cance s
A icle
Dual Ta ge ing o BRAF and mTOR Signaling in
Melanoma Cells wi h Py idinyl
Imidazole Compounds
Ve onika Palušo á1,2, Te eza Renzo á1, Amandine Ve lande 1, Te eza Vaclo á1,
Michaela Medko á1, Linda Ce lo á1, Mi osla a Sedláˇcko á1, Hana Hˇ íbko á1,
I a Slanino á1, Mi iama K u á1, Vladimí Ro ekl 1,2 , Hana Uhlíˇ o á3,4 , Ane a Kˇ ížo á4,
Radim Chmelík3,4 , Pa el Veselý4, Michaela K a ˇcíko á5, Lukáš T an í ek 6,
Kay Oli e Schink 7,8 and S jepan Uld ijan 1,2,*
1Facul y o Medicine, Masa yk Uni e si y, Kamenice 753/5, 625 00 B no, Czech Republic;
e [email p o ec ed] (V.P.); [email p o ec ed] (T.R.); [email p o ec ed] (A.V.);
[email p o ec ed] (T.V.); [email p o ec ed] (M.M.); [email p o ec ed] (L.C.);
[email p o ec ed] (M.S.); [email p o ec ed] (H.H.); [email p o ec ed] (I.S.);
[email p o ec ed] (M.K.); [email p o ec ed] (V.R.)
2In e na ional Clinical Resea ch Cen e , S . Anne’s Uni e si y Hospi al B no, Pekaˇ ská664/53, 656 91 B no,
Czech Republic
3Ins i u e o Physical Enginee ing, Facul y o Mechanical Enginee ing, B no Uni e si y o Technology,
Technická2896/2, 616 69 B no, Czech Republic; [email p o ec ed].cz (H.U.);
[email p o ec ed].cz (R.C.)
4CEITEC—Cen al Eu opean Ins i u e o Technology, B no Uni e si y o Technology, Pu kyˇno a 656/123,
612 00 B no, Czech Republic; [email p o ec ed].cz (A.K.); [email p o ec ed].cz (P.V.)
5Na ional Cen e o Biomolecula Resea ch, Masa yk Uni e si y, Kamenice 753/5, 625 00 B no,
Czech Republic; [email p o ec ed]
6CEITEC—Cen al Eu opean Ins i u e o Technology, Masa yk Uni e si y, Kamenice 753/5, 625 00 B no,
Czech Republic; [email p o ec ed]
7Cen e o Cance Cell Rep og amming, Facul y o Medicine, Uni e si y o Oslo, Mon ebello, N-0379 Oslo,
No way; Kay.Oli e .Schink@ - esea ch.no
8
Depa men o Molecula Cell Biology, Ins i u e o Cance Resea ch, Oslo Uni e si y Hospi al, Mon ebello,
N-0379 Oslo, No way
*Co espondence: [email p o ec ed]
Recei ed: 29 Feb ua y 2020; Accep ed: 5 June 2020; Published: 10 June 2020


Abs ac :
BRAF inhibi o s can delay he p og ession o me as a ic melanoma, bu esis ance usually
eme ges, leading o elapse. D ugs simul aneously a ge ing wo o mo e pa hways essen ial o cance
g ow h could slow o p e en he de elopmen o esis an clones. He e, we iden i ied py idinyl
imidazole compounds SB202190, SB203580, and SB590885 as dual inhibi o s o c i ical p oli e a i e
pa hways in human melanoma cells bea ing he V600E ac i a ing mu a ion o BRAF kinase. We ound
ha he d ugs simul aneously dis up he BRAF V600E-d i en ex acellula signal- egula ed kinase
(ERK) mi ogen-ac i a ed p o ein kinase (MAPK) ac i i y and he mechanis ic a ge o apamycin
complex 1 (mTORC1) signaling in melanoma cells. Py idinyl imidazole compounds di ec ly inhibi
BRAF V600E kinase. Mo eo e , hey in e e e wi h he endolysosomal compa men , p omo ing
he accumula ion o la ge acidic acuole-like esicles and dynamic changes in mTOR signaling.
A ansien inc ease in mTORC1 ac i i y is ollowed by he en ichmen o he Ragula o complex
p o ein p18/LAMTOR1 a con ac si eso la ge esiclesand delocaliza ion o mTOR om helysosomes.
The induced dis up ion o he endolysosomal pa hway no only dis up s mTORC1 signaling, bu
also ende s melanoma cells sensi i e o endoplasmic e iculum (ER) s ess. Ou indings iden i y
new ac i i ies o pha macologically ele an small molecule compounds and p o ide a biological
a ionale o he de elopmen o an i-melanoma he apeu ics based on he py idinyl imidazole co e.
Cance s 2020,12, 1516; doi:10.3390/cance s12061516 www.mdpi.com/jou nal/cance s
Cance s 2020,12, 1516 2 o 24
Keywo ds:
melanoma; BRAF V600E; BRAF inhibi o ; small molecule d ug; py idinyl imidazole;
endosome; lysosome; mTORC1; ER s ess
1. In oduc ion
Malignan melanoma is agg essi e cance a ec ing he skin and o he issues whe e
pigmen -p oducing melanocy es eside. Al hough melanoma accoun s o less han 5% o all
de ma ologic umo s, i is esponsible o app oxima ely 80% o all dea hs om skin cance s [
1
].
The main isk ac o o he de elopmen o melanoma is o e exposu e o sola UV adia ion. The UV
ligh can induce cance -p omo ing mu a ions ha can s imula e melanocy e g ow h independen ly
om ex e nal s imuli [
2
,
3
]. Mos human melanomas can be placed in o wo subg oups based on he
ype o mu a ion d i ing he ERK MAPK signaling pa hway and cell p oli e a ion [4,5]. Abou a hal
o melanoma pa ien s ca y a po en ac i a ing mu a ion V600E in he BRAF kinase, which ende s he
no mally dime ic BRAF kinase enzyma ically ac i e as a monome , independen o ups eam signaling.
The second-la ges g oup o melanoma pa ien s bea s ac i a ing mu a ions in he NRAS p o ein.
The c ucial ole o BRAF-d i en oncogenic ERK signaling in melanoma s imula ed he p eclinical
and clinical de elopmen o a la ge numbe o s uc u ally di e en RAF inhibi o s [
6
]. Ta ge ing
mu an BRAF can lead o a s ong esponse in melanoma pa ien s. S ill, un o una ely, he cance s
commonly acqui e o he p o-su i al mu a ions compensa ing o he deple ion o BRAF ac i i y [
7
]
and causing esis ance o ea men [
8
]. In BRAF inhibi o - esis an melanoma cells, a combina ion o
MEK inhibi o s wi h phospha idylinosi ol 3-kinase (PI3K) o mechanis ic a ge o apamycin (mTOR)
inhibi o s can signi ican ly dec ease cance cell su i al [
9
,
10
]. Fu he mo e, in mouse models o
BRAF-mu a ed melanoma, inhibi ion o PI3K coope a ed wi h ERK pa hway inhibi ion o o es all he
onse o MAPK pa hway inhibi o esis ance [11].
Ac i a ion o ecep o y osine kinases by g ow h ac o s s imula es bo h MAPK and
PI3K/AKT/mTOR signaling pa hways o coo dina e cell g ow h, p oli e a ion, and su i al [
12
].
The mTOR se ine/ h eonine p o ein kinase is a co e componen o wo unc ionally dis inc p o ein
complexes—mTOR complex 1 (mTORC1) and 2 (mTORC2). Complex 1 balances he anabolic and
ca abolic p ocesses in esponse o g ow h ac o s wi h ATP, oxygen, and nu ien a ailabili y. When
ac i e, mTORC1 s imula es p o eosyn hesis and inhibi s ca abolic p ocesses such as au ophagy [
13
].
Small GTPases Rheb (Ras homolog en iched in b ain) and Rag media e he ac i a ion o mTORC1 [
14
].
