he s, hei ole du ing cance p og ession has been unco e ed, poin ing ou cannabinoids
as a po en ial a ge o disease managemen . He e, we show ha a CB2 ecep o agonis
syn he ic cannabinoid wi h no psychoac i e e ec s, URB447, educes se e al p ome as a ic
p ope ies o cance cells. Finally, daily ea men wi h URB447 a ec s umo ea u es o
melanoma, leading o educed me as a ic g ow h in he li e .
We ha e epo ed a dose-dependen educ ion in melanoma and CRC cell iabili y
when exposed o URB447. These indings a e in line wi h ecen s udies using a new CB2
agonis epo ing he same end [
25
]. We obse ed ha his dec eased cell iabili y is
pa ly media ed by umo cell apop osis. P e ious s udies om o he g oups ha e linked
CB2 ecep o agonis s wi h his phenomenon, poin ing ou o Caspase 3 and 7 inc ease
as esponsible o he obse ed pheno ype, along wi h PARP exp ession s imula ion [
26
],
which may accoun o he epo ed obse a ions. Mo eo e , CB2 s imula ion leads o
an iapop o ic p o ein Bcl-2 educ ion [
27
], he e o e, acili a ing cell dea h. A second
mechanism ha seems o be in ol ed in cance cell educed iabili y. URB447 ea men
al e ed cell cycle in bo h models, leading o G0/G1 cell cycle a es , accompanied wi h
dec ease in S phase cell coun s. This inding goes along a ecen s udy epo ing G1 phase
a es in CB2 s imula ed CRC cance cells. Fu he mo e, hey ound educed le els o
cyclins in CB2 s imula ed colo ec al CRC cells [
26
].Mo eo e , CDK4 exp ession was also
educed when agonizing a CB2 ecep o in a glioblas oma cance model [
28
], which may
explain he esul s ob ained using URB447.
The agg essi eness o cance cells inc eases when hey unde go EMT and, he e o e,
acqui e inc eased mo ili y o lea e he p ima y lesion and colonize dis an o gans. In e -
es ingly, URB447 impai ed cance cell mig a ion in a dose-dependen end. Thus, he
in ol emen o CB2 ecep o in he egula ion o umo cell mig a ion needs u he a en-
ion. In his ega d, he pa ial CB2 ecep o agonis , CBD, showed an i-mig a o y e ec s on
panc ea ic cance [
29
], in line wi h ou obse a ions. In e es ingly, CBD blocked epi helial
g ow h ac o media ed lung cance cell mig a ion
in i o
con olling he le els o EMT
genes, such as imen in [
30
], suppo ing ou obse a ions. Mo eo e , he CB2 ecep o
agonis educed MMP-9 sec e ion in dend i ic cells [31], impai ing hei mig a ion, which
may also occu in cance cells. Rega ding he exp ession o MMPs, URB447 did no al e he
sec e ion o me allop o eases a e 24 h incuba ion. Howe e , se um s a a ion o umo
cells could help elucida e he e ec o URB447 in MMP sec e ion, since 1% se um exhibi ed
MMP ac i i y, which may unco e he di e ences gene a ed by URB447.
In i o
, URB447 educed he me as a ic bu den in a melanoma model o li e me as a-
sis. In de ail, daily ea men wi h 1 mg/kg URB447 impai ed me as a ic g ow h in he li e
in 25% in melanoma. The epo ed educ ion in cell iabili y and cell cycle a es migh
pa ially media e he obse ed e ec
in i o
. Mo eo e , p oapop o ic ac ion o URB447
migh help educe li e me as a ic a ea. Apa om he di ec e ec o URB447 in umo
cells, a po en ial ac ion in he colonized o gan mus be aken in o accoun . In his ega d,
he syn he ic a ypical cannabinoid Abn-CBD, a cannabidiol (CBD) de i a i e is e ec i e in
educing li e ela ed in lamma ion and subsequen li e damage du ing non-alcoholic
a y li e disease [
32
]. In line wi h his epo , a selec i e CB2 agonis p o ec ed he li e
om bile duc liga ion media ed inju y h ough he impai men o he in lamma o y e-
sponse [
33
]. Simila ly, he inhibi ion o he in lamma o y esponse is in ol ed in he CB2
ecep o media ed li e p o ec ion du ing alcohol- ela ed inju y [
34
]. Rega ding he non-
pa enchymal cells in he li e ,
in i o
knock ou o he CB2 ecep o d o e he augmen ed
in lamma o y esponse o hepa ic s ella e cells and inc eased li e damage, wo sening he
CCl4 p omo ed li e ib osis [
35
]. The e o e, URB447 migh educe he in lamma o y and
ib o ic s a us o he li e , impai ing immune supp ession and umo -associa ed collagen
accumula ion, hus, slowing down umo g ow h.