To ac i a e he mTORC1 complex, Rheb mus be in a GTP-bound s a e and p oximi y o mTORC1 [
15
].
Insu icien g ow h ac o signaling, low glucose condi ions, o low oxygen condi ions p omo e he
dissocia ion o he TSC1/2 p o ein complex. Released TSC2 causes a swi ch o Rheb in o i s inac i e
GDP-bound s a e, p e en ing he ac i a ion o mTORC1 [
16
,
17
]. In e es ingly, TSC2 ansloca ion o
he lysosome appea s o be a uni e sal cellula esponse o s ess s imuli [18].
C i ical ac o s enabling he mTOR complex 1 ancho ing o endomemb anes a e he Rag amily o
small GTPases and a sca old p o ein complex called Ragula o [
19
,
20
]. Fou ela ed Rag GTPases, RagA,
RagB, RagC, and RagD, a e exp essed in mammalian cells. They o m RagA/B–RagC/D he e odime s,
in which RagA o RagB physically in e ac wi h RagC o RagD [
20
,
21
]. The ec ui men o mTORC1 o
he lysosome depends on he nucleo ide-bound s a e o Rags, which o m a docking si e o Rap o , an
essen ial subuni o mTORC1 [
22
]. In amino acid-s a ed condi ions, RagA/B a e bound o GDP, which
is apidly exchanged wi h GTP when amino acid le els a e es o ed [
20
,
23
]. The sensing o amino
acid le els akes place in he lysosomal lumen, and indi idual amino acids ha e hei speci ic senso s,
which ac ups eam o Rag GTPases [24,25].
The PI3K/AKT/mTOR and RAF/MEK/ERK signaling cascades o m nume ous eedback loops and
in e connec a mul iple poin s o c oss alk. Inhibi ion o one pa hway can be pa ially compensa ed
by he enhanced ac i i y o he o he , implying ha dual a ge ing o bo h pa hways may imp o e
ea men e icacy and lead o be e clinical ou comes [
26
]. In he cu en s udy, we show ha
Cance s 2020,12, 1516 3 o 24
py idinyl imidazole compounds a e capable o simul aneously a ge ing he BRAF oncogene and
mTORC1 signaling in human melanoma cells. They di ec ly inhibi he BRAF kinase ac i i y and, a
he same ime, in e e e wi h he endolysosomal compa men , leading o a loss o mTORC1 lysosomal
localiza ion and ac i i y. The py idinyl imidazole compounds supp ess he g ow h and p oli e a ion
o melanoma cells and sensi ize hem o addi ional s ess s imuli. These indings p o ide a biological
a ionale o u he de elopmen o py idinyl imidazole an i-melanoma d ugs.
2. Resul s
2.1. Py idinyl Imidazole p38 MAPK Inhibi o s Dis up BRAF V600E-D i en ERK Signaling in Human
Melanoma Cells
Py idinyl imidazole inhibi o s SB202190 and SB203580 ha e been widely used in biomedical
esea ch as selec i e chemical p obes o p38 MAPK biological ac i i y, despi e epo s sugges ing
addi ional kinase a ge s and cell ype-speci ic p38-independen e ec s o hese compounds [
27
–
30
].
We analyzed he biological ac i i y o SB202190 in A375 melanoma cells and ound ha SB202190,
bu no a s uc u ally dis inc p38 inhibi o SB239063, s ongly a ec ed he g ow h o melanoma cell
cul u es, sugges ing a p38-independen an ip oli e a i e e ec o he py idinyl imidazole compound
in human melanoma cells (Figu e 1A).
Themos commond i e so melanomap oli e a iona eNRASandBRAFmu a ions, cons i u i ely
ac i a ing he ERK MAPK pa hway in abou 80% o umo s [
4
,
5
]. In e es ingly, some epo s
sugges ed ha py idinyl imidazole compounds could ac i a e ERK signaling by p omo ing CRAF
(RAF-1) ac i i y [
31
–
33
]. A small-molecule lib a y sc een using bioluminescence esonance ene gy
ans e -based biosenso s iden i ied SB202190 and SB203580 as po en ac i a o s o RAF dime iza ion,
which migh explain he epo ed ERK pa hway ac i a ion in esponse o SB203580 [
34
]. We, he e o e,
es ed he possibili y ha he py idinyl imidazole p38 inhibi o s could di ec ly modula e RAF kinase
ac i i y and ERK signaling in melanoma cells. We analyzed ERK-dependen ansc ip ion in A375
cells, bea ing he mos common ac i a ing mu a ion o BRAF kinase (V600E), s ably ans ec ed wi h
a ecen ly de eloped ERK ac i i y luci e ase epo e cons uc [
35
]. Su p isingly, we ound ha
SB202190 s ongly inhibi ed ERK-d i en luci e ase ac i i y in his sys em, as po en ly as MEK kinase
inhibi o s U0126 and PD184352 ha we e used as posi i e con ols (Figu e 1B).
Nex , we ea ed A375 cells wi h inc easing concen a ions o SB202190 o SB203580 and analyzed
ERK pa hway ac i i y by Wes e n blo ing, using MEK and ERK phospho-speci ic an ibodies. Speci ic
MEK inhibi o PD184352 se ed as a posi i e con ol. Bo h py idinyl imidazole compounds induced a
dose-dependen dec ease in he le els o ac i e ERK and MEK kinases (Figu e 1C). The expe imen
was epea ed h ee imes (addi ional Wes e n blo s a e a ailable in Figu e S1), and we de e mined
he ela i e P-MEK/MEK and P-ERK/ERK a ios be ween phospho yla ed (ac i e) and o al MEK and
ERK kinase le els. The esul s p esen ed in Figu e S2 indica e ha bo h compounds could inhibi ERK
pa hway ac i i y in A375 cells, bu SB202190 a ec ed he pa hway mo e po en ly han SB203580. The
ac ha bo h MEK and ERK ac i i y was dec eased sugges ed ha he py idinyl imidazole compounds
a ge he ERK signaling pa hway ups eam o MEK kinase.
The inhibi o y e ec o SB202190 on ERK ac i i y was obse ed in human melanoma cell lines
ca ying BRAF V600E mu a ion (A375, G361, Colo-800), bu no in melanoma cells wi h NRAS
mu a ions (MEL-JUSO, SK-MEL-30, IPC-298) (Figu e 1D). This esul indica ed ha py idinyl imidazole
p38 inhibi o s migh ac as inhibi o s o mu an BRAF, bu no wild ype CRAF kinase, which ac i a es
MEK in cells bea ing mu a ed NRAS. Impo an ly, wo s uc u ally un ela ed small-molecule p38
inhibi o s SB239063 and BIRB796 did no a ec ERK ac i i y in melanoma cells (Figu e 1D). The esul s
o wo addi ional independen eplica es o his expe imen a e a ailable in Figu e S1.
Cance s 2020,12, 1516 4 o 24
Cance s 2020, 12, x 4 o 25
(h ps://imagej.ne /Fiji). The esul s p esen ed in Figu e 1E sugges ha SB202190 could inhibi he
ac i i y o endogenous BRAF V600E p o ein immunop ecipi a ed om A375 melanoma cells.
The possibili y ha he p38 MAPK inhibi o s SB202190 and SB203580 migh a ge mu an BRAF
kinase was indi ec ly suppo ed by he ac ha a s uc u ally ela ed py idinyl imidazole de i a i e
SB590885 was de eloped as a BRAF-speci ic inhibi o [36]. When we compa ed in he 3-(4,5-
dime hyl hiazol-2-yl)-2,5-diphenyl e azolium b omide (MTT) assays he e ec o SB202190 and
SB590885 on he p oli e a ion o a panel o melanoma cell lines, as expec ed, we obse ed ha BRAF-
mu a ed melanoma cell lines we e mo e sensi i e o he compounds han NRAS-mu a ed melanoma
cells (Figu e S3). BRAF-inhibi o emu a enib se ed as a posi i e con ol. In e es ingly, highe
concen a ions o SB590885 also nega i ely a ec ed he g ow h o NRAS-mu a ed cell lines,
indica ing he possibili y o addi ional, BRAF-independen , cy o oxic ac i i y o he py idinyl
imidazole compounds in melanoma cells (Figu e S3).
Figu e 1.
Py idinyl imidazole p38 mi ogen-ac i a ed p o ein kinase (MAPK) inhibi o s SB202190 and
SB203580 dis up mu an BRAF kinase ac i i y in human melanoma cells. (
A
) The p oli e a ion o A375
cells was measu ed by low cy ome y du ing 4-day cul i a ion. The numbe o cells was compa ed
be ween con ols (DMSO- ea ed) and cells ea ed wi h SB202190 (SB202; 15
µ
M) and SB239063 (SB239;
15
µ
M). The p esen ed da a (mean +SD) we e ob ained in h ee independen expe imen s. (
B
) Rela i e
ERK ac i i y in A375 cells s ably ans ec ed wi h an ERK ac i i y luci e ase epo e plasmid. Cells
we e ea ed o 24 h wi h SB202190 (10
µ
M) and MEK inhibi o s U0126 (10
µ
M) and PD184352 (PD; 1
µ
M). Th ee independen expe imen s we e pe o med. Resul s a e p esen ed as mean ela i e ERK
ac i i y +SD. (
C
) Le els o phospho yla ed MEK and ERK kinase we e analyzed by Wes e n blo .
A375 cells we e ea ed o one hou wi h inc easing concen a ions o SB202190 (SB202) and SB203580
(SB203). MEK inhibi o PD184352 (PD; 100 nM) was used as a posi i e con ol. The ela i e a io
be ween P-ERK and he o al ERK le els is also indica ed o each sample. (
D
) ERK ac i i y was
analyzed by Wes e n blo in melanoma cell lines bea ing BRAF (A375, G361, COLO-800) o NRAS
(MEL-JUSO, SK-MEL-30, IPC-298) mu a ions. Cells we e ea ed o 24 h wi h he p38 inhibi o s
SB202190 (SB202; 10
µ
M), SB239063 (SB239; 10
µ
M), and BIRB796 (BIRB; 10
µ
M). The ela i e a io
be ween P-ERK and he o al ERK le els is also indica ed o each sample. (
E
)
In i o
BRAF kinase
assay using kinase-dead MEK as a subs a e and endogenous V600E BRAF kinase immunop ecipi a ed
om A375 cells. SB202190 was used a 5
µ
M. Da a we e ob ained in h ee independen expe imen s.
Resul s a e p esen ed as ela i e alues, mean +SD. **** deno es p<0.0001.
Cance s 2020,12, 1516 5 o 24
Nex , we pe o med an
in i o
BRAF kinase ac i i y assay using a ecombinan kinase-dead
MEK p o ein as a subs a e. Th ee independen expe imen s we e pe o med, and he le els o
MEK phospho yla ion we e de e mined by Wes e n blo ing and quan i ied using ImageJ/Fiji (h ps:
//imagej.ne /Fiji). The esul s p esen ed in Figu e 1E sugges ha SB202190 could inhibi he ac i i y o
endogenous BRAF V600E p o ein immunop ecipi a ed om A375 melanoma cells.
The possibili y ha he p38 MAPK inhibi o s SB202190 and SB203580 migh a ge mu an
BRAF kinase was indi ec ly suppo ed by he ac ha a s uc u ally ela ed py idinyl imidazole
de i a i e SB590885 was de eloped as a BRAF-speci ic inhibi o [
36
]. When we compa ed in he
3-(4,5-dime hyl hiazol-2-yl)-2,5-diphenyl e azolium b omide (MTT) assays he e ec o SB202190
and SB590885 on he p oli e a ion o a panel o melanoma cell lines, as expec ed, we obse ed ha
BRAF-mu a ed melanoma cell lines we e mo e sensi i e o he compounds han NRAS-mu a ed
melanoma cells (Figu e S3). BRAF-inhibi o emu a enib se ed as a posi i e con ol. In e es ingly,
highe concen a ions o SB590885 also nega i ely a ec ed he g ow h o NRAS-mu a ed cell lines,
indica ing he possibili y o addi ional, BRAF-independen , cy o oxic ac i i y o he py idinyl imidazole
compounds in melanoma cells (Figu e S3).
2.2. SB202190-Induced Vacuoles in Melanoma Cells Ha e an Endocy ic O igin
Among he e ec s epo ed o he p38 MAPK inhibi o s, SB202190 and SB203580, was he
o ma ion o la ge acuole-like s uc u es. Some epo s linked he pheno ype o he dis up ion o
au ophagy, which was la e shown o be p38-independen [
30
,
37
]. In ou expe imen s, bo h compounds
induced s ong cy oplasmic acuoliza ion in A375 melanoma cells (Figu e 2A). We, he e o e, analyzed
in de ail his pheno ype and i s possible con ibu ion o he g ow h-inhibi o y ac i i y o he py idinyl
imidazole d ugs in melanoma cells. Analysis o indi idual ames o a ime-lapse eco ding om
a phase-con as mic oscope e ealed ha he la ge acuoles induced by SB202190 in melanoma
cells migh o m by he usion o smalle esicles (Figu e S4). Elec on mic oscopy analysis o
SB202190- ea ed A375 cells showed ha he la ge acuoles a e mos ly de oid o dense s uc u es
(Figu e 2B). The low mass densi y o he acuole-like s uc u es was also con i med using quan i a i e
phase imaging (Figu e 2C).
The abo e esul s indica ed he possibili y ha SB202190-induced acuole-like s uc u es could
o m in esponse o a dis up ion o esicula endocy ic anspo , a molecula pa hway ha is
esponsible o he ac i e anspo o memb ane p o eins, including ecep o s egula ing g ow h
signaling, and o he up ake o i al nu ien s om he ex acellula en i onmen [
38
]. Small molecule
luo escen dyes such as ac idine o ange o LysoT acke G een DND-26 s ain acidic compa men s in
li ing cells, including endosomes and lysosomes. Bo h dyes eadily ma ked he SB202190-induced
acuoles in A375 melanoma cells, suppo ing he possibili y o hei endolysosomal o igin (Figu e 3A,B).
Using con ocal luo escence mic oscopy, we obse ed ha only a small p opo ion o SB202190-induced
acuoles wi h a diame e la ge han 1
µ
m exp ess ea ly endosomal ma ke RAB5 (Figu e 3C). In
con as , mos SB202190-induced acuoles wi h a diame e la ge han 1
µ
m exp ess la e endosome
ma ke RAB7 a hei su ace (Figu e 3D), indica ing ha he majo i y o enla ged acuoles o igina ed
om he la e endosome.
Mo eo e , suc ose-induced osmo ic s ess o he small molecule inhibi o 5-(N-E hyl-N-
isop opyl)amilo ide (EIPA), which inhibi s a o m o endocy osis called mac opinocy osis, p e en ed
he o ma ion o la ge acuoles in SB202190- ea ed cells (Figu e 3E). The nega i e impac o EIPA
on RAB7-posi i e acuole-like esicles was also obse ed using luo escence mic oscopy (Figu e S5).
These da a u he suppo ed he endocy ic o igin o he enla ged esicles. Fu he mo e, li e-cell
imaging indica ed ha mos SB202190-induced acuoles could no be ecycled back o he plasma
memb ane, sugges ing a block in la e s ages o endosomal a icking (Video S1).

Cance s 2020,12, 1516 6 o 24
Cance s 2020, 12, x 6 o 25
Figu e 2. Py idinyl imidazole compounds induce acuoliza ion o cy oplasm in BRAF-mu a ed
human melanoma cells. A375 cells we e ea ed wi h DMSO (con ol), SB202190 (SB202; 10 μM), and
SB203580 (SB203; 10 μM). (A) Phase-con as ligh mic oscopy o li ing cells a e 12 h ea men wi h
py idinyl imidazole p38 MAPK inhibi o s. Scale ba : 45 µm. The ep esen a i e g aph shows he mean
numbe o acuoles pe cell quan i ied using ImageJ/Fiji ( ind maxima—b igh spo s abo e a ce ain
h eshold). Dying ounded cells we e excluded om he analysis. Simila esul s we e ob ained in
h ee independen expe imen s. (B) The con en o acuole-like esicles was isualized by elec on
mic oscopy 24 h pos - ea men wi h SB202190 (SB202). Con ol and SB202(1)—scale ba 5 µm.