Pha maceu icals 2022,15, 1166 8 o 11
Fu he s udies will un a el he ole o li e non-pa enchymal cells upon URB447 ea -
men and he egula ion o he immune esponse unde he CB2-s imula ed mic oen i onmen .
4. Ma e ials and Me hods
4.1. Animals
C57BL/6J male mice (6–8 weeks old) we e ob ained om Cha les Ri e (Ba celona,
Spain). Ins i u ional guidelines and na ional laws o expe imen al ca e guidelines we e
ollowed o animal housing, ca e and expe imen al condi ions. Animal condi ions we e
ul illed wi h unlimi ed ood and wa e a ailabili y. All he conduc ed
in i o
expe i-
men s we e app o ed by he Basque Coun y Uni e si y E hical Commi ee (CEID) and by
ins i u ional, na ional and in e na ional guidelines o animals use in esea ch ac i i ies.
4.2. Cell Lines and Reagen s
The mouse malignan melanoma B16-F10 and colon ca cinoma MCA38, bo h syngeneic
wi h C57BL/6J mice we e ob ained om he Ame ican Type Cul u e Collec ion (ATCC, LGC
S anda ds SLU). MCA38 cells we e cul u ed in RPMI-1640 medium and B16-F10 cells we e
main ained in DMEM medium. To ge he comple e medium, media we e supplemen ed
wi h hea -inac i a ed 10% e al bo ine se um (FBS), penicillin (100 U/mL), s ep omycin
(100
µ
g/mL) and ampho e icin B (0.25
µ
g/mL). All he eagen s we e pu chased om
The mo Fishe Scien i ic (Wal ham, MA, USA). Cance cell lines we e disca ded a e
en passages and subs i u ed by new ba ches. The syn he ic cannabinoid URB447 was
pu chased om Cayman Chemicals (Ann A bo , MI, USA).
4.3. Cell Viabili y
In o al, 5
×
10
3
/well cance cells we e cul u ed in 96-well pla es and supplemen ed
wi h comple e g ow h medium o 24 h. A e wa ds, di e en concen a ions o URB447
anging om 10
µ
M o 50
µ
M we e added o cance cell cul u es in 1% FBS supplemen ed
medium. Con ol cells we e ea ed wi h 0.1% DMSO ( inal concen a ion) dilu ed in
1% FBS con aining esh medium. T ea men e ec i eness was measu ed a e 24 and
48 h
, incuba ing umo cells o 2 h wi h P es oBlue cell iabili y eagen (The mo Fishe
Scien i ic (Wal ham, MA, USA)) ollowing manu ac u e ’s indica ions.
4.4. Apop o ic Cell De e mina ion
Apop osis analysis was ca ied ou by cul u ing 3
×
10
5
cance cells in 6-well pla es
o 18 h in comple e medium. The medium was changed and cells we e ea ed wi h
10, 25 and 50
µ
M URB447 in esh medium supplemen ed wi h 1% FBS o 24 h (con ol
cells we e ea ed wi h 0.1% DMSO). The supe na an was collec ed and a ached cells
we e ypsinized and added in o he same ube as loa ing cells. A e washing wo imes
wi h PBS, Dead Cell Apop osis Ki wi h Annexin V FITC and PI (The mo Fishe Scien i ic
(Wal ham, MA, USA)) was used o quan i y he apop osis media ed by URB447, ollowing
manu ac u e ’s ins uc ions. Apop osis was e alua ed by low cy ome y using he Gallios
cy ome e (Beckman Coul e , B ea, CA, USA).
4.5. Cell Cycle Analysis
Fo cell cycle analysis, 3
×
10
5
cance cells we e cul u ed in 6-well pla es o 18 h in
comple e medium. The medium was changed and cells we e ea ed wi h 10, 25 and 50
µ
M
URB447 in esh medium supplemen ed wi h 1% FBS o 24 h (0.1% DMSO was used as
ehicle ea men o con ol cells). A ached cells we e collec ed h ough ypsiniza ion and
subjec ed o phospha e-bu e ed saline (PBS) washes p io o ixa ion using 70% E hanol
o 30 min a 4 deg ees. E hanol was elimina ed om cells h ough PBS washes p io o
cell s aining using p opidium iodide (PI) con aining FxCycle PI/RNase Solu ion (The mo
Fishe Scien i ic (Wal ham, MA, USA)) ollowing he manu ac u e ’s indica ions. Finally,
he URB447 media ed pe u ba ion o cell cycle was e alua ed by low cy ome y using he
Gallios cy ome e (Beckman Coul e , B ea, CA, USA).