SB202(2)—scale ba 1 µm. Two independen expe imen s showed simila esul s. (C) Quan i a i e
phase-imaging analysis o cellula d y mass in melanoma cells ea ed o 12 h wi h SB202190. The
ze o le el o d y mass densi y was de ined as he densi y o he obse a ion medium. The a eal
densi y dis ibu ion o he d y mass was quan i ied in p o iles ( igh panels) indica ed by da k lines.
Red a ows highligh he posi ion o acuoles. Two independen digi al holog aphy mic oscopy
expe imen s showed simila esul s.
The abo e esul s indica ed he possibili y ha SB202190-induced acuole-like s uc u es could
o m in esponse o a dis up ion o esicula endocy ic anspo , a molecula pa hway ha is
Figu e 2.
Py idinyl imidazole compounds induce acuoliza ion o cy oplasm in BRAF-mu a ed
human melanoma cells. A375 cells we e ea ed wi h DMSO (con ol), SB202190 (SB202; 10
µ
M), and
SB203580 (SB203; 10
µ
M). (
A
) Phase-con as ligh mic oscopy o li ing cells a e 12 h ea men wi h
py idinyl imidazole p38 MAPK inhibi o s. Scale ba : 45
µ
m. The ep esen a i e g aph shows he
mean numbe o acuoles pe cell quan i ied using ImageJ/Fiji ( ind maxima—b igh spo s abo e
a ce ain h eshold). Dying ounded cells we e excluded om he analysis. Simila esul s we e
ob ained in h ee independen expe imen s. (
B
) The con en o acuole-like esicles was isualized by
elec on mic oscopy 24 h pos - ea men wi h SB202190 (SB202). Con ol and SB202(1)—scale ba 5
µ
m.
SB202(2)—scale ba 1
µ
m. Two independen expe imen s showed simila esul s. (
C
) Quan i a i e
phase-imaging analysis o cellula d y mass in melanoma cells ea ed o 12 h wi h SB202190. The ze o
le el o d y mass densi y was de ined as he densi y o he obse a ion medium. The a eal densi y
dis ibu ion o he d y mass was quan i ied in p o iles ( igh panels) indica ed by da k lines. Red a ows
highligh he posi ion o acuoles. Two independen digi al holog aphy mic oscopy expe imen s
showed simila esul s.
Cance s 2020,12, 1516 7 o 24
Cance s 2020, 12, x 7 o 25
esponsible o he ac i e anspo o memb ane p o eins, including ecep o s egula ing g ow h
signaling, and o he up ake o i al nu ien s om he ex acellula en i onmen [38]. Small
molecule luo escen dyes such as ac idine o ange o LysoT acke G een DND-26 s ain acidic
compa men s in li ing cells, including endosomes and lysosomes. Bo h dyes eadily ma ked he
SB202190-induced acuoles in A375 melanoma cells, suppo ing he possibili y o hei
endolysosomal o igin (Figu e 3A,B). Using con ocal luo escence mic oscopy, we obse ed ha only
a small p opo ion o SB202190-induced acuoles wi h a diame e la ge han 1 µm exp ess ea ly
endosomal ma ke RAB5 (Figu e 3C). In con as , mos SB202190-induced acuoles wi h a diame e
la ge han 1 µm exp ess la e endosome ma ke RAB7 a hei su ace (Figu e 3D), indica ing ha he
majo i y o enla ged acuoles o igina ed om he la e endosome.
Mo eo e , suc ose-induced osmo ic s ess o he small molecule inhibi o 5-(N-E hyl-N-
isop opyl)amilo ide (EIPA), which inhibi s a o m o endocy osis called mac opinocy osis, p e en ed
he o ma ion o la ge acuoles in SB202190- ea ed cells (Figu e 3E). The nega i e impac o EIPA on
RAB7-posi i e acuole-like esicles was also obse ed using luo escence mic oscopy (Figu e S5).
These da a u he suppo ed he endocy ic o igin o he enla ged esicles. Fu he mo e, li e-cell
imaging indica ed ha mos SB202190-induced acuoles could no be ecycled back o he plasma
memb ane, sugges ing a block in la e s ages o endosomal a icking (Video S1).
Figu e 3. Vacuole-like esicles induced by py idinyl imidazole compounds in melanoma cells ha e
an endolysosomal o igin. A375 cells we e ea ed wi h SB202190 (SB202; 15 μM) o he equi alen
amoun o ehicle (DMSO) in con ol. (A) Cells we e ea ed wi h SB202190 o 20 h and s ained wi h
ac idine o ange (5 µg/mL) o 15 min. Scale ba : 50 µm. The g aph shows he ela i e change in he
yellow s ained a ea in esponse o SB202190. Simila esul s we e ob ained in wo independen
expe imen s. (B) Cells we e ea ed wi h SB202190 o 20 h and s ained wi h LysoT acke G een (50
nM) o 15 min. Scale ba : 50 µm. The g aph shows he ela i e change o he LysoT acke G een
signal in esponse o SB202190. Simila esul s we e ob ained in wo independen expe imen s. (C)
The con ocal mic oscopy de ec ion o endogenous ea ly endosomal ma ke RAB5 a e 24 h ea men
wi h SB202190. Scale ba : 20 µm. The g aph shows he numbe o RAB5-posi i e acuoles pe cell
wi h a diame e la ge han 1 µm. Simila esul s we e ob ained in h ee independen expe imen s.
(D) Decon ol ed wide- ield luo escence mic oscopy imaging o EGFP- agged la e
endosomal/lysosomal ma ke RAB7A a e 24 h SB202190 ea men . Scale ba : 10 µm. The diame e
Figu e 3.
Vacuole-like esicles induced by py idinyl imidazole compounds in melanoma cells ha e an
endolysosomal o igin. A375 cells we e ea ed wi h SB202190 (SB202; 15
µ
M) o he equi alen amoun
o ehicle (DMSO) in con ol. (
A
) Cells we e ea ed wi h SB202190 o 20 h and s ained wi h ac idine
o ange (5
µ
g/mL) o 15 min. Scale ba : 50
µ
m. The g aph shows he ela i e change in he yellow
s ained a ea in esponse o SB202190. Simila esul s we e ob ained in wo independen expe imen s.
(B) Cells we e ea ed wi h SB202190 o 20 h and s ained wi h LysoT acke G een (50 nM) o 15 min.
Scale ba : 50
µ
m. The g aph shows he ela i e change o he LysoT acke G een signal in esponse o
SB202190. Simila esul s we e ob ained in wo independen expe imen s. (
C
) The con ocal mic oscopy
de ec ion o endogenous ea ly endosomal ma ke RAB5 a e 24 h ea men wi h SB202190. Scale ba :
20
µ
m. The g aph shows he numbe o RAB5-posi i e acuoles pe cell wi h a diame e la ge han
1
µ
m. Simila esul s we e ob ained in h ee independen expe imen s. (
D
) Decon ol ed wide- ield
luo escence mic oscopy imaging o EGFP- agged la e endosomal/lysosomal ma ke RAB7A a e 24
h SB202190 ea men . Scale ba : 10
µ
m. The diame e o RAB7-posi i e s uc u es was de e mined,
and he numbe o acuoles la ge han 1
µ
m pe cell was plo ed in he g aph. The expe imen was
pe o med h ee imes wi h simila esul s. (
E
) E ec on acuoliza ion was isualized by b igh - ield
images in cells ea ed o 20 h wi h SB202190 alone and in combina ion wi h suc ose (0.5 M) o EIPA (50
µ
M). Scale ba : 50
µ
m. The g aph shows he numbe o acuoles pe cell, quan i ied using ImageJ/Fiji
( ind maxima—b igh spo s abo e a ce ain h eshold). Dying ounded cells we e excluded om he
analysis. The expe imen was epea ed h ee imes wi h a simila esponse o he addi ion o suc ose
and EIPA.
2.3. Py idinyl Imidazole Compounds Pa ly Mimic PIK y e Inhibi ion in Melanoma Cells
The appa en de ec in he endocy ic pa hway p omo ing a ma ked inc ease in he olume
o endolysosomes led us o sea ch he li e a u e o a simila pheno ype. Inhibi ion o he FYVE
inge -con aining phosphoinosi ide kinase (PIK y e) had been p e iously epo ed o induce ex ensi e
cy oplasmic acuoliza ion in some cell ypes [
39
,
40
]. In A375 melanoma cells, he pheno ype induced
by a small-molecule PIK y e inhibi o YM201636 was s ikingly simila o ha induced by SB202190,
including he cha ac e is ic accumula ion o la ge esicles (Figu e 4A). As he BRAF inhibi o SB590885
bea s signi ican s uc u al simila i y o p38 MAPK inhibi o s SB202190 and SB203580, we hypo hesized
ha i migh also be capable o a ge ing endolysosomal a icking. Indeed, we also obse ed he
Cance s 2020,12, 1516 8 o 24
accumula ion o la ge acuole-like esicles in SB590885- ea ed A375 melanoma cells (Figu e 4A).
Fu he mo e, a small molecule inhibi o o acuola H+ATPase ba ilomycin A1 ha can p e en he
o ma ion o acuoles in esponse o PIK y e inhibi ion [
41
] also blocked he acuoliza ion induced by
py idinyl imidazole compounds (Figu e 4A).
Cance s 2020, 12, x 8 o 25
o RAB7-posi i e s uc u es was de e mined, and he numbe o acuoles la ge han 1 μm pe cell
was plo ed in he g aph. The expe imen was pe o med h ee imes wi h simila esul s. (E) E ec
on acuoliza ion was isualized by b igh - ield images in cells ea ed o 20 h wi h SB202190 alone
and in combina ion wi h suc ose (0.5 M) o EIPA (50 μM). Scale ba : 50 µm. The g aph shows he
numbe o acuoles pe cell, quan i ied using ImageJ/Fiji ( ind maxima—b igh spo s abo e a ce ain
h eshold). Dying ounded cells we e excluded om he analysis. The expe imen was epea ed h ee
imes wi h a simila esponse o he addi ion o suc ose and EIPA.
2.3. Py idinyl Imidazole Compounds Pa ly Mimic PIK y e Inhibi ion in Melanoma Cells
The appa en de ec in he endocy ic pa hway p omo ing a ma ked inc ease in he olume o
endolysosomes led us o sea ch he li e a u e o a simila pheno ype. Inhibi ion o he FYVE inge -
con aining phosphoinosi ide kinase (PIK y e) had been p e iously epo ed o induce ex ensi e
cy oplasmic acuoliza ion in some cell ypes [39,40]. In A375 melanoma cells, he pheno ype induced
by a small-molecule PIK y e inhibi o YM201636 was s ikingly simila o ha induced by SB202190,
including he cha ac e is ic accumula ion o la ge esicles (Figu e 4A). As he BRAF inhibi o
SB590885 bea s signi ican s uc u al simila i y o p38 MAPK inhibi o s SB202190 and SB203580, we
hypo hesized ha i migh also be capable o a ge ing endolysosomal a icking. Indeed, we also
obse ed he accumula ion o la ge acuole-like esicles in SB590885- ea ed A375 melanoma cells
(Figu e 4A). Fu he mo e, a small molecule inhibi o o acuola H+ ATPase ba ilomycin A1 ha can
p e en he o ma ion o acuoles in esponse o PIK y e inhibi ion [41] also blocked he
acuoliza ion induced by py idinyl imidazole compounds (Figu e 4A).
Figu e 4.
Pheno ype induced in melanoma cells by py idinyl imidazole compounds pa ly mimics
PIK y e inhibi ion. A375 cells we e ea ed 24 h wi h py idinyl imidazole inhibi o s SB202190 (SB202; 15
µ
M),SB590885(SB590; 5
µ
M),andPIK y einhibi o YM201636(YM;1
µ
M).(
A
)Aphase-con as analysis
o he acuoliza ion induced by he inhibi o s and he e ec o he co ea men wi h ba ilomycin A (Ba A;
50 nM). Scale ba : 50
µ
m. Vacuoles pe cell we e quan i ied using ImageJ/Fiji ( ind maxima—b igh
spo s abo e a ce ain h eshold) and p esen ed in he g aph. Dying ounded cells we e excluded
om he analysis. Th ee independen expe imen s showed a simila e ec o he addi ion o Ba A.
(
B
) Con ocal mic oscopy analysis o luo escen p o ein-labeled endosomal ma ke (mChe y-Endo-14).
Scale ba : 20
µ
m. The a ea o mChe y-posi i e acuola s uc u es was quan i ied in each ea men
and plo ed in he g aph. Th ee independen expe imen s showed simila esul s. (
C
) Con ocal
luo escence mic oscopy de ec ion o endogenous phospha idylinosi ol 3,5-bisphospha e (PI(3,5)P2).
Scale ba : 10
µ
m. Th ee images o di e en pa s o he same specimen we e acqui ed and analyzed
using ImageJ/Fiji. Images we e h esholded by he signal in ensi y, and he amoun o PI(3,5)P2 oci pe
cell was de e mined. Simila esul s we e ob ained in wo independen expe imen s.
Cance s 2020,12, 1516 9 o 24
Nex , we ec opically exp essed he mChe y- agged endosomal ma ke RhoB (mChe y-Endo-14)
in A375 melanoma cells. Using con ocal luo escence mic oscopy, we ound ha i localized o he
memb anes o esicles induced by he inhibi o o PIK y e as well as esicles induced by he py idinyl
imidazole compounds (Figu e 4B). PIK y e is esponsible o he syn hesis o phospha idylinosi ol
3,5-bisphospha e (PI(3,5)P2) [
42
], and his lipid appea s o be c i ical o he p ope ma u a ion o
endosomes [
43
]. To ou su p ise, he cellula s aining pa e ns de ec ed by immuno luo escence wi h
an an i-PI(3,5)P2 an ibody in YM201636- and py idinyl imidazole- ea ed A375 cells we e e y simila
(Figu e 4C). Collec i ely, hese esul s sugges ed ha he py idinyl imidazole compounds and he
PIK y e inhibi o YM201636 caused a e y simila de ec in endocy osis, po en ially by a ge ing
simila signaling pa hways. Ne e heless, whe he hese changes migh impac on he g ow h and
su i al o cance cells emained unclea .
2.4. Py idinyl Imidazole Compounds Induce Changes in mTOR Subcellula Localiza ion in A375
Melanoma Cells
B idges e al. epo ed ha Rap o , an essen ial subuni o mTORC1, could in e ac wi h PI(3,5)P2,
and PIK y e was necessa y o he subcellula localiza ion and ac i a ion o mTORC1 in 3T3-L1
adipocy es [
40
]. The ac i a ion o mTORC1 equi es he ansloca ion o he mTOR kinase o he
lysosomalsu ace [
22
]. Thep ocessin ol esin e ac ions be ween lysosomal -ATPaseand apen ame ic
p o ein complex called Ragula o [
44
]. This complex possesses a guanine nucleo ide exchange ac o
(GEF) ac i i y owa ds he Rag GTPases ha can ec ui mTORC1 o ac i a ion a he lysosomal
su ace [19,45,46].