Pha maceu icals 2022,15, 1166 9 o 11
4.6. Wound Healing Assay
Cance cells we e cul u ed a 2
×
10
5
cells/well in 24-well pla es in comple e medium
o 24 h. B16-F10 and MCA38 cells we e incuba ed wi h 25
µ
g/mL and 1
µ
g/mL My omicin
C, espec i ely (Fishe Scien i ic, Mad id, Spain) o 2 h. Then, a sc a ch was made using he
200
µ
L ip, ollowed by h ee washes o elimina e de ached cells. Finally, con ol cells we e
incuba ed wi h 0.5% DMSO whe eas ea ed cells we e incuba ed wi h 50, 25 and
10 µM
URB447 in 1% FBS supplemen ed medium. Pic u es we e aken a he ime o ea men
addi ion (T0) and a e 24 h (T 24 h). The o al wound a ea was compa ed be ween ha o
T0 and T24. Resul s a e shown as he pe cen al o wound a ea closed.
4.7. Zymog aphy
The sec e ion o MMPs was de e mined by gela in zymog aphy as p e iously de-
sc ibed. A o al 2
×
10
5
cells /mL we e cul u ed o 24 h in comple e medium supple-
men ed wi h 10% FBS. Then, he medium was changed o 1% FBS con aining medium
and ea men s we e added o 24 h. Finally, con ol and URB447- ea ed cell supe na an s
we e collec ed and cen i uged o 5 min a 4000 pm. Supe na an s we e un in 1% gela in
con aining 10% bis-ac ylamide gels. Fo gela in diges ion, gels we e incuba ed o e nigh
in de eloping bu e ollowed by s aining in Coomassie Blue solu ion (BioRad, He cules,
CA, USA). Diges ed bands we e quan i ied using Image J2 so wa e (Na ional Ins i u es o
Heal h, Be hesda, MD, USA).
4.8. In Vi o Li e Me as asis Assay
C57BL/6J male mice (6–8 weeks old) we e anes he ized using Xilazyn/Ke amin so-
lu ion. A hin cu was made in he le lank unde he ibs o expose he spleen. Then,
2
×
10
5
B16-F10 cells we e injec ed in he dis al pole o he spleen o each mouse dilu ed in
100
µ
L PBS (2
×
10
6
cels/mL concen a ion). The spleen was ca e ully eloca ed and he
wound was closed. Mice we e ea ed wi h in ape i oneal injec ions o 1 mg/kg URB447
(200
µ
L/mouse) om day 1 o day 10 and sac i iced 14 days a e umo cell inocula ion
(3% URB447 in DMSO, 97% PBS). Con ol animals we e ea ed wi h ehicle solu ion
(3% DMSO in PBS). All he p ocedu es in ol ing animals we e app o ed by he Uni e si y
o he Basque Coun y and he Basque Go e nmen E hics commi ee (e hical app o al
code M20_2020_315).
Au ho Con ibu ions:
Concep ualiza ion, A.B. and D.A.-A.; me hodology, A.B. and B.A.; o mal
analysis, A.B. and B.A.; in es iga ion, A.B. and B.A.; esou ces, A.B., B.A. and D.A.-A.; da a cu a ion,
A.B. and B.A.; w i ing—o iginal d a p epa a ion, A.B.; w i ing— e iew and edi ing, B.A., A.D. and
D.A.-A.; supe ision, D.A.-A.; p ojec adminis a ion, A.B. and D.A.-A.; unding acquisi ion, A.D.
and D.A.-A. All au ho s ha e ead and ag eed o he published e sion o he manusc ip .
Funding:
This esea ch was unded by EITB Ma a oia-BIOEF (BIO18/IC/003), and he Spanish
Minis y o Science and Inno a ion (MINECOR20/P66/AEI/10.13039/501100011033).
Ins i u ional Re iew Boa d S a emen :
The animal s udy p o ocol was app o ed by he E hics
Commi ee o The Uni e si y o he Basque Coun y and he Basque Go e nmen (M20_2020_315).
In o med Consen S a emen : No applicable.
Da a A ailabili y S a emen : Da a is con ained wi hin he a icle.
Acknowledgmen s:
The au ho s hank he his ological sample p epa a ion ca ied ou by C is ina
Tobillas om he Cell Biology and His ology Depa men .
Con lic s o In e es : The au ho s decla e no con lic o in e es .