We hypo hesized ha changes induced in he endolysosomal compa men o melanoma cells
in esponse o PIK y e inhibi ion and py idinyl imidazole compounds migh in e e e wi h mTOR
subcellula localiza ion. To s udy his possibili y, we exp essed in A375 cells EGFP- agged lysosomal
Ragula o complex p o ein p18/LAMTOR1 and analyzed i s colocaliza ion wi h endogenous mTOR
kinase using luo escence mic oscopy. P o ein p18/LAMTOR1 se es as a sca old o he assembly o he
Ragula o –Rag GTPase complex and is esponsible o i s ancho ing o he lysosomal memb ane [
47
].
The mTOR-p18/LAMTOR1 colocaliza ion pa e n was also analyzed in cells ea ed wi h d ugs
a ge ing he mTORC1 kinase (Rapamycin), p38 MAPK (BIRB-796), and ERK MAPK signaling (U0126,
Vemu a enib). E en hough he A375 melanoma cells we e g own in ull media, he PIK y e inhibi o ,
as well as he py idinyl imidazole compounds SB202190 and SB590885, all seemed o dis up lysosomal
mTOR a ge ing as he mTOR s aining pa e n changed om do -like s uc u es o di use cy oplasmic
s aining (Figu e 5A). Impo an ly, a swi ch o mTOR di use s aining pa e n upon he ex ended
ea men was no obse ed in esponse o he p38 MAPK inhibi o BIRB796, he MEK kinase
inhibi o U0126, o he BRAF inhibi o emu a enib. The esul s indica ed ha he obse ed e ec o
py idinyl imidazole compounds was likely no linked o hei capaci y o inhibi p38 and ERK MAPK
signaling pa hways (Figu e 5A). In e es ingly, he esul s o he con ocal mic oscopy sugges ed ha
p18/LAMTOR1 migh no be e enly dis ibu ed on he su ace o enla ged endolysosomes induced by
py idinyl imidazoles. The Ragula o complex p o ein appea ed o o m clus e s on he su ace o he
la ge acuole like s uc u es (Figu e 5A). A de ailed ime-lapse mic oscopy analysis indica ed ha
p18/LAMTOR1 migh p e e en ially clus e a he in e ace o he la ge acuoles (Video S2).
The mTORC1 ac i i y di ec ly egula es he subcellula localiza ion o he ansc ip ion ac o EB
(TFEB), a mas e egula o o lysosomal biogenesis [
48
]. S a a ion, dis up ion o lysosomal unc ion,
and pha macological inhibi ion o mTORC1 can s imula e TFEB-dependen ansc ip ion by p omo ing
TFEB nuclea ansloca ion [
49
,
50
]. In e es ingly, PIK y e inhibi ion was also shown o induce nuclea
accumula ion o TFEB [
51
,
52
]. In ou expe imen s, he PIK y e inhibi o YM201636 and py idinyl
imidazole inhibi o s SB202190 and SB590885 all p omo ed nuclea localiza ion o TFEB (Figu e 5B),
indica ing ha he d ug-induced dis up ion o mTOR lysosomal e he ing could inhibi mTORC1
ac i i y in melanoma cells.
Cance s 2020,12, 1516 16 o 24
4. Ma e ials and Me hods
4.1. Cell Cul u e and T ea men s
Human melanoma cell lines A375, G361, COLO-800, MEL-JUSO, SK-MEL-30, and IPC-298 we e
pu chased om he Eu opean Collec ion o Cell Cul u es (ECACC; Salisbu y, UK). All cell lines we e
main ained a 37
◦
C in a humidi ied a mosphe e con aining 5% CO2. IPC-298, MEL-JUSO, COLO-800,
and A375 cells we e cul u ed in RPMI-1640 (Sigma-Ald ich, P ague, Czech Republic). G361 cells we e
p opaga ed in McCoy
0
s 5a (The mo Fishe Scien i ic, P ague, Czech Republic) and SK-MEL-30 in
Dulbecco’s modi ied Eagle’s medium (The mo Fishe Scien i ic). G ow h media we e supplemen ed
wi h 10% e al bo ine se um (FBS), 2 mM L-glu amine, penicillin (100 IU/mL), and s ep omycin
(100
µ
g/mL). The epo e cell line o measu ing ERK pa hway ac i i y was p epa ed by s able
ans ec ion o A375 cells wi h pK ox24(MapE k)Luc plasmid cons uc [
35
]. Cells we e egula ly
checked o mycoplasma con amina ion using Mycoplasma De ec ion Ki (Bio ool, Munich, Ge many)
and DAPI s aining ollowed by luo escence mic oscopy.
The ollowing compounds we e used o cell ea men s: SB202190, SB239063, suc ose, EIPA,
hapsiga gin, and unicamycin (Sigma-Ald ich); U0126 (Wako Chemicals, Neuss, Ge many); SB203580,
BIRB796, PD184352, YM201636, and apamycin (Selleckchem, Munich, Ge many); emu a enib
(Tinib-Tools, Olomouc, Czech Republic); SB590885 (MedChemExp ess, Monmou h Junc ion, NJ, USA);
and pu omycin (Cayman Chemical, Ann A bo , MI, USA). S ock solu ions o he compounds we e
p epa ed in dime hyl sul oxide (DMSO). Inhibi o s ocks we e dilu ed in p e-wa med cell cul u e
medium and added o he cells. Con ols ecei ed he co esponding amoun o he ehicle.
4.2. Wes e n Blo ing
Cells we e lysed in 2
×
Laemli sample bu e , and p o eins in o al cell lysa es we e sepa a ed by
SDS-polyac ylamide gel elec opho esis (10 o 15% ac ylamide) using mini e ical elec opho esis
uni SE250 (Hoe e , Hollis on, MA, USA). P o eins we e ans e ed o poly inylidene luo ide (PVDF)
memb anes (Me ck Millipo e, P ague, Czech Republic) in he T ans-Blo SD semi-d y ans e sys em
(Bio-Rad, P ague, Czech Republic). Memb anes we e blocked wi h 5% non- a milk in is-bu e ed
saline +0.1% Tween 20 (TBST) o one hou a oom empe a u e and incuba ed wi h p ima y an ibodies
o e nigh a 4
◦
C. The nex day, memb anes we e washed 3
×
10 min in TBST and incuba ed wi h
seconda y an ibodies o one hou a oom empe a u e. P o eins o in e es we e isualized wi h
enhanced chemiluminescence (ECL) subs a e (The mo Fishe Scien i ic) in he G:BOX de ec ion sys em
(Syngene, Camb idge, UK). The in ensi y o bands was quan i ied using ImageJ/Fiji. O iginal da a a e
a ailable in Figu e S7.
P ima yan ibodies used o Wes e nblo : mousean i-
α
- ubulin (B-7; sc-5286), abbi an i-pMEK1/2
(sc-7995), goa an i-MEK1 (C-18; sc-219), mouse an i-BRAF (F-7; sc-5284) (San a C uz Bio echnology),
abbi an i-p70 S6K (#2708), abbi an i-phospho-p70 S6K (#9234), abbi an i-phospho-S6 Se 235/236
(#4858), abbi an i-S6(#2217), abbi an i-phospho-ERK1/2T202/Y204 (#4370), abbi an i-ERK1/2(#9102)
(Cell Signaling Technology, Dan e s, MA, USA), mouse an i-Pu omycin (MABE343) (Sigma-Ald ich),
and mouse an i-PCNA (PC-10), kindly p o ided by D . Boˇ i oj Voj ˇešek (Masa yk Memo ial Cance
Ins i u e, B no, Czech Republic). Seconda y an ibodies conjuga ed o ho se adish pe oxidase (HRP):
donkey an i- abbi (sc-2357), an i-mouse (sc-516102), and an i-goa (sc-2020) (San a C uz Bio echnology,
Heidelbe g, Ge many). All an ibodies we e used acco ding o he manu ac u e s’ ecommenda ions.
4.3. In Vi o Kinase Assays
Melanoma cells (A375) we e seeded a 10 cm pla e, washed once wi h ice-cold phospha e-bu e ed
saline (PBS), sc aped ou , and lysed as p e iously desc ibed [
82
,
83
]. The lysa e was cen i uga ed a
13,000
×
g o 25 min a 4
◦
C. Mouse an i-BRAF (sc-5284; San a C uz Bio echnology) an ibody was
added (1
µ
g pe each sample) o he lysa e and incuba ed o 1–2 h a 4
◦
C on a slow o a o . P o ein G
Sepha ose 4 Fas Flow beads (GE Heal hca e, Chicago, IL, USA) we e added and incuba ed o ano he

Cance s 2020,12, 1516 17 o 24
2–3 h a 4
◦
C on a slow o a o . Beads–immune complexes we e washed as p e iously desc ibed [
82
,
83
].
The kinase assay was pe o med in he p esence o 20
µ
M ATP and 500 ng o a ecombinan human
inac i e MEK-1 as he subs a e (Li e Technologies, P ague, Czech Republic). A co esponding amoun
o DMSO (con ol) o SB202190 (5 µM) was added o he eac ion pe o med o 40 min a 30 ◦C wi h
gen le agi a ion. The kinase eac ions we e s opped by he addi ion o 20
µ
L 2
×
SDS sample bu e ,
analyzed using Wes e n blo , and densi y o bands was quan i ied ia ImageJ/Fiji.
4.4. T ansien T ans ec ions and Fluo escence Mic oscopy
The day a e seeding he cells o a glass co e slip, cells we e ans ec ed using Tu boFec
ans ec ion eagen (The mo Fishe Scien i ic) o FuGENE HD (P omega, Madison, WI, USA) ollowing
he manu ac u e ’s ins uc ions. Twen y- ou hou s pos ans ec ions, cells we e ea ed o he
indica ed ime, ixed wi h 4% pa a o maldehyde, pe meabilized wi h 0.1% T i on X, and incuba ed
wi h p ima y an ibodies a 4
◦
C o e nigh . Seconda y an ibodies we e used a oom empe a u e o
one hou . Then, co e slips we e s ained wi h DAPI (sc-3598; San a C uz Bio echnology) and washed
h ee imes wi h PBS. Images we e acqui ed using an in e ed con ocal mic oscope Ca l Zeiss LSM 700
(Jena, Ge many) wi h Plan-Apoch oma 63x/1.4 Oil DIC M27 objec i e (Zeiss) and p ocessed in ZEISS
ZEN Mic oscope So wa e o ImageJ/Fiji. Quan i ica ion was pe o med using a leas h ee echnical
eplica es o ake in o accoun he a iabili y owing o di e ences in mic oscopy se ings. All analyses
we e pe o med in a leas wo independen expe imen s.
Li e cell imaging: Melanoma cells (A375) we e seeded on Ma Tek 35 mm glass-bo om dishes
(Ma Tek Co po a ion, Ashland, MA, USA), and he nex day ans ec ed using FuGENE HD (P omega)
acco ding o he manu ac u e ’s ins uc ions. Cells we e washed ou hou s la e and ea ed wi h
he inhibi o (SB202190, 15
µ
M) o 24 h. Fo imaging, he media was eplaced wi h wa m Li e Cell
Imaging bu e (In i ogen) con aining he inhibi o (SB202190, 15
µ
M) and supplemen ed wi h 20 mM
glucose. Li e cell imaging was pe o med on he Del a ision OMX V4 mic oscope (GE Heal hca e)
equipped wi h h ee wa e -cooled PCO.edge sCMOS came as, a solid-s a e ligh sou ce, and lase -based
au o ocus. Hea ed s age and an objec i e hea e (20/20 Technologies, Wilming on, NC, USA) p o ided
en i onmen al con ol condi ions. Images we e decon ol ed using so WoRx so wa e and p ocessed
in ImageJ/Fiji [84,85].
Dyes used o cell s aining: LysoT acke G een DND-26 (The mo Fishe Scien i ic), Ac idine
O ange hemi(zinc chlo ide) sal (A6014, Sigma-Ald ich).
The ollowing p ima y an ibodies we e used o immuno luo escence: abbi an i-mTOR (#2983),
abbi an i-Rab5 (#3547; Cell Signaling Technology), mouse an i-P dIns(3,5)P2 (Z-P035; Echelon
Biosciences, Sal Lake Ci y, UT, USA). An i- abbi Alexa Fluo 594/488 (A11012/A11008) and an i-mouse
Alexa Fluo 488 (A11001) (Li e Technologies) we e used as seconda y an ibodies.
Lis o plasmids: mChe y-Endo-14 (Addgene, #55040), EGFP-Rab7A (Addgene, #28407),
N1-p18-EGFP (Addgene, #42334), and EGFP-N1-TFEB (Addgene, #38119).
4.5. Quan i a i e Phase Imaging Analysis Using Cohe ence-Con olled Holog aphic Mic oscopy
T ansmi ed-ligh cohe ence-con olled holog aphic mic oscope (CCHM) was used o e alua e
he dis ibu ion o he d y mass (DM) (e.g., p o eins, ca bohyd a es, a s, amino acids, and nucleic
acids) densi y in A375 cells and he d ug-induced acuoles. The mic oscope was designed and buil a
he Ins i u e o Physical Enginee ing (IPE) and he Cen al Eu opean Ins i u e o Technology (CEITEC),
B no Uni e si y o Technology. The se up o he mic oscope and he p inciple behind he image
econs uc ion ha e p e iously been desc ibed in de ail [
86
]. The CCHM quan i a i e phase images a e
o med by he phase shi be ween he objec and e e ence wa e de ec ed using he in e e ence o ligh .
A e e y image poin , he phase shi alue is di ec ly p opo ional o he cell d y-mass densi y [87].
Fo ob aining he images, we used 20
×
/NA =0.4 objec i es, a digi al CCD came a As opix
1.4, and so wa e de eloped a he IPE and CEITEC. The ligh sou ce was a halogen lamp spec ally
es ic ed by an in e e ence il e (FWHM =10 nm, maximum ansmissi i y a 650 nm).
Cance s 2020,12, 1516 18 o 24
Melanoma cells (A375) we e obse ed li e in he imaging chambe wi h 5% CO2. Be o e imaging,
he cul u e medium was eplaced wi h an obse a ion medium: Eagle’s minimal essen ial medium
wi hou phenol ed (bu e ed o pH 7.4) (Sigma-Ald ich), supplemen ed wi h 10 % FBS and only
one- hi d o he no mal concen a ion o sodium bica bona e.
4.6. Elec on Mic oscopy
A375 melanoma cells we e seeded on 35 mm pla es, g own o 24 h and ha es ed 12 h a e
ea men , washed h ee imes in 0.1 M cacodyla e bu e (pH 6.98), and ixed o 2.5 h in 3%
glu a aldehyde solu ion (p epa ed in 0.1 M cacodyla e bu e con aining 0.2 M saccha ose). A e wa d,
pos - ixa ion was pe o med in 1% osmium e oxide in 0.1 M cacodyla e bu e o 2.5 h a oom
empe a u e. Indi idual sec ions we e s ained wi h 2.5% u anyl ace a e (6 min), lead ci a e (3 min),
and obse ed using ansmission elec on mic oscopy (Philips Mo gagni, FEI Company, Eindho en,
The Ne he lands).
4.7. Quan i ica ion o In acellula Me aboli es Using NMR Spec oscopy
Ace oni ile ex ac ion was employed o quench cell me abolism and o ex ac low molecula
weigh compounds om A375 melanoma cells quan i a i ely [
83
,
88
]. Following emo al o ace oni ile
ia acuum concen a ion, d ied ex ac s we e esuspended in 550
µ
L o D
2
O (Sigma-Ald ich)
con aining 0.005% sodium 3-( ime hylsilyl)-p opiona e-2,2,3,3-d4 (TSP) (Sigma-Ald ich) used as bo h
chemical shi e e ence and in e nal s anda d o me aboli e quan i ica ion.
In o ma ion on he concen a ion o me aboli es in indi idual samples was de i ed om olumes
o co esponding signals in 1D 1H NMR spec um. The assignmen o signals in he NMR spec a
o indi idual samples o a me aboli e was achie ed ia a compa ison o a sample spec um wi h
spec a o pu e me aboli es (Sigma-Ald ich). The 1D 1H spec a we e measu ed a 700 MHz using a
B uke A ance III NMR spec ome e (B uke , Bille ica, MA, USA) equipped wi h a iple esonance
oom empe a u e p obe using he zgp pulse sequence (s anda d B uke pulse p og am lib a y). All
spec a we e acqui ed a 20
◦
C and p ocessed using TopSpin 3.2 (B uke ). To make he compa ison o
me aboli e concen a ion p o iles among a ious samples possible, he signal in ensi ies in indi idual
samples we e no malized o o al p o ein concen a ion.
4.8. Flow Cy ome y and MTT P oli e a ion Assay
Fo cell iabili y assay, melanoma cells we e collec ed 48 h pos - ea men and washed once
wi h ice-cold PBS. Cell pelle s we e esuspended in ice-cold PBS, and 1
µ
g/mL p opidium iodide (PI)
(Sigma-Ald ich) was added o he suspension and cell luo escence was measu ed using he A une
Acous ic Focusing Cy ome e (The mo Fishe Scien i ic). As li e cells exclude PI, he pe cen age o
dead cells was calcula ed based on he p opo ion o PI-posi i e cells in he popula ion.
Up ake o BSA was measu ed in A375 melanoma cells, seeded in densi y 200,000 cells pe well on
a six-well pla e. The nex day, cells we e ea ed wi h es ed d ugs o one hou , and hen 10 ug/mL o
DQ Red BSA (The mo Fishe Scien i ic, D12051) was added o he medium o 30 min. A e wa d,
cells we e chased o 75 min in DQ-BSA ee medium (wi h inhibi o s) and collec ed. Flow cy ome y
analysis was pe o med on LSR II low cy ome e (BD Biosciences, San Jose, CA, USA) using FACS
Di a (BD Biosciences) so wa e.
MTT assay was used o measu e cellula me abolic ac i i y as an indica o o cell iabili y
and p oli e a ion o melanoma cell lines (A375, G361, COLO-800, MEL-JUSO, SK-MEL-30,
IPC-298). Cells we e seeded a a densi y o 1–2
×
10
3
cells/well in a 96-well pla e and g own
o e nigh . A e ea men wi h inhibi o s o 48 h, cells we e incuba ed wi h 0.5 mg/mL MTT
(3-(4,5-dime hyl hiazol-2-yl)-2,5-diphenyl e azolium b omide) o 4 h a 37
◦
C. A e wa d, cells we e
cen i uga ed, and he wa e -insoluble o mazan p oduc was dissol ed in DMSO (200
µ
L/well). The
abso bance a 570 nm was de e mined using a mic opla e eade Ve saMax (Molecula De ices, San
Jose, CA, USA).
Cance s 2020,12, 1516 19 o 24
4.9. S a is ical Analysis
The analyses we e pe o med using G aphPad P ism 7 (G aphPad So wa e, San Diego, CA, USA).
Th ee o mo e independen expe imen s we e pe o med o each da a se , ep esen ed as mean +SD.
S a is ical analysis was done using S uden ’s - es o analysis o a iance (ANOVA) when mul iple
samples we e compa ed. Values o * p<0.05, ** p<0.01, *** p<0.001, and **** p<0.0001 we e
conside ed s a is ically signi ican .
5. Conclusions
We iden i ied py idinyl imidazole compounds SB2020190, SB203580, and SB590885 as dual
inhibi o s o mu an BRAF kinase and mTOR signaling in melanoma cells. The dual a ge ing o
essen ial p o-g ow h pa hways in melanoma cells indica es he po en ial o he de elopmen o BRAF
inhibi o s based on he py idinyl imidazole co e ha could be less p one o he de elopmen o acqui ed
d ug esis ance. Mo eo e , he dis up ion o he endolysosomal compa men by py idinyl imidazole
d ugs can sensi ize melanoma cells o ER s esso s, u he unde sco ing hei he apeu ic po en ial.
Supplemen a y Ma e ials:
The ollowing a e a ailable online a h p://www.mdpi.com/2072-6694/12/6/1516/s1,
Figu e S1: Addi ional independen eplica es o all Wes e n blo s p esen ed in his s udy; Figu e S2: Py idinyl
imidazole compounds inhibi ERK signaling in A375 melanoma cells; Figu e S3: Analysis o he p oli e a ion o
BRAF- and NRAS-mu a ed melanoma cell lines in he p esence o py idinyl imidazole compounds; Figu e S4:
Cohe ence-con olled holog aphic mic oscopy; Figu e S5: Immuno luo escence mic oscopy; Figu e S6. Nu ien
up ake was nega i ely a ec ed in A375 melanoma cells ea ed wi h py idinyl imidazole compounds; Figu e S7:
Unc opped o iginal Wes e n blo s; Video S1: SB202190 dis up s la e endosomal a icking in melanoma cells:
Decon ol ed wide- ield li e-cell luo escence mic oscopy o A375 melanoma cells ansien ly ans ec ed wi h a
plasmid cons uc encoding EGFP-labeled la e endosomal ma ke RAB7A (EGFP-Rab7A); Video S2: p18/LAMTOR1
clus e s a con ac si es o SB202190-induced acuole-like esicles: Li e-cell imaging was pe o med using A375
cells ansien ly ans ec ed wi h a plasmid cons uc encoding EGFP-labeled LAMTOR1 (N1-p18-EGFP).
Au ho Con ibu ions:
Concep ualiza ion, V.P. and S.U.; alida ion, V.P., T.R., M.K. (Michaela K a ˇc
í
ko
á
), and
S.U.; o mal analysis, V.P., S.U., and K.O.S.; in es iga ion, V.P., T.R., T.V., M.M., L.C., M.K. (Mi iama K u
á
),
M.S., H.H., I.S., H.U., A.K., V.R., K.O.S., and S.U.; esou ces, A.V., L.T., R.C., M.S., and P.V.; w i ing—o iginal
d a p epa a ion, V.P. and S.U.; w i ing— e iew & edi ing, V.P. and S.U.; isualiza ion, V.P., T.R., and K.O.S.;
supe ision, V.R., L.T., R.C., P.V., K.O.S., and S.U. All au ho s ha e ead and ag eed o he published e sion o
he manusc ip .
Funding:
This esea ch was suppo ed by he Eu opean Regional De elopmen Fund—P ojec ENOCH (No.
CZ.02.1.01/0.0/0.0/16_019/0000868), he Speci ic Uni e si y Resea ch (MUNI/A/1087/2018), he Czech Science
Founda ion (18-01396S), and he Czech-Bioimaging: Na ional In as uc u e o Biological and Medical Imaging
(LM2018129). L.T. was suppo ed by a g an om he Minis y o Heal h o he Czech Republic (NV19-08-00450).
Access o he NMR in as uc u e was suppo ed by a g an om he MEYS (CIISB – LM2018127).
Acknowledgmen s:
We wan o hank Dob omila Klemo
á
o elec on mic oscopy sample p epa a ion; Lucie
Šupol
í
ko
á
o echnical assis ance; Boˇ i oj Voj ˇešek (Masa yk Memo ial Cance Ins i u e, B no, Czech Republic)
o he an i-PCNA an ibody; and Pa el K ejˇc
í
, Michael Da idson, Qing Zhong, Da id Saba ini, and Shawn
Fe guson o p o iding plasmid cons uc s. The Flow Cy ome y Co e Facili y a Oslo Uni e si y Hospi al is
g a e ully acknowledged o help wi h he DQ-BSA up ake assay. We also hank he Co e Facili y o Ad anced
Ligh Mic oscopy a Oslo Uni e si y Hospi al o help wi h li e-cell imaging.
Con lic s o In e es : The au ho s decla e no con lic o in e es .
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